Preface

Chapter 9 **Autologous Platelet-Rich Plasma and Mesenchymal Stem Cells for the Treatment of Chronic Wounds 149**

Chapter 10 **The Wound Healing Responses and Corneal Biomechanics after**

Chapter 12 **Facilitation of Wound Healing Following Laparoscopic and**

Rebekah Amarini, Sufan Chien and Girish J. Kotwal

Chapter 13 **Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal Substitutes in Chronic**

Silvia Izzo, Lidia De Felice and Bruno Salvati

Chapter 14 **The Impact of Biofilm Formation on Wound Healing 235**

Juan José Santivañez Palomino, Arturo Vergara and Manuel

**Conventional Abdominal Surgery with Dressings, Patches,**

Raffaele Capoano, Rita Businaro, Besar Kolce, Andrea Biancucci,

Rafael A. Mendoza, Ji-Cheng Hsieh and Robert D. Galiano

Peter A. Everts

**VI** Contents

Cadena

**Antibiotics, etc. 203**

**Phlebostatic Ulcers 215**

**Keratorefractive Surgery 181** Wenjing Wu and Yan Wang

Chapter 11 **Open Abdomen: The Surgeons' Challenge 189**

Wound healing and its treatment are subjects that have been discussed for centuries in the medical literature. Wounds are everywhere, occurring in the young and elderly and in hos‐ pital and at home, and affect patients in every clinical specialty around the world. With an improved understanding of the wound healing process, regenerative treatments have been developed. In the historical process, the pathophysiology of wound healing was better un‐ derstood, especially with advances in cellular and molecular techniques. The factors affect‐ ing wound healing are: size, site, and shape of the wound, injury method, agents used and recurring trauma, foreign objects, hematoma or seroma, heat, amount of oxygen, smoking, infection, nutritional factors, medicines, radiotherapy, and systemic diseases.

In the chapter by Fernando Pereira Beserra et al., the authors review the pathway in the skin healing cascade, relating the major chemical inflammatory mediators, cellular and molecular. Local and systemic factors that interfere with healing and disorders associated with tissue repair deficiency in chronic inflammations, burns, and hypertrophy are also demonstrated. In the third chapter by Christian Agyare et al., biomarkers are discussed relevant to the wound healing process. Non-healing wounds are also identified, where biomarker-guided approaches may be of clinical importance in their management. The fourth chapter by Mo‐ hammad Reza Farahpour examines medicinal plants in wound healing and shows wound healing effects by different mechanisms, such as modulation in wound healing, decreasing bacterial count, improving collagen deposition, increasing fibroblasts and fibrocytes, etc.

The fifth chapter by Victor Y. A. Barku deals with plant secondary metabolite antioxidants and briefly reviews antioxidant properties of medicinal plants to highlight the important roles medicinal plants play in wound healing. The sixth chapter by Juin-Hong Cherng dis‐ cusses the detailed mechanisms and efficacy of natural polysaccharides in accelerating the wound healing process, thereby encouraging the advanced strategies for future wound management. The seventh chapter by Aragona Salvatore Emanuele et al. provides an inter‐ esting overview of wound healing: from tissue repair to tissue regeneration. They define wound repair as the incomplete regeneration of the original tissue with hyperproduction of organized collagen, which can lead to the production of new tissue with an 80% similarity to the original tissue.

The eighth chapter by Diego Caicedo and Jesús Devesa focuses on a large amount of experi‐ mental and clinical evidence on the action of growth hormones in wound repair and ana‐ lyzes how the physiological rhythm of growth hormone secretion influences this process. It also looks at one of the most important signaling pathways that mediates the effects of growth hormones on tissue regeneration. The ninth chapter by Peter A. Everts deals with both platelet-rich plasma and mesenchymal stem cell applications. These have the potential

to become effective and ideal autologous biological cell-based therapies, which can be ap‐ plied to chronic wounds to effectively change the wound bed microenvironment to enable and accelerate wound closure.

The tenth chapter by Yan Wang and Wenjing Wu discusses wound healing and the biome‐ chanics of corneal refractive procedures to better understand corneal wound healing from the biomechanical viewpoint. This is mandatory if refractive surgery is ever to achieve more predictable and safer refractive results. The eleventh chapter by Manuel Cadena and Juan José Santivañez provides a comprehensive review of the open abdomen. This is the most challenging of wounds that a surgeon faces because of the metabolic, physiological, and dy‐ namic implications that this condition entails. The twelfth chapter by Girish J. Kotwal et al. discusses facilitation of wound healing with current general wound care, following laparo‐ scopic and conventional surgery with dressings, patches, antibiotics, etc. The last chapter by Raffaele Capoano et al. addresses multidisciplinary approaches to the stimulation of wound healing and use of dermal substitutes in chronic phlebostatic ulcers.

The purpose of presenting this book is to provide an insight into current perspectives on wound healing processes.

> **Kamil Hakan Dogan** Selcuk University Faculty of Medicine Department of Forensic Medicine Konya, Turkey

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: An Overview of Wound Healing**

Wound is the deterioration of the normal integrity of the body by the physical damage of any agent. Erosion, ulcer, and fissure expressions are used in wound statement. Erosion is an expression that determines the focal epidermis losses that do not go into the dermis. Fissure is the tissue loss that determines vertical fractures in the form of cracks and it can hold the epidermis and/or dermis. Ulcers are focal wounds with the dermis and tissue loss in the epidermis. Ulcers may become chronic and may cause difficult treatment for clinicians. The wound healing progress depends on many factors ranging from the general condition of the patient to the treatment and the cause of the wound. Wound healing, complications, and scar development

One of the results of advances in medical technology is increased longevity; associated with this is an increased prevalence of chronic diseases and consequently chronic wounds. There is a need to provide an evidence-based approach to the management of chronic wounds. The amount of knowledge about the processes of wound healing has significantly increased in recent years. It has become more difficult to select the most appropriate therapy for a specific type of wound. Wound healing overlaps into the many disciplines of medicine in general. Dermatologists, surgeons, internists, and geriatricians are becoming increasingly involved in the field of wound care. General practitioners and family physicians are frequently required to treat acute and chronic wounds.

There are two types of wounds: acute and chronic. After trauma or excisional surgery, acute wounds result. If the wound does not heal within 6 weeks, it is chronic. The factors such as involvement of underlying structures, depth of wound, primary wound care, and tissue use are effective in chronic wound formation. The main reason is inadequate circulation in all of the circumstances. Infection; trauma; thermal, chemical, and electrical burns; foreign bodies; postoperative dehiscence; diabetic ulcers; pressure sores; and trophic changes following spinal injury are common etiologies [1]. There are regeneration and tissue repair processes

include multifactorial and highly complex pathophysiological components.

**Introductory Chapter: An Overview of Wound Healing**

DOI: 10.5772/intechopen.84494

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Kamil Hakan Dogan

Kamil Hakan Dogan

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.84494

#### **Introductory Chapter: An Overview of Wound Healing Introductory Chapter: An Overview of Wound Healing**

DOI: 10.5772/intechopen.84494

#### Kamil Hakan Dogan Kamil Hakan Dogan

to become effective and ideal autologous biological cell-based therapies, which can be ap‐ plied to chronic wounds to effectively change the wound bed microenvironment to enable

The tenth chapter by Yan Wang and Wenjing Wu discusses wound healing and the biome‐ chanics of corneal refractive procedures to better understand corneal wound healing from the biomechanical viewpoint. This is mandatory if refractive surgery is ever to achieve more predictable and safer refractive results. The eleventh chapter by Manuel Cadena and Juan José Santivañez provides a comprehensive review of the open abdomen. This is the most challenging of wounds that a surgeon faces because of the metabolic, physiological, and dy‐ namic implications that this condition entails. The twelfth chapter by Girish J. Kotwal et al. discusses facilitation of wound healing with current general wound care, following laparo‐ scopic and conventional surgery with dressings, patches, antibiotics, etc. The last chapter by Raffaele Capoano et al. addresses multidisciplinary approaches to the stimulation of wound

The purpose of presenting this book is to provide an insight into current perspectives on

**Kamil Hakan Dogan**

Konya, Turkey

Selcuk University Faculty of Medicine Department of Forensic Medicine

healing and use of dermal substitutes in chronic phlebostatic ulcers.

and accelerate wound closure.

VIII Preface

wound healing processes.

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.84494

### **1. Introduction**

Wound is the deterioration of the normal integrity of the body by the physical damage of any agent. Erosion, ulcer, and fissure expressions are used in wound statement. Erosion is an expression that determines the focal epidermis losses that do not go into the dermis. Fissure is the tissue loss that determines vertical fractures in the form of cracks and it can hold the epidermis and/or dermis. Ulcers are focal wounds with the dermis and tissue loss in the epidermis. Ulcers may become chronic and may cause difficult treatment for clinicians. The wound healing progress depends on many factors ranging from the general condition of the patient to the treatment and the cause of the wound. Wound healing, complications, and scar development include multifactorial and highly complex pathophysiological components.

One of the results of advances in medical technology is increased longevity; associated with this is an increased prevalence of chronic diseases and consequently chronic wounds. There is a need to provide an evidence-based approach to the management of chronic wounds. The amount of knowledge about the processes of wound healing has significantly increased in recent years. It has become more difficult to select the most appropriate therapy for a specific type of wound. Wound healing overlaps into the many disciplines of medicine in general. Dermatologists, surgeons, internists, and geriatricians are becoming increasingly involved in the field of wound care. General practitioners and family physicians are frequently required to treat acute and chronic wounds.

There are two types of wounds: acute and chronic. After trauma or excisional surgery, acute wounds result. If the wound does not heal within 6 weeks, it is chronic. The factors such as involvement of underlying structures, depth of wound, primary wound care, and tissue use are effective in chronic wound formation. The main reason is inadequate circulation in all of the circumstances. Infection; trauma; thermal, chemical, and electrical burns; foreign bodies; postoperative dehiscence; diabetic ulcers; pressure sores; and trophic changes following spinal injury are common etiologies [1]. There are regeneration and tissue repair processes

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

involving a number of molecular and cellular events for the reconstitution of damaged tissue. The exudative, proliferative, and extracellular matrix remodeling phases are sequential events that occur during wound healing. These events involve soluble mediators, blood cells, and parenchymal cells. Tissue edema develops after injury. In the proliferative stage, the area of tissue injury is reduced by fibroplasia and contracting myofibroblasts. Angiogenesis and reepithelialization may still be observed at this stage. Endothelial cells can differentiate into mesenchymal components. A set of signaling proteins are reported to have role in this process [2]. Chronic wounds are a major health problem. There are several local and systemic factors that affect wound healing. Management of a patient with a chronic wound requires close cooperation of physicians and other healthcare workers from related departments. Wound assessment is vital for evaluating the effectiveness of planned treatment in chronic wounds. Accurate and comprehensive wound assessment depends on meticulous and consistent clinical observation and on the use of quantitative measurement methods.

**References**

London: Springer-Verlag; 2012. pp. 73-92

[1] Bhattacharya V, Agarwal NK, Bhattacharya S. Measurement of wound healing and tissue repair. In: Mani R, Romanelli M, Shukla V, editors. Measurements in Wound Healing.

Introductory Chapter: An Overview of Wound Healing http://dx.doi.org/10.5772/intechopen.84494 3

[2] Gonzalez AC, Costa TG, Andrade ZA, Medrado ARAP. Wound healing—A literature

review. Anais Brasileiros de Dermatologia. 2016;**91**(5):614-620

The most important point in the treatment of chronic wounds is to determine the causes of the wound, if possible, to eliminate the causes and to provide a suitable environment for the wound healing mechanisms of the body to work. In order for a wound to heal, there should be no circulatory problems in the area of the wound, and there should be plenty of clean blood flow, elimination or reduction of the discharge of the wound, removal of the wound, and pressure of the wound (if it is pressured, pressed, or pressed by any object such as shoes). Dead tissue and foreign bodies in the wound should be removed. Chronic wounds can be treated by conventional treatment methods. However, this may prolong the treatment period or make it difficult. The modern wound care products used today eliminate the deficiencies in the wound healing process and accelerate the healing by correcting the healing stage where the wound is inserted. A majority of these products are tools and equipment that helps in healing. These products allow the wound to heal in a shorter time and with minimal cosmetic loss. These products provide a moist environment for wound healing, prevent and treat infection, control discharge, and reduce the odor and pain caused by the wound. They reduce the frequency of dressing and provide acceptable esthetic appearance and functionality by the patient in their daily life. Although at first glance the unit costs may seem high, they reduce the total cost of treatment by reducing infection and shortening the wound healing time.

There are many publications about wound healing, but this book intends to give an overview of the current perspectives on wound healing, to be useful to practice care in wound healing and for improving the quality of life. It is considered that this book will be useful for clinicians who are interested with wound care. I gratefully acknowledge the help and support of the authors from five continents and nine countries of the world who contributed to this book.

### **Author details**

Kamil Hakan Dogan Address all correspondence to: drhakan2000@gmail.com Selcuk University, Turkey

### **References**

involving a number of molecular and cellular events for the reconstitution of damaged tissue. The exudative, proliferative, and extracellular matrix remodeling phases are sequential events that occur during wound healing. These events involve soluble mediators, blood cells, and parenchymal cells. Tissue edema develops after injury. In the proliferative stage, the area of tissue injury is reduced by fibroplasia and contracting myofibroblasts. Angiogenesis and reepithelialization may still be observed at this stage. Endothelial cells can differentiate into mesenchymal components. A set of signaling proteins are reported to have role in this process [2]. Chronic wounds are a major health problem. There are several local and systemic factors that affect wound healing. Management of a patient with a chronic wound requires close cooperation of physicians and other healthcare workers from related departments. Wound assessment is vital for evaluating the effectiveness of planned treatment in chronic wounds. Accurate and comprehensive wound assessment depends on meticulous and consistent clini-

The most important point in the treatment of chronic wounds is to determine the causes of the wound, if possible, to eliminate the causes and to provide a suitable environment for the wound healing mechanisms of the body to work. In order for a wound to heal, there should be no circulatory problems in the area of the wound, and there should be plenty of clean blood flow, elimination or reduction of the discharge of the wound, removal of the wound, and pressure of the wound (if it is pressured, pressed, or pressed by any object such as shoes). Dead tissue and foreign bodies in the wound should be removed. Chronic wounds can be treated by conventional treatment methods. However, this may prolong the treatment period or make it difficult. The modern wound care products used today eliminate the deficiencies in the wound healing process and accelerate the healing by correcting the healing stage where the wound is inserted. A majority of these products are tools and equipment that helps in healing. These products allow the wound to heal in a shorter time and with minimal cosmetic loss. These products provide a moist environment for wound healing, prevent and treat infection, control discharge, and reduce the odor and pain caused by the wound. They reduce the frequency of dressing and provide acceptable esthetic appearance and functionality by the patient in their daily life. Although at first glance the unit costs may seem high, they reduce the total cost of

There are many publications about wound healing, but this book intends to give an overview of the current perspectives on wound healing, to be useful to practice care in wound healing and for improving the quality of life. It is considered that this book will be useful for clinicians who are interested with wound care. I gratefully acknowledge the help and support of the authors from five continents and nine countries of the world who contributed to this book.

cal observation and on the use of quantitative measurement methods.

treatment by reducing infection and shortening the wound healing time.

Address all correspondence to: drhakan2000@gmail.com

**Author details**

Kamil Hakan Dogan

Selcuk University, Turkey

2 Wound Healing - Current Perspectives


**Chapter 2**

Provisional chapter

**Regulatory Mechanisms and Chemical Signaling of**

DOI: 10.5772/intechopen.81731

Wound healing is a highly complex biological process composed of three overlapping phases: inflammatory, proliferative, and remodeling. The acute inflammatory response has being an integral role in tissue healing and fundamental for the homeostasis and reestablishment. This phase depends on the interaction of cytokines, growth factors, chemokines, and chemical mediators from cells to perform regulatory events and complex interactions of the extracellular matrix, extracellular molecules, soluble mediators, various resident cells such as fibroblasts and keratinocytes, and infiltrated leukocyte subtypes that act to restore or replace the integrity of the skin. If this well-orchestrated response becomes deregulated, the wound can become chronic or progressively fibrotic, with both outcomes impairing tissue function, which can ultimately lead to organ failure and death. In this chapter, we will review the pathway in the skin healing cascade, relating the major chemical inflammatory mediators, cellular and molecular, as well as demonstrating the local and systemic factors that interfere in healing and disorders associated with tissue

repair deficiency in chronic inflammations, burns and hypertrophy.

Keywords: wound healing, inflammation, cytokines, growth factors, chemokines

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Regulatory Mechanisms and Chemical Signaling of

**Mediators Involved in the Inflammatory Phase of**

Mediators Involved in the Inflammatory Phase of

**Cutaneous Wound Healing**

Cutaneous Wound Healing

Lucas Fernando Sérgio Gushiken,

Lucas Fernando Sérgio Gushiken,

http://dx.doi.org/10.5772/intechopen.81731

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Fernando Pereira Beserra,

Fernando Pereira Beserra,

Maria Fernanda Hussni and Cláudia Helena Pellizzon

Maria Fernanda Hussni and Cláudia Helena Pellizzon

Abstract

#### **Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous Wound Healing** Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous Wound Healing

DOI: 10.5772/intechopen.81731

Fernando Pereira Beserra, Lucas Fernando Sérgio Gushiken, Maria Fernanda Hussni and Cláudia Helena Pellizzon Fernando Pereira Beserra, Lucas Fernando Sérgio Gushiken, Maria Fernanda Hussni and Cláudia Helena Pellizzon

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81731

Abstract

Wound healing is a highly complex biological process composed of three overlapping phases: inflammatory, proliferative, and remodeling. The acute inflammatory response has being an integral role in tissue healing and fundamental for the homeostasis and reestablishment. This phase depends on the interaction of cytokines, growth factors, chemokines, and chemical mediators from cells to perform regulatory events and complex interactions of the extracellular matrix, extracellular molecules, soluble mediators, various resident cells such as fibroblasts and keratinocytes, and infiltrated leukocyte subtypes that act to restore or replace the integrity of the skin. If this well-orchestrated response becomes deregulated, the wound can become chronic or progressively fibrotic, with both outcomes impairing tissue function, which can ultimately lead to organ failure and death. In this chapter, we will review the pathway in the skin healing cascade, relating the major chemical inflammatory mediators, cellular and molecular, as well as demonstrating the local and systemic factors that interfere in healing and disorders associated with tissue repair deficiency in chronic inflammations, burns and hypertrophy.

Keywords: wound healing, inflammation, cytokines, growth factors, chemokines

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### 1. Introduction

#### 1.1. Skin wound healing

Skin is the largest organ of all vertebrates, and it is very important to protect the organism against external damage [1]. When the loss of structural integrity of the skin occurs, the organism starts the wound healing process, involving some coordinated, interdependent, and overlapping mechanisms—such as inflammation, cell proliferation, reepithelialization of wounded area, and extracellular matrix remodeling—to restructure the skin homeostasis [2, 3]. The initial mechanism of wound healing is the fibrin clot synthesis to avoid bleeding and to keep the local hemostasis, leading to the platelet retention and activation of local vascular mediators [4, 5]. From now on, there is the dilatation of the local vessels due to the release of histamine and serotonin, as well as the increase of vessel permeability, improving the leukocyte migration to the wounded area and starting the inflammatory process. In the first 5 days after the lesion, neutrophils are attracted to the region, removing pathogenic antigens and dead tissue through phagocytosis and protease secretion. After 3 days, there is the macrophage migration to the wounded area, with the maintenance of inflammatory response [5]. Due to the tissue destruction, the local keratinocytes release interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), essential cytokines in the inflammatory mechanism, through recruitment and activation of leukocytes in the region and with important roles coordinating other wound healing mechanisms. With these facts, keratinocytes, macrophages, platelets, and endothelial cells of wounded area release some mediators such as growth factors (EGF, FGF, PDGF, TGF-β), cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ), and chemokines, which will control other subsequent mechanisms in skin wound healing [6, 7].

Model Mediator Target/signaling protein Biologic effect References

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

and GM-CSF and PMN

occludin, claudin-1, claudin2, claudin-3, and

12-rMuIL-12/collagen structure and alignment

extracellular domain

plasminogen activator

growth factor–β and TGF-

(collagen accumulation at the wound sites), TGF-1,

claudin-5

(ECD)

VCAM-1 IL-1α Il-1β MIP-1α

Neutrophil depletion exhibited significantly accelerated reepithelialization, without altering the macrophage infiltration or the collagen content in the wound bed

http://dx.doi.org/10.5772/intechopen.81731

Epinephrine altered the neutrophil (PMN) dependent inflammatory response to a cutaneous wound through an IL-6 mediated mechanism via β2 adrenergic receptor-

IL -1β induced increased claudin -1 expression in cell

IL-12 induced a rapid onset and higher metabolic activity in wounded skin at

Not change the wound healing and inflammatory

Delayed angiogenesis and collagen deposition, by the reduced expression of angiogenic and fibrogenic

IL-1 β interacts com PA by

TGF-β1inibits functional

less IL-8. Much less IL-8 in stimulated fetal fibroblasts

dependent

culture

early time

speed

growth factors Reduced inflammatory

than in adults

process

CD301b-expressing subpopulation of macrophages is critical for activation of reparative

Inflammation, fibrosis, neovascularization, and regeneration of parenchymal cells were affected by the receptor

response

tPA

tPA

IL-8 Fetal fibroblasts produced

Dovi et al. [51]

7

Kim et al. [52]

Rozlomiy and Markov [53]

Li et al. [54]

Fuentes et al. [55]

Lin et al. [56]

Lian et al. [57]

Liechty et al. [58]

Shook et al. [59]

Ishida et al. [31]

Neutrophil MPO, macrophages, and

Epinephrine IL-6, IL-1β, TNF-α, IL-1α,

IL-1β Human recombinant IL-1β,

IL-12 Recombinant murine IL-

IL-1β and TGF-β1 IL-1β TGF-β1/tissue type

CD301b macrophage IL-10, platelet-derived

CX3CL1 and CX3CR1 MPO, Hydroxyproline

β1

VEGF

(tPa)

EFG EGFR/vaccination

IL-6 IL-6/ICAM-1

Platelet-derived growth factor (PDGF)

collagens

In vivo BALB/c mice

In vivo C57BL/6 mice

In vivo C57Bl/wild-type

mice

In vivo BALB/C mice

In vivo BALB/C mice

In vivo

In vitro Keratinocytes

In vitro Human and mice fibroblasts

In vivo C57/Bl6 mice and Mgl2DTR/GFP mice

In vivo Levels and role of chemokine CX3CL1 (fractalkine) and its receptor CX3CR1 in mouse model

BALB/C mice and IL-6 KO mice

Therefore, the inflammatory mechanism is an important step to the correct and well-coordinated wound healing, modulating the subsequent mechanisms of healing. Furthermore, the comprehension of inflammatory response can lead to new treatments to wound repair and decrease of healing disorders like hypertrophic scars, keloids, chronic inflammation, skin infections, and unwounded lesions [8].

### 2. Materials and methods

The search for this chapter was carried out on PubMed, Scopus, and Web of Science until June 2018, using "inflammation", "inflammatory process", "skin wound healing", "cytokines", "chemokines"


Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous… http://dx.doi.org/10.5772/intechopen.81731

1. Introduction

1.1. Skin wound healing

6 Wound Healing - Current Perspectives

unwounded lesions [8].

In vivo BALB/c mice

2. Materials and methods

Skin is the largest organ of all vertebrates, and it is very important to protect the organism against external damage [1]. When the loss of structural integrity of the skin occurs, the organism starts the wound healing process, involving some coordinated, interdependent, and overlapping mechanisms—such as inflammation, cell proliferation, reepithelialization of wounded area, and extracellular matrix remodeling—to restructure the skin homeostasis [2, 3]. The initial mechanism of wound healing is the fibrin clot synthesis to avoid bleeding and to keep the local hemostasis, leading to the platelet retention and activation of local vascular mediators [4, 5]. From now on, there is the dilatation of the local vessels due to the release of histamine and serotonin, as well as the increase of vessel permeability, improving the leukocyte migration to the wounded area and starting the inflammatory process. In the first 5 days after the lesion, neutrophils are attracted to the region, removing pathogenic antigens and dead tissue through phagocytosis and protease secretion. After 3 days, there is the macrophage migration to the wounded area, with the maintenance of inflammatory response [5]. Due to the tissue destruction, the local keratinocytes release interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), essential cytokines in the inflammatory mechanism, through recruitment and activation of leukocytes in the region and with important roles coordinating other wound healing mechanisms. With these facts, keratinocytes, macrophages, platelets, and endothelial cells of wounded area release some mediators such as growth factors (EGF, FGF, PDGF, TGF-β), cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ), and chemokines, which will control other subsequent mechanisms in skin wound healing [6, 7].

Therefore, the inflammatory mechanism is an important step to the correct and well-coordinated wound healing, modulating the subsequent mechanisms of healing. Furthermore, the comprehension of inflammatory response can lead to new treatments to wound repair and decrease of healing disorders like hypertrophic scars, keloids, chronic inflammation, skin infections, and

The search for this chapter was carried out on PubMed, Scopus, and Web of Science until June 2018, using "inflammation", "inflammatory process", "skin wound healing", "cytokines", "chemokines"

Model Mediator Target/signaling protein Biologic effect References

The absence of CD4 and CD8 lymphocytes changes in cytokine expression and inflammatory cell infiltrate, but does not influence wound breaking strength, collagen content, or angiogenesis

Chen et al. [50]

and CXCL-1 IL-4

and CCL-2, IL-4

CD4 cells IL1β, IL-6, IL-17, IFN-γ,

CD8 cells IL1β, IL-6, TNF-α, CXCL-1


7


Model Mediator Target/signaling protein Biologic effect References

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

COX-2 COX-1 and COX-2 Selective inhibition of

Stat1, Smad2, P-Smad2, Smad3, Smad7, α-Tubulin, VEGF, CD3, IL-12p35, IL-12p40, IL-18, COLIAI

— EGF, VEGF, IGF and FGF Growth factors accelerated

IFN-γ and TGF-β1 MPO, TGF-β, Stat1, P-

TLR4 CD3+, T cells, Ki67, NF-κB,

Vγ4 T cells IL-17A, IGF-1, CCL20, NF-

23p19

p-p38, and p-JNK

κB p65, p-NF-κB p6, STAT3, p-STAT3, IL-1β, IL-

In vitro Human skin fibroblasts CCD966- SK and HaCaT keratinocytes In vivo BALB/C mice

In vivo SKH-1 mice

In vivo BALB/c mice

In vivo Wistar rats

In vivo C3H/Hej TLR4 deficient and wildtype C3H/HeOuj

mice In vitro NHEK and THP1 cell line

In vivo C57BL/6 mice Fibroblasts and keratinocytes IL-19 IRF8 coordinates M1 macrophage population (decrease of M1 mediators IL-1 β, IL-6, TNF-α, iNOS), with no interference in M2 macrophage mediators (arg-1, mrc-1, IL-10)

http://dx.doi.org/10.5772/intechopen.81731

expression in fibroblasts and KGF induces IL-19 expression in keratinocytes Sun et al. [69]

9

Blomme et al. [70]

Ishida et al. [71]

de Masi et al. [72]

Chen et al. [73]

Li et al. [74]

keratinocyte proliferation. IL-19 induces keratinocyte

COX-2 and nonselective inhibition of COX-1 and COX-2 did not affect the healing of sutured surgical incisions in mouse skin.

Crosstalk between the IFNγ/Stat1 and TGF-β1/Smad signaling pathways in the skin wound healing.

the healing process promoting greater angiogenic activity and accelerated fibroplasia and the deposition of type I

TLR4 is activated in early skin wound healing, with a functional mutation of TLR4 results in altered inflammatory cell infiltration, differential cytokine production, and impaired wound closure, besides IL-1β production by injured keratinocytes is induced through the TLR4 p38/JNK pathway

Mechanistic link between Vγ4 T cell-derived IL-17A, epidermal IL-1β/IL-23, DETC-derived IGF-1, and wound healing responses

in the skin

collagen

KGF promotes

migration

IL-19 IL-19 upregulates KGF

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous… http://dx.doi.org/10.5772/intechopen.81731

Model Mediator Target/signaling protein Biologic effect References

C3, C3a

TNF-α mRNA

complement system (CS), Signal transducer, activator of transcription 4 (STAT4), Leukocyte infiltration, C5a,

Macrophages, IL-10, IL-6, aFGF, bFGF, TGF-1, PDGF,

IL-1β p38, MAPK, ERK IL-1β stimulates PTGS2 in

Number of macrophages, neutrophils, and cells positive for activated caspase-3, VEGF-A, or

TGF-β1

γδ T Cell FGF-7, FGF-10, IGF-1,

JAML

IL-6 and IL-4 — IL6 and ICOS-ICOSL

Macrophage M1 and M2 IRF-8 IRF-8 is an inflammatory

PIC1 loaded into the derma CELL did reduce the number of inflammatory cells in the wound bed

kCYC/mice mast cell increased.

IL-10: increased, bFGF decreased in kCYC/mice. IL-10 plays an important role in delayed wound

fibroblast and p38-MAPK in other cells. PGE2 activates INHBA

important in inflammatory

Inflammatory phase: reduced formation of vascularized granulation tissue, leading to minimized scar formation. Phase of tissue formation: severe hemorrhage in the wound. Wound closure did

No significant impact in tissue maturation phases.

macrophages had a non-

healing

phase

not occur.

inflammatory transcriptomic profile and demonstrated that inhibition of IL-17 in mice accelerated normal and delayed

healing.

mediator.

wound healing.

γδT Lymphocytes stimulate the gene and protein expression of important mediators in acute healing model

signaling the skin wound healing in mutant mice

Inhibition of IRF-8 impairs

IL-17 Ly6cloMHCIIhi

— IGF-1, IL-4, IL-6, and IL-3

Cunnion et al. [60]

Kimura et al. [61]

Arai et al. [62]

Lania et al. [63]

Lucas et al. [64]

Rodero et al. [65]

Xu et al. [66]

Maeda et al. [67]

Guo et al. [68]

Peptide inhibitor of complement C1 (PIC1)

In vivo kCYC/mice IL-10 Mast cells migrating,

IL-4, IL-12, IL-6, IGF-1 and IFN-α e γ

Inducible diphtheria toxin receptor + diphtheria toxin injections + mice lacking the TGF-b receptor type II (TbRII), Depletion of macrophages

Ly6cloMHCIIhi macrophage

In vivo db/db mice as a diabetic skin wound

8 Wound Healing - Current Perspectives

model.

In vivo C57BL/6J mice

In vivo Sprague Dawley

In vivo

In vivo C57Bl/6J mice

In vivo C57BL/6mice

/ ICOSL -?-

In vivo

Mutant mice ICOS

C57BL/6 mice and human acute wounds

Mouse model with conditional depletion of macrophages

Rats


9


and "growth factors" as keywords. The articles published in the last 20 years were considered

Model Mediator Target/signaling protein Biologic effect References

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

IL-27 (dendritic cells) Keratin-6 (keratinocytes) IL-27 is synthesized by

infected wounds. Mixidin2 and mixidin3 modulated inflammatory signaling with antiinflammatory activity

http://dx.doi.org/10.5772/intechopen.81731

dendritic cells and modulates keratinocyte proliferation, migration, and differentiation after Yang et al. [82]

11

skin injury

Wound healing is an extremely dynamic and interactive biological process involving complex interactions of extracellular matrix, extracellular molecules, soluble mediators, multiple resident cells (fibroblasts and keratinocytes), and subtypes of infiltrating leukocytes that together act to restore integrity of the damaged tissue and replace the lost. This process comprises three sequential and overlapping stages, regardless of the amount of injured tissue: hemostasis/ inflammation; cell proliferation and matrix repair; and reepithelialization and remodeling of

The inflammatory phase, hemostasis, leukocyte migration, and the beginning of the tissue repair cascade occur. Initially, in response to inflammatory agents, there is reduction of blood flow by vasoconstriction, and with extravasation of blood from the injured vessel, platelets are activated causing the coagulation process to begin [10]. During this process, there is a progressive increase in vascular permeability to migrant cells and biologically active substances. From this process, essential elements for the physiological continuation of healing appear: a fibrin framework, necessary for the migration of the cells that will reach the lesion site, and pro and

Neutrophils are the first immune cells recruited into wounded tissue to play a role in reestablishing tissue homeostasis through pathogen phagocytosis and macrophage recruitment as well as excessive neutrophil activity which can contribute to the development of

anti-inflammatory chemo/cytokines that will aid in cell activation and migration [11].

3.1. Mediators involved in the inflammatory phase of wound healing

Table 1. Chemical mediators involved in the inflammatory response of skin wound healing.

scar tissue, which involve complex biochemical and cellular mechanisms [9].

(1998–2018). The results are displayed in Table 1.

3. Results and discussion

keratinocytes In vivo

In vivo

mice

BALB/c female mice

B57BL6/J, B57BL6/ NJ, CD301bGFP-DTR, and Il27Ra/

3.2. Neutrophils and macrophages

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous… http://dx.doi.org/10.5772/intechopen.81731 11


Table 1. Chemical mediators involved in the inflammatory response of skin wound healing.

and "growth factors" as keywords. The articles published in the last 20 years were considered (1998–2018). The results are displayed in Table 1.

### 3. Results and discussion

Model Mediator Target/signaling protein Biologic effect References

MIP-2, KC, IL-17A, MCP-1,

PPAR-γ, TNF-α, VEGF,

IL-4 from mast cells IL-4, MCP-1 Increased MCP-1

collagen 1

Mast cells TNF-α, MIP-2, VEGF,

miR-31 NF-kB, STAT3, RAS/

FGF-2

MAPK

IL-6, IL-8, TNF-α STAT3, p38, JNK, EGFR STAT3, p38, JNK, and NF-

RANTES

IL-6, STAT3 IL-6 induces keratinocyte

migration indirectly, through the STAT3 activation cascade in fibroblasts, with the synthesis of a fibroblastderived factor

MIP-2, KC, IL-17A (neutrophil attractors) were increased in JA18KO mice. MCP-1 and RANTES (macrophage and lymphocyte attractors) were decreased in Ja18KO

Decrease of neutrophil apoptosis in Ja18KO mice. iNKT ciNKT cells promote skin wound healing by regulating neutrophil

Increase of TNF-α in PPAR-γ KO mice and delay in wound healing

chemoattractant activity in mast cell migration. Increase of IL-4 synthesis

IL-4 stimulates fibroblast

Decrease of neutrophil infiltration in KO mice. Increase of FGF-2 in KO

Mast cells modulate neutrophil infiltration in wound site, with unlikely influence in proliferative phase of wound healing

Increase of miR-31 in wound edge keratinocytes during inflammation through NF-kB and STAT3

kB activation lead to IL-6, IL-8, and TNF-a increase in

pathways miR-31 regulates keratinocyte migration through RAS/MAPK

pathway

mice.

apoptosis

by mast cells

activation

mice.

Gallucci et al. [75]

Tanno et al. [76]

Chen et al. [77]

Trautmann et al. [78]

Egozi et al. [79]

Shi et al. [80]

Han et al. [81]

IL-6 from keratinocytes and fibroblasts

Invariant natural killer T cells (iNKT) and neutrophils

PPAR-γ from macrophages

In vitro Primary keratinocytes and fibroblasts of IL-6 KO or C57BL/6 mice

In vivo

In vivo PPAR-γ KO mice and C57BL/6 J male

mice

In vivo Human incision model

In vivo WBB6F1/J-KitW/ KitW–v mast cell KO female mice and WBB6F1 female

mice

In vivo miR-31 loss-offunction mice

In vitro Primary normal human

Ja18KO (iNKT celldeficient) mice and C57BL/6 mice

10 Wound Healing - Current Perspectives

#### 3.1. Mediators involved in the inflammatory phase of wound healing

Wound healing is an extremely dynamic and interactive biological process involving complex interactions of extracellular matrix, extracellular molecules, soluble mediators, multiple resident cells (fibroblasts and keratinocytes), and subtypes of infiltrating leukocytes that together act to restore integrity of the damaged tissue and replace the lost. This process comprises three sequential and overlapping stages, regardless of the amount of injured tissue: hemostasis/ inflammation; cell proliferation and matrix repair; and reepithelialization and remodeling of scar tissue, which involve complex biochemical and cellular mechanisms [9].

The inflammatory phase, hemostasis, leukocyte migration, and the beginning of the tissue repair cascade occur. Initially, in response to inflammatory agents, there is reduction of blood flow by vasoconstriction, and with extravasation of blood from the injured vessel, platelets are activated causing the coagulation process to begin [10]. During this process, there is a progressive increase in vascular permeability to migrant cells and biologically active substances. From this process, essential elements for the physiological continuation of healing appear: a fibrin framework, necessary for the migration of the cells that will reach the lesion site, and pro and anti-inflammatory chemo/cytokines that will aid in cell activation and migration [11].

#### 3.2. Neutrophils and macrophages

Neutrophils are the first immune cells recruited into wounded tissue to play a role in reestablishing tissue homeostasis through pathogen phagocytosis and macrophage recruitment as well as excessive neutrophil activity which can contribute to the development of nonhealing wounds. They play a central role in both killing microbes and promoting wound healing [12]. These cells are extremely important in the inflammatory process, but once recruited into wound sites in such large numbers with exacerbated cytokine secretion, overproduction of reactive oxygen species (ROS), causing extracellular matrix (ECM) and cell membrane damage, and resulting in premature cell senescence [13].

Since prolonged presence of proinflammatory cytokines may prevent resolution and both pro and anti-inflammatory cytokines are necessary for wound healing, sequential delivery of pro and anti-inflammatory cytokines could be an interesting strategy for improving chronic wound healing [17]. For example, IL-22, considered a proinflammatory cytokine, helped the wound healing of diabetic mice by inducing keratinocyte proliferator and signal transduction and activation of transcription 3 (STAT3) [24]. In this same context but in other tissues, to improve bone repair, decellularized bone was engineered to sequentially release IL-4 (proinflammatory cytokine) and implanted in mice at the site of injury. The sequential release promoted macrophage polarization to switch from a pro to an anti-inflammatory phenotype,

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

http://dx.doi.org/10.5772/intechopen.81731

13

IL-10 is a regulatory cytokine, which can be secreted by many kinds of immune cells, including Th1, Th2, Th17, Treg, and CD8+ T cells, B cells, dendritic cells, macrophages, NK cells, eosinophils, neutrophils, basophils, and MCs, as well as nonimmune cells including keratinocytes. This cytokine is considered an anti-inflammatory cytokine because it is capable of inhibiting the production of other proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [26]. In addition to its potent anti-inflammatory effects, IL-10 has been shown to regulate fibrogenic cytokines, such as transforming growth factor-β (TGF-β), as a part of its role in the regulation

Chemokines are small molecules that induce chemotaxis and activation of certain subsets of leukocytes. They are classified into four types: CC chemokines, CXC chemokines, C chemokines, and CX3C chemokine. Chemokines play important roles in wound healing and are important for maintaining skin homeostasis, and their disruption can result in skin pathologies [28]. They also play important roles in establishing microenvironment in which migratory immune cells,

CX3CL1 is expressed by inflamed endothelial cells and epithelial cells, including macrophage, keratinocytes, and vascular smooth muscle cells, whereas CX3CR1 is mainly expressed by neutrophils, monocytes, mast cells, T cells, and NK cells [30]. In the cutaneous wound healing, CX3CL1 has been shown to be expressed by macrophages and endothelial cells, while CX3CR1 is expressed by macrophages and fibroblasts. Decreased expression of macrophage-related cytokines, such as TGF-β and VEGF, and reduced deposition and α-smooth muscle actin and

Growth factors are naturally occurring endogenous mediators capable of controlling the control of cell growth, proliferation, migration, and differentiation [32]. Once bound specifically to its receptor, the ligand-receptor interaction is able to activate intracellular signal transduction

together with skin-resident cells, cause prolonged inflammation [29].

collagen were shown in the injured skin of CX3CR1/ mice [31].

pathways that regulate different cellular functions [33].

resulting in improved wound healing [25].

of tissue remodeling [27].

3.4. Chemokines

3.5. Growth factors

Monocyte-derived macrophages are often considered to be the most important immune cell type in this process. In intact skin, these cells are the most abundant cell types performing sentinel and homeostatic function. The monocytes migrate from vascular circulation to wound. Both infiltrating and resident macrophages on skin are activated by local signals and developed into several subpopulations defined by their different functional phenotypes [14]. Many studies have confirmed that macrophages are critical for proper skin wound healing [15–17]. Upon initial infiltration, proinflammatory macrophages, also called M1, are also responsible for removing cellular debris, damaged matrix, microbes, and neutrophils [17].

### 3.3. Cytokines

Wound healing is regulated by growth factors and cytokines that are essential not only in the inflammatory process but also in the cell proliferation and maintenance in the repair process by various mechanisms [18].

Cytokines released by neutrophils during apoptosis are chemotactic for monocytes, which start to arrive 5–6 h post injury. IL-1β is a key interleukin of antimicrobial response by inflammatory response amplification; it stimulates leukocyte recruitment, the release of acute phase proteins, and the increase of permeability of blood vessels [19]. Some authors consider this cytokine as part of a proinflammatory positive feedback loop that sustains a persistent proinflammatory wound macrophage phenotype, contributing to impaired healing of diabetic wounds [18].

TNF-α is a second proinflammatory cytokine that contributes to a chronic wound state. It acts on several stages of leukocyte recruitment mechanism, neutrophils and macrophages, inducing molecular adhesion regulation, chemokine production, and metalloproteinase matrix, as well as tissue inhibitors of metalloproteinases [20]. Interleukin IL-6 is a soluble proinflammatory mediator with pleiotropic activities in inflammation, hematopoiesis, and immune responses [21]. Together with TNF-α and IL-1β, IL-6 is present in high concentrations in inflammatory processes. After IL-6 is secreted into the area of injury at the beginning of the inflammatory process, it is directed to the liver through the bloodstream, transmitting the information and inducing the hepatocytes to produce certain inflammatory agents [22]. Another important cytokine in the inflammatory phase is IL-8, which also acts as a chemokine (CXCL8). This cytokine is mainly produced by monocytes and in smaller amounts by fibroblasts, endothelial cells, keratinocytes, melanocytes, hepatocytes, and chondrocytes. It usually receives stimuli from other cytokines, such as IL-1, TNF-α, and IFN-γ [23]. The main action of IL-8 is the migration to cells of the immune system, mainly neutrophils, also determining an increase in the expression of endothelial adhesion molecules cells [13].

Since prolonged presence of proinflammatory cytokines may prevent resolution and both pro and anti-inflammatory cytokines are necessary for wound healing, sequential delivery of pro and anti-inflammatory cytokines could be an interesting strategy for improving chronic wound healing [17]. For example, IL-22, considered a proinflammatory cytokine, helped the wound healing of diabetic mice by inducing keratinocyte proliferator and signal transduction and activation of transcription 3 (STAT3) [24]. In this same context but in other tissues, to improve bone repair, decellularized bone was engineered to sequentially release IL-4 (proinflammatory cytokine) and implanted in mice at the site of injury. The sequential release promoted macrophage polarization to switch from a pro to an anti-inflammatory phenotype, resulting in improved wound healing [25].

IL-10 is a regulatory cytokine, which can be secreted by many kinds of immune cells, including Th1, Th2, Th17, Treg, and CD8+ T cells, B cells, dendritic cells, macrophages, NK cells, eosinophils, neutrophils, basophils, and MCs, as well as nonimmune cells including keratinocytes. This cytokine is considered an anti-inflammatory cytokine because it is capable of inhibiting the production of other proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6 [26]. In addition to its potent anti-inflammatory effects, IL-10 has been shown to regulate fibrogenic cytokines, such as transforming growth factor-β (TGF-β), as a part of its role in the regulation of tissue remodeling [27].

### 3.4. Chemokines

nonhealing wounds. They play a central role in both killing microbes and promoting wound healing [12]. These cells are extremely important in the inflammatory process, but once recruited into wound sites in such large numbers with exacerbated cytokine secretion, overproduction of reactive oxygen species (ROS), causing extracellular matrix (ECM) and cell

Monocyte-derived macrophages are often considered to be the most important immune cell type in this process. In intact skin, these cells are the most abundant cell types performing sentinel and homeostatic function. The monocytes migrate from vascular circulation to wound. Both infiltrating and resident macrophages on skin are activated by local signals and developed into several subpopulations defined by their different functional phenotypes [14]. Many studies have confirmed that macrophages are critical for proper skin wound healing [15–17]. Upon initial infiltration, proinflammatory macrophages, also called M1, are also responsible

Wound healing is regulated by growth factors and cytokines that are essential not only in the inflammatory process but also in the cell proliferation and maintenance in the repair process

Cytokines released by neutrophils during apoptosis are chemotactic for monocytes, which start to arrive 5–6 h post injury. IL-1β is a key interleukin of antimicrobial response by inflammatory response amplification; it stimulates leukocyte recruitment, the release of acute phase proteins, and the increase of permeability of blood vessels [19]. Some authors consider this cytokine as part of a proinflammatory positive feedback loop that sustains a persistent proinflammatory wound macrophage phenotype, contributing to impaired healing of diabetic

TNF-α is a second proinflammatory cytokine that contributes to a chronic wound state. It acts on several stages of leukocyte recruitment mechanism, neutrophils and macrophages, inducing molecular adhesion regulation, chemokine production, and metalloproteinase matrix, as well as tissue inhibitors of metalloproteinases [20]. Interleukin IL-6 is a soluble proinflammatory mediator with pleiotropic activities in inflammation, hematopoiesis, and immune responses [21]. Together with TNF-α and IL-1β, IL-6 is present in high concentrations in inflammatory processes. After IL-6 is secreted into the area of injury at the beginning of the inflammatory process, it is directed to the liver through the bloodstream, transmitting the information and inducing the hepatocytes to produce certain inflammatory agents [22]. Another important cytokine in the inflammatory phase is IL-8, which also acts as a chemokine (CXCL8). This cytokine is mainly produced by monocytes and in smaller amounts by fibroblasts, endothelial cells, keratinocytes, melanocytes, hepatocytes, and chondrocytes. It usually receives stimuli from other cytokines, such as IL-1, TNF-α, and IFN-γ [23]. The main action of IL-8 is the migration to cells of the immune system, mainly neutrophils, also determining an

increase in the expression of endothelial adhesion molecules cells [13].

membrane damage, and resulting in premature cell senescence [13].

for removing cellular debris, damaged matrix, microbes, and neutrophils [17].

3.3. Cytokines

wounds [18].

by various mechanisms [18].

12 Wound Healing - Current Perspectives

Chemokines are small molecules that induce chemotaxis and activation of certain subsets of leukocytes. They are classified into four types: CC chemokines, CXC chemokines, C chemokines, and CX3C chemokine. Chemokines play important roles in wound healing and are important for maintaining skin homeostasis, and their disruption can result in skin pathologies [28]. They also play important roles in establishing microenvironment in which migratory immune cells, together with skin-resident cells, cause prolonged inflammation [29].

CX3CL1 is expressed by inflamed endothelial cells and epithelial cells, including macrophage, keratinocytes, and vascular smooth muscle cells, whereas CX3CR1 is mainly expressed by neutrophils, monocytes, mast cells, T cells, and NK cells [30]. In the cutaneous wound healing, CX3CL1 has been shown to be expressed by macrophages and endothelial cells, while CX3CR1 is expressed by macrophages and fibroblasts. Decreased expression of macrophage-related cytokines, such as TGF-β and VEGF, and reduced deposition and α-smooth muscle actin and collagen were shown in the injured skin of CX3CR1/ mice [31].

#### 3.5. Growth factors

Growth factors are naturally occurring endogenous mediators capable of controlling the control of cell growth, proliferation, migration, and differentiation [32]. Once bound specifically to its receptor, the ligand-receptor interaction is able to activate intracellular signal transduction pathways that regulate different cellular functions [33].

PDGF plays a crucial role in the healing process in both chronic and normal wounds. This growth factor is released from degranulating platelets following an injury into the wound fluid [34]. PDGF stimulates mitogenicity and chemotaxis of cells, such as neutrophils, macrophages, fibroblasts, and smooth muscle cells to the site of the wound, initiating the inflammatory process stage [35]. Its function has already been described during the stage of epithelialization of wound healing by upregulating the production of growth factors, such as insulin-like growth factor (IGF)-1 and thrombospondin-1, in turn IGF-1 increases the motility of keratinocyte cells and thrombospondin-1 inhibits proteolytic and enzymatic degradation of PDGF [36].

4. Conclusion

Conflict of interest

Author details

Fernando Pereira Beserra<sup>1</sup>

Cláudia Helena Pellizzon<sup>1</sup>

(UNESP), São Paulo, Brazil

References

University (UNESP), São Paulo, Brazil

10.1016/j.jss.2011.07.007

The authors declare no conflict of interests.

\* \*Address all correspondence to: claudia@ibb.unesp.br

Increasing scientific knowledge has contributed to define highly coordinated molecular and cellular events involved in the cutaneous wound healing. Recent findings show that there is a clear correlation between the stage of the wound and its effectiveness in the healing process, and endogenous mediators, such as cytokines, chemokines, and growth factors, indicate important and crucial steps in the normal healing and are useful as prognostic indicators. Although much is known about the cellular and molecular basis of normal skin healing, there are still avenues of research left to unravel that will guide us to better therapies, new therapeutic targets, and strategies for the skin wound treatment, especially chronic wounds.

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

, Lucas Fernando Sérgio Gushiken<sup>1</sup>

1 Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University

2 Undergraduate Student School of Veterinary Medicine and Animal Science, São Paulo State

[1] Fikru A, Makonnen E, Eguale T, Debella A, Mekonnen GA. Evaluation of in vivo wound healing activity of methanol extract of Achyranthes aspera L. Journal of Ethnophar-

[2] Martins VL, Caley M, O'Toole EA. Matrix metalloproteinases and epidermal wound repair. Cell and Tissue Research. 2013;351:255-268. DOI: 10.1007/s00441-012-1410-z

[3] Agha R, Ogawa R, Pietramaggiori G, Orgill DP. A review of the role of mechanical forces in cutaneous wound healing. The Journal of Surgical Research. 2011;171:700-708. DOI:

macology. 2012;143:469-474. DOI: 10.1016/j.jep.2012.06.049

, Maria Fernanda Hussni<sup>2</sup> and

http://dx.doi.org/10.5772/intechopen.81731

15

Angiogenesis is an extremely important process in normal development and tissue homeostasis and repair, besides contributes directly also to various forms of pathology, such as tumor development and metastasis, psoriasis, rheumatoid arthritis, and wet macular degeneration [37]. One of the most important proangiogenic mediators is vascular endothelial growth factor (VEGF), responsible for stimulating new blood vessels formation, tissue proliferation, migration, differentiation, and survival, which contribute to the angiogenesis process, in addition to influencing the repair and wound closure and granulation tissue formation [38]. The VEGF family has several members, and one of its members such as VEGF-A begins the process of wound healing promoting biological events linked to angiogenesis and migration of endothelial cells [39]. Administration of VEGF-A has been reported to restore impairment of angiogenesis in diabetic ischemic limbs in an animal model as well as to improve the reepithelialization process of diabetic wounds [40].

Epidermal growth factor (EGF) stimulates proliferation and differentiation of various cells, including fibroblasts, endothelial cells, and epithelial cells, and shows mitogenic and migratory activity on the edge keratinocytes of the lesions [41]. EGF participates in this mechanism, which is considered essential in the cutaneous wound healing, which begins a few hours after the injury, but presents a more evident activity in the proliferative phase of wound healing, and continues until the extracellular matrix remodeling phase [42].

Another growth factor that involves healing process activity is the family of fibroblast growth factors (FGF), which have already been reported to play crucial events in the wound healing process [43]. FGFs are secreted by keratinocytes, fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, and mast cells [44]. During an acute cutaneous wound process, it has been reported an increase in the production of FGF-2 and that they are responsible for formation of granulation tissue, reepithelialization, and tissue remodeling [45]. Moreover, functions such as synthesis, deposition of various constituents of the extracellular matrix, and increased motility of keratinocytes are regulated by FGF-2 [46].

Transforming growth factor type-β (TGF-β) activity in the healing process was analyzed by being one of the proteins with the greatest spectrum of activities, with effect on cell proliferation, differentiation and production of extracellular matrix, and immunological modulation [46, 47]. Moreover, TGF-β has many biological activities and is thought to be a particularly important contributor to fibrosis, angiogenesis, and tissue repair. This growth factor can also influence T cells, including Th17 and Treg cells, as well as B cells, dendritic cells, NK cells, neutrophils, and eosinophils [48, 49].

### 4. Conclusion

PDGF plays a crucial role in the healing process in both chronic and normal wounds. This growth factor is released from degranulating platelets following an injury into the wound fluid [34]. PDGF stimulates mitogenicity and chemotaxis of cells, such as neutrophils, macrophages, fibroblasts, and smooth muscle cells to the site of the wound, initiating the inflammatory process stage [35]. Its function has already been described during the stage of epithelialization of wound healing by upregulating the production of growth factors, such as insulin-like growth factor (IGF)-1 and thrombospondin-1, in turn IGF-1 increases the motility of keratinocyte cells and

Angiogenesis is an extremely important process in normal development and tissue homeostasis and repair, besides contributes directly also to various forms of pathology, such as tumor development and metastasis, psoriasis, rheumatoid arthritis, and wet macular degeneration [37]. One of the most important proangiogenic mediators is vascular endothelial growth factor (VEGF), responsible for stimulating new blood vessels formation, tissue proliferation, migration, differentiation, and survival, which contribute to the angiogenesis process, in addition to influencing the repair and wound closure and granulation tissue formation [38]. The VEGF family has several members, and one of its members such as VEGF-A begins the process of wound healing promoting biological events linked to angiogenesis and migration of endothelial cells [39]. Administration of VEGF-A has been reported to restore impairment of angiogenesis in diabetic ischemic limbs in an animal model as well as to improve the reepithelialization

Epidermal growth factor (EGF) stimulates proliferation and differentiation of various cells, including fibroblasts, endothelial cells, and epithelial cells, and shows mitogenic and migratory activity on the edge keratinocytes of the lesions [41]. EGF participates in this mechanism, which is considered essential in the cutaneous wound healing, which begins a few hours after the injury, but presents a more evident activity in the proliferative phase of wound healing,

Another growth factor that involves healing process activity is the family of fibroblast growth factors (FGF), which have already been reported to play crucial events in the wound healing process [43]. FGFs are secreted by keratinocytes, fibroblasts, endothelial cells, smooth muscle cells, chondrocytes, and mast cells [44]. During an acute cutaneous wound process, it has been reported an increase in the production of FGF-2 and that they are responsible for formation of granulation tissue, reepithelialization, and tissue remodeling [45]. Moreover, functions such as synthesis, deposition of various constituents of the extracellular matrix, and increased motility

Transforming growth factor type-β (TGF-β) activity in the healing process was analyzed by being one of the proteins with the greatest spectrum of activities, with effect on cell proliferation, differentiation and production of extracellular matrix, and immunological modulation [46, 47]. Moreover, TGF-β has many biological activities and is thought to be a particularly important contributor to fibrosis, angiogenesis, and tissue repair. This growth factor can also influence T cells, including Th17 and Treg cells, as well as B cells, dendritic cells, NK cells,

and continues until the extracellular matrix remodeling phase [42].

thrombospondin-1 inhibits proteolytic and enzymatic degradation of PDGF [36].

process of diabetic wounds [40].

14 Wound Healing - Current Perspectives

of keratinocytes are regulated by FGF-2 [46].

neutrophils, and eosinophils [48, 49].

Increasing scientific knowledge has contributed to define highly coordinated molecular and cellular events involved in the cutaneous wound healing. Recent findings show that there is a clear correlation between the stage of the wound and its effectiveness in the healing process, and endogenous mediators, such as cytokines, chemokines, and growth factors, indicate important and crucial steps in the normal healing and are useful as prognostic indicators. Although much is known about the cellular and molecular basis of normal skin healing, there are still avenues of research left to unravel that will guide us to better therapies, new therapeutic targets, and strategies for the skin wound treatment, especially chronic wounds.

### Conflict of interest

The authors declare no conflict of interests.

### Author details

Fernando Pereira Beserra<sup>1</sup> , Lucas Fernando Sérgio Gushiken<sup>1</sup> , Maria Fernanda Hussni<sup>2</sup> and Cláudia Helena Pellizzon<sup>1</sup> \*

\*Address all correspondence to: claudia@ibb.unesp.br

1 Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), São Paulo, Brazil

2 Undergraduate Student School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), São Paulo, Brazil

### References


[4] Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature. 2008;453:314-321. DOI: 10.1038/nature07039

[18] Zhao R, Liang H, Clarke E, Jackson C, Xue M. Inflammation in chronic wounds. International Journal of Molecular Sciences. 2016;17:1-14. DOI: 10.3390/ijms17122085

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

http://dx.doi.org/10.5772/intechopen.81731

17

[19] MacLeod AS, Rudolph R, Corriden R, Ye I, Garijo O, Havran WL. Skin-resident T cells sense ultraviolet radiation-induced injury and contribute to DNA repair. Journal of Immu-

[20] Gragnani A, Müller BR, da Silva IDCG, de Noronha SMR, Ferreira LM. Keratinocyte growth factor, tumor necrosis factor-alpha and interleukin-1 beta gene expression in cultured fibroblasts and keratinocytes from burned patients. Acta Cirurgica Brasileira. 2013;28:551-558

[21] Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold

[22] Scheller J, Chalaris A, Schmidt-arras D, Rose-john S. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochimica et Biophysica Acta. 2011;1813:878-888.

[23] Mayadas TN, Cullere X, Lowell CA. The multifaceted functions of neutrophils. Annual Review of Pathology Mechanisms of Disease. 2014;9:181-218. DOI: 10.1146/annurev-pathol-

[24] Avitabile S, Odorisio T, Madonna S, Eyerich S, Guerra L, Eyerich K, et al. Interleukin-22 promotes wound repair in diabetes by improving keratinocyte pro-healing functions. The Journal of Investigative Dermatology. 2015;135:2862-2870. DOI: 10.1038/jid.2015.278 [25] Spiller KL, Nassiri S, Witherel CE, Anfang RR, Ng J, Nakazawa KR, et al. Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds. Biomaterials. 2015;37:194-207. DOI:

[26] Saraiva M, O'Garra A. The regulation of IL-10 production by immune cells. Nature

[27] Yamamoto T, Eckes B, Krieg T. Effect of Interleukin-10 on the gene expression of type I collagen, fibronectin, and decorin in human skin fibroblasts: Differential regulation by transforming growth factor-β and monocyte chemoattractant protein-1. Biochemical and Biophysical Research Communications. 2001;281:200-205. DOI: 10.1006/bbrc.2001.4321

[28] Nakayama T, Watanabe Y, Oiso N, Higuchi T, Shigeta A, Mizuguchi N, et al. Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1. Journal of Immunology. 2010;185:

[29] Sugaya M. Chemokines and skin diseases. Archivum Immunologiae et Therapiae Exp-

[30] White GE, Greaves DR. Fractalkine: A survivor's guide chemokines as antiapoptotic mediators. Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:589-594. DOI:

erimentalis (Warsz). 2015;63:109-115. DOI: 10.1007/s00005-014-0313-y

papers3://publication/uuid/29D2218B-4653-4A36-AF38-4DAC5ACDC4E0

nology. 2014;192:5695-5702. DOI: 10.4049/jimmunol.1303297

Spring Harbor Perspectives in Biology. 2014;6:a016295

DOI: 10.1016/j.bbamcr.2011.01.034

10.1016/j.biomaterials.2014.10.017.Sequential

6472-6479. DOI: 10.4049/jimmunol.0904126

10.1161/ATVBAHA.111.237412

Reviews. Immunology. 2010;10:170-181. DOI: 10.1038/nri2711

020712-164023


[18] Zhao R, Liang H, Clarke E, Jackson C, Xue M. Inflammation in chronic wounds. International Journal of Molecular Sciences. 2016;17:1-14. DOI: 10.3390/ijms17122085

[4] Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration.

[5] Tziotzios C, Profyris C, Sterling J. Cutaneous scarring: Pathophysiology, molecular mechanisms, and scar reduction therapeutics. Journal of the American Academy of Dermatol-

[6] Reinke JM, Sorg H. Wound repair and regeneration. European Surgical Research. 2012;49:

[7] Singer AJ, Clark RAF. Cutaneous wound healing. The New England Journal of Medicine.

[8] Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: Molecular and cellular mechanisms. The Journal of Investigative Dermatology. 2017;127:514-525. DOI: 10.1038/sj.

[9] Boateng JS, Matthews KH, Stevens HNE, Eccleston GM. Wound healing dressings and drug delivery systems: A review. Journal of Pharmaceutical Sciences. 2008;97:2892-2923.

[10] Martin P, Feng Y. Inflammation: Wound healing in zebrafish. Nature. 2009;459:921-923.

[11] Morin C, Roumegous A, Carpentier G, Barbier-Chassefière V, Garrigue-Antar L, Caredda S, et al. Modulation of inflammation by Cicaderma ointment accelerates skin wound healing. The Journal of Pharmacology and Experimental Therapeutics. 2012;343:115-124.

[12] Wilgus TA, Roy S, McDaniel JC. Neutrophils and wound repair: Positive actions and negative reactions. Advances in Wound Care. 2013;2:379-388. DOI: 10.1089/wound.2012.0383

[13] Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflamma-

[14] Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nature

[15] Nagaoka T, Kaburagi Y, Hamaguchi Y, Hasegawa M, Takehara K, Steeber DA, et al. Delayed wound healing in the absence of intercellular adhesion molecule-1 or L-selectin expression. The American Journal of Pathology. 2000;157:237-247. DOI: 10.1016/S0002-9440(10)

[16] Mori R, Kondo T, Ohshima T, Ishida Y, Mukaida N. Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration. The FASEB

[17] Larouche J, Sheoran S, Maruyama K, Martino MM. Immune regulation of skin wound healing: Mechanisms and novel therapeutic targets. Advances in Wound Care. 2018;7(7):

tion. Nature Reviews. Immunology. 2013;13:159-175. DOI: 10.1038/nri3399

Reviews. Immunology. 2012;11:723-737. DOI: 10.1038/nri3073.Protective

Nature. 2008;453:314-321. DOI: 10.1038/nature07039

ogy. 2012;66:13-24. DOI: 10.1016/j.jaad.2011.08.035

1999;341:738-746. DOI: 10.1056/NEJM199909023411006

35-43. DOI: 10.1159/000339613

jid.5700701

16 Wound Healing - Current Perspectives

64534-8

Journal. 2002;16:963-974

209-231. DOI: 10.1089/wound.2017.0761

DOI: 10.1002/jps.21210

DOI: 10.1038/459921a

DOI: 10.1124/jpet.111.188599


[31] Ishida Y, Gao J-L, Murphy PM. Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function. Journal of Immunology. 2008;180:569-579. DOI: 10.4049/jimmunol.180.1.569

[44] Nie C, Yang D, Xu J, Si Z, Jin X, Zhang J. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. Cell Transplanta-

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

http://dx.doi.org/10.5772/intechopen.81731

19

[45] Jaferian S, Soleymaninejad M, Negahdari B, Eatemadi A. Stem cell, biomaterials and growth factors therapy for hepatocellular carcinoma. Biomedicine and Pharmacotherapy.

[46] Lee SH, Lee JH, Cho KH. Effects of human adipose-derived stem cells on cutaneous wound healing in nude mice. Annals of Dermatology. 2011;23:150-155. DOI: 10.5021/

[47] Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. The FASEB Journal.

[48] Biernacka A, Dobaczewski M, Frangogiannis NG. TGF-b signaling in fibrosis. Growth

[49] Sanjabi S, Oh SA, Li MO. Regulation of the immune response by TGF-B: From conception to autoimmunity and infection. Cold Spring Harbor Perspectives in Biology. 2017;9:

[50] Chen L, Mehta ND, Zhao Y, DiPietro LA. Absence of CD4 or CD8 lymphocytes changes infiltration of inflammatory cells and profiles of cytokine expression in skin wounds, but does not impair healing. Experimental Dermatology. 2014;23:189-194. DOI: 10.1021/nn300902w.

[51] Dovi JV, He LK, DiPietro L. Accelerated wound closure in neutrophil-depleted mice.

[52] Kim MH, Gorouhi F, Ramirez S, Granick JL, Byrne BA, Soulika AM, et al. Catecholamine stress alters neutrophils trafficking and impairs wound healing by beta2 adrenergic receptor mediated upregulation of IL-6. The Journal of Investigative Dermatology. 2014;134:

[53] Rozlomiy VL, Markov AG. Effect of interleukin-1beta on the expression of tight junction proteins in the culture of HaCaT keratinocytes. Bulletin of Experimental Biology and

[54] Li J, Bower AJ, Vainstein V, Gluzman-Poltorak Z, Chaney EJ, Marjanovic M, et al. Effect of recombinant interleukin-12 on murine skin regeneration and cell dynamics using in vivo multimodal microscopy. Biomedical Optics Express. 2015;6:4277. DOI: 10.1364/BOE.6.004277

[55] Fuentes D, Chacón L, Casacó A, Ledón N, Fernández N, Iglesias A, et al. Effects of an epidermal growth factor receptor-based cancer vaccine on wound healing and inflammation processes in murine experimental models. International Wound Journal. 2014;11:

[56] Lin ZQ. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. Journal of Leukocyte Biology. 2003;73:

Journal of Leukocyte Biology. 2003;73:448-455. DOI: 10.1189/jlb.0802406

tion. 2011;20:205-216. DOI: 10.3727/096368910X520065

2017;88:1046-1053. DOI: 10.1016/j.biopha.2017.01.154

Factors. 2011;29:196-202. DOI: 10.3109/08977194.2011.595714

2004;18:816-827. DOI: 10.1096/fj.03-1273rev

a022236. DOI: 10.1101/cshperspect.a022236

809-817. DOI: 10.1038/jid.2013.415.Catecholamine

98-103. DOI: 10.1111/j.1742-481X.2012.01074.x

713-721. DOI: 10.1189/jlb.0802397

Medicine. 2010;149:280-283

ad.2011.23.2.150

Release


[44] Nie C, Yang D, Xu J, Si Z, Jin X, Zhang J. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. Cell Transplantation. 2011;20:205-216. DOI: 10.3727/096368910X520065

[31] Ishida Y, Gao J-L, Murphy PM. Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function. Journal of

[32] Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair and Regeneration. 2008;16:585-601. DOI:

[33] Koria P. Delivery of growth factors for tissue regeneration and wound healing. BioDrugs.

[34] Uysal AC, Mizuno H, Tobita M, Ogawa R, Hyakusoku H. The effect of adipose-derived stem cells on ischemia-reperfusion injury: Immunohistochemical and ultrastructural evaluation. Plastic and Reconstructive Surgery. 2009;124:804-815. DOI: 10.1097/PRS.0b013e3181b17bb4

[35] Mardani M, Asadi-Samani M, Rezapour S, Rezapour P. Evaluation of bred fish and seawater fish in terms of nutritional value, and heavy metals. Journal of Chemical and Pharma-

[36] Mardani M, Mahmoud B, Moradmand JS, Salehi A, Davoodi M, Ghobadi S, et al. Comparison of the descurainia sophia and levostatin effect on the ldl cholesterol reduction, a clinical trial study. Journal of Chemical and Pharmaceutical Sciences. 2016;9:1329-1333

[37] Nagy JA, Dvorak AM, Dvorak HF. Vascular hyperpermeability, angiogenesis, and stroma generation. Cold Spring Harbor Perspectives in Medicine. 2012;2:a006544. DOI: 10.1101/

[38] Kim HR, Lee JH, Kim KW, Kim BM, Lee SH. The relationship between synovial fluid VEGF and serum leptin with ultrasonographic findings in knee osteoarthritis. International Journal of Rheumatic Diseases. 2016;19:233-240. DOI: 10.1111/1756-185X.12486

[39] Huang SP, Hsu CC, Chang SC, Wang CH, Deng SC, Dai NT, et al. Adipose-derived stem cells seeded on acellular dermal matrix grafts enhance wound healing in a murine model of a full-thickness defect. Annals of Plastic Surgery. 2012;69:656-662. DOI: 10.1097/SAP.

[40] Song SH, Lee MO, Lee JS, Jeong HC, Kim HG, Kim WS, et al. Genetic modification of human adipose-derived stem cells for promoting wound healing. Journal of Dermatolog-

[41] Singh B, Carpenter G, Coffey RJ. EGF receptor ligands: Recent advances. F1000Research.

[42] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Phys-

[43] Ghafarzadeh M, Eatemadi A. Clinical efficacy of liposome-encapsulated Aloe vera on melasma treatment during pregnancy. Journal of Cosmetic and Laser Therapy. 2017;19:

ical Science. 2012;66:98-107. DOI: 10.1016/j.jdermsci.2012.02.010

2016;5:2270. DOI: 10.12688/f1000research.9025.1

181-187. DOI: 10.1080/14764172.2017.1279329

iological Reviews. 2003;83:835-870

Immunology. 2008;180:569-579. DOI: 10.4049/jimmunol.180.1.569

2012;26:163-175. DOI: 10.2165/11631850-000000000-00000

10.1111/j.1524-475X.2008.00410.x

18 Wound Healing - Current Perspectives

ceutical Sciences. 2016;9:1277-1283

cshperspect.a006544

0b013e318273f909


[57] Lian X, Yang L, Gao Q, Yang T. IL-1α is a potent stimulator of keratinocyte tissue plasminogen activator expression and regulated by TGF-β1. Archives of Dermatological Research. 2008;300:185-193. DOI: 10.1007/s00403-007-0828-8

[68] Guo Y, Yang Z, Wu S, Xu P, Peng Y, Yao M. Inhibition of IRF8 negatively regulates macrophage function and impairs cutaneous wound healing. Inflammation. 2017;40:

Regulatory Mechanisms and Chemical Signaling of Mediators Involved in the Inflammatory Phase of Cutaneous…

http://dx.doi.org/10.5772/intechopen.81731

21

[69] Sun D, Yeh C-H, So E, Wang L, Wei T, Chang M, et al. Interleukin (IL)-19 promoted skin wound healing by increasing fibroblast keratinocyte growth factor expression. Cytokine.

[70] Blomme EAG, Chinn KS, Hardy MM, Casler JJ, Kim SH, Opsahl AC, et al. Selective cyclooxygenase-2 inhibition does not affect the healing of cutaneous full-thickness incisional wounds in SKH-1 mice. The British Journal of Dermatology. 2003;148:211-223.

[71] Ishida Y, Kondo T, Takayasu T, Iwakura Y, Mukaida N. The essential involvement of cross-talk between IFN- and TGF- in the skin wound-healing process. Journal of Immu-

[72] De Masi ECDJ, Campos ACL, De Masi FDJ, Ratti MAS, Ike IS, De Masi RDJ. A influência de fatores de crescimento na cicatrização de feridas cutâneas de ratas. Brazilian Journal of

[73] Chen L, Guo S, Ranzer MJ, Dipietro LA. Toll-like receptor 4 plays an essential role in early skin wound healing. Journal of Investigative Dermatology. 2013;133:258-267. DOI:

[74] Li Y, Wang Y, Zhou L, Liu M, Liang G, Yan R, et al. Vγ4 T cells inhibit the pro-healing functions of dendritic epidermal T cells to delay skin wound closure through IL-17A.

[75] Gallucci RM, Sloan DK, Heck JM, Murray AR, O'Dell SJ. Interleukin 6 indirectly induces keratinocyte migration. The Journal of Investigative Dermatology. 2004;122:764-772. DOI:

[76] Tanno H, Kawakami K, Kanno E, Suzuki A, Takagi N, Yamamoto H, et al. Invariant NKT cells promote skin wound healing by preventing a prolonged neutrophilic inflammatory response. Wound Repair and Regeneration. 2017;25:805-815. DOI: 10.1111/wrr.12588

[77] Chen H, Shi R, Luo B, Yang X, Qiu L, Xiong J, et al. Macrophage peroxisome proliferatoractivated receptor B deficiency delays skin wound healing through impairing apoptotic cell clearance in mice. Cell Death and Disease. 2015;6:1-13. DOI: 10.1038/cddis.2014.544

[78] Trautmann A, Toksoy A, Engelhardt E, Bröcker E-B, Gillitzer R. Mast cell involvement in normal human skin wound healing: Expression of monocyte chemoattractant protein-1 is correlated with recruitment of mast cells which synthesize interleukin-4 in vivo. The Journal of Pathology. 2000;190:100-106. DOI: 10.1002/(SICI)1096-9896(200001)190:1<100::AID-PATH496>3.0.

nology. 2004;172:1848-1855. DOI: 10.4049/jimmunol.172.3.1848

Otorhinolaryngology. 2016;82:512-521. DOI: 10.1016/j.bjorl.2015.09.011

Frontiers in Immunology. 2018;9:1-19. DOI: 10.3389/fimmu.2018.00240

68-78. DOI: 10.1007/s10753-016-0454-8

DOI: 10.1046/j.1365-2133.2003.05065.x

10.1038/jid.2012.267.Toll-like

10.1111/j.0022-202X.2004.22323.x

CO;2-Q

2013;62:360-368. DOI: 10.1016/j.cyto.2013.03.017


[68] Guo Y, Yang Z, Wu S, Xu P, Peng Y, Yao M. Inhibition of IRF8 negatively regulates macrophage function and impairs cutaneous wound healing. Inflammation. 2017;40: 68-78. DOI: 10.1007/s10753-016-0454-8

[57] Lian X, Yang L, Gao Q, Yang T. IL-1α is a potent stimulator of keratinocyte tissue plasminogen activator expression and regulated by TGF-β1. Archives of Dermatological

[58] Liechty KW, Crombleholme TM, Cass DL, Martin B, Adzick NS. Diminished interleukin-8 (IL-8) production in the fetal wound healing response. The Journal of Surgical Research.

[59] Shook B, Xiao E, Kumamoto Y, Iwasaki A, Horsley V. CD301b+ macrophages are essential for effective skin wound healing. The Journal of Investigative Dermatology. 2016;136:

[60] Cunnion KM, Krishna NK, Pallera HK, Pineros-Fernandez A, Rivera MG, Hair PS, et al. Complement activation and STAT4 expression are associated with early inflammation in

[61] Kimura T, Sugaya M, Blauvelt A, Okochi H, Sato S. Delayed wound healing due to increased interleukin-10 expression in mice with lymphatic dysfunction. Journal of Leu-

[62] Arai KY, Ono M, Kudo C, Fujioka A, Okamura R, Nomura Y, et al. IL-1β stimulates activin βA mRNA expression in human skin fibroblasts through the MAPK pathways, the nuclear factor-κB pathway, and prostaglandin E2. Endocrinology. 2011;152:3779-3790.

[63] Lania BG, Morari J, De Souza AL, Da Silva MN, De Almeida AR, Veira-Damiani G, et al. Topical use and systemic action of green and roasted coffee oils and ground oils in a cutaneous incision model in rats (Rattus norvegicus albinus). PLoS One. 2017;12:1-17.

[64] Lucas T, Waisman A, Ranjan R, Roes J, Krieg T, Muller W, et al. Differential roles of macrophages in diverse phases of skin repair. Journal of Immunology. 2010;184:3964-3977.

[65] Rodero MP, Hodgson SS, Hollier B, Combadiere C, Khosrotehrani K. Reduced IL-17a expression distinguishes a Ly6c lo MHCIIhimacrophage population promoting wound healing. The Journal of Investigative Dermatology. 2013;133:783-792. DOI: 10.1038/jid.

[66] Xu P, Fu X, Xiao N, Guo Y, Pei Q, Peng Y, et al. Involvements of γδT lymphocytes in acute and chronic skin wound repair. Inflammation. 2017;40:1416-1427. DOI: 10.1007/s10753-

[67] Maeda S, Fujimoto M, Matsushita T, Hamaguchi Y, Takehara K, Hasegawa M. Inducible costimulator (ICOS) and ICOS ligand signaling has pivotal roles in skin wound healing via cytokine production. The American Journal of Pathology. 2011;179:2360-2369. DOI:

diabetic wounds. PLoS One. 2017;12:1-19. DOI: 10.1371/journal.pone.0170500

Research. 2008;300:185-193. DOI: 10.1007/s00403-007-0828-8

kocyte Biology. 2013;94:137-145. DOI: 10.1189/jlb.0812408

1998;77:80-84. DOI: 10.1006/jsre.1998.5345

20 Wound Healing - Current Perspectives

1885-1891. DOI: 10.1038/ng.3641.Punctuated

DOI: 10.1210/en.2011-0255

DOI: 10.1371/journal.pone.0188779

DOI: 10.4049/jimmunol.0903356

10.1016/j.ajpath.2011.07.048

2012.368

017-0585-6


[79] Egozi EI, Ferreira AM, Burns AL, Gamelli RL, Dipietro LA. Mast cells modulate the inflammatory but not the proliferative response in healing wounds. Wound Repair and Regeneration. 2003;11:46-54. DOI: 10.1046/j.1524-475X.2003.11108.x

**Chapter 3**

**Provisional chapter**

**Biomarkers of Wound Healing**

**Biomarkers of Wound Healing**

DOI: 10.5772/intechopen.80222

The prevalence of conditions that eventually result in poor wound healing abounds as humans advance in age. With the increased possibility of wounds not healing comes a leap in morbidity and mortality with its accompanying socioeconomic impact. It is therefore relevant to understand what accounts for aberrant wound healing and more importantly the molecular markers involved in this pathological state. There are known events associated with the wound healing process, spanning from cellular involvement to the role of specific proteins such as cytokines and growth factors that are significant biomarkers in the wound healing process. This chapter discusses biomarkers relevant to the wound healing process, and these biomarkers go a long way to help identify and stratify nonhealing patients for whom biomarker-guided approaches may be of impor-

The concept of biomarkers has existed from the time of the inception of ayurvedic medicine, just around the seventh century when the sweetness of urine was linked to diabetes even though the terminology had not been developed then [1]. The perspective of what constitutes the definition of a biomarker is somewhat diverse. Biomarkers (biological markers) are generally biomolecules whose qualitative and quantitative presence provides an indication of the state of a biological system. A more exhaustive definition as provided by the World Health Organization (WHO) led joint venture on chemical safety that describes a biomarker as any substance, structure, or process that can be measured in the body or its products that can

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Christian Agyare, Newman Osafo and

Christian Agyare, Newman Osafo and

http://dx.doi.org/10.5772/intechopen.80222

tance clinically in their management.

**Keywords:** wound, biomarkers, cytokines, growth factors, proteases

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Yaw Duah Boakye

**Abstract**

**1. Introduction**

Yaw Duah Boakye


**Chapter 3 Provisional chapter**

#### **Biomarkers of Wound Healing Biomarkers of Wound Healing**

Christian Agyare, Newman Osafo and Yaw Duah Boakye Christian Agyare, Newman Osafo and Yaw Duah Boakye

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80222

#### **Abstract**

[79] Egozi EI, Ferreira AM, Burns AL, Gamelli RL, Dipietro LA. Mast cells modulate the inflammatory but not the proliferative response in healing wounds. Wound Repair and

[80] Shi J, Ma X, Su Y, Song Y, Tian Y, Yuan S, et al. MiR-31 mediates inflammatory signaling to promote re-epithelialization during skin wound healing. The Journal of Investigative

[81] Han HM, Ko S, Cheong MJ, Bang JK, Seo CH, Luchian T, et al. Myxinidin2 and myxinidin3 suppress inflammatory responses through STAT3 and MAPKs to promote wound

[82] Yang B, Suwanpradid J, Sanches-Lagunes R, Choi HW, Hoang P, Wang D, et al. IL-27 facilitates skin wound healing through induction of epidermal proliferation and host defense. The Journal of Investigative Dermatology. 2017;137:1166-1175. DOI: 10.1016/j.

Regeneration. 2003;11:46-54. DOI: 10.1046/j.1524-475X.2003.11108.x

Dermatology. 2018;18:31857-31858. DOI: 10.1016/j.jid.2018.03.1521

clinbiochem.2015.06.023.Gut-Liver

22 Wound Healing - Current Perspectives

healing. Oncotarget. 2017;8:87582-87597. DOI: 10.18632/oncotarget.20908

The prevalence of conditions that eventually result in poor wound healing abounds as humans advance in age. With the increased possibility of wounds not healing comes a leap in morbidity and mortality with its accompanying socioeconomic impact. It is therefore relevant to understand what accounts for aberrant wound healing and more importantly the molecular markers involved in this pathological state. There are known events associated with the wound healing process, spanning from cellular involvement to the role of specific proteins such as cytokines and growth factors that are significant biomarkers in the wound healing process. This chapter discusses biomarkers relevant to the wound healing process, and these biomarkers go a long way to help identify and stratify nonhealing patients for whom biomarker-guided approaches may be of importance clinically in their management.

DOI: 10.5772/intechopen.80222

**Keywords:** wound, biomarkers, cytokines, growth factors, proteases

### **1. Introduction**

The concept of biomarkers has existed from the time of the inception of ayurvedic medicine, just around the seventh century when the sweetness of urine was linked to diabetes even though the terminology had not been developed then [1]. The perspective of what constitutes the definition of a biomarker is somewhat diverse. Biomarkers (biological markers) are generally biomolecules whose qualitative and quantitative presence provides an indication of the state of a biological system. A more exhaustive definition as provided by the World Health Organization (WHO) led joint venture on chemical safety that describes a biomarker as any substance, structure, or process that can be measured in the body or its products that can

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

influence or predict the incidence of outcome or disease [2]. The application of biomarkers has attained a vital and grounded position in clinical research, usually as predictors of the clinical outcomes for a varied number of disease conditions and their management [3].

tissues also serve as reservoirs for the polypeptide [11]. The actions of IL-1 span from systemic changes in the neurological, hematologic, endocrinologic, and metabolic systems to some local effects that are particularly relevant in wound healing [12]. By influencing both destructive and repair processes, it contributes the mesenchymal tissue remodeling, and it does so by influencing quite a number of cells. First of all, it stimulates capillary endothelial cells to produce chemokines such as MCP-1 and also cause an upregulation of the synthesis of vascular adhesion molecules such as ICAM-1, VCAM-1, and E-selectin [13, 14]. The combined effect of these two actions is to cause the infiltration of the injury site with mononuclear cells, thus setting the stage for inflammatory response. The expression of matrix metalloproteases (MMPs) from resident fibroblasts is also under the control of IL-1. The call of MMPs to play results in the degradation of the extracellular matrix to allow for enhanced monocyte migration. It also leads to a down-modulation of the inflammatory response as MMPs degrade IL-1. Inhibiting the IL-1 pathway through the use of recombinant antibodies and macrophages from IL-1 receptor knockout mice appeared to turn the tables around as far as the wound microenvironment is concerned by inducing a switch from pro-inflammatory to healingassociated macrophage phenotypes and growth factors [14]. Therefore, there is a negative

Biomarkers of Wound Healing

25

http://dx.doi.org/10.5772/intechopen.80222

Interleukin 6 is described as the chief contributor to the stimulation of a majority of the acute-phase proteins during inflammation. IL-6-deficient transgenic mice (IL-6 KO) therefore showed a substantial delayed cutaneous wound healing relative to the wild-type control ani-

Based on similar animal model studies on IL-6 knockout mice and the administration of recombinant murine IL-6 protein, IL-6 was found to be essential in stimulating the mitogenic activity of keratinocytes, an action that has been linked to scar formation as well as exerting a chemo-attractive action on neutrophils [6]. These effects seek to kick-start the wound healing process. However, a study conducted to determine the indicators of inflammation in the pathogenesis of diabetic foot ulcers identified a positive correlation between high serum IL-6 levels in diabetic patients with foot ulcers and low serum IL-6 levels in those without foot

This is not surprising as IL-6 has a reputation for dictating the transition from acute to chronic

Tumor necrosis factor alpha (TNF-α) is a key pro-inflammatory cytokine involved in the early phase of most inflammatory events in the body. Employing mouse models, the expression of TNF-α at detectable levels was discovered to happen just after wound creation and sees an increase in the first several hours until it reaches a peak within 24 hours after which it returns to the basal level [17]. Vascular endothelial cells, keratinocytes, and fibroblasts are the major sources of TNF-α which cause an initiation of the inflammatory phase of the wound

implication for wound healing in the absence of high expression of IL-1.

mals by about threefold, the time required for healing [15].

ulcers. This implicates its effect on poor wound healing [16].

**2.3. Tumor necrosis factor-α (TNF-α)**

inflammation systemically by its stimulatory effects on T and B cells.

**2.2. Interleukin 6 (IL-6)**

Extensive scientific investigation into the mechanism of wound healing has revealed that the traditional guides in the determination of the wound healing potential, i.e., erythrocyte sedimentation rate (ESR) and C-reactive protein, do not yield enough positive and negative predictive values [4]. In lieu of the scientific evidence available, the focus has shifted to cytokines, chemokines, and proteases which hold the greatest potential as biomarkers [4].

### **2. Cytokines**

Cytokines are proteins of relatively low molecular weight that are secreted to influence or modulate the behavior of immune cells and also other cells [5]. Crucial among them include interleukins, lymphokines, and other signaling molecules such as interferons and tissue necrosis factor (TNF-α). It has been long considered and corroborated by scientific evidence that pro-inflammatory cytokines such as interleukins 1α (IL-1α), 1β (IL-1β), and 6 (IL-6) and TNF-α play essential roles in wound healing process such as the stimulation of keratinocyte and fibroblast proliferation, modulation of immune response, synthesis and breakdown of extracellular matrix proteins, and the chemotaxis of fibroblast to the wound site [6].

Grellner et al. [7, 8] in their work to quantitatively analyze pro-inflammatory cytokines in human skin wounds realized an upregulation of the expression of IL-1α, IL-1β, IL-6, and TNF-α in the inflammatory phase of the wound healing process. The levels of these proinflammatory cytokines (TNF-α, IL-1, and IL-6) were higher in nonhealing wounds than healing wounds owing to the fact that nonhealing wounds stay in the inflammatory phase of wound healing process [4]. Bilder et al. [9] also report an increase in the levels of IL-8 in chronic nonhealing wounds as opposed to those with a healing potential. Ligi et al. [10] upon the assessment of several studies which evaluated the level expression of cytokines and chemokines in the microenvironment of a chronic ulcer alluded to a heightened pro-inflammatory condition in a nonhealing wound, thus corroborating other studies. It was however noted that the level of cytokines detectable does not necessarily correlate to its bioactivity due to antiinflammatory cytokines whose presence counteracts the activity of these pro-inflammatory cytokines [10]. There are also specific cytokine inhibitors and proteolytic enzymes that also act on these cytokines to mask their bioavailability [10]. Patel et al. [4] also report the inconsistency in wound and serum levels of cytokines which poses a challenge in its use as reliable biomarkers of nonhealing wounds.

#### **2.1. Interleukin 1 (IL-1)**

The IL-1 family of cytokines is made up of two pro-inflammatory cytokines, namely, IL-*α* and IL-*β*. Interleukin 1 is primarily sourced from macrophages in the event of injury, infection, and antigenic challenge although the epidermal, epithelial, lymphoid, and vascular tissues also serve as reservoirs for the polypeptide [11]. The actions of IL-1 span from systemic changes in the neurological, hematologic, endocrinologic, and metabolic systems to some local effects that are particularly relevant in wound healing [12]. By influencing both destructive and repair processes, it contributes the mesenchymal tissue remodeling, and it does so by influencing quite a number of cells. First of all, it stimulates capillary endothelial cells to produce chemokines such as MCP-1 and also cause an upregulation of the synthesis of vascular adhesion molecules such as ICAM-1, VCAM-1, and E-selectin [13, 14]. The combined effect of these two actions is to cause the infiltration of the injury site with mononuclear cells, thus setting the stage for inflammatory response. The expression of matrix metalloproteases (MMPs) from resident fibroblasts is also under the control of IL-1. The call of MMPs to play results in the degradation of the extracellular matrix to allow for enhanced monocyte migration. It also leads to a down-modulation of the inflammatory response as MMPs degrade IL-1. Inhibiting the IL-1 pathway through the use of recombinant antibodies and macrophages from IL-1 receptor knockout mice appeared to turn the tables around as far as the wound microenvironment is concerned by inducing a switch from pro-inflammatory to healingassociated macrophage phenotypes and growth factors [14]. Therefore, there is a negative implication for wound healing in the absence of high expression of IL-1.

#### **2.2. Interleukin 6 (IL-6)**

influence or predict the incidence of outcome or disease [2]. The application of biomarkers has attained a vital and grounded position in clinical research, usually as predictors of the clinical

Extensive scientific investigation into the mechanism of wound healing has revealed that the traditional guides in the determination of the wound healing potential, i.e., erythrocyte sedimentation rate (ESR) and C-reactive protein, do not yield enough positive and negative predictive values [4]. In lieu of the scientific evidence available, the focus has shifted to cytokines,

Cytokines are proteins of relatively low molecular weight that are secreted to influence or modulate the behavior of immune cells and also other cells [5]. Crucial among them include interleukins, lymphokines, and other signaling molecules such as interferons and tissue necrosis factor (TNF-α). It has been long considered and corroborated by scientific evidence that pro-inflammatory cytokines such as interleukins 1α (IL-1α), 1β (IL-1β), and 6 (IL-6) and TNF-α play essential roles in wound healing process such as the stimulation of keratinocyte and fibroblast proliferation, modulation of immune response, synthesis and breakdown of

Grellner et al. [7, 8] in their work to quantitatively analyze pro-inflammatory cytokines in human skin wounds realized an upregulation of the expression of IL-1α, IL-1β, IL-6, and TNF-α in the inflammatory phase of the wound healing process. The levels of these proinflammatory cytokines (TNF-α, IL-1, and IL-6) were higher in nonhealing wounds than healing wounds owing to the fact that nonhealing wounds stay in the inflammatory phase of wound healing process [4]. Bilder et al. [9] also report an increase in the levels of IL-8 in chronic nonhealing wounds as opposed to those with a healing potential. Ligi et al. [10] upon the assessment of several studies which evaluated the level expression of cytokines and chemokines in the microenvironment of a chronic ulcer alluded to a heightened pro-inflammatory condition in a nonhealing wound, thus corroborating other studies. It was however noted that the level of cytokines detectable does not necessarily correlate to its bioactivity due to antiinflammatory cytokines whose presence counteracts the activity of these pro-inflammatory cytokines [10]. There are also specific cytokine inhibitors and proteolytic enzymes that also act on these cytokines to mask their bioavailability [10]. Patel et al. [4] also report the inconsistency in wound and serum levels of cytokines which poses a challenge in its use as reliable

The IL-1 family of cytokines is made up of two pro-inflammatory cytokines, namely, IL-*α* and IL-*β*. Interleukin 1 is primarily sourced from macrophages in the event of injury, infection, and antigenic challenge although the epidermal, epithelial, lymphoid, and vascular

extracellular matrix proteins, and the chemotaxis of fibroblast to the wound site [6].

outcomes for a varied number of disease conditions and their management [3].

chemokines, and proteases which hold the greatest potential as biomarkers [4].

**2. Cytokines**

24 Wound Healing - Current Perspectives

biomarkers of nonhealing wounds.

**2.1. Interleukin 1 (IL-1)**

Interleukin 6 is described as the chief contributor to the stimulation of a majority of the acute-phase proteins during inflammation. IL-6-deficient transgenic mice (IL-6 KO) therefore showed a substantial delayed cutaneous wound healing relative to the wild-type control animals by about threefold, the time required for healing [15].

Based on similar animal model studies on IL-6 knockout mice and the administration of recombinant murine IL-6 protein, IL-6 was found to be essential in stimulating the mitogenic activity of keratinocytes, an action that has been linked to scar formation as well as exerting a chemo-attractive action on neutrophils [6]. These effects seek to kick-start the wound healing process. However, a study conducted to determine the indicators of inflammation in the pathogenesis of diabetic foot ulcers identified a positive correlation between high serum IL-6 levels in diabetic patients with foot ulcers and low serum IL-6 levels in those without foot ulcers. This implicates its effect on poor wound healing [16].

This is not surprising as IL-6 has a reputation for dictating the transition from acute to chronic inflammation systemically by its stimulatory effects on T and B cells.

#### **2.3. Tumor necrosis factor-α (TNF-α)**

Tumor necrosis factor alpha (TNF-α) is a key pro-inflammatory cytokine involved in the early phase of most inflammatory events in the body. Employing mouse models, the expression of TNF-α at detectable levels was discovered to happen just after wound creation and sees an increase in the first several hours until it reaches a peak within 24 hours after which it returns to the basal level [17]. Vascular endothelial cells, keratinocytes, and fibroblasts are the major sources of TNF-α which cause an initiation of the inflammatory phase of the wound healing by promoting the recruitment of inflammatory leukocytes. TNF-α is also involved in the regulation of the activity of fibroblasts, keratinocytes, and vascular endothelial cells as well as in modulating synthesis of extracellular matrix proteins and matrix metalloproteinase [17, 18]. Based on diabetic models, an increase in TNF-α level coupled with decrease in IL-10 that has anti-inflammatory properties results in sustained expression of chemokines CXCL2 and CCL2 and leads to continuous infiltration of leucocytes to the injury site. This ultimately prolongs the inflammation and reduces the wound healing potential [19].

points to no local growth factor deficiency in chronic leg ulcers with the possible exception of TGF [6]. Trengove et al. [26] after studying wound fluids from both healing and nonhealing wounds arrive at similar conclusion that poor wound healing may be due to inflammatory

Biomarkers of Wound Healing

27

http://dx.doi.org/10.5772/intechopen.80222

Platelet-derived growth factors (PDGFs) are made up of a family of homodimeric or heterodimeric growth factors, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD [27]. PDGF has been established to have chemotactic role for cells that migrate to the healing wound site such as fibroblasts, neutrophils, and monocytes. It was actually the very first growth factor shown to have this function [28]. It additionally stimulates the proliferation of fibroblast and the deposition of extracellular matrix. In vitro studies have also revealed that it stimulates insulin growth factor (IGF) release in fibroblasts which is vital to the initiation of the repair process [28]. Lastly, it stimulates fibroblasts to contract collagen matrices and induces the myofibroblast phenotype in the implicated cells. It has thus been established to be a major player in the wound healing and has formed the basis for studies into its clinical

Owing to the close proximity of the expression sites of the PDGF, which is predominantly in the epidermis, and its receptors which are also in the dermis and granulating tissue, a paracrine mechanism has been suggested for its action [6, 29]. However, unlike other growth factors like fibroblast growth factor (FGF) and vascular epithelial growth factor (VEGF) that see an overexpression in the microenvironment or at the site of a healing wound or one in a granulation phase, the increase in the expression of PDGF-BB is without this spatial limitation as its levels in plasma also increases. It does make it potentially useful as the biomarker in

The action of proteases and their inhibitors goes a long way to influence the equilibrium between extracellular matrix (ECM) degradation and deposition which is responsible for the coordinated and timely healing of wounds [30]. There is an overwhelming wealth of evidence to suggest that nonhealing wounds are characterized by an increase in the levels of proteases and an imbalance in the protease/protease inhibitor levels [30, 31]. This manifests as a persistence of proteolysis and degradation of the extracellular matrix causing wound healing to delay. Significant among these proteases are the matrix metalloproteases (MMPs) [32]. MMPs are part of a family of zinc endopeptidase which essentially help in the degradation of provisional extracellular matrix, facilitate the migration of inflammatory cells to the wound site, remodel the granulation tissue, and modulate angiogenesis [28]. MMP activity as measured using Azocoll assay was found to be significantly elevated in chronic wounds as compared to

mediators rather than a deficiency of growth factors.

application in the treatment of wound healing disorders.

acute wounds, thus implicating it poor wound healing [26].

wound healing [10].

**4. Proteases**

**3.1. Platelet-derived growth factors (PDGF)**

#### **2.4. Transforming growth factor (TGF)**

Transforming growth factor describes the superfamily for pluripotent cytokines which have very important functions to perform during disease, homeostasis, development, and repair. These sets of proteins are structurally related, but functionally distinguishable and relevant among them for wound healing are the isoforms TGF-β 1–3 [20]. The roles of these isoforms in the wound healing process can be both distinct and overlapping. However, the overall nature of their contribution to the wound healing has generated some controversy and thus is among the most studied molecules involved in the process [6]. Transforming growth factor β1 (TNF-β1) however has the widest spectrum of actions, affecting all manners of cell types that are involved in all stages of wound healing. These effects have been reported to be both positive and negative [21]. Historically, the synthesis of TNF-β1 from keratinocytes, platelets, and macrophages is upregulated right after injury, and this is crucial for initiating inflammation and granulation tissue formation. In addition, TNF-β1 contributes to the chemotactic migration of cells during wound repair. Some proteases such as MMP-1, MMP-2, MMP-3, and MMP-9 are also under the control of TNF-β1 [6, 22]. Based on human studies, TNF-β1 was found to stimulate the production of extracellular matrix molecules, including collagens and fibronectin, which strengthen the repaired wound. In spite of this knowledge, available evidence goes to raise questions about the true effects of TNF-β1 levels on wound healing [23]. Wound healing in Smad knockout mice, which have the signaling pathway of TNF-β1 blocked, was rather accelerated to the surprise of the investigators. In similar fashion, TNF-β1 knockout mice showed demonstrated reepithelialization during incisional wound repair, in comparison with wild-type mice. The consensus in the face of current evidence is that the selective inhibition of TNF-β1 in some cells may prove beneficial [24].

### **3. Growth factors**

The growth factors are essentially responsible for the initiation of the proliferation stage of the wound healing process. The platelet-derived growth factor (PDGF), transforming growth factors (TGF-, TGF-), insulin growth factor (IGF-1), fibroblast growth factor (FGF), and granulocyte-macrophage colony-stimulating factors (GM-CSF) are examples of growth factors whose roles in wound healing as well as their possible use as biomarkers have been studied extensively based on their expressed levels [25]. In spite of the fact that insight about ideal levels and the spatiotemporal distribution of growth factors is far from complete, available data points to no local growth factor deficiency in chronic leg ulcers with the possible exception of TGF [6]. Trengove et al. [26] after studying wound fluids from both healing and nonhealing wounds arrive at similar conclusion that poor wound healing may be due to inflammatory mediators rather than a deficiency of growth factors.

### **3.1. Platelet-derived growth factors (PDGF)**

Platelet-derived growth factors (PDGFs) are made up of a family of homodimeric or heterodimeric growth factors, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD [27]. PDGF has been established to have chemotactic role for cells that migrate to the healing wound site such as fibroblasts, neutrophils, and monocytes. It was actually the very first growth factor shown to have this function [28]. It additionally stimulates the proliferation of fibroblast and the deposition of extracellular matrix. In vitro studies have also revealed that it stimulates insulin growth factor (IGF) release in fibroblasts which is vital to the initiation of the repair process [28]. Lastly, it stimulates fibroblasts to contract collagen matrices and induces the myofibroblast phenotype in the implicated cells. It has thus been established to be a major player in the wound healing and has formed the basis for studies into its clinical application in the treatment of wound healing disorders.

Owing to the close proximity of the expression sites of the PDGF, which is predominantly in the epidermis, and its receptors which are also in the dermis and granulating tissue, a paracrine mechanism has been suggested for its action [6, 29]. However, unlike other growth factors like fibroblast growth factor (FGF) and vascular epithelial growth factor (VEGF) that see an overexpression in the microenvironment or at the site of a healing wound or one in a granulation phase, the increase in the expression of PDGF-BB is without this spatial limitation as its levels in plasma also increases. It does make it potentially useful as the biomarker in wound healing [10].

### **4. Proteases**

healing by promoting the recruitment of inflammatory leukocytes. TNF-α is also involved in the regulation of the activity of fibroblasts, keratinocytes, and vascular endothelial cells as well as in modulating synthesis of extracellular matrix proteins and matrix metalloproteinase [17, 18]. Based on diabetic models, an increase in TNF-α level coupled with decrease in IL-10 that has anti-inflammatory properties results in sustained expression of chemokines CXCL2 and CCL2 and leads to continuous infiltration of leucocytes to the injury site. This ultimately

Transforming growth factor describes the superfamily for pluripotent cytokines which have very important functions to perform during disease, homeostasis, development, and repair. These sets of proteins are structurally related, but functionally distinguishable and relevant among them for wound healing are the isoforms TGF-β 1–3 [20]. The roles of these isoforms in the wound healing process can be both distinct and overlapping. However, the overall nature of their contribution to the wound healing has generated some controversy and thus is among the most studied molecules involved in the process [6]. Transforming growth factor β1 (TNF-β1) however has the widest spectrum of actions, affecting all manners of cell types that are involved in all stages of wound healing. These effects have been reported to be both positive and negative [21]. Historically, the synthesis of TNF-β1 from keratinocytes, platelets, and macrophages is upregulated right after injury, and this is crucial for initiating inflammation and granulation tissue formation. In addition, TNF-β1 contributes to the chemotactic migration of cells during wound repair. Some proteases such as MMP-1, MMP-2, MMP-3, and MMP-9 are also under the control of TNF-β1 [6, 22]. Based on human studies, TNF-β1 was found to stimulate the production of extracellular matrix molecules, including collagens and fibronectin, which strengthen the repaired wound. In spite of this knowledge, available evidence goes to raise questions about the true effects of TNF-β1 levels on wound healing [23]. Wound healing in Smad knockout mice, which have the signaling pathway of TNF-β1 blocked, was rather accelerated to the surprise of the investigators. In similar fashion, TNF-β1 knockout mice showed demonstrated reepithelialization during incisional wound repair, in comparison with wild-type mice. The consensus in the face of current evidence is that the

prolongs the inflammation and reduces the wound healing potential [19].

selective inhibition of TNF-β1 in some cells may prove beneficial [24].

The growth factors are essentially responsible for the initiation of the proliferation stage of the wound healing process. The platelet-derived growth factor (PDGF), transforming growth factors (TGF-, TGF-), insulin growth factor (IGF-1), fibroblast growth factor (FGF), and granulocyte-macrophage colony-stimulating factors (GM-CSF) are examples of growth factors whose roles in wound healing as well as their possible use as biomarkers have been studied extensively based on their expressed levels [25]. In spite of the fact that insight about ideal levels and the spatiotemporal distribution of growth factors is far from complete, available data

**2.4. Transforming growth factor (TGF)**

26 Wound Healing - Current Perspectives

**3. Growth factors**

The action of proteases and their inhibitors goes a long way to influence the equilibrium between extracellular matrix (ECM) degradation and deposition which is responsible for the coordinated and timely healing of wounds [30]. There is an overwhelming wealth of evidence to suggest that nonhealing wounds are characterized by an increase in the levels of proteases and an imbalance in the protease/protease inhibitor levels [30, 31]. This manifests as a persistence of proteolysis and degradation of the extracellular matrix causing wound healing to delay. Significant among these proteases are the matrix metalloproteases (MMPs) [32]. MMPs are part of a family of zinc endopeptidase which essentially help in the degradation of provisional extracellular matrix, facilitate the migration of inflammatory cells to the wound site, remodel the granulation tissue, and modulate angiogenesis [28]. MMP activity as measured using Azocoll assay was found to be significantly elevated in chronic wounds as compared to acute wounds, thus implicating it poor wound healing [26].

Proteases as biomarkers for wound healing hold the key to transform clinical approach to the management of wounds. For example, the appropriateness of using protease-modulating dressing and tissue-engineered products, scaffolds, and skin grafts for the treatment can be made by the determination of the levels of proteases [33].

**Author details**

Christian Agyare<sup>1</sup>

**References**

ehc/ehc222.htm

ehc/ehc155.htm

2000;**113**:251-264

1964-1974

\*, Newman Osafo<sup>2</sup>

\*Address all correspondence to: cagyare.pharm@knust.edu.gh

Nkrumah University of Science and Technology, Kumasi, Ghana

Nkrumah University of Science and Technology, Kumasi, Ghana

Journal of Molecular Biomarkers Diagnosis. 2011;**2**:118

Journal of Wound Care. 2016;**25**(1):46-55

USA: Williams & Wilkins, Philadelphia; 2006

Physiological Reviews. 2003;**83**(3):835-870

and Yaw Duah Boakye<sup>1</sup>

Biomarkers of Wound Healing

29

http://dx.doi.org/10.5772/intechopen.80222

1 Faculty of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutics, Kwame

2 Faculty of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology, Kwame

[1] Karley D, Gupta D, Tiwari A. Biomarkers: The future of medical science to detect cancer.

[2] WHO International Programme on Chemical Safety Biomarkers in Risk Assessment: Validity and Validation. 2001. Retrieved from: http://www.inchem.org/documents/ehc/

[3] WHO International Programme on Chemical Safety Biomarkers and Risk Assessment: Concepts and Principles. 1993. Retrieved from: http://www.inchem.org/documents/ehc/

[4] Patel S, Maheshwari A, Chandra A. Biomarkers for wound healing and their evaluation.

[5] Cytokine. In: Stedman's Medical Dictionary. 28th ed. Wolters Kluwer Health, Lippincott:

[6] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines.

[7] Grellner W, Georg T, Wilske J. Quantitative analysis of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) in human skin wounds. Forensic Science International.

[8] Grellner W, Vieler S, Madea B. Transforming growth factors (TGF-alpha and TGF-beta1) in the determination of vitality and wound age: Immunohistochemical study on human

[9] Bilder D, Schober M, Perrimon N. Integrated activity of PDZ protein complexes regu-

[10] Ligi D, Mosti G, Croce L, Raffetto JD, Mannello F. Chronic venous disease—Part I: Inflammatory biomarkers in wound healing. Biochimica et Biophysica Acta. 2016;**1862**(10):

skin wounds. Forensic Science International. 2005;**153**:174-180

lates epithelial polarity. Nature Cell Biology. 2003;**5**:53-58

### **5. Matrix metalloproteinase**

Matrix metalloproteinases (MMPs) are a group of endopeptidase that are zinc and calcium dependent and are usually divided into six groups depending on the substrate they act on. These MMPs consist of collagenases (MMP-1, MMP-3, MMP-8); gelatinases (MMP-2, MMP-9); stromelysins (MMP-3, MMP-10); matrilysins (MMP-7, MMP-26); membrane-type MMPs (MT-MMP) like MMP-14, MMP-15, MMP-16, and MMP-24; and other MMPs (MMP-11, MMP-12, MMP-19, MMP-20, MMP-22, MMP-23, MMP-28) [34].

Various MMPs are relevant to the wound healing process at varied points, and the tight control of their proteolytic activity is also essential to conduct the different events of wound healing [36]. MMPs are however generally involved in the inflammatory, proliferative, and remodeling phases of the wound healing process by modulating cytokine/chemokine activity by activating them enzymatically or influencing their availability by cleaving them from cell surface. Additionally, the actions of MMPs involve the breakdown of proteins part of the cellcell and cell-extracellular matrix interaction [35, 36].

In terms of the predictive roles of MMPs' level for the wound healing process, some studies have focused on the MMP-1 to tissue inhibitor of metalloproteinase (TIMP-1) ratio. In one study, for instance, a significant correlation was found between a high ratio of MMP-1/ TIMP-1 and good healing (r = 0.65, p = 0.008) with receiver operator curve (ROC) analysis showing an MMP-1/TIMP-1 ratio of 0.39 being the best predictive value for wound healing. High levels of MMP-8 and MMP-9 also appear to have negative predictive value for the process of wound healing [32].

### **6. Conclusion**

With the growing research into the therapeutic benefits of biomarkers comes the challenge of identifying biomarkers that satisfy the required characteristics for use clinically. It is prudent to validate new biomarkers affecting the wound healing process by employing innovative, simple, and cost-effective molecular approaches to determine the type, level, and activity of all potential biomarkers. With the advent of trendsetting technical knowhow in defining diseases and other biological processes, it has become increasingly possible to identify and characterize novel biomarkers of the wound healing process. Continuing the research into identification of new biomarkers affecting the wound healing process is imperative since it will eventually have weighty health benefits on patients and offer a relevant guide to wound management. This will significantly lower the risks of microbial colonization and invasion of wounds and loss of structural function as a result of chronic wounds.

### **Author details**

Proteases as biomarkers for wound healing hold the key to transform clinical approach to the management of wounds. For example, the appropriateness of using protease-modulating dressing and tissue-engineered products, scaffolds, and skin grafts for the treatment can be

Matrix metalloproteinases (MMPs) are a group of endopeptidase that are zinc and calcium dependent and are usually divided into six groups depending on the substrate they act on. These MMPs consist of collagenases (MMP-1, MMP-3, MMP-8); gelatinases (MMP-2, MMP-9); stromelysins (MMP-3, MMP-10); matrilysins (MMP-7, MMP-26); membrane-type MMPs (MT-MMP) like MMP-14, MMP-15, MMP-16, and MMP-24; and other MMPs (MMP-11,

Various MMPs are relevant to the wound healing process at varied points, and the tight control of their proteolytic activity is also essential to conduct the different events of wound healing [36]. MMPs are however generally involved in the inflammatory, proliferative, and remodeling phases of the wound healing process by modulating cytokine/chemokine activity by activating them enzymatically or influencing their availability by cleaving them from cell surface. Additionally, the actions of MMPs involve the breakdown of proteins part of the cell-

In terms of the predictive roles of MMPs' level for the wound healing process, some studies have focused on the MMP-1 to tissue inhibitor of metalloproteinase (TIMP-1) ratio. In one study, for instance, a significant correlation was found between a high ratio of MMP-1/ TIMP-1 and good healing (r = 0.65, p = 0.008) with receiver operator curve (ROC) analysis showing an MMP-1/TIMP-1 ratio of 0.39 being the best predictive value for wound healing. High levels of MMP-8 and MMP-9 also appear to have negative predictive value for the pro-

With the growing research into the therapeutic benefits of biomarkers comes the challenge of identifying biomarkers that satisfy the required characteristics for use clinically. It is prudent to validate new biomarkers affecting the wound healing process by employing innovative, simple, and cost-effective molecular approaches to determine the type, level, and activity of all potential biomarkers. With the advent of trendsetting technical knowhow in defining diseases and other biological processes, it has become increasingly possible to identify and characterize novel biomarkers of the wound healing process. Continuing the research into identification of new biomarkers affecting the wound healing process is imperative since it will eventually have weighty health benefits on patients and offer a relevant guide to wound management. This will significantly lower the risks of microbial colonization and invasion of

wounds and loss of structural function as a result of chronic wounds.

made by the determination of the levels of proteases [33].

MMP-12, MMP-19, MMP-20, MMP-22, MMP-23, MMP-28) [34].

cell and cell-extracellular matrix interaction [35, 36].

cess of wound healing [32].

**6. Conclusion**

**5. Matrix metalloproteinase**

28 Wound Healing - Current Perspectives

Christian Agyare<sup>1</sup> \*, Newman Osafo<sup>2</sup> and Yaw Duah Boakye<sup>1</sup>

\*Address all correspondence to: cagyare.pharm@knust.edu.gh

1 Faculty of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutics, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

2 Faculty of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

### **References**


[11] Chamberlain SC, Leiferman ME, Frisch EK, et al. Interleukin expression after injury and the effects of interleukin-1 receptor antagonist. PLoS One. 2013;**8**(8):e71631

[25] Olczyk P, Mencner Ł, Komosinska-Vassev K. The role of the extracellular matrix components in cutaneous wound healing. BioMed Research International. 2014;**2014**:747584,

Biomarkers of Wound Healing

31

http://dx.doi.org/10.5772/intechopen.80222

[26] Trengove NJ, Stacey MC, Macauley S, et al. Analysis of the acute and chronic wound environments: The role of proteases and their inhibitors. Wound Repair and Regeneration.

[27] Rönnstrand L. Signaling by the platelet-derived growth facto receptor family. In: Bradshaw RA, Dennis EA, editors. Handbook of Cell Signaling. Cambridge, Massachusetts, USA:

[28] Diagn JMB, Gupta A. Wound healing: Assessment by various markers. Journal of

[29] Pastar I, Stojadinovic O, Yin NC, et al. Epithelialization in wound healing: A comprehen-

[30] McCarty SM, Percival SL. Proteases and delayed wound healing. Advances in Wound

[31] Demidova-Rice TN, Hamblin MR, Herman IM. Acute and impaired wound healing: Pathophysiology and current methods for drug delivery, part 1: Normal and chronic wounds: Biology, causes, and approaches to care. Advances in Skin & Wound Care.

[32] Iuonut AM, Dindelegan GC, Ciuce C. Proteases as biomarkers in wound healing.

[33] International Consensus. The Role of Proteases in Wound Healing Diagnostics. An

[34] Ligi D, Mosti G, Croce L, Raffetto JD, Mannello F. Chronic venous disease—Part II: Proteolytic biomarkers in wound healing. Biochimica et Biophysica Acta. 2016;**1862**(10):

[35] Caley MP, Martins VLC, O'Toole EA. Metalloproteinases and wound healing. Advances

[36] Jablonska-Trypuc A, Matejczyk M, Rosochacki S. Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs. Journal of Enzyme Inhibition and Medicinal Chemistry. 2016;**31**(1):177-183

Expert Working Group Review. London: Wounds International; 2011. p. 10

8 pages

1999;**7**(6):442-452

Academic Press. p. 427

Care. 2013;**2**(8):438-447

2012;**25**(7):304-314

1900-1908

Molecular Biomarkers. 2016;**8**(2):8-10

sive review. Advances in Wound Care. 2014;**3**(7):445-464

Temporomandibular Journal. 2011;**61**(1-2):65-73

in Wound Care. 2015;**4**(4):225-234


[25] Olczyk P, Mencner Ł, Komosinska-Vassev K. The role of the extracellular matrix components in cutaneous wound healing. BioMed Research International. 2014;**2014**:747584, 8 pages

[11] Chamberlain SC, Leiferman ME, Frisch EK, et al. Interleukin expression after injury and

[12] Doersch KM, DelloStritto DJ, Newell-Rogers MK. The contribution of interleukin-2 to effective wound healing. Experimental Biology and Medicine. 2017;**242**(4):384-396 [13] Cook-Mills JM, Marchese ME, Abdala-Valencia H. Vascular cell adhesion molecule-1 expression and signaling during disease: Regulation by reactive oxygen species and

[14] Lobmann R, Zemlin C, Motzkau M, Reschke K, Lehnert H. Expression of matrix metalloproteinases and growth factors in diabetic foot wounds treated with a protease absor-

[15] Gallucci RM, Simeonova PP, Matheson JM, Kommineni C, Guriel JL, Sugawara T, Luster MI. Impaired cutaneous wound healing in interleukin-6–deficient and immunosup-

[16] Sallam AWA, El-Sharawy MHA. Role of interleukin-6 (IL-6) and indicators of inflammation in the pathogenesis of diabetic foot ulcers. Australian Journal of Basic and Applied

[17] Ritsu M, Kawakami K, Kanno E, Tanno H, Ishii K. Critical role of tumor necrosis factor- a in the early process of wound healing in skin. Journal of Dermatology and Dermatologic

[18] Mast BA, Schultz GS. Interactions of cytokines, growth factors, and proteases in acute

[19] Sen CK.Wound healing essentials: Let there be oxygen. Wound Repair and Regeneration.

[20] Huang T, Schor LS, Hinck PA. Biological activity differences between TGF-β1 and TGFβ3 correlate with differences in the rigidity and arrangement of their component mono-

[21] Wang XJ, Han G, Owens P, Siddiqui Y, Li AG. Role of TGF beta-mediated inflammation in cutaneous wound healing. The Journal of Investigative Dermatology. Symposium

[22] Manicone AM, McGuire JK. Matrix metalloproteinases as modulators of inflammation.

[23] Goncalves MBB, Rabeh SAN, Tercariol CAS. The contribution of distance learning to the knowledge of nursing lecturers regarding assessment of chronic wounds. Revista

[24] Tejiram S, Kavalukas SL, Shupp JW, Barbul A. Wound healing. Wound Healing Bio-

Seminars in Cell and Developmental Biology. 2008;**19**(1):34-41

Latino-Americana de Enfermagem. 2015;**23**(1):122-129

and chronic wounds. Wound Repair and Regeneration. 1996;**4**:411-420

bent dressing. Journal of Diabetes and its Complications. 2006;**20**(5):329-335

the effects of interleukin-1 receptor antagonist. PLoS One. 2013;**8**(8):e71631

antioxidants. Antioxidants and Redox Signaling. 2011;**15**(6):1607-1638

pressed mice. FASEB Journal. 2000;**14**(15):2525-2531

Sciences. 2012;**6**(6):430-435

30 Wound Healing - Current Perspectives

Surgery. 2017;**21**(1):14-19

mers. Biochemistry. 2014;**53**(36):5737-5749

Proceedings. 2006;**11**(1):112-117

materials. 2016:3-39

2009;**17**:1-18


**Chapter 4**

**Provisional chapter**

**Medicinal Plants in Wound Healing**

**Medicinal Plants in Wound Healing**

DOI: 10.5772/intechopen.80215

Wound healing process is known as interdependent cellular and biochemical stages which are in trying to improve the wound. Wound healing can be defined as stages which is done by body and delayed in wound healing increases chance of microbial infection. Improved wound healing process can be performed by shortening the time needed for healing or lowering the inappropriate happens. The drugs were locally or systemically administrated in order to help wound healing. Antibiotics, antiseptics, desloughing agents, extracts, etc. have been used in order to wound healing. Some synthetic drugs are faced with limitations because of their side effects. Plants or combinations derived from plants are needed to investigate identify and formulate for treatment and management of wound healing. There is increasing interest to use the medicinal plants in wound healing because of lower side effects and management of wounds over the years. Studies have shown that medicinal plants improve wound healing in diabetic, infected and opened wounds. The different mechanisms have been reported to improve the wound healing by medicinal plants. In this chapter, some medicinal plants and the reported mechanisms will be discussed.

**Keywords:** antibacterial, animal studies, inflammatory phase, medicinal plants,

Wound healing is defined as a collection of complex process which comprises different compounds including soluble mediators, blood cells, extracellular matrix, and parenchymal cells [1, 2]. Wound healing is divided into stages including inflammation process, tissue formation, and tissue remodeling. The inflammatory phase involves different stages such as platelet accumulation, coagulation, and leukocyte migration. Re-epithelialization, angiogenesis, fibroplasia, and wound contraction are stages for tissue formation. Remodeling phase may be

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Mohammad Reza Farahpour

Mohammad Reza Farahpour

**Abstract**

wound healing

**1. Introduction**

http://dx.doi.org/10.5772/intechopen.80215

#### **Medicinal Plants in Wound Healing Medicinal Plants in Wound Healing**

#### Mohammad Reza Farahpour Mohammad Reza Farahpour

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80215

#### **Abstract**

Wound healing process is known as interdependent cellular and biochemical stages which are in trying to improve the wound. Wound healing can be defined as stages which is done by body and delayed in wound healing increases chance of microbial infection. Improved wound healing process can be performed by shortening the time needed for healing or lowering the inappropriate happens. The drugs were locally or systemically administrated in order to help wound healing. Antibiotics, antiseptics, desloughing agents, extracts, etc. have been used in order to wound healing. Some synthetic drugs are faced with limitations because of their side effects. Plants or combinations derived from plants are needed to investigate identify and formulate for treatment and management of wound healing. There is increasing interest to use the medicinal plants in wound healing because of lower side effects and management of wounds over the years. Studies have shown that medicinal plants improve wound healing in diabetic, infected and opened wounds. The different mechanisms have been reported to improve the wound healing by medicinal plants. In this chapter, some medicinal plants and the reported mechanisms will be discussed.

DOI: 10.5772/intechopen.80215

**Keywords:** antibacterial, animal studies, inflammatory phase, medicinal plants, wound healing

### **1. Introduction**

Wound healing is defined as a collection of complex process which comprises different compounds including soluble mediators, blood cells, extracellular matrix, and parenchymal cells [1, 2]. Wound healing is divided into stages including inflammation process, tissue formation, and tissue remodeling. The inflammatory phase involves different stages such as platelet accumulation, coagulation, and leukocyte migration. Re-epithelialization, angiogenesis, fibroplasia, and wound contraction are stages for tissue formation. Remodeling phase may be

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lasted for 1 month, and the dermis may respond to injury with the production of collagen and matrix proteins and then returns to its pre-injury phenotype [3, 4].

acid and a type of polysaccharide [19] that help wound healing process. *Aloe vera* extract shows beneficial effects on wound healing by decreasing the inflammatory phase and supplying more mature granulation tissue which finally promotes healing and may be caused to produce a sound well-remodeled scar [16]. The *Aloe vera* leaf gel has beneficial effects on wound healing by antioxidant properties which can be attributed to some compounds including indoles, and alkaloids [20]. The spectrophotometric analyses show that *Aloe vera* contains non-flavonoid polyphenols compounds phytosterols, and indoles that may encourage the symptoms related with diabetes [20]. These compounds also shows antibacterial properties which may help to alleviate the wound healing in infected wounds. Chitra et al. [21] have reported the different mechanisms for wound healing of *Aloe vera* which mainly attributed to

Medicinal Plants in Wound Healing

35

http://dx.doi.org/10.5772/intechopen.80215

*Anethum graveolens L.* (dill) (Apiaceae) is known as one of the most popular medicinal plants in all over world. *Anethum graveolens* is known to have some properties such as antimicrobial, antidiabetic and anti-inflammatory that can improve wound healing [22]. Some compounds including cis-carvone, limonene, α-phellandrene, and anethofuran are major compounds in dill essential oil [23]. Alpha-phellandrene is other major compounds in dill essential oil which may decrease bacterial growth and colonization and is to be beneficial in infected wounds [24, 25].

Eucalyptus is also known as Dinkum oil and is belonging to family *Myrtaceae*. Eucalyptus contains some compounds such as cineole which is also known as eucalyptol. It not only contains cineole but contains other compounds such as pinene, camphene, and phellandrene, citronellal, geranyl acetate. It is traditionally used for skin care including burns, blisters, her-

*Securigera securidaca*, a native plant of Iran, has traditionally been used in the southern part of Iran in order to treatment the diabetes. It is commonly used in order to treat the wound healing. Flavonoids and coumarins are broadly used as major constituents in aerial parts, of *Securigera securidaca* that act as strong antioxidants [27]. It is also known to have antibacterial

*Trigonella foenum-graecum*, is so called fenugreek, is extensively used in preparations the Ayurveda and also known to have effects antiulcer action and hypocholesterolaemic effects. Fenugreek (*Trigonella foenum-graecum*) has commonly been used as a condiment and in food preparations. Fenugreek is known to have hypoglycemic effects [29]. Fenugreek seeds have some polysaccharides such as diosgenin, yamogenin, gitogenin, tigogenin, and neotigogens. Saponins can produce steroidal effects which can decrease inflammation in the body. Other bioactive constituents of fenugreek are including mucilage, volatile oils, flavonoids and

enhancing collages turnover rate and level of lysyl oxidase.

pes, cuts, wounds, skin infections and insect bites [26].

properties that improve wound healing in infected wounds [28].

**2.3.** *Anethum graveolens*

**2.4. Eucalyptus**

**2.5.** *Securigera securidaca*

**2.6. Trigonella foenum**

The different treatments are used in order to treat the wound healing. The different treatments have locally and systemically been used in order to help wound healing. The different agents are used in order to wound healing including antibiotics and antiseptics, desloughing agents (chemical debridement, e.g., hydrogen peroxide, eusol and collagenase ointment, wound healing promoters, some substances such as tissue extracts, vitamins, and minerals and a number of plant products [5]. Medicinal plants heal wound healing process by promoting blood clotting, fighting against infection and accelerating wound healing. It can be stated plants and chemical agents obtained from plants improve treatment and manage wound healing [5]. Medicinal plants show wound healing effects by the different mechanisms, such as modulation in wound healing, decreasing bacterial count, improving collagen deposition, increasing fibroblasts and fibrocytes, etc. In this chapter, we will describe different mechanisms in medicinal plants.

### **2. Medicinal plants**

#### **2.1. Cinnamon**

*Cinnamomum verum*, cinnamon, belongs to the *Lauraceae* family. Cinnamon has been traditionally used in traditional systems of medicine. Cinnamon bark is used as spice, condiment and flavoring agent. It has some properties such as antioxidant, antiulcer, antimicrobial, antidiabetic, hypoglycemic, hypolipidemic and anti-inflammatory activity [6], which can be beneficial in types of wound such as diabetic and infected wounds. In addition to mentioned properties, cinnamon is known to have significant levels of polyphenols that may enhance glucose uptake in animals [7]. It increases glucose transporters-1 (GLUT-1) mRNA levels in mice adipocytes [8]. Studies have shown that cinnamon alcoholic and aqueous extracts accelerating the wound healing by their antioxidant properties [9, 10]. In this association, other studies have shown that faulted antioxidant system causes to increase oxidative stress which damages proteins, nucleotides, lipid levels and delays wound healing [11, 12]. On the other hand, anti-inflammatory effects of cinnamon components including cinnamaldehyde [13], 2-hydroxycinnamaldehyde [14] and quercetin [15] can help to accelerating wound healing.

#### **2.2.** *Aloe vera*

*Aloe vera* is a native plant in Africa and is so called lily of the desert or the plant of immortality. The *Aloe vera* extract has some beneficial properties which can decrease inflammation; enhance mature granulation tissue and resulting in help to accelerate wound healing [16]. It also decreases the blood glucose which can be beneficial in diabetic wounds [17]. Topical administration of *Aloe vera* gel is beneficial tool in healing minor burns and application of the *Aloe vera* gel is harmless as hypersensitive reactions to it are rare. However, *Aloe vera* gel may have harmful effects on severe burns and may actually prevent healing [18]. Gels have been traditionally found which contain 96% of water and essential oil, amino acids, minerals, vitamins, enzymes and glycoproteins. In addition, *Aloe vera* extract promotes the wound healing process because of its anti-inflammatory property. Because *Aloe vera* extract contains tannic acid and a type of polysaccharide [19] that help wound healing process. *Aloe vera* extract shows beneficial effects on wound healing by decreasing the inflammatory phase and supplying more mature granulation tissue which finally promotes healing and may be caused to produce a sound well-remodeled scar [16]. The *Aloe vera* leaf gel has beneficial effects on wound healing by antioxidant properties which can be attributed to some compounds including indoles, and alkaloids [20]. The spectrophotometric analyses show that *Aloe vera* contains non-flavonoid polyphenols compounds phytosterols, and indoles that may encourage the symptoms related with diabetes [20]. These compounds also shows antibacterial properties which may help to alleviate the wound healing in infected wounds. Chitra et al. [21] have reported the different mechanisms for wound healing of *Aloe vera* which mainly attributed to enhancing collages turnover rate and level of lysyl oxidase.

### **2.3.** *Anethum graveolens*

lasted for 1 month, and the dermis may respond to injury with the production of collagen and

The different treatments are used in order to treat the wound healing. The different treatments have locally and systemically been used in order to help wound healing. The different agents are used in order to wound healing including antibiotics and antiseptics, desloughing agents (chemical debridement, e.g., hydrogen peroxide, eusol and collagenase ointment, wound healing promoters, some substances such as tissue extracts, vitamins, and minerals and a number of plant products [5]. Medicinal plants heal wound healing process by promoting blood clotting, fighting against infection and accelerating wound healing. It can be stated plants and chemical agents obtained from plants improve treatment and manage wound healing [5]. Medicinal plants show wound healing effects by the different mechanisms, such as modulation in wound healing, decreasing bacterial count, improving collagen deposition, increasing fibroblasts and fibrocytes, etc. In this chapter, we will describe different mechanisms in medicinal plants.

*Cinnamomum verum*, cinnamon, belongs to the *Lauraceae* family. Cinnamon has been traditionally used in traditional systems of medicine. Cinnamon bark is used as spice, condiment and flavoring agent. It has some properties such as antioxidant, antiulcer, antimicrobial, antidiabetic, hypoglycemic, hypolipidemic and anti-inflammatory activity [6], which can be beneficial in types of wound such as diabetic and infected wounds. In addition to mentioned properties, cinnamon is known to have significant levels of polyphenols that may enhance glucose uptake in animals [7]. It increases glucose transporters-1 (GLUT-1) mRNA levels in mice adipocytes [8]. Studies have shown that cinnamon alcoholic and aqueous extracts accelerating the wound healing by their antioxidant properties [9, 10]. In this association, other studies have shown that faulted antioxidant system causes to increase oxidative stress which damages proteins, nucleotides, lipid levels and delays wound healing [11, 12]. On the other hand, anti-inflammatory effects of cinnamon components including cinnamaldehyde [13], 2-hydroxycinnamaldehyde [14] and quercetin [15] can help to accelerating wound healing.

*Aloe vera* is a native plant in Africa and is so called lily of the desert or the plant of immortality. The *Aloe vera* extract has some beneficial properties which can decrease inflammation; enhance mature granulation tissue and resulting in help to accelerate wound healing [16]. It also decreases the blood glucose which can be beneficial in diabetic wounds [17]. Topical administration of *Aloe vera* gel is beneficial tool in healing minor burns and application of the *Aloe vera* gel is harmless as hypersensitive reactions to it are rare. However, *Aloe vera* gel may have harmful effects on severe burns and may actually prevent healing [18]. Gels have been traditionally found which contain 96% of water and essential oil, amino acids, minerals, vitamins, enzymes and glycoproteins. In addition, *Aloe vera* extract promotes the wound healing process because of its anti-inflammatory property. Because *Aloe vera* extract contains tannic

matrix proteins and then returns to its pre-injury phenotype [3, 4].

**2. Medicinal plants**

34 Wound Healing - Current Perspectives

**2.1. Cinnamon**

**2.2.** *Aloe vera*

*Anethum graveolens L.* (dill) (Apiaceae) is known as one of the most popular medicinal plants in all over world. *Anethum graveolens* is known to have some properties such as antimicrobial, antidiabetic and anti-inflammatory that can improve wound healing [22]. Some compounds including cis-carvone, limonene, α-phellandrene, and anethofuran are major compounds in dill essential oil [23]. Alpha-phellandrene is other major compounds in dill essential oil which may decrease bacterial growth and colonization and is to be beneficial in infected wounds [24, 25].

#### **2.4. Eucalyptus**

Eucalyptus is also known as Dinkum oil and is belonging to family *Myrtaceae*. Eucalyptus contains some compounds such as cineole which is also known as eucalyptol. It not only contains cineole but contains other compounds such as pinene, camphene, and phellandrene, citronellal, geranyl acetate. It is traditionally used for skin care including burns, blisters, herpes, cuts, wounds, skin infections and insect bites [26].

#### **2.5.** *Securigera securidaca*

*Securigera securidaca*, a native plant of Iran, has traditionally been used in the southern part of Iran in order to treatment the diabetes. It is commonly used in order to treat the wound healing. Flavonoids and coumarins are broadly used as major constituents in aerial parts, of *Securigera securidaca* that act as strong antioxidants [27]. It is also known to have antibacterial properties that improve wound healing in infected wounds [28].

### **2.6. Trigonella foenum**

*Trigonella foenum-graecum*, is so called fenugreek, is extensively used in preparations the Ayurveda and also known to have effects antiulcer action and hypocholesterolaemic effects. Fenugreek (*Trigonella foenum-graecum*) has commonly been used as a condiment and in food preparations. Fenugreek is known to have hypoglycemic effects [29]. Fenugreek seeds have some polysaccharides such as diosgenin, yamogenin, gitogenin, tigogenin, and neotigogens. Saponins can produce steroidal effects which can decrease inflammation in the body. Other bioactive constituents of fenugreek are including mucilage, volatile oils, flavonoids and amino acid, alkaloids. The other active ingredient found in fenugreek is 4-hydroxyisoleucine [30]. It has been reported that fenugreek releases anti-inflammatory substance into wound region and decreases inflammation [31]. In addition, antimicrobial properties of fenugreek may increase its anti-inflammatory responses. A study has shown that flavonoids and triterpenoids may promote the wound healing process because of its antimicrobial properties [32]. Fenugreek is known to have antioxidant properties which can accelerate wound healing [33]. The kinetics of wound contraction and epithelialization were improved in a significant level from topical administration of the fenugreek seed [34].

**2.11. Linumu sitatissimum**

**2.12. Moltkia coerulea**

**2.13. Ribwort plantain**

**2.14. Rosemary officinalis**

**2.15.** *Allium sativum*

Flaxseed (*Linumu sitatissimum*) is one of oldest cultivated plant and is often cultivated for its fiber and oil. Flaxseed and its derivatives are known as rich sources of the essential fatty acid and alpha-linolenic acid, which are biological precursor for omega-3 and fatty acids such as eicosapentaenoic which may improve wound healing. Dogoury et al. [38] reported that topical administration of *Chamomilla recutita* and *Linumu sitatissimum* could decrease inflammatory phase and enhance the proliferative stage. They have also advised to consider *Chamomilla recutita* and *Linumu sitatissimum* as alternative agents for nitrofurazone ointment in wound healing process.

Medicinal Plants in Wound Healing

37

http://dx.doi.org/10.5772/intechopen.80215

*Moltkia coerulea* is considered as one of most important plants in *Boraginaceae* that is belonging to *Lithospermeae* subfamily [40]. It is known to have some properties such as antioxidant and antibacterial effects, because of large amounts of flavonoids and phenols [41] which may accelerate wound healing. Farahpour et al. [42] have shown that topical administration of *Moltkia coerulea* improved well-formed clot in wound area, down-regulated the inflammation by exerting antioxidant properties, increased vascularization, promoted the collagen synthe-

*Ribwort plantain* (Plantaginaceae) is a perennial plant species with a worldwide distribution and large ecological amplitude. It is also known to have antibacterial properties [43, 44]. Studies have shown that *Ribwort plantain* accelerates epithelialization and wound contraction [45, 46]. Farahpour and Heydari [47] have shown that antioxidant properties reduce inflam-

Rosemary is belonging to the mint family which is known to have antioxidant properties because of its compounds including carnosic acid, carnosol, rosmarinic acid, diterpene, triterpenoid, phenolic acid and flavonoids [48]. It is also known to have anti-inflammatory [49] and anti-microbial properties [50] which may promote wound healing. In addition, its essential oil contains major levels of terpenoids, limonene, 1, 8-cineol, carnosic acid, rosemarinic acid and α-pinene, that can reduce inflammatory phase and can accelerate the healing process by promoting the proliferation stage [51]. Abu-Al-Basal [52] reported that rosmarinus aqueous extract accelerates wound healing by closure of the wound area and full-thickness epidermal regeneration and organization in diabetic BALB/c mice. Nejati et al. [53] have reported that topical application of rosemary ointment significantly decreased inflammatory cells, increased fibroblast migration and also increased wound contraction in wound healing in infected rat model.

*Allium sativum* L. (Amaryllidaceae) is a member of the lily family which contains high levels of alliin, allyl cysteine, allyl disulfide, and allicin and has powerful antioxidant agents [54].

sis by up-regulating the fibroblasts and fibrocytes cells proliferation.

mation and increase wound contraction in rabbits.

### **2.7. Nelumbo nucifera**

*Nelumbo nucifera* is belonging to family Nymphaeaceae which is so called Kamal in Hindi and Lotus in English. It has mud with large flower and is extensively used as natural and traditional healers. Its leaves are known to have wound healer effects [35]. It is reported that methanolic extract of *Nelumbo nucifera* rhizomes in the formulation of ointment could improve types of wound model in rats. This effect was studied in excision wound model, incision wound model and dead space wound model in the different concentrations of 5 and 10% w/w ointment. The both concentrations could significantly improve wound models. The both concentrations could improve contracting activity, wound close time, tensile strength, regeneration of tissue at the wound site and lysyl oxidase activity. The observed effects were similar to standard drugs [36].

#### **2.8. Neem**

Neem leaf extracts and essential oil from seeds are known to have antimicrobial effect which may be beneficial in the infected wounds. In addition, it can be stated that neem maintains wound and lesion free from secondary infections through reducing bacterial population. Clinical studies have shown that neem extract prevents inflammation and subsequently increases wound healing [5]. Neem leaf extracts and oil from seeds show antimicrobial effect which is mainly attributed to its compounds including margosic acid, glycerides of fatty acids, butyric acid and trace valeric acid [35].

#### **2.9. Chamomile**

*Chamomilla recutita* is so called as chamomile and is belonging to the *Asteraceae* family. It contains some substances such as chamazulene, alpha bisabolol, bisabolol oxides, spiroethers, and flavonoids which induce therapeutic effects [37]. It is also known to have anti-inflammatory which decreases inflammation during infected wounds [37]. Gholami Dogoury et al. [38] have shown that topical administration of *Chamomilla recutita* could decrease inflammatory phase and increase the proliferative stage. They have also advised to consider *Chamomilla recutita* as safe alternative chemicals for nitrofurazone ointment in wound healing process.

#### **2.10. Bael**

Bael which is so called *Aegle marmelos* which is belonging to family *Rutaceae*. It contains carbohydrates, protein, volatile oil, tanines, vitamin C and vitamin A. two alkaloids Omethylhalfordional and isopentylhalfordinol. It is traditionally used to treat wound healing properties [39].

### **2.11. Linumu sitatissimum**

amino acid, alkaloids. The other active ingredient found in fenugreek is 4-hydroxyisoleucine [30]. It has been reported that fenugreek releases anti-inflammatory substance into wound region and decreases inflammation [31]. In addition, antimicrobial properties of fenugreek may increase its anti-inflammatory responses. A study has shown that flavonoids and triterpenoids may promote the wound healing process because of its antimicrobial properties [32]. Fenugreek is known to have antioxidant properties which can accelerate wound healing [33]. The kinetics of wound contraction and epithelialization were improved in a significant level

*Nelumbo nucifera* is belonging to family Nymphaeaceae which is so called Kamal in Hindi and Lotus in English. It has mud with large flower and is extensively used as natural and traditional healers. Its leaves are known to have wound healer effects [35]. It is reported that methanolic extract of *Nelumbo nucifera* rhizomes in the formulation of ointment could improve types of wound model in rats. This effect was studied in excision wound model, incision wound model and dead space wound model in the different concentrations of 5 and 10% w/w ointment. The both concentrations could significantly improve wound models. The both concentrations could improve contracting activity, wound close time, tensile strength, regeneration of tissue at the wound site and lysyl oxidase activity. The observed effects were similar to standard drugs [36].

Neem leaf extracts and essential oil from seeds are known to have antimicrobial effect which may be beneficial in the infected wounds. In addition, it can be stated that neem maintains wound and lesion free from secondary infections through reducing bacterial population. Clinical studies have shown that neem extract prevents inflammation and subsequently increases wound healing [5]. Neem leaf extracts and oil from seeds show antimicrobial effect which is mainly attributed to its compounds including margosic acid, glycerides of fatty

*Chamomilla recutita* is so called as chamomile and is belonging to the *Asteraceae* family. It contains some substances such as chamazulene, alpha bisabolol, bisabolol oxides, spiroethers, and flavonoids which induce therapeutic effects [37]. It is also known to have anti-inflammatory which decreases inflammation during infected wounds [37]. Gholami Dogoury et al. [38] have shown that topical administration of *Chamomilla recutita* could decrease inflammatory phase and increase the proliferative stage. They have also advised to consider *Chamomilla recutita* as safe alternative chemicals for nitrofurazone ointment in wound healing process.

Bael which is so called *Aegle marmelos* which is belonging to family *Rutaceae*. It contains carbohydrates, protein, volatile oil, tanines, vitamin C and vitamin A. two alkaloids Omethylhalfordional

and isopentylhalfordinol. It is traditionally used to treat wound healing properties [39].

from topical administration of the fenugreek seed [34].

acids, butyric acid and trace valeric acid [35].

**2.7. Nelumbo nucifera**

36 Wound Healing - Current Perspectives

**2.8. Neem**

**2.9. Chamomile**

**2.10. Bael**

Flaxseed (*Linumu sitatissimum*) is one of oldest cultivated plant and is often cultivated for its fiber and oil. Flaxseed and its derivatives are known as rich sources of the essential fatty acid and alpha-linolenic acid, which are biological precursor for omega-3 and fatty acids such as eicosapentaenoic which may improve wound healing. Dogoury et al. [38] reported that topical administration of *Chamomilla recutita* and *Linumu sitatissimum* could decrease inflammatory phase and enhance the proliferative stage. They have also advised to consider *Chamomilla recutita* and *Linumu sitatissimum* as alternative agents for nitrofurazone ointment in wound healing process.

### **2.12. Moltkia coerulea**

*Moltkia coerulea* is considered as one of most important plants in *Boraginaceae* that is belonging to *Lithospermeae* subfamily [40]. It is known to have some properties such as antioxidant and antibacterial effects, because of large amounts of flavonoids and phenols [41] which may accelerate wound healing. Farahpour et al. [42] have shown that topical administration of *Moltkia coerulea* improved well-formed clot in wound area, down-regulated the inflammation by exerting antioxidant properties, increased vascularization, promoted the collagen synthesis by up-regulating the fibroblasts and fibrocytes cells proliferation.

### **2.13. Ribwort plantain**

*Ribwort plantain* (Plantaginaceae) is a perennial plant species with a worldwide distribution and large ecological amplitude. It is also known to have antibacterial properties [43, 44]. Studies have shown that *Ribwort plantain* accelerates epithelialization and wound contraction [45, 46]. Farahpour and Heydari [47] have shown that antioxidant properties reduce inflammation and increase wound contraction in rabbits.

### **2.14. Rosemary officinalis**

Rosemary is belonging to the mint family which is known to have antioxidant properties because of its compounds including carnosic acid, carnosol, rosmarinic acid, diterpene, triterpenoid, phenolic acid and flavonoids [48]. It is also known to have anti-inflammatory [49] and anti-microbial properties [50] which may promote wound healing. In addition, its essential oil contains major levels of terpenoids, limonene, 1, 8-cineol, carnosic acid, rosemarinic acid and α-pinene, that can reduce inflammatory phase and can accelerate the healing process by promoting the proliferation stage [51]. Abu-Al-Basal [52] reported that rosmarinus aqueous extract accelerates wound healing by closure of the wound area and full-thickness epidermal regeneration and organization in diabetic BALB/c mice. Nejati et al. [53] have reported that topical application of rosemary ointment significantly decreased inflammatory cells, increased fibroblast migration and also increased wound contraction in wound healing in infected rat model.

#### **2.15.** *Allium sativum*

*Allium sativum* L. (Amaryllidaceae) is a member of the lily family which contains high levels of alliin, allyl cysteine, allyl disulfide, and allicin and has powerful antioxidant agents [54]. Farahpour et al. [55] have shown that topical administration of *Allium sativum* accelerated wound healing because of its preliminary impact on mast-cell distribution and increased collagen synthesis and up-regulated angiogenesis, and improved the healing process by increasing the intra-cytoplasmic carbohydrate ratio.

from high dose administration of *Pistacia atlantica* suggests that dosing higher concentration contains more constituents that plays major role in shortening healing time. In other study, Farahpour and Fathollahpour [71] have shown that ointment prepared from flaxseed and pistachio oil decreased polymorphonuclear and mononuclear cell distribution, improved new

Medicinal Plants in Wound Healing

39

http://dx.doi.org/10.5772/intechopen.80215

*Astragalus* is known as Huang Qi in China and contains polysaccharides, saponins, flavonoids, amino acids and trace elements. *Astragalus* had high potential in wound healing and its mechanism was by preventing inflammation, accelerating cell cycle and promoting the

*Morinda citrifolia* Linn (Rubiaceae) is so called noni or Indian mulberry. A significant enhance in the wound-healing activity has reported in the animals treated with the *Morinda citrifolia* extract in comparison to animals receiving the placebo control treatments. *Morinda citrifolia* extract improves wound healing by decreasing wound size and time to epithelialization [14].

Lucidone is a natural compound in *Lindera erythrocarpa* Makino which is known to have some properties such as antioxidant, anti-inflammatory, neuroprotective and anti-vital efficacies [73]. It has reported that Lucidone prevents free radical-induced oxidative stress and inflammation in human skin HaCaT cells [74]. Lucidone maintains human skin keratinocytes against UVAinduced DNA damage and mitochondrial dysfunction. Lucidone promoted wound healing by cooperation of keratinocyte/fibroblast/endothelial cell growth and migration and macrophage inflammation by PI3K/AKT, Wnt/β-catenin and NF-κB signaling cascade activation [75].

Genistein is one of the most important isoflavones in legumes and has estrogen-like effects [76] antioxidative effects by regulating antioxidant enzyme activities such as super oxide dismutase, heme oxygenase-1 and glutathione peroxidase [77]. Studies have reported that dietary supplementation of genistein improved the regular wound healing process by regulating the antioxidant defense system and pro-inflammatory cytokines [78]. Treatment with genistein improved NLRP3 inflammasome in the basal level and alleviated inflammation and antioxidant defense system at early stage of wound healing in diabetic mice [79]. Eo et al. [79] have also reported that genistein improved wound healing by modulating in inflammation

*Asiaticoside* is a glycoside compound which is commonly used in order to wound healing. A study has shown that topical application of 0.4% solution of asiaticoside on the wound of

vessel formation and fibroblast distribution in injured rabbits.

secretion of repair factors in wound healing model [72].

and oxidative stress during inflammatory stage.

**2.20. Astragalus membranaceus**

**2.21.** *Morinda citrifolia* **Linn**

**2.22. Lucidone**

**2.23. Genistein**

**2.24. Asiaticoside**

#### **2.16.** *Vitis vinifera*

Grape *Vitis vinifera* is belonging to *Vitaceae* family and contains vitamin E, linoleic acid, oligomer pro-anthocyanidins compounds and phenolic compounds such as flavonoids, phenolic acids and antioxidants [56] stilbenes and anthocyanins [57]. Active compounds present have beneficial effects including anti-inflammatory and wound healing [58], antimicrobial and diabetes properties [59]. Nejati and Farahpour [60] have shown that *Vitis vinifera* accelerated wound healing process by increasing neovascularization, fibroblast migration and epithelialization and can stimulate the enclosure of burn wounds.

#### **2.17.** *Calendula officinalis*

*Calendula officinalis L.* is so called pot marigold and is one of the medicinal plants in the *Asteraceae* family. Phytochemical evaluations of *Calendula officinalis* showed the presence of the flavonoids, flavonol glycosides, coumarins, saponins, triterpenes, alcohol triterpenes, fatty acid esters, carotenoids, essential oils, hydrocarbons, and fatty acids [61, 62]. Some studies have reported biological activities in *Calendula officinalis* including wound healing and anti-inflammatory effects [61, 62]. Farahpour [63] showed that *Calendula officinalis* aerial part hydroalcoholic extracts, have antinociceptive and anti-inflammatory activities in chemical pain and anti-inflammatory tests.

#### **2.18.** *Curcuma longa*

Turmeric (*Curcuma longa L.*) is known as turmeric and is belonging to *Zingiberaceae* family [64]. Turmeric extract contain major amounts of mineral dyes, curcumin, curcuminoids, phenolic compounds and volatile oils including turmerone, atlantone and zingiberene [65]. Farahpour et al. [66] showed that topical application of differential levels of hydroethanolic extract of turmeric rhizome remarkably accelerated wound healing activity by increasing in the rate of wound contraction and re-epithelialization, tensile strength value and collagen deposition in rat as an *in vivo* experimental wound models, and suggested to use various types of wounds in animal and human beings.

#### **2.19.** *Pistacia atlantica*

The *Pistacia atlantica* is belonging to *Anacardiaceae* family and is known to have anti-inflammatory, antibacterial, antimicrobial properties [67, 68]. Haghdoost et al. [69] have shown that *Pistacia atlantica* has beneficial effect on burn wound healing. Farahpour et al. [70] shown that the different levels of *Pistacia atlantica* decreased the healing time, improved the wound contraction, up-regulated hydroxyproline content and increased the neovascularization. They have also reported that *Pistacia atlantica* increased collagen deposition simultaneously by up-regulating the mast cells and fibroblast distribution. Finally, obtaining better results from high dose administration of *Pistacia atlantica* suggests that dosing higher concentration contains more constituents that plays major role in shortening healing time. In other study, Farahpour and Fathollahpour [71] have shown that ointment prepared from flaxseed and pistachio oil decreased polymorphonuclear and mononuclear cell distribution, improved new vessel formation and fibroblast distribution in injured rabbits.

#### **2.20. Astragalus membranaceus**

*Astragalus* is known as Huang Qi in China and contains polysaccharides, saponins, flavonoids, amino acids and trace elements. *Astragalus* had high potential in wound healing and its mechanism was by preventing inflammation, accelerating cell cycle and promoting the secretion of repair factors in wound healing model [72].

### **2.21.** *Morinda citrifolia* **Linn**

*Morinda citrifolia* Linn (Rubiaceae) is so called noni or Indian mulberry. A significant enhance in the wound-healing activity has reported in the animals treated with the *Morinda citrifolia* extract in comparison to animals receiving the placebo control treatments. *Morinda citrifolia* extract improves wound healing by decreasing wound size and time to epithelialization [14].

#### **2.22. Lucidone**

Farahpour et al. [55] have shown that topical administration of *Allium sativum* accelerated wound healing because of its preliminary impact on mast-cell distribution and increased collagen synthesis and up-regulated angiogenesis, and improved the healing process by increas-

Grape *Vitis vinifera* is belonging to *Vitaceae* family and contains vitamin E, linoleic acid, oligomer pro-anthocyanidins compounds and phenolic compounds such as flavonoids, phenolic acids and antioxidants [56] stilbenes and anthocyanins [57]. Active compounds present have beneficial effects including anti-inflammatory and wound healing [58], antimicrobial and diabetes properties [59]. Nejati and Farahpour [60] have shown that *Vitis vinifera* accelerated wound healing process by increasing neovascularization, fibroblast migration and epitheliali-

*Calendula officinalis L.* is so called pot marigold and is one of the medicinal plants in the *Asteraceae* family. Phytochemical evaluations of *Calendula officinalis* showed the presence of the flavonoids, flavonol glycosides, coumarins, saponins, triterpenes, alcohol triterpenes, fatty acid esters, carotenoids, essential oils, hydrocarbons, and fatty acids [61, 62]. Some studies have reported biological activities in *Calendula officinalis* including wound healing and anti-inflammatory effects [61, 62]. Farahpour [63] showed that *Calendula officinalis* aerial part hydroalcoholic extracts, have antinociceptive and anti-inflammatory activities in chemical

Turmeric (*Curcuma longa L.*) is known as turmeric and is belonging to *Zingiberaceae* family [64]. Turmeric extract contain major amounts of mineral dyes, curcumin, curcuminoids, phenolic compounds and volatile oils including turmerone, atlantone and zingiberene [65]. Farahpour et al. [66] showed that topical application of differential levels of hydroethanolic extract of turmeric rhizome remarkably accelerated wound healing activity by increasing in the rate of wound contraction and re-epithelialization, tensile strength value and collagen deposition in rat as an *in vivo* experimental wound models, and suggested to use various types of wounds

The *Pistacia atlantica* is belonging to *Anacardiaceae* family and is known to have anti-inflammatory, antibacterial, antimicrobial properties [67, 68]. Haghdoost et al. [69] have shown that *Pistacia atlantica* has beneficial effect on burn wound healing. Farahpour et al. [70] shown that the different levels of *Pistacia atlantica* decreased the healing time, improved the wound contraction, up-regulated hydroxyproline content and increased the neovascularization. They have also reported that *Pistacia atlantica* increased collagen deposition simultaneously by up-regulating the mast cells and fibroblast distribution. Finally, obtaining better results

ing the intra-cytoplasmic carbohydrate ratio.

zation and can stimulate the enclosure of burn wounds.

**2.16.** *Vitis vinifera*

38 Wound Healing - Current Perspectives

**2.17.** *Calendula officinalis*

pain and anti-inflammatory tests.

in animal and human beings.

**2.19.** *Pistacia atlantica*

**2.18.** *Curcuma longa*

Lucidone is a natural compound in *Lindera erythrocarpa* Makino which is known to have some properties such as antioxidant, anti-inflammatory, neuroprotective and anti-vital efficacies [73]. It has reported that Lucidone prevents free radical-induced oxidative stress and inflammation in human skin HaCaT cells [74]. Lucidone maintains human skin keratinocytes against UVAinduced DNA damage and mitochondrial dysfunction. Lucidone promoted wound healing by cooperation of keratinocyte/fibroblast/endothelial cell growth and migration and macrophage inflammation by PI3K/AKT, Wnt/β-catenin and NF-κB signaling cascade activation [75].

### **2.23. Genistein**

Genistein is one of the most important isoflavones in legumes and has estrogen-like effects [76] antioxidative effects by regulating antioxidant enzyme activities such as super oxide dismutase, heme oxygenase-1 and glutathione peroxidase [77]. Studies have reported that dietary supplementation of genistein improved the regular wound healing process by regulating the antioxidant defense system and pro-inflammatory cytokines [78]. Treatment with genistein improved NLRP3 inflammasome in the basal level and alleviated inflammation and antioxidant defense system at early stage of wound healing in diabetic mice [79]. Eo et al. [79] have also reported that genistein improved wound healing by modulating in inflammation and oxidative stress during inflammatory stage.

#### **2.24. Asiaticoside**

*Asiaticoside* is a glycoside compound which is commonly used in order to wound healing. A study has shown that topical application of 0.4% solution of asiaticoside on the wound of streptozotocin-induced diabetic rats could improve the tensile strength, hydroxyproline content, protein content and epithelialization and accelerate facilitating the wound healing [80]. Another study has shown that 0.2% solution of asiaticoside increased hydroxyproline, tensile strength and quick healing. It also promoted angiogenesis collagen formation, remodeling of the collagen matrix and stimulated of glycosaminoglycan synthesis in a rat wound chamber model [81]. Antioxidants have major important role in the wound healing process that may improve wound healing by antioxidant property.

**References**

**22**:407-412

2004;**36**:119-125

**17**:1027-1032

in Aging. 2007;**2**:219-225

Botany. 2012;**24**:1-26

2015;**17**:1-9

Regenerative Medicine. 2012;**6**:218-218

DOI: 10.1016/j.molimm 2010:12-24

healing. Vet Scan. 2008;**3**(1):1-24

[1] Bullers S, Berry H, Ingham E, Southgate J. The resolution of inflammation during the regeneration of biological scaffolds by human tissue. Journal of Tissue Engineering and

Medicinal Plants in Wound Healing

41

http://dx.doi.org/10.5772/intechopen.80215

[2] Pesce M, Patruno A, Speranza L, Reale M. Extremely low frequency electromagnetic field and wound healing: Implication of cytokines as biological mediators. European

[3] Castillo-Briceno P, Bihan D, Nilges M, Hamaia S, Meseguer J, Garcia-Ayala A, et al. Arole for specific collagen motifs during wound healing and inflammatory response of fibroblasts in the teleost fishgil the adseabream. Molecular Immunology. 2011;**48**:826-834.

[4] Bainbridge P. Wound healing and the role of fibroblasts. Journal of Wound Care. 2013;

[5] Raina R, Prawez S, Verma PK, Pankaj NK. Medicinal plants and their role in wound

[6] Vaibhavi J, Rakesh R, Pankaj K. Cinnamon: A pharmacological review. Journal of

[7] Qin B, Nagasaki M, Ren M, Bajotto G, Oshida Y, Sato Y. Cinnamon extract prevents the insulin resistance induced by a high-fructose diet. Hormone and Metabolic Research.

[8] Cao H, Gravesc DJ, Anderson RA. Cinnamon extract regulates glucose transporter and insulin-signaling gene expression in mouse adipocytes. Phytomedicine. 2010;

[9] Kamath JV, Rana AC, Chowdhury AR. Pro-healing effect of Cinnamomum zeylanicum

[10] Farahpour MR, Habibi M. Evaluation of the wound healing activity of an ethanolic

[11] Rahman K. Studies on free radicals, antioxidants, and co-factors. Clinical Interventions

[12] Sharma P, Jha AB, Dubey RS, Pessarakli M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Journal of

[13] Yang D, Liang XC, Shi Y, Sun Q, Liu D, Liu W, Zhang H. Anti-oxidative and antiinflammatory effects of cinnamaldehyde on protecting high glucose-induced damage in cultured dorsal root ganglion neurons of rats. Chinese Journal of Integrative Medicine.

[14] Lee SH, Lee SY, Son DJ, Lee H, Yoo HS, Song S, Oh KW, Han DC, Kwon BM, Hong JT. Inhibitory effect of 2′-hydroxycinnamaldehyde on nitric oxide production through

extract of Ceylon cinnamon in mice. Veterinary Medicine. 2012;**57**:53-57

Cytokine Network. 2013;**24**:1-10. DOI: 10.1684/ecn.2013. 0332

Advanced Science and Engineering Research. 2010;**1**:19-23

bark. Phytotherapy Research. 2003;**17**:970-972

#### **2.25. Curcumin**

Curcumin is a phenolic compound which is isolated from *Curcuma longa Linn* [82] and used for its various biological and therapeutic properties. It also has antioxidant, anti-inflammatory, antimutagenic, anticarcinogenic, anti-infective and anticoagulant effects [82]. Curcumin can improve wound healing by its anti-inflammatory, anti-oxidant and anti-infectious properties and also because of the prevention of STAT, TNF-α, cyclin D1, COX-2, NF-κB, IL (1β, 6, 8) expressions, and down-regulation of MMP- 8 expression [83]. Curcumin also increases collagen deposition, tissue remodeling, fibroblast proliferation, granulation tissue formation and vascular density [82]. It also prevented the growth of dangerous pathogens like methicillin-resistant *Staphylococcus aureus* (MRSA) [84], P*. gingivalis, P. intermedia, F. nucleatum,* and *T. denticola* [85].

### **3. Conclusion**

In this chapter, the possible mechanisms were described. We only mentioned some medicinal plants. The most medicinal plants act through antioxidant and antibacterial properties. However, some medicinal herbs and especially active compounds act by gene expression. It cannot certainly be stated efficiency medicinal plants in improving wound healing, but they have major potential for improving wound healing. The use of active compounds is a new strategy to improve the wound healing. Medicinal plants and active compounds help to decrease the inflammation. Future studies will be needed to determine the more mechanisms.

### **Conflict of interest**

None.

### **Author details**

Mohammad Reza Farahpour

Address all correspondence to: mrf78s@gmail.com

Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran

### **References**

streptozotocin-induced diabetic rats could improve the tensile strength, hydroxyproline content, protein content and epithelialization and accelerate facilitating the wound healing [80]. Another study has shown that 0.2% solution of asiaticoside increased hydroxyproline, tensile strength and quick healing. It also promoted angiogenesis collagen formation, remodeling of the collagen matrix and stimulated of glycosaminoglycan synthesis in a rat wound chamber model [81]. Antioxidants have major important role in the wound healing process that may

Curcumin is a phenolic compound which is isolated from *Curcuma longa Linn* [82] and used for its various biological and therapeutic properties. It also has antioxidant, anti-inflammatory, antimutagenic, anticarcinogenic, anti-infective and anticoagulant effects [82]. Curcumin can improve wound healing by its anti-inflammatory, anti-oxidant and anti-infectious properties and also because of the prevention of STAT, TNF-α, cyclin D1, COX-2, NF-κB, IL (1β, 6, 8) expressions, and down-regulation of MMP- 8 expression [83]. Curcumin also increases collagen deposition, tissue remodeling, fibroblast proliferation, granulation tissue formation and vascular density [82]. It also prevented the growth of dangerous pathogens like methicillin-resistant *Staphylococcus aureus* (MRSA) [84], P*. gingivalis, P. intermedia, F. nucleatum,* and *T. denticola* [85].

In this chapter, the possible mechanisms were described. We only mentioned some medicinal plants. The most medicinal plants act through antioxidant and antibacterial properties. However, some medicinal herbs and especially active compounds act by gene expression. It cannot certainly be stated efficiency medicinal plants in improving wound healing, but they have major potential for improving wound healing. The use of active compounds is a new strategy to improve the wound healing. Medicinal plants and active compounds help to decrease the inflammation. Future studies will be needed to determine the more mechanisms.

improve wound healing by antioxidant property.

**2.25. Curcumin**

40 Wound Healing - Current Perspectives

**3. Conclusion**

**Conflict of interest**

**Author details**

Mohammad Reza Farahpour

Islamic Azad University, Urmia, Iran

Address all correspondence to: mrf78s@gmail.com

Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch,

None.


inhibition of NF-κB activation in RAW 264.7 cells. Biochemical Pharmacology. 2005; **69**:791-799

[28] Amin GH. Popular Medicinal Plants of Iran. 2nd ed. Tehran: Tehran University of

Medicinal Plants in Wound Healing

43

http://dx.doi.org/10.5772/intechopen.80215

[29] Hasan M, Rahman M. Effect of fenugreek on Type 2 diabetic patients. International

[30] Acharya SN, Srichamoren A, Basu S, Ooraikul B, Basu T. Improvement in the nutraceutical properties of fenugreek (Trigonella foenum-graecum L.) Songklanakarin J. Science

[31] Muhammed DO, Salih NA. Effect of application of fenugreek (Trigonella foenum-graecum) on skin wound healing in rabbits. AL-Qadisiyah Journal of Veterinary Medicine

[32] Badri PN, Renu S. Role of medicinal plants in wound healing. Research Journal of Medi-

[33] Kaur C, Kapoor HC. Anti-oxidant activity and total phenolic content of some Asian vegetables. International Journal of Food Science and Technology. 2002;**37**:153-161

[34] Sumitra M, Manikandan P, Suguna L, Hittar GCE. Study of dermal wound healing activity of Trigonella foenum graceum seeds in rats. Journal of Clinical Biochemistry and

[35] Sabale P, Bhimani B, Prajapati C, Sabale V. An overview of medicinal plants as wound

[36] Mukherjee PK, Mukherjee K, Pal M, Saha BP. Wound healing potential of Nelumba

[37] Mann C, Staba EJ. The chemistry, pharmacology, and commercial formulations of chamomile. In: Craker LE, Simon JE, editors. Herbs, Spices, and Medicinal Plants: Recent Advances in Botany, Horticulture and Pharmacology. Vol. 1. Phoenix, AZ: Oryx Press;

[38] Gholami Dogoury H, Farahpour MR, Amniattalab A. Comparison effect of chamomile (*Chamomilla Recutita*) hydroethanolic extract and flaxseed oil (*Linum Ustatissum*) alone and simultaneous administration with nitrofurazone in wound healing process. Indian

[39] Jaswanth A, Loganathan V, Manimaran S, Rukmani S. Wound healing activity of Aegle

[40] Azimova SS, Glushenkova AI. Lipids, lipophilic components and essential oils from plant sources. New York: Springer Science Business Medical LLC; 2012. p. 163

[41] Koncic MZ, Kremer D, Gruz J, et al. Antioxidant and antimicrobial properties of Moltkia petraea (Tratt.) Griseb. Flower, leaf and stem infusions. Food and Chemical Toxicology.

[42] Farahpour MR, Dilmaghanian A, Faridy M, Karashi E. Topical Moltkia coerulea hydroethanolic extract accelerates the repair of excision wound in a rat model. Chinese Journal

healers. Journal of Applied Pharmaceutical Science. 2012;**2**(11):143-150

nucifera (Nymphaceae) rhizome extract. Phytomedicine. 2000;**7**:66-73

Journal of Fundamental and Applied Life Sciences. 2015;**10**:2231-6345

marmelos. Indian Journal of Pharmaceutical Sciences. 2001;**63**:41-44

Journal of Environmental Research and Public Health. 2016;**10**:251-255

Medical Sciences Publications; 2005. p. 240

and Technology. 2006;**28**(1):1-9

Sciences. 2012;**2**:86-93

cinal Plants. 2011;**5**:392-405

Nutrition. 2008;**200**:59-67

1986. pp. 235-280

2010;**48**:1537-1542

of Traumatology. 2016;**19**:97-103


[28] Amin GH. Popular Medicinal Plants of Iran. 2nd ed. Tehran: Tehran University of Medical Sciences Publications; 2005. p. 240

inhibition of NF-κB activation in RAW 264.7 cells. Biochemical Pharmacology. 2005;

[15] Caddeo C, Díez-Sales O, Pons R, Fernàndez-Busquets X, Fadda AM, Manconi M. Topical anti-inflammatory potential of quercetin in lipid-based nanosystems: In vivo and in vitro

[16] Abdel Hamid AAM, Solaiman MFM. Effect of topical aloe vera on the process of healing of full-thickness skin burn: A histological and immunohistochemical study. Journal of

[17] Daburkar M, Lohar V, Rathore AS, Bhutada P, Tangadpaliwar S. An in vivo and in vitro investigation of the effect of Aloe vera gel ethanolic extract using animal model with

diabetic foot ulcer. Journal of Pharmacy and Bioallied Sciences. 2014;**6**(3):205-212

[18] Schmidt JM, Greenspoon JS. *Aloe vera* dermal wound gel is associated with a delay in

[19] Schäfer M, Werner S. Oxidative stress in normal and impaired wound repair. Phar-

[20] Nejatzadeh-Barandozi F. Antibacterial activities and antioxidant capacity of Aloe vera. Organic and Medicinal Chemistry Letters. 2013;**3**:5 http://www.orgmedchemlett.com/

[21] Chitra P, Sajithlal GB, Chandrakasan G. Influence of Aloe vera on collagen turnover in healing of dermal wounds in rats. Indian Journal of Experimental Biology. 1998;

[22] Heamalatha S, Swarnalatha S, Divya M, Gandhi-Lakshmi R, Ganga-Devi A, Gomathi E. Pharmacognostical, pharmacological, investigation on Anethum graveolens Linn: A review. Research Journal of Pharmaceutical, Biological and Chemical Sciences.

[23] Radulescu V, Popescu ML, Ilies DC. Chemical composition of the volatile oil from different plant parts of *Anethum graveolens L*.(Umbelliferae) cultivated in Romania. Farmácia.

[24] Singh G, Maurya S, Lampasona MP, Catalan C. Chemical constituents, antimicrobial investigations, and antioxidative potentials of *Anethum graveolens L.* essential oil and

[25] Zhang JH, Sun HL, Chen SY, Zeng L, Wang TT. Anti-fungal activity, mechanism studies on α-Phellandrene and nonanal against Penicillium cyclopium. Botanlcal Study.

[26] Hukkeri VT, Karadi RV, Akki KS, Savadi RV, Jaiprakash B, Kuppast J, Patil MB. Wound healing property of Eucalyptus globulus leaf extract. Indian Drugs. 2002;**39**:481-483 [27] Tofighi Z, Asgharian P, Goodarzi S, Hadjiakhoondi A, Ostad SN, Yassa N. Potent cytotoxic flavonoids from Iranian Securigera securidaca. Medicinal Chemistry Research.

acetone extract. Journal of Food Science. 2005;**70**:208-215

Histology & Histopathology. 2015;**2**:1-9. DOI: 10.7243/2055-091X-2-3

wound healing. Obstetrics and Gynecology. 1991;**78**:115-117

macological Research. 2008;**58**:165-171

evaluation. Pharmacological Research. 2014;**1**:959-968

**69**:791-799

42 Wound Healing - Current Perspectives

content/3/1/5

**36**:896-901

2011;**2**:564-574

2010;**58**:594-600

2017;**58**:13-21

2014;**23**:1718-1724


[43] Kumar VP, Chauhan NS, Padh H, Rajani M. Search for antibacterial and antifungal agents from selected Indian medicinal plants. Journal of Ethnopharmacology. 2006;**107**:182-188

[56] Mureşan A, Alb C, Suciu S, Clichici S, Filip A, Login C. Studies on antioxidant effects of the red grapes seed extract from Vitis Vinifera, Burgund Mare, Recaş in pregnant rats.

Medicinal Plants in Wound Healing

45

http://dx.doi.org/10.5772/intechopen.80215

[57] Maier T, Schieber A, Kammerer DR, Carle R. Residues of grape (Vitis vinifera L.) seed oil production as a valuable source of phenolic antioxidants. Food Chemistry. 2009;

[58] Hemmati AA, Aghel N, Rashidi I, Gholampur-Aghdami A. Topical grape (Vitis vinifera) seed extract promotes repair of full thickness wound in rabbit. International Wound

[59] Xia E-Q, Deng G-F, Guo Y-J, Li H-B. Biological activities of polyphenols from grapes.

[60] Nejati H, Farahpour MR. Effect of topical red grape seed hydroethanol extract on burn wound healing in rats. Indian Journal of Chemical Technology. 2014;**6**:2340-2346 2014 [61] Parente LML, Júnior L, de Souza R, Tresvenzol LMF, Vinaud MC, de Paula JR, et al. Wound healing and anti-inflammatory effect in animal models of pot marigold growing

in Brazil. Evidence-Based Complementary and Alternative Medicine. 2012;**2012** [62] Preethi KC, Kuttan R. Wound healing activity of flower extract of calendula Officinalis. Journal of Basic and Clinical Physiology and Pharmacology. 2009;**20**(1):73-80

[63] Farahpour MR. Antioxidant activity, Antinociceptive and anti-inflammatory effects of pot marigold hydroalcoholic extract on experimental animals. International Journal of

[64] Chan EWC, Lim YY, Wong SK, Lim KK, Tan SP, Lianto FS, Yong MY. Effects of different drying methods on the antioxidant properties of leaves and tea of ginger species. Journal

[65] Li S, Yuan W, Deng G, Wang P, Yang P, Aggarwal BB. Chemical composition and product quality control of turmeric (Curcuma longa L.). Journal of Pharmaceutical Crops.

[66] Farahpour MR, Emami P, Jangkhahe GS. In vitro antioxidant properties and wound healing activity of hydroethanolic turmeric rhizome extract (Zingiberaceae). International

[67] Ali-Shtayeh M, Abu Ghdeib SI. Antifungal activity of plant extracts against dermato-

[68] Magiatis P, Melliou E, Skaltsounis AL, Chinou IB, Mitaku S. Chemical composition and antimicrobial activity of the essential oils of Pistacia lentiscus var. chia. Planta Medicine.

[69] Haghdoost F, Baradaran Mahdavi MM, Zandifar A, Sanei MH, Zolfaghari B, Javanmard SH. *Pistacia atlantica* resin has a dose-dependent effect on angiogenesis and skin burn wound healing in rat, Evidence-Based Complementary and Alternative Medicine; 2013.

Journal of Pharmacy and Pharmaceutical Sciences. 2014;**8**:474-478

8 p. http://dx.doi.org/10.1155/2013/893425. Article ID 893425

International Journal of Molecular Sciences. 2010;**11**(2):622-646

Acta Physiologica Hungarica. 2010;**97**(2):240-246

PharmTech Research. 2014;**6**(5):1640-1646

of Food Chemistry. 2009;**113**:166-172

phytes. Mycoses. 1999;**42**:665-672

2011;**2**:28-54

1999;**65**:749-752

**112**(3):551-559

Journal. 2011;**8**(5):514-520


[56] Mureşan A, Alb C, Suciu S, Clichici S, Filip A, Login C. Studies on antioxidant effects of the red grapes seed extract from Vitis Vinifera, Burgund Mare, Recaş in pregnant rats. Acta Physiologica Hungarica. 2010;**97**(2):240-246

[43] Kumar VP, Chauhan NS, Padh H, Rajani M. Search for antibacterial and antifungal agents from selected Indian medicinal plants. Journal of Ethnopharmacology. 2006;**107**:182-188

[44] Braga FG, Bouzada MLM, Fabri RL, de O Matos M, Moreira FO, Scio E, Coimbra ES. Antileishmanial and antifungal activity of plants used in traditional medicine in Brazil.

[45] Scortichini M, Rossi MP. Preliminary in vitro evaluation of the antimicrobial activity of terpenes and terpenoids towards Erwinia amylovora (Burrill) Winslow et al. Journal of

[46] Sasidharan S, Nilawatyi R, Xavier R, Latha LY, Amala R. Wound healing potential of Elaeis guineensis Jacq leaves in an infected albino rat model. Molecules. 2010;**15**:3186-3199

[47] Farahpour MR, Heydari A. Wound healing effect of hydroethanolic extract of *Ribwort plantain* leaves in rabbits. Research Opinions in Animal and Veterinary Sciences. 2014;**5**

[48] Erkan N, Ayranci G, Ayranci E. Antioxidant activities of rosemary (Rosmarinus Officinalis L.) extract, black seed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic

[49] Mengoni ES, Vichera G, Rigano LA, Rodriguez-Puebla ML, Galliano SR, Cafferata EE, Pivetta OH, Moreno S, Vojnov AA. Suppression of COX-2, IL-1β and TNF-α expression and leukocyte infiltration in inflamed skin by bioactive compounds from Rosmarinus

[50] Moreno S, Scheyer T, Romano CS, Vojnov AA. Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition. Free Radical Research.

[51] Jiang Y, Wu N, Fu YJ, Wang W, Luo M, Zhao CJ, Zu YG, Liu XL. Chemical composition and antimicrobial activity of the essential oil of rosemary. Environmental Toxicology

[52] Abu-Al-Basal MA. Healing potential of Rosmarinus officinalis L. on full-thickness excision cutaneous wounds in alloxan-induceddiabetic BALB/c mice. Journal of

[53] Nejati H, Farahpour MR, Neyriz NM. Topical rosemary officinalis essential oil improves wound healing against disseminated Candida albicans infection in rat model.

[54] Chung LY. The antioxidant properties of garlic compounds: Allyl cysteine, alliin, allicin,

[55] Farahpour MR, Hesaraki S, Faraji D, Zeinalpour R, Aghaei M. Hydroethanolic *Allium sativum* extract accelerates excision wound healing: Evidence for roles of mast-cell infiltration and intracytoplasmic carbohydrate ratio. Brazilian Journal of Pharmaceutical

Comparative Clinical Pathology. 2015. DOI: 10.1007/s00580-015-2086-z

and allyl disulfide. Journal of Medicinal Food. 2006;**9**:205-213

Sciences. 2017. http://dx.doi.org/10.1590/s2175-97902017000115079

Journal of Ethnopharmacology. 2007;**111**:396-402

acid and sesamol. Food Chemistry. 2008;**110**(1):76-82

officinalis L. Fitoterapia. 2011;**82**(3):414-421

and Pharmacology. 2011;**32**(1):63-68

Ethnopharmacology. 2010;**131**:443-450

Applied Microbiology. 1991;**71**:109-112

(3):143-147

44 Wound Healing - Current Perspectives

2006;**40**(2):223-231


[70] Farahpour MR, Mirzakhani N, Doostmohammadi J, Ebrahimzadeh M. Hydroethanolic Pistacia atlantica hulls extract improved wound healing process; evidence for mast cells infiltration, angiogenesis and RNA stability. International Journal of Surgery. 2015; **17**:88-98

[83] Ghosh S, Banerjee S, Sil PC. The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. Food and Chemical Toxicology.

Medicinal Plants in Wound Healing

47

http://dx.doi.org/10.5772/intechopen.80215

[84] Mun SH, Joung DK, Kim YS, Kang OH, Kim SB, et al. Synergistic antibacterial effect of curcumin against methicillin resistant Staphylococcus aureus. Phytomedicine. 2013;

[85] Izui S, Sekine S, Maeda K, Kuboniwa M, Takada A. Antibacterial activity of curcumin against periodontopathic bacteria. Journal of Periodontology. 2016;**87**(1):83-90

2015;**83**:111-124

**20**(8-9):714-718


[83] Ghosh S, Banerjee S, Sil PC. The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. Food and Chemical Toxicology. 2015;**83**:111-124

[70] Farahpour MR, Mirzakhani N, Doostmohammadi J, Ebrahimzadeh M. Hydroethanolic Pistacia atlantica hulls extract improved wound healing process; evidence for mast cells infiltration, angiogenesis and RNA stability. International Journal of Surgery. 2015;

[71] Farahpour MR, Fathollahpour S. Topical co-administration of flaxseed and pistachio ointment promoted wound healing; evidence for histopathological features. Compara-

[72] Zhao B, Zhang X, Han W, Cheng J, Qin Y.Wound healing effect of an *Astragalus membranaceus* polysaccharide and its mechanism. Molecular Medicine Reports. 2017;**15**:4077-4083 [73] Chen W, Wang S, Chiu C, Tseng C, Lin C, Wang H, Lee J. Lucidone suppresses hepatitis C virus replication by Nrf2-mediated heme oxygenase-1 induction. Antimicrobial

[74] Kumar KS, Yang H, Tsai Y, Hung P, Chang S, Lo H, Shen P, Chen S, Wang H, Wang S. Lucidone protects human skin keratinocytes against free radical-induced oxidative damage and inflammation through the up-regulation of HO-1/Nrf2 antioxidant genes and down-regulation of NF-κB signaling pathway. Food and Chemical Toxicology.

[75] Yang H, Tsai Y, Korivi M, Chang C, Hseu Y. Lucidone promotes the cutaneouswound healing process via activation of the PI3K/AKT, Wnt/β-catenin and NF-κB signaling

[76] Wang TT, Sathyamoorthy N, Phang JM. Molecular effects of genistein on estrogen recep-

[77] Mezei O, Banz WJ, Steger RW, Peluso MR, Winters TA, Shay N. Soy isoflavones exert antidiabetic and hypolipidemic effects through the PPAR pathways in obese Zucker rats

[78] Park E, Lee SM, Jung IK, Lim Y, Kim J.Effects of genistein on early-stage cutaneous wound healing. Biochemical and Biophysical Research Communications. 2011;**410**:514-519 [79] Eo H, Lee H-J, Lim Y. Ameliorative effect of dietary genistein on diabetes induced hyper-inflammation and oxidative stress during early stage of wound healing in alloxan induced diabetic mice. Biochemical and Biophysical Research Communications.

[80] Maquart FX, Chastang F, Simeon A, Birembaut P, Gillery P, et al. Triterpenes from Centella asiatica stimulate extracellular matrix accumulation in rat experimental wounds.

[81] Rosen H, Blumenthal A, McCallum J. Effect of asiaticoside on wound healing in the rat. Proceedings of the Society for Experimental Biology and Medicine. 1967;**125**(1):279-280

[82] Mahmood K, Zia KM, Zuber M, Salman M, Anjum MN. Recent developments in curcumin and curcumin based polymeric materials for biomedical applications: A review.

International Journal of Biological Macromolecules. 2015;**81**:877-890

and murine RAW 264.7 cells. The Journal of Nutrition. 2003;**133**:1238-1243

tive Clinical Pathology. 2015. DOI: 10.1007/s00580-015-2097-9

pathways. Biochimica et Biophysica Acta. 1864;**2017**:151-168

tor mediated pathways. Carcinogenesis. 1996;**17**:271-275

European Journal of Dermatology. 1999;**9**(4):289-296

Agents and Chemotherapy. 2013;**57**:1180-1191

**17**:88-98

46 Wound Healing - Current Perspectives

2013;**59**:55-66

2016;**478**:1021-1027


**Chapter 5**

**Provisional chapter**

**Wound Healing: Contributions from Plant Secondary**

**Wound Healing: Contributions from Plant Secondary** 

Plants by their genetic makeup possess an innate ability to synthesize a wide variety of phytochemicals that help them to perform their normal physiological functions and/or to protect themselves from microbial pathogens and animal herbivores. The synthesis of these phytochemicals presents the plants their natural tendency to respond to environmental stress conditions. These phytochemicals are classified either as primary or secondary metabolites. The secondary metabolites have been identified in plants as alkaloids, terpenoids, phenolics, anthraquinones, and triterpenes. These plant-based compounds are believed to have diverse medicinal properties including antioxidant properties. Plants have therefore been a potential source of antioxidants which have received a great deal of attention since increased oxidative stress has been identified as a major causative factor in the development and progression of several life-threatening diseases, including neurodegenerative and cardiovascular diseases and wound infection. Consequently, many medicinal plants have been cited and known to effect wound healing and antioxidant properties. This chapter briefly reviews antioxidant properties of medicinal plants

to highlight the important roles medicinal plants play in wound healing.

**Keywords:** wound healing, antioxidants, phytochemicals, reactive oxygen species,

Plant-derived drugs have been part of the human race in the healthcare for thousands of years [1]. Throughout the world, a huge percentage of population depends upon the use of plantbased medicine because of their easy availability and also due to the lack of better healthcare

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.81208

**Metabolite Antioxidants**

**Metabolite Antioxidants**

http://dx.doi.org/10.5772/intechopen.81208

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Victor Y.A. Barku

**Abstract**

plants

**1. Introduction**

Victor Y.A. Barku

#### **Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants**

DOI: 10.5772/intechopen.81208

Victor Y.A. Barku Victor Y.A. Barku

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81208

#### **Abstract**

Plants by their genetic makeup possess an innate ability to synthesize a wide variety of phytochemicals that help them to perform their normal physiological functions and/or to protect themselves from microbial pathogens and animal herbivores. The synthesis of these phytochemicals presents the plants their natural tendency to respond to environmental stress conditions. These phytochemicals are classified either as primary or secondary metabolites. The secondary metabolites have been identified in plants as alkaloids, terpenoids, phenolics, anthraquinones, and triterpenes. These plant-based compounds are believed to have diverse medicinal properties including antioxidant properties. Plants have therefore been a potential source of antioxidants which have received a great deal of attention since increased oxidative stress has been identified as a major causative factor in the development and progression of several life-threatening diseases, including neurodegenerative and cardiovascular diseases and wound infection. Consequently, many medicinal plants have been cited and known to effect wound healing and antioxidant properties. This chapter briefly reviews antioxidant properties of medicinal plants to highlight the important roles medicinal plants play in wound healing.

**Keywords:** wound healing, antioxidants, phytochemicals, reactive oxygen species, plants

### **1. Introduction**

Plant-derived drugs have been part of the human race in the healthcare for thousands of years [1]. Throughout the world, a huge percentage of population depends upon the use of plantbased medicine because of their easy availability and also due to the lack of better healthcare

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

alternatives. Plant-based medicines or herbal medicines have been effective and safe traditional methods practiced in many countries including China, India, and most African countries for the treatment of various diseases [2]. A large number of plant extracts, concoctions, poultices, decoctions, or pastes are equally used in many countries for treatment of diseases, cuts, wounds, and burns. Thus, since antiquity, several medicinal plants and plant-based strategies are widely known for their significant role in wound healing and skin regeneration as well as their therapeutic applications [2].

it can be deeper, extending into subcutaneous tissue with damage to other structures such as

Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants

http://dx.doi.org/10.5772/intechopen.81208

51

Wound can result through accident or intentional etiology or as a result of a disease process. Wounding, irrespective of the cause and whatever the form, damages the tissue and disrupts

Based on the underlying cause of wound creation, wounds may be classified into two main groups: open and closed wounds. In open wounds, the skin is broken, and the underlying tissue is exposed to the outside environment allowing blood to leave the body. These are wounds in which there is loss of superficial surface covering the tissue such as loss of skin. Such wounds are opened to invasion by microorganisms [15]. Open wounds consist of abrasion or glazes, laceration, incision, puncture, avulsion, cuts, blisters, penetration, and gunshot wounds. In closed wounds, the skin is intact, and the underlying tissue is not directly exposed to the outside world. The superficial surface covering the wound is not lost. The wound occurs under the surface of the skin without affecting the skin and hence does not involve any external bleeding. Infection of these wounds is rare, and it may resolve without any treatment if it is not extensive. Examples of closed wounds are contusion (bruises), hematomas, and crush injuries. Wound can also be classified as either internal or external based on the wound origin. Internal wounds result from impaired immune and nervous system functions and/or decreased supply of blood, oxygen, or nutrients to that area, such as in cases of chronic medical illness (diabetes, atherosclerosis, and deep vein thrombosis). External wounds are usually caused by penetrating objects or non-penetrating trauma. Penetrating wounds result from trauma that breaks through the full thickness of the skin, reaching down to the underlying tissue and organs, and include stab wounds (trauma from sharp objects, such as knives), skin cuts, surgical wounds (intentional cuts in the skin to perform surgical procedures), and gunshot

Non-penetrating wounds are usually the result of blunt trauma or friction with other surfaces; the wound does not break through the skin and may include abrasions (scraping of the outer skin layer), lacerations (a tear-like wound), contusions (swollen bruises due to accumulation of blood and dead cells under the skin), and concussions (damage to the underlying organs

Depending on the healing time, wound can further be classified as either acute or chronic wounds [14]. Acute wounds heal uneventfully (with no complications) in the predicted amount of time, while chronic wounds take a longer time to heal and might have some

The presence of foreign material and bacteria leads to another way to classify wounds. A wound that has dirt, fragments of the causative agent, bacteria, or other foreign materials is determined to be contaminated or infected. A wound with no foreign materials or debris

tendons, muscles, vessels, nerves, parenchymal organs, and bones [12, 13].

the local environment within it [14].

wounds (wounds resulting from firearms).

inside is determined to be clean [15].

complications.

and tissue on the head with no significant external wound).

**2.1. Types of wounds**

Wound is an injury that damages the dermal layer of the skin. Several factors contribute to wound generation, e.g., accidental traumas or surgery, and in certain cases, this dermal injury may have a devastating outcome [2, 3]. Wound healing is the natural process which leads to restore the structural and functional integrities of injured tissues. It involves several biochemical and cellular pathways, in order to repair the lesions and to restore the physiological conditions. Fortunately, the human body has the inbuilt capacity to promote this repairing process. However, there can be impairment of this sophisticated repairing process leading to chronic or non-healing wounds, which may result in severe clinical complications or even patient death. Deficiencies in nutritional factors which are essential in cellular differentiation, immune functioning, and collagen formation may result to the failure of wound healing process [4]. Additionally, oxygen- and nitrogen-centered reactive species are known to play crucial roles in regulating healing [2, 5]. Hence, high concentrations of these reactive species are present in wound sites. Unfortunately, these substances can induce harmful effects on cells and tissues and even promote oxidative stress that generates lipid peroxidation, damage of deoxyribonucleic acid (DNA), and enzyme inactivation, including free radical scavenger enzymes [6]. This necessitates the involvement or use of antioxidants which may represent potential therapeutic tools to enhance and accelerate wound healing process.

Several phytoconstituents such as triterpenes, alkaloids, and polyphenols show antioxidant and antimicrobial effects and are able to promote one or more mechanisms of the reparative process [7]. Accordingly, numerous plant extracts have been employed to promote wound healing with a high degree of success [8]. Many wound healing medicinal plants have been investigated to possess antioxidant properties. In other dimensions, numerous studies conducted over the years showed the great potential of plants in promoting wound healing, by virtue of their high contents in antioxidant properties. This document therefore intends to throw more light on the existing literature on wound healing potentials of medicinal plants and their antioxidant properties.

### **2. Wound**

A physical, chemical, thermal, microbial, or immunological action on the living tissue may result in disruption of a cellular, anatomical, and functional continuity of the living tissue [9]. This phenomenon results in an injury to the skin or the underlying tissue or organ termed a wound. A wound is therefore damage or disruption to the normal anatomical structure and function [10, 11]. This can range from a simple break in the epithelial integrity of the skin or it can be deeper, extending into subcutaneous tissue with damage to other structures such as tendons, muscles, vessels, nerves, parenchymal organs, and bones [12, 13].

Wound can result through accident or intentional etiology or as a result of a disease process. Wounding, irrespective of the cause and whatever the form, damages the tissue and disrupts the local environment within it [14].

### **2.1. Types of wounds**

alternatives. Plant-based medicines or herbal medicines have been effective and safe traditional methods practiced in many countries including China, India, and most African countries for the treatment of various diseases [2]. A large number of plant extracts, concoctions, poultices, decoctions, or pastes are equally used in many countries for treatment of diseases, cuts, wounds, and burns. Thus, since antiquity, several medicinal plants and plant-based strategies are widely known for their significant role in wound healing and skin regeneration

Wound is an injury that damages the dermal layer of the skin. Several factors contribute to wound generation, e.g., accidental traumas or surgery, and in certain cases, this dermal injury may have a devastating outcome [2, 3]. Wound healing is the natural process which leads to restore the structural and functional integrities of injured tissues. It involves several biochemical and cellular pathways, in order to repair the lesions and to restore the physiological conditions. Fortunately, the human body has the inbuilt capacity to promote this repairing process. However, there can be impairment of this sophisticated repairing process leading to chronic or non-healing wounds, which may result in severe clinical complications or even patient death. Deficiencies in nutritional factors which are essential in cellular differentiation, immune functioning, and collagen formation may result to the failure of wound healing process [4]. Additionally, oxygen- and nitrogen-centered reactive species are known to play crucial roles in regulating healing [2, 5]. Hence, high concentrations of these reactive species are present in wound sites. Unfortunately, these substances can induce harmful effects on cells and tissues and even promote oxidative stress that generates lipid peroxidation, damage of deoxyribonucleic acid (DNA), and enzyme inactivation, including free radical scavenger enzymes [6]. This necessitates the involvement or use of antioxidants which may represent

potential therapeutic tools to enhance and accelerate wound healing process.

Several phytoconstituents such as triterpenes, alkaloids, and polyphenols show antioxidant and antimicrobial effects and are able to promote one or more mechanisms of the reparative process [7]. Accordingly, numerous plant extracts have been employed to promote wound healing with a high degree of success [8]. Many wound healing medicinal plants have been investigated to possess antioxidant properties. In other dimensions, numerous studies conducted over the years showed the great potential of plants in promoting wound healing, by virtue of their high contents in antioxidant properties. This document therefore intends to throw more light on the existing literature on wound healing potentials of medicinal plants

A physical, chemical, thermal, microbial, or immunological action on the living tissue may result in disruption of a cellular, anatomical, and functional continuity of the living tissue [9]. This phenomenon results in an injury to the skin or the underlying tissue or organ termed a wound. A wound is therefore damage or disruption to the normal anatomical structure and function [10, 11]. This can range from a simple break in the epithelial integrity of the skin or

as well as their therapeutic applications [2].

50 Wound Healing - Current Perspectives

and their antioxidant properties.

**2. Wound**

Based on the underlying cause of wound creation, wounds may be classified into two main groups: open and closed wounds. In open wounds, the skin is broken, and the underlying tissue is exposed to the outside environment allowing blood to leave the body. These are wounds in which there is loss of superficial surface covering the tissue such as loss of skin. Such wounds are opened to invasion by microorganisms [15]. Open wounds consist of abrasion or glazes, laceration, incision, puncture, avulsion, cuts, blisters, penetration, and gunshot wounds. In closed wounds, the skin is intact, and the underlying tissue is not directly exposed to the outside world. The superficial surface covering the wound is not lost. The wound occurs under the surface of the skin without affecting the skin and hence does not involve any external bleeding. Infection of these wounds is rare, and it may resolve without any treatment if it is not extensive. Examples of closed wounds are contusion (bruises), hematomas, and crush injuries.

Wound can also be classified as either internal or external based on the wound origin. Internal wounds result from impaired immune and nervous system functions and/or decreased supply of blood, oxygen, or nutrients to that area, such as in cases of chronic medical illness (diabetes, atherosclerosis, and deep vein thrombosis). External wounds are usually caused by penetrating objects or non-penetrating trauma. Penetrating wounds result from trauma that breaks through the full thickness of the skin, reaching down to the underlying tissue and organs, and include stab wounds (trauma from sharp objects, such as knives), skin cuts, surgical wounds (intentional cuts in the skin to perform surgical procedures), and gunshot wounds (wounds resulting from firearms).

Non-penetrating wounds are usually the result of blunt trauma or friction with other surfaces; the wound does not break through the skin and may include abrasions (scraping of the outer skin layer), lacerations (a tear-like wound), contusions (swollen bruises due to accumulation of blood and dead cells under the skin), and concussions (damage to the underlying organs and tissue on the head with no significant external wound).

Depending on the healing time, wound can further be classified as either acute or chronic wounds [14]. Acute wounds heal uneventfully (with no complications) in the predicted amount of time, while chronic wounds take a longer time to heal and might have some complications.

The presence of foreign material and bacteria leads to another way to classify wounds. A wound that has dirt, fragments of the causative agent, bacteria, or other foreign materials is determined to be contaminated or infected. A wound with no foreign materials or debris inside is determined to be clean [15].

### **3. Wound healing**

Wound healing is a complex and dynamic process of replacing devitalized and missing cellular structures and tissue layers. The wound healing process can be divided into three or four distinct basic phases. Inflammatory, fibroblastic or proliferation, and maturation or remodeling constitutes the three-phase division [16, 17]. In the four-phase concept, there are the hemostasis phase, the inflammatory phase, the proliferation phase, and the remodeling phase. In the three-phase approach, the hemostasis phase is contained within the inflammatory phase [18]. The normal physiology of wound healing depends on low levels of reactive oxygen species (ROS) and oxidative stress [19, 20]. An overexposure to oxidative stress leads to impaired wound healing. Free radicals are highly unstable molecules, and ROS are a form of free radicals that include the oxygen atom as well as reactive molecules such as superoxides and peroxides. Although normally formed as a by-product of metabolism and are reactive to invading organisms, overproduction leads to an increased load of free radicals and ROS known as oxidative stress. Free radicals attack and remove electrons from all types of molecules in the cell, including nucleic acids in DNA, proteins, and polyunsaturated fatty acids in cell membranes or organelle membranes. When free radicals attack proteins, they break peptide bonds in the protein backbone, changing the protein structure and altering its functionality [21]. All of these processes are detrimental to the proliferation of new cells in the healing process of epithelial wounds. ROS are likely needed at some basal level for wound healing. The importance of ROS to wound healing is illustrated by studies demonstrating that total suppression of oxidant production results in impaired healing, just as excessive amounts of oxidants do. ROS have also been implicated as important mediators of cell signaling and inflammation in wound repair. Although ROS production is physiologic, excessive production can be harmful.

the antioxidant defense system. It is the accumulated damage due to free radical activity in the human body. Excessive amounts of ROS may be a primary cause of biomolecular oxidation. The ROS have the ability to attack numerous molecules in the membrane that contain carbon–carbon double bonds (C〓C). For instance, polyunsaturated fatty acids are particularly sensitive to free radicals. The free radicals are destructive to these molecules including proteins and lipids through oxidation [25]. As a result, ROS have the potential of causing peroxidation of membrane lipids, aggression of tissue membranes and proteins, or damage to DNA and enzyme and generally by oxidizing low-density lipoproteins (LDL). This may result in significant damage to cell structure, contributing to various diseases, such as cancer, stroke, diabetes, arthritis, hemorrhagic shock, coronary artery diseases, cataract, cancer, and acquired immune deficiency syndrome (AIDS) as well as age-related degenerative brain diseases [26]. Under normal circumstances, the cell can reduce the impact of these free radicals and ROS by an endogenous system, i.e., by the body's natural antioxidant defense mechanisms. Physiologic antioxidant defenses include the ROS-detoxifying enzymes superoxide dismutase (SOD), catalase, glutathione peroxidases, and peroxiredoxins [27]. However, the following factors or conditions may contribute to the overproduction of ROS and antioxidant depletion: the mitochondrial electron transport chain; excessive stimulation of nicotinamide adenine dinucleotide phosphate (NADPH); exposure to environmental pollutants such as cigarette smoke, ultraviolet (UV) rays, radiation and toxic chemicals which weaken the body's defense system; and exposure to explosion-generated shock waves [28, 29]. It becomes evidently clear that the devastating impact of ROS can only be reduced successfully through exogenous systems. There is therefore the need to provide the body with a constant supply of antioxidants through dietary supplementation. Antioxidants are postulated to help control wound oxidative stress and thereby accelerate wound healing. They are important mediators in regulating the damage that is potentially incurred by biological molecules such as DNA, protein, lipids,

Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants

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53

Antioxidants are substances that prevent oxidation to occur. They are compounds that detoxify ROS to prevent their damaging effects through multi-mechanisms. Antioxidants may offer resistance against the oxidative stress by scavenging free radicals, inhibiting lipid peroxidation and thus preventing disease. Antioxidants have the ability to prevent, delay, or ameliorate many of the effects of free radicals. During certain diseased state, as well as during aging, there is a need to boost the antioxidant abilities, thereby potentiating the immune mechanism [30]. The antioxidants preserve and stimulate the function of immune cells against homeo-

Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ) are commonly employed as preservatives or additives by pharmaceutical, cosmetic, and food companies [32]. The free radicals are known to be scavenged by these synthetic antioxidants. However, reports on the involvement of synthetic antioxidants in chronic diseases and their adverse side effects leading to carcinogenicity have restricted their use in foods. Therefore, attention has been focused on natural

and body tissue in the presence of reactive species.

antioxidants mainly from plant sources [33, 34].

**3.2. Antioxidants**

static disturbances [31].

#### **3.1. Reactive oxygen species (ROS)**

Oxidation is a basic part of the aerobic life and our metabolism. The body uses oxygen (O2 ) to produce energy by oxidizing glucose. In the biochemical process involving oxygen, i.e., during oxidation, many highly unstable reactive molecules called free radicals are produced. The free radicals are atoms or molecules having odd number of electrons. Atoms of oxygen or nitrogen having central unpaired electron are called reactive oxygen or nitrogen species [22, 23]. These species are natural by-products produced by the normal metabolism of oxygen in living organisms. These reactive oxygen species (ROS) are various forms of activated oxygen which causes oxidative damage. They include free radicals such as superoxide anion radicals (O2 ) <sup>−</sup>, hydroxyl radicals (OH˙), and non-free radical species such as peroxyl radicals (O<sup>2</sup> ) −2 and singlet oxygen ( 1 O2 ) which are various forms of activated oxygen generated in the body [24].

In small amounts, these ROS can be beneficial as signal transducers and growth regulators. However, during oxidative stress, large or excessive amounts of these ROS can be produced and may be dangerous and harmful to the body. The free radicals have the potential to damage biological tissues by disrupting cell membranes. This then affects the ability of the cell to transport substances across the membranes. The immune system is vulnerable to oxidative stress. Oxidative stress refers to an imbalance between the production of free radicals and the antioxidant defense system. It is the accumulated damage due to free radical activity in the human body. Excessive amounts of ROS may be a primary cause of biomolecular oxidation. The ROS have the ability to attack numerous molecules in the membrane that contain carbon–carbon double bonds (C〓C). For instance, polyunsaturated fatty acids are particularly sensitive to free radicals. The free radicals are destructive to these molecules including proteins and lipids through oxidation [25]. As a result, ROS have the potential of causing peroxidation of membrane lipids, aggression of tissue membranes and proteins, or damage to DNA and enzyme and generally by oxidizing low-density lipoproteins (LDL). This may result in significant damage to cell structure, contributing to various diseases, such as cancer, stroke, diabetes, arthritis, hemorrhagic shock, coronary artery diseases, cataract, cancer, and acquired immune deficiency syndrome (AIDS) as well as age-related degenerative brain diseases [26]. Under normal circumstances, the cell can reduce the impact of these free radicals and ROS by an endogenous system, i.e., by the body's natural antioxidant defense mechanisms. Physiologic antioxidant defenses include the ROS-detoxifying enzymes superoxide dismutase (SOD), catalase, glutathione peroxidases, and peroxiredoxins [27]. However, the following factors or conditions may contribute to the overproduction of ROS and antioxidant depletion: the mitochondrial electron transport chain; excessive stimulation of nicotinamide adenine dinucleotide phosphate (NADPH); exposure to environmental pollutants such as cigarette smoke, ultraviolet (UV) rays, radiation and toxic chemicals which weaken the body's defense system; and exposure to explosion-generated shock waves [28, 29]. It becomes evidently clear that the devastating impact of ROS can only be reduced successfully through exogenous systems. There is therefore the need to provide the body with a constant supply of antioxidants through dietary supplementation. Antioxidants are postulated to help control wound oxidative stress and thereby accelerate wound healing. They are important mediators in regulating the damage that is potentially incurred by biological molecules such as DNA, protein, lipids, and body tissue in the presence of reactive species.

#### **3.2. Antioxidants**

) to

)

and singlet oxygen

) −2 <sup>−</sup>, hydroxyl

**3. Wound healing**

52 Wound Healing - Current Perspectives

**3.1. Reactive oxygen species (ROS)**

( 1 O2

Wound healing is a complex and dynamic process of replacing devitalized and missing cellular structures and tissue layers. The wound healing process can be divided into three or four distinct basic phases. Inflammatory, fibroblastic or proliferation, and maturation or remodeling constitutes the three-phase division [16, 17]. In the four-phase concept, there are the hemostasis phase, the inflammatory phase, the proliferation phase, and the remodeling phase. In the three-phase approach, the hemostasis phase is contained within the inflammatory phase [18]. The normal physiology of wound healing depends on low levels of reactive oxygen species (ROS) and oxidative stress [19, 20]. An overexposure to oxidative stress leads to impaired wound healing. Free radicals are highly unstable molecules, and ROS are a form of free radicals that include the oxygen atom as well as reactive molecules such as superoxides and peroxides. Although normally formed as a by-product of metabolism and are reactive to invading organisms, overproduction leads to an increased load of free radicals and ROS known as oxidative stress. Free radicals attack and remove electrons from all types of molecules in the cell, including nucleic acids in DNA, proteins, and polyunsaturated fatty acids in cell membranes or organelle membranes. When free radicals attack proteins, they break peptide bonds in the protein backbone, changing the protein structure and altering its functionality [21]. All of these processes are detrimental to the proliferation of new cells in the healing process of epithelial wounds. ROS are likely needed at some basal level for wound healing. The importance of ROS to wound healing is illustrated by studies demonstrating that total suppression of oxidant production results in impaired healing, just as excessive amounts of oxidants do. ROS have also been implicated as important mediators of cell signaling and inflammation in wound repair.

Although ROS production is physiologic, excessive production can be harmful.

oxidative damage. They include free radicals such as superoxide anion radicals (O2

) which are various forms of activated oxygen generated in the body [24].

radicals (OH˙), and non-free radical species such as peroxyl radicals (O<sup>2</sup>

Oxidation is a basic part of the aerobic life and our metabolism. The body uses oxygen (O2

produce energy by oxidizing glucose. In the biochemical process involving oxygen, i.e., during oxidation, many highly unstable reactive molecules called free radicals are produced. The free radicals are atoms or molecules having odd number of electrons. Atoms of oxygen or nitrogen having central unpaired electron are called reactive oxygen or nitrogen species [22, 23]. These species are natural by-products produced by the normal metabolism of oxygen in living organisms. These reactive oxygen species (ROS) are various forms of activated oxygen which causes

In small amounts, these ROS can be beneficial as signal transducers and growth regulators. However, during oxidative stress, large or excessive amounts of these ROS can be produced and may be dangerous and harmful to the body. The free radicals have the potential to damage biological tissues by disrupting cell membranes. This then affects the ability of the cell to transport substances across the membranes. The immune system is vulnerable to oxidative stress. Oxidative stress refers to an imbalance between the production of free radicals and Antioxidants are substances that prevent oxidation to occur. They are compounds that detoxify ROS to prevent their damaging effects through multi-mechanisms. Antioxidants may offer resistance against the oxidative stress by scavenging free radicals, inhibiting lipid peroxidation and thus preventing disease. Antioxidants have the ability to prevent, delay, or ameliorate many of the effects of free radicals. During certain diseased state, as well as during aging, there is a need to boost the antioxidant abilities, thereby potentiating the immune mechanism [30]. The antioxidants preserve and stimulate the function of immune cells against homeostatic disturbances [31].

Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ) are commonly employed as preservatives or additives by pharmaceutical, cosmetic, and food companies [32]. The free radicals are known to be scavenged by these synthetic antioxidants. However, reports on the involvement of synthetic antioxidants in chronic diseases and their adverse side effects leading to carcinogenicity have restricted their use in foods. Therefore, attention has been focused on natural antioxidants mainly from plant sources [33, 34].

#### **3.3. Plants as important sources of natural antioxidants in wound healing**

There is great interest in the use of natural products, which include compounds derived from fruits, plants, and herbs. Plants have an innate ability to synthesize a wide variety of phytochemicals. Plants do not only provide the carbohydrates, proteins, and fats necessary in the diet of man and other animals but also produce a vast range of organic materials to perform their normal physiological functions and to protect themselves from microbial pathogens and animal herbivores and to respond to environmental stress conditions. Hence plants accumulate a range of low- and high-molecular weight secondary metabolites that play important roles in ROS metabolism and avoidance of uncontrolled oxidation of essential biomolecules.

hence are able to reduce highly oxidizing free radicals by forming less reactive flavonoid radicals. As a result, they are able to prevent lipid peroxidation which is one of the most important actions of free radicals that leads to cellular membrane damage and, ultimately, to cell death [44]. Flavonoids are also able to scavenge nitric oxide which forms in combination with superoxide free radicals the highly damaging peroxynitrite and also to inhibit xanthine oxidase, an important biological source of superoxide radicals that can react with hydrogen

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55

Other flavonoids such as quercetin, kaempferol, myristin, apigenin, and luteolin also have antioxidative activity in many in vitro studies [45]. It has been observed that anthocyanins, which were one of the main antioxidant components in red wine, were the most effective, both in scavenging ROS and in inhibiting lipoprotein oxidation [46]. Quercetin is also known to have inhibited iron-catalyzed Fenton reaction (reaction between superoxide radicals with hydrogen

The exogenous (dietary) antioxidants are mainly derived from food and medicinal plants, such as fruits, vegetables, cereals, mushrooms, beverages, flowers, spices, and traditional medicinal herbs [47]. A large number of plant species and their phytochemicals have diverse medicinal properties, and almost majority of these plants have been found to possess excel-

peroxide to produce a more toxic hydroxyl radical [44].

lent antioxidant activity within in vitro assays.

**Figure 1.** Flow chart showing the different classifications of antioxidants.

peroxide).

Consequently, plants have efficient complex enzymatic and non-enzymatic antioxidant defense systems to avoid the toxic effects of free radicals. Enzymatic systems include SOD, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), while non-enzymatic systems consist of low-molecular weight antioxidants (ascorbic acid, glutathione, proline, carotenoids, phenolic acids, flavonoids, etc.) and high-molecular weight secondary metabolites such as tannins [35, 36]. **Figure 1** gives a summary of the different classifications of antioxidants.

Among all secondary metabolites, phenolic compounds have been mentioned to be largely the contributory compound to antioxidant activity of plants since they have shown promising antioxidant activity in many in vivo and in vitro studies. Phenolic compounds exhibit a considerable free radical scavenging (antioxidant) activity, which is determined by their reactivity as hydrogen or electron-donating agents, the stability of the resulting antioxidant-derived radical, their reactivity with other antioxidants, and finally their metal-chelating properties [37, 38]. Similarly, polyphenols derived from plants are of great importance because of their potential antioxidant and antimicrobial properties [39]. Plant phenolics are mainly classified into five major groups as phenolic acids, flavonoids, lignans, stilbenes, and tannins. These classes of phytochemicals are found to have excellent antioxidant activity and are widely available for the treatment of a multitude of cutaneous ailments. Many studies have presented plants to possess great potential for wound healing because they are versatile as antioxidant and antimicrobial sources. Medicinal plants and their active compounds have been used in medicine since ancient times and are well known for their abilities to promote wound healing and prevent infection without grave side effects [40].

Flavonoids are a group of polyphenolic compounds with known properties which include free radical scavenging, inhibition of hydrolytic and oxidative enzymes, and anti-inflammatory action [41]. The best-described property of almost every group of flavonoids is their capacity to act as antioxidants. The flavones and catechins seem to be the most powerful flavonoids for protecting the body against reactive oxygen species. Flavonoids may have an additive effect to the endogenous scavenging compounds. Many in vitro studies have demonstrated the potent peroxyl radical scavenging abilities of flavonoids, which contribute to inhibiting lipid peroxidation and oxidation of LDL [42].

Flavonoids are known to possess protective effects in biological systems due to their capacity to transfer free radical electrons, chelate metal catalysts, activate antioxidant enzymes, reduce alpha-tocopherol radicals, and inhibit oxidases [43]. Flavonoids have lower redox potentials hence are able to reduce highly oxidizing free radicals by forming less reactive flavonoid radicals. As a result, they are able to prevent lipid peroxidation which is one of the most important actions of free radicals that leads to cellular membrane damage and, ultimately, to cell death [44]. Flavonoids are also able to scavenge nitric oxide which forms in combination with superoxide free radicals the highly damaging peroxynitrite and also to inhibit xanthine oxidase, an important biological source of superoxide radicals that can react with hydrogen peroxide to produce a more toxic hydroxyl radical [44].

**3.3. Plants as important sources of natural antioxidants in wound healing**

54 Wound Healing - Current Perspectives

There is great interest in the use of natural products, which include compounds derived from fruits, plants, and herbs. Plants have an innate ability to synthesize a wide variety of phytochemicals. Plants do not only provide the carbohydrates, proteins, and fats necessary in the diet of man and other animals but also produce a vast range of organic materials to perform their normal physiological functions and to protect themselves from microbial pathogens and animal herbivores and to respond to environmental stress conditions. Hence plants accumulate a range of low- and high-molecular weight secondary metabolites that play important roles in ROS metabolism and avoidance of uncontrolled oxidation of essential biomolecules.

Consequently, plants have efficient complex enzymatic and non-enzymatic antioxidant defense systems to avoid the toxic effects of free radicals. Enzymatic systems include SOD, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), while non-enzymatic systems consist of low-molecular weight antioxidants (ascorbic acid, glutathione, proline, carotenoids, phenolic acids, flavonoids, etc.) and high-molecular weight secondary metabolites such as tannins [35, 36]. **Figure 1** gives a summary of the different classifications of antioxidants.

Among all secondary metabolites, phenolic compounds have been mentioned to be largely the contributory compound to antioxidant activity of plants since they have shown promising antioxidant activity in many in vivo and in vitro studies. Phenolic compounds exhibit a considerable free radical scavenging (antioxidant) activity, which is determined by their reactivity as hydrogen or electron-donating agents, the stability of the resulting antioxidant-derived radical, their reactivity with other antioxidants, and finally their metal-chelating properties [37, 38]. Similarly, polyphenols derived from plants are of great importance because of their potential antioxidant and antimicrobial properties [39]. Plant phenolics are mainly classified into five major groups as phenolic acids, flavonoids, lignans, stilbenes, and tannins. These classes of phytochemicals are found to have excellent antioxidant activity and are widely available for the treatment of a multitude of cutaneous ailments. Many studies have presented plants to possess great potential for wound healing because they are versatile as antioxidant and antimicrobial sources. Medicinal plants and their active compounds have been used in medicine since ancient times and are well known for their abilities to promote wound healing

Flavonoids are a group of polyphenolic compounds with known properties which include free radical scavenging, inhibition of hydrolytic and oxidative enzymes, and anti-inflammatory action [41]. The best-described property of almost every group of flavonoids is their capacity to act as antioxidants. The flavones and catechins seem to be the most powerful flavonoids for protecting the body against reactive oxygen species. Flavonoids may have an additive effect to the endogenous scavenging compounds. Many in vitro studies have demonstrated the potent peroxyl radical scavenging abilities of flavonoids, which contribute to inhibiting

Flavonoids are known to possess protective effects in biological systems due to their capacity to transfer free radical electrons, chelate metal catalysts, activate antioxidant enzymes, reduce alpha-tocopherol radicals, and inhibit oxidases [43]. Flavonoids have lower redox potentials

and prevent infection without grave side effects [40].

lipid peroxidation and oxidation of LDL [42].

Other flavonoids such as quercetin, kaempferol, myristin, apigenin, and luteolin also have antioxidative activity in many in vitro studies [45]. It has been observed that anthocyanins, which were one of the main antioxidant components in red wine, were the most effective, both in scavenging ROS and in inhibiting lipoprotein oxidation [46]. Quercetin is also known to have inhibited iron-catalyzed Fenton reaction (reaction between superoxide radicals with hydrogen peroxide).

The exogenous (dietary) antioxidants are mainly derived from food and medicinal plants, such as fruits, vegetables, cereals, mushrooms, beverages, flowers, spices, and traditional medicinal herbs [47]. A large number of plant species and their phytochemicals have diverse medicinal properties, and almost majority of these plants have been found to possess excellent antioxidant activity within in vitro assays.

**Figure 1.** Flow chart showing the different classifications of antioxidants.

The natural function of vitamin E which is present in vegetable oils, nuts, and other fatty plant-based foods is to prevent oxidation. Vitamin E therefore acts as antioxidant when consumed. It helps to prevent degradation of cell membranes in regions containing C〓C bonds. Some other antioxidants operate by different mechanisms, reacting with oxygen molecules (O2 ) to prevent the production of free radicals. Additionally, there are numerous dietary antioxidants that can be consumed which contribute to an enhanced cellular protection. Ascorbic acid, for example, effectively scavenges ROS and resynthesizes α-tocopherol [48].

biochemical indicators of antioxidant parameters using 10% (w/w) ointment of 50% ethanol extract showed a remarkable wound healing activity. In the study of uninfected wounds, epithelization period was reduced from 24.66 ± 0.97 for the control group treated with blank ointment to 12.16 ± 0.36 for the group treatment. Similarly, in the case of infected wounds with *Staphylococcus epidermidis*, the percentage of wound contraction was significantly enhanced. Also, the extract significantly increased superoxide dismutase and catalase and reduced glutathione when compared with the control group of infected and uninfected wounds [11].

Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants

http://dx.doi.org/10.5772/intechopen.81208

57

*Limonia acidissima* Linn. is used traditionally in India for the treatment of tumors, asthma, wounds, cardiac debility, and hepatitis. Wound healing investigations using the excision, incision, and dead space wound models were carried out on the 200 and 400 mg/kg methanol extract doses. The wound contracted progressively when treated with the extracts. In the wounding healing, results for the incision and dead space models, breaking strength, hydroxyproline, and granulation tissue weight, as well as SOD and catalase all increased significantly (p < 0.05), following treatment with the extract and standard drug, when compared with the control group. Thus, the extract not only promoted wound healing but also exerted

*Marrubium vulgare* L. (Lamiaceae) is a gray-leaved herbaceous perennial medicinal plant well known for several pharmaceutical activities. It is traditionally employed against respiratory infections such as bronchitis, coughs, and asthma. An experiment carried out on the hydroalcoholic leaf extract showed a good activity, with the half maximal effective concentration (EC50) of 38.56 ± 0.10 μg/mL (DPPH assay). A preliminary MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide-tetrazolium dye] test with 5 μg/mL concentration was noncytotoxic and able to improve fibroblast growth. This capability was subsequently confirmed by the results of in vitro wound healing test that led to the conclusion that *M. vulgare* might

*Morinda citrifolia* (Noni) has been traditionally used to treat broken bones, deep cuts, bruises, sores, and wounds. It is also reported to have a broad range of nutritional and therapeutic values for cancer, infection, arthritis, diabetes, asthma, hypertension, and pain. Investigating the effect of *M. citrifolia* leaves on experimental wounds and lipid peroxide levels in rats resulted in ample evidences confirming that *M. citrifolia* enhances wound healing by acting on various phases of the wound healing process. There was a significant increase in wound contraction rate, skin breaking strength that reflected increased collagen levels. The findings from the investigation also showed a decrease in lipid peroxide level in the *M. citrifolia*-treated group [54].

*Musa paradisiaca* (plantain) is a crop in the genus *Musa* that is indigenous to tropical and subtropical countries. To validate the ethnotherapeutic claims of the plant in skin diseases, wound healing activity was studied, besides antioxidant activity to understand the mechanism of wound healing activity. Methanol and hexane extracts of *M. paradisiaca* peel were used to evaluate the biological activity (antioxidant and wound healing) on the regenerative process of the epithelial tissue. The two extracts showed a good inhibition of DPPH radical; the hexane has an activity of 94.25% and methanol 87.33% at a concentration of 125 μg/mL compared with BHT 43.22% as a control. Wound closure was significantly more advanced in the treated groups (methanol 94.62%, hexane 88.39%) compared with control groups 81.75% at

antioxidant activity [53].

be effective against skin injuries [2].

Similarly, a number of plants and plant isolates have been reported to protect free radicalinduced damage in various experimental models. In recent times, focus on plant research has increased all over the world, and a large body of evidences has been collected to show the immense potential of medicinal plants used in various traditional systems. Green tea, for example, contains catechin components that are known to stimulate antioxidant activity by scavenging free radicals, inhibiting pro-oxidant enzymes and stimulating antioxidant enzymes [49]. Majority of these plants are endowed with free radical scavenging molecules, such as vitamins, terpenoids, phenolic acids, lignins, stilbenes, tannins, flavonoids, quinones, coumarins, alkaloids, amines, betalains, and other metabolites, which are rich in antioxidant activities [50].

Various plant products have been used in the treatment of wounds over the years. Wound healing phytochemical compounds fight infection, promote blood clotting, and accelerate the healing process. Numerous phytochemical compounds have been identified and synthesized from medicinal plants that have unique properties associated with the mechanism of wound healing. Interestingly, many of these wound healing plants investigated displayed antioxidant potential as their major unique properties. A plethora of examples of medicinal plants appears in literature to have shown both wound healing and antioxidant properties.

*Clausena anisata* (Willd) Hook. (Rutaceae) is a shrub widely used in many parts of West Africa including Ghana as therapeutic alternatives for the management of wounds and treatment of other bacterial and fungal infections. In a study conducted on the ethanol leaf, extract of *C. anisata* was found to exhibit antioxidant property with the half maximal inhibitory concentration (IC50) of 32.9 μg/mL. The extract enhanced the rate of wound closure and also exhibited high influence on proliferation of fibroblasts and levels of fibrous connective tissues in the wound bed [51].

*Croton bonplandianum* has been credited with potential to cure liver diseases, swelling of the body, cure against ring worms, and skin diseases. An investigation on the ethanolic and aqueous extracts of the dried leaves of *C. bonplandianum* on experimental excision wounds inflicted on Wistar Albino rats of either sex showed a definite, positive effect on wound healing, with significant increase in wound contraction. Antioxidant property of the extracts was also confirmed by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and nitric oxide scavenging activity [52].

*Leucas lanata* Wall. ex Benth. (Lamiaceae) is a medicinal plant whose juice has been traditionally used by local peoples to treat stomachache, headache, whooping cough, and as an antidote for reptile poison. Fresh leaves are applied externally for wound healing and for absorbing pus when placed on the affected area. A study designed to evaluate wound healing potential of *L. lanata* through the excision wound model and functional changes in biochemical indicators of antioxidant parameters using 10% (w/w) ointment of 50% ethanol extract showed a remarkable wound healing activity. In the study of uninfected wounds, epithelization period was reduced from 24.66 ± 0.97 for the control group treated with blank ointment to 12.16 ± 0.36 for the group treatment. Similarly, in the case of infected wounds with *Staphylococcus epidermidis*, the percentage of wound contraction was significantly enhanced. Also, the extract significantly increased superoxide dismutase and catalase and reduced glutathione when compared with the control group of infected and uninfected wounds [11].

The natural function of vitamin E which is present in vegetable oils, nuts, and other fatty plant-based foods is to prevent oxidation. Vitamin E therefore acts as antioxidant when consumed. It helps to prevent degradation of cell membranes in regions containing C〓C bonds. Some other antioxidants operate by different mechanisms, reacting with oxygen molecules

) to prevent the production of free radicals. Additionally, there are numerous dietary antioxidants that can be consumed which contribute to an enhanced cellular protection. Ascorbic

Similarly, a number of plants and plant isolates have been reported to protect free radicalinduced damage in various experimental models. In recent times, focus on plant research has increased all over the world, and a large body of evidences has been collected to show the immense potential of medicinal plants used in various traditional systems. Green tea, for example, contains catechin components that are known to stimulate antioxidant activity by scavenging free radicals, inhibiting pro-oxidant enzymes and stimulating antioxidant enzymes [49]. Majority of these plants are endowed with free radical scavenging molecules, such as vitamins, terpenoids, phenolic acids, lignins, stilbenes, tannins, flavonoids, quinones, coumarins, alkaloids, amines, betalains, and other metabolites, which are rich in antioxidant activities [50].

Various plant products have been used in the treatment of wounds over the years. Wound healing phytochemical compounds fight infection, promote blood clotting, and accelerate the healing process. Numerous phytochemical compounds have been identified and synthesized from medicinal plants that have unique properties associated with the mechanism of wound healing. Interestingly, many of these wound healing plants investigated displayed antioxidant potential as their major unique properties. A plethora of examples of medicinal plants

*Clausena anisata* (Willd) Hook. (Rutaceae) is a shrub widely used in many parts of West Africa including Ghana as therapeutic alternatives for the management of wounds and treatment of other bacterial and fungal infections. In a study conducted on the ethanol leaf, extract of *C. anisata* was found to exhibit antioxidant property with the half maximal inhibitory concentration (IC50) of 32.9 μg/mL. The extract enhanced the rate of wound closure and also exhibited high influence on proliferation of fibroblasts and levels of fibrous connective tissues in the

*Croton bonplandianum* has been credited with potential to cure liver diseases, swelling of the body, cure against ring worms, and skin diseases. An investigation on the ethanolic and aqueous extracts of the dried leaves of *C. bonplandianum* on experimental excision wounds inflicted on Wistar Albino rats of either sex showed a definite, positive effect on wound healing, with significant increase in wound contraction. Antioxidant property of the extracts was also confirmed by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and nitric oxide scavenging activity [52].

*Leucas lanata* Wall. ex Benth. (Lamiaceae) is a medicinal plant whose juice has been traditionally used by local peoples to treat stomachache, headache, whooping cough, and as an antidote for reptile poison. Fresh leaves are applied externally for wound healing and for absorbing pus when placed on the affected area. A study designed to evaluate wound healing potential of *L. lanata* through the excision wound model and functional changes in

appears in literature to have shown both wound healing and antioxidant properties.

acid, for example, effectively scavenges ROS and resynthesizes α-tocopherol [48].

(O2

56 Wound Healing - Current Perspectives

wound bed [51].

*Limonia acidissima* Linn. is used traditionally in India for the treatment of tumors, asthma, wounds, cardiac debility, and hepatitis. Wound healing investigations using the excision, incision, and dead space wound models were carried out on the 200 and 400 mg/kg methanol extract doses. The wound contracted progressively when treated with the extracts. In the wounding healing, results for the incision and dead space models, breaking strength, hydroxyproline, and granulation tissue weight, as well as SOD and catalase all increased significantly (p < 0.05), following treatment with the extract and standard drug, when compared with the control group. Thus, the extract not only promoted wound healing but also exerted antioxidant activity [53].

*Marrubium vulgare* L. (Lamiaceae) is a gray-leaved herbaceous perennial medicinal plant well known for several pharmaceutical activities. It is traditionally employed against respiratory infections such as bronchitis, coughs, and asthma. An experiment carried out on the hydroalcoholic leaf extract showed a good activity, with the half maximal effective concentration (EC50) of 38.56 ± 0.10 μg/mL (DPPH assay). A preliminary MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide-tetrazolium dye] test with 5 μg/mL concentration was noncytotoxic and able to improve fibroblast growth. This capability was subsequently confirmed by the results of in vitro wound healing test that led to the conclusion that *M. vulgare* might be effective against skin injuries [2].

*Morinda citrifolia* (Noni) has been traditionally used to treat broken bones, deep cuts, bruises, sores, and wounds. It is also reported to have a broad range of nutritional and therapeutic values for cancer, infection, arthritis, diabetes, asthma, hypertension, and pain. Investigating the effect of *M. citrifolia* leaves on experimental wounds and lipid peroxide levels in rats resulted in ample evidences confirming that *M. citrifolia* enhances wound healing by acting on various phases of the wound healing process. There was a significant increase in wound contraction rate, skin breaking strength that reflected increased collagen levels. The findings from the investigation also showed a decrease in lipid peroxide level in the *M. citrifolia*-treated group [54].

*Musa paradisiaca* (plantain) is a crop in the genus *Musa* that is indigenous to tropical and subtropical countries. To validate the ethnotherapeutic claims of the plant in skin diseases, wound healing activity was studied, besides antioxidant activity to understand the mechanism of wound healing activity. Methanol and hexane extracts of *M. paradisiaca* peel were used to evaluate the biological activity (antioxidant and wound healing) on the regenerative process of the epithelial tissue. The two extracts showed a good inhibition of DPPH radical; the hexane has an activity of 94.25% and methanol 87.33% at a concentration of 125 μg/mL compared with BHT 43.22% as a control. Wound closure was significantly more advanced in the treated groups (methanol 94.62%, hexane 88.39%) compared with control groups 81.75% at 15 days. The results suggested that extracts obtained with methanol has potential to stimulate the healing process in a close relation to antioxidant properties more that hexane extracts [55].

**Acknowledgements**

**Conflict of interest**

**Author details**

Victor Y.A. Barku

**References**

Author declared no conflict of interest.

Address all correspondence to: vbarku@ucc.edu.gh

and Pharmaceutical Sciences. 2014;**2**:100-103

Wound Care. 2015;**4**:560-582

cytokines? Nitric Oxide. 2002;**7**:1-10

Journal of Pharmaceutics. 2004;**284**:1-12

pharmacology. 2012;**143**:469-474

Review. 2003;**8**:359-377

Molecules. 2017;**22**:1851. DOI: 10.3390/molecules22111851T

Department of Chemistry, School of Physical Sciences, University of Cape Coast, Ghana

[1] Balachandar R, Saran Prakash L, Ashok Kumar K, Ragavi A, Gurumoorthy P.Antioxidant activity and wound healing potential of selected medicinal plants. Journal of Chemical

Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants

http://dx.doi.org/10.5772/intechopen.81208

59

[2] Amri B, Martino EID, Vitulo EID, Corana F, Ben-Kaâb LB, Rui M, et al. *Marrubium vulgare* L. leave extract: Phytochemical composition, antioxidant and wound healing properties.

[3] Frykber RG, Banks J. Challenges in the treatment of chronic wounds. Advances in

[4] Mackay D, Miller AL. Nutritional support for wound healing. Alternative Medicine

[5] Schwentker A, Vodovotz Y, Weller R, Billiar TR. Nitric oxide and wound repair: Role of

[6] Edwards J, Howley P, Cohen IK. In vitro inhibition of human neutrophil elastase by oleic acid albumin formulations from derivatized cotton wound dressings. International

[7] Somashekar Shetty B. Wound healing and indigenous drugs: Role as antioxidants: A review. Research and Reviews: Journal of Medical and Health Sciences. 2013;**2**(2):5-15 [8] Fikru A, Makonnen E, Eguale T, Debella A, Mekonnen GA. Evaluation of in vivo wound healing activity of methanol extract of *Achyranthes aspera* L. Journal of Ethno-

I am grateful to the University of Cape Coast for their support.

*Phaleria macrocarpa* is a traditional medicinal plant from New Guinea, Papua Island, and Indonesia. It is used to treat cancer, diabetes, ulcers, and hypercholesterolemia. Topical application of *P. macrocarpa* fruit extracts on skin excision wounds in rats resulted in an improved wound contraction rate and considerable reduction in healing time. The extract showed significant healing effect on excision wounds and demonstrated an important role in the inflammation process by increasing antioxidant enzyme activities, thereby accelerating the wound healing process and reducing tissue injury [56].

*Polygonatum odoratum* is an important herbal medicine used in folk medicine for the treatment of various elements. Its leaf extract is known to have possessed strong antioxidant, antibacterial, and anti-breast cancer activity. Topical application of ethanol leaf extract of this plant on the rate of wound healing closure using male Sprague Dawley rats in an excision wound healing model significantly accelerated the rate of wound healing with less inflammatory cells and more collagen with angiogenesis [57].

*Sphaeranthus amaranthoides* is a medicinal plant used in folklore medicine in India for the treatment of skin diseases. The evaluation of antioxidant activity of the methanol extract and its flavonoid fraction by using DPPH free radical scavenging activity, total antioxidant capacity, and total phenolic content showed variable degrees of antioxidant activity. When wound healing activity was studied in excision wound model in rats, both the methanol extract and the flavonoid fraction exhibited better wound healing activity than the standard drug (silver sulfadiazine). The methanolic extract and flavonoid fraction significantly enhanced the rate of wound contraction and the period of epithelialization comparable to silver sulfadiazine [58].

In my study of wound healing medicinal plants, 26 wound healing plants used among the people of Kpando Traditional Area for effective wound healing have been identified. In vitro investigations on four of these plants, namely, *Anogeissus leiocarpus*, *Amaranthus spinosus*, *Corchorus olitorius*, and *Combretum dolichopetalum*, exhibited wound healing efficacies and antioxidant properties.

The enhanced wound healing potency of various herbal extracts, therefore, may be partly attributed to free radical scavenging action of the phytoconstituents present in plant extracts.

### **4. Conclusions**

Many plants used traditionally in treatment of wound possess antioxidant activity. It is evidently clear that wound healing and repair are accelerated by applying plant extracts that are rich in antioxidant phytochemicals. The assertion made that wound healing and antioxidant activity coexist, to some extent, can be confirmed. Researchers are encouraged to intensify their search for plants for the treatment of wounds with novel antioxidant activity that could be beneficial in therapeutic practice.

### **Acknowledgements**

15 days. The results suggested that extracts obtained with methanol has potential to stimulate the healing process in a close relation to antioxidant properties more that hexane extracts [55]. *Phaleria macrocarpa* is a traditional medicinal plant from New Guinea, Papua Island, and Indonesia. It is used to treat cancer, diabetes, ulcers, and hypercholesterolemia. Topical application of *P. macrocarpa* fruit extracts on skin excision wounds in rats resulted in an improved wound contraction rate and considerable reduction in healing time. The extract showed significant healing effect on excision wounds and demonstrated an important role in the inflammation process by increasing antioxidant enzyme activities, thereby accelerating the wound

*Polygonatum odoratum* is an important herbal medicine used in folk medicine for the treatment of various elements. Its leaf extract is known to have possessed strong antioxidant, antibacterial, and anti-breast cancer activity. Topical application of ethanol leaf extract of this plant on the rate of wound healing closure using male Sprague Dawley rats in an excision wound healing model significantly accelerated the rate of wound healing with less inflammatory

*Sphaeranthus amaranthoides* is a medicinal plant used in folklore medicine in India for the treatment of skin diseases. The evaluation of antioxidant activity of the methanol extract and its flavonoid fraction by using DPPH free radical scavenging activity, total antioxidant capacity, and total phenolic content showed variable degrees of antioxidant activity. When wound healing activity was studied in excision wound model in rats, both the methanol extract and the flavonoid fraction exhibited better wound healing activity than the standard drug (silver sulfadiazine). The methanolic extract and flavonoid fraction significantly enhanced the rate of wound contraction and the period of epithelialization comparable to

In my study of wound healing medicinal plants, 26 wound healing plants used among the people of Kpando Traditional Area for effective wound healing have been identified. In vitro investigations on four of these plants, namely, *Anogeissus leiocarpus*, *Amaranthus spinosus*, *Corchorus olitorius*, and *Combretum dolichopetalum*, exhibited wound healing efficacies and

The enhanced wound healing potency of various herbal extracts, therefore, may be partly attributed to free radical scavenging action of the phytoconstituents present in plant extracts.

Many plants used traditionally in treatment of wound possess antioxidant activity. It is evidently clear that wound healing and repair are accelerated by applying plant extracts that are rich in antioxidant phytochemicals. The assertion made that wound healing and antioxidant activity coexist, to some extent, can be confirmed. Researchers are encouraged to intensify their search for plants for the treatment of wounds with novel antioxidant activity that could

healing process and reducing tissue injury [56].

58 Wound Healing - Current Perspectives

cells and more collagen with angiogenesis [57].

silver sulfadiazine [58].

antioxidant properties.

**4. Conclusions**

be beneficial in therapeutic practice.

I am grateful to the University of Cape Coast for their support.

### **Conflict of interest**

Author declared no conflict of interest.

### **Author details**

Victor Y.A. Barku

Address all correspondence to: vbarku@ucc.edu.gh

Department of Chemistry, School of Physical Sciences, University of Cape Coast, Ghana

### **References**


[9] Ammar I, Bardaa S, Mzid M, Sahnoun Z, Rebaii T, Attia H, et al. Antioxidant, antibacterial land in vivo dermal wound healing effects of Opuntia flower extracts. International Journal of Biological Macromolecules. 2015;**81**:483-490

[24] Visioli F, Keaney JF, Halliwell B. Antioxidants and cardiovascular disease; pancrease or

Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants

http://dx.doi.org/10.5772/intechopen.81208

61

[25] Tosun M, Ercisli S, Sengul M, Oezr H, Polat T, Ozturk E. Antioxidant properties and phenolic content of eight salvia species from Turkey. Biological Research. 2009;**42**:175-181

[26] Parr A, Bolwell GP. PhEnols in the plant and in man: The potential for possible nutritional enhancement of the diet by modifying the phenols content or profile. Journal of

[27] Arouma OI. Nutrition and health aspect of free radical and antioxidants. Food and

[28] Elsayed NM, Gorbunov NV. Interplay between high energy impulse noise (blast) and

[29] Valkov M, Leibfritz D, Moncol J, Cronin MTD, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. The International

[30] Devasagayam TPA, Sainis KB.Immune system and antioxidants, especially those derived from Indian medicinal plants. Indian Journal of Experimental Biology. 2002;**40**:639-655 [31] De la Fuente M, Victor M. Antioxidants as modulators of immune function. Immunology

[32] Nguyen Q, Eun J. Antioxidant activity of solvent extracts from Vietnamese medicinal

[33] Dehghan G, Shafiee A, Ghahremani M, Ardestani S, Abdollahi M. Antioxidant potential of various extracts from Ferulaszovitsiana in relation to their phenolic contents.

[34] Kai-Wei L, Chiung-Hui L, Huang-Yao T, Horng-Huey K, Bai-Luh W. Antioxidant prenylflavonoids from *Artocarpus communis* and *Artocarpus elasticus*. Food Chemistry. 2009;

[35] Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy Reviews. 2010;**4**(8):118-126. DOI: 10.4103/

[36] Kasote DM, Katyare SS, Hegde MV, Bae H. Significance of antioxidant potential of plants and its relevance to therapeutic applications. International Journal of Biological

[37] Tuadhar ET, Rao A. Plasma protein oxidation and total antioxidant power in premenstrual syndrome. Asian Pacific Journal of Tropical Medicine. 2010;**3**(3):237-240

[38] Wojdylo A, Oszmianski J, Czmerys R. Antioxidant phenolic compounds in 32 selected

[39] Kumbhare MR, Guleha V, Sivakumar T. Estimation of total phenolic content, cytotoxicity and in vitro antioxidant activity of stem bark of Moringa oleifera. Asian Pacific

tonics for tired sheep. Cardiovascular Research. 2000;**47**(3):409

the Science of Food and Agriculture. 2000;**80**:985-1015

antioxidants in the lung. Toxicology. 2003;**189**:63-67

Journal of Biochemistry & Cell Biology. 2006;**7**(1):45-78

plants. Journal of Medicinal Plant Research. 2011;**5**(13):2798-2811

Chemical Toxicology. 1994;**32**:671-683

and Cell Biology. 2000;**78**(1):n49-n54

Pharmaceutical Biology. 2007;**45**(9):1-9

Sciences. 2015;**11**(8):982-991. DOI: 10.7150/ijbs.12096

herbs. Food Chemistry. 2007;**105**:940-949

Journal of Tropical Disease. 2012; **2**(2):144-150

**115**:558-562

0973-7847.70902


[24] Visioli F, Keaney JF, Halliwell B. Antioxidants and cardiovascular disease; pancrease or tonics for tired sheep. Cardiovascular Research. 2000;**47**(3):409

[9] Ammar I, Bardaa S, Mzid M, Sahnoun Z, Rebaii T, Attia H, et al. Antioxidant, antibacterial land in vivo dermal wound healing effects of Opuntia flower extracts. International

[10] Paarakh PM, Chansouria JPN, Khosa RL. Wound healing activity of *Annona muricata*

[11] Dixit V, Verma P, Agnihotri P, Paliwal AK, Rao CV, Husain T. Antimicrobial, antioxidant and wound healing properties of *Leucas lanata* Wall. ex Benth. The Journal of Phyto-

[12] Kayode OA. Epidemiological study on wound distribution pattern in horses presented at two veterinary clinics in south west, Nigeria between 2007-2010. Journal of Dairy, Veterinary & Animal Research. 2017;**5**(4):00148. DOI: 10.15406/jdvar.2017.05.00148 [13] Alonso JE, Lee J, Burgess AR, Browner BD. The management of complex orthopaedic

[14] Velnar T, Bailey T, Smrkolj V. The wound healing process: An overview of the cellular and molecular mechanisms. The Journal of International Medical Research. 2009;

[15] Open Wound Basics. https://www.woundcarecenters.org/article/wound-basics/open-

[16] Gilmore MA.Phases of wound healing. Dimensions in Oncology Nursing. 1991;**5**(3):32-34 [17] Mercandetti M. Wound Healing and Repair, 2017. Available from: https://emedicine.

[18] Maxson S, Lopez EA, Yoo D, Danilkovitch-Miagkova A, Leroux MA. Concise review: Role of mesenchymal stem cells in wound repair. Stem Cells Translational Medicine.

[19] Dunnill C, Patton T, Brennan J, Barrett J, Dryden M, Cooke J, et al. Reactive oxygen species (ROS) and wound healing: The functional role of ROS and emerging ROSmodulating technologies for augmentation of the healing process. International Wound

[20] Nita M, Grzybowski A. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxidative Medicine and Cellular Longevity.

[21] Underdown MJ. Antioxidants and wound healing [thesis]. East Tennessee: East Ten-

[22] Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature.

[23] Sathya M, Kkilavani R. Phytochemical screening and in vitro antioxidant activity of *Saccharum spontaneum Linn*. International Journal of Pharmaceutical Sciences Review

injuries. The Surgical Clinics of North America. 1996;**76**(4):879-903

Journal of Biological Macromolecules. 2015;**81**:483-490

extract. Journal of Pharmacy Research. 2009;**2**:404-406

pharmacology. 2015;**4**(1):9-16

60 Wound Healing - Current Perspectives

medscape.com/article/1298129-overview

Journal. 2017;**14**:89-96. DOI: 10.1111/iwj.12557

2016;**2016**:3164734. DOI: 10.1155/2016/3164734

**37**:1528-1542

wound-basics

2012;**1**(2):142-149

nessee University; 2013

2000;**408**(6809):239-247

and Research. 2013;**18**(11):75-79


[40] Budovsky A, Yarmolinsky L, Ben-Shabat S. Effect of medicinal plants on wound healing. Wound Repair and Regeneration. 2015;**23**:171-183

[54] Pandurang Rasal VP, Sinnathambi A, Ashok P, Yeshmaina S. Wound healing and antioxidant activities of *Morinda citrifolia* leaf extract in rats. Iranian Journal of Pharmacology

Wound Healing: Contributions from Plant Secondary Metabolite Antioxidants

http://dx.doi.org/10.5772/intechopen.81208

63

[55] Canales-Aguirre A, Carvallo-Aceves A, Manzano-Chávez L, Padilla-Camberos E, Lugo-Cervantes E. Wound healing and antioxidant activities of extracts from *Musa paradisiaca*

[56] Abood WN, Al-Henhena NA, Abood AN, Al-Obaidi MMJ, Ismail S, Abdulla MA, et al. Wound-healing potential of the fruit extract of Phaleria macrocarpa. Bosnian Journal of

[57] Mughrabi FF, Hashim H, Mahmood AA, Suzy SM, Salmah I, Zahra AA, et al. Acceleration of wound healing activity by *Polygonatum odoratum* leaf extract in rats. Journal of

[58] Geethalakshmi R, Sakravarthi C, Kritika T, Arul Kirubakaran M, Sarada DVL. Evaluation of antioxidant and wound healing potentials of *Sphaeranthus amaranthoides* Burm.f. Bio

Medicinal Plant Research. 2014;**8**(13):523-528. DOI: 10.5897/JMPR10.451

Med Research International. 2013;**2013**:7. DOI: 10.1155/2013/607109

L. peel. Planta Medica. 2008;**74**:PD9. DOI: 10.1055/s-0028-1084684

and Therapeutics. 2008;**7**:49-52

Basic Medical Sciences. 2015;**15**(2):25-30


[54] Pandurang Rasal VP, Sinnathambi A, Ashok P, Yeshmaina S. Wound healing and antioxidant activities of *Morinda citrifolia* leaf extract in rats. Iranian Journal of Pharmacology and Therapeutics. 2008;**7**:49-52

[40] Budovsky A, Yarmolinsky L, Ben-Shabat S. Effect of medicinal plants on wound healing.

[41] Frankel EN, Waterhouse AL, Kinsella JE. Inhibition of human LDL oxidation by resve-

[42] Castelluccio C, Paganga G, Melikian N, Bolwell GP, Pridham J, Sampson J, et al. Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants.

[43] Hirano R, Sasamoto W, Matsumoto A, Itakura H, Igarashi O, Kondo K. Antioxidant ability of various flavonoids against DPPH radicals and LDL oxidation. Journal of Nutri-

[44] Cuyckens F, Claeys M. Mass spectrometry in structural analysis of flavonoids. Journal of

[45] Frankel EN, Kanner J, German JB, Parks E, Kinsella JE. Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet. 1993;**341**:454-457

[46] Ghiselli A, Nardini M, Baldi A, Scaccini C. Antioxidant activity of different phenolic fractions separated from an Italian red wine. Journal of Agricultural and Food Chemistry.

[47] Dong-Pi X, Li Y, Meng X, Zhou T, Zhou Y, Zheng J, et al. Natural antioxidants in foods and medicinal plants: Extraction, assessment and resources. International Journal of

[48] Williamson J, Hughes CM, Davison GW. Exogenous plant-based nutraceutical supplementation and peripheral cell mononuclear DNA damage following high intensity exer-

[49] Modi AJ, Khadabadi SS, Deokate UA, Farooqui IA, Deore SL, Gangwani MR. *Argyreia speciosa* Linn. F: Phytochemistry, pharmacognosy and pharmacological studies. Journal

[50] Amutha Iswarya Devi J, Kottai Muthu A. Phytochemical screening, antioxidant activities and total phenolic content of ethanolic extract from whole plant of *Saccharum spontaneum (*Linn.). International Journal of Chemical and Pharmaceutical Sciences. 2014;**5**(2):112-118

[51] Agyepong N, Agyare C, Ossei PPS, Duah Boakye Y. Antioxidant and in vivo wound healing activities of *Clausena anisata*. European Journal of Medicinal Plants. 2015;**10**(2):1-8

[52] Divya S, Naveen Krishna K, Ramachandran S, Dhanaraju MD. Wound healing and in vitro antioxidant activities of *Croton bonplandianum* leaf extract in rats. Global Journal

[53] Ilango K, Chitra V. Wound healing and anti-oxidant activities of the fruit pulp of *Limonia Acidissima* Linn (Rutaceae) in rats. Tropical Journal of Pharmaceutical Research.

Wound Repair and Regeneration. 2015;**23**:171-183

tional Science and Vitaminology (Tokyo). 2001;**47**:357-362

Molecular Sciences. 2017;**18**:96. DOI: 10.3390/ijms18010096

cise. Antioxidants. 2018;**7**:70. DOI: 10.3390/antiox7050070

of Pharmacognosy and Phytotherapy. 2010;**293**:34-42

of Pharmacology. 2011;**5**(3):159-163

2010;**9**(3):223-230

ratrol. Lancet. 1995;**341**:454-457

62 Wound Healing - Current Perspectives

FEBS Letters. 1995;**368**:188-192

Mass Spectrometry. 2004;**39**:1-15

1998;**46**:361-367


**Chapter 6**

**Provisional chapter**

**The Strategies of Natural Polysaccharide in Wound**

**The Strategies of Natural Polysaccharide in Wound** 

Severe or chronic wounds related to diseases or serious incidents have received big attention from not only a scientific standpoint but also a business perspective. Therefore, an effective treatment to abridge the long-term hospitalization of severe wound becomes indispensable. Glycosaminoglycan (GAG), one of the extracellular matrix molecules produced by fibroblasts, participates in cell-cell and cell-matrix interactions, in cell proliferation and migration, and in cytokine and growth factor signaling associated with all phases of wound recovery. Natural polysaccharide, for example, calcium alginate, which consists of mainly differing ratios of d-mannuronic and l-guluronic acid and rich of calcium ions, has been demonstrated to functionalize the glycosaminoglycan activity during wound healing. Once the trigger of the underlying wound healing mechanisms was understood, it should be possible to find ways to enhance and resolve the wound healing process in the patient with conditions and may lead to the potential for treatment

**Keywords:** glycosaminoglycan (GAG), extracellular matrix (ECM) molecules, cytokines,

Wound injuries are the most common health problem people faced in decades and continuously demand advanced wound management strategies to obtain optimal healing. Wound injuries can range from small wounds caused by daily activities to chronic or severe wounds caused by diseases or serious incidents. Besides the type of wound being treated, the effectiveness of wound management also involves a better understanding of different factors such as

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.80812

**Healing**

**Healing**

Juin-Hong Cherng

Juin-Hong Cherng

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80812

alternatives in the future clinical field.

natural polysaccharide, wound healing

#### **The Strategies of Natural Polysaccharide in Wound Healing The Strategies of Natural Polysaccharide in Wound Healing**

DOI: 10.5772/intechopen.80812

#### Juin-Hong Cherng Juin-Hong Cherng

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80812

#### **Abstract**

Severe or chronic wounds related to diseases or serious incidents have received big attention from not only a scientific standpoint but also a business perspective. Therefore, an effective treatment to abridge the long-term hospitalization of severe wound becomes indispensable. Glycosaminoglycan (GAG), one of the extracellular matrix molecules produced by fibroblasts, participates in cell-cell and cell-matrix interactions, in cell proliferation and migration, and in cytokine and growth factor signaling associated with all phases of wound recovery. Natural polysaccharide, for example, calcium alginate, which consists of mainly differing ratios of d-mannuronic and l-guluronic acid and rich of calcium ions, has been demonstrated to functionalize the glycosaminoglycan activity during wound healing. Once the trigger of the underlying wound healing mechanisms was understood, it should be possible to find ways to enhance and resolve the wound healing process in the patient with conditions and may lead to the potential for treatment alternatives in the future clinical field.

**Keywords:** glycosaminoglycan (GAG), extracellular matrix (ECM) molecules, cytokines, natural polysaccharide, wound healing

### **1. Introduction**

Wound injuries are the most common health problem people faced in decades and continuously demand advanced wound management strategies to obtain optimal healing. Wound injuries can range from small wounds caused by daily activities to chronic or severe wounds caused by diseases or serious incidents. Besides the type of wound being treated, the effectiveness of wound management also involves a better understanding of different factors such as

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the healing process and the physical-chemical properties of the available dressings [1]. Mostly in small wounds, tissue injuries will heal completely with normal healing phases within weeks [2]. On the other hand, severe or chronic wounds are hard to heal within months or a year and often reoccur with persistent inflammation [3], which represent major challenges to patients medically and financially. Therefore, proper wound dressings that have the ability to accelerate the wound healing phases and reduce the healing time simultaneously are required to overcome this problem.

Among the various molecules secreted by ECM, the GAG has partners that have significant roles in the control of the all wound healing phases, either acute wound or severe wound. Those molecules participate in cell-cell and cell-matrix interactions, in cell proliferation and migration, and in cytokine and growth factor signaling, thus locally modulating their biologic activities. In an acute wound, the healing progresses through the normal phases of wound healing (hemostasis, inflammation, proliferation, and remodeling) within weeks, while in a severe wound, those phases do not progress normally, mostly within months or years, thus needing an appropriate supplementary treatment to enhance the healing process. Therefore, GAG or GAG-containing material treatment is expected to become a key to turning and accelerating not only acute wound but also especially severe or chronic wound healing problems.

The Strategies of Natural Polysaccharide in Wound Healing

http://dx.doi.org/10.5772/intechopen.80812

67

Hemostasis is the beginning of the wound healing process and may be defined as the interaction between platelets and vessel of vascular injury. The vital mediators of hemostasis are fibrin, platelets, and blood vessels. In the first 1 or 2 hours after injury, wound repair starts with the formation of a fibrin matrix through the proteolytic cleavage of fibrinogen by thrombin, and fibrin directly binds to platelets to produce a clot [17–21]. The α granules of platelets release numerous growth factors, such as PDGF, TGF-α, TGF-β, bFGF, IGF-1, and VEGF [22, 23]. Further, PDGF and IGF-1 call up and activate the fibroblasts as well as synthesize GAG and collagen to lead the

The enzymes of the fibrinolytic resist the clot formation. On the other hand, serpins ensure that excessive fibrinolytic activity does not occur. The ECM contains a network of scaffold proteins that are linked by GAG. GAG, especially HS, plays a key role as anticoagulants that have important acts to manage the regulations of many of the serpins [15]. HS represents 50–90% of the total GAG content [25] and is only in contact with blood when an injury occurs [26, 27]. HS has been identified binding with more than 100 proteins involved in hemostasis, many growth factors, proteins involved in lipid metabolism, and proteins of the ECM [28]. In addition, HS maintains hemostasis as an effective mediator of angiogenesis at the surface of

Summarily, the hemostasis phase begins the healing process, generates blood clot formation which maintains the structure of vessels, and provides a temporary matrix, secreting cytokines and other growth factors, in order to prepare the wound bed for the next phase of the

The inflammatory response is elicited by infection or tissue injury involved in the distribution of blood components (plasma and leukocytes) to the damage site [29, 30]. This phase occurs in the next 24–48 hours after injury on average accompanied by inflammation symptoms, such as redness, body heat, swelling, and pain around the wounded place [31]. Once the bleeding is controlled, the key cells of the inflammatory response such as neutrophils, macrophages, and

**2.1. Role of glycosaminoglycan in acute wound healing**

migration and proliferation of the cells into the wound site [24, 25].

*2.1.1. Hemostasis phase of wound healing*

endothelial cells [4].

healing process.

*2.1.2. Inflammatory phase of wound healing*

Glycosaminoglycans (GAG) are extracellular matrix molecules that have significant roles in the control of the all wound healing phases, either an acute wound or severe wound, such as an effective mediator of angiogenesis and inflammation [4, 5] and promote the wound recovery by leading to rapid granulation, vascularization, and reepithelialization [6]. As a GAG-rich content, the use of natural polysaccharide as wound dressing-based material is proposed to enhance the healing phases, especially to abridge the long-term healing mechanism in severe wound injury, determined by several studies and clinical trials. This chapter will further discuss the detail mechanisms and efficacy of natural polysaccharide in accelerating the wound healing process, thereby intended to encourage the advanced strategies for future wound management.

### **2. The involvement of glycosaminoglycan during wound healing**

Generally, wound healing has been represented with the complexity and overlapping of its phases. These processes integrate a dynamic interaction between cells and extracellular matrix (ECM) that trigger tissue or organ regeneration. The significant roles of ECM and its components during each stage of the healing process are represented by structural matrix provision and function of signal transduction compliance in the dynamic of biological reactions during each stage of the healing process [7, 8–11].

ECM provides structural and functional integrity to connective tissues and organs [12]; yet its synthesis and deposition mainly occur in response to growth factors, cytokines, and mechanical signals mediated via cell surface receptors [13]. In the case of wound healing, ECM consists of at least four major classes [8]: (1) structural proteins such as the collagens and elastin; (2) multidomain adhesive glycoproteins such as fibronectin, vitronectin, and laminin; (3) glycosaminoglycan (GAG) such as hyaluronic acid (HA), proteoglycans (PGs) including versican, syndecans, glypicans, aggrecan, and perlecan (chondroitin sulfate (CS)/dermatan sulfate (DS), and heparin sulfate (HS))—and keratin sulfate (KS), often in large amounts; and (4) matricellular proteins such as secreted protein acidic and rich in cysteine (SPARC, also known as osteonectin and BM-40), thrombospondins 1 and 2, tenascin C and X, and osteopontin.

GAG is the important constituent of the extracellular matrix found on cell surfaces [14] and widely distributed in connective tissues. GAG is composed of characteristic repeating disaccharides, with specific monosaccharides sulfated at each of C<sup>2</sup> , C<sup>3</sup> , C<sup>4</sup> , and C<sup>6</sup> [15]. These compounds are highly anionic polymers, which interact with many cationic species (such as ions and proteins) due to the presence of the carboxylic acid and sulfate functional groups [16]. The negative ion charge of GAG molecules carries was considered substantial in many biological processes.

Among the various molecules secreted by ECM, the GAG has partners that have significant roles in the control of the all wound healing phases, either acute wound or severe wound. Those molecules participate in cell-cell and cell-matrix interactions, in cell proliferation and migration, and in cytokine and growth factor signaling, thus locally modulating their biologic activities. In an acute wound, the healing progresses through the normal phases of wound healing (hemostasis, inflammation, proliferation, and remodeling) within weeks, while in a severe wound, those phases do not progress normally, mostly within months or years, thus needing an appropriate supplementary treatment to enhance the healing process. Therefore, GAG or GAG-containing material treatment is expected to become a key to turning and accelerating not only acute wound but also especially severe or chronic wound healing problems.

#### **2.1. Role of glycosaminoglycan in acute wound healing**

#### *2.1.1. Hemostasis phase of wound healing*

the healing process and the physical-chemical properties of the available dressings [1]. Mostly in small wounds, tissue injuries will heal completely with normal healing phases within weeks [2]. On the other hand, severe or chronic wounds are hard to heal within months or a year and often reoccur with persistent inflammation [3], which represent major challenges to patients medically and financially. Therefore, proper wound dressings that have the ability to accelerate the wound healing phases and reduce the healing time simultaneously are required

Glycosaminoglycans (GAG) are extracellular matrix molecules that have significant roles in the control of the all wound healing phases, either an acute wound or severe wound, such as an effective mediator of angiogenesis and inflammation [4, 5] and promote the wound recovery by leading to rapid granulation, vascularization, and reepithelialization [6]. As a GAG-rich content, the use of natural polysaccharide as wound dressing-based material is proposed to enhance the healing phases, especially to abridge the long-term healing mechanism in severe wound injury, determined by several studies and clinical trials. This chapter will further discuss the detail mechanisms and efficacy of natural polysaccharide in accelerating the wound healing process,

thereby intended to encourage the advanced strategies for future wound management.

Generally, wound healing has been represented with the complexity and overlapping of its phases. These processes integrate a dynamic interaction between cells and extracellular matrix (ECM) that trigger tissue or organ regeneration. The significant roles of ECM and its components during each stage of the healing process are represented by structural matrix provision and function of signal transduction compliance in the dynamic of biological reac-

ECM provides structural and functional integrity to connective tissues and organs [12]; yet its synthesis and deposition mainly occur in response to growth factors, cytokines, and mechanical signals mediated via cell surface receptors [13]. In the case of wound healing, ECM consists of at least four major classes [8]: (1) structural proteins such as the collagens and elastin; (2) multidomain adhesive glycoproteins such as fibronectin, vitronectin, and laminin; (3) glycosaminoglycan (GAG) such as hyaluronic acid (HA), proteoglycans (PGs) including versican, syndecans, glypicans, aggrecan, and perlecan (chondroitin sulfate (CS)/dermatan sulfate (DS), and heparin sulfate (HS))—and keratin sulfate (KS), often in large amounts; and (4) matricellular proteins such as secreted protein acidic and rich in cysteine (SPARC, also known as osteonectin and BM-40), thrombospondins 1 and 2, tenascin C and X, and osteopontin.

GAG is the important constituent of the extracellular matrix found on cell surfaces [14] and widely distributed in connective tissues. GAG is composed of characteristic repeating disaccha-

are highly anionic polymers, which interact with many cationic species (such as ions and proteins) due to the presence of the carboxylic acid and sulfate functional groups [16]. The negative ion charge of GAG molecules carries was considered substantial in many biological processes.

, C<sup>3</sup> , C<sup>4</sup>

, and C<sup>6</sup>

[15]. These compounds

**2. The involvement of glycosaminoglycan during wound healing**

tions during each stage of the healing process [7, 8–11].

rides, with specific monosaccharides sulfated at each of C<sup>2</sup>

to overcome this problem.

66 Wound Healing - Current Perspectives

Hemostasis is the beginning of the wound healing process and may be defined as the interaction between platelets and vessel of vascular injury. The vital mediators of hemostasis are fibrin, platelets, and blood vessels. In the first 1 or 2 hours after injury, wound repair starts with the formation of a fibrin matrix through the proteolytic cleavage of fibrinogen by thrombin, and fibrin directly binds to platelets to produce a clot [17–21]. The α granules of platelets release numerous growth factors, such as PDGF, TGF-α, TGF-β, bFGF, IGF-1, and VEGF [22, 23]. Further, PDGF and IGF-1 call up and activate the fibroblasts as well as synthesize GAG and collagen to lead the migration and proliferation of the cells into the wound site [24, 25].

The enzymes of the fibrinolytic resist the clot formation. On the other hand, serpins ensure that excessive fibrinolytic activity does not occur. The ECM contains a network of scaffold proteins that are linked by GAG. GAG, especially HS, plays a key role as anticoagulants that have important acts to manage the regulations of many of the serpins [15]. HS represents 50–90% of the total GAG content [25] and is only in contact with blood when an injury occurs [26, 27]. HS has been identified binding with more than 100 proteins involved in hemostasis, many growth factors, proteins involved in lipid metabolism, and proteins of the ECM [28]. In addition, HS maintains hemostasis as an effective mediator of angiogenesis at the surface of endothelial cells [4].

Summarily, the hemostasis phase begins the healing process, generates blood clot formation which maintains the structure of vessels, and provides a temporary matrix, secreting cytokines and other growth factors, in order to prepare the wound bed for the next phase of the healing process.

### *2.1.2. Inflammatory phase of wound healing*

The inflammatory response is elicited by infection or tissue injury involved in the distribution of blood components (plasma and leukocytes) to the damage site [29, 30]. This phase occurs in the next 24–48 hours after injury on average accompanied by inflammation symptoms, such as redness, body heat, swelling, and pain around the wounded place [31]. Once the bleeding is controlled, the key cells of the inflammatory response such as neutrophils, macrophages, and lymphocytes assemble into the wound site, simultaneously release a large number of active mediators (cytokines and growth factors), and thus stimulate the inflammatory phase [32–34].

contraction [42]. During this phase, new epithelium forms along with the transition of granulation tissue to a mature scar. This process is accompanied by high mechanic strength of the formed tissue, reduction of capillary amounts by combining into bigger blood vessels, lowering cell density and metabolic activity of the tissue, and lowering the content of GAG [43, 44]. The mechanic strength of the formed tissue equals 25% related to the dermis and equals 80% related to the unchanged tissue after many months of reconstruction [43, 45, 46]. Considering that GAG activities are able to reduce the inflammatory responses and ECM deposition in the early phases of wound healing, a proper wound handling in the beginning of injury with a GAG-rich-containing material is expected to heal the wound more closely to normal skin and

The Strategies of Natural Polysaccharide in Wound Healing

http://dx.doi.org/10.5772/intechopen.80812

69

Severe or chronic wounds, hard-to-heal wounds related to diseases or serious incidents, were also commonly encountered instead of acute wounds. Mostly, this issue has been associated with the aging of the population. Unlike acute wounds, the treatment and management of severe wounds represent major challenges to patients medically and financially, resulting in

Severe wounds are frequently characterized by persistent injury and prolonged inflammation, high incidence of bacterial biofilms, and excessive proteolysis [3]. Impairment of macrophage function and angiogenic response is also suggested, which are mostly related to severe wounds healing process [47, 48]. Due to the prolonged inflammation, an excessive recruitment of inflammatory cells to the wound bed will be incurred and produced by the large numbers of neutrophils. It is known that neutrophils can eliminate damaged tissue from the temporary matrix of the wound site and prevent microbial infection. On the other hand, however, the unmanageable neutrophils' potential to kill pathogens also can lead to excessive protease production that initiates significant tissue damage to the host which is harmful to wound healing as they cause degradation of the ECM and growth factors [16]. Furthermore, the inefficient cell proliferation due to ECM molecule degradation within the wound leads to impaired angiogenesis that indicates further wound bed defacement and impaired healing. Hence, in order to conquer this issue, the prevention of prolonged inflammation is a goal

GAG has been found to bind to neutrophils, macrophages, and lymphocytes which are the key cells of the inflammatory response. The effect of excessive protease production caused by too many activated neutrophils in the wound site can be inhibited by electrostatic binding with certain anionic polymers such as GAG or functionalized dextrans [16]. The highly anionic nature of GAG that was expected would be ion pairing with the cationic neutrophils to interfere the activity of these cationic proteins via charge interactions. Therefore, it may be possible that by this mechanism the excessive neutrophil recruitment is reduced and the

However, after serious tissue injury, the glycanases and proteases can destroy GAG [49]. The lack of GAG in severe wounds can be fixed with the addition of GAG-containing material, such as a natural polysaccharide, directly into the wound site as a wound dressing. With the

wound can pass from the inflammatory stage to the next stage of healing.

**2.2. Role of glycosaminoglycan in severe or chronic wound healing**

reduce the period of this phase.

strategy in severe wound therapy.

a long-term recovery.

HA, a non-sulfated GAG of the ECM, is involved in a significant process of the inflammatory phase. During this phase, HA assembles in the wound bed and regulates early inflammation to modulate inflammatory cell and fibroblast cell migration, pro-inflammatory cytokine synthesis, and the phagocytosis of invading microbes [5]. Moreover, HA may bind and improve the efficiency of chemokines to neutrophils. Butler et al. revealed that HA appeared able to present stimuli to neutrophils [35]. HA on the endothelial surface was increased as well as the efficiency of recruitment of neutrophils. In the inflammatory phase, neutrophils collagenase and elastase eliminate damaged tissue from the temporary matrix of the wound site, while monocytes transform into macrophages and phagocytose fragments of denatured ECM debriding the wound site and inactivating any source of microbial infection through the activity of secreted proteases.

At sites of inflammation, the low-molecular-weight HA fragments (accumulated from degradation of high-molecular-weight HA) can initiate Toll-like receptor 2 and Toll-like receptor 4 induction of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β [36]. Furthermore, the growth factors and cytokines released by the inflammatory cells induce the migration and proliferation of fibroblast and keratinocyte, which synthesize the levels of HA. All along the reepithelialization process, where epithelial cells migrate across the new tissue to form a barrier between the wound and the environment, the level of HA was found significantly elevated [37]. The secretion of cytokines such as TGF-β, PDGF, FGF-2, IL-1, and TNF-α modulate collagen deposition by fibroblasts and penetration of new blood vessels into the wound site.

#### *2.1.3. Proliferation phase of wound healing*

During the proliferation phase of wound healing, over the next 2 or 3 days and lasts for about 2 weeks thereafter, the layer of a new matrix by fibroblasts restores the tissue at the wound site. The other mesenchymal cells also enter the inflammatory site of the wound in response to growth factors that are necessary for the stimulation of cell proliferation [38]. Moreover, fibroblasts, endothelial cells, and keratinocytes produce IGF-I, FGF-2, TGF-β, PDGF, and VEGF, promoting cell migration and proliferation, matrix synthesis, and angiogenesis.

The fibroblasts synthesize collagen and PGs. Both of them act to form an unstructured connective tissue medium that provides new cells to migrate. A number of PGs were presented in the wound site, and their GAG side chains were involved in the stabilization and activation of growth factors [15]. Sulfated PGs with CS and DS contribute in collagen polymerization [39], and HS PGs on cells can create anchors to the surrounding matrix [40]. The PGs provide a matrix for cellular attachment, and some PGs (hyalectans) form ternary complexes with HA hydrating the tissue promoting cell survival and migration above the granulation tissue to cover the wound site.

#### *2.1.4. Remodeling phase of wound healing*

Remodeling is the final phase of wound healing which is achieved over longer periods of up to a year after the initial wound injury [41]. This phase is characterized by wound surface contraction [42]. During this phase, new epithelium forms along with the transition of granulation tissue to a mature scar. This process is accompanied by high mechanic strength of the formed tissue, reduction of capillary amounts by combining into bigger blood vessels, lowering cell density and metabolic activity of the tissue, and lowering the content of GAG [43, 44]. The mechanic strength of the formed tissue equals 25% related to the dermis and equals 80% related to the unchanged tissue after many months of reconstruction [43, 45, 46]. Considering that GAG activities are able to reduce the inflammatory responses and ECM deposition in the early phases of wound healing, a proper wound handling in the beginning of injury with a GAG-rich-containing material is expected to heal the wound more closely to normal skin and reduce the period of this phase.

#### **2.2. Role of glycosaminoglycan in severe or chronic wound healing**

lymphocytes assemble into the wound site, simultaneously release a large number of active mediators (cytokines and growth factors), and thus stimulate the inflammatory phase [32–34]. HA, a non-sulfated GAG of the ECM, is involved in a significant process of the inflammatory phase. During this phase, HA assembles in the wound bed and regulates early inflammation to modulate inflammatory cell and fibroblast cell migration, pro-inflammatory cytokine synthesis, and the phagocytosis of invading microbes [5]. Moreover, HA may bind and improve the efficiency of chemokines to neutrophils. Butler et al. revealed that HA appeared able to present stimuli to neutrophils [35]. HA on the endothelial surface was increased as well as the efficiency of recruitment of neutrophils. In the inflammatory phase, neutrophils collagenase and elastase eliminate damaged tissue from the temporary matrix of the wound site, while monocytes transform into macrophages and phagocytose fragments of denatured ECM debriding the wound site and inactivating any source of microbial infection through the activ-

At sites of inflammation, the low-molecular-weight HA fragments (accumulated from degradation of high-molecular-weight HA) can initiate Toll-like receptor 2 and Toll-like receptor 4 induction of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β [36]. Furthermore, the growth factors and cytokines released by the inflammatory cells induce the migration and proliferation of fibroblast and keratinocyte, which synthesize the levels of HA. All along the reepithelialization process, where epithelial cells migrate across the new tissue to form a barrier between the wound and the environment, the level of HA was found significantly elevated [37]. The secretion of cytokines such as TGF-β, PDGF, FGF-2, IL-1, and TNF-α modulate collagen deposition by fibroblasts and penetration of new blood vessels into the wound site.

During the proliferation phase of wound healing, over the next 2 or 3 days and lasts for about 2 weeks thereafter, the layer of a new matrix by fibroblasts restores the tissue at the wound site. The other mesenchymal cells also enter the inflammatory site of the wound in response to growth factors that are necessary for the stimulation of cell proliferation [38]. Moreover, fibroblasts, endothelial cells, and keratinocytes produce IGF-I, FGF-2, TGF-β, PDGF, and VEGF,

The fibroblasts synthesize collagen and PGs. Both of them act to form an unstructured connective tissue medium that provides new cells to migrate. A number of PGs were presented in the wound site, and their GAG side chains were involved in the stabilization and activation of growth factors [15]. Sulfated PGs with CS and DS contribute in collagen polymerization [39], and HS PGs on cells can create anchors to the surrounding matrix [40]. The PGs provide a matrix for cellular attachment, and some PGs (hyalectans) form ternary complexes with HA hydrating the tissue promoting cell survival and migration above the granulation tissue to

Remodeling is the final phase of wound healing which is achieved over longer periods of up to a year after the initial wound injury [41]. This phase is characterized by wound surface

promoting cell migration and proliferation, matrix synthesis, and angiogenesis.

ity of secreted proteases.

68 Wound Healing - Current Perspectives

cover the wound site.

*2.1.4. Remodeling phase of wound healing*

*2.1.3. Proliferation phase of wound healing*

Severe or chronic wounds, hard-to-heal wounds related to diseases or serious incidents, were also commonly encountered instead of acute wounds. Mostly, this issue has been associated with the aging of the population. Unlike acute wounds, the treatment and management of severe wounds represent major challenges to patients medically and financially, resulting in a long-term recovery.

Severe wounds are frequently characterized by persistent injury and prolonged inflammation, high incidence of bacterial biofilms, and excessive proteolysis [3]. Impairment of macrophage function and angiogenic response is also suggested, which are mostly related to severe wounds healing process [47, 48]. Due to the prolonged inflammation, an excessive recruitment of inflammatory cells to the wound bed will be incurred and produced by the large numbers of neutrophils. It is known that neutrophils can eliminate damaged tissue from the temporary matrix of the wound site and prevent microbial infection. On the other hand, however, the unmanageable neutrophils' potential to kill pathogens also can lead to excessive protease production that initiates significant tissue damage to the host which is harmful to wound healing as they cause degradation of the ECM and growth factors [16]. Furthermore, the inefficient cell proliferation due to ECM molecule degradation within the wound leads to impaired angiogenesis that indicates further wound bed defacement and impaired healing. Hence, in order to conquer this issue, the prevention of prolonged inflammation is a goal strategy in severe wound therapy.

GAG has been found to bind to neutrophils, macrophages, and lymphocytes which are the key cells of the inflammatory response. The effect of excessive protease production caused by too many activated neutrophils in the wound site can be inhibited by electrostatic binding with certain anionic polymers such as GAG or functionalized dextrans [16]. The highly anionic nature of GAG that was expected would be ion pairing with the cationic neutrophils to interfere the activity of these cationic proteins via charge interactions. Therefore, it may be possible that by this mechanism the excessive neutrophil recruitment is reduced and the wound can pass from the inflammatory stage to the next stage of healing.

However, after serious tissue injury, the glycanases and proteases can destroy GAG [49]. The lack of GAG in severe wounds can be fixed with the addition of GAG-containing material, such as a natural polysaccharide, directly into the wound site as a wound dressing. With the rich source of GAG at the wound environment and a better understanding of GAG roles in healing processes, it has been possible to formulate therapeutic strategies which are expected to accelerate severe wound healing.

dressing, a severe wound injury in swine model treated with this material exhibited a rapid reepithelialization and less scar formation, which appeared with a smooth wound, compared to commonly used wound dressing [62]. The ability of natural polysaccharide reduces scar formation in severe wound injury due to its rich content of GAG which was known to promote wound healing and leads to rapid granulation, vascularization, and reepithelialization, thus yielding a minimum scar formation certainly [6]. As well, once the dressing is attached with the wound, an ion exchange reaction occurs between the calcium in the alginate and the sodium in the exudate, producing a soluble gel which in turn helps maintain a moist wound environment [63]. A moist wound environment will prevent the scab's formation and facili-

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71

Regarding immune system activation, the release of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, IFN-γ, and TNF-α, after wound injury also takes an important part during the wound healing process. Various crucial processes at the wound site, such as stimulation of keratinocyte and fibroblast proliferation, synthesis and breakdown of ECM proteins, and regulation of immune response, were handled with these cytokines [64]. Their expressions were shown to be intensely upregulated during the inflammatory phase of healing and

Natural polysaccharide, by its oligosaccharides (β-glucan, xyloglucan, chitin, pectin, d-mannuronic, and l-guluronic), can stimulate human cells to produce cytokines [66, 67]. Particularly, the mechanism of β-glucan is mediated by several receptors including dectin-1 receptor, Toll-like receptors (2, 4, 6), complement receptor 3, scavenger receptor, and lactosylceramide [68]. Once binding to the dectin-1, as the most important receptor, β-glucan stimulates the production of many cytokines or activates other immune and nonimmune reaction mechanisms [69]. Martins et al. demonstrated that a polysaccharide-rich fraction of *Agaricus brasiliensis* is able to regulate the host response by activating both pro- and antiinflammatory mechanisms, thus increasing the production of TNF-α and IL-1β by human monocytes through modulation of Toll-like receptor 4 and Toll-like receptor 2 expression [70]. In addition, even after TLR blockade, these polysaccharides still activated the monocytes to produce considerable levels of IFN-γ, IL-1β, and IL-10. TNF-α and IFN-γ were recognized as the important agents of the anti-mycobacterial cytokine cascade, and IL-10 was considered as an inhibitory cytokine which is important to the adequate balance between inflammatory and immunopathological responses [71]. On the other hand, IL-1β is known as a critical mediator of inflammation which is involved in neutrophil mobilization, cellular adhesion to the endothelium, and white blood cell infiltration [72, 73]. Zhao et al. determined the wound healing effect of an *Astragalus membranaceus* polysaccharide treatment and its mechanism through in vitro and in vivo studies. The results showed that this polysaccharide was able to promote human skin fibroblast propagation and accelerate cell cycle progression, as well as the reepithelialization, revascularization, and cytokine secretion of TGF-β1, bFGF, and EGF which significantly confirmed the accelerated wound closure in mouse wound model [74]. TGF-β1 is an important promoter in the fibroblast proliferation and the secretion of ECM and inhibits its degradation, while EGF and bFGF are important stimulators in the formation of reepithelialization and keratinocyte migration in wound healing [75]. Moreover, the pain and the mechanism of pain signals including peripheral and central processing are also related to the modulation of TGF-β, which is implicated in the pathogenesis of keloids and hypertrophic

tate the growth and migration of cells to optimize the formation of new tissues.

strongly reduced after wound healing was impaired [65].

## **3. Natural polysaccharide in wound healing**

### **3.1. The properties of natural polysaccharide**

Glycosaminoglycan (GAG) has been shown to perform significant roles in cell signaling and development, angiogenesis, anticoagulation, and co-receptors for growth factors, which belong to the control of the all wound healing phases, both of acute wound and severe wound. GAG is an enormous complex of carbohydrate molecules that interact with several proteins involved in physiological and pathological processes [50, 51]. GAG, with a molecular weight roughly around 10–100 kDa, is a linear negatively charged polysaccharide. This electrostatic characteristic is useful for managing the excessive protease production through ion pairing with the cationic neutrophils and interfering the activity of these cationic proteins via charge interactions [16]. Once the excessive neutrophil recruitment is reduced as well as the excessive protease production, the wound can pass efficiently from the inflammatory stage to the next stage of healing, especially for severe wound injury.

Polysaccharides, especially natural polysaccharide, have been extensively used in wounddressing development due to their properties such as being biocompatible, nonimmunogenic, and antimicrobial [52–54]. They appeared as abundant sources in many different forms of plants and production in the body. Based on their availability, a natural polysaccharide with different chemical structures and physical properties represents a large source of materials for progressive applications in the future, especially in the domain of biomaterials for the medical field [55, 56].

Containing a beta-1,3-d-glucan linker, natural polysaccharides contribute to the wound healing process because of their ability to stimulate immune system activation by activating macrophages that clean up the wound site after injury [57]. The macrophage is one of the major inflammatory cells in wounds. It has many substantial functions during wound healing, such as host defense, the promotion and resolution of inflammation, the removal of apoptotic cells, and the support of cell proliferation and tissue restoration following injury [58]. In several studies, natural polysaccharides have been shown to enhance macrophage cytotoxic activity against tumor cells and microorganisms and activate phagocytic activity by escalated reactive oxygen species (ROS) and nitric oxide (NO) production [59–61]. These abilities are useful for enhancing the quality of the wound healing process.

### **3.2. The effect of natural polysaccharide structure in wound healing: animal studies**

Alginate, chitosan, and hyaluronic acid are mainly natural polysaccharides that are considered as good candidates for the management of wounds in decades. Alginate, as a prominent example of a natural polysaccharide with the abundance of GAG, has been utilized to become platforms used for fabricating wound dressing materials. Spun as calcium alginate wound dressing, a severe wound injury in swine model treated with this material exhibited a rapid reepithelialization and less scar formation, which appeared with a smooth wound, compared to commonly used wound dressing [62]. The ability of natural polysaccharide reduces scar formation in severe wound injury due to its rich content of GAG which was known to promote wound healing and leads to rapid granulation, vascularization, and reepithelialization, thus yielding a minimum scar formation certainly [6]. As well, once the dressing is attached with the wound, an ion exchange reaction occurs between the calcium in the alginate and the sodium in the exudate, producing a soluble gel which in turn helps maintain a moist wound environment [63]. A moist wound environment will prevent the scab's formation and facilitate the growth and migration of cells to optimize the formation of new tissues.

rich source of GAG at the wound environment and a better understanding of GAG roles in healing processes, it has been possible to formulate therapeutic strategies which are expected

Glycosaminoglycan (GAG) has been shown to perform significant roles in cell signaling and development, angiogenesis, anticoagulation, and co-receptors for growth factors, which belong to the control of the all wound healing phases, both of acute wound and severe wound. GAG is an enormous complex of carbohydrate molecules that interact with several proteins involved in physiological and pathological processes [50, 51]. GAG, with a molecular weight roughly around 10–100 kDa, is a linear negatively charged polysaccharide. This electrostatic characteristic is useful for managing the excessive protease production through ion pairing with the cationic neutrophils and interfering the activity of these cationic proteins via charge interactions [16]. Once the excessive neutrophil recruitment is reduced as well as the excessive protease production, the wound can pass efficiently from the inflammatory stage to the next

Polysaccharides, especially natural polysaccharide, have been extensively used in wounddressing development due to their properties such as being biocompatible, nonimmunogenic, and antimicrobial [52–54]. They appeared as abundant sources in many different forms of plants and production in the body. Based on their availability, a natural polysaccharide with different chemical structures and physical properties represents a large source of materials for progressive applications in the future, especially in the domain of biomaterials for the medical

Containing a beta-1,3-d-glucan linker, natural polysaccharides contribute to the wound healing process because of their ability to stimulate immune system activation by activating macrophages that clean up the wound site after injury [57]. The macrophage is one of the major inflammatory cells in wounds. It has many substantial functions during wound healing, such as host defense, the promotion and resolution of inflammation, the removal of apoptotic cells, and the support of cell proliferation and tissue restoration following injury [58]. In several studies, natural polysaccharides have been shown to enhance macrophage cytotoxic activity against tumor cells and microorganisms and activate phagocytic activity by escalated reactive oxygen species (ROS) and nitric oxide (NO) production [59–61]. These abilities are useful for

**3.2. The effect of natural polysaccharide structure in wound healing: animal studies**

Alginate, chitosan, and hyaluronic acid are mainly natural polysaccharides that are considered as good candidates for the management of wounds in decades. Alginate, as a prominent example of a natural polysaccharide with the abundance of GAG, has been utilized to become platforms used for fabricating wound dressing materials. Spun as calcium alginate wound

to accelerate severe wound healing.

70 Wound Healing - Current Perspectives

**3. Natural polysaccharide in wound healing**

stage of healing, especially for severe wound injury.

enhancing the quality of the wound healing process.

field [55, 56].

**3.1. The properties of natural polysaccharide**

Regarding immune system activation, the release of pro-inflammatory cytokines, such as IL-1β, IL-6, IL-8, IFN-γ, and TNF-α, after wound injury also takes an important part during the wound healing process. Various crucial processes at the wound site, such as stimulation of keratinocyte and fibroblast proliferation, synthesis and breakdown of ECM proteins, and regulation of immune response, were handled with these cytokines [64]. Their expressions were shown to be intensely upregulated during the inflammatory phase of healing and strongly reduced after wound healing was impaired [65].

Natural polysaccharide, by its oligosaccharides (β-glucan, xyloglucan, chitin, pectin, d-mannuronic, and l-guluronic), can stimulate human cells to produce cytokines [66, 67]. Particularly, the mechanism of β-glucan is mediated by several receptors including dectin-1 receptor, Toll-like receptors (2, 4, 6), complement receptor 3, scavenger receptor, and lactosylceramide [68]. Once binding to the dectin-1, as the most important receptor, β-glucan stimulates the production of many cytokines or activates other immune and nonimmune reaction mechanisms [69]. Martins et al. demonstrated that a polysaccharide-rich fraction of *Agaricus brasiliensis* is able to regulate the host response by activating both pro- and antiinflammatory mechanisms, thus increasing the production of TNF-α and IL-1β by human monocytes through modulation of Toll-like receptor 4 and Toll-like receptor 2 expression [70]. In addition, even after TLR blockade, these polysaccharides still activated the monocytes to produce considerable levels of IFN-γ, IL-1β, and IL-10. TNF-α and IFN-γ were recognized as the important agents of the anti-mycobacterial cytokine cascade, and IL-10 was considered as an inhibitory cytokine which is important to the adequate balance between inflammatory and immunopathological responses [71]. On the other hand, IL-1β is known as a critical mediator of inflammation which is involved in neutrophil mobilization, cellular adhesion to the endothelium, and white blood cell infiltration [72, 73]. Zhao et al. determined the wound healing effect of an *Astragalus membranaceus* polysaccharide treatment and its mechanism through in vitro and in vivo studies. The results showed that this polysaccharide was able to promote human skin fibroblast propagation and accelerate cell cycle progression, as well as the reepithelialization, revascularization, and cytokine secretion of TGF-β1, bFGF, and EGF which significantly confirmed the accelerated wound closure in mouse wound model [74]. TGF-β1 is an important promoter in the fibroblast proliferation and the secretion of ECM and inhibits its degradation, while EGF and bFGF are important stimulators in the formation of reepithelialization and keratinocyte migration in wound healing [75]. Moreover, the pain and the mechanism of pain signals including peripheral and central processing are also related to the modulation of TGF-β, which is implicated in the pathogenesis of keloids and hypertrophic scarring [76]. The use of calcium alginate dressing for severe wound injury treatment in the animal model demonstrated high levels of TGF-β1, TGF-β2, and TGF-β3, suggesting that it might contribute to reduced pain perception [62].

**5. Conclusion and future perspective**

Dewi Sartika MS for her help in documentation.

Address all correspondence to: i72bbb@gmail.com

National Defense Medical Center, Taipei, Taiwan, R.O.C.

medical applications.

**Acknowledgements**

**Conflict of interest**

**Author details**

Juin-Hong Cherng1,2,3

Center, Taipei, Taiwan, R.O.C.

Health Sciences, Taipei, Taiwan, R.O.C.

and/or publication of this article.

Wound dressings have a significant function in the management of wound recovery and have been continuously developed upon to improve the quality of the healing process. In this respect, a natural polysaccharide, with GAG-rich content, has been shown as a potential candidate to enhance the healing process of the wound, especially a severe wound, due to its outstanding properties. The detailed mechanism of natural polysaccharide involvement in wound healing was presented in this chapter, and it is expected to raise further wound management strategies. For example, recently, 3D bioprinting was expanded in tissue engineering for personalized regenerative medicine; hence, natural polysaccharide can be considerably utilized as the bio-ink for the printing of various types of structures as scaffolds as the desired function. The combined therapeutic potential of natural polysaccharide and proper development technique would be a promising potential not only in the wound management field but also the other

The Strategies of Natural Polysaccharide in Wound Healing

http://dx.doi.org/10.5772/intechopen.80812

73

The author would like to thank Dr. Chih-Hsin Wang (Department of Plastic and Reconstructive Surgery), Dr. Cheng-Che Liu (Department and Graduate Institute of Physiology and Biophysics), and Dr. Yi-Wen Wang (Department and Graduate Institute of Biology and Anatomy) at the Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (ROC) for their helpful discussion during this chapter writing, as well as thanks to

The author declared no potential conflicts of interest with respect to the research, authorship,

1 Department and Graduate Institute of Biology and Anatomy, National Defense Medical

2 General Clinical Research Center for New Drug Trial, Tri-Service General Hospital,

3 Department of Gerontological Health Care, National Taipei University of Nursing and

#### **3.3. The application of natural polysaccharide in clinical trials**

There are also several clinical trials of natural polysaccharide for wound repair. A natural polysaccharide that contains rich GAG has been widely used in the medical field as electrospun regenerative materials in the act of matrices that mimic tissues which are being replaced during wound healing. HA-based dressings have been used for chronic wound ulcer treatment of various etiologies, burns, and epithelial surgical wounds. The results revealed that HA significantly upgraded the healing process of wounds compared to traditional standards of care [77]. In line with this result, chitosan and alginate, fabricated as gelling fiber dressing, have been examined to accelerate the healing of patients with chronic non-healing wounds [78, 79]. This dressing has the ability to gel when in contact with wound fluid, less painful to remove, suitable for moderate to high exudate, reduced bioburden, and maintain hemostasis. Taken together, all of these benefits nominate natural polysaccharide as an advisable material in accelerating the wound healing process.

### **4. Limitation to using natural polysaccharide in wound healing**

Generally, natural polysaccharide has demonstrated considerable merit as a treatment for chronic wounds for their anti-inflammatory and moist wound environment preservation abilities. Despite, especially for atopy people (the people with genetic tendency to develop allergic diseases), a natural polysaccharide may induce the immune system to overreact and cause irritation due to its heterogeneous complex structure. Hence, the control of the molecular weight of natural polysaccharide is expected to overcome this limitation. Through the selection of desirable molecular weight, we could simplify or remove the excessive part of natural polysaccharide that may cause the hypersensitivity reaction. Additionally, in the dry wound, these properties may also lead to the inefficiency of the wound healing process. They may cause dehydration to the dry wound, thus reducing blood flow and the epithelial cells' migration ability around the wound site which interrupts the creation of new tissue. As evidence, reepithelialization of the wound site is more rapid under moist conditions than under dry ones with natural polysaccharide wound dressing treatment [80–82]. With controllable molecular weight, it is probable that the potential of natural polysaccharide in accelerating the wound healing process can be utilized as well into several types of wound injury and patient background.

A natural polysaccharide is the element of human dermal ECM [83–86]. As naturally occurring compounds, they have been demonstrated as a great potential for medical, pharmaceutical, and biomedical applications, including wound dressings, biomaterials, and tissue regeneration, due to their economical, less toxic, and favorable compatibility profile. However, possessing a lack of protein structure, natural polysaccharide exhibits a very poor bio-stability and difficulty to assemble a "matrix" to bridge the damaged tissue during wound healing process, therefore facilitating wound contraction and leading to scar formation [87–89]. To address this limitation, natural polysaccharide has been combined with the other natural polymers or synthetic polymers to yield the desired bioactive material.

### **5. Conclusion and future perspective**

Wound dressings have a significant function in the management of wound recovery and have been continuously developed upon to improve the quality of the healing process. In this respect, a natural polysaccharide, with GAG-rich content, has been shown as a potential candidate to enhance the healing process of the wound, especially a severe wound, due to its outstanding properties. The detailed mechanism of natural polysaccharide involvement in wound healing was presented in this chapter, and it is expected to raise further wound management strategies. For example, recently, 3D bioprinting was expanded in tissue engineering for personalized regenerative medicine; hence, natural polysaccharide can be considerably utilized as the bio-ink for the printing of various types of structures as scaffolds as the desired function. The combined therapeutic potential of natural polysaccharide and proper development technique would be a promising potential not only in the wound management field but also the other medical applications.

### **Acknowledgements**

scarring [76]. The use of calcium alginate dressing for severe wound injury treatment in the animal model demonstrated high levels of TGF-β1, TGF-β2, and TGF-β3, suggesting that it

There are also several clinical trials of natural polysaccharide for wound repair. A natural polysaccharide that contains rich GAG has been widely used in the medical field as electrospun regenerative materials in the act of matrices that mimic tissues which are being replaced during wound healing. HA-based dressings have been used for chronic wound ulcer treatment of various etiologies, burns, and epithelial surgical wounds. The results revealed that HA significantly upgraded the healing process of wounds compared to traditional standards of care [77]. In line with this result, chitosan and alginate, fabricated as gelling fiber dressing, have been examined to accelerate the healing of patients with chronic non-healing wounds [78, 79]. This dressing has the ability to gel when in contact with wound fluid, less painful to remove, suitable for moderate to high exudate, reduced bioburden, and maintain hemostasis. Taken together, all of these benefits nominate natural polysaccharide as an advisable material in accelerating the wound healing process.

**4. Limitation to using natural polysaccharide in wound healing**

Generally, natural polysaccharide has demonstrated considerable merit as a treatment for chronic wounds for their anti-inflammatory and moist wound environment preservation abilities. Despite, especially for atopy people (the people with genetic tendency to develop allergic diseases), a natural polysaccharide may induce the immune system to overreact and cause irritation due to its heterogeneous complex structure. Hence, the control of the molecular weight of natural polysaccharide is expected to overcome this limitation. Through the selection of desirable molecular weight, we could simplify or remove the excessive part of natural polysaccharide that may cause the hypersensitivity reaction. Additionally, in the dry wound, these properties may also lead to the inefficiency of the wound healing process. They may cause dehydration to the dry wound, thus reducing blood flow and the epithelial cells' migration ability around the wound site which interrupts the creation of new tissue. As evidence, reepithelialization of the wound site is more rapid under moist conditions than under dry ones with natural polysaccharide wound dressing treatment [80–82]. With controllable molecular weight, it is probable that the potential of natural polysaccharide in accelerating the wound healing process can be utilized as well into several types of wound injury and patient background.

A natural polysaccharide is the element of human dermal ECM [83–86]. As naturally occurring compounds, they have been demonstrated as a great potential for medical, pharmaceutical, and biomedical applications, including wound dressings, biomaterials, and tissue regeneration, due to their economical, less toxic, and favorable compatibility profile. However, possessing a lack of protein structure, natural polysaccharide exhibits a very poor bio-stability and difficulty to assemble a "matrix" to bridge the damaged tissue during wound healing process, therefore facilitating wound contraction and leading to scar formation [87–89]. To address this limitation, natural polysaccharide has been combined with the other natural

polymers or synthetic polymers to yield the desired bioactive material.

might contribute to reduced pain perception [62].

72 Wound Healing - Current Perspectives

**3.3. The application of natural polysaccharide in clinical trials**

The author would like to thank Dr. Chih-Hsin Wang (Department of Plastic and Reconstructive Surgery), Dr. Cheng-Che Liu (Department and Graduate Institute of Physiology and Biophysics), and Dr. Yi-Wen Wang (Department and Graduate Institute of Biology and Anatomy) at the Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (ROC) for their helpful discussion during this chapter writing, as well as thanks to Dewi Sartika MS for her help in documentation.

### **Conflict of interest**

The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

### **Author details**

Juin-Hong Cherng1,2,3

Address all correspondence to: i72bbb@gmail.com

1 Department and Graduate Institute of Biology and Anatomy, National Defense Medical Center, Taipei, Taiwan, R.O.C.

2 General Clinical Research Center for New Drug Trial, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, R.O.C.

3 Department of Gerontological Health Care, National Taipei University of Nursing and Health Sciences, Taipei, Taiwan, R.O.C.

### **References**

[1] Morgan DA. Wounds—What should a dressing formulary include? Hospital Pharmacist. 2002;**9**:261-266

[15] Melrose J. Glycosaminoglycans in wound healing. Bone and Tissue Regeneration

The Strategies of Natural Polysaccharide in Wound Healing

http://dx.doi.org/10.5772/intechopen.80812

75

[16] Peplow PV. Glycosaminoglycan: A candidate to stimulate the repair of chronic wounds.

[17] Mosesson MW, Siebenlist KR, Meh DA. The structure and biological features of fibrinogen and fibrin. Annals of the New York Academy of Sciences. 2001;**936**:11-30. DOI:

[18] Ofosu FA, Nyarko KA. Human platelet thrombin receptors: Roles in platelet activation. Hematology/Oncology Clinics of North America. 2000;**14**:1185-1198. DOI: 10.1016/

[19] Scheraga HA, Laskowski M. The fibrinogen-fibrin conversion. Advances in Protein

[20] Blomback B. Studies on the action of thrombotic enzymes on bovine fibrinogen as mea-

[21] Blomback B, Hessel B, Hogg D, Therkildsen L. A two-step fibrinogen-fibrin transition in

[22] Reinke JM, Sorg H. Wound repair and regeneration. European Surgical Research. 2012;

[23] Shah JMY, Omar E, Pai DR, Sood S. Cellular events and biomarkers of wound healing. Indian Journal of Plastic Surgery. 2012;**45**(2):220-228. DOI: 10.4103/0970-0358.101282 [24] Olczyk P, Ramos P, Biernas M, Komosinska-Vassev K, Stojko J, Pilawa B. Microwave saturation of complex EPR spectra and free radicals of burnt skin treated with apitherapeutic agent. Evidence-Based Complementary and Alternative Medicine. 2013;**2013**:545201.

[25] Reitsma S, Slaaf DW, Vink H, van Zandvoort MA, oude Egbrink MG. The endothelial glycocalyx: Composition, functions, and visualization. Pflügers Archiv. 2007;**454**:

[26] Shworak NW, Kobayashi T, de Agostini A, Smits NC. Anticoagulant heparin sulfate to not clot—or not? Progress in Molecular Biology and Translational Science. 2010;**93**:

[27] Mertens G, Cassiman JJ, Van den Berghe H, Vermylen J, David G. Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties. The Journal of Biological Chemistry.

[28] Conrad HE. Heparin-binding Proteins. San Diego, CA: Academic Press; 1998

[29] Majno G, Joris I. Cells, Tissues, and Disease. London: Oxford University Press; 2004

[30] Vinay K, Cotran RS, Robbins SL. Robbins Basic Pathology. Philadelphia, PA: Saunders;

Thrombosis and Haemostasis. 2005;**94**:4-16. DOI: 10.1160/TH04-12-0812

Chemistry. 1957;**12**:1-19. DOI: 10.1016/S0065-3233(08)60115-1

sured by N-terminal analysis. Arkiv för Kemi. 1958;**12**:321-335

blood coagulation. Nature. 1978;**275**:501-505. DOI: 10.1038/275501a0

Insights. 2016;**7**:29-50. DOI: 10.4137/BTRI.S38670

10.1111/j.1749-6632.2001.tb03491.x

**49**:35-43. DOI: 10.1159/000339613

DOI: 10.1155/2013/545201

1992;**267**:20435-20443

2003

345-359. DOI: 10.1007/s00424-007-0212-8

153-178. DOI: 10.1016/S1877-1173(10)93008-1

S0889-8588(05)70178-7


[15] Melrose J. Glycosaminoglycans in wound healing. Bone and Tissue Regeneration Insights. 2016;**7**:29-50. DOI: 10.4137/BTRI.S38670

**References**

2002;**9**:261-266

74 Wound Healing - Current Perspectives

DOI: 10.1383/surg.20.5.114.14626

DOI: 10.1016/S0142-9612(02)00100-X

1524-475X.2009.00466.x

10.1111/j.1067-1927.2004.012302.x

2014;**11**:159-163. DOI: 10.1111/j.1742-481X.2012.01057.x

200708020-00001

ph.53.030191.001113

University Press; 1992

[1] Morgan DA. Wounds—What should a dressing formulary include? Hospital Pharmacist.

[2] Percival JN. Classification of wounds and their management. Surgery. 2002;**20**(5):114-117.

[3] Demidova-Rice TN, Hamblin MR, Herman IM. Acute and impaired wound healing: Pathophysiology and current methods for drug delivery, part 1: Normal and chronic wounds: Biology, causes, and approaches to care. Advances in Skin & Wound Care.

[4] Shriver Z, Liu D, Sasisekharan R. Emerging views of heparin sulfate glycosaminoglycan structure/activity relationships modulating dynamic biological functions. Trends in

[5] Chen WYJ, Abatangelo G. Functions of hyaluronan in wound repair. Wound Repair and

[6] Kirker KR, Luo Y, Nielson JH, Shelby J, Prestwich GD. Glycosaminoglycan hydrogel films as bio-interactive dressings for wound healing. Biomaterials. 2002;**23**:3661-3671.

[7] Raghow R. The role of extracellular matrix in postinflammatory wound healing and fibrosis. The FASEB Journal. 1994;**8**(11):823-831. DOI: 10.1096/fasebj.8.11.8070631

[8] Schultz GS, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair and Regeneration. 2009;**17**(2):153-162. DOI: 10.1111/j.

[9] Hodde JP, Johnson CE. Extracellular matrix as a strategy for treating chronic wounds. American Journal of Clinical Dermatology. 2007;**8**(2):61-66. DOI: 10.2165/00128071-

[10] Tran KT, Griffith L, Wells A. Extracellular matrix signaling through growth factor receptors during wound healing. Wound Repair and Regeneration. 2004;**12**(3):262-268. DOI:

[11] Frenkel JS. The role of hyaluronan in wound healing. International Wound Journal.

[12] Carey DJ. Control of growth and differentiation of vascular cells by extracellular matrix proteins. Annual Review of Physiology. 1991;**53**:161-177. DOI: 10.1146/annurev.

[13] Boudreau NJ, Jones PL. Extracellular matrix and integrin signalling: The shape of things to come. The Biochemical Journal. 1999;**339**(3):481-488. DOI: 10.1042/bj3390481

[14] D'Ardenne AJ. Ground substance. In: McGee JOD, Isaacson PG, Wright NA, editors. Oxford Textbook of Pathology: Principles of Pathology. 1st ed. London: Oxford

Cardiovascular Medicine. 2002;**12**:71-77. DOI: 10.1016/S1050-1738(01)00150-5

Regeneration. 1999;**7**(2):79-89. DOI: 10.1046/j.1524-475X.1999.00079.x

2012;**25**(7):304-314. DOI: 10.1097/01.ASW.0000416006.55218.d0


[31] Busti AJ, Hooper JS, Amaya CJ, Kazi S. Effects of perioperative antiinflammatory and immunomodulating therapy on surgical wound healing. Pharmacotherapy. 2005;**25**(11): 1566-1591. DOI: 10.1592/phco.2005.25.11.1566

[46] Vedrenne N, Coulomb B, Danigo A, Bont'e F, Desmouli'ere A. The complex dialogue between (myo)fibroblasts and the extracellular matrix during skin repair processes and

The Strategies of Natural Polysaccharide in Wound Healing

http://dx.doi.org/10.5772/intechopen.80812

77

[47] Maruyama K, Asai J, Ii M, Thorne T, Losordo DW, D'Amore PA. Decreased macrophage number and activation lead to reduced lymphatic vessel formation and contribute to impaired diabetic wound healing. The American Journal of Pathology. 2007;**170**:

[48] Galiano RD, Tepper OM, Pelo CR, et al. Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. The American Journal of Pathology.

[49] Eming SA, Krieg T, Davidson JM. Inflammation in wound repair: Molecular and cellular mechanisms. The Journal of Investigative Dermatology. 2007;**127**:514-525. DOI: 10.1038/

[50] Jackson RL, Busch SJ, Cardin AD. Glycosaminoglycans: Molecular properties, protein interactions, and role in physiological processes. Physiological Reviews. 1991;**71**:

[51] Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate. Advances in Carbohydrate Chemistry and Biochemistry. 2001;**57**:159-206. DOI: 10.1016/

[52] Chen JP, Chang GY, Chen JK. Electrospun collagen/chitosan nanofibrous membrane as wound dressing. Colloids and Surfaces A: Physicochemical and Engineering Aspects.

[53] Noh HK, Lee SW, Kim JM, Oh JE, Kim KH, Chung CP, et al. Electrospinning of chitin nanofibers: Degradation behavior and cellular response to normal human keratinocytes and fibroblasts. Biomaterials. 2006;**27**:3934-3944. DOI: 10.1016/j.biomaterials.2006.03.016

[54] Cai Z, Mo X, Zhang K, Fan L, Yin A, He C, et al. Fabrication of chitosan/silk fibroin composite nanofibers for wound-dressing applications. International Journal of Molecular

[55] Dumitriu S. Polysaccharides as biomaterials. In: S. Dumitriu, editor. Polymeric Biomaterials.

[56] Rinaudo M. Characterization and properties of some polysaccharides used as biomaterials. Macromolecular Symposia. 2006;**245-246**:549. DOI: 10.1002/masy.200651379

[57] Su CH, Sun CS, Juan SW, Hu CH, Ke WT, Sheu MT. Fungal mycelia as the source of chitin and polysaccharides and their applications as skin substitutes. Biomaterials.

[58] Koh TJ, DiPietro LA. Inflammation and wound healing: The role of the macrophage. Expert Reviews in Molecular Medicine. 2011;**13**(23). DOI: 10.1017/S1462399411001943

ageing. Pathologie Biologie. 2012;**60**(1):20-27. DOI: 10.1016/j.patbio.2011.10.002

1178-1191. DOI: 10.2353/ajpath.2007.060018

481-539. DOI: 10.1152/physrev.1991.71.2.481

2008;**313**:183-188. DOI: 10.1016/j.colsurfa.2007.04.129

Sciences. 2010;**11**:3529-3539. DOI: 10.3390/ijms11093529

1997;**18**:1169-1174. DOI: 10.1016/S0142-9612(97)00048-3

New York: Marcel Dekker; 2002. pp. 1-61

sj.jid.5700701

S0065-2318(01)57017-1

2004;**164**:1935-1947. DOI: 10.1016/S0002-9440(10)63754-6


[46] Vedrenne N, Coulomb B, Danigo A, Bont'e F, Desmouli'ere A. The complex dialogue between (myo)fibroblasts and the extracellular matrix during skin repair processes and ageing. Pathologie Biologie. 2012;**60**(1):20-27. DOI: 10.1016/j.patbio.2011.10.002

[31] Busti AJ, Hooper JS, Amaya CJ, Kazi S. Effects of perioperative antiinflammatory and immunomodulating therapy on surgical wound healing. Pharmacotherapy. 2005;**25**(11):

[32] Broughton G II, Janis JE, Attinger CE. The basic science of wound healing. Plastic and Reconstructive Surgery. 2006;**117**(7):12S-34S. DOI: 10.1097/01.prs.0000225430.42531.c2 [33] Campos ACL, Groth AK, Branco AB. Assessment and nutritional aspects of wound healing. Current Opinion in Clinical Nutrition and Metabolic Care. 2008;**11**(3):281-288. DOI:

[34] Gosain A, DiPietro LA. Aging and wound healing. World Journal of Surgery. 2004;**28**:

[35] Butler LM, Rainger GE, Nash GB. A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods.

Experimental Cell Research. 2009;**315**:3433-3441. DOI: 10.1016/j.yexcr.2009.08.012 [36] Campo GM, Avenoso A, Campo S, D'Ascola A, Nastasi G, Calatroni A. Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes. Biochimie. 2010;**92**(2):204-215. DOI: 10.1016/j.biochi.2009.10.006 [37] Clark RAF. Overview and general considerations of wound repair. In: Clark RAF, Henson PM, editors. The Molecular and Cellular Biology of Wound Repair. Boston, MA:

[38] Martin P. Wound healing—Aiming for perfect skin regeneration. Science. 1997;**276**(5309):

[39] Hascall VC. Proteoglycans: Structure and Function. New York, USA: Plenum Press; 1981 [40] Lark MW, Culp LA. Cell-Matrix Interaction: Biochemistry of Close and Tight-Focal Adhesive Contacts of Fibroblasts to Extracellular Matrices. New York, USA: Mercel

[41] MacKay D, Miller AL. Nutritional support for wound healing. Alternative Medicine

[42] Sethi KK, Yannas IV, Mudera V, Eastwood M, McFarland C, Brown RA. Evidence for sequential utilization of fibronectin, vitronectin, and collagen during fibroblast mediated collagen contraction. Wound Repair and Regeneration. 2002;**10**(6):397-408. DOI:

[43] Diegelmann RF, Evans MC. Wound healing: An overview of acute, fibrotic and delayed

[44] Hoffman M, Harger A, Lenkowski A, Hedner U, Roberts HR, Monroe DM. Cutaneous wound healing is impaired in hemophilia B. Blood. 2006;**108**(9):3053-3060. DOI: 10.1182/

[45] Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration.

1566-1591. DOI: 10.1592/phco.2005.25.11.1566

10.1097/MCO.0b013e3282fbd35a

Springer; 1998

76 Wound Healing - Current Perspectives

Decker; 1987

Review. 2003;**8**(4):359-377

blood-2006-05-020495

10.1046/j.1524-475X.2002.10609.x

healing. Frontiers in Bioscience. 2004;**9**:283-289

Nature. 2008;**453**(7193):314-321. DOI: 10.1038/nature07039

321-326. DOI: 10.1007/s00268-003-7397-6

75-81. DOI: 10.1126/science.276.5309.75


[59] Park HR, Hwang D, Suh HJ, Yu KW, Kim TY, Shin KS. Anti-tumor and anti-metastatic activities of rhamnogalacturonan-II-type polysaccharide isolated from mature leaves of green tea via activation of macrophages and natural killer cells. International Journal of Biological Macromolecules. 2017;**99**:179-186. DOI: 10.1016/j.ijbiomac.2017.02.043

[71] Cavalcanti YVN, Brelaz MCA, Neves JKAL, Ferraz JC, Pereira VRA. Role of TNF-alpha, IFN-gamma, and IL-10 in the development of pulmonary tuberculosis. Pulmonary

The Strategies of Natural Polysaccharide in Wound Healing

http://dx.doi.org/10.5772/intechopen.80812

79

[72] Dinarello CA. Biologic basis for interleukin-1 in disease. Blood. 1996;**87**(6):2095-2147

[73] Echeverry S, Shi XQ, Haw A, Liu H, Zhang ZW, Zhang J. Transforming growth factorbeta1 impairs neuropathic pain through pleiotropic effects. Molecular Pain. 2009;**5**:16.

[74] Zhao B, Zhang X, Han W, Cheng J, Qin Y. Wound healing effect of an Astragalus membranaceus polysaccharide and its mechanism. Molecular Medicine Reports. 2017;**15**:

[75] Thorey IS, Hinz B, Hoeflich A, Kaesler S, Bugnon P, Elmlinger M, et al. Transgenic mice reveal novel activities of growth hormone in wound repair, angiogenesis, and myofibroblast differentiation. The Journal of Biological Chemistry. 2004;**279**:26674-26684. DOI:

[76] Zhu Y, Colak T, Shenoy M, Liu L, Mehta K, Pai R, et al. Transforming growth factor beta induces sensory neuronal hyperexcitability, and contributes to pancreatic pain and hyperalgesia in rats with chronic pancreatitis. Molecular Pain. 2012;**8**:65. DOI: 10.

[77] Voigt J, Driver VR. Hyaluronic acid derivatives and their healing effect on burns, epithelial surgical wounds, and chronic wounds: A systematic review and meta-analysis of randomized controlled trials. Wound Repair and Regeneration. 2012;**20**:317-331. DOI:

[78] Mason S, Clarke C. A multicentred cohort evaluation of a chitosan gelling fiber dressing. British Journal of Nursing. 2015;**24**:869-876. DOI: 10.12968/bjon.2015.24.17.868

[79] Ausili E, Paolucci V, Triarico S, et al. Treatment of pressure sores in spina bifida patients with calcium alginate and foam dressings. European Review for Medical and Phar-

[80] Daamen WF, van Moerkerkn HT, Hafmans T, Buttafoco L, Poot AA, Veerkamp JH, et al. Preparation and evaluation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds for tissue engineering. Biomaterials. 2003;**24**:4001-4009. DOI: 10.1016/

[81] Lamberg SI, Stoolmiller AC. Glycosaminoglycans. A biochemical and clinical review. Journal of Investigative Dermatology. 1974;**63**:433-449. DOI: 10.1111/1523-1747.ep12680346

[82] Ma L, Gao C, Mao Z, Zhou J, Shen J, Hu X, et al. Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials. 2003;**24**:4833-4841. DOI:

Medicine. 2012;**2012**:745483. DOI: 10.1155/2012/745483

DOI: 10.1186/1744-8069-5-16

10.1074/jbc.M311467200

1186/1744-8069-8-65

S0142-9612(03)00273-4

10.1016/S0142-9612(03)00374-0

10.1111/j.1524-475X.2012.00777.x

macological Sciences. 2013;**17**(12):1642-1647

4077-4083. DOI: 10.3892/mmr.2017.648


[59] Park HR, Hwang D, Suh HJ, Yu KW, Kim TY, Shin KS. Anti-tumor and anti-metastatic activities of rhamnogalacturonan-II-type polysaccharide isolated from mature leaves of green tea via activation of macrophages and natural killer cells. International Journal of

Biological Macromolecules. 2017;**99**:179-186. DOI: 10.1016/j.ijbiomac.2017.02.043

bial functions. mBio. 2016:e01820. DOI: 10.1128/mBio.01820-16

10.1016/j.bcp.2017.09.003

78 Wound Healing - Current Perspectives

annurev.pp.42.060191.003251

017-0102-2

10.1097/01.all.0000113681.18759.5e

iwj.12813

[60] Wagener J, MacCallum DM, Brown GD, Gown NAR. *Candida albicans* chitin increases arginase-1 activity in human macrophages, with an impact on macrophage antimicro-

[61] Lundahl MLE, Scanlan EM, Lavelle EC. Therapeutic potential of carbohydrates as regulators of macrophage activation. Biochemical Pharmacology. 2017;**146**:23-41. DOI:

[62] Wang CH, Chang SJ, Tzeng YS, Shih YJ, Adrienne C, Chen SG, et al. Enhanced woundhealing performance of a phyto-polysaccharide-enriched dressing-a preclinical small and large animal study. International Wound Journal. 2017;**14**:1359-1369. DOI: 10.1111/

[63] Kannon GA, Garrett AB. Moist wound healing with occlusive dressings. A clinical review. Dermatologic Surgery. 1995;**21**:583-590. DOI: 10.1111/j.1524-4725.1995.tb00511.x

[64] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines.

[65] Hubner G, Brauchle M, Smola H, Madlener M, Fassler R, Werner S. Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-

[66] Iwamoto M, Kurachi M, Nakashima T, Kim D, Yamaguchi K, Oda T, et al. Structure– activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells. FEBS Letters. 2005;**579**:4423-4429. DOI: 10.1016/j.febslet.2005.07.007

[67] Ryan CA, Farmer EE. Oligosaccharide signals in plants: A current assessment. Annual Review of Plant Physiology and Plant Molecular Biology. 1991;**42**:651-674. DOI: 10.1146/

[68] Bloebaum RM, Grant JA, Sur S. Immunomodulation: The future of allergy and asthma treatment. Current Opinion in Allergy and Clinical Immunology. 2004;**4**:63-67. DOI:

[69] Sun L, Zhao Y. The biological role of dectin-1 in immune response. International Reviews

[70] Martins PR, Soares AMV, Domeneghini AVSP, Golim MA, Kaneno R. Agaricus brasiliensis polysaccharides stimulate human monocytes to capture *Candida albicans*, express tolllike receptors 2 and 4, and produce pro-inflammatory cytokines. Journal of Venomous Animals and Toxins including Tropical Diseases. 2017;**23**:17. DOI: 10.1186/s40409-

of Immunology. 2007;**26**:349-364. DOI: 10.1080/08830180701690793

Physiological Reviews. 2003;**83**:835-870. DOI: 10.1152/physrev.00031.2002

treated mice. Cytokine. 1996;**8**(7):548-556. DOI: 10.1006/cyto.1996.0074


[83] Price RD, Berry MG, Navsaria HA. Hyaluronic acid: The scientific and clinical evidence. Journal of Plastic, Reconstructive & Aesthetic Surgery. 2007;**60**:1110-1119. DOI: 10.1016/j. bjps.2007.03.005

**Chapter 7**

**Provisional chapter**

**From Tissue Repair to Tissue Regeneration**

**From Tissue Repair to Tissue Regeneration**

DOI: 10.5772/intechopen.81291

In Regeneration 3.0, the priority is to combine the anti-inflammatory activity of the nine proteins acting as growth factors in the bovine colostrum, the homeostatic, angiogenic and reorganizational activities of the matrix, the modulation of collagen synthesis and the remodeling of the epithelium. The choice of bovine colostrum and its associated properties was the basis for the design of devices that could also offer those properties: barrier action, anti-inflammatory action and pain reduction, reduction and absorption of exudates, combating of bacterial and fungal proliferation, antioxidant action and hydration and protection against skin diseases and dermatosis. We now know the key players in the wound healing process and we have new molecules available to act on them, but the future must necessarily lie in the transfer of molecules and information between the endothelium, ECM and cell membrane, which can be directed toward tissue regeneration if the resident stem cells have the chance of communicating and interacting with new therapeutic models, all this without forgetting that the human being is at the center

**Keywords:** wound healing, tissue regeneration, bovine colostrum, stem cells,

The complexity of the wound healing process is increasingly understood and characterized. Until recently, the wound healing mechanism was interpreted as a fibroproliferative response with the aim of producing a cicatricial reaction (repair), with different mechanisms than those seen in a fetal environment, in which the scope of the healing process is tissue regeneration. However, recent awareness of the biological pathways and cell classes

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Aragona Salvatore Emanuele, Mereghetti Giada,

Aragona Salvatore Emanuele, Mereghetti Giada,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Ferrari Alessio and Giorgio Ciprandi

Ferrari Alessio and Giorgio Ciprandi

http://dx.doi.org/10.5772/intechopen.81291

of research and scientific evolution.

aimed protocol

**1. Introduction**

**Abstract**


#### **Chapter 7 Provisional chapter**

#### **From Tissue Repair to Tissue Regeneration From Tissue Repair to Tissue Regeneration**

DOI: 10.5772/intechopen.81291

Aragona Salvatore Emanuele, Mereghetti Giada, Ferrari Alessio and Giorgio Ciprandi Aragona Salvatore Emanuele, Mereghetti Giada, Ferrari Alessio and Giorgio Ciprandi

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81291

#### **Abstract**

[83] Price RD, Berry MG, Navsaria HA. Hyaluronic acid: The scientific and clinical evidence. Journal of Plastic, Reconstructive & Aesthetic Surgery. 2007;**60**:1110-1119. DOI: 10.1016/j.

[84] Vats A, Tolley NS, Polak JM, Gough JE. Scaffolds and biomaterials for tissue engineering: A review of clinical applications. Clinical Otolaryngology and Allied Sciences. 2003;

[85] West DC, Shaw DM, Lorenz P, Adzick NS, Longaker MT. Fibrotic healing of adult and late gestation fetal wounds correlates with increased hyaluronidase activity and removal of hyaluronan. The International Journal of Biochemistry & Cell Biology. 1997;**29**:201-210.

[86] Zhong SP, Zhang YZ, Lim CT. Tissue scaffolds for skin wound healing and dermal reconstruction. Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology.

[87] Chansiripornchai P, Pramatwinai C, Rungsipipat A, Ponsamart S, Nakchat O. The efficiency of polysaccharide gel extracted from fruit-hulls of durian (*Durio zibethinus* L.) for wound healing in pig skin. Acta Horticulturae. 2005;**679**:37-43. DOI: 10.17660/

[88] Winter GD, Scales JT. Effect of air drying and dressing on the surface of a wound. Nature.

[89] Suzuki Y, Nishimura Y, Tanihara M, Suzuki K, Nakamura T, Shimizu Y, et al. Evaluation of a novel alginate gel dressing: Cytotoxicity to fibroblasts in vitro and foreign-body reaction in pig skin in vivo. Journal of Biomedical Materials Research. 1998;**39**:317-322.

DOI: 10.1002/(SICI)1097-4636(199802)39:2<317::AID-JBM20>3.0.CO;2-8

**28**:165-172. DOI: 10.1046/j.1365-2273.2003.00686.x

DOI: 10.1016/S1357-2725(96)00133-1

2010;**2**:510-525. DOI: 10.1002/wnan.100

1963;**197**(5):91-92. DOI: 10.1038/197091b0

ActaHortic.2005.679.5

bjps.2007.03.005

80 Wound Healing - Current Perspectives

In Regeneration 3.0, the priority is to combine the anti-inflammatory activity of the nine proteins acting as growth factors in the bovine colostrum, the homeostatic, angiogenic and reorganizational activities of the matrix, the modulation of collagen synthesis and the remodeling of the epithelium. The choice of bovine colostrum and its associated properties was the basis for the design of devices that could also offer those properties: barrier action, anti-inflammatory action and pain reduction, reduction and absorption of exudates, combating of bacterial and fungal proliferation, antioxidant action and hydration and protection against skin diseases and dermatosis. We now know the key players in the wound healing process and we have new molecules available to act on them, but the future must necessarily lie in the transfer of molecules and information between the endothelium, ECM and cell membrane, which can be directed toward tissue regeneration if the resident stem cells have the chance of communicating and interacting with new therapeutic models, all this without forgetting that the human being is at the center of research and scientific evolution.

**Keywords:** wound healing, tissue regeneration, bovine colostrum, stem cells, aimed protocol

### **1. Introduction**

The complexity of the wound healing process is increasingly understood and characterized. Until recently, the wound healing mechanism was interpreted as a fibroproliferative response with the aim of producing a cicatricial reaction (repair), with different mechanisms than those seen in a fetal environment, in which the scope of the healing process is tissue regeneration. However, recent awareness of the biological pathways and cell classes

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

characterizing the various phases of the wound healing process and current attention toward biomaterials and possible new applications for stem cells, together with the use of bioengineered tissues, have led to a reinterpretation of this process from the perspective of regenerative medicine, intended as the possibility of recreating a tissue as similar as possible to the original.

Current understanding of some of the tissue repair mechanisms enables the application of therapeutic models that have become an established part of everyday practice. The healing process takes place over three complex phases: management of the inflammatory process, cell proliferation (excessive or impaired), and extracellular matrix (ECM) remodeling. Wound repair is characterized by the incomplete regeneration of the original tissue with hyperproduction of organized collagen, which can lead to the production of new tissue with an 80% similarity to the original tissue. Impaired host management of this process leads to an abnormal fibroproliferative response, causing the production of hypertrophic or keloid scars. While some of the mechanisms are already known, new discoveries in the field of molecular and cellular mechanisms enable us to hypothesize other tissue healing management mechanisms and to apply new therapeutic models to achieve results beyond those currently possible. As far back as 2013, Aragona and Marazzi and colleagues [1] published an article in the Italian edition of Surgical Tribune discussing new research in the treatment of skin lesions in regenerative medicine, establishing the bases for the interpretation of the inflammatory process that directly involve some of the cell classes naturally involved in the inflammatory process underlying wound regeneration.

Current gains in knowledge of tissue regeneration, and above all of stem cells and their behavior, open new ground and suggest new future therapeutic models. Research into the effects of electromagnetic fields on stem cells in particular indicates that a paradigm shift is within our grasp. With a little imagination, we can visualize ourselves as an avatar observing nature and the universe: we can close our eyes and listen to the sounds and energy emitted by cells, or open them to observe phenomena that yesterday, we thought a world away. We begin this journey through the study of the inflammatory process by taking a look at in-vitro experimental models, fundamental above all in understanding the molecular, genetic and cellular patterns of the various tissues.

### **2. The inflammatory model of skin lesions. From experimental model to humans**

*"Why, sir, his hide is so tanned with his trade that he will keep out water a great while, and your water is a sore decayer of your whoreson dead body."*

*W. Shakespeare, Hamlet, Act 5, Scene 1.*

regeneration process. This innovation over animal models makes it possible to establish the role and concentration of all the humoral factors (cytokines and growth factors), genetic factors (genes expressed in the various phases of the process), cellular factors and, above all, the fibroblasts and collagen associated with the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), fundamental for extracellular matrix (ECM) remodeling. It also enables the concept of tissue regeneration to take over from tissue repair

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 83

The literature is full of studies meticulously describing the inflammatory process of wounds. It should be remembered that modulation of this process and its impact on the proliferation, differentiation and function of inflammatory cells is aimed at controlling that very inflammation. Our research had the objective of framing all these observations and this scientific knowledge in the biological context in which the process takes place, within its specific indi-

The shift of perspective from inflammation to regeneration involves the systemic treatment of patients with skin and mucosal wounds and local anti-inflammatory treatment involving modulation of the endothelial and ECM inflammation, the anti-inflammatory cytokines and the MMPs. **Figure 1** highlights the switch between the wound repair and regeneration pathway and the chronic inflammation pathway that feeds the chronicity of the skin lesions. Initially, platelet activation leads to the release and activation of TGF-beta 1, PDGF, TNF-alpha and IL1, with the recruitment of neutrophils deputized to natural debridement and to regulation of the expression of the adhesion molecules. The neutrophils are followed by macrophages, the cell population now at the center of attention in wound healing research. These ensure sustained debridement, the release of proinflammatory cytokines, and the potentiation of the fibropro-

The role of macrophages deserves a chapter of its own in relation to their division into the M1 population, which can eliminate invading microorganisms and promote the inflammatory response during the initial inflammatory process, and subsequently the M2 population, during the resolution of the inflammatory process. By losing its reactivity to inflammatory stimuli, the increasingly studied M2 population becomes capable of eliminating damaged

This all takes place within the dynamic structure of the ECM, which can be defined as "the submerged world where everything is possible" thanks to the equilibrium between the glycoprotein, molecular and cellular components assured by the aqueous component that, in light of the latest knowledge, seems to enable communication between the cells and the ECM and could explain some amplification mechanisms of the healing process. The authors had the opportunity to test an in vitro model on both epidermal and Dry Eye Syndrome cell cultures. In both trials, the primary objective was to have a cellular, ultrastructural and biochemical model similar to real life in which to cause an injury similar to that under investigation. The biological injury, whether damage to the epidermis and dermis or creation of an area of dehydration on the eye surface, recreated the natural pathophysiological environment, and the in vitro model offers a faithful reflection. *"Efficacy of a New Ocular Surface Modulator in Restoring* 

as the primary objective of research and clinical practice.

liferative response in the context of chronic inflammation.

cells and tissues, promoting neoangiogenesis and tissue remodeling.

vidual reactivity.

Understanding the inflammation process is enabled by the design and creation of in vitro and in vivo cellular models, with the primary objective of establishing the effect of the molecules of biological agents on the inflammatory process associated with the tissue repair and regeneration process. This innovation over animal models makes it possible to establish the role and concentration of all the humoral factors (cytokines and growth factors), genetic factors (genes expressed in the various phases of the process), cellular factors and, above all, the fibroblasts and collagen associated with the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), fundamental for extracellular matrix (ECM) remodeling. It also enables the concept of tissue regeneration to take over from tissue repair as the primary objective of research and clinical practice.

characterizing the various phases of the wound healing process and current attention toward biomaterials and possible new applications for stem cells, together with the use of bioengineered tissues, have led to a reinterpretation of this process from the perspective of regenerative medicine, intended as the possibility of recreating a tissue as similar as pos-

Current understanding of some of the tissue repair mechanisms enables the application of therapeutic models that have become an established part of everyday practice. The healing process takes place over three complex phases: management of the inflammatory process, cell proliferation (excessive or impaired), and extracellular matrix (ECM) remodeling. Wound repair is characterized by the incomplete regeneration of the original tissue with hyperproduction of organized collagen, which can lead to the production of new tissue with an 80% similarity to the original tissue. Impaired host management of this process leads to an abnormal fibroproliferative response, causing the production of hypertrophic or keloid scars. While some of the mechanisms are already known, new discoveries in the field of molecular and cellular mechanisms enable us to hypothesize other tissue healing management mechanisms and to apply new therapeutic models to achieve results beyond those currently possible. As far back as 2013, Aragona and Marazzi and colleagues [1] published an article in the Italian edition of Surgical Tribune discussing new research in the treatment of skin lesions in regenerative medicine, establishing the bases for the interpretation of the inflammatory process that directly involve some of the cell classes naturally involved in the inflammatory process

Current gains in knowledge of tissue regeneration, and above all of stem cells and their behavior, open new ground and suggest new future therapeutic models. Research into the effects of electromagnetic fields on stem cells in particular indicates that a paradigm shift is within our grasp. With a little imagination, we can visualize ourselves as an avatar observing nature and the universe: we can close our eyes and listen to the sounds and energy emitted by cells, or open them to observe phenomena that yesterday, we thought a world away. We begin this journey through the study of the inflammatory process by taking a look at in-vitro experimental models, fundamental above all in understanding the molecular, genetic and

**2. The inflammatory model of skin lesions. From experimental model** 

*"Why, sir, his hide is so tanned with his trade that he will keep out water a great while, and your water* 

Understanding the inflammation process is enabled by the design and creation of in vitro and in vivo cellular models, with the primary objective of establishing the effect of the molecules of biological agents on the inflammatory process associated with the tissue repair and

*W. Shakespeare, Hamlet, Act 5, Scene 1.*

sible to the original.

82 Wound Healing - Current Perspectives

underlying wound regeneration.

cellular patterns of the various tissues.

*is a sore decayer of your whoreson dead body."*

**to humans**

The literature is full of studies meticulously describing the inflammatory process of wounds. It should be remembered that modulation of this process and its impact on the proliferation, differentiation and function of inflammatory cells is aimed at controlling that very inflammation. Our research had the objective of framing all these observations and this scientific knowledge in the biological context in which the process takes place, within its specific individual reactivity.

The shift of perspective from inflammation to regeneration involves the systemic treatment of patients with skin and mucosal wounds and local anti-inflammatory treatment involving modulation of the endothelial and ECM inflammation, the anti-inflammatory cytokines and the MMPs.

**Figure 1** highlights the switch between the wound repair and regeneration pathway and the chronic inflammation pathway that feeds the chronicity of the skin lesions. Initially, platelet activation leads to the release and activation of TGF-beta 1, PDGF, TNF-alpha and IL1, with the recruitment of neutrophils deputized to natural debridement and to regulation of the expression of the adhesion molecules. The neutrophils are followed by macrophages, the cell population now at the center of attention in wound healing research. These ensure sustained debridement, the release of proinflammatory cytokines, and the potentiation of the fibroproliferative response in the context of chronic inflammation.

The role of macrophages deserves a chapter of its own in relation to their division into the M1 population, which can eliminate invading microorganisms and promote the inflammatory response during the initial inflammatory process, and subsequently the M2 population, during the resolution of the inflammatory process. By losing its reactivity to inflammatory stimuli, the increasingly studied M2 population becomes capable of eliminating damaged cells and tissues, promoting neoangiogenesis and tissue remodeling.

This all takes place within the dynamic structure of the ECM, which can be defined as "the submerged world where everything is possible" thanks to the equilibrium between the glycoprotein, molecular and cellular components assured by the aqueous component that, in light of the latest knowledge, seems to enable communication between the cells and the ECM and could explain some amplification mechanisms of the healing process. The authors had the opportunity to test an in vitro model on both epidermal and Dry Eye Syndrome cell cultures. In both trials, the primary objective was to have a cellular, ultrastructural and biochemical model similar to real life in which to cause an injury similar to that under investigation. The biological injury, whether damage to the epidermis and dermis or creation of an area of dehydration on the eye surface, recreated the natural pathophysiological environment, and the in vitro model offers a faithful reflection. *"Efficacy of a New Ocular Surface Modulator in Restoring* 

It enables us to approach the real target and model, human beings (relevance), and to determine and distinguish between differences in the response (reliability) and to reproduce the response in vivo (predictivity). The model is reproducible (the tests can be repeated with similar results) and the biological response of the trial products can be confirmed at different

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 85

For researchers working with skin lesions every day, the in vitro experimental model makes it possible to develop a clinical intuition and investigate the hypothesized role of a therapeutic substances or agents, while the use of data from in vitro models and the testing of potential medications enables a decision on whether or not to develop them and use them in subse-

As noted above, the repair and regeneration process consists of three phases (modulation of the inflammatory process, modulation of cell proliferation, and modulation of extracellular matrix remodeling), and regenerative medicine must be based on cell therapies, engineered tissues and biological products formulated by clinical research and able to mimic and repro-

Before entering into the merit of the in vitro model, it is worth asking what diagnostic factors are predictive of an evolution toward healing. Clinical factors are the first to be considered: underlying conditions, comorbidities, nutritional status, and medical treatments. In relation to humoral factors, can proinflammatory cytokines, MMPs, growth factors and macrophages be measured or tested in a recalcitrant skin ulcer? Can they be made the diagnostic target of an inflammation that develops toward healing or toward chronicity? All these targets may be measurable and are – or could be – predictive, but we are still far from their use in clinical

In the author's opinion, it should not be forgotten that humans are variable individuals: we know more than ever how humans become ill, but we also know that the repair, regeneration

In vitro models of skin lesions enable us to establish the behavior of the innate immune system in the first, inflammatory phase, which lasts from 2 hours to 5 days. Cell migration and inter-cellular and cell–cell matrix adhesion have been observed (and are discussed below), and the following markers have been quantified: IL-1a, IL-8, TNF-alpha, IFN-A1, HSP-70 (Heat protein shock 70),

After 1 week, in both the experimental model and in vivo, the cell proliferation phase begins. This involves granulation of the newly formed tissue and modulation of the cell population (transition from M1 to M2, blockage of monocytes, increase in fibroblasts and deposition of type III and VII collagen), with a process defined as wound retraction and re-epithelialization. In this phase we can measure VGEF-C, CTNNB1 (gene expression of cadherin production),

The remodeling phase involves an increase in collagen deposition and in its tensile strength, with the substitution of collagen type III by type I; this should not necessarily be encouraged, as it leads to cicatricial retraction and repair rather than regeneration of the original tissue.

MAP3K8, NK, CD68, MMP-2, MMP-9, ADAM15, ADAMTS8, ITGA1, ITGB2, and RPSA.

quent in vivo studies to add substance and support to the clinical studies.

duce the natural repair and regeneration process.

practice to establish the most suitable treatment.

and healing process is highly individual.

FLNB, TMP2, BPI, FN1, and DECORIN.

times (reproducibility).

**Figure 1.** Highlights the switch between the wound repair and regeneration pathway and the chronic inflammation pathway that feeds the chronicity of the skin lesions (M. Meloni- Vitroscreen Milano Italy 2017).

*Epithelial Changes in an in Vitro Model of Dry Eye Syndrome"* Barabino and colleagues, Current Eye Research, in press [2].

The process of learning about the biological reality examined in vitro and the scientific observation necessary to recreate the metabolic, structural and ultrastructural conditions of a cell/tissue/ organ model make it possible to understand and reproduce the biological mechanism involved. Above all, however – and this is the revolutionary part for the translation of research to clinical practice – they make it possible to predict quantitatively the model's response, and hence the natural response of the disorder under study, following treatment with a given biological substance. This means understanding if that biological substance – that drug – actually works.

For skin lesions, the in vitro model has an absolute value, because animal models suffer from important biological interferences. For investigation of the mechanism of action, the in vitro method opens up:

"the marvelous opportunity to discover something different, something unknown before now, because using a biological model guarantees the predictive value of the generated data and permits us to measure what cannot or should not be measured in vivo.

*"Measure what can be measured, and make measurable what cannot be measured" (Galileo).*

"The in vitro model, therefore is not a test, but an experimental model, and in skin lesions it enables a better understanding of the mechanisms of action, the collection of quantitative information that could not be obtained in any other way, and the creation of a body of evidence, case by case." Marisa Meloni, CEO Vitroscreen, Milano, Italy.

It enables us to approach the real target and model, human beings (relevance), and to determine and distinguish between differences in the response (reliability) and to reproduce the response in vivo (predictivity). The model is reproducible (the tests can be repeated with similar results) and the biological response of the trial products can be confirmed at different times (reproducibility).

For researchers working with skin lesions every day, the in vitro experimental model makes it possible to develop a clinical intuition and investigate the hypothesized role of a therapeutic substances or agents, while the use of data from in vitro models and the testing of potential medications enables a decision on whether or not to develop them and use them in subsequent in vivo studies to add substance and support to the clinical studies.

As noted above, the repair and regeneration process consists of three phases (modulation of the inflammatory process, modulation of cell proliferation, and modulation of extracellular matrix remodeling), and regenerative medicine must be based on cell therapies, engineered tissues and biological products formulated by clinical research and able to mimic and reproduce the natural repair and regeneration process.

Before entering into the merit of the in vitro model, it is worth asking what diagnostic factors are predictive of an evolution toward healing. Clinical factors are the first to be considered: underlying conditions, comorbidities, nutritional status, and medical treatments. In relation to humoral factors, can proinflammatory cytokines, MMPs, growth factors and macrophages be measured or tested in a recalcitrant skin ulcer? Can they be made the diagnostic target of an inflammation that develops toward healing or toward chronicity? All these targets may be measurable and are – or could be – predictive, but we are still far from their use in clinical practice to establish the most suitable treatment.

*Epithelial Changes in an in Vitro Model of Dry Eye Syndrome"* Barabino and colleagues, Current

**Figure 1.** Highlights the switch between the wound repair and regeneration pathway and the chronic inflammation

pathway that feeds the chronicity of the skin lesions (M. Meloni- Vitroscreen Milano Italy 2017).

The process of learning about the biological reality examined in vitro and the scientific observation necessary to recreate the metabolic, structural and ultrastructural conditions of a cell/tissue/ organ model make it possible to understand and reproduce the biological mechanism involved. Above all, however – and this is the revolutionary part for the translation of research to clinical practice – they make it possible to predict quantitatively the model's response, and hence the natural response of the disorder under study, following treatment with a given biological substance. This means understanding if that biological substance – that drug – actually works. For skin lesions, the in vitro model has an absolute value, because animal models suffer from important biological interferences. For investigation of the mechanism of action, the in vitro

"the marvelous opportunity to discover something different, something unknown before now, because using a biological model guarantees the predictive value of the generated data

"The in vitro model, therefore is not a test, but an experimental model, and in skin lesions it enables a better understanding of the mechanisms of action, the collection of quantitative information that could not be obtained in any other way, and the creation of a body of evi-

and permits us to measure what cannot or should not be measured in vivo.

dence, case by case." Marisa Meloni, CEO Vitroscreen, Milano, Italy.

*"Measure what can be measured, and make measurable what cannot be measured" (Galileo).*

Eye Research, in press [2].

84 Wound Healing - Current Perspectives

method opens up:

In the author's opinion, it should not be forgotten that humans are variable individuals: we know more than ever how humans become ill, but we also know that the repair, regeneration and healing process is highly individual.

In vitro models of skin lesions enable us to establish the behavior of the innate immune system in the first, inflammatory phase, which lasts from 2 hours to 5 days. Cell migration and inter-cellular and cell–cell matrix adhesion have been observed (and are discussed below), and the following markers have been quantified: IL-1a, IL-8, TNF-alpha, IFN-A1, HSP-70 (Heat protein shock 70), MAP3K8, NK, CD68, MMP-2, MMP-9, ADAM15, ADAMTS8, ITGA1, ITGB2, and RPSA.

After 1 week, in both the experimental model and in vivo, the cell proliferation phase begins. This involves granulation of the newly formed tissue and modulation of the cell population (transition from M1 to M2, blockage of monocytes, increase in fibroblasts and deposition of type III and VII collagen), with a process defined as wound retraction and re-epithelialization. In this phase we can measure VGEF-C, CTNNB1 (gene expression of cadherin production), FLNB, TMP2, BPI, FN1, and DECORIN.

The remodeling phase involves an increase in collagen deposition and in its tensile strength, with the substitution of collagen type III by type I; this should not necessarily be encouraged, as it leads to cicatricial retraction and repair rather than regeneration of the original tissue.

We can thus determine the presence and role of IL-1a, IL-8, HPS70, NK and BPI in the injured tissue in the inflammatory phase, of IL1a-IL-8, TNF-alpha, MMP2, MMP9, ADAM15 and ADAMTS8 in the re-epithelialization phase, of ITGA1 and ITGbeta2 in the cell migration and adhesion phase, and of VEGF-C, CTNNB1, DNAI1, FLNB, and TPM2 in the subsequent phase, in which remodeling begins.

causes the production of hypertrophic scars or keloids. New scientific knowledge in the fields of molecular and cellular mechanisms allows us to hypothesize other healing tissue process

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 87

Chronic non-responding skin lesions are incurred by a defect in ECM remodeling (third stage of the regeneration process). Abnormal collagen deposition blocks the action of fibroblasts and re-epithelialization is halted, resulting in an inflammatory process that becomes chronic due to the humoral (cytokines) and cellular components present that prevent the lesion from healing. The proinflammatory cytokines produced by the cell populations involved in wound healing trigger, promote and regulate the process by stimulating these cells (macrophages) to act. Any disruption of this combined and synergistic action between cytokines and M1 and M2 macrophages lays the basis for non-healing. In fact, M1 macrophages secrete IL1, IL6, IL12, TNF-alpha and MMPs, which in turn stimulate, amplify and regulate the proinflammatory phase preparatory to the next phase, where the switch to M2

In the chronic lesion, proteases alter the granulation tissue, stopping cell migration for the purpose of scarring. Histologic data demonstrating the altered regulation of synthesis and

Under the microscope, the endothelium-ECM-cell complex resembles a dynamic world in continuous movement, with genetically encoded interactions and biological pathways aimed at recovering the normal physiology of the damaged tissue (ES Aragona). This is the basis for

Attention is given to the role of MMPs, the enzymes that degrade the extracellular matrix, and to their balancing by their inhibitors (TIMPs) and cytokines, especially in local arterial and venous diseases. The cytokine TNF-alpha and gelatinase MMP-9, which are significantly over-expressed in both endothelial inflammation conditions and damaged venous vascular

Endothelial cells play an important role in early wound repair, thanks to their ability to stimulate the inflammatory process. They produce large amounts of TNF-alpha, which can be quantified through intracellular mobility receptors for hyaluronic acid (ICAM). At the same time, endothelial function restoration begins with the restoration of endothelial glycosaminoglycans linked to the reduction of MMP9 and the block of MMPs through the action of natural or organic derivatives of the hydrodynamic substance hyaluronic acid. This increases the water level, thus enabling the zinc at the core of the MMPs to be blocked, triggering phase

TNF-alpha is an important mediator during the inflammatory phase and, with TGF-beta, activates the expression of MMP9. In in-vitro cell cultures, TNF-alpha is over-produced in the first 24 hours, and the proinflammatory function is preparatory to the repair process. TNFalpha inhibits the collagen-alpha-1 gene in fibroblasts, stimulates the fibroblasts to produce

management procedures and apply new therapeutic models.

produces TGF-beta and IL10.

collagen synthesis is salient.

walls, are investigated in particular.

repair and regeneration.

**3.2. Scientific background of the operating rationale**

the work method studied and applied in our Centre.

collagen and promotes angiogenesis. (**Figure 2**).

The biological processes involved in the various phases of cicatrization are complex and often concomitant. The inflammatory phase, the keratinocyte migration phase, the proliferation phase, the formation of new tissue, and remodeling are all associated with a number of morphological and biochemical changes that can be quantified in the various skin layers through the choice of relevant markers.

The observation of these pathophysiological phenomena and processes in nature leads us to hypothesize new therapeutic procedures. The ability to detect and quantify the biological agents involved in the process, and to study their behavior, could enable the development of new therapeutic strategies.

Innovation means searching and researching.

Researching means having intuitions that can be tested.

Testing means using scientific processes that can validate the research.

Skin lesions, precisely because they are an expression of a systemic pathological status, lead us to use the experimental model to validate future therapies based on awareness of the fine molecular and cellular mechanisms. Understanding the phenomena inherent to the acute and chronic inflammatory process as a defense mechanism activated by the body leads to an understanding of how people can become ill and recover. Reproducing these phenomena in vitro and validating them and comparing them with in vivo data enables the evaluation of any identified therapeutic agent and its possible use in clinical practice.

It can today be asserted that the skin is a sophisticated immune surveillance system acting through its network of epithelial cells, lymphocytes and AgP cells as well as its resident microbiota, the alteration of which can trigger serious consequences. Future experimental models will be characterized by studies in this sense, with great benefits for medical research, pharmaceuticals and cosmetics.

### **3. The inflammatory process in healing tissue in vivo. Molecular and cellular components involved**

### **3.1. Changes in the healing process**

Wound repair involves the partial regeneration of the original tissue, with hyperproduction of organized collagen, which can lead to the production of new tissue with an 80% similarity to the original tissue. Abnormal host management involves a fibroproliferative response that causes the production of hypertrophic scars or keloids. New scientific knowledge in the fields of molecular and cellular mechanisms allows us to hypothesize other healing tissue process management procedures and apply new therapeutic models.

Chronic non-responding skin lesions are incurred by a defect in ECM remodeling (third stage of the regeneration process). Abnormal collagen deposition blocks the action of fibroblasts and re-epithelialization is halted, resulting in an inflammatory process that becomes chronic due to the humoral (cytokines) and cellular components present that prevent the lesion from healing. The proinflammatory cytokines produced by the cell populations involved in wound healing trigger, promote and regulate the process by stimulating these cells (macrophages) to act. Any disruption of this combined and synergistic action between cytokines and M1 and M2 macrophages lays the basis for non-healing. In fact, M1 macrophages secrete IL1, IL6, IL12, TNF-alpha and MMPs, which in turn stimulate, amplify and regulate the proinflammatory phase preparatory to the next phase, where the switch to M2 produces TGF-beta and IL10.

In the chronic lesion, proteases alter the granulation tissue, stopping cell migration for the purpose of scarring. Histologic data demonstrating the altered regulation of synthesis and collagen synthesis is salient.

### **3.2. Scientific background of the operating rationale**

We can thus determine the presence and role of IL-1a, IL-8, HPS70, NK and BPI in the injured tissue in the inflammatory phase, of IL1a-IL-8, TNF-alpha, MMP2, MMP9, ADAM15 and ADAMTS8 in the re-epithelialization phase, of ITGA1 and ITGbeta2 in the cell migration and adhesion phase, and of VEGF-C, CTNNB1, DNAI1, FLNB, and TPM2 in the subsequent

The biological processes involved in the various phases of cicatrization are complex and often concomitant. The inflammatory phase, the keratinocyte migration phase, the proliferation phase, the formation of new tissue, and remodeling are all associated with a number of morphological and biochemical changes that can be quantified in the various skin layers through

The observation of these pathophysiological phenomena and processes in nature leads us to hypothesize new therapeutic procedures. The ability to detect and quantify the biological agents involved in the process, and to study their behavior, could enable the development of

Skin lesions, precisely because they are an expression of a systemic pathological status, lead us to use the experimental model to validate future therapies based on awareness of the fine molecular and cellular mechanisms. Understanding the phenomena inherent to the acute and chronic inflammatory process as a defense mechanism activated by the body leads to an understanding of how people can become ill and recover. Reproducing these phenomena in vitro and validating them and comparing them with in vivo data enables the evaluation of any identified therapeutic agent and its possible use in clinical

It can today be asserted that the skin is a sophisticated immune surveillance system acting through its network of epithelial cells, lymphocytes and AgP cells as well as its resident microbiota, the alteration of which can trigger serious consequences. Future experimental models will be characterized by studies in this sense, with great benefits for medical research,

Wound repair involves the partial regeneration of the original tissue, with hyperproduction of organized collagen, which can lead to the production of new tissue with an 80% similarity to the original tissue. Abnormal host management involves a fibroproliferative response that

**3. The inflammatory process in healing tissue in vivo. Molecular** 

phase, in which remodeling begins.

86 Wound Healing - Current Perspectives

the choice of relevant markers.

new therapeutic strategies.

pharmaceuticals and cosmetics.

**and cellular components involved**

**3.1. Changes in the healing process**

practice.

Innovation means searching and researching.

Researching means having intuitions that can be tested.

Testing means using scientific processes that can validate the research.

Under the microscope, the endothelium-ECM-cell complex resembles a dynamic world in continuous movement, with genetically encoded interactions and biological pathways aimed at recovering the normal physiology of the damaged tissue (ES Aragona). This is the basis for the work method studied and applied in our Centre.

Attention is given to the role of MMPs, the enzymes that degrade the extracellular matrix, and to their balancing by their inhibitors (TIMPs) and cytokines, especially in local arterial and venous diseases. The cytokine TNF-alpha and gelatinase MMP-9, which are significantly over-expressed in both endothelial inflammation conditions and damaged venous vascular walls, are investigated in particular.

Endothelial cells play an important role in early wound repair, thanks to their ability to stimulate the inflammatory process. They produce large amounts of TNF-alpha, which can be quantified through intracellular mobility receptors for hyaluronic acid (ICAM). At the same time, endothelial function restoration begins with the restoration of endothelial glycosaminoglycans linked to the reduction of MMP9 and the block of MMPs through the action of natural or organic derivatives of the hydrodynamic substance hyaluronic acid. This increases the water level, thus enabling the zinc at the core of the MMPs to be blocked, triggering phase repair and regeneration.

TNF-alpha is an important mediator during the inflammatory phase and, with TGF-beta, activates the expression of MMP9. In in-vitro cell cultures, TNF-alpha is over-produced in the first 24 hours, and the proinflammatory function is preparatory to the repair process. TNFalpha inhibits the collagen-alpha-1 gene in fibroblasts, stimulates the fibroblasts to produce collagen and promotes angiogenesis. (**Figure 2**).

**Figure 2.** The phases of tissue regeneration and the involvement of cell types, micromolecules and extracellular matrix proteins.

**Figure 3.** Cytokines pathways in M1-like and M2-like phenotypes.

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 89

**Figure 4.** The type of macrophages, their differentiation and the role in tissue repair.

The reduction in M1 macrophages and hence in TNF-alpha, associated with the block of monocytes and the increased production of fibroblasts and hence of collagen, creates the foundation for the M1-M2 switch and the start of the repair and regeneration process in an ever-dynamic and constantly changing cytokine and cellular pool (ES Aragona).

### **4. Macrophage regulation in wound healing**

The role of M1 macrophages in the inflammatory process and of M2 macrophages in the repair and regeneration process is still debated, especially in the event of a disrupted M1/M2 ratio, which produces various pathological effects related to the chronification of inflammation and associated disorders. Naturally, in this case our focus is on non-healing.

It is worth repeating that M1 macrophages are proinflammatory cells to all effects, producing proinflammatory cytokines (IL1, IL6, IL12, TNF-a and oxidative metabolites (NO and SAD) (3, 4) which are involved in defense of the host and in the debridement process. (**Figures 3 and 4**).

The M2 population is stimulated above all by the drop in M1, IL4 and IL13; this is the key for the remodeling process, which follows or accompanies the switching-off and termination of the inflammatory process.

**Figure 3.** Cytokines pathways in M1-like and M2-like phenotypes.

The reduction in M1 macrophages and hence in TNF-alpha, associated with the block of monocytes and the increased production of fibroblasts and hence of collagen, creates the foundation for the M1-M2 switch and the start of the repair and regeneration process in an

**Figure 2.** The phases of tissue regeneration and the involvement of cell types, micromolecules and extracellular matrix

The role of M1 macrophages in the inflammatory process and of M2 macrophages in the repair and regeneration process is still debated, especially in the event of a disrupted M1/M2 ratio, which produces various pathological effects related to the chronification of inflamma-

It is worth repeating that M1 macrophages are proinflammatory cells to all effects, producing proinflammatory cytokines (IL1, IL6, IL12, TNF-a and oxidative metabolites (NO and SAD) (3, 4) which are involved in defense of the host and in the debridement process.

The M2 population is stimulated above all by the drop in M1, IL4 and IL13; this is the key for the remodeling process, which follows or accompanies the switching-off and termination of the

ever-dynamic and constantly changing cytokine and cellular pool (ES Aragona).

tion and associated disorders. Naturally, in this case our focus is on non-healing.

**4. Macrophage regulation in wound healing**

(**Figures 3 and 4**).

proteins.

88 Wound Healing - Current Perspectives

inflammatory process.

**Figure 4.** The type of macrophages, their differentiation and the role in tissue repair.

One of the keys for interpreting M1/M2 switching is the genotyping and receptor typing of the two populations and how they react to both chemical and energy stimuli, a concept we will return to below. The M2 population is divided into three subclasses, M2a, M2b and M2c. M2a is stimulated by the cytokines Il4/IL13 and IL4Ra. Leibovich and colleagues [3] also characterized a fourth group, M2d. This is involved in the diminishment of inflammation and the upregulation of IL10 and VEGF, like subtype M2a, which is associated with low levels of TNFa and IL12.

The RMC's data on the etiological causes of skin lesions confirm the etiology of such a lesion, as reported in the studies provided by the various centers involved. The success of the guidelines and protocols will only be maintained if we carry on managing the inflammatory pro-

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 91

The chronic skin wound outpatient clinic involves experts in various disciplines for the treat-

Current knowledge of gross and ultrastructural skin anatomy enables the stages of the regeneration process to be followed and highlights the importance of the extracellular matrix. This

**Picture 1.** Case report. A 82-year-old man patient with cerebral and peripheral and diabetic circulatory pluripatologies. Appearance of internal perimalleolar lesion right with clear infection and with positivity to E. Coli and Pseudomonas

cess with a view to regeneration (**Picture 1**).

Autoimmune and rheumatic lesions; (**Picture 5**)

Vascular lesions; (**Picture 2**) Pressure lesions; (**Picture 3**)

Burns and scars. (**Picture 7**)

healing process in 3 months.

Diabetic foot lesions; (**Picture 4**)

Post-surgical skin lesions; (**Picture 6**)

ment of:

M1/M2 polarization seems to depend on two transcription regulators, the interferon regulatory factors IRF5 and IRF4. A correlation has been demonstrated between IFR5, high levels of M1 and inflammation and between IRF4 and M2, both linked to specific gene expressions and modulated by various substances. The latter include adenosine, which modifies the membrane's response to the M1-M2 switch, hence modifying the intracellular and ECM information signal [3].

## **5. A new approach to the treatment of skin lesions with regenerative medicine**

### **5.1. The anti-inflammatory regenerative medicine (AIMED) protocol: the importance of inflammatory process modulation in triggering the tissue regeneration phase**

The care and treatment of non-healing wounds is a major challenge for specialized centers. These wounds have a significant impact on health expenditure and a profound effect on the wellbeing of both patients and their families.

The skin is an important barrier. It protects us from numerous agents that would otherwise cause more frequent and more severe damage to our bodies. The skin system is a defense mechanism that aims to maintain a balance and involves various molecular, cellular, immune, endocrine, and neurological mechanisms. The understanding of these mechanisms has led to the development of numerous new drugs and medical devices for skin diseases.

Numerous authors have investigated the phases of wound repair processes and regeneration. The perfect picture is that of a complex system of humoral, cellular, molecular and ultrastructural regulation, described as a cohesive orchestra. This is the basis of the regeneration process, but it often stalls and impairs healing.

The Centre for Regenerative Medicine (now RMC) was set up by the Istituto Clinico Humanitas Mater Domini in July 2015 to focus on chronic wounds and high morbidity (infections, pain, complications), given their social implications. It has created a working group, now promoted by the Multidisciplinary Association for Wellbeing and Regeneration (AMbeR), involving numerous professionals working on the treatment and care of people with chronic skin and mucous membrane diseases. The team's research was the first step in collaboration and cooperation with other specialized centers, universities and organizations throughout Italy. Its attention is focused on the most important and decisive area of skin lesion management: modulation of the inflammatory process.

The RMC's data on the etiological causes of skin lesions confirm the etiology of such a lesion, as reported in the studies provided by the various centers involved. The success of the guidelines and protocols will only be maintained if we carry on managing the inflammatory process with a view to regeneration (**Picture 1**).

The chronic skin wound outpatient clinic involves experts in various disciplines for the treatment of:

Vascular lesions; (**Picture 2**)

One of the keys for interpreting M1/M2 switching is the genotyping and receptor typing of the two populations and how they react to both chemical and energy stimuli, a concept we will return to below. The M2 population is divided into three subclasses, M2a, M2b and M2c. M2a is stimulated by the cytokines Il4/IL13 and IL4Ra. Leibovich and colleagues [3] also characterized a fourth group, M2d. This is involved in the diminishment of inflammation and the upregulation of IL10 and VEGF, like subtype M2a, which is associated with low levels of

M1/M2 polarization seems to depend on two transcription regulators, the interferon regulatory factors IRF5 and IRF4. A correlation has been demonstrated between IFR5, high levels of M1 and inflammation and between IRF4 and M2, both linked to specific gene expressions and modulated by various substances. The latter include adenosine, which modifies the membrane's response to the M1-M2 switch, hence modifying the intracellular and ECM informa-

**5. A new approach to the treatment of skin lesions with regenerative** 

**5.1. The anti-inflammatory regenerative medicine (AIMED) protocol: the importance of inflammatory process modulation in triggering the tissue regeneration phase**

The care and treatment of non-healing wounds is a major challenge for specialized centers. These wounds have a significant impact on health expenditure and a profound effect on the

The skin is an important barrier. It protects us from numerous agents that would otherwise cause more frequent and more severe damage to our bodies. The skin system is a defense mechanism that aims to maintain a balance and involves various molecular, cellular, immune, endocrine, and neurological mechanisms. The understanding of these mechanisms has led to

Numerous authors have investigated the phases of wound repair processes and regeneration. The perfect picture is that of a complex system of humoral, cellular, molecular and ultrastructural regulation, described as a cohesive orchestra. This is the basis of the regeneration

The Centre for Regenerative Medicine (now RMC) was set up by the Istituto Clinico Humanitas Mater Domini in July 2015 to focus on chronic wounds and high morbidity (infections, pain, complications), given their social implications. It has created a working group, now promoted by the Multidisciplinary Association for Wellbeing and Regeneration (AMbeR), involving numerous professionals working on the treatment and care of people with chronic skin and mucous membrane diseases. The team's research was the first step in collaboration and cooperation with other specialized centers, universities and organizations throughout Italy. Its attention is focused on the most important and decisive area of skin lesion management:

the development of numerous new drugs and medical devices for skin diseases.

TNFa and IL12.

90 Wound Healing - Current Perspectives

tion signal [3].

**medicine**

wellbeing of both patients and their families.

process, but it often stalls and impairs healing.

modulation of the inflammatory process.

Pressure lesions; (**Picture 3**)

Diabetic foot lesions; (**Picture 4**)

Autoimmune and rheumatic lesions; (**Picture 5**)

Post-surgical skin lesions; (**Picture 6**)

Burns and scars. (**Picture 7**)

Current knowledge of gross and ultrastructural skin anatomy enables the stages of the regeneration process to be followed and highlights the importance of the extracellular matrix. This

**Picture 1.** Case report. A 82-year-old man patient with cerebral and peripheral and diabetic circulatory pluripatologies. Appearance of internal perimalleolar lesion right with clear infection and with positivity to E. Coli and Pseudomonas healing process in 3 months.

**Picture 2.** Case report. A 71-year-old man patient with vascular lesion present for 6 years and resistant to therapies. Healing process in 5 months.

**Picture 3.** Case report. A 69-year-old patient with IV stade decubitus injury. Healing process in 35 days.

has the appearance of a semi-fluid gel, and contains enzymes, hormones and vitamins and a dense network of macromolecular complexes (GAGs, proteoglycans and glycoproteins). The cells are immersed in this active substance, whose electromagnetic properties are fundamental for the life of the cells themselves as well as the water in the human body. The coherence of the electromagnetic diffusion is essential for correct intercell and cell-ECM harmony.

ensuring the balance of all the components involved in the life cycle of the regenerative phase. The regeneration of skin that has been damaged by multiple etiological factors is made possible by its ability to interact with the outside, and especially the essence of the tissue, that is constantly renewed and capable of repairing and reacting to lesions due to the presence of epidermal stem cells in the dermis and epidermis. In patients with recalcitrant skin lesions, the presence of comorbidities such as chronic disease, diabetes, vascular insufficiency, peripheral edema secondary to heart failure, malnutrition, bedsores, and infections can affect the body's ability to respond to treatment, but can also have a negative effect on the inflammatory modulation process itself, triggering a chronic phase that feeds the non-

**Picture 5.** Case report. A 78- year-old patient with arthritis, left hand with inflammationand abscess. Healing process

**Picture 4.** Case report. A 63-year-old patient with infected diabetic food. . Healing process in 2 months.

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 93

healing of the lesions.

in 3 months.

The other aspect highlighted by research into the modulation of the inflammatory process is that the ECM determines the process of differentiation, proliferation and cell migration,

**Picture 4.** Case report. A 63-year-old patient with infected diabetic food. . Healing process in 2 months.

**Picture 5.** Case report. A 78- year-old patient with arthritis, left hand with inflammationand abscess. Healing process in 3 months.

**Picture 3.** Case report. A 69-year-old patient with IV stade decubitus injury. Healing process in 35 days.

the electromagnetic diffusion is essential for correct intercell and cell-ECM harmony.

has the appearance of a semi-fluid gel, and contains enzymes, hormones and vitamins and a dense network of macromolecular complexes (GAGs, proteoglycans and glycoproteins). The cells are immersed in this active substance, whose electromagnetic properties are fundamental for the life of the cells themselves as well as the water in the human body. The coherence of

**Picture 2.** Case report. A 71-year-old man patient with vascular lesion present for 6 years and resistant to therapies.

Healing process in 5 months.

92 Wound Healing - Current Perspectives

The other aspect highlighted by research into the modulation of the inflammatory process is that the ECM determines the process of differentiation, proliferation and cell migration, ensuring the balance of all the components involved in the life cycle of the regenerative phase. The regeneration of skin that has been damaged by multiple etiological factors is made possible by its ability to interact with the outside, and especially the essence of the tissue, that is constantly renewed and capable of repairing and reacting to lesions due to the presence of epidermal stem cells in the dermis and epidermis. In patients with recalcitrant skin lesions, the presence of comorbidities such as chronic disease, diabetes, vascular insufficiency, peripheral edema secondary to heart failure, malnutrition, bedsores, and infections can affect the body's ability to respond to treatment, but can also have a negative effect on the inflammatory modulation process itself, triggering a chronic phase that feeds the nonhealing of the lesions.

**5.2. Personal experience; materials and methods**

**5.3. The foundation of the RMC**

patients and their families.

hospital research institutes with which they work.

for the transferral of the results of this research to clinical practice.

The regenerative medicine outpatient clinic (RMC) for the treatment of recalcitrant lesions was established in Castellanza in the summer of 2015. In 2017–2018, 869 patients (286 men and 583 women) were treated at the RMC, for a total of 1718 treatments. Even before applying national and international guidelines on the management of acute and chronic wounds the main activity was the formation of a multidisciplinary team sharing the same philosophy of care: to put patients, their inner world and their families at the heart of the process. The clinical research already practiced by some members of the RMC team was the driving force for the development of hospital treatment and home care models as a therapeutic continuum and

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 95

*"There are skills and abilities, and then there's a subtle strength that the patients transmit to you, to say that everything we are doing has given them a better quality of life (Giada Mereghetti, RMC Coordinator).* The Regenerative Medicine Centre (RMC) was set up in June 2015 in Castellanza, a town in the province of Varese in Lombardy, near the border with the province of Milan, on the basis of the skills and motivation of a group of professionals with a common objective. The RMC forms part of a much larger project, which aims to create a close-knit network of professionals to act as national and international spokespersons for a new way of looking at skin wounds. This ambitious project initially brought together professionals with different skills but who were united by a single mission: to confront and photograph the world of difficult wounds, broadening the objective beyond the usual common goal of mere treatment, and involving

Motivation is the main characteristic common to all project members: the same members who (initially only through ideological discussions) later actively contributed to the RMC's construction, putting an ideal and an objective into practice. Each of the individual professionals, with their important personal experience of the medical world and with years upon years of study in their various scientific disciplines, decided to contribute their knowledge, experience and abilities to the construction of a new organizational model and the creation of a closeknit network of activities, which they hope will spread through the entire country. Each of the members has embraced the philosophy of caregiving, associating respect for the clinical priorities of the patients and their families with the use of national and international methods and guidelines for clinical research and the continuous evolution of the field of wound management. From this perspective, all the professionals making up the RMC outpatient team, with their individual duties and respect for the shared guidelines and protocols, have opened up their perspective of wounds in relation to constant interaction with the university and

The RMC is close to achieving its main research objective in relation to the treatment of wounds: namely, gaining knowledge of the inflammatory process resolution mechanisms in tissue repair and regeneration. Its ambition is an organizational model which takes its professionals outside the hospital walls to enable their scientific and human knowledge to be transferred to and shared with the key players in this project: the patients and their families.

**Picture 6.** Case report. A 57-old-patient with dehiscence of the surgical wound. Application of NPWT therapy and Healing process in 2 months.

**Picture 7.** Case report. A 24 old-patient with II and III degree burns on the face and upper limb.

These stages are always present in all types of lesions and physiological process phases, and their timescales, interaction, genetic, humoral, cellular, and ultrastructural mechanisms (which are the basis for the regeneration of injured tissues, in both in vitro models and live models) are all understood. In this article, the authors underline some aspects of the repair and regeneration process in relation to the rationale of the proposed AIMED Model and cohesion between clinical research activities and their translation to methodology. Key roles are played by fibrin deposition and hydration of the matrix by hyaluronic acid, stimulating the production of fibroblasts, while other cell types (granulocytes, monocytes, M1 macrophages and cytokines) play a role in the shutdown of the inflammatory proliferative phase (endothelial cells, fibroblasts and keratinocytes) and the ECM remodeling phase.

### **5.2. Personal experience; materials and methods**

The regenerative medicine outpatient clinic (RMC) for the treatment of recalcitrant lesions was established in Castellanza in the summer of 2015. In 2017–2018, 869 patients (286 men and 583 women) were treated at the RMC, for a total of 1718 treatments. Even before applying national and international guidelines on the management of acute and chronic wounds the main activity was the formation of a multidisciplinary team sharing the same philosophy of care: to put patients, their inner world and their families at the heart of the process. The clinical research already practiced by some members of the RMC team was the driving force for the development of hospital treatment and home care models as a therapeutic continuum and for the transferral of the results of this research to clinical practice.

#### **5.3. The foundation of the RMC**

**Picture 7.** Case report. A 24 old-patient with II and III degree burns on the face and upper limb.

Healing process in 2 months.

94 Wound Healing - Current Perspectives

lial cells, fibroblasts and keratinocytes) and the ECM remodeling phase.

These stages are always present in all types of lesions and physiological process phases, and their timescales, interaction, genetic, humoral, cellular, and ultrastructural mechanisms (which are the basis for the regeneration of injured tissues, in both in vitro models and live models) are all understood. In this article, the authors underline some aspects of the repair and regeneration process in relation to the rationale of the proposed AIMED Model and cohesion between clinical research activities and their translation to methodology. Key roles are played by fibrin deposition and hydration of the matrix by hyaluronic acid, stimulating the production of fibroblasts, while other cell types (granulocytes, monocytes, M1 macrophages and cytokines) play a role in the shutdown of the inflammatory proliferative phase (endothe-

**Picture 6.** Case report. A 57-old-patient with dehiscence of the surgical wound. Application of NPWT therapy and

*"There are skills and abilities, and then there's a subtle strength that the patients transmit to you, to say that everything we are doing has given them a better quality of life (Giada Mereghetti, RMC Coordinator).*

The Regenerative Medicine Centre (RMC) was set up in June 2015 in Castellanza, a town in the province of Varese in Lombardy, near the border with the province of Milan, on the basis of the skills and motivation of a group of professionals with a common objective. The RMC forms part of a much larger project, which aims to create a close-knit network of professionals to act as national and international spokespersons for a new way of looking at skin wounds. This ambitious project initially brought together professionals with different skills but who were united by a single mission: to confront and photograph the world of difficult wounds, broadening the objective beyond the usual common goal of mere treatment, and involving patients and their families.

Motivation is the main characteristic common to all project members: the same members who (initially only through ideological discussions) later actively contributed to the RMC's construction, putting an ideal and an objective into practice. Each of the individual professionals, with their important personal experience of the medical world and with years upon years of study in their various scientific disciplines, decided to contribute their knowledge, experience and abilities to the construction of a new organizational model and the creation of a closeknit network of activities, which they hope will spread through the entire country. Each of the members has embraced the philosophy of caregiving, associating respect for the clinical priorities of the patients and their families with the use of national and international methods and guidelines for clinical research and the continuous evolution of the field of wound management. From this perspective, all the professionals making up the RMC outpatient team, with their individual duties and respect for the shared guidelines and protocols, have opened up their perspective of wounds in relation to constant interaction with the university and hospital research institutes with which they work.

The RMC is close to achieving its main research objective in relation to the treatment of wounds: namely, gaining knowledge of the inflammatory process resolution mechanisms in tissue repair and regeneration. Its ambition is an organizational model which takes its professionals outside the hospital walls to enable their scientific and human knowledge to be transferred to and shared with the key players in this project: the patients and their families.

### **5.4. Main objectives of the RMC**

• To respect the mission of managing patients with any acute or chronic lesion affecting the skin or mucous membranes of any etiology (vascular, diabetic, rheumatological, traumatic) while respecting their humanness as a whole.

• Prevention and management of complications;

struction and/or skin grafts;

Emergency Department);

professionals;

• Health education.

(E.S. Aragona – 2017).

residential care homes (RSA));

• Promotion of community outreach initiatives;

bactericide and stimulus for tissue regeneration;

• Ultrasound treatment of skin lesions to remove necrotic and fibrinous tissue, acting as a

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 97

• Surgical procedures for biopsies, surgical debridement, and removal of lesions with recon-

• Creation of protocols on the basis of technological innovations and monitoring of markers; • Consultancy and cooperation with all in-house services (Inpatient wards and Accident &

• Consultancy and cooperation with community services (GPs, integrated home care (ADI),

• Cooperation with the training department to create a network of consistent, competent

The Castellanza RMC has a 360° structure, in which the specialists taking charge of care injuries collaborate in important phases of patient assessment and management of both the injuries and the patients as a whole. After this phase of diagnostic classification, the patients are provided with multilevel treatment that applies all the steps detailed in international guidelines,

**Figure 5.** AIMED - Anti Inflammaging regenerative medicine: operating protocol for chronic wound management

• Documentation of the assessments and procedures through shared records;


#### **5.5. Clinical care journey at the RMC**

The patient journey in the RMC thus requires careful management involving numerous professionals with distinct areas and responsibilities. Patients go through a well-defined process that should give them a sense of competence, humanity and harmony, making them feel an active part of the group alongside the medical workers treating them. These workers act through the application of protocols and guidelines detailing the treatment and use of the main advanced dressings in relation to the different wound types, which are drawn up on the basis of the main clinical studies found in the literature and others, published more recently, followed directly by the RMC. The RMC makes use of both its own professionals and those employed by the institutes in which it works, as well as of partners and disciplines whose goal is to channel their energies toward the better treatment of patients with skin, cartilage and mucous membrane lesions.

#### **5.6. Care and assistance in the RMC**

Given the main objective and mission of the RMC, which can be summarized as the management of patients with any acute or chronic lesion affecting the skin or mucous membranes and who have non-healing ulcers or wounds of different etiologies (vascular, diabetic, rheumatological, traumatic), the RMC's activities can be classified in the following areas:


**5.4. Main objectives of the RMC**

96 Wound Healing - Current Perspectives

complementary treatments.

**5.5. Clinical care journey at the RMC**

**5.6. Care and assistance in the RMC**

• Assessment of the lesion;

• Overall assessment of the patient;

• Application of validated, shared protocols;

while respecting their humanness as a whole.

• To respect the mission of managing patients with any acute or chronic lesion affecting the skin or mucous membranes of any etiology (vascular, diabetic, rheumatological, traumatic)

• To standardize the mechanisms for stimulating regenerative and reparative biochemical and cell processes leading to the healing of lesions, making use of the abilities of the team's

• To create new therapeutic models at the base of regenerative medicine to exploit synthetic biological molecules, engineered tissues and cell therapies that could reproduce the body's

• To remain part of a much wider project that focuses attention on skin, cartilage and mucous membrane lesions and places patients at the center of an innovative, barrier-free patient journey. • To take care of the patient from diagnosis through treatment and follow-up, providing services as needed on the basis of clinical indications and integrating them with innovative

The patient journey in the RMC thus requires careful management involving numerous professionals with distinct areas and responsibilities. Patients go through a well-defined process that should give them a sense of competence, humanity and harmony, making them feel an active part of the group alongside the medical workers treating them. These workers act through the application of protocols and guidelines detailing the treatment and use of the main advanced dressings in relation to the different wound types, which are drawn up on the basis of the main clinical studies found in the literature and others, published more recently, followed directly by the RMC. The RMC makes use of both its own professionals and those employed by the institutes in which it works, as well as of partners and disciplines whose goal is to channel their energies toward the better treatment of patients with skin, cartilage and mucous membrane lesions.

Given the main objective and mission of the RMC, which can be summarized as the management of patients with any acute or chronic lesion affecting the skin or mucous membranes and who have non-healing ulcers or wounds of different etiologies (vascular, diabetic, rheumato-

logical, traumatic), the RMC's activities can be classified in the following areas:

• Management and removal of the cause leading to the formation of the lesion;

clinicians alongside the public and private institutes with which RMC works.

own wound repair mechanisms in everyday clinical practice.

The Castellanza RMC has a 360° structure, in which the specialists taking charge of care injuries collaborate in important phases of patient assessment and management of both the injuries and the patients as a whole. After this phase of diagnostic classification, the patients are provided with multilevel treatment that applies all the steps detailed in international guidelines,

**Figure 5.** AIMED - Anti Inflammaging regenerative medicine: operating protocol for chronic wound management (E.S. Aragona – 2017).

but with an additional, innovative perspective that gives importance to anti-inflammatory and regenerative activity.

*the acute and chronic wounds with an alkaline pH have a lower rate of cure than the wounds with a pH close to neutral. The wound healing process slows down when the pH is high, under alkaline* 

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 99

Intact skin has a pH between 4.8 and 6, depending on the area in question. The pH of wounds cannot be easily measured, but literature data demonstrate that a wound pH of around 4 can

In *The effects of pH on wound healing, biofilms, and antimicrobial efficacy* published in Wound Repair Regen. 2014 (March) [5], Percival et al. attribute wound pH with an important role in the activity of MMPs, TIMPs and fibroblasts and in collagen production. pH also interferes with bacterial proliferation and the patient's immunological response, and its monitoring and control is one of the strategies used to trigger the healing process. In *The effect of pH on the Extracellular Matrix and Biofilms,* published in Adv. Wound Care, Jul 1,4 [6], Jones, Cochrane and Percival provide an overview of the role of pH and its effect on the ECM and biofilm in connection with wound healing. Chronic lesions have an alkaline pH, while the pH tends

The model involves the use of commercially available products chosen on the basis of their

The authors of the present article have started an observational study of a class III medical device following a study of the bacterial load of the lesion and of certain bacterial strains (*Staphylococcus aureus, Staph. epidermidis, Escherichia coli* and *Pseudomonas aeruginosa*) that associated a pH of 4.5–5.00 with the mechanical removal of bacteria and protease due to the presence in its composition of d-mannose, copper sulfate, zinc and other components that are

The topical treatment involves two phases. The first, enzymatic and mechanical debridement (the technique preferred by the authors), is the crucial moment in wound management, eliminating the mechanical and biochemical causes that can perpetuate the inflammatory process and preparing the wound for the action of biological and physical substances that can trigger a local anti-inflammatory action and catalyze the proliferation of cell populations, ensuring

The lesions are always treated with the local application of medical devices, biophysical

Even though it is described as part of step 2, systemic treatment is applied from the very start of the wound's management under a 360°, polyvalent protocol depending on the type and

properties, their contact time and the duration of their action on the wound.

*conditions. (Levine).*

trigger a more rapid wound healing process.

toward acidity during the healing process.

*Polyhexanide with betaine surfactant.*

part of the authors' know-how. STEP 2A—Topical Treatment

good hydration in the wound bed.

therapies and cell therapies.

severity of the lesion.

STEP 2B—Systemic Treatment

*Hypochlorous acid.*

The AIMED operating protocol provides local treatment of skin lesions and a general evaluation of the patient, with particular attention to the preliminary assessment of the causes of lesion). (**Figure 5)**. The model enables the dynamic partnership of all professionals working with RMC specialists to ensure a simple, interruption-free patient journey in the Institute. An example of this is the in-house cooperation with the Cardiology and Hemodynamics Dept. for vascular lesions of arterial origin, which are evaluated within a multidisciplinary team where, from their first visit to the clinic, patients are guided through a diagnostic angiography journey involving a vascular rehabilitation process. In these revascularized patients the care model also focuses on the risks of reperfusion and the production of a proinflammatory state with increased peripheral oxidation and potential necrosis of the tissues affected by the critical ischemia.

### **6. Description of the lesion model under the AIMED protocol**

The lesions are classified by type and stage in accordance with international guidelines, and the various types of advanced treatment are assessed and selected in relation to the type of lesion and the operating protocol, with attention to pharmacoeconomics.

Five basic operating protocol steps.

Step 1: Preparation of the lesion for treatment. This includes cleansing and combating infections using current methods [4].

Step 2: Topical and systemic treatment protocols to modulate the inflammatory phase and trigger the regenerative phase.

Step 3: Biophysical therapies to stimulate regeneration.

Step 4: Cell therapies.

Step 5: Surgical therapies.

These steps are discussed below in detail.

STEP 1—Wound Preparation

The first step is deep cleansing of the wound and modifying its pH. This important step requires careful management: cleaning the wound of nonviable tissue, fibrin, protease, bacteria and biofilm or cellular debris can eliminate potential causes of non-healing. This is followed by modification of the lesion's pH.

*The pH value within the microenvironment of the wound directly and indirectly influences all biochemical reactions that take place in the healing process. It has been shown that the pH of the surface of a wound plays an important role in wound healing and helps to control the infection and increase antimicrobial activity, the release of oxygen, angiogenesis, protease activity and bacterial toxicity. The pH value influences cellular events that regulate the healing process of wounds. It was observed that*  *the acute and chronic wounds with an alkaline pH have a lower rate of cure than the wounds with a pH close to neutral. The wound healing process slows down when the pH is high, under alkaline conditions. (Levine).*

Intact skin has a pH between 4.8 and 6, depending on the area in question. The pH of wounds cannot be easily measured, but literature data demonstrate that a wound pH of around 4 can trigger a more rapid wound healing process.

In *The effects of pH on wound healing, biofilms, and antimicrobial efficacy* published in Wound Repair Regen. 2014 (March) [5], Percival et al. attribute wound pH with an important role in the activity of MMPs, TIMPs and fibroblasts and in collagen production. pH also interferes with bacterial proliferation and the patient's immunological response, and its monitoring and control is one of the strategies used to trigger the healing process. In *The effect of pH on the Extracellular Matrix and Biofilms,* published in Adv. Wound Care, Jul 1,4 [6], Jones, Cochrane and Percival provide an overview of the role of pH and its effect on the ECM and biofilm in connection with wound healing. Chronic lesions have an alkaline pH, while the pH tends toward acidity during the healing process.

The model involves the use of commercially available products chosen on the basis of their properties, their contact time and the duration of their action on the wound.

*Polyhexanide with betaine surfactant.*

### *Hypochlorous acid.*

but with an additional, innovative perspective that gives importance to anti-inflammatory and

The AIMED operating protocol provides local treatment of skin lesions and a general evaluation of the patient, with particular attention to the preliminary assessment of the causes of lesion). (**Figure 5)**. The model enables the dynamic partnership of all professionals working with RMC specialists to ensure a simple, interruption-free patient journey in the Institute. An example of this is the in-house cooperation with the Cardiology and Hemodynamics Dept. for vascular lesions of arterial origin, which are evaluated within a multidisciplinary team where, from their first visit to the clinic, patients are guided through a diagnostic angiography journey involving a vascular rehabilitation process. In these revascularized patients the care model also focuses on the risks of reperfusion and the production of a proinflammatory state with increased periph-

The lesions are classified by type and stage in accordance with international guidelines, and the various types of advanced treatment are assessed and selected in relation to the type of

Step 1: Preparation of the lesion for treatment. This includes cleansing and combating infec-

Step 2: Topical and systemic treatment protocols to modulate the inflammatory phase and

The first step is deep cleansing of the wound and modifying its pH. This important step requires careful management: cleaning the wound of nonviable tissue, fibrin, protease, bacteria and biofilm or cellular debris can eliminate potential causes of non-healing. This is fol-

*The pH value within the microenvironment of the wound directly and indirectly influences all biochemical reactions that take place in the healing process. It has been shown that the pH of the surface of a wound plays an important role in wound healing and helps to control the infection and increase antimicrobial activity, the release of oxygen, angiogenesis, protease activity and bacterial toxicity. The pH value influences cellular events that regulate the healing process of wounds. It was observed that* 

eral oxidation and potential necrosis of the tissues affected by the critical ischemia.

**6. Description of the lesion model under the AIMED protocol**

lesion and the operating protocol, with attention to pharmacoeconomics.

regenerative activity.

98 Wound Healing - Current Perspectives

Five basic operating protocol steps.

tions using current methods [4].

trigger the regenerative phase.

Step 4: Cell therapies.

Step 5: Surgical therapies.

STEP 1—Wound Preparation

These steps are discussed below in detail.

lowed by modification of the lesion's pH.

Step 3: Biophysical therapies to stimulate regeneration.

The authors of the present article have started an observational study of a class III medical device following a study of the bacterial load of the lesion and of certain bacterial strains (*Staphylococcus aureus, Staph. epidermidis, Escherichia coli* and *Pseudomonas aeruginosa*) that associated a pH of 4.5–5.00 with the mechanical removal of bacteria and protease due to the presence in its composition of d-mannose, copper sulfate, zinc and other components that are part of the authors' know-how.

### STEP 2A—Topical Treatment

The topical treatment involves two phases. The first, enzymatic and mechanical debridement (the technique preferred by the authors), is the crucial moment in wound management, eliminating the mechanical and biochemical causes that can perpetuate the inflammatory process and preparing the wound for the action of biological and physical substances that can trigger a local anti-inflammatory action and catalyze the proliferation of cell populations, ensuring good hydration in the wound bed.

The lesions are always treated with the local application of medical devices, biophysical therapies and cell therapies.

#### STEP 2B—Systemic Treatment

Even though it is described as part of step 2, systemic treatment is applied from the very start of the wound's management under a 360°, polyvalent protocol depending on the type and severity of the lesion.

Administration of a dietary supplement containing a well-balanced mix of serratio-peptidase, escin, bromelain and selenium. This supplement has proteolytic, fibrinolytic, anti-edema and draining properties, as well as an antioxidant action.

It has been demonstrated that polarized light at 590 nm stimulates angiogenesis and growth factors, while at 830 nm it activates the cells involved in wound healing; a dose of 20 J/cm2 stimulates increased collagen deposition, an increase in myofibroblasts and a better ultra-

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 101

The Lumiheal protocol involves the use of broadband wavelengths (blue, green, yellow/ orange from 450 nm to 610 nm) from a light emitting diode to amplify the physical effect of stimulation of the regenerative processes in the injured area due to the emission of photons in the form of fluorescence. The LumiHeal Protocol has been applied over the last 3 months in 8 patients with complicated treatment-resistant infected wounds. The improvement in the

High intensity, variable frequency magnetic fields for outpatient and home treatment. As

• Are anti-inflammatory, through modulation of the profile of cytokines produced by proin-

• Are angiogenic, with increased proliferation of endothelial cells and FGF-2 (fibroblast

PRP is a powerful concentrate of growth factors that stimulate tissue regeneration and is used to treat damaged tissues. Under current Italian legislation, the PRP used by the RMC must be

• **Improve the microcirculation**, with increased collagen production [21, 22].

**Picture 8.** Case report. A 74-old-patient with vascular ulcers of the lower limbs treated with PRP.

structural organization of the wound healing process [9–14].

wounds is documented by photographic evidence [15–20].

**Pulsed electromagnetic fields (PEMF)**

already demonstrated in our first patients, they:

flammatory cells (IL-1, NGF, ROS, IL8);

STEP 4—Cell Therapies

Platelet-rich plasma (PRP)

growth factor), improving microcirculation;

prepared in a local transfusion center (**Picture 8**) [23–26].

*2. LumiHeal*

The rationale for the prescription of this supplement is:


Administration of low-dose cytokines to combat the inflammatory process.

Patients are prescribed with 3–6 months of treatment with low-dose cytokines, formulated with a kinetic system called Sequential Kinetic Activation (SKA), and containing:


Low dose therapy is an important part of wound management because it introduces into clinical practice a strategic concept for future therapies: the disease may be the result of an altered concentration of messenger or signal molecules (hormones, cytokines, neurotransmitters) for cellular activity, and in this case the modulation of these molecules can restore the disrupted balances, enabling healing [8].

STEP 3—Biophysical Therapies

### **Photobiomodulation**

During the process the wound is subjected to dual-type light frequencies for 8–20 minutes.

*1. Polarized light*

RMC is the first center in Italy to use a unique light source that is:


It has been demonstrated that polarized light at 590 nm stimulates angiogenesis and growth factors, while at 830 nm it activates the cells involved in wound healing; a dose of 20 J/cm2 stimulates increased collagen deposition, an increase in myofibroblasts and a better ultrastructural organization of the wound healing process [9–14].

### *2. LumiHeal*

Administration of a dietary supplement containing a well-balanced mix of serratio-peptidase, escin, bromelain and selenium. This supplement has proteolytic, fibrinolytic, anti-edema and

• Bacteriostatic action in uninfected lesions and antibiotic therapy in infected lesions;

Patients are prescribed with 3–6 months of treatment with low-dose cytokines, formulated

**2.** IL-10 - adjustment of the anti-inflammatory process in chronic diseases with reduction of

Low dose therapy is an important part of wound management because it introduces into clinical practice a strategic concept for future therapies: the disease may be the result of an altered concentration of messenger or signal molecules (hormones, cytokines, neurotransmitters) for cellular activity, and in this case the modulation of these molecules can restore the

During the process the wound is subjected to dual-type light frequencies for 8–20 minutes.

• Low-energy, reaching the wound with a constant intensity, producing bio-stimulating

Administration of low-dose cytokines to combat the inflammatory process.

**1.** Anti IL-1 - regulation and suppression of the inflammatory response.

RMC is the first center in Italy to use a unique light source that is:

• Polychromatic, with a wavelength ranging from 480 to 3400 nm;

• Incoherent, with out-of-phase waves delivering low-intensity light;

with a kinetic system called Sequential Kinetic Activation (SKA), and containing:

draining properties, as well as an antioxidant action.

• Anti-edema and draining action;

• Reduction of secretions;

100 Wound Healing - Current Perspectives

• Promotion of healing [7].

**3.** IL-4 - Th1-Th2 switch control.

STEP 3—Biophysical Therapies

**Photobiomodulation**

*1. Polarized light*

effects.

disrupted balances, enabling healing [8].

• Polarized, propagating in parallel planes;

• Pain relief;

IL-6.

The rationale for the prescription of this supplement is:

• Anti-inflammatory action (proteolytic and fibrinolytic);

The Lumiheal protocol involves the use of broadband wavelengths (blue, green, yellow/ orange from 450 nm to 610 nm) from a light emitting diode to amplify the physical effect of stimulation of the regenerative processes in the injured area due to the emission of photons in the form of fluorescence. The LumiHeal Protocol has been applied over the last 3 months in 8 patients with complicated treatment-resistant infected wounds. The improvement in the wounds is documented by photographic evidence [15–20].

### **Pulsed electromagnetic fields (PEMF)**

High intensity, variable frequency magnetic fields for outpatient and home treatment. As already demonstrated in our first patients, they:


### STEP 4—Cell Therapies

Platelet-rich plasma (PRP)

PRP is a powerful concentrate of growth factors that stimulate tissue regeneration and is used to treat damaged tissues. Under current Italian legislation, the PRP used by the RMC must be prepared in a local transfusion center (**Picture 8**) [23–26].

**Picture 8.** Case report. A 74-old-patient with vascular ulcers of the lower limbs treated with PRP.

#### Lipogems

Method for obtaining, through adipose liposuction, micro-fractured tissue for autologous use, which is reapplied to patients with skin lesions to further stimulate the cell regeneration process [27–30].

These articles affirm what we wrote in the introduction: inflammation has a major role in the wound healing process, in which disabling chronic diseases add to local systemic effects such as tissue hypoxia and pH changes, post-revascularization damage, cell aging and infections. Therapeutic resources take account of the numerous techniques and resources available, with

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 103

In this research process we began with a definition: **Repair 1.0**, signifying a dressing process involving the use of advanced dressings. This type of dressing is required to maintain an adequate wound moisture level, to be partly or totally occlusive, and to passively absorb the exudate, with a function determined by the patient's metabolism and biological "performance".

**Repair 2.0**, in contrast, involves the use of bioactive dressings with a biological action on the wound (hyaluronic acid, collagen, silver, etc.). From this perspective, the RMC investigated a sterile gauze dressing in which the role of the bioactive substances (hyaluronic acid, carnosine) is specifically defined in the literature, and involves mechanical protection of the lesion

Zhao et al. examine the causes of nonhealing wounds, and attribute the greatest responsibility to the inflammatory process. That study's relevance to the present article is its affirmation of the role of nitric oxide in the repair process and the well-known harm caused by ROS that, through systemic or topical treatment with antioxidants (carnosine), can be turned around in

In this context, we began working with bioactive substances with innovative properties (in relation to both composition and biological action) in comparison with their competitors. This potential innovation lies in the use of bovine colostrum, that, when stabilized through industrial processes to a pH of 6.8, assures the compound's stability and its action against the tissue acidosis found in damaged tissues. This has positive consequences for the modulation of the inflammatory process and the tissue repair process as well as on the ability to stimulate the cellular and ultrastructural regenerative process. (Bagnara G: *Le cellule staminali*, Cap 11. Ed

In **Regeneration 3.0**, the priority is to combine the anti-inflammatory activity of the nine proteins acting as growth factors in the bovine colostrum, the homeostatic, angiogenic and reorganizational activities of the matrix, the modulation of collagen synthesis and the remodeling of the epithelium. The choice of bovine colostrum and its associated properties was the basis for the design of devices that could also offer those properties: barrier action, antiinflammatory action and pain reduction, reduction and absorption of exudates, combating of bacterial and fungal proliferation, antioxidant action and hydration and protection against

The purpose of this test is to compare the efficacy of two medical devices in the repairing of wounds simulating this situation in vitro by making a cut on cell monolayer of human

particular attention to growth factors.

non-responding lesions [31].

skin diseases and dermatosis.

This is the culmination of an in-vitro and an in-vivo test.

**7.2. In Vitro comparative evaluation of wound healing activity of medical**

Esculapio 2017).

(gauze) combined with a direct anti-inflammatory action.

STEP 5—Surgical Therapies

At the RMC, surgical therapies for the repair of skin lesions are standardized. They involve the use of skin substitutes that promote the production of a structured collagen matrix, enabling better angiogenesis. When monitoring the application of skin substitutes, the focus is on the skin's reparative capacity as well as the risk of tissue rejection. The removal of any necrosis when cleaning the wound is essential to prepare it for autologous or heterologous grafts that fully integrate into the patient's dermis, and signs of rejection must be managed promptly.

### **7. From repair to regeneration: Regeneration 3.0**

### **7.1. The Prometheus project**

The RMC's clinical experience in cell therapies is based on the traumatic extraction methods that underlie the preparation of platelet-rich plasma and the extraction and centrifugation of adipose tissue (lipoaspiration) with the aid of modern technology. The results of their use in a treatment pathway that accompanies patients in their management are remarkable.

In relation to the use and performance of mesenchymal cells (immunomodulatory, paracrine and regenerative activity) in the RMC's clinical research, the multidisciplinary team were united by the realization that mesenchymal cells cannot be used in therapy and there is a need to optimize a daily therapy that has a similar effect on cell and tissue biostimulation. The Prometheus Project - Alfakjn Wound Care has been embraced by the RMC because it is *innovative* and because it anticipates the next frontier for regenerative medicine: *specific, individualized cell therapies.* Growth factors have made an explosive breakthrough into clinical practice, and the decision to focus on the quality and efficacy of the therapies containing them is strategic for the near future.

A careful analysis of the components of and claims made for the medical devices trialed by the RMC for the treatment of superficial and deep wounds and lesions through a clinical research joint venture has highlighted the need for a scientific value to be attributed to the rationale for using these devices, within the framework of a multidisciplinary activity intended to give added value to the device's action in regenerative medicine. The objective of the RMC's study was to offer an innovative solution to the current difficulties in managing nonhealing skin lesions. To do this, we first tried to answer a question: Do difficult wounds exist, or is it simply that we do not know how to treat them?

Our research is based on two articles, published in 2016 (Zhao R et al.: Inflammation in Chronic Wounds. Int J Mol Sci) and 2017 (Han G et al.: Chronic Wound Healing: A Review of current Management and Treatments. Adv. Ther) [12, 13].

These articles affirm what we wrote in the introduction: inflammation has a major role in the wound healing process, in which disabling chronic diseases add to local systemic effects such as tissue hypoxia and pH changes, post-revascularization damage, cell aging and infections. Therapeutic resources take account of the numerous techniques and resources available, with particular attention to growth factors.

Lipogems

process [27–30].

STEP 5—Surgical Therapies

102 Wound Healing - Current Perspectives

**7.1. The Prometheus project**

is strategic for the near future.

that we do not know how to treat them?

current Management and Treatments. Adv. Ther) [12, 13].

Method for obtaining, through adipose liposuction, micro-fractured tissue for autologous use, which is reapplied to patients with skin lesions to further stimulate the cell regeneration

At the RMC, surgical therapies for the repair of skin lesions are standardized. They involve the use of skin substitutes that promote the production of a structured collagen matrix, enabling better angiogenesis. When monitoring the application of skin substitutes, the focus is on the skin's reparative capacity as well as the risk of tissue rejection. The removal of any necrosis when cleaning the wound is essential to prepare it for autologous or heterologous grafts that fully integrate into the patient's dermis, and signs of rejection must be managed promptly.

The RMC's clinical experience in cell therapies is based on the traumatic extraction methods that underlie the preparation of platelet-rich plasma and the extraction and centrifugation of adipose tissue (lipoaspiration) with the aid of modern technology. The results of their use in a

In relation to the use and performance of mesenchymal cells (immunomodulatory, paracrine and regenerative activity) in the RMC's clinical research, the multidisciplinary team were united by the realization that mesenchymal cells cannot be used in therapy and there is a need to optimize a daily therapy that has a similar effect on cell and tissue biostimulation. The Prometheus Project - Alfakjn Wound Care has been embraced by the RMC because it is *innovative* and because it anticipates the next frontier for regenerative medicine: *specific, individualized cell therapies.* Growth factors have made an explosive breakthrough into clinical practice, and the decision to focus on the quality and efficacy of the therapies containing them

A careful analysis of the components of and claims made for the medical devices trialed by the RMC for the treatment of superficial and deep wounds and lesions through a clinical research joint venture has highlighted the need for a scientific value to be attributed to the rationale for using these devices, within the framework of a multidisciplinary activity intended to give added value to the device's action in regenerative medicine. The objective of the RMC's study was to offer an innovative solution to the current difficulties in managing nonhealing skin lesions. To do this, we first tried to answer a question: Do difficult wounds exist, or is it simply

Our research is based on two articles, published in 2016 (Zhao R et al.: Inflammation in Chronic Wounds. Int J Mol Sci) and 2017 (Han G et al.: Chronic Wound Healing: A Review of

treatment pathway that accompanies patients in their management are remarkable.

**7. From repair to regeneration: Regeneration 3.0**

In this research process we began with a definition: **Repair 1.0**, signifying a dressing process involving the use of advanced dressings. This type of dressing is required to maintain an adequate wound moisture level, to be partly or totally occlusive, and to passively absorb the exudate, with a function determined by the patient's metabolism and biological "performance".

**Repair 2.0**, in contrast, involves the use of bioactive dressings with a biological action on the wound (hyaluronic acid, collagen, silver, etc.). From this perspective, the RMC investigated a sterile gauze dressing in which the role of the bioactive substances (hyaluronic acid, carnosine) is specifically defined in the literature, and involves mechanical protection of the lesion (gauze) combined with a direct anti-inflammatory action.

Zhao et al. examine the causes of nonhealing wounds, and attribute the greatest responsibility to the inflammatory process. That study's relevance to the present article is its affirmation of the role of nitric oxide in the repair process and the well-known harm caused by ROS that, through systemic or topical treatment with antioxidants (carnosine), can be turned around in non-responding lesions [31].

In this context, we began working with bioactive substances with innovative properties (in relation to both composition and biological action) in comparison with their competitors. This potential innovation lies in the use of bovine colostrum, that, when stabilized through industrial processes to a pH of 6.8, assures the compound's stability and its action against the tissue acidosis found in damaged tissues. This has positive consequences for the modulation of the inflammatory process and the tissue repair process as well as on the ability to stimulate the cellular and ultrastructural regenerative process. (Bagnara G: *Le cellule staminali*, Cap 11. Ed Esculapio 2017).

In **Regeneration 3.0**, the priority is to combine the anti-inflammatory activity of the nine proteins acting as growth factors in the bovine colostrum, the homeostatic, angiogenic and reorganizational activities of the matrix, the modulation of collagen synthesis and the remodeling of the epithelium. The choice of bovine colostrum and its associated properties was the basis for the design of devices that could also offer those properties: barrier action, antiinflammatory action and pain reduction, reduction and absorption of exudates, combating of bacterial and fungal proliferation, antioxidant action and hydration and protection against skin diseases and dermatosis.

This is the culmination of an in-vitro and an in-vivo test.

### **7.2. In Vitro comparative evaluation of wound healing activity of medical**

The purpose of this test is to compare the efficacy of two medical devices in the repairing of wounds simulating this situation in vitro by making a cut on cell monolayer of human



The animals were individually housed, their backs have been shaved, cleaned with alcohol, the skin has been gently pulled up and placed between two round ceramic magnetic

**Figure 7.** In vitro comparative evaluation of wound healing activity of medical devices - Cell Growth . Alfakjn ResearchCenter

weight of 2.4 g and 1000G magnetic forces; this process creates a compressive pressure of 50 mmHg between the two magnets. Then the animals have been divided into two groups

Control group: three IR cycles have been performed in 3 mice to initiate decubitus ulcer formation. A single IR cycle consists of a 12-hour period of magnet placement, followed by a release of rest period of 12 hours. After the 3 IR cycles, the animals have been sacrificed.

) and are 5 mm thick, with an average

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 105

plates which have a 12-mm diameter (113 mm2

**Picture 9.** Initiate decubitus ulcer formation in mice to test colostrum derivative therapy.

Milano 2018 by Bio Basic Europe SRL. Via A. Panizzi,10 Milano Italy.

as follows:

**Figure 6.** In vitro comparative evaluation of wound healing activity of medical devices, Alfakjn ResearchCenter Milano 2018 by Bio Basic Europe SRL. Via A. Panizzi,10 Milano Italy.

fibroblasts (Hude) and then evaluating the approximation of the edges of the cut in cells treated with the two medical devices, in comparison to untreated cells. In order to select the concentrations of 2 medical devices to be used for the test r (not cytotoxic concentrations for the cells), a preliminary MTT cell cultures of fibroblasts was performed. The cells were treated with scalar concentrations of the two medical devices (as low as 1 mg/ml and subsequent dilutions 1: 2) and untreated cells were used as negative control. Based on the obtained results concentrations of 2 medical devices of 1–0.25–0.15 mg/ml were chosen to continue the test. After making a cut on the cell monolayer of confluent fibroblasts (simulation of a wound), the cells were treated with the chosen concentrations of the two medical devices, as negative control untreated cells were used and, as internal quality control one standard with known activity of wound healing activity. Therefore, we have performed a morphologic evaluation of the monolayer by microscopy and a measurement of IL-8 levels. From the morphological evaluation a net approach of the flaps of the monolayer was observed in the plates treated with the various concentrations of Colostrum Gel. The dosage dell'IL8 showed significant decrease in the% of IL8 to concentrations of 1 and 0.25 mg/ml by both tested medical devices, showing therefore a comparable anti-inflammatory action on fibroblasts (**Figure 6**). These results indicate that the effectiveness of the active ingredients present in the product have a different target than the reduction of the inflammatory response. The obtained results have showed the effectiveness in wound healing of the medical device Colostrum Gel, compared to the medical device Gel no active. The medical device Gel Herpes no active is in fact shows a "nutrient" activity on cells but it is not able to stimulate the repair of the damage (cutting), this latter activity is due to the presence of actives ingredients present in the formula of Colostrum Gel. Colostrum Gel reduced IL8 production by fibroblasts and contains active ingredients. Those stimulate wound healing (simulation in vitro by cutting the monolayer of cultured fibroblasts and evidence of the approximation of the edges of the cut and almost total closure of the same) (**Figure 7**).

#### **7.3. Topical use of Colostro AIM 4% fluid cream in a murine model of pressure ulcers**

This study reports the development of a murine model of pressure ulcers by using externally placed magnets to create the ischemic events of ischemia reperfusion (IR) injury.

**Figure 7.** In vitro comparative evaluation of wound healing activity of medical devices - Cell Growth . Alfakjn ResearchCenter Milano 2018 by Bio Basic Europe SRL. Via A. Panizzi,10 Milano Italy.

The animals were individually housed, their backs have been shaved, cleaned with alcohol, the skin has been gently pulled up and placed between two round ceramic magnetic plates which have a 12-mm diameter (113 mm2 ) and are 5 mm thick, with an average weight of 2.4 g and 1000G magnetic forces; this process creates a compressive pressure of 50 mmHg between the two magnets. Then the animals have been divided into two groups as follows:

fibroblasts (Hude) and then evaluating the approximation of the edges of the cut in cells treated with the two medical devices, in comparison to untreated cells. In order to select the concentrations of 2 medical devices to be used for the test r (not cytotoxic concentrations for the cells), a preliminary MTT cell cultures of fibroblasts was performed. The cells were treated with scalar concentrations of the two medical devices (as low as 1 mg/ml and subsequent dilutions 1: 2) and untreated cells were used as negative control. Based on the obtained results concentrations of 2 medical devices of 1–0.25–0.15 mg/ml were chosen to continue the test. After making a cut on the cell monolayer of confluent fibroblasts (simulation of a wound), the cells were treated with the chosen concentrations of the two medical devices, as negative control untreated cells were used and, as internal quality control one standard with known activity of wound healing activity. Therefore, we have performed a morphologic evaluation of the monolayer by microscopy and a measurement of IL-8 levels. From the morphological evaluation a net approach of the flaps of the monolayer was observed in the plates treated with the various concentrations of Colostrum Gel. The dosage dell'IL8 showed significant decrease in the% of IL8 to concentrations of 1 and 0.25 mg/ml by both tested medical devices, showing therefore a comparable anti-inflammatory action on fibroblasts (**Figure 6**). These results indicate that the effectiveness of the active ingredients present in the product have a different target than the reduction of the inflammatory response. The obtained results have showed the effectiveness in wound healing of the medical device Colostrum Gel, compared to the medical device Gel no active. The medical device Gel Herpes no active is in fact shows a "nutrient" activity on cells but it is not able to stimulate the repair of the damage (cutting), this latter activity is due to the presence of actives ingredients present in the formula of Colostrum Gel. Colostrum Gel reduced IL8 production by fibroblasts and contains active ingredients. Those stimulate wound healing (simulation in vitro by cutting the monolayer of cultured fibroblasts and evidence of the approximation of the edges of the cut and almost total closure

**Figure 6.** In vitro comparative evaluation of wound healing activity of medical devices, Alfakjn ResearchCenter Milano

2018 by Bio Basic Europe SRL. Via A. Panizzi,10 Milano Italy.

104 Wound Healing - Current Perspectives

**7.3. Topical use of Colostro AIM 4% fluid cream in a murine model of pressure ulcers**

This study reports the development of a murine model of pressure ulcers by using externally placed magnets to create the ischemic events of ischemia reperfusion (IR) injury.

of the same) (**Figure 7**).

Control group: three IR cycles have been performed in 3 mice to initiate decubitus ulcer formation. A single IR cycle consists of a 12-hour period of magnet placement, followed by a release of rest period of 12 hours. After the 3 IR cycles, the animals have been sacrificed.

**Picture 9.** Initiate decubitus ulcer formation in mice to test colostrum derivative therapy.

Group B: three IR cycles have been performed in each mouse to initiate decubitus ulcer formation. A topical administration of 200 mg of AIM LIFEIN- SIDE 4% (AI13–002-B) has been applied on the backs of each mouse the day before the first IR cycle and at the end of each compressive cycle. After the 3 IR cycles, the animals have been sacrificed. Skin samples of each mouse have been collected from the treated area, fixed in 10% phosphate-buffered formalin and wax embedded. 2 μm thickness sections were obtained and collected on silanizated slides and stained by hematoxylin- eosin. The samples were then observed with an optical microscope Nikon 80i, fitted with a digital camera (**Picture 9**).

### **8. Results**


**Picture 10.** Wide ulcerative area of the epidermis; marked spongiosis of basal layer (intercellular bridges appear very prominent); marked diffuse inflammatory infiltrate; conjunctival edema; vascular hyperemia; presence of extravasal

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 107

**Picture 11.** Macroscopic analysis of dorsal skin revealed: skin ulcers with necrosis areas and edema.

erythrocytes.

The most significant aspect with a view to new tissue regeneration therapies and hence the control of the inflammatory process were the results in relation to the disappearance of the wound and the reduction of the inflammatory process. This requires the future consideration of the action time of the medical device and its contact time with the damaged tissue.

The results in relation to clinical healing take account of chronic conditions defined as nonresponders and their good management from the time of diagnosis. The data on patient compliance with the use of the medical device reveal the absence of any symptoms or side effects, ensuring the patient's safety and boosting the device's reliability and efficacy.

#### **8.1. Analysis of results and validity of the protocol**

The RMC treats patients with chronic wounds of various etiologies. The AIMED model treatment has been applied in 85% of cases. Analysis of the 360° wound management process has revealed remarkable results in relation to:

Group B: three IR cycles have been performed in each mouse to initiate decubitus ulcer formation. A topical administration of 200 mg of AIM LIFEIN- SIDE 4% (AI13–002-B) has been applied on the backs of each mouse the day before the first IR cycle and at the end of each compressive cycle. After the 3 IR cycles, the animals have been sacrificed. Skin samples of each mouse have been collected from the treated area, fixed in 10% phosphate-buffered formalin and wax embedded. 2 μm thickness sections were obtained and collected on silanizated slides and stained by hematoxylin- eosin. The samples were then observed with an optical

**1.** Control group: 3 IR cycles Skin macroscopic analysis Macroscopic analysis of dorsal skin revealed the presence of mild skin lesions, edema and signs of necrosis of the epidermis. (Photo 10). Histological analysis of dorsal skin samples stained by hematoxylin–eosin showed: wide ulcerative area of the epidermis; marked spongiosis of basal layer (intercellular bridges appear very prominent); marked diffuse inflammatory infiltrate; conjunctival edema; vascular hyperemia; presence of extravasal erythrocytes (**Picture 10**). **2.** Group B: 3 IR cycles+Colostro AIM at the end of each compressive cycle and the day before the first IR cycle. Skin macroscopic analysis. Macroscopic analysis of dorsal skin revealed: skin ulcers with necrosis areas and edema (**Picture 11**). Histological analysis of dorsal skin samples stained by hematoxylin–eosin showed: wide ulcerative area of the epidermis; when present epidermis is hyperplasic, with not regular thickness, marked spongiosis of basal layer and presence of lymphocytes; marked diffuse inflammatory infiltrate; conjunctival edema; vascular hyperemia; presence of extravasal

The most significant aspect with a view to new tissue regeneration therapies and hence the control of the inflammatory process were the results in relation to the disappearance of the wound and the reduction of the inflammatory process. This requires the future consideration

The results in relation to clinical healing take account of chronic conditions defined as nonresponders and their good management from the time of diagnosis. The data on patient compliance with the use of the medical device reveal the absence of any symptoms or side effects,

The RMC treats patients with chronic wounds of various etiologies. The AIMED model treatment has been applied in 85% of cases. Analysis of the 360° wound management process has

of the action time of the medical device and its contact time with the damaged tissue.

ensuring the patient's safety and boosting the device's reliability and efficacy.

**8.1. Analysis of results and validity of the protocol**

revealed remarkable results in relation to:

microscope Nikon 80i, fitted with a digital camera (**Picture 9**).

**8. Results**

106 Wound Healing - Current Perspectives

erythrocytes (**Picture 12**).

**Picture 10.** Wide ulcerative area of the epidermis; marked spongiosis of basal layer (intercellular bridges appear very prominent); marked diffuse inflammatory infiltrate; conjunctival edema; vascular hyperemia; presence of extravasal erythrocytes.

**Picture 11.** Macroscopic analysis of dorsal skin revealed: skin ulcers with necrosis areas and edema.

**Picture 12.** Wide ulcerative area of the epidermis; when present epidermis is hyperplasic, with not regular thickness, marked spongiosis of basal layer and presence of lymphocytes; marked diffuse inflammatory infiltrate; conjunctival edema; vascular hyperemia; presence of extravasal erythrocytes.

processes and remodeling of the ECM. At the same time, they highlight how the abnormal evolution of the inflammatory process to a chronic condition involves abnormal cellularity, inappropriate collagen deposit and the presence of protease, preventing re-epithelialization

**Picture 13.** A 73-year-old patient with heart failure and lower limb ulcers. Healing process with the AIMED method in

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 109

Knowledge of the biological pathways at an ultramolecular and cellular level enables the identification of various areas where clinical research could intervene with biological drugs or biophysical therapies to influence the healing pathways of non-responding chronic wounds or stimulate the metabolic or regenerative processes, blocking the mechanisms leading to chronicity and, in particular, intervening in the chronic inflammatory process. In this case, in vitro tests could help by enabling new biological compounds to be tested on cellular models of skin damage. We have demonstrated that colostrum is paradigmatic of the therapeutic philosophy adopted by the RMC, in the sense that it is capable of reducing levels of proinflammatory cytokines and protease (MMP-9), blocking M1 activity and stimulating the activity of fibroblasts, resulting in the production of type III and VII collagen to aid regeneration. Analysis of the results obtained with low dose therapy and the effects of biophysical therapies (photobiomodulation and PEMF) could provide guidance on aspects that the authors consider to be of current and future interest: modulation of nitric oxide in vasodilation and the provision of regenerative molecules (PEMF), and the reduction of the inflammatory component (IL-6 and C-reactive protein). Analysis of the current literature suggests that reduction of the inflammatory component is the key to regenerative recovery of chronic nonresponding wounds, now that we have a better understating of their pathogenesis and pathophysiological processes. The adoption of a 360° rather than sequential wound management model is based on the authors' choices and experience, and has a firm scientific basis. We believe that

this model ensures that patients receive the best possible care and attention.

and regeneration of the lesion. (**Picture 13**).

3 and a half months.


Another important aspect of the RMC's activity is the constant back-and-forth between the results obtained through clinical observation and the analysis of the collected data through clinical research, in the light of a possible reinterpretation in a future scenario focusing on two key aspects of the healing of chronic wounds: anti-inflammatory action and regenerative action [31, 32].

In this article, the authors lay down the scientific basis for a chronic wound healing process involving an appropriate sequence of the modulation of the inflammatory and proliferative

**Picture 13.** A 73-year-old patient with heart failure and lower limb ulcers. Healing process with the AIMED method in 3 and a half months.

processes and remodeling of the ECM. At the same time, they highlight how the abnormal evolution of the inflammatory process to a chronic condition involves abnormal cellularity, inappropriate collagen deposit and the presence of protease, preventing re-epithelialization and regeneration of the lesion. (**Picture 13**).

**Picture 12.** Wide ulcerative area of the epidermis; when present epidermis is hyperplasic, with not regular thickness, marked spongiosis of basal layer and presence of lymphocytes; marked diffuse inflammatory infiltrate; conjunctival

• Best wound management with a greater awareness of dressing protocols and the use of

Another important aspect of the RMC's activity is the constant back-and-forth between the results obtained through clinical observation and the analysis of the collected data through clinical research, in the light of a possible reinterpretation in a future scenario focusing on two key aspects of the healing of chronic wounds: anti-inflammatory action and regenerative

In this article, the authors lay down the scientific basis for a chronic wound healing process involving an appropriate sequence of the modulation of the inflammatory and proliferative

existing medications on the market, integrated into the AIMED model;

edema; vascular hyperemia; presence of extravasal erythrocytes.

• Reduced complications and signs of comorbidity;

• Better compliance and patient and family satisfaction;

• Rapid healing; • Pain reduction;

108 Wound Healing - Current Perspectives

action [31, 32].

• Reduced health expenditure;

• Reduced rate of recurrence.

Knowledge of the biological pathways at an ultramolecular and cellular level enables the identification of various areas where clinical research could intervene with biological drugs or biophysical therapies to influence the healing pathways of non-responding chronic wounds or stimulate the metabolic or regenerative processes, blocking the mechanisms leading to chronicity and, in particular, intervening in the chronic inflammatory process. In this case, in vitro tests could help by enabling new biological compounds to be tested on cellular models of skin damage. We have demonstrated that colostrum is paradigmatic of the therapeutic philosophy adopted by the RMC, in the sense that it is capable of reducing levels of proinflammatory cytokines and protease (MMP-9), blocking M1 activity and stimulating the activity of fibroblasts, resulting in the production of type III and VII collagen to aid regeneration.

Analysis of the results obtained with low dose therapy and the effects of biophysical therapies (photobiomodulation and PEMF) could provide guidance on aspects that the authors consider to be of current and future interest: modulation of nitric oxide in vasodilation and the provision of regenerative molecules (PEMF), and the reduction of the inflammatory component (IL-6 and C-reactive protein). Analysis of the current literature suggests that reduction of the inflammatory component is the key to regenerative recovery of chronic nonresponding wounds, now that we have a better understating of their pathogenesis and pathophysiological processes. The adoption of a 360° rather than sequential wound management model is based on the authors' choices and experience, and has a firm scientific basis. We believe that this model ensures that patients receive the best possible care and attention.

### **9. Conclusions**

The molecular pond is in a state of constant agitation and turbulence, with the molecules spinning and vibrating and bouncing off one another…

Quantum medicine returns to that concept of electromagnetic fields, the energy of which can change the very essence of nature. The concept that emerges is that the cell can undergo a

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 111

Stem cells will be mentioned only briefly, as it will be left to other authors to present the latest data. Stem cells deriving from adipose tissue have now become part of everyday clinical practice. However, their results in the treatment of skin wounds are not yet unequivocal, as the greatest obstacle they encounter, directly after implantation, is the inflammatory reaction of the host. They have a migratory capacity that enables them to reach the target site through the blood, enabling rapid access to the entire body. They are then captured by the target organs through complex interactions with endothelial cells that enable them to leave the circulation

Given this premise, stem cells could offer an opportunity for the regenerative treatment of skin lesions, but only if used according to holistic principles. The authors explain their view by returning to the concept of self-healing of the cell in a context such as tissue regeneration, where cell proliferation and differentiation are specific and fundamental processes. After transplantation, stem cells work only if they can communicate with the stem cells already resident in the tissue, acting as a starter and stimulating the existing cells through the cell membrane. In 2012, Yamanaka won the Nobel prize for a 2006 study on the induction of stem cells from fibroblasts through cell manipulation, observing that adult cells can be reprogrammed to become pluripotent. The limitation of this technique is the low efficiency of the

Today, stem cells can be incubated and stimulated with platelet lysate, which stimulates their proliferation, or with biological agents, but they can also be reactivated in vivo through lowintensity magnetic fields affecting the matrix, membrane and cytoskeleton. Following external stimulation from receptors, the microtubules immersed in the intracellular water vibrate and transmit information to and from the nucleus through signal molecules, just as a dipole transmits the signal beyond the point where it was generated, amplifying the response and turning the cell membrane into a center for signal processing and communication with the outside world. The microtubule is a "molecular cable" that enables the system to memorize information (*C. Ventura*). All this is made possible by the presence of water, which is essential to enable the humoral and cellular components to perform their roles and the microtubules to spread a dynamic network sensitive to even the slightest signal alteration: a "conscious" network that modulates recognition and communication through the signification of coded

Pluripotent stem cells differentiate thanks to an epigenetic code comprising a molecular network that turns specific genes on and off. The information carried by the molecules is only a part of all the information that reaches the cells, of which a large part arrives with magnetic and sound fields. Ventura and colleagues differentiated embryonal stem cells from heart tissue cells by subjecting them to low frequency (50 Hertz), low intensity (0.6 mm tesla) electromagnetic

*"The cells communicate with each other using information carried by molecules, which act over a short range, or transported by electromagnetic waves and sounds transmitted over a long range and targeted* 

differentiation process and the oncogenic risk caused by the use of viral vectors.

self-healing process if it receives the right information: this is the new advance.

for tissue regeneration.

messages.

fields.

*precisely to the molecules" (Pier Maria Biava).*

**Life on the Edge – J. McFadden and J. Al-Khalili**

### **10. The future of regeneration**

The study of biological molecules has enabled a glimpse of a possible new key for the interpretation of biological phenomena linked to management of the inflammatory process, and some such molecules could be prototypes for others still to come. The greater availability of water molecules around more hydrophilic molecules and the better organization of the body's water seem to produce a greater and better biological response.

*"We could interpret the disease as a loss of some levels of cohesive hierarchy between the domains, with a consequent loss of the electromagnetic control exerted over the biomolecules" (E. Del Giudice).*

In the study of the hydrodynamic behavior of numerous molecules, it always comes back to the endothelium, the ECM and the cells themselves. The role of the cell membrane seems worthy of attention, as its structure enables substances to travel or be transported into the cell, but the functional properties of the membrane that we know today make it the protagonist of a new biological culture, in which chemistry meets anatomy and anatomy is subject to physical stimuli that can modify its essence [33–36].

The inner cell is packed with a thick, intricate network of microtubules formed by well-known proteins (tubulin). This network, called the **cytoskeleton**, has a complicated and constantly changing dynamic structure and function: some branches form, others break down and disappear, others extend in multiple directions.

Most intracellular metabolic reactions take place along the branches of the cytoskeleton. Its structure is thus fundamental for biochemical functionality (a new functional concept), which marks a continuum between the cell nucleus, the membrane and the outside of the cell, protected by another important structure, the glycocalyx. When the cell dies, its cytoskeleton breaks down. This highly dynamic behavior is difficult to understand, but there have been a number of studies of both the biochemistry involved and the energy and charge transport capacity along the microtubules (Davydov, 1982 and references reported therein).

The cytoskeleton is a system of canals (microtubules) in which substances are transported and information is transmitted. This is made possible because all its molecules, including water (which makes up 80% or more of the cytoskeleton by weight), have a dipole moment – in other words, an electrically charged spatial distribution involving a positively charged pole and a negatively charged pole. **All macromolecules become biologically active only if they are immersed in an aqueous matrix.** This demonstrates the predominant role of water in living beings. Intuition is transformed into scientific data and becomes reality.

Quantum medicine returns to that concept of electromagnetic fields, the energy of which can change the very essence of nature. The concept that emerges is that the cell can undergo a self-healing process if it receives the right information: this is the new advance.

**9. Conclusions**

110 Wound Healing - Current Perspectives

The molecular pond is in a state of constant agitation and turbulence, with the molecules

The study of biological molecules has enabled a glimpse of a possible new key for the interpretation of biological phenomena linked to management of the inflammatory process, and some such molecules could be prototypes for others still to come. The greater availability of water molecules around more hydrophilic molecules and the better organization of the

*"We could interpret the disease as a loss of some levels of cohesive hierarchy between the domains, with a consequent loss of the electromagnetic control exerted over the biomolecules" (E. Del Giudice).*

In the study of the hydrodynamic behavior of numerous molecules, it always comes back to the endothelium, the ECM and the cells themselves. The role of the cell membrane seems worthy of attention, as its structure enables substances to travel or be transported into the cell, but the functional properties of the membrane that we know today make it the protagonist of a new biological culture, in which chemistry meets anatomy and anatomy is subject to physi-

The inner cell is packed with a thick, intricate network of microtubules formed by well-known proteins (tubulin). This network, called the **cytoskeleton**, has a complicated and constantly changing dynamic structure and function: some branches form, others break down and dis-

Most intracellular metabolic reactions take place along the branches of the cytoskeleton. Its structure is thus fundamental for biochemical functionality (a new functional concept), which marks a continuum between the cell nucleus, the membrane and the outside of the cell, protected by another important structure, the glycocalyx. When the cell dies, its cytoskeleton breaks down. This highly dynamic behavior is difficult to understand, but there have been a number of studies of both the biochemistry involved and the energy and charge transport capacity along the microtubules (Davydov, 1982 and references

The cytoskeleton is a system of canals (microtubules) in which substances are transported and information is transmitted. This is made possible because all its molecules, including water (which makes up 80% or more of the cytoskeleton by weight), have a dipole moment – in other words, an electrically charged spatial distribution involving a positively charged pole and a negatively charged pole. **All macromolecules become biologically active only if they are immersed in an aqueous matrix.** This demonstrates the predominant role of water in

living beings. Intuition is transformed into scientific data and becomes reality.

body's water seem to produce a greater and better biological response.

spinning and vibrating and bouncing off one another…

**Life on the Edge – J. McFadden and J. Al-Khalili**

cal stimuli that can modify its essence [33–36].

appear, others extend in multiple directions.

reported therein).

**10. The future of regeneration**

Stem cells will be mentioned only briefly, as it will be left to other authors to present the latest data. Stem cells deriving from adipose tissue have now become part of everyday clinical practice. However, their results in the treatment of skin wounds are not yet unequivocal, as the greatest obstacle they encounter, directly after implantation, is the inflammatory reaction of the host. They have a migratory capacity that enables them to reach the target site through the blood, enabling rapid access to the entire body. They are then captured by the target organs through complex interactions with endothelial cells that enable them to leave the circulation for tissue regeneration.

Given this premise, stem cells could offer an opportunity for the regenerative treatment of skin lesions, but only if used according to holistic principles. The authors explain their view by returning to the concept of self-healing of the cell in a context such as tissue regeneration, where cell proliferation and differentiation are specific and fundamental processes. After transplantation, stem cells work only if they can communicate with the stem cells already resident in the tissue, acting as a starter and stimulating the existing cells through the cell membrane. In 2012, Yamanaka won the Nobel prize for a 2006 study on the induction of stem cells from fibroblasts through cell manipulation, observing that adult cells can be reprogrammed to become pluripotent. The limitation of this technique is the low efficiency of the differentiation process and the oncogenic risk caused by the use of viral vectors.

Today, stem cells can be incubated and stimulated with platelet lysate, which stimulates their proliferation, or with biological agents, but they can also be reactivated in vivo through lowintensity magnetic fields affecting the matrix, membrane and cytoskeleton. Following external stimulation from receptors, the microtubules immersed in the intracellular water vibrate and transmit information to and from the nucleus through signal molecules, just as a dipole transmits the signal beyond the point where it was generated, amplifying the response and turning the cell membrane into a center for signal processing and communication with the outside world. The microtubule is a "molecular cable" that enables the system to memorize information (*C. Ventura*). All this is made possible by the presence of water, which is essential to enable the humoral and cellular components to perform their roles and the microtubules to spread a dynamic network sensitive to even the slightest signal alteration: a "conscious" network that modulates recognition and communication through the signification of coded messages.

Pluripotent stem cells differentiate thanks to an epigenetic code comprising a molecular network that turns specific genes on and off. The information carried by the molecules is only a part of all the information that reaches the cells, of which a large part arrives with magnetic and sound fields. Ventura and colleagues differentiated embryonal stem cells from heart tissue cells by subjecting them to low frequency (50 Hertz), low intensity (0.6 mm tesla) electromagnetic fields.

*"The cells communicate with each other using information carried by molecules, which act over a short range, or transported by electromagnetic waves and sounds transmitted over a long range and targeted precisely to the molecules" (Pier Maria Biava).*

Molecular information comprises a concerto that enables life to begin and maintain its balance. It is a chemical system governed by electromagnetic forces. The body's water enables this electromagnetic regulation of the biochemistry, as described in the studies of Emilio Del Giudice on the dynamics of water. Cohesive water (in which the molecules are held together by smaller energy forms) oscillates at a given frequency and attracts molecules that resonate at the same frequency. These molecules interact chemically and produce a new form of energy that, in turn, "reconditions" the magnetic field, modifying its frequency and causing the emergence of its information content of various levels of complexity, which tells the water molecules what to do.

itself in the skin wound, an expression of it all. Bacterial infections, which are difficult to combat due to antibiotic resistance, greatly complicate the roles of our innate immune system and lymphocytes. Reading between the lines of routine blood tests, the patient's discomfort can often be sensed through information on the nitrogen balance and the hemoglobin value. The lack of new antibiotics and the impossibility of treating patients at home with hospital medications mean that new, more biological and more physical pathways must be investigated to interact with and defeat bacteria. The results of Montagnier and colleagues suggest that exposing bacteria to electromagnetic fields and hence altering their genetic code or forcing

We now know the key players in the wound healing process and we have new molecules available to act on them, but the future must necessarily lie in the transfer of molecules and information between the endothelium, ECM and cell membrane, which can be directed toward tissue regeneration if the resident stem cells have the chance of communicating and interacting with new therapeutic models; all this without forgetting the human being, at the

, Ferrari Alessio2

and Giorgio Ciprandi<sup>3</sup>

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 113

their membranes to become more water permeable could lead to their implosion.

\*, Mereghetti Giada1

1 Center of Regenerative Medicine—Humanitas Mater Domini, Castellanza, VA, Italy

[1] Aragona SE et al. La Medicina Rigenerativa: Le nuove ricerche per la cura delle lesioni

[2] Barabino S et al. Efficacy of a new ocular surface modulator in restoring epithelial changes in an in vitro model of dry eye syndrome. Current Eye Research. Mar 2017;**42**(3):358-363

[3] Ferrante CJ, Leibovich SJ. Regulation of Macrophage Polarization and Wound Healing.

[4] Shultz GS et al. Wound bed preparation a systematic approach to wound management.

[5] Percival SL et al. The effects of pH on wound healing, biofilms, and antimicrobial effi-

center of research and scientific evolution [37, 38].

\*Address all correspondence to: saadmaswood@gmail.com

2 Alfakjn Research Center, Valenza Po, Alessandria, Italy

Advanced Wound Care. Feb 2012;**1**(1):10-16

3 Allergy Clinic, Casa di Cura Villa Montallegro, Genoa, Italy

cutanee. Surgical Tribune Ed Italiana. Gen-Feb 10-14. 2013;**1**

Wound Repair and Regeneration. 2003;**11**(suppl 1):S1-S28

cacy. Wound Repair and Regeneration. Mar-Apr 2014;**22**(2):174-186

**Author details**

**References**

Aragona Salvatore Emanuele1

"We could interpret disease as a loss, by the body's water, of some levels of cohesive hierarchy between the domains, with a consequent loss of the electromagnetic control exerted over the biomolecules (Emilio Del Giudice).

While awaiting the new frontiers and conquests that the use of stem cells will open up in the field of cell regeneration, today it is possible to introduce biological therapies tailor-made for each individual patient. The biological molecules used in the preclinical and clinical phase enable greater communication between the patient's biological components (endothelium, matrix, cell), thanks to their greater hydrodynamic capacity and the formation of cohesive, organized water, which modulates the components of the inflammatory process and directs it toward tissue regeneration and healing of skin wounds.

*Later I looked again, and before my eyes a door stood open in Heaven, and in my ears was the voice with the ring of a trumpet, which I had heard at first, speaking to me and saying, "Come up here, and I will show you what must happen in the future."*

#### *Revelation 3.4*

In conclusion, this article has briefly presented our current knowledge of the modulation of the inflammatory process. It first discussed the possibility of following the process in in-vitro models – a valuable option, both for the knowledge they provide and for the possibility of learning more about the behavior of biological agents in relation to tissue regeneration. It then followed the process from a molecular perspective, delving into the "magma" of the pro- and anti-inflammatory cytokines and concentrating on what needs to be blocked in order to reduce the inflammatory process (TNF-alpha and IL1), without losing sight of the structure of the ECM, which remains the main target and the place in which the newly formed tissue is remodeled. It went on to discuss current results in relation to the possible clinical application of stem cells in regenerative medicine, highlighting the role of biological water as a transducer of molecular and energy information perceived by the stem cells, as well as the role of the cell membrane which, in the presence of water and in concert with the complex of molecular" cables" (the cytoskeleton), becomes a signal and information processing center involving receptors, adhesion molecules, the ECM structure and cell populations, with a "chimera" effect that is subject to both known and undiscovered physicochemical laws.

We would like to end with some practical considerations. The wound healing process is a complex process intertwined with the biological mechanisms causing individuals to become ill. Systemic and local factors combine to cause the process to become chronic and perpetuate itself in the skin wound, an expression of it all. Bacterial infections, which are difficult to combat due to antibiotic resistance, greatly complicate the roles of our innate immune system and lymphocytes. Reading between the lines of routine blood tests, the patient's discomfort can often be sensed through information on the nitrogen balance and the hemoglobin value. The lack of new antibiotics and the impossibility of treating patients at home with hospital medications mean that new, more biological and more physical pathways must be investigated to interact with and defeat bacteria. The results of Montagnier and colleagues suggest that exposing bacteria to electromagnetic fields and hence altering their genetic code or forcing their membranes to become more water permeable could lead to their implosion.

We now know the key players in the wound healing process and we have new molecules available to act on them, but the future must necessarily lie in the transfer of molecules and information between the endothelium, ECM and cell membrane, which can be directed toward tissue regeneration if the resident stem cells have the chance of communicating and interacting with new therapeutic models; all this without forgetting the human being, at the center of research and scientific evolution [37, 38].

### **Author details**

Molecular information comprises a concerto that enables life to begin and maintain its balance. It is a chemical system governed by electromagnetic forces. The body's water enables this electromagnetic regulation of the biochemistry, as described in the studies of Emilio Del Giudice on the dynamics of water. Cohesive water (in which the molecules are held together by smaller energy forms) oscillates at a given frequency and attracts molecules that resonate at the same frequency. These molecules interact chemically and produce a new form of energy that, in turn, "reconditions" the magnetic field, modifying its frequency and causing the emergence of its information content of various levels of complexity, which tells the water

"We could interpret disease as a loss, by the body's water, of some levels of cohesive hierarchy between the domains, with a consequent loss of the electromagnetic control exerted over the

While awaiting the new frontiers and conquests that the use of stem cells will open up in the field of cell regeneration, today it is possible to introduce biological therapies tailor-made for each individual patient. The biological molecules used in the preclinical and clinical phase enable greater communication between the patient's biological components (endothelium, matrix, cell), thanks to their greater hydrodynamic capacity and the formation of cohesive, organized water, which modulates the components of the inflammatory process and directs it

*Later I looked again, and before my eyes a door stood open in Heaven, and in my ears was the voice with the ring of a trumpet, which I had heard at first, speaking to me and saying, "Come up here, and I will* 

In conclusion, this article has briefly presented our current knowledge of the modulation of the inflammatory process. It first discussed the possibility of following the process in in-vitro models – a valuable option, both for the knowledge they provide and for the possibility of learning more about the behavior of biological agents in relation to tissue regeneration. It then followed the process from a molecular perspective, delving into the "magma" of the pro- and anti-inflammatory cytokines and concentrating on what needs to be blocked in order to reduce the inflammatory process (TNF-alpha and IL1), without losing sight of the structure of the ECM, which remains the main target and the place in which the newly formed tissue is remodeled. It went on to discuss current results in relation to the possible clinical application of stem cells in regenerative medicine, highlighting the role of biological water as a transducer of molecular and energy information perceived by the stem cells, as well as the role of the cell membrane which, in the presence of water and in concert with the complex of molecular" cables" (the cytoskeleton), becomes a signal and information processing center involving receptors, adhesion molecules, the ECM structure and cell populations, with a "chimera"

effect that is subject to both known and undiscovered physicochemical laws.

We would like to end with some practical considerations. The wound healing process is a complex process intertwined with the biological mechanisms causing individuals to become ill. Systemic and local factors combine to cause the process to become chronic and perpetuate

molecules what to do.

112 Wound Healing - Current Perspectives

*Revelation 3.4*

biomolecules (Emilio Del Giudice).

toward tissue regeneration and healing of skin wounds.

*show you what must happen in the future."*

Aragona Salvatore Emanuele1 \*, Mereghetti Giada1 , Ferrari Alessio2 and Giorgio Ciprandi<sup>3</sup>


### **References**


[6] Jones EM, Cochrane CA, Percival SL. The effect of pH on the extracellular matrix and biofilms. Advances in Wound Care. 2015;**4**:431-439

[20] Houreld NN, Ayuk SM, Abrahamse H. Expression of genes in normal fibroblast cells(WS1) in response to irradiation at 660 nm. Journal of Photochemistry and

From Tissue Repair to Tissue Regeneration http://dx.doi.org/10.5772/intechopen.81291 115

[21] Aragona SE et al. Electromagnetic field in control tissue regeneration, pelvic pain, neuroinflammation and modulation of non – neuronal cells. Journal of Biological Regulators

[22] Costin GE, Birlea SA, Norris DA. Trends in wound repair repair: cellular and molecular basis of regenerative therapy using electromagnetic fields. Current Molecular Medicine.

[23] Everts PA, Knape JT, Weibrich G, Schonberger JP, Hoffmann J, Overdevest EP, et al. Platelet-rich plasma and platelet gel: A review. The Journal of Extra-Corporeal Technology.

[24] Christgau M, Moder D, Hiller KA, Dada A, Schmitz G, Schmalz G. Growth factors and cytokines in autologous platelet concentrate and their correlation to periodontal regen-

[25] Marx RE. Platelet-rich plasma (PRP): What is PRP and what is not PRP? Implant

[26] Alio JL, Arnalich-Montiel F, Rodriguez AE. The role of "eye platelet rich plasma" (E-PRP) for wound healing in ophthalmology. Current Pharmaceutical Biotechnology.

[28] Otero-Vinas M, Falanga V. Mesenchymal stem cell in chronic wounds: The spectrum

[29] Gaur M et al. Mesenchimal stem cellfrom adipose tissue in clinical application for dermatological indications and skin aging. International Journal of Molecular Sciences.

[30] Carlo T, Valeria C, Carlo V. Adipose tissue and mesenchymal stem cells: State of the art and lipogems® technology development. Current Stem Cell Reports. 2016;**2**:304-312 [31] Zhao R et al. Inflammation in chronic wounds. International Journal of Molecular

[32] Han G et al. Chronic wound healing: A rewiew of currente management and treatments.

[33] Mario BP, Silvia C, Federica F, Eva B, Liza L, Domenico R, et al. Stem cell differentiation stage factors from zebrafish embryo: A novel strategy to modulate the fate of normal and pathological human (stem) cells. Current Pharmaceutical Biotechnology. 2015;**16**:782-792

[34] Ventura C et al. Melodie cellulari—Elettromagnetismo, musica e suono della voce per parlare alle dinamiche più profonde della nostra biologia la medicina biologica. 2017. vol. 3

from basic to advanced therapy. Advances in Wound Care. 2016;**5**:215-222

eration outcomes. Journal of Clinical Periodontology. 2006;**33**:837-845

[27] Bagnara G. Le Cellule Staminali, Cap 11. Ed Esculapio. 2017

Photobiology. 2014;**130**:146-152

2012;**12**:14-26

2006;**38**:174-187

Dentistry. 2001;**10**:225-228

2012;**13**:1257-1265

2017;**18**(1):208

Sciences. 2016;**17**:E2085

Advances in Therapy

and Homeostatic Agents. 2017;**31**(2 Suppl):219-225


[6] Jones EM, Cochrane CA, Percival SL. The effect of pH on the extracellular matrix and

[7] Aragona SE et al. Le Lesioni cutanee vascolari. Journal of Clinical Medicine and Therapy.

[8] Aragona SE et al. Chronic inflammation therapy combined with low dose cytokines in the treatment of chronic ulcers of lower limbs. Journal of Biological Regulators and

[9] Dreifke MB et al. Current wound healing procedures and potential care. Materials Science & Engineering, C: Materials for Biological Applications. 2015;**48**:651-662

[10] Khan I et al. Biophysycal approaches for oral wound healing: Emphasis on photobio-

[11] Aragona SE et al. Photobiomodulation with polarized light in the treatment of cutaneous and mucosal ulcerative lesions. Journal of Biological Regulators and Homeostatic

[12] Lipovsky A, Nitzan Y, Lubart R. A possible mechanism for visible light-induced wound

[13] Corazza AV, Jorge J, Kuraci C, Bagnato VS. Photobiomodulation on the angiogenesis of skin wounds in rats using different light sources. Photomedicine and Laser Surgery.

[14] Yadav A, Gupta A. Noninvasive red and near-infrared wavelenght-induced photobiomodulation: Promotin impaired wound healing. Photodermatology, Photoimmunology

[15] Aragona SE et al. Photobiomodulation in the treatment of chronic non–responding wounds. Journal of Biological Regulators and Homeostatic Agents. 2017;**31**(2 Suppl):201-205

[16] Kuffler DP. Photobiomodulation in promoting wound healing: A review. Regenerative

[17] Peplow PV, Chung TY, Baxter GD. Photodynamic modulation of wound healing: A review of human and animals studies. Photomedicine and Laser Surgery. Mar 2012;**30**(3):118-148

[18] Ranjbar R, Takhtfooladi MA. The effects of photobiomodulation therapy on staphilococcus aureus infected surgical wounds in diabetic rats. A microbiolgical, histopathological,

[19] Medeiros JL, Nicolau RA, Nicola EM, dos santos JN, Pinheiro AL. Healing of surgical wounds made with lambda 970-nm diode laser associated or not with laser phototherapy (lambda 655 nm) or polarized light (lambda 400-2000 nm). Photomedicine and Laser

and biomechanical study. Acta Cirúrgica Brasileira. Aug 2016;**31**(8):498-504

biofilms. Advances in Wound Care. 2015;**4**:431-439

Homeostatic Agents. 2017;**31**(2 Suppl):193-200

Agents. 2017;**31**(2 Suppl):213-218

and Photomedicine. 2017;**33**:4-13

Medicine. 2016;**11**:107-122

Surgery. Aug 2010;**28**(4):489-496

2007;**25**:102-106

modulation. Advances in Wound Care. 2015;**4**(12):724-737

healing. Lasers in Surgery and Medicine. 2008;**40**:509-514

2017;**19**(2)

114 Wound Healing - Current Perspectives


[35] Montagnier L, Aissa J, DelGiudice E, Lavallee C, Tedeschi A, Vitiello G. DNA waves and water. Journal of Physics Conference Series. 2011;**306**:012007

**Chapter 8**

**Provisional chapter**

**Growth Hormone (GH) and Wound Healing**

**Growth Hormone (GH) and Wound Healing**

DOI: 10.5772/intechopen.80978

Wound healing is complex and numerous factors overlap perfectly with the goal of wound closure. Among them, we will focus on a large amount of experimental and clinical evidence on the action of GH in wound repair. We will analyze how the physiological rhythm of GH secretion influences this process, and also one of the most important signaling pathways that mediate the effects of GH on tissue regeneration. The role of IGF-1 and the factors that stimulate GH secretion and that have also been shown to improve healing will also be reviewed. In addition, it will be analyzed the cellular senescence process, which plays a key role in nonhealing wounds associated with chronic diseases. The benefit of GH in this last circumstance is especially important. The lesions associated with catabolic states, mainly burns, are considered a delicate situation in which it is extraordinarily difficult to act with growth factors due to the fragile situation of these patients, often children. The positive action of GH in these states will also be described. In summary, we will analyze many evidences about the beneficial effects of GH and its

**Keywords:** wound healing, growth hormone, tissue regeneration, IGF-1, cellular

Wound healing represents a major challenge in medicine due to its complexity and potential severity. It is a sequential process that requires the perfect interaction of many factors and cell types. As is well known, the key aspects in wound healing are the growth of granulation tissue and the proliferation and migration of keratinocytes at the edges of the wound. For this, a series of cytokines and growth factors arriving from blood and others produced locally, act in an autocrine or paracrine manner, orchestrating the communication between cells and

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Diego Caicedo and Jesús Devesa

Diego Caicedo and Jesús Devesa

http://dx.doi.org/10.5772/intechopen.80978

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

main secretagogues in the healing of wounds.

senescence, chronic diseases, catabolic states, secretagogues


#### **Growth Hormone (GH) and Wound Healing Growth Hormone (GH) and Wound Healing**

DOI: 10.5772/intechopen.80978

#### Diego Caicedo and Jesús Devesa Diego Caicedo and Jesús Devesa

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80978

#### **Abstract**

[35] Montagnier L, Aissa J, DelGiudice E, Lavallee C, Tedeschi A, Vitiello G. DNA waves and

[36] DelGiudice E, Preparata G, Fleischmann M. QED coherence and electrolyte solutions.

[37] Ader R, Cohen N. Psychoneuroimmunology: Conditioning and stress. Annual Review

[38] Ader R, Cohen N, Felten D. Psychoneuroimmunology: Interactions between the nervous

water. Journal of Physics Conference Series. 2011;**306**:012007

The Journal of Electroanalytical Chemistry. 2000;**482**:110-116

system and the immune system. Lancet. 1995;**345**(8942):99-103

of Psychology. 1993;**44**:53-85

116 Wound Healing - Current Perspectives

Wound healing is complex and numerous factors overlap perfectly with the goal of wound closure. Among them, we will focus on a large amount of experimental and clinical evidence on the action of GH in wound repair. We will analyze how the physiological rhythm of GH secretion influences this process, and also one of the most important signaling pathways that mediate the effects of GH on tissue regeneration. The role of IGF-1 and the factors that stimulate GH secretion and that have also been shown to improve healing will also be reviewed. In addition, it will be analyzed the cellular senescence process, which plays a key role in nonhealing wounds associated with chronic diseases. The benefit of GH in this last circumstance is especially important. The lesions associated with catabolic states, mainly burns, are considered a delicate situation in which it is extraordinarily difficult to act with growth factors due to the fragile situation of these patients, often children. The positive action of GH in these states will also be described. In summary, we will analyze many evidences about the beneficial effects of GH and its main secretagogues in the healing of wounds.

**Keywords:** wound healing, growth hormone, tissue regeneration, IGF-1, cellular senescence, chronic diseases, catabolic states, secretagogues

### **1. Introduction**

Wound healing represents a major challenge in medicine due to its complexity and potential severity. It is a sequential process that requires the perfect interaction of many factors and cell types. As is well known, the key aspects in wound healing are the growth of granulation tissue and the proliferation and migration of keratinocytes at the edges of the wound. For this, a series of cytokines and growth factors arriving from blood and others produced locally, act in an autocrine or paracrine manner, orchestrating the communication between cells and

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

regulating the healing process [1–4]. In the normal repair of a tissue, the resident cells have the mission of producing these cytokines and growth factors that cooperate in their repair function. In fact, it has been discovered that some of them promote cell proliferation, angiogenesis, and synthesis of extracellular matrix (ECM) [5]. Therefore, the direct application in the wound of specific stimulating peptides is expected to increase the healing of chronic ulcers until now considered as incurable.

We currently know that growth hormone (GH) is a pleiotropic factor capable of acting positively in many organs and tissues. For years, the use of GH for wound healing has been investigated [6, 7]. For example, the use of recombinant human GH as an anabolic treatment in burns to accelerate wound healing is already classic [8, 9]. Patients with severe burns who were treated with systemic GH improved both their healing and their survival [10, 11]. More recently, a number or studies have shown that GH is a promising agent in the acceleration of wound healing [12, 13]. In addition to the stimulation of granulation tissue formation, GH increases collagen deposition, and facilitates epithelialization [14, 15]. This effect of GH has been seen in experimental models of undernourished rats, in which the administration of the hormone made the granulation tissue to grow in previously induced wounds [16]. Similar results have been found in GH-transgenic mice models [17]. Although contradictory data can be found in the literature on this particular action of GH, most studies support its benefit, and evidence of its positive effects (**Table 1**) will be described widely later in the text. It should be noticed that GH activation normally is produced in morbid conditions as catabolic or chronic diseases, and it may have no effect in healthy subjects. For example, when we artificially produce an injury in normal individuals, there are no differences about the speed of wound healing between GH-treated subjects and controls [18]. Furthermore, some data suggest that systemic GH treatment is detrimental for wound healing in healthy individuals [19]. The same is found in healthy people if we try to stimulate the immune system with GH [20]. There is much evidence to support an angiogenic effect of GH in patients with critical limb ischemia that suffer usually from ischemic ulcers; or its benefit in aging or in diabetes mellitus (DM) [21, 22]. The latter data show the specific role that the hormone can play depending on the morbid condition of the patient. **Figures 1**–**3** show the evolution of a patient with critical ischemia of the lower limbs, suffering from an ulcer, before and after 8 weeks of treatment with subcutaneous GH administration (0.4 mg/day).

The regulation of metabolic factors acting on wound healing is well known, albeit some aspects have still to be elucidated. Growth hormone-releasing hormone (GHRH) and Ghrelin are some of the most important factors controlling not only the synthesis and release of GH from the pituitary gland, but also regulating the GH receptor (GHR) and its function [23, 24]. As it will be detailed further, both hormones have also been described as having the ability to improve the healing process. Although this is not the aim of this review, at this point we cannot forget the association between Klotho and the GH/IGF-1 axis [22], especially during aging.

The key problem is to find the best way for the hormone to be administered, and the best vehicle to carry it out. However, while for GH both systemic and local administration have been demonstrated to be effective in the healing of wounds, in the case of IGF-1, the main mediator of GH actions, systemic, unlike local use, has no effect, probably because GH can have direct actions that are added to its indirect actions stimulating other growth factors involved in

wound healing. It has to be highlighted that the concentration of GH, when applied locally, and the dose, when a systemic administration is chosen, are also of importance and may determine the final effect and/or the appearance of complications. Systemic GH may increase the collagen production and mechanical strength of wounds [15, 25]. It has been reported

**Figure 1.** Five minutes reactive hyperemia test. Response to artificially induced ischemia in an affected limb with Chronic limb-threatening ischemia and a nonhealing wound. The limb is compressed until losing the flow for 5 minutes. Results after 8 weeks of systemic GH treatment. RHT: reactive hyperemia test; RHT0: ankle pressure at baseline; RHT30″: ankle pressure at 30 seconds; RHT1′-2′-3′-4′-5′: ankle pressure at 1, 2, 3, 4 and 5 minutes. y axis: mm Hg (data obtained from

**A.** Inflammation phase

• Angiogenesis:

**C.** Remodeling phase

the GHAS trial).

• Stimulates the recruitment of inflammatory cells: monocytes and T-lymphocytes by

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 119

increasing MCP-1, without changing neutrophil count.

**B.** Proliferation phase: granulation tissue formation (dose-dependent) • Diffuse wound occupation of fibroblasts and myofibroblast.

• Increase fibroblasts proliferation along with total collagen deposition.

• Increase proliferation and migration of keratinocytes, accelerating epithelization.

• High doses of GH can delay wound closure (overgrowth of granulation tissue).

• Diffuse wound occupation of inflammatory cells.

• Increase secretion of ECM: scaffold function.

◊ Boosts the formation of capillaries.

• It could improve neurogenic response.

◊ Directly, or indirectly: VEGF, FGF, or SDF-1.

• Accelerates the remodeling of the granulation tissue.

**Table 1.** Key points about evidence of GH and wound healing.

◊ Attraction of endothelial cells from the bone marrow.

**A.** Inflammation phase

regulating the healing process [1–4]. In the normal repair of a tissue, the resident cells have the mission of producing these cytokines and growth factors that cooperate in their repair function. In fact, it has been discovered that some of them promote cell proliferation, angiogenesis, and synthesis of extracellular matrix (ECM) [5]. Therefore, the direct application in the wound of specific stimulating peptides is expected to increase the healing of chronic ulcers until now

We currently know that growth hormone (GH) is a pleiotropic factor capable of acting positively in many organs and tissues. For years, the use of GH for wound healing has been investigated [6, 7]. For example, the use of recombinant human GH as an anabolic treatment in burns to accelerate wound healing is already classic [8, 9]. Patients with severe burns who were treated with systemic GH improved both their healing and their survival [10, 11]. More recently, a number or studies have shown that GH is a promising agent in the acceleration of wound healing [12, 13]. In addition to the stimulation of granulation tissue formation, GH increases collagen deposition, and facilitates epithelialization [14, 15]. This effect of GH has been seen in experimental models of undernourished rats, in which the administration of the hormone made the granulation tissue to grow in previously induced wounds [16]. Similar results have been found in GH-transgenic mice models [17]. Although contradictory data can be found in the literature on this particular action of GH, most studies support its benefit, and evidence of its positive effects (**Table 1**) will be described widely later in the text. It should be noticed that GH activation normally is produced in morbid conditions as catabolic or chronic diseases, and it may have no effect in healthy subjects. For example, when we artificially produce an injury in normal individuals, there are no differences about the speed of wound healing between GH-treated subjects and controls [18]. Furthermore, some data suggest that systemic GH treatment is detrimental for wound healing in healthy individuals [19]. The same is found in healthy people if we try to stimulate the immune system with GH [20]. There is much evidence to support an angiogenic effect of GH in patients with critical limb ischemia that suffer usually from ischemic ulcers; or its benefit in aging or in diabetes mellitus (DM) [21, 22]. The latter data show the specific role that the hormone can play depending on the morbid condition of the patient. **Figures 1**–**3** show the evolution of a patient with critical ischemia of the lower limbs, suffering from an ulcer, before and after 8 weeks of treatment with subcutaneous GH adminis-

The regulation of metabolic factors acting on wound healing is well known, albeit some aspects have still to be elucidated. Growth hormone-releasing hormone (GHRH) and Ghrelin are some of the most important factors controlling not only the synthesis and release of GH from the pituitary gland, but also regulating the GH receptor (GHR) and its function [23, 24]. As it will be detailed further, both hormones have also been described as having the ability to improve the healing process. Although this is not the aim of this review, at this point we cannot forget the association between Klotho and the GH/IGF-1 axis [22], especially during

The key problem is to find the best way for the hormone to be administered, and the best vehicle to carry it out. However, while for GH both systemic and local administration have been demonstrated to be effective in the healing of wounds, in the case of IGF-1, the main mediator of GH actions, systemic, unlike local use, has no effect, probably because GH can have direct actions that are added to its indirect actions stimulating other growth factors involved in

considered as incurable.

118 Wound Healing - Current Perspectives

tration (0.4 mg/day).

aging.

	- Diffuse wound occupation of fibroblasts and myofibroblast.
	- Increase fibroblasts proliferation along with total collagen deposition.
	- Increase secretion of ECM: scaffold function.
	- Increase proliferation and migration of keratinocytes, accelerating epithelization.
	- Angiogenesis:
		- ◊ Boosts the formation of capillaries.
		- ◊ Directly, or indirectly: VEGF, FGF, or SDF-1.
		- ◊ Attraction of endothelial cells from the bone marrow.
	- It could improve neurogenic response.
	- High doses of GH can delay wound closure (overgrowth of granulation tissue).
	- Accelerates the remodeling of the granulation tissue.

**Table 1.** Key points about evidence of GH and wound healing.

**Figure 1.** Five minutes reactive hyperemia test. Response to artificially induced ischemia in an affected limb with Chronic limb-threatening ischemia and a nonhealing wound. The limb is compressed until losing the flow for 5 minutes. Results after 8 weeks of systemic GH treatment. RHT: reactive hyperemia test; RHT0: ankle pressure at baseline; RHT30″: ankle pressure at 30 seconds; RHT1′-2′-3′-4′-5′: ankle pressure at 1, 2, 3, 4 and 5 minutes. y axis: mm Hg (data obtained from the GHAS trial).

wound healing. It has to be highlighted that the concentration of GH, when applied locally, and the dose, when a systemic administration is chosen, are also of importance and may determine the final effect and/or the appearance of complications. Systemic GH may increase the collagen production and mechanical strength of wounds [15, 25]. It has been reported

that systemic GH administration could accelerate the split-thickness skin defect in pigs [7]. However, systemic use of GH may induce side effects that must be considered when using this way. Such collateral effects are dependent on dose and time of administration. Although the topical use of GH seems to be better to reduce the possibility of side effects, unfortunately, this way of administration also present some deficiencies. Nevertheless, GH therapy has also the advantage of its relatively low cost. To produce growth factors for medical use in nonhealing wounds is costly, and hence, increasing the production of these factors by local GH

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 121

GH actions on wound healing have been evaluated in different studies from the macroscopic

During the inflammatory phase of skin wounds in mice, GH stimulated the recruitment of inflammatory cells after 3 days of topical treatment, allowing to improve the degradation of the injured tissue [26]. Monocytes, monocyte chemoattractant protein-1 (MCP-1), and T-lymphocytes play a key role in the control of the healing process. GH is a strong inductor of these cells [27–29] and activates human monocyte chemotaxis and migration [29]. A low dose of exogenous GH administration induces the expression of MCP-1 mRNA up to eight-fold [28]. However, as it will be described further, the stimulation of these immune cells by GH not only benefits inflammatory phase, but also angiogenic and neurogenic responses [19]. After analyzing the areas of wounds in the inflammatory phase when GH is used topically in mice, GH-treated mice increased the number of macrophages by about 15%, and the number

The effects of GH on the immune system have been extensively analyzed. In a model of peritonitis, GH reduced bacterial counts in the peritoneal layer and increased the number of exudative neutrophils [30]. Furthermore, GH increases the thymic mass in patients infected with human immunodeficiency virus (HIV), and the number of CD4+ T-lymphocytes [31]. In

A study in male mice in which an incision wound occurred showed that local administration of GH led to increased cellular infiltration in the wound area, mainly occupied by inflammatory cells, fibroblasts, and myofibroblasts, while in the control group (who did not receive the hormone) this type of cellular infiltration was only observed at the edges of the wound. This finding indicates that GH, directly or indirectly, had accelerated the migration and recruit-

Fibroblasts play a key role in all aspects of this process. In response to early injury signals, fibroblasts proliferate and migrate into the wound. They significantly contribute to the synthesis of the extracellular matrix (ECM), providing a scaffold for cellular ingrowth [32]. In addition, fibroblasts secrete various important cytokines with both autocrine and paracrine

**2. Experimental and clinical evidences of GH action on wound** 

of lymphocytes by 50% without changing neutrophil recruitment [26].

these cases, GH was able to restore immune function.

ment of cells, such as fibroblasts, to the site of injury [26].

effects [33–36]. This concept is schematized in **Figure 4**.

administration, could be more cost-effective.

and microscopic points of view.

**healing**

**Figure 2.** Evolution in the same patient that in **Figure 1** of the ankle-brachial index (ABI), calculated as the rate of the arterial ankle pressure divided by the arterial brachial pressure, and the arterial pressure at the ankle (measured in mmHg). Results show a positive evolution in angiogenesis, parallel to the wound evolution and the 5 minutes RHT (data obtained from the GHAS trial).

**Figure 3.** Picture of a nonhealing wound in the same patient as in **Figure 1** suffering from Chronic limb-threatening ischemia. Evolution after 8 weeks of systemic administration of GH: (A) baseline picture and (B) final picture (data obtained from the GHAS trial).

that systemic GH administration could accelerate the split-thickness skin defect in pigs [7]. However, systemic use of GH may induce side effects that must be considered when using this way. Such collateral effects are dependent on dose and time of administration. Although the topical use of GH seems to be better to reduce the possibility of side effects, unfortunately, this way of administration also present some deficiencies. Nevertheless, GH therapy has also the advantage of its relatively low cost. To produce growth factors for medical use in nonhealing wounds is costly, and hence, increasing the production of these factors by local GH administration, could be more cost-effective.

## **2. Experimental and clinical evidences of GH action on wound healing**

GH actions on wound healing have been evaluated in different studies from the macroscopic and microscopic points of view.

During the inflammatory phase of skin wounds in mice, GH stimulated the recruitment of inflammatory cells after 3 days of topical treatment, allowing to improve the degradation of the injured tissue [26]. Monocytes, monocyte chemoattractant protein-1 (MCP-1), and T-lymphocytes play a key role in the control of the healing process. GH is a strong inductor of these cells [27–29] and activates human monocyte chemotaxis and migration [29]. A low dose of exogenous GH administration induces the expression of MCP-1 mRNA up to eight-fold [28]. However, as it will be described further, the stimulation of these immune cells by GH not only benefits inflammatory phase, but also angiogenic and neurogenic responses [19]. After analyzing the areas of wounds in the inflammatory phase when GH is used topically in mice, GH-treated mice increased the number of macrophages by about 15%, and the number of lymphocytes by 50% without changing neutrophil recruitment [26].

The effects of GH on the immune system have been extensively analyzed. In a model of peritonitis, GH reduced bacterial counts in the peritoneal layer and increased the number of exudative neutrophils [30]. Furthermore, GH increases the thymic mass in patients infected with human immunodeficiency virus (HIV), and the number of CD4+ T-lymphocytes [31]. In these cases, GH was able to restore immune function.

A study in male mice in which an incision wound occurred showed that local administration of GH led to increased cellular infiltration in the wound area, mainly occupied by inflammatory cells, fibroblasts, and myofibroblasts, while in the control group (who did not receive the hormone) this type of cellular infiltration was only observed at the edges of the wound. This finding indicates that GH, directly or indirectly, had accelerated the migration and recruitment of cells, such as fibroblasts, to the site of injury [26].

Fibroblasts play a key role in all aspects of this process. In response to early injury signals, fibroblasts proliferate and migrate into the wound. They significantly contribute to the synthesis of the extracellular matrix (ECM), providing a scaffold for cellular ingrowth [32]. In addition, fibroblasts secrete various important cytokines with both autocrine and paracrine effects [33–36]. This concept is schematized in **Figure 4**.

**Figure 3.** Picture of a nonhealing wound in the same patient as in **Figure 1** suffering from Chronic limb-threatening ischemia. Evolution after 8 weeks of systemic administration of GH: (A) baseline picture and (B) final picture (data

**Figure 2.** Evolution in the same patient that in **Figure 1** of the ankle-brachial index (ABI), calculated as the rate of the arterial ankle pressure divided by the arterial brachial pressure, and the arterial pressure at the ankle (measured in mmHg). Results show a positive evolution in angiogenesis, parallel to the wound evolution and the 5 minutes RHT (data

obtained from the GHAS trial).

obtained from the GHAS trial).

120 Wound Healing - Current Perspectives

GH can act directly on endothelial cells through the GHR, or indirectly, by increasing others growth factors such as VEGF, FGF, or SDF-1; in this way, the hormone facilitates the proliferation, migration, and formation of endothelial cell tubes, as well as the attraction of that type of

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 123

The formation of blood vessels is already observed 7 days after the local administration of GH in mice. Again, the dose utilized is important, since at 10−<sup>7</sup> M doses of GH a higher number of blood vessels was produced in the granulation tissue, compared to the control group and the group treated with GH 10–8 M [26]. A similar effect was also observed after 14 days of treatment, indicating that GH maintained its proangiogenic effect during the 2 weeks of application.

All these results point out that GH is a member of those molecules that have pleiotropic actions on skin cells, and confirm previous research showing that after an injury to the skin, the process of wound healing is accelerated in GH-transgenic mice overexpressing GH [19]. In the latter study, full-thickness incisional and excisional wounds developed a highly vascularized granulation tissue. However, the bursting strength of these injuries did not increase. In these injured mice, wound closure was even delayed as a result of increased granulation tissue formation, demonstrating that, on one hand, GH can grow this essential tissue for healing, but on the other hand, at high doses, the overgrowth of granulation tissue can even delay wound closure. The authors of this study also support the fact that this action of GH on healing is probably not mediated via IGF-1 [19], in contrast to previous studies that hypothesized a direct role of IGF-1, induced by GH, in healing wounds [44, 45]. Currently, many evidences support the fact that circulating IGF-1 does not affect the wound, but that IGF-1 produced locally by fibroblasts, macrophages, and endothelial cells is the responsible for wound healing [46, 47]. Nevertheless, topically applied GH also increases the concentration of IGF-1 mRNA in the granulation tissue in vivo [48]. In any case, if the effect of IGF-1 on wound healing occurs as a consequence of the local production of IGF-1, induced by GH within the wound, it is equally important the fact that the topical administration of GH can facilitate the healing of wounds.

Furthermore, as it will be described later, some of the hormones related to the control of GH secretion, as GHRH and Ghrelin, have also shown positive effects on wound healing, show-

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is considered one of the most relevant intracellular signaling pathways utilized by hormones, growth factors, and cytokines to carry out their cellular actions [49], and it is also involved in wound healing [50]. Cell proliferation, migration, differentiation, and apoptosis are mediated

Basically, the regulation of the JAK/STAT pathway is carried out by various mechanisms such as tyrosine phosphatase, internalization-degradation of signaling molecules, receptor antagonists, and inhibitors such as inhibitors of activated STAT proteins (PIAS) or suppressors of

by this pathway [51]. When its control is altered, this promotes chronic inflammation.

ing, once again, the strong influence of GH on wound healing.

**2.1. Signaling pathways in wound healing**

*2.1.1. The JAK/STAT signaling pathway*

cytokine signaling proteins (SOCS) [52].

cells from the bone marrow through the CXCR4 receptor for SDF-1 [43].

**Figure 4.** Schematic description of the effects of GH on a wound during the early inflammatory process and stages after it. Lastly, GH also induces the acceleration of the granulation tissue and the wound is healed. Blue arrows indicate stimulation.

The role of GH in accelerating the granulation tissue has been described in previous work [37]. The cell recruitment along with collagen deposition was also accelerated in response to GH during the phase of granulation tissue. An increased mitosis and migration of keratinocyte were found after 7 days of the incision in mice treated with the hormone, parallel to the secretion of ECM to give consistency to the aforementioned granulation tissue [26]. In this study, it was observed that GH accelerated the migration and proliferation of these cells already in the first week of treatment [26], but also the analysis of the samples showed that topical treatment with GH, regardless of the concentration used, increased the total collagen deposition after 7 and 14 days of treatment. That is, GH therapy not only accelerated the remodeling of the granulation tissue, but also the epithelization, with a more stratified epidermis. Another study showed that the systemic application of GH stimulated the formation of granulation tissue in wounds of malnourished rats [16].

In vitro studies on plates coated with Matrigel® with endothelial cells have shown that GH produces a mitogen effect, which affects cell morphology, increases ECM and boosts the formation of structures similar to capillaries [38]. Some data supporting the action of GH on collagen deposition have been described in patients with acromegaly, in whom the excess of GH determines severe cardiac damage with fibrosis [22].

The role of GH in fibroblast proliferation is crucial for the wound healing process [39]. In one study, when GH was applied topically, fibroblast proliferation increased significantly, as indicated by a tetrazolium-based colorimetric assay. However, the increase in proliferation differed according to the concentration of GH, being 2.5 IU/L the best dose to stimulate the proliferation of fibroblasts [40].

Angiogenesis plays a key role during the granulation phase and tissue remodeling, as new vessels are required for the progression of wound healing. Endothelial cells express the GHR [41], and the participation of GH in the latter process has been widely demonstrated and reviewed [21, 22, 42]. Moreover, GH-transgenic mice show an increase in blood vessels during tissue repair [19].

GH can act directly on endothelial cells through the GHR, or indirectly, by increasing others growth factors such as VEGF, FGF, or SDF-1; in this way, the hormone facilitates the proliferation, migration, and formation of endothelial cell tubes, as well as the attraction of that type of cells from the bone marrow through the CXCR4 receptor for SDF-1 [43].

The formation of blood vessels is already observed 7 days after the local administration of GH in mice. Again, the dose utilized is important, since at 10−<sup>7</sup> M doses of GH a higher number of blood vessels was produced in the granulation tissue, compared to the control group and the group treated with GH 10–8 M [26]. A similar effect was also observed after 14 days of treatment, indicating that GH maintained its proangiogenic effect during the 2 weeks of application.

All these results point out that GH is a member of those molecules that have pleiotropic actions on skin cells, and confirm previous research showing that after an injury to the skin, the process of wound healing is accelerated in GH-transgenic mice overexpressing GH [19]. In the latter study, full-thickness incisional and excisional wounds developed a highly vascularized granulation tissue. However, the bursting strength of these injuries did not increase. In these injured mice, wound closure was even delayed as a result of increased granulation tissue formation, demonstrating that, on one hand, GH can grow this essential tissue for healing, but on the other hand, at high doses, the overgrowth of granulation tissue can even delay wound closure. The authors of this study also support the fact that this action of GH on healing is probably not mediated via IGF-1 [19], in contrast to previous studies that hypothesized a direct role of IGF-1, induced by GH, in healing wounds [44, 45]. Currently, many evidences support the fact that circulating IGF-1 does not affect the wound, but that IGF-1 produced locally by fibroblasts, macrophages, and endothelial cells is the responsible for wound healing [46, 47]. Nevertheless, topically applied GH also increases the concentration of IGF-1 mRNA in the granulation tissue in vivo [48]. In any case, if the effect of IGF-1 on wound healing occurs as a consequence of the local production of IGF-1, induced by GH within the wound, it is equally important the fact that the topical administration of GH can facilitate the healing of wounds.

Furthermore, as it will be described later, some of the hormones related to the control of GH secretion, as GHRH and Ghrelin, have also shown positive effects on wound healing, showing, once again, the strong influence of GH on wound healing.

### **2.1. Signaling pathways in wound healing**

### *2.1.1. The JAK/STAT signaling pathway*

The role of GH in accelerating the granulation tissue has been described in previous work [37]. The cell recruitment along with collagen deposition was also accelerated in response to GH during the phase of granulation tissue. An increased mitosis and migration of keratinocyte were found after 7 days of the incision in mice treated with the hormone, parallel to the secretion of ECM to give consistency to the aforementioned granulation tissue [26]. In this study, it was observed that GH accelerated the migration and proliferation of these cells already in the first week of treatment [26], but also the analysis of the samples showed that topical treatment with GH, regardless of the concentration used, increased the total collagen deposition after 7 and 14 days of treatment. That is, GH therapy not only accelerated the remodeling of the granulation tissue, but also the epithelization, with a more stratified epidermis. Another study showed that the systemic application of GH stimulated the formation of granulation

**Figure 4.** Schematic description of the effects of GH on a wound during the early inflammatory process and stages after it. Lastly, GH also induces the acceleration of the granulation tissue and the wound is healed. Blue arrows indicate

In vitro studies on plates coated with Matrigel® with endothelial cells have shown that GH produces a mitogen effect, which affects cell morphology, increases ECM and boosts the formation of structures similar to capillaries [38]. Some data supporting the action of GH on collagen deposition have been described in patients with acromegaly, in whom the excess of

The role of GH in fibroblast proliferation is crucial for the wound healing process [39]. In one study, when GH was applied topically, fibroblast proliferation increased significantly, as indicated by a tetrazolium-based colorimetric assay. However, the increase in proliferation differed according to the concentration of GH, being 2.5 IU/L the best dose to stimulate the

Angiogenesis plays a key role during the granulation phase and tissue remodeling, as new vessels are required for the progression of wound healing. Endothelial cells express the GHR [41], and the participation of GH in the latter process has been widely demonstrated and reviewed [21, 22, 42]. Moreover, GH-transgenic mice show an increase in blood vessels during

tissue in wounds of malnourished rats [16].

proliferation of fibroblasts [40].

tissue repair [19].

stimulation.

122 Wound Healing - Current Perspectives

GH determines severe cardiac damage with fibrosis [22].

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is considered one of the most relevant intracellular signaling pathways utilized by hormones, growth factors, and cytokines to carry out their cellular actions [49], and it is also involved in wound healing [50]. Cell proliferation, migration, differentiation, and apoptosis are mediated by this pathway [51]. When its control is altered, this promotes chronic inflammation.

Basically, the regulation of the JAK/STAT pathway is carried out by various mechanisms such as tyrosine phosphatase, internalization-degradation of signaling molecules, receptor antagonists, and inhibitors such as inhibitors of activated STAT proteins (PIAS) or suppressors of cytokine signaling proteins (SOCS) [52].

Indirect examples of the implication of this pathway in wound healing are the relationship of the same with the immune system, the main actor during healing. Inhibitors of the JAK/STAT signaling pathway are currently used to treat autoimmune diseases, including psoriasis and rheumatoid arthritis [53].

GHR is a transmembrane protein belonging to the family of receptors of class I cytokines, which homodimerizes after its binding to the ligand and signals through the family of tyrosine kinases of JAK2 and the recruitment of transcriptional factors of the STAT type, in particular isoforms 3 and 5. Intracellular signaling involves the activation by phosphorylation of different intracellular proteins, including IRS-1, MAPK, and phosphatidylinositol 3-kinase (PI3-K) [54]. Intracellular signaling induced by GH is downregulated by the family of cytokine signaling suppressors (SOCS) [55].

Different members of this pathway have been described, but their main mission is to transmit extracellular information, via specific receptors binding of the ligand and phosphorylation, to the nucleus. To describe the functioning of the latter pathway is out of the scope of this review, and we will focus on those aspects related to wound healing.

### *2.1.1.1. Role of JAK/STAT in wound healing*

Fibroblasts, endothelial cells, keratinocytes, and macrophages, are some of the cells in which cytokines and growth factors, using the JAK/STAT pathway, play a key role during the wound repair process. Pathological conditions can affect the normal functioning of this pathway, delaying the closure of the wound, and leading to the development of a chronic wound [56]. For example, Feng and colleagues studied the gene expression pattern of seven SOCSs members in tissue collected from chronic venous leg ulcer patients; they found significantly higher mRNA levels of SOCS3 and SOCS4 in chronic nonhealing ulcers as compared to healing/healed ulcers [57]. In chronic wounds, it is required that the JAK/STAT pathway be upregulated, especially in cases when its normal functioning has been compromised; more specifically in an environment of senescent cells or DM where there is a reduced growth factor/receptor signaling [53].

since in the absence of SOCS3, IL-6 acts decreasing tumor necrosis factor alpha (TNFα), by inhibiting the STAT3 signaling. It has to be highlighted that immune response, crucial in wound healing, is modulated by IL6 [59]. SOCS4 and SOCS5 have also been linked to EGF signaling, regulating its receptor (EGFR), and affecting its signaling capacity in senescent

**Figure 5.** Activation of the GHR by GH, administered subcutaneously or secreted by the pituitary gland (systemic) or locally applied on the wound (topical). (1) After the interaction between GH and its receptor (GHR) a cascade of signaling pathways is initiated by JAK2 activation leading to the expression of a number of genes. (2) The GHR may be internalized together with GH and translocated to the nucleus, where it may also activate gene expression. (3) GH and GHR may suffer a lysosomal degradation after being internalized, but also, depending on the tissue, the hormone may suffer a specific proteolytic cleavage giving origin to vasoinhibins (4) which may block angiogenesis and arteriogenesis. (5) SOCS are expressed after GHR translocation to the nucleus of the cell, and they act by inhibiting GH signaling, directly or affecting the translocation of the GHR to the nucleus. (6, 7) Cells may express GH that acts in an autocrine (6) or paracrine (7) manner. This cellular production of the hormone may lead to an interaction with the membrane GHR (8) impeding the effects of endocrine, or exogenously administered hormone, or topically applied; these auto/paracrine GHs even may produce the desensitization of GHR, therefore impeding GH actions at this level. On the left of the figure, signals induced by GH on the expression of several genes leading to several positive effects, such as angiogenesis. On the right of the figure, it can be seen that some pathological situations, such as diabetes mellitus (DM) or peripheral ischemia lead to inflammation. In this situation, Il-1β, TNF-α or endotoxins, induce the expression or activation of SOCS which block GH signaling pathways. Blue arrows: stimulation; red arrows and squares, inhibition. +: activation; −: inhibition.

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 125

The effects of the JAK/STAT pathway seem to be time-dependent: positive and protective in

Upregulation of the STAT genes and activation of the STAT proteins have been directly linked

Further investigations into cellular and molecular mechanisms and signaling pathways involved in wound healing, and methods of activating senescent cells through various treatments will add possible benefits on this process in the future; therefore GH, the anti-aging

All organisms have an adaptive mechanism, and several of their functions are synchronized to environmental factors and possess biological clocks that endogenously estimate the time. Consequently, functions such as the sleep-wake cycle and secretion of various hormones

early phases, while negative and inhibitory in the later chronic phase [60].

to wound healing in intestinal epithelial cells [61].

**2.2. GH, circadian rhythm disorders, and wound healing**

factor for excellence, could be one of them.

fibroblast cells [53].

As known, GH is one of the growth factors using the JAK/STAT pathway as an intracellular signaling pathway for exerting its actions. In fact, the inhibition of this GH signaling by SOCS members has been defended as a key factor affecting GH effect along with vasoinhibins. Moreover, GH induces the expression of CIS and SOCS1–3, which suggests that these proteins may also play a physiological role in the regulation of GH secretion. SOCS2 seems to act downregulating GH activity [58]. In this sense, pro-inflammatory cytokines such as IL-1B or TNFα, and endotoxins, which are frequently increased in inflammatory states such as DM or in patients suffering from severe peripheral ischemia, may induce SOCS proteins which could lead to a GH insensitivity. Nevertheless, SOCS2 has been found that paradoxically may upregulate GH signaling at high concentrations in mice [58]. Thus, both uncontrolled inflammation and infection at wounds may block the action of growth factors using this pathway, and delay the healing (**Figure 5**).

Although not properly known, the JAK/STAT signaling is regulated by SOCS proteins, having an influence on the action of cytokines and growth factors, as well as on the cells involved in the wound repair process [52]. SOCS have been related to inflammatory diseases,

Indirect examples of the implication of this pathway in wound healing are the relationship of the same with the immune system, the main actor during healing. Inhibitors of the JAK/STAT signaling pathway are currently used to treat autoimmune diseases, including psoriasis and

GHR is a transmembrane protein belonging to the family of receptors of class I cytokines, which homodimerizes after its binding to the ligand and signals through the family of tyrosine kinases of JAK2 and the recruitment of transcriptional factors of the STAT type, in particular isoforms 3 and 5. Intracellular signaling involves the activation by phosphorylation of different intracellular proteins, including IRS-1, MAPK, and phosphatidylinositol 3-kinase (PI3-K) [54]. Intracellular signaling induced by GH is downregulated by the family of cyto-

Different members of this pathway have been described, but their main mission is to transmit extracellular information, via specific receptors binding of the ligand and phosphorylation, to the nucleus. To describe the functioning of the latter pathway is out of the scope of this

Fibroblasts, endothelial cells, keratinocytes, and macrophages, are some of the cells in which cytokines and growth factors, using the JAK/STAT pathway, play a key role during the wound repair process. Pathological conditions can affect the normal functioning of this pathway, delaying the closure of the wound, and leading to the development of a chronic wound [56]. For example, Feng and colleagues studied the gene expression pattern of seven SOCSs members in tissue collected from chronic venous leg ulcer patients; they found significantly higher mRNA levels of SOCS3 and SOCS4 in chronic nonhealing ulcers as compared to healing/healed ulcers [57]. In chronic wounds, it is required that the JAK/STAT pathway be upregulated, especially in cases when its normal functioning has been compromised; more specifically in an environment of senescent cells or DM where there is a reduced growth

As known, GH is one of the growth factors using the JAK/STAT pathway as an intracellular signaling pathway for exerting its actions. In fact, the inhibition of this GH signaling by SOCS members has been defended as a key factor affecting GH effect along with vasoinhibins. Moreover, GH induces the expression of CIS and SOCS1–3, which suggests that these proteins may also play a physiological role in the regulation of GH secretion. SOCS2 seems to act downregulating GH activity [58]. In this sense, pro-inflammatory cytokines such as IL-1B or TNFα, and endotoxins, which are frequently increased in inflammatory states such as DM or in patients suffering from severe peripheral ischemia, may induce SOCS proteins which could lead to a GH insensitivity. Nevertheless, SOCS2 has been found that paradoxically may upregulate GH signaling at high concentrations in mice [58]. Thus, both uncontrolled inflammation and infection at wounds may block the action of growth factors using this pathway, and delay the healing (**Figure 5**).

Although not properly known, the JAK/STAT signaling is regulated by SOCS proteins, having an influence on the action of cytokines and growth factors, as well as on the cells involved in the wound repair process [52]. SOCS have been related to inflammatory diseases,

review, and we will focus on those aspects related to wound healing.

rheumatoid arthritis [53].

124 Wound Healing - Current Perspectives

kine signaling suppressors (SOCS) [55].

*2.1.1.1. Role of JAK/STAT in wound healing*

factor/receptor signaling [53].

**Figure 5.** Activation of the GHR by GH, administered subcutaneously or secreted by the pituitary gland (systemic) or locally applied on the wound (topical). (1) After the interaction between GH and its receptor (GHR) a cascade of signaling pathways is initiated by JAK2 activation leading to the expression of a number of genes. (2) The GHR may be internalized together with GH and translocated to the nucleus, where it may also activate gene expression. (3) GH and GHR may suffer a lysosomal degradation after being internalized, but also, depending on the tissue, the hormone may suffer a specific proteolytic cleavage giving origin to vasoinhibins (4) which may block angiogenesis and arteriogenesis. (5) SOCS are expressed after GHR translocation to the nucleus of the cell, and they act by inhibiting GH signaling, directly or affecting the translocation of the GHR to the nucleus. (6, 7) Cells may express GH that acts in an autocrine (6) or paracrine (7) manner. This cellular production of the hormone may lead to an interaction with the membrane GHR (8) impeding the effects of endocrine, or exogenously administered hormone, or topically applied; these auto/paracrine GHs even may produce the desensitization of GHR, therefore impeding GH actions at this level. On the left of the figure, signals induced by GH on the expression of several genes leading to several positive effects, such as angiogenesis. On the right of the figure, it can be seen that some pathological situations, such as diabetes mellitus (DM) or peripheral ischemia lead to inflammation. In this situation, Il-1β, TNF-α or endotoxins, induce the expression or activation of SOCS which block GH signaling pathways. Blue arrows: stimulation; red arrows and squares, inhibition. +: activation; −: inhibition.

since in the absence of SOCS3, IL-6 acts decreasing tumor necrosis factor alpha (TNFα), by inhibiting the STAT3 signaling. It has to be highlighted that immune response, crucial in wound healing, is modulated by IL6 [59]. SOCS4 and SOCS5 have also been linked to EGF signaling, regulating its receptor (EGFR), and affecting its signaling capacity in senescent fibroblast cells [53].

The effects of the JAK/STAT pathway seem to be time-dependent: positive and protective in early phases, while negative and inhibitory in the later chronic phase [60].

Upregulation of the STAT genes and activation of the STAT proteins have been directly linked to wound healing in intestinal epithelial cells [61].

Further investigations into cellular and molecular mechanisms and signaling pathways involved in wound healing, and methods of activating senescent cells through various treatments will add possible benefits on this process in the future; therefore GH, the anti-aging factor for excellence, could be one of them.

### **2.2. GH, circadian rhythm disorders, and wound healing**

All organisms have an adaptive mechanism, and several of their functions are synchronized to environmental factors and possess biological clocks that endogenously estimate the time. Consequently, functions such as the sleep-wake cycle and secretion of various hormones exhibit a rhythm with a characteristic period of ~24 hours (the so-called circadian rhythms). There is a relationship between feeding, the organs involved in food intake, metabolic networks, and circadian physiology. One of the most important endocrine axes involved in circadian rhythm is the axis Ghrelin-GH-IGF-1. The coordinating role of these hormones lies in regulating appetite, behavior, growth, and cell proliferation, with a clear influence in the metabolic regulation of nutrients and all those processes dependent of them, as it is wound healing. Some hormones have been implied in the regulation of circadian GH production, as cortisol, thyrotropin (TSH), and insulin, in addition to some important neurotransmitters [62].

**3. IGF-1 and wound healing**

participation of IGF-1 [27].

tal animals [85].

neous level.

sion. These limit its clinical usefulness.

IGF-1 is considered as the main mediator of GH actions, and it has been considered as the "authentic" GH, at least for growing, although GH exerts many actions directly without the

Growth Hormone (GH) and Wound Healing http://dx.doi.org/10.5772/intechopen.80978 127

IGF-1 is a polypeptide structural and functionally similar to insulin. It is produced in the liver and practically all extrahepatic tissues, and its production depends not only of GH, but also is strongly influenced by the nutritional status of the organism, at least in the liver. The local production of IGF-1 has been shown to regulate many physiological and pathophysiological states such as fetal development, atherosclerosis, and tissue repair. During tissue repair,

In wounds, IGF-1 increases protein production and cell proliferation and migration, which are crucial in the healing process [81, 82]. IGF-1 expression is enhanced in subcutaneous [5], and incisional [83] wounds, and in postburn injuries [84]. Some studies have shown that the administration of exogenous IGF-I enhanced protein synthesis in severely burned experimen-

Moreover, the levels of this growth factor are reduced in the wound environment of diabetic patients. Wound-related parameters as proteins, DNA, hydroxyproline, and macrophages have been shown to be decreased as a consequence of diabetes. After 14 days of treatment with IGF-1 in rats with diabetes produced by streptozotocin, it was observed that the total values of hydroxyproline, DNA, proteins, and macrophages increased by 48, 52, 31, and 40%, respectively [5]. These data support the fact that the suppression of IGF-1 and the macrophage function impairment within the wound environment by the diabetic state are responsible, at

In this context, the relationship between the IGF-1 receptor (IGF-1R) and the estrogen receptor (ER) is of interest. Locally administered IGF-1 promotes wound repair in an estrogen-deprived animal model, the ovariectomized (Ovx) mouse, mainly by dampening the local inflammatory response and promoting re-epithelialization. Using specific IGF-1R and ER antagonists it has been shown how IGF-1-mediated effects on re-epithelialization were directly mediated by IGF-1R [86]. In contrast, the anti-inflammatory effects of IGF-1 were predominantly mediated by ERs, in particular ERa (**Figure 6**). When ERa-null mice were used, IGF-1 could not promote healing and local inflammation increased [86]. These findings illustrate the great complexity of interactions between growth factors at the cuta-

Recent data on the systemic administration of IGF-1 have shown an apparent lack of effect in wound healing. Therefore, perhaps only the IGF-1 produced locally by fibroblasts and macrophages contributes to the regulation of wound healing [46, 47], although it is also possible that the dose used and the type of administration do not have been the most appropriate in this case. If the systemic IGF-1 is ineffective in wound healing, topical administration of IGF-1 could be considered, as other growth factors such as EGF, TGFβ, or the own GH. In addition, IGF-1 systemic administration produces mild complications as hypoglycemia and hypoten-

IGF-1 is secreted by platelets, macrophages, and fibroblasts of the wound [5].

least in part, for the delay of wound healing in this disease.

Although GH is mainly released by the anterior pituitary gland, there is a peripheral GH production in practically all the organism, highly dependent on developmental stages, at the level of tissues as nervous system, or the immune, cardiovascular, gonadal, and musculoskeletal system. This peripheral GH plays an autocrine and paracrine role [63]. In humans, plasma GH shows a circadian pattern of secretion, different according to sex and age; during puberty, the hormone reaches its highest plasma values, but once puberty ends, the secretion of the hormone begins to decline until being practically undetectable in elder people [64].

The circadian pattern of GH is affected by nutritional status (caloric intake), age, stress, sex, physical exercise, and lack of sleep. Nutritional status is a key determinant in the regulation of GH secretion; thus, while fasting increases the frequency of GH secretion pulses, while IGF-1 levels decrease, in obesity the opposite occurs, at least during childhood [65]. During fasting, GHR are downregulated [66, 67]. Evidence for a circadian effect on the reduction of human GH gene expression has been demonstrated in response to excess caloric intake [68], and obesity has been associated to the suppression of circulating GH [69].

The highest pulse amplitude of GH secretion is observed during the REM phase of the sleep, while sleep deprivation leads to a strong inhibition of nocturnal GH secretion [70, 71].

Several studies have shown that prolonged sleep deprivation, with the subsequent stress, leads to a reduction in body mass, elevated energy metabolism, changes in circulating hormones, and loss of immune system integrity [72]. Stress mediators act on immune cells to modulate the production of key regulatory cytokines [73, 74]. Thus, circadian rhythm disorders affect the levels of IL-1, IL-2, IL-6, TNFα, natural killer cells, adrenocorticotropic hormone (ACTH), cortisol, GH, and melatonin, all of them playing a key role in wound healing [75–77]. Melatonin has potential effects on the immune system, as inhibition of pineal melatonin synthesis with propranolol or pinealectomy results in immunosuppression and negative effects on wound healing [78]. Yet another study found that melatonin improved wound healing when given at night, coinciding with its normal circadian period of secretion [79].

Notwithstanding all these data, some studies disagree with the concept that sleep exerts a predominant influence on GH release and its effects whatever the conditions be, as it seems to occur compensatory mechanisms promoting GH pulses during wakefulness [80].

Thus, GH influences on wound healing progression. Physiologic circadian rhythm, with higher levels of the hormone during the night, will make a faster healing of wounds during the night, and the alteration of this pattern by different factors might exert a deleterious effect on wound healing via GH and others hormones related to it, although compensatory mechanisms have been described in the long-term.
