Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes

*Mohammad Bayat and Sufan Chien*

## **Abstract**

Wound healing is an active and compound biological course which can be divided into four steps: hemostasis, inflammation, proliferation, and remodeling. Diabetes mellitus induces weakened wound healing by disturbing one or more of the biological functions of these steps. Diabetic foot ulcers result from the simultaneous action of multiple disturbing causes. Mesenchymal stem cells, especially autologous ones, are easily accessible with noninvasive methods and have been shown to provide a regenerative microenvironment at wound sites. Despite current knowledge, major hurdles remain to be overcome in order to achieve effective therapeutic effects. Photobiomodulation is the use of light to reduce pain and inflammation and stimulate healing and the proliferation of stem cells, which would be very useful in increasing stem cell function and in regenerative medicine. The current study analyzes the results of studies using separate and combined administrations of stem cells and photobiomodulation on diabetic wound healing in patients and animal models. We hypothesize that the combined application of photobiomodulation and stem cells will accelerate the repair process and assist the healing of foot ulcers in diabetes mellitus patients.

**Keywords:** wound healing, diabetes mellitus, diabetic foot ulcers, mesenchymal stem cells, adipose tissue-derived stem cells, photobiomodulation

## **1. Introduction**

Diabetes mellitus (DM) is the most important cause of illness and death, affecting 422 million adults worldwide [1]. Epidemiological studies of DM in the U.S. have shown that almost one out of every three people in the U.S. is prone to preDM or suffering from DM. The Centers for Disease Control and Prevention (CDC) recently reported that more than 100 million adults in the U.S. have DM or pre-DM. In 2015, a total of 30.3 million people of all ages, or 9.4% of the U.S. populace, were reported to have DM. Moreover, a total of 33.9% of the U.S. adults aged 18 years or older (84.1 million people) had pre-DM in 2015. Almost half (48.3%) of adults aged 65 years or older have pre-DM which, if left untreated, will develop into Type 2 DM within 5 years [2]. Of the entire population of the U.S., 33% are predicted to be afflicted by DM by the year 2050 [3]. Diabetic foot ulcer (DFU) is still the predominant cause of hospitalization for patients with DM,

and DM is the chief reason for more than 50% of nontraumatic leg amputations. Obviously, these operations increase the death ratio [3].

In this chapter notes are provided about the following subjects: 2, acute wound healing in healthy subjects; 3, a mechanistic approach to wound healing in DM; 4, DFU;5, administration of stem cells in DFUs; 6, adipose tissue-derived stem cells (ADSC); 7, regenerative potential of ADSC; 8, PBM and its effects on cells and stem cells; 9, how the combined application of photobiomodulation (PBM) and ADSCs accelerates healing in DFU; and 10, finally we will deliver our conclusions in section 10.

## **2. Acute normal skin injury repair course in healthy subjects**

The acute normal skin injury repair course can be separated into four overlying steps: 1. coagulation; 2. inflammation; 3. proliferation; and 4. remodeling. During the first step, blood-clotting actions preclude extreme hemorrhage and deliver temporary protection to the injured area. The development of inflammation directs the use of leukocytes, neutrophils, and macrophages; the creation of growth factors; and the stimulation of fibroblasts, keratinocytes, and angiogenesis. Achievement of the proliferation step in wound repair directs the creation of extracellular matrix (ECM), i.e. rich, vascularized granulation tissue. Lastly, ECM maturation and cell apoptosis direct the creation of scar tissue with physical features that are similar to unwounded skin [4]. The repair of an acute skin injury comprises synchronized cellular and molecular responses. First, immune cells migrate to the injury site, then they initiate pathogen clearance, while also participating in the repair course. Cut epidermal borders upregulate wound-related genes, thereby allowing mutual cell migration. Local and blood-borne fibroblasts increase and migrate to produce wound granulation tissue, provide organization and signaling clues, and deliver new ECM. Some fibroblasts differentiate into myofibroblasts to help wound closure. The wound bed is perfused with oxygen and nutrients through new blood vessels derived by angiogenesis [5].

## **3. A mechanistic approach to wound healing in DM**

DM causes the repair course directed to a non-healing wound (chronic wound or ulcer) to lag, resulting in practical restrictions, gait trouble, and contamination. The weakening of repair in DM patients is well-known, but the connection between pathophysiology and weakened skin injury repair in DM is still an unidentified etiology. The repair course requires cooperation between inflammatory cells and biochemical mediators encouraged by many elements. Nevertheless, alterations in the cellular and biochemical elements and accomplishments are concerns associated with wound healing failure in DM patients. Neutrophils, monocytes, macrophages, keratinocytes, fibroblasts, T and B cells, mast cells, and endothelial cells all contribute to wound repair and dynamically to the creation and regulation of various cytokines and growth factors. Monocytes, which later transform into macrophages, are the principal manufacturers of pro-inflammatory cytokines, including interleukin-1 (IL) -1β, tumor necrosis factor (TNF)-α, IL-6 and cytokines, and growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF) - β in both healthy and diabetic subjects. Neutrophils, such as T and B cells, are also important producers of TNF-α and IL-10 cells among others, keratinocytes, fibroblasts, mast cells, and endothelial cells which participate in the production of VEGF, IGF-1, and TGF-β.

#### *Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.96905*

Macrophages are fundamental providers in healing. Hyperglycemia and oxidative stress alter the epigenetic code that results in alterations to the polarization and plastination of macrophages. Dysregulated macrophage polarization is one of the key hindrances to wound repair. Investigations have revealed that in DM, a compound function is included at the molecular level which is accountable for hindered wound repair. Actions like the continued production of pro-inflammatory cytokines, weakened angiogenic response and microvascular difficulties, weakened macrophage and neutrophils function, weakened keratinocytes and fibroblast migration, and increased and weakened creation of healing-associated elements like decreased growth factor creation have been reported in animal simulations of DM. The steps of the remedial course in diabetic sufferers are also delayed (in the inflammatory stage) by other elements as well as specific metabolic insufficiencies, weakened functional responses like hypoxia due to glycation of hemoglobin, and changes in red blood cell membranes and the tapering of blood vessels. Hypoxia reduces the oxygen stream to wounds because of tapering blood vessels. Hemoglobin glycation causes a lack of nutrients and oxygen to tissue, which further interrupts the repair course. Diabetic wounds continuously stimulate the unfolded protein response (UPR) and increase expression of pro-inflammatory chemokine in comparison with normal wounds. Native ischemia because of microvascular problems in DM significantly delays the repair course [6].

Reduced IGF-1 and TGF-β values at sites of tissue injury have been described in both diabetic animals and human (h) s with DM and are accountable for delayed repair to skin injuries. TGF-β employs and encourages the motivation of inflammatory cells, including neutrophils, macrophages, and lymphocytes, as well as keratinocytes, fibroblasts, and the creation of growth factors, which hasten neovascular formation, and the creation and delayed deterioration of ECMs. The decreased attentiveness of TGF-β has been described in skin injury repair in diabetic subjects. Many studies have proven that matrix metalloproteinase (MMP)-encoding genes have a TGF-β1-dependent preventative component in the promoter region, which down-regulates expression of the gene. Reduced TGF-β values and improved expression of MMPs induce the extreme deterioration of growth factors. Accompanied by MMP-encoding genes, transcription factors like Smad-2, Smad-3, and Smad-4 also trigger and suppress TGF-β target genes. TGF-β1 triggers Smad-2 and 3 for the creation of collagen. Reductions in TGF-β1 values augment the use of triggered inflammatory cells to hinder progression from the inflammatory step to the proliferation step in the repair course of diabetic wounds. Elevated TGF-β3 values are supposed to reduce TGF-β1 values in diabetic subjects, which leads to augmented macrophage action and reduced collagen creation. In DM, elevated glucose levels increase macrophage action, directing more reactive oxygen species (ROS) to extend the inflammatory step. Reduced values and expression of these growth factors weaken and extend the skin injury repair course in DM [6].

## **4. DFU**

Disturbances in the coordination of glucose homeostasis induce hyperglycemic prominence and result in the initiation of certain metabolic pathways that, in their unusual situation, lead to the progression of vascular deficiency, nerve damage caused by ulcerations in inferior limbs because of changed patterns of plantar pressure, and consequently foot abnormalities. Abuse to the foot produced by trauma to the affected area remains hidden to the patient because of damage to afferent sensory nerves [7]. Diabetic neuropathy results in foot muscular inequality, inadequate feeling in the skin, and ultimately foot irregularities that lead to augmented force applied to the skin when walking. Collectively, the above-mentioned occurrences are accompanied by foot ischemia [8] and DFU formation.

When a foot ulcer develops, the foot is at increased risk for aggressive infection, and as soon as it is combined with a peripheral artery occlusive disease, the sufferer will have dangerous foot ischemia [8]. Thus, the etiology for DFU is composite. Disruption of harmony in glucose homeostasis causes hyperglycemic status, results in activation of certain metabolic pathways which in their abnormal state subsequently leads to development of vascular insufficiency, nerve damages headed by ulceration in lower extremity due to plantar pressures and foot deformity. Staphylococcus is the most common infectious bacterium [9]. A diabetic foot infection may be a warning limb complaint. Infection is identified by the occurrence or augmented ratio of inflammation markers. Frequently, these markers are less noticeable than anticipated. Imaging investigations can identify or better define profound, soft tissue-infected areas and are regularly required to detect pathological results in bone. The primary bactericidal cure as well as the length of cure are observational. There is a considerable delay in DFU injury repair that has been correlated to many irregularities [9]. Today, DM is the chief origin of nontraumatic amputations in the U.S. Generally, around 5% of DM patients develop DFUs, and 1% of them wind up with an amputation. Around 60% of diabetic patients will develop neuropathy, ultimately leading to a DFU. The danger of a DFU is augmented in people with flatfoot, as they apply uneven pressure across the foot, leading to local inflammation in risky areas of the foot. The yearly occurrence of DFU ranges from 9.1 to 26.1 million cases globally, and about 15% to 25% of DM patients will develop a DFU sometime during their lifespan.

As the number of newly identified DM cases rises annually, the occurrence of DFU is also destined to rise. DFUs are accountable for higher medical charges than any other diabetic difficulty. The usual cost of curing one DFU is \$8,000, that of an infected DFU is \$17,000, and that of a chief amputation is \$45,000. Over 80,000 amputations are done yearly on diabetic patients in the U.S., and approximately 50% of patients with amputations will develop ulcers and infections in the other foot within 1.5 years. Sadly, 58% of people with DM will experience a second amputation 3–5 years after the first one. Furthermore, the prevalence of death occurring 3 years after a first amputation has been estimated to be as high as 20%–50%, and these statistics have not altered considerably in the past 30 years despite major developments in the medicinal and surgical management of DM patients [10]. Management of DFUs is mainly based on severity (score), blood vessel status, and the existence of contamination. Inhibiting the reappearance of DFUs remains a chief medical objective [11]. Numerous novel cures correlated to these aberrations have been discovered in wound repair with differing achievements [9].

#### **5. Administration of mesenchymal stem cells (MSC) in DFUs**

As previously described, DFUs are one of the more frequent and severe difficulties of DM, as wound repair is weakened in the diabetic foot. Investigations concentrated on comprehensively understanding these functions could allow for a precisely directed cure for DFUs. The main treatments for DFUs are currently wound debridement, weight off-loading, neovascularization, and contamination treatment. Nevertheless, some DFUs are extremely impervious to routine cures, and the development of wound repair remains to be the goal of numerous cure policies. Novel cure choices such as bioengineered skin substitutes, ECM proteins, cytokines,

#### *Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.96905*

and negative pressure wound therapy, have been developed as supplementary remedies for DFUs [12]. Stem cell therapies have appeared as top-notch cure methods with the possibility of returning tissue to its pre-injury state.

The use of cellular therapy in the treatment of skin injuries is presently a dynamic field of research. Multi-potent adult stem cells are an attractive option for cell therapy, as they have a high possibility of proliferation and the capability of differentiating into diverse cell types and creating a range of cytokines and growth factors essential to wound repair. This study concentrated on the involvement of three types of adult stem cell populations through a skin injury repair course and their beneficial possibilities for use in cell therapy.

Endothelial progenitor cells (EPCs) are endothelial precursors involved in the revascularization of injured tissue and tissue repair. Their vascular repairing potentials have been described in a range of translational and human investigations into ischemic illnesses, together with myocardial infarction, stroke, and peripheral arterial illness. Furthermore, numerous articles have stated that EPC engraftment can enhance wound repair by improving new blood vessel formation in granulation tissue. It has been reported that the administrated EPCs released a variety of wound repair-related growth factors and cytokines, thus encouraging the implementation of monocyte/ macrophage and exciting endogenous new blood vessel formation during the course of skin injury repair. Another study showed that the transplantation of human cluster of differentiation (CD) 133+ progenitor cells into streptozotocin-induced diabetic mice amplified the wound closure rate and capillary density in granulation tissues. These results suggest that EPC engraftment would be favorable for the cure of skin wounds, specifically chronic wounds which are often connected with reduced peripheral blood flow and continue to be tough to heal using existing beneficial tactics [13].

Bone marrow-derived mesenchymal stem cells (BM-MSCs), comprise another talented nominee for the reparation or substitution of injured tissue. BM-MSCs have the ability to differentiate into numerous lineages, such as endothelial cells, neural cells, and hepatocytes, among others. Furthermore, research has shown that BM-MSCs participate in wound repair by differentiating into numerous cutaneous cell types. It has further been reported that BM-MSCs differentiate into keratinocytes, endothelial cells, pericytes, and monocytes. One study reported that BM-MSCs significantly improved wound repair in both diabetic and nondiabetic mice; BM-MSC-treated wounds displayed augmented wound contraction by discharging proangiogenic elements including VEGF and angiopoietin-1. Analysis of paracrine elements released from BM-MSCs with real-time polymerase chain reaction (PCR) and of BM-MSC-CM by enzyme-linked immunosorbent assay (ELISA) showed that BM-MSCs secreted VEGF, IGF-1, epidermal growth factor (EGF), keratinocyte growth factor (KGF), angiopoietin-1, and stromal derived factor (SDF)-1. These paracrine elements from MSC-condition media (CM) displayed a pronounced influence in utilizing CD14<sup>+</sup> monocytes, keratinocytes, and endothelial cells in injured tissue, thus encouraging the skin injury repair course [13].

## **6. Adipose tissue-derived stem cells (ADSCs)**

ADSCs are placed inside the stromal vascular fraction of adipose tissue. They have the ability to differentiate into adipogenic, osteogenic, chondrogenic, and myogenic cells when they are cultivated in particular culture circumstances. New information has shown the possible effects of ADSCs on new blood vessel formation in ischemic illness animal simulation. ADSCs discharge numerous powerful antigenic elements and were also shown to collaborate in angiogenesis by differentiating into endothelial cells in an *in vivo* study. The engraftment of ADSCs is reported to

encourage wound contraction and enhance blood perfusion in injured skin. When ADSCs were cultivated in hypoxic circumstances, they released VEGF 5-time more than in normoxic circumstances [13].

In regenerative medicine, adult stem cells are the greatest encouraging cell types for cell-based therapies. Human adipose tissue has been presented as a novel origin for multipotent stem cells. These so-named ADSCs are considered perfect for use in regenerative therapies. Their chief benefit over MSC extracted from other origins, e.g., from bone marrow, is that they can be simply and repeatable collected using negligibly aggressive methods with little injury. ADSCs are multipotent and can differentiate into numerous cell types. Interestingly, ADSCs are categorized by immunosuppressive properties and have little immunogenicity. Their discharge of trophic elements make compulsory the healing and regenerative results in an extensive variety of administrations. Generally, these specific characteristics of ADSCs make them very much applicable for medical uses. Therefore, the beneficial probability of ADSCs is huge [13].

## **7. Regenerative potential of adipose tissue-derived stem cells**

The beneficial impacts of ADSCs have been determined to be valuable in regenerative therapies for many illnesses. Specifically, ADSCs can be collected, handled, and cultured in a nominally aggressive, yet calm and persuasive method, and they have the great probability of differentiating into mature cells along the mesodermal, ectodermal, and endodermal lineages. Throughout recent years, crucial advancements have been made concerning the separation, morphological features, molecular biology, and in vitro differentiation potential of stem cells, and it has become clear that ADSCs might facilitate beneficial effects. Not only do they act as tissue-specific progenitor cells, but they also participate in a number of chief functions, e.g., paracrine-mediated signaling of angiogenesis, inflammation, cell homing, and cell survival. The above-mentioned essential results have assisted us in gradually closing the hole between basic knowledge and clinical application; meanwhile, ADSCs have been used in clinical trials all over the globe, presenting as harmless and realistic options in a range of simulations.

Nevertheless, before ADSCs can be used in conventional medical administrations, numerous obvious queries associated with ADSCs must be resolved. With the intention of fully appreciating the fundamental functions which control ADSCs, future experimentations should, for example, concentrate on additional accurate markers for the improved and source-precise classification of ADSCs. Moreover, the genetic alteration of ex vivo-cultivated cells should not be ignored, and the controllers concerning differentiation, migration, and cell viability after in vivo engraftment must be clarified. Furthermore, as the scientific comprehension of the regenerative capabilities and, therefore, the potential uses of ADSCs increases, the possible dangerous threats must be addressed, and the supervisory outline that directs their medical usage must be established. Presently, precise supervisory instructions are set by the country in which treatment occurs. Clearly, the worldwide standardization of rules of use is crucial. In conclusion, scientific advancements, supervisory rules, and a commercial substructure are all vital factors in the development and conversion of this talented MSC origin [14].

Conversely, there are some methodological questions for the application of MSCs and ADSCs for skin regeneration in DM patients, as discussed below.

1.The elevated extracellular glucose density in diabetic wounds leads to the collection of advanced glycosylation end products (AGEs). The creation

*Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.96905*

of AGEs prevents proliferation, leads to hADSCs apoptosis, prevents the differentiation, proliferation, and homeostasis of ADSCs into endothelial cells, and also prevents the production of collagen protein, ultimately hindering wound repair [15].


## **8. PBM and its effect on cells and stem cells**

The term "LASER" originated as an acronym for "light amplification by stimulated emission of radiation." Laser radiation could encourage a photobiomodulatory impact on cells and tissues, participating in a concentrating inflection of cell behaviors, increasing the courses of tissue repair. PBM, also recognized by its former term low-level laser therapy (LLLT), is a safe technique that participates in pain reduction and decreases inflammation, along with improving cure and tissue healing. It also encourages cell propagation and increases stem cell differentiation [24]. PBM is a fast-developing technology applied many medical situations where stimulus of repair, decrease in pain and inflammation, and renovation of action are needed. While skin is obviously exposed to light more than any other organ, it still reacts fine to red and near-infrared wavelengths. The photons are absorbed by mitochondrial chromophores in skin cells. Therefore, electron transport, adenosine triphosphate nitric oxide release, blood flow, ROS increase, and various signaling paths are triggered. Stem cells can be activated, permitting augmented tissue repair and healing [25]. PBM, with its above-mentioned properties, can mediate numerous illnesses and circumstances, such as DM, brain damage, spinal cord injury, dermatological circumstances, oral annoyance, and diverse fields in dentistry. Most studies have reported a rise in the propagation ratio of radiated cells [24]. PBM definitely controlled the in vitro propagation of the ADSC examined, and new in

vitro documents have been presented where PBM meaningfully augmented hAD-SCs cell survival in comparison to control and PBM-treated hBM-MSC groups [26].

## **9. How the combined application of PBM and ADSCs can accelerate DFU healing**

Concerning the low viability ratio of ADSCs transplanted onto a wound, the application of some superior pretreatment agents not only makes available a good biological circumstance for the transplanted ADSC, but also encourages their propagation, differentiation, and paracrine capabilities and causes them to discharge more cytokines and growth factors [27]. Because PBM can augment the proliferation ratio of cultivated ADSCs [26], it can be considered as an effective approach for the preconditioning of ADSCs in in vitro situations preceding ADSC transplantation. In Zare et al. study [26] both in vitro human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and h adipose-derived stem cells (hADSCs) were irradiated with 36 protocols using two different laser types (helium-neon [He-Ne] and diodes), four different laser wavelengths (HeNe laser, 630 nm, 810 nm, 630 + 810 nm); three different energy densities (0.6 J/cm2 , 1.2 J/cm<sup>2</sup> , 2.4 J/cm<sup>2</sup> ); and three different PBM times (1, 2, and 3). A total of 1 × 104 MSCs were seeded in each well of a 24 well-plate. Next, the He–Ne laser at 632.8 nm, (IR-2000; IAEA, Tehran, Iran), red laser at 630 nm, NIR laser at 810 nm, and 630 nm +810 nm (NILTVIR202 Noura Instruments, Tehran, Iran) were applied. It should be mentioned that immediately after switched on the laser machine, it was ready for PBM therapy. In order to ensure exposure of the entire well (15.6 mm well) to PBM, the He–Ne laser emission was expanded by an optic culminator and the spot size of the red and NIR lasers were increased by a cone shaped pine hole culminator. Control MSCs did not receive PBM. **Table 1** lists the PBM protocol specifications.

Zare et al. study [26] demonstrated that PBM with the combined 630 + 810 nm lasers significantly stimulated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which was measured the effects of PBM on MSC viability, and significantly decreased population doubling time (PDT) and apoptosis rate of hBM-MSCs and hASDCs in vitro. There were no pharmacological side effects of PBM on MSC as evidenced by measuring apoptosis rate of MSCs. Zare et al. reported new in vitro evidence where PBM administered at 630 nm (one and two times, 0.6 and 1.2 J/cm2 ) and 630 + 810 nm (three times, 2.4 J/cm2 ) significantly increased hADSC cell viability compared to its control and the PBM-treated hBM-MSC groups. PBM-based medical trials and experiments will display new uses for PBM and MSC remedies [28].


#### **Table 1.** *Specifications of the photobiomodulation (PBM) protocol.*

*Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.96905*

Accordingly, Ahmadi et al. examined the efficiency of several preconditioned ADSCs and PBM regimes on healing an infected ischemic delayed-healing wound in type 1 diabetic rats. Their study included five groups of rats: (1) control, (2) control ADSCs [diabetic ADSCs were engrafted into the wound bed], (3) ADSCs + PBM in vivo (diabetic ADSCs were transplanted into the wound, followed by in vivo PBM therapy), (4) ADSCs + PBM in vitro, and (5) ADSCs + PBM in vitro + in vivo.

Ahmadi et al. for in vitro study seeded a total of 1 × 104 passage-4 ADSCs in each well of a 24-well plate for each of three groups: healthy control ADSC, diabetic control ADSC, and experimental diabetic ADSC. Here, red laser alone plus infrared laser alone (NILTVIR202 Noura Instruments, Tehran, Iran) at two energy densities (1.2 J/cm<sup>2</sup> and 2.4 J/cm<sup>2</sup> ) were used to irradiate the ADSC every other day for three sessions according to a previously published protocol. Ahmadi et al. found that diabetic ADSCs preconditioned with PBM had significantly increased the MSC viability, and significantly decreased PDT, and apoptotic rate of ADSCs in comparison with diabetic ADSCs. **Table 2** lists the in vitro, and in vivo PBM parameters. The control ADS did not receive PBM. The wounds of the rats in groups 3 and 5 were subjected to PBM in vivo (**Figure 1**).


**Table 2.**

*Specifications of in vitro and in vivo photobiomodulation parameters.*

#### **Figure 1.**

*A photo of the wound, photobiomodulation (PBM) target points, and adipose tissue -derived stem cell (ADSCs) injection points.*

**Table 2** lists the complete specifications of the PBM protocols for invitro and in vivo studies. There were no pharmacological side effects of PBM on MSC in Ahmadi et al. study as evidenced by histological examination of wounds.

Groups 3 and 5 showed significant reductions in bacterial contamination compared to groups 1 and 2. Groups 2, 3, 4, and 5 showed significantly enhanced wound contraction ratios in comparison with group 1. Groups 2–5 displayed

*Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.96905*

significantly increased wound strength compared to group 1.In most cases, group 5 had significantly better results than groups 2, 3, and 4. Ahmadi et al. concluded that preconditioning diabetic ADSCs with PBM in vitro plus PBM in vivo significantly accelerated healing in the diabetic rat model of an ischemic infected delayed-healing wound [29]. In other related studies, the same results were reported. Khosravi et al. reported that the in vitro preconditioning of hADSCs with PBM significantly amplified bone repair in a rat model of critical size femoral defect in vivo [30]. Liao et al. explored the therapeutic potential of hADSCs preconditioned with PBM. Cultured ADSCs were treated with PBM. In addition, a mouse photoaged skin simulation was proven by UVB radiation. Liao et al. concluded that PBM is a persuasive bioenhancer of ADSCs and may improve the healing possibility of ADSCs for medical use [31]. While few studies give some evidence for the positive effects of PBM alone for wounds in diabetic patients [32], or PBM plus skin grafts for burn ulcers in diabetic patients [33, 34], there have been no clinical trials using human models to show stem cells plus PBM as an effective agent in wound care regimes to date. Further well designed clinical trials are necessary to determine the true value of ADSCs plus PBM in routine wound care regimes for patients with DM.

## **10. Conclusions**

Present knowledge dictates that when an organ is healthy, the inflammatory phase of wound healing is well orchestrated, lasting only a few days, and the steps of tissue repair proceed normally. However, when an organ is hyperglycemic, as with DM, the inflammatory process is extended, the integrity of the skin is not restored, and DFU occurs. DFUs are a serious clinical problem and affect millions of people around the world. They need repetitious cures, impose extensive medical expenses, and create a major economic burden on healthcare systems worldwide. Thus, much work has been concentrated on evolving new healing approaches for wound treatment. Preclinical studies have shown that preconditioning diabetic ADSCs with PBM in vitro significantly increases ADSC function over that of diabetic ADSCs. Preconditioning diabetic ADSC with PBM significantly hastened healing in ischemic MRSA–infected, delayed-healing wounds in rats with type one DM compared to the control, ADSC alone, and ADSC plus PBM-in vivo rats. The combined administration of preconditioned diabetic-ADSC with PBM plus PBM therapy in vivo demonstrated a significantly superior effect compared to other treatment protocols [29].

Whereas our hypothesis (combined application of PBM and stem cells can accelerate repairing process and assist healing DFU in animal models and patients) was confirmed through preclinical studies [29, 30, 31], we suggest further animal and clinical trial investigations be conducted in order to provide more documentation. Hopefully these outcomes would help the use of ADSCs plus PBM as a routine treatment protocol for the healing of severe DFU in patients with DM.

We confirm there were no conflicts of interest.

*Recent Advances in Wound Healing*

## **Author details**

Mohammad Bayat1,2\* and Sufan Chien2

1 Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Price Institute of Surgical Research, University of Louisville, and Noveratech LLC of Louisville, United States

\*Address all correspondence to: bayat\_m@yahoo.com

© 2021 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.

*Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.96905*

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[20] M.M. Martino, K. Maruyama, G.A. Kuhn, T. Satoh, O. Takeuchi, R. Müller, S. Akira, Inhibition of IL-1R1/MyD88 signalling promotes mesenchymal stem cell-driven tissue regeneration, Nature communications. 2016; 7: 11051. DOI: 10.1038/ncomms11051

[21] W. Lin, L. Xu, S. Zwingenberger, E. Gibon, S.B. Goodman, G. Li, Mesenchymal stem cells homing to improve bone healing, Journal of orthopaedic translation. 2017; 9: 19-27. DOI: 10.1016/j.jot.2017.03.002

[22] Y. Wu, L. Chen, P.G. Scott, E.E. Tredget, Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis, Stem cells. 2007; 25: 2648-2659. DOI: 10.1634/ stemcells.2007-0226

[23] G. Muhammad, J. Xu, J.W. Bulte, A. Jablonska, P. Walczak, M. Janowski, Transplanted adipose-derived stem cells can be short-lived yet accelerate healing of acid-burn skin wounds: a multimodal imaging study, Scientific reports. 2017; 7: 1-11. DOI: 10.1038/ s41598-017-04484-0

[24] C. Dompe, L. Moncrieff, J. Matys, K. Grzech-Leśniak, I. Kocherova, A. Bryja, M. Bruska, M. Dominiak, P. Mozdziak,

T.H.I. Skiba, Photobiomodulation— Underlying Mechanism and Clinical Applications, Journal of Clinical Medicine. 2020; 9: 1724. DOI: 10.3390/ jcm9061724

[25] P. Avci, A. Gupta, M. Sadasivam, D. Vecchio, Z. Pam, N. Pam, M.R. Hamblin, Low-level laser (light) therapy (LLLT) in skin: stimulating, healing, restoring, Seminars in cutaneous medicine and surgery, NIH Public Access., 2013: 41. PMID: 24049929.

[26] F. Zare, A. Moradi, S. Fallahnezhad, S.K. Ghoreishi, A. Amini, S. Chien, M. Bayat, Photobiomodulation with 630 plus 810 nm wavelengths induce more in vitro cell viability of human adipose stem cells than human bone marrow-derived stem cells, J Photochem Photobiol B. 2019;201: 111658. DOI: 10.1016/j.jphotobiol.2019.111658

[27] P. Li, X. Guo, A review: therapeutic potential of adipose-derived stem cells in cutaneous wound healing and regeneration, Stem Cell Research & Therapy. 2018; 9:) 302. doi: 10.1186/ s13287-018-1044-5.

[28] T. Kushibiki, T. Hirasawa, S. Okawa, M. Ishihara, Low reactive level laser therapy for mesenchymal stromal cells therapies, Stem cells international. 2015;2015:974864. doi: 10.1155/2015/974864.

[29] H. Ahmadi, A. Amini, F. Fadaei Fathabady, A. Mostafavinia, F. Zare, R. Ebrahimpour-Malekshah, M.N. Ghalibaf, M. Abrisham, F. Rezaei, R. Albright, S.K. Ghoreishi, S. Chien, M. Bayat, Transplantation of photobiomodulationpreconditioned diabetic stem cells accelerates ischemic wound healing in diabetic rats, Stem Cell Res Ther. 2010; 11: 494. doi: 10.1186/s13287-020-01967-2

[30] A. Khosravipour, A. Amini, R. Masteri Farahani, F. Zare, A. Mostafavinia, S. Fallahnezhad, S. Akbarzade, P. Ava, M. Asgari, A.

*Combined Administration of Stem Cells and Photobiomodulation on Wound Healing in Diabetes DOI: http://dx.doi.org/10.5772/intechopen.96905*

Mohammadbeigi, F. Rezaei, S.K. Ghoreishi, S. Chien, M. Bayat, Preconditioning adipose-derived stem cells with photobiomodulation significantly increased bone healing in a critical size femoral defect in rats, Biochem Biophys Res Commun. 2020; 531 : 105-111. 10.1016/j.bbrc.2020.07.048.

[31] X. Liao, S.H. Li, G.H. Xie, S. Xie, L.L. Xiao, J.X. Song, H.W. Liu, Preconditioning With Low-Level Laser Irradiation Enhances the Therapeutic Potential of Human Adipose-derived Stem Cells in a Mouse Model of Photoaged Skin, Photochemistry and photobiology 94 (2018) 780-790. doi: 10.1111/php.12912.

[32] M.C. Feitosa, A.F. Carvalho, V.C. Feitosa, I.M. Coelho, R.A. Oliveira, E. Arisawa, Effects of the Low-Level Laser Therapy (LLLT) in the process of healing diabetic foot ulcers, Acta Cir Bras. 2015; 30: 852-857. doi: 10.1590/ S0102-865020150120000010.

[33] M. Dahmardehei, N. Kazemikhoo, R. Vaghardoost, S. Mokmeli, M. Momeni, M.A. Nilforoushzadeh, F. Ansari, A. Amirkhani, Effects of low level laser therapy on the prognosis of split-thickness skin graft in type 3 burn of diabetic patients: a case series, Lasers Med Sci. 2016; 31: 497-502. doi: 10.1007/ s10103-016-1896-9.

[34] R. Vaghardoost, M. Momeni, N. Kazemikhoo, S. Mokmeli, M. Dahmardehei, F. Ansari, M.A. Nilforoushzadeh, P. Sabr Joo, S. Mey Abadi, S. Naderi Gharagheshlagh, S. Sassani, Effect of low-level laser therapy on the healing process of donor site in patients with grade 3 burn ulcer after skin graft surgery (a randomized clinical trial), Lasers Med Sci.2018; 33: 603-607. doi: 10.1007/s10103-017-2430-4.

## **Chapter 5** Chronic Venous Ulcer

*Walid A.M. Ganod*

## **Abstract**

This chapter sheds light on the cause and effect of chronic venous ulcers (CVUs) and the therapeutic procedures used to treat them. In the last two decades, many changes have occurred in the strategy of wound management through the development of adjunctive therapy that supports wound healing. Eventually, the latest development in platelet concentration technology produced platelet-rich fibrin (PRF). It was categorized as the second-generation platelet concentration family after platelet-rich plasma (PRP). Venous leg ulcers (VLUs) account for 70% of all leg ulcers and are estimated to affect 1% of the population; prevalence increases with age. The chronicity and refractory nature of venous ulcers have a great effect on the quality of life (QoL) and work productivity of patients, in addition to the expenditure of significant medical resources and efforts. Therefore, the goal of VLU management is to induce rapid healing without recurrence, which mainly helps to improve QoL. The first therapeutic procedure used in the treatment of VLU was compression therapy, in which the application of effective graduated compression decreased the overload in the venous system and venous reflux. Furthermore, it accelerated the capillary blood flow and decreased capillary fluid leakage, which alleviated limb edema.

**Keywords:** venous ulcer, ambulatory venous hypertension, chronic venous insufficiency, compression therapy, platelet-rich fibrin

## **1. Introduction**

Chronic leg ulcers (CLUs) are chronic wounds that do not show a tendency to heal within a reasonable period. This period can determine the state of a chronic wound; if there is no tendency to heal after 3 months or if the wound does not fully heal after 12 months, the ulcer is determined to be chronic [1]. The aforementioned period is not a fixed number; it is governed by other factors, such as ulcer ethology, size, and so on [2].

In general, ulcers can be described in many ways; for instance, ulcers have a fullthickness wound, lack a source of re-epithelization in the center, and show poor tendency to heal.

The most common clinical cause of CLUs is venous insufficiency followed by arterial insufficiency, diabetes, or a combination of two or more of these factors [3].

The Wound Healing Society described chronic wounds as "a silent epidemic disorder" correlated to the percentage of the public with this condition. In the United States, approximately 6.5 million patients suffer from chronic nonhealed wounds. Therefore, two million working days are lost annually. In addition, in the United Kingdom, the annual incidence of leg ulcers has been estimated to be 3.5 per 1000 individuals [4].

Venous insufficiency is considered the most common cause of leg ulcers, accounting for 70% of leg ulcers. Inline arterial diseases and mixed venous and arterial disorders account for 10 and 15% of ulcers, respectively. There is a major challenge in the assessment and diagnosis of CLU in regard to miscellaneous disorders such as vasculitis and hematological diseases. These kinds of disorders represent the remaining 5% of the causes of CLUs [5].

Scottish guidelines define a chronic venous leg ulcer as "an open lesion between the knee and the ankle joint that remains unhealed for at least 4 weeks and occurs in the presence of venous disease" [6].

## **2. Anatomy of venous system in lower limbs**

The anatomical variation and nonuniform nomenclature of the lower limb vein system, especially in the literature, supported the constitution of the International Interdisciplinary Committee in 2001 to perform adjustment and uniformity of the anatomical terminology of lower limb veins (**Figure 1**) [8].

The veins of the lower limb can be classified into three systems: superficial, deep, and perforator veins (**Figure 2**). These veins are arranged into two main compartments: superficial and deep compartments. A superficial compartment is present between the skin and muscular fascia, which contain superficial veins. A deep compartment that contains deep veins is present under the deep fascia. The perforator veins are connected to the superficial and deep system [9]. **Figure 3** shows that there is another compartment within the superficial compartment enclosing the saphenous vein, which is called the saphenous compartment.

The principle method for venous return from the lower limb is through the deep vein system, which pairs below the knee and accompanies arteries and then joins to form the popliteal vein that completely ascends as the femoral vein. The main veins in the superficial system that are the target of many venous therapies are the great saphenous vein and small saphenous veins, which are connected with communicator veins [10].

**Figure 1.** *Diagram of the superficial venous system of lower limbs [7].*

*Chronic Venous Ulcer DOI: http://dx.doi.org/10.5772/intechopen.97709*

**Figure 2.** *Normal vein valve.*

#### **Figure 3.** *Ultrasound image of the saphenous compartment.*

The perforator veins have normal unidirectional flow from the superficial to deep system, and there are more than 150 perforators in the lower extremities, but most of them are inactive in the normal state. Perforator veins on the medial aspect of the leg represent the most clinically important perforators in chronic vein insufficiency [11].

Valves of lower extremity veins are anatomical features that have clinical importance in cases of incompetence of these valves, transmission of venous pressure to skin venules, and development of skin changes [12].

## **3. Physiology of lower limb venous system**

The venous system functions to support circulation by the venous return mechanism, as 60–80% of blood volume rests in the venous system (25% in the splanchnic network and other residual volumes in postcapillary venules). Therefore, venous return must be equal to cardiac output to maintain homeostasis of tissue perfusion [13]. Many factors have a role in venous return mechanisms, such as central pumps, pressure gradients, venous valves, and muscle pumps (peripheral pumps) [7].

Venous valves are distributed mainly in the distal vein circulation of the lower limb to overcome the effect of gravity and break down the hydrostatic pressure of

#### **Figure 4.**

*Schematic summarization of the relationship between pressure and volume in the lower extremities while walking and standing. Note the efficacy of the calf muscle pumping mechanism that leads to a decrease in venous volume and pressure with walking and a slight delay in increasing venous pressure opposite to venous blood volume in the standing position. Alternation in this relationship results in high ambulatory venous pressure.*

the blood column into segments. Valve closure is a passive mechanism involving a gradient pressure difference between the supra- and infravalvular segments and normal retrograde flow that lasts for less than 0.5 seconds, which is enough time to close cusps completely [12].

Calf muscle contraction (gastrocnemius and soleus) is an essential part of the mechanism of venous return, and it has been estimated that approximately 60% of venous return from the lower limb depends on the ejection force of the calf muscle. The net result of serial contraction of the calf muscle during exercise produces a streamline and unidirectional blood in the deep venous system toward the heart and improves cardiac output [12].

The efficacy of calf muscle pumps is dependent on the strength of the muscle, range of movement of the ankle joint, and competence of vein valves. We hypothesize that atrophy of the calf muscle decreases the strength of contraction, resulting in a reduction in venous return and chronic vein insufficiency that underlie the pathogenesis of venous ulcers [14].

Accumulation of blood in peripheral venous circulation during rest leads to elevation of venous pressure, especially with the standing position, while contraction of the calf muscle will decrease the venous pressure to a suitable baseline. The measurement of the drop in superficial venous system pressure after exercise is called *ambulatory venous pressure* (AVP), which is an indicator of calf muscle pump function, and an elevation above 30 mm Hg has a linear relationship with leg ulcers (**Figure 4**) [15].

### **4. Pathophysiology of venous ulcer**

The pathophysiology behind chronic leg venous ulcers is still unclear. *Ambulatory venous hypertension* (AVH) is the essential pathological factor behind venous ulcers. Venous incompetence can result from immobility, ineffective pumping

#### *Chronic Venous Ulcer DOI: http://dx.doi.org/10.5772/intechopen.97709*

mechanisms of the calf muscles, and venous valve dysfunction. In addition, venous valve dysfunction that results from venous thrombosis, phlebitis, or trauma leads to alterations in venous hemodynamics and precipitates venous hypertension [16].

Subsequently, chronic blood stasis of the lower limb venous system causes further capillary damage with inflammatory process activation. Leukocyte activation, endothelial damage, platelet aggregation, and intracellular edema are highly related to venous ulcer development and impaired wound healing [17].

## **4.1 Ambulatory venous hypertension**

The calf muscle pump consists of the calf muscle, a superficial venous system, a deep venous system, and perforators that connect both systems. The out-flow vein of this pump is the popliteal vein. Failure of the calf muscle pump to decrease AVP leads to persistent elevation of postexercise pressure or AVH [15].

Therefore, one or more of the following pathological situations can lead to calf muscle pump dysfunction and AVH.

### *4.1.1 Reflux in superficial veins system*

The cause behind the reflux or incompetent valve in superficial veins is still ambiguous. Currently, there is a discussion on congenital and acquired factors that may be behind the structural changes in valve cusps.

The reflux in superficial veins can be compensated with calf muscle contraction if perforator valves are competent. Secondary incompetent valves of deep and perforator veins are likely to occur with large-volume reflux post sapheno-femoral or sapheno-popliteal incompetence [12].

#### *4.1.2 Reflux/obstruction in deep veins system*

Post thrombotic damage to deep veins will result in obstruction, reflux, or both and can even lead to reflux in superficial and perforator veins later (*post thrombotic syndrome*) [12].

#### *4.1.3 Incompetent medial calf perforator*

An outward flow of incompetent perforators more than 500 milliseconds and equal to or more than 3.5 millimeters in size will have a significant hemodynamic effect with high AVH and skin changes [12].

Muscular dysfunction of the calf muscle, fixed ankle joint, and prolonged immobilization will lead to blood stasis and venous hypertension as a result of pump mechanism failure [18]. In clinical practice, these pathologies present in combination with multilevel involvement in a large group of patients.

#### **4.2 Chronic venous disorder and chronic venous insufficiency**

According to updated terminology of chronic venous disorders in the VEIN-TERM transatlantic interdisciplinary consensus document, chronic venous disorder (CVD) is defined as a wide spectrum of functional and morphological abnormalities that involve the vein system from telangiectasia to venous ulcers (C1–C6 clinical classes). The term chronic venous insufficiency (CVI) is reserved for advanced CVD (C3–C6 clinical classes) and includes moderate to severe edema, skin changes, or venous ulcers [19].

CVI is classified into two types. Primary chronic venous insufficiency occurs due to weakness or degenerative changes in wall or venous valves that started as reflux

in superficial veins and proceed to perforators and deep veins later due to overload that led to dilatation in the venous wall [20].

Secondary chronic venous insufficiency, known as *post-thrombotic syndrome*, is secondary to acute DVT and later sequelae that can lead to reflux, obstruction, or both in deep veins. Additionally, it could be secondary to superficial thrombophlebitis or arteriovenous fistula [19].

## **4.3 Revised CEAP classification of chronic venous disorders (CVDs)**

The need for clinical assessment, evaluation, and stage identification methods of CVD supported the presentation of the CEAP classification at the American Venous Forum annual meeting in 1994, which was revised in 2004 [21].

CEAP classification is a method for categorizing CVD based on: Clinical manifestations Ethological factors Anatomical distribution of disease Pathophysiological process behind this disorder (**Table 1**)


*\* Each clinical class is further subclassed as "S" if symptomatic and "A" if asymptomatic.*

*\* The symptoms include aching, pain, tightness, skin irritation, heaviness, muscle cramps, and other symptoms relating to venous disorders.*

## **Table 1.**

## **5. Epidemiology of venous ulcer**

According to the Edinburgh study, a cross-sectional study of a random sample, VLUs represent approximately 70% of all leg ulcers and affect 1% of the population; prevalence increases with age [22].

Development in the diagnosis and early management of varicose veins, especially with significant reflux, can decrease the prevalence of venous ulcers by 50%, as superficial vein insufficiency represents 50% of the causes of leg ulcers. The management of risk factors such as obesity has a strong relationship with venous ulcers [23].

Based on estimates of the San Diego epidemiologic study, more than 11 million men and 22 million women between the ages of 40 and 80 years in the United States have varicose veins, and more than two million adults have advanced CVD with skin changes or ulcers [24]. The incidence of postthrombotic venous ulcers has not changed in the past two decades for women and has recently increased in men [25].

## **6. Clinical presentation and diagnosis of CVI and venous ulcer**

There is a wide spectrum of differential diagnoses for ulcers in the lower limbs. Therefore, proper management depends on determining the etiology of ulcers and managing them. Venous ulcers are the most common cause of lower limb ulcers, followed by arterial and diabetic ulcers. There are distinctive clinical presentation and physical examination findings that can help to differentiate venous ulcers from other lower extremity disorders [26].

The diagnosis of venous ulcers is generally clinical; this step in the diagnosis of venous ulcers is often neglected by physicians. Diagnosis is based on radiology reports such as color duplex ultrasonography and venography, which may be helpful in doubtful cases [27].

Inspection and palpation are essential parts of the examination and should be used to search for signs of venous disorder. Auscultation for bruit is particularly helpful in those with vascular malformation and arteriovenous fistula [27]. Examination is always performed with patients in a standing position and should focus on the size and distribution of varicose veins.

Eklof et al. defined signs present in the clinical part of CEAP classification that suggested CVI (**Figures 5**–**7**) [21]:

*Lower limb edema:* Venous hypertension edema unilaterally starts at the ankle and pitting pattern and worsens in the evening.

*Eczema:* Erythematous dermatitis, which is usually distributed on varicose veins because of uncontrolled CVD but can be seen anywhere in response to local management.

*Skin pigmentation:* Extravasated blood due to venous hypertension in the small vein leads to intradermal accumulation of hemosiderin, which causes brownish darkening of the skin around the ankle region and sometimes the leg.

*Lipodermatosclerosis (LDS):* This is defined by Eklof et al. as localized chronic inflammation with fibrosis in skin and subcutaneous tissue and may progress to scarring and contracture in the Achilles tendon [21]. Most authors agree that LDS is highly suggestive of severe CVI and provides clues about the poor prognosis of wound healing. LDS frequently leads to the development of venous ulcers in many cases [18].

*Atrophic blanche (white atrophy):* This is smooth, white atrophic plaque surrounded by dilated capillary and sometimes hyperpigmentation. It is also a sign of severe CVI and should be distinguished from healed ulcers by history, as it develops

**Figure 5.** *Atrophie blanche.*

**Figure 6.** *Lipodermatosclerosis.*

independently. Ulcerated atrophie blanche can be extremely painful and has a low tendency for healing [18].

*Venous ulcer:* Gillespie mentioned the most current updated definition for venous ulcer as *"a full thickness defect of the skin, located in the lower leg, typically with pigmentation and/or skin changes and presence or history of venous disease (documented* 

*history of DVT, documented axial venous reflux or deep vein obstruction) in the absence of another condition that could be the essential cause of the ulcer"* [28].

A positive history of previous DVT events, family history of varicose veins, or previous intervention to the venous system in line with good clinical examination help to clarify the diagnosis in up to 76% of cases of venous ulcers [18]. The choice of investigation should be based on the severity of the problem and management plan. The noninvasive method is usually used to evaluate patients for venous ablation or preoperative surgery for perforators; invasive diagnostic methods should be used for patients who need complex operations, such as valve reconstruction or venous bypass [12].

The palpable pedal pulse or measurement of ankle-to-brachial blood pressure ratio (ankle/brachial index [ABI]) is one of the critical points in diagnosis of venous ulcer, as it differentiates venous ulcer from arterial ulcer and determines if there is any association of ischemic degree that contraindicated to compression therapy, which is the traditional management approach for venous ulcers. Culture swabs and investigations for vasculitis and connective tissue diseases such as rheumatoid arthritis are helpful in the diagnosis of difficult cases and resistant ulcers [18].

The atypical appearance of ulcers, such as nodular growth, everted edges, deterioration, or delayed healing with appropriate treatment, are indications for biopsy to exclude malignant transformation [29].

Duplex scanning is currently the gold standard for the evaluation of patients with CVIs. It has high sensitivity and specificity in the diagnosis of superficial and deep venous system disorders, and it provides information about the patency of the deep venous system, diameter of the vein, and flow rate. The real-time color duplex scan makes the orientation of venous flow much easier and provides information about reflux in the superficial, deep, or perforator veins. Through interpretation of all previous dates, the differentiation between primary and secondary CVIs is easy. In addition, duplex scans today have an important role in endovenous procedures [23].

Phlebography, such as ascending or descending phlebography, is not a first-line diagnostic tool in cases of venous ulcers and is preserved for evaluation of the venous system before complex procedures such as valve reconstruction or bypass, as it can provide information about the level of obstruction in the deep venous system and the state of valves [23]. CT angiogram is a useful tool for the assessment of the pelvic vein and inferior vena cava (IVC), especially before venous stenting, and MR venogram is preferred for vein malformation cases.

## **7. Management of venous ulcer**

The management of CVUs is a major challenge in terms of healing, preventing recurrence and minimizing social and economic effects. In the Western world, approximately 1% of the annual healthcare balance is expended on venous ulcer care [22]. The future world vision directed more towards the prevention rather than the management of venous ulcers becomes more expensive over time, thus standing in the way of the 2009 Pacific Vascular Symposium's goal to decrease incidence of venous ulcer by 50% in the next 10 years [30].

The key for the management of venous ulcers is reduced AVH, which leads to a decrease in edema and inflammatory reactions in the leg, resulting in stimulated healing of ulcers and preventing recurrence if optimum venous pressure is maintained. The correction of vein disorders that lead to venous hypertension is an important step in addition to ulcer care [31]. The management of venous ulcers includes conservative (lifestyle modification, compression therapy, and ulcer care) and surgical (surgical cover of ulcer and surgical elimination of venous hypertension) procedures.

#### **7.1 Conservative management**

### *7.1.1 Modification of lifestyle*

Theoretically, moderate exercise concentrated on mobility of the ankle joint and contraction of the calf muscle (peripheral heart) are beneficial in decreasing venous congestion of the lower limbs and hemodynamics. Although there is not a lot of evidence confirming the effect of exercise on healing venous ulcers, supervised moderate exercise should be considered as adjuvant to main treatment for CVI and venous ulcer.

Another important procedure that is not practical for patients is leg elevation at or above the level of the heart, which can decrease venous pressure around the ankle nearly to zero, resulting in an improvement in lower limb swelling and an increased ulcer healing rate. Leg elevation, if associated with compression therapy, can decrease ulcer recurrence [31].

#### *7.1.2 Compression therapy*

Compression therapy is still the cornerstone of CVI and venous ulcer care. It is defined as an applied external pressure on a specific lower limb area to overcome gravity and hydrostatic pressure in veins. The mechanism of action has not been fully understood until now. It depends on preserving interfacing pressure and stiffness (increase of interface pressure with activity as increased limb circumference by muscle contraction).

In applying compression to a patient in a normal standing position, an external pressure of 35–40 mmHg will narrow the vein; however, if pressure exceeds

#### *Chronic Venous Ulcer DOI: http://dx.doi.org/10.5772/intechopen.97709*

60 mmHg, it will lead to occluding of the vein. As such, optimum external graduated pressure between 35 and 40 mmHg will improve venous pumping function and microcirculation. In addition, it lowers the level of inflammatory mediators, such as alpha tumor necrosis factor, which causes tissue damage. Therefore, compression promotes ulcer healing [31].

The Unna boot developed in 1885 is the oldest modality of compression therapy. Other more familiar modalities include compressive bandages, compression stockings, and intermittent pneumatic devices. LaPlace's law states that the pressure in the cylinder is inversely related to the radius with uniform tension on the wall, so this modality of compression will provide graduated pressure that is the highest at the ankle, resulting in the cephalic direction of venous flow [32].

A recent Cochrane review found that venous ulcers heal more rapidly with the application of compression therapy than without compression therapy and that high-grade compression with a three- or four-layer bandage or short stretch bandage is better than other systems that deliver low pressure [33].

A meta-analysis out of the United Kingdom found that high-grade compression (sub-bandage pressure 35–40 mmHg at ankle) by standardized four-layer bandage technique shows shorter healing time than short stretch bandages [34]. The average healing rate is approximately 60–70% at 12–24 weeks in various types of compression models [35].

Brien et al. stated that four-layer bandaging is the most effective method for the management of venous ulcers, with a healing rate of 54% at 3 months in a randomized control trial conducted on 200 patients. In addition, they recommend using it routinely in the management of patients with uncomplicated venous ulcers. Additionally, it can decrease the rate of recurrence if maintained lifelong [36].

The following sections discuss the technique and components of these systems, also known as the *Charing Cross Hospital Bandage*, according to recommendations from the Scottish Intercollegiate Guidelines Network (SIGN) guidelines [37] and the International Leg Ulcer Advisory Board (**Figure 8**) [38].

First layer: The padding layer involves application of orthopedic cotton in a spiral fashion with minimal overlap from the base of toes to just under the knee to protect the bony prominence and absorb exudate. In patients with ankle circumference less than 18 cm, an additional layer is needed as an artificial increase in circumference.

Second layer: This is a layer of cotton crepe bandage that oversmooths the first layer and has the last effect in compression. It is applied in a spiral fashion with 50% overlap.

#### **Figure 8.**

*Component of four-layer bandaging. (1) Orthopedic cotton; (2) cotton crepe bandage; (3) elastic extensible bandage; and (4) elastic cohesive bandage.*

#### *Recent Advances in Wound Healing*

Third layer: This is an elastic extensible bandage applied by figure eight winding with 50% extension from base of toes to just under the knee (it provides sub-bandage pressure = 17 mmHg). The ankle joint is kept in dorsiflexion or at a 90-degree angle.

Fourth layer: This layer is an elastic cohesive bandage applied in a spiral fashion with 50% overlap and 50% extension (adds remaining 23 mmHg sub-bandage pressure) (**Figure 9**).

The disadvantage of the four-layer compression bandage is that it needs trained physicians to apply the optimum pressure, whereas compression stockings can be used by the patient and removed at night [39].

Intermittent pneumatic compression is expensive and requires immobilization of the patient. Therefore, it is reserved for bedridden patients who cannot tolerate continuous compression therapy [40].

**Figure 9.** *Four-layer compression bandaging steps.*

## *7.1.3 Ulcer care*

Tap water can be used to clean venous ulcers. There is no advantage observed with the use of physiological saline and recommended deep debridement for recalcitrant chronic venous leg ulcers to remove fibrosis that arrests the healing process, but the use of chemical or enzymatic debridement has no special advantage [23].

A meta-analysis of 42 randomized controlled trials showed no major difference between dressing types and expensive hydrocolloid dressings. Medical evidence does not support increased healing with hydrocolloid dressings compared to lowercost, simple nonadherent dressings. Without clear evidence that supports the use of certain dressings over others, the choice of dressings for venous ulcers can be directed by cost, ease of application, and patient and physician preference [41].

There is no evidence to support that the use of topical antibiotics has a positive effect on the management of infected venous ulcers or promotes healing. A Cochrane review on the use of silver-containing topical material concluded that there is insufficient evidence to support its use in infected venous ulcers. Other articles support avoiding topical application because it sensitizes the skin and recommend managing clinically infected venous ulcers with systemic antibiotics [29].

## **7.2 Surgical management**

## *7.2.1 Surgical cover of ulcer (skin graft)*

Skin grafting may be used for patients with large or refractory venous ulcers that do not show signs of healing within 4–6 weeks with standard care [29]. However, skin grafting is not effective if there is persistent edema, which is common with venous insufficiency, and the underlying venous disease is not addressed. A Cochrane review found few high-quality studies to support the use of skin grafting for the treatment of venous ulcers [42].

## *7.2.2 Surgery for venous insufficiency*

The role of surgery is to reduce venous hypertension, promote healing, and prevent ulcer recurrence. Surgical options for the treatment of venous insufficiency include ablation of the saphenous vein, interruption of the perforating veins with subfascial endoscopic surgery, stenting of iliac vein obstruction, and removal of incompetent superficial veins with phlebectomy, stripping, sclerotherapy, or laser therapy [43].

Scottish guidelines state that there is no evidence to support surgical intervention for venous insufficiency prior to standard management (compression) for healing venous ulcers. One study showed a significant difference in recurrence in favor of surgery [23].

## **7.3 Platelet concentrates**

Platelet concentrates are autologous material prepared from venous blood after various processing of blood samples. Generally, it depends on the centrifugation principle to separate the whole blood sample into red blood cells that heavily precipitate down and concentrate other elements that can be used topically or via infiltration for therapeutic purposes [44].

Platelet concentrates were first presented 20 years ago and were developed with the aim of using blood protein elements as a biological source of growth factors to promote the angiogenesis process and stimulate cells involved in the healing process, such as fibroblasts, neutrophils, and mesenchymal stem cells [45].

#### *Recent Advances in Wound Healing*

Platelet-rich fibrin (PRF) is a natural fibrin matrix developed by Choukroun et al. in France through new technology that is characterized by a simple and open access technique without anticoagulant or bovine thrombin. Just immediate centrifugation of patients' blood samples leads to conversion of fibrinogen to fibrin by physiological thrombin; this slow polymerization of fibrin charges it by platelets, leucocytes, and cytokines to give us autologous biomaterials from platelets and immune cells to support healing [46].

The protocol for preparing PRF is very simple. Blood is extracted from the patient and placed in a glass-coated tube without anticoagulant and immediately centrifuged. Time is an important factor, as the coagulation cascade starts within minutes via activation of platelets through contact with the glass tube in absence of an anticoagulant. Then, physiological thrombin transforms the fibrinogen to a fibrin network charged with active platelets and cytokines that will take the middle portion of the tube between the precipitated red blood cells layer at the bottom and acellular plasma at the top [47].

Any delay in blood handling will lead to the start of coagulation without separation of the blood component, and fibrin will be formed in a diffuse way in all tubes, resulting in a blood clot and not a PRF clot (**Figures 10**–**12**) [48].

From clinical data, note the ability of PRF to induce healing without any inflammatory excess. Dohan et al. stated that the PRF process not only activates platelets but also activates leucocytes to release important cytokines in response to artificial inflammation induced by these techniques. An initial investigation revealed that PRF also functions as an immune node to increase defense mechanisms and control inflammatory responses, which explains the decrease in surgical site infection treated by PRF because of trapped cytokines in fibrine networks [49].

PRF contains three main components that are important to tissue healing.

The first of these components is the host cells, which constitute the main difference between PRF and previous-generation PRP, as PRF incorporates not only platelets but also incorporates active leucocytes that have a role in anti-infection and regulation of immunity. The natural three-dimensional fibrin network is a

**Figure 10.** *Centrifuge device used for PRF.*

**Figure 11.** *PRF clot at middle of tube.*

**Figure 12.** *PRF membranes on the surface of the ulcer.*

second component that does not work as a server for host cells only but can also promote cell invasion and help in tissue regeneration. The last items in these structures are the natural growth factors that have an important biological role in the healing process, as platelet-derived growth factor (PDGF) is an essential growth factor for cell migration, differentiation, and proliferation. Vascular endothelial growth factor (VEGF) is also important for the angiogenesis process in granulation tissue, and other growth factors, such as TGF-beta, epidermal growth factor, and insulin-like growth factor, are important for wound healing (**Figures 13** and **14**) [45, 50].

Yazawa et al. stated that the concentration of growth factors in PRF was three times greater than that in PRP due to the use of fibrine as a drug delivery system for growth factors, which helped in the slow release of natural factors over a period of approximately 1 week [51].

#### **Figure 13.**

*A male patient, 45 years old, with secondary CVI of the left leg with two ulcers treated with four-layer compression bandages and PRF membrane applied on the proximal ulcer only.*

#### **Figure 14.**

*A 55-year-old female patient with primary CVI of the left leg with ulcers treated with four-layer compression bandages and PRF.*

## **8. Conclusion**


## **Acknowledgements**

Staff of Scientific Board of Vascular Surgery Department, Faculty of Medicine, Zagazig University, Egypt.

Rwida Nori Alati, Zliten Medical College, Asmarya Islamic University.

*Chronic Venous Ulcer DOI: http://dx.doi.org/10.5772/intechopen.97709*

## **Conflict of interest**

No conflict of interest.

## **Author details**

Walid A.M. Ganod Vascular Surgery Unit, Zliten Medical Center, Zliten Medical College, Asmarya Islamic University, Zliten, Libya

\*Address all correspondence to: walid.ganod@gmail.com

© 2021 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.

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## Section 3
