*3.4.1. Cell‐mediated repair*

*In vitro* studies have shown that MSCs possess phenotypic properties resembling native dermal fibroblasts or myoblasts [85]. Furthermore, BM‐MSCs may accelerate wound closure by differentiating into epidermal keratinocytes and other skin cells [15, 23, 79, 86, 87]. Recent studies have shown that MSCs undergo trans‐differentiation into keratinocyte, epidermal cells and microvascular endothelial cells when cultured under defined culture conditions [30] and express keratinocyte‐specific protein (KSP) [79, 88, 89]. MSCs therefore could be utilised for wound healing by transplanting aggregated MSCs into the injured tissue to increase collagen deposition and improve epithelisation [80]. They can also differentiate into other skin cells such as endothelial cells, keratin 14‐positive cells and pericytes, when localised to blood vessels and dermis [86], sebaceous glands and hair follicles [23].

## *3.4.2. Secretory‐mediated repair–role of mesenchymal stem cell‐conditioned media (MSC‐CM)*

Paracrine signalling of BM‐MSCs is the major mechanism by which these cells contribute to wound repair, in which their secretory products impact on inflammation, fibrotic proliferation and angiogenesis [18]. Many studies have reported that MSC‐CM is the supernatant from MSC *in vitro culture* significantly promotes wound healing by affecting the pivotal steps of the repair process. The components of MSC‐CM have accelerated epithelialisation and via chemotaxis recruit endothelial cells and macrophages to the injured site *in vivo* [21]. The MSC‐CM recruits both epidermal keratinocytes and dermal fibroblasts to the wound site *in vitro* [21, 23]. As well as to its activity as chemoattractant, MSC paracrine secretions serve as regulators for cell migration in response to wounding leading to faster wound closure by regulating dermal fibroblast migration [90]. MSC secretory mitogenic molecules stimulate the proliferation of keratinocytes, dermal fibroblasts and endothelial cells [91]. Conditioned medium derived from the cell culture of MSCs (MSC‐CM) contains all the effector biomolecules which could be effectively utilised in tissue regeneration and wound healing by promoting migration, proliferation and differentiation of human skin cells such as fibroblast and keratinocytes. Collectively, these data suggest MSC‐CM may represent a novel therapeutic strategy for wound therapy [25].

### **3.5. Biologically active substances secreted by mesenchymal stem cells**

The potential of MSCs in regenerative medicine and wound healing has been reflected by their secretion of biomolecules including growth factors, cytokines and chemokines [18, 21, 25]. Some 36 biomolecules have been reported to be released by human MSCs (h‐MSCs) which act in concert to promote the wound‐healing process [66].

### *3.5.1. Growth factors*

paracrine signalling have both been implicated as mechanisms by which MSCs recruit other host cells in all steps of healing process to improve tissue repair [23]. To better understand the role of MSCs in wound healing, we have divided their participation in repair into two major

*In vitro* studies have shown that MSCs possess phenotypic properties resembling native dermal fibroblasts or myoblasts [85]. Furthermore, BM‐MSCs may accelerate wound closure by differentiating into epidermal keratinocytes and other skin cells [15, 23, 79, 86, 87]. Recent studies have shown that MSCs undergo trans‐differentiation into keratinocyte, epidermal cells and microvascular endothelial cells when cultured under defined culture conditions [30] and express keratinocyte‐specific protein (KSP) [79, 88, 89]. MSCs therefore could be utilised for wound healing by transplanting aggregated MSCs into the injured tissue to increase collagen deposition and improve epithelisation [80]. They can also differentiate into other skin cells such as endothelial cells, keratin 14‐positive cells and pericytes, when localised to blood vessels

*3.4.2. Secretory‐mediated repair–role of mesenchymal stem cell‐conditioned media (MSC‐CM)*

**3.5. Biologically active substances secreted by mesenchymal stem cells**

in concert to promote the wound‐healing process [66].

The potential of MSCs in regenerative medicine and wound healing has been reflected by their secretion of biomolecules including growth factors, cytokines and chemokines [18, 21, 25]. Some 36 biomolecules have been reported to be released by human MSCs (h‐MSCs) which act

Paracrine signalling of BM‐MSCs is the major mechanism by which these cells contribute to wound repair, in which their secretory products impact on inflammation, fibrotic proliferation and angiogenesis [18]. Many studies have reported that MSC‐CM is the supernatant from MSC *in vitro culture* significantly promotes wound healing by affecting the pivotal steps of the repair process. The components of MSC‐CM have accelerated epithelialisation and via chemotaxis recruit endothelial cells and macrophages to the injured site *in vivo* [21]. The MSC‐CM recruits both epidermal keratinocytes and dermal fibroblasts to the wound site *in vitro* [21, 23]. As well as to its activity as chemoattractant, MSC paracrine secretions serve as regulators for cell migration in response to wounding leading to faster wound closure by regulating dermal fibroblast migration [90]. MSC secretory mitogenic molecules stimulate the proliferation of keratinocytes, dermal fibroblasts and endothelial cells [91]. Conditioned medium derived from the cell culture of MSCs (MSC‐CM) contains all the effector biomolecules which could be effectively utilised in tissue regeneration and wound healing by promoting migration, proliferation and differentiation of human skin cells such as fibroblast and keratinocytes. Collectively, these data suggest MSC‐CM may represent a novel therapeutic strategy for

mechanisms: (1) cell‐mediated repair and (2) secretory‐mediated repair (**Figure 1**).

and dermis [86], sebaceous glands and hair follicles [23].

*3.4.1. Cell‐mediated repair*

108 Wound Healing - New insights into Ancient Challenges

wound therapy [25].

Human MSCs secrete a wide range of growth factors that play a significant role in the wound‐ healing process. These are angiopoietins (ANGPT), connective tissue growth factors (CTGFs), epidermal growth factor (EGF), fibroblast growth factors (FGFs), insulin‐like growth factors (IGF), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet‐derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF) and scatter factors which are a family of growth factors also known as plasminogen‐related growth factors (PRGFs), which include two members: hepatocyte growth factor (HGF) also referred to as plasminogen‐related growth factor‐1 (PRGF‐1) and macrophage‐stimulating protein (MSP) which is also known as scatter factor‐2 (SF‐2) or hepatocyte growth factor‐like protein (HGFL) (**Table 2**).



MSCs secrete a wide spectrum of growth factors. These biological substances participate in wound healing from early stages starting with haemostasis and coagulation and ending with remodelling. These growth factors promote angiogenesis, accelerate proliferation and also migration of endothelial cells. In addition, they are involved in the contraction phase, ending at the last stages of remodelling, leading to wound healing in the absence of scar formation.

**Table 2.** The main growth factors secreted by MSCs and their roles and functions in the wound‐healing process.

### *3.5.2. Cytokines*

Cytokines are small proteins secreted by many cell types which affect the activity of other cells including immune cells; they include interleukins, lymphokines and other signalling biomo‐ lecules including interferons and tumour necrosis factor [40]. Here, they are categorised into groups depending on their role in the wound‐healing process (**Table 3**).


MSCs secrete a wide range of cytokines. These secretions initiate and terminate the inflammatory phase and accelerate proliferation and also migration of endothelial cells. In addition, they are involved in the contraction phase, ending at the last stages of remodelling, leading to wound healing in the absence of scar formation.

**Table 3.** The main cytokines secreted by MSCs and their roles and functions in the wound‐healing process.

### *3.5.3. Chemokines*

**Growth factors**

HGF or PRGF‐ 1 **Function(s) References**

[96]

[97]

[99]

[40]

[40]

[21, 22]

[40, 100]

microvascular endothelial cells and their adherence molecule expression PDGF Stimulates DNA synthesis, attracts fibroblasts to wound sites, enhances their production of

> The first chemotactic growth factor participating in migration of fibroblasts, monocytes and neutrophils into the skin wound, subsequently stimulating the production of extracellular

Enhances keratinocytes to migrate, proliferate and produce matrix metalloproteinase and

differentiation of keratinocytes and macrophages. plays an integral role in inflammation,

Activins which are members of TGF‐β family act as enhancers for granulation tissue

Activin B supports wound repair and regeneration of hair follicles, promotes wound

VEGF Regulates angiogenesis [20, 40]

MSCs secrete a wide spectrum of growth factors. These biological substances participate in wound healing from early stages starting with haemostasis and coagulation and ending with remodelling. These growth factors promote angiogenesis, accelerate proliferation and also migration of endothelial cells. In addition, they are involved in the contraction phase, ending at the last stages of remodelling, leading to wound healing in the absence of scar formation.

**Table 2.** The main growth factors secreted by MSCs and their roles and functions in the wound‐healing process.

groups depending on their role in the wound‐healing process (**Table 3**).

Cytokines are small proteins secreted by many cell types which affect the activity of other cells including immune cells; they include interleukins, lymphokines and other signalling biomo‐ lecules including interferons and tumour necrosis factor [40]. Here, they are categorised into

VEGF‐α promotes wound closure [21, 22] Promotes proliferation of endothelial cells [11]

It inhibits fibrosis and promotes re‐epithelialisation [98]

TGF‐β1 activates keratinocytes and macrophages, while suppressing T lymphocytes [94] TGF‐β3 stimulates remodelling [11]

collagenase, collagen and glycosaminoglycan

110 Wound Healing - New insights into Ancient Challenges

stimulates new blood vessel formation

and release of chemotactic cytokines

closure

*3.5.2. Cytokines*

matrix and the induction of a myofibroblast phenotype

MSP Accelerates cell migration and proliferation with regulation of proliferation and

TGF Enhances proliferation of epithelial cells, expression of antimicrobial peptides

proliferation and the remodelling phases of the healing process

fibroblasts and the induction of extracellular matrix deposition

Chemokines are a subtype of small cytokines responsible for stimulating chemotaxis and extravasation of leukocytes; hence, referred to as so, they are called chemotactic cytokines [40]. Human MSCs release several chemokines that participate in wound healing such as IL‐8 and its receptor (CXCL8), macrophage chemoattractant protein‐1 (MCP‐1) and its receptor (CCL2), macrophage inflammatory protein‐1‐alpha and macrophage inflammatory protein‐1‐beta (MIP‐1α and MIP‐1β) and stromal‐derived factor 1 (SDF‐1) (**Table 4**).


MSCs secrete some chemokines. These biological substances participate in wound healing from early stages starting with haemostasis and coagulation and ending with remodelling. They also participate in the inflammatory phase and accelerate proliferation and also migration of endothelial cells. In addition, they are involved in the contraction phase, ending at the last stages of remodelling, leading to wound healing in the absence of scar formation.

**Table 4.** The main chemokines secreted by MSCs and their roles and functions in the wound‐healing process.

### *3.5.4. Mesenchymal stem cell exosomes (MSC‐EXOSOME)*

Exosomes are tiny vesicles (30–100 nm in diameter) present in blood and urine and perhaps all other biological fluids that may also be collected from *in vitro* cell culture [112, 113]. Originating from endosomal sections and released from the plasma membrane into the extracellular environment these vesicles participate in coagulation, intracellular communica‐ tion and signalling and cytoplasmic cleaning [112–115]. It has been reported that MSC‐ EXOSOME repair renal injury indicating that MSC‐EXOSOME as a potential mechanism that could be harnessed for wound healing [116, 117]. With respect to wound healing, MSC‐ EXOSOME plays an important role in collagen synthesis, the acceleration of cell migration and proliferation and in the formation of new and mature blood vessel [113]. Exosome healing action could be attributed to its ability to transfer RNA, miRNA and proteins such as Wint‐4 into the injured tissues which participate in skin repair by promoting re‐epithelialisation and cell proliferation as well as through the activation of β‐catenin which plays a pivotal role in skin development and wound healing [116]. Additionally, MSC‐EXOSOME has been shown to accelerate wound healing through mediating pathway signalling of some genes such as Akt, ERK and STAT3 as well as by enhancing the expression of important growth factors, Such as; HGF, IGF‐1, NGF and SDF‐1 which collectively accelerate migration and proliferation of fibroblasts in normal and diabetic wounds [118]. Moreover, MSC‐EXOSOME reduces levels of pro‐apoptotic Bax thereby inhibiting apoptosis of skin cells such keratinocytes and fibroblasts [117, 119]. Collectively, these data suggest the MSC‐EXOSOMES thus play a significant role in wound healing.

The application of MSC‐CM or MSC‐EXOSOME onto chronic wounds either by direct injection or by designing biological dressings enriched with MSC‐CM collected from autologous MSCs may therefore provide a valuable therapeutic strategy.

The participation of MSC secretions in wound healing is summarised in **Figure 2**.

**Figure 2.** Participation of MSC secretions in wound‐healing phases and events. MSCs secrete a wide spectrum of growth factors, cytokines and chemokines. These biological substances participate in wound healing from early stages starting with haemostasis and coagulation and ending with remodelling. These secretions initiate and terminate the inflammatory phase and promote angiogenesis, accelerate proliferation and also migration of endothelial cells. In addi‐ tion, they are involved in the contraction phase, ending at the last stages of remodelling, leading to wound healing in the absence of scar formation.

### **3.6. Benefits of the use of mesenchymal stem cells in treating wounds**

As we previously mentioned, MSCs have been considered safe irrespective their use in different clinical indications, since no critical adverse or side effect of MSCs has been shown when used therapeutically in humans [74, 75, 120]. Interestingly, patients with conditions such as sepsis [106], acute limb ischemia, acute GvHD [78, 120], critical myocardial infarction, Crohn's disease, acute tubular necrosis and multiple sclerosis can obtain benefit from the use of MSC therapies with no reported contraindications [120].

### *3.6.1. Immunomodulatory features of MSCs*

its receptor (CXCL8), macrophage chemoattractant protein‐1 (MCP‐1) and its receptor (CCL2), macrophage inflammatory protein‐1‐alpha and macrophage inflammatory protein‐1‐beta

**Chemokines Function(s) References**

MCP‐1 MCP‐1 and its receptor (CCL2) are primarily involved in macrophage infiltration [66, 105]

MIP‐1 MIP‐1α and MIP‐1β promote wound closure [21, 22]

SDF‐1 Plays a role in regulating skin homeostasis and tissue remodelling [40]

ending at the last stages of remodelling, leading to wound healing in the absence of scar formation.

**Table 4.** The main chemokines secreted by MSCs and their roles and functions in the wound‐healing process.

MSCs secrete some chemokines. These biological substances participate in wound healing from early stages starting with haemostasis and coagulation and ending with remodelling. They also participate in the inflammatory phase and accelerate proliferation and also migration of endothelial cells. In addition, they are involved in the contraction phase,

Exosomes are tiny vesicles (30–100 nm in diameter) present in blood and urine and perhaps all other biological fluids that may also be collected from *in vitro* cell culture [112, 113]. Originating from endosomal sections and released from the plasma membrane into the extracellular environment these vesicles participate in coagulation, intracellular communica‐ tion and signalling and cytoplasmic cleaning [112–115]. It has been reported that MSC‐ EXOSOME repair renal injury indicating that MSC‐EXOSOME as a potential mechanism that could be harnessed for wound healing [116, 117]. With respect to wound healing, MSC‐ EXOSOME plays an important role in collagen synthesis, the acceleration of cell migration and proliferation and in the formation of new and mature blood vessel [113]. Exosome healing action could be attributed to its ability to transfer RNA, miRNA and proteins such as Wint‐4 into the injured tissues which participate in skin repair by promoting re‐epithelialisation and cell proliferation as well as through the activation of β‐catenin which plays a pivotal role in skin development and wound healing [116]. Additionally, MSC‐EXOSOME has been shown to accelerate wound healing through mediating pathway signalling of some genes such as Akt, ERK and STAT3 as well as by enhancing the expression of important growth factors, Such as;

Enhances the migration of epithelial cells *in vitro* [25, 111]

Inflammation regulatory chemokines in the wound‐healing process [40]

MIP‐1α and MIP‐1β increase macrophage trafficking [105]

Promotes wound closure [21, 22] Induces cell migration [74]

[110]

(MIP‐1α and MIP‐1β) and stromal‐derived factor 1 (SDF‐1) (**Table 4**).

(CXCL8) act as a chemoattractant

*3.5.4. Mesenchymal stem cell exosomes (MSC‐EXOSOME)*

for neutrophils

112 Wound Healing - New insights into Ancient Challenges

IL‐8 Increases keratinocyte proliferation and stimulate re‐epithelialisation in human skin grafts, both in *vitro* and in *vivo*. IL‐8 and its receptor

> In 2000, Liechty et al. [121] were the first to recognise that MSCs possess unique immunologic features allowing them to persist in a xenogeneic environment and modulate the immune

response. They have the potential to reduce inflammation and enhance repair wounds [122]. The exact mechanism by which MSCs modulate the immune system is not fully understood. The potential mechanism includes cell‐to‐cell direct contact and by the secretion of immune suppressive factors, interacting with other immune cells such as T lymphocytes, B lympho‐ cytes, dendritic cells (DC) and natural killer cells (NKCs) [123]. In 2013, Patel et al. [74] reported that MSCs suppress both the activation and proliferation of lymphocytes in response to allogeneic antigens as well as enhancing the development of CD8+ regulatory T cells (Treg) in the suppression of an allogeneic lymphocyte response. Additional immune suppressive activities of MSC include inhibition of differentiation of peripheral blood monocyte progenitor cells and CD34+ haematopoietic progenitor cells (HPCs) into antigen‐presenting cells (APCs) [124]. MSCs also inhibit the proliferation of NK cells mediated by IL‐2 or IL‐15 [125]. MSCs have been shown to exert other immunomodulatory activities including altering the prolifer‐ ation and activation of B cells, IgG production, antibody secretion, chemoattractant behaviour, and reducing the expression of CD40, and CD86 and major histocompatibility complex class II (MHC‐II) [126].

The ability of MSCs to modulate T cell and their proliferation, participation and activity [127] and suppress the proliferation of B cells [128] and NK cells [125] is well documented. By attenuating the function of these cells, MSCs reduce the pro‐fibrotic process [129]. Importantly, by the secretion of Prostaglandin E2 (PGE2) [102, 103] and 1L‐10 [11, 40, 106], MSCs regulate macrophage and lymphocyte function [30]. For instance, PGE2 attenuates mitogenesis and proliferation of T cells in the wound [124] acting as co‐parameter in regulating the transition from Th1 cells into Th2 cells [130]. On the other hand, IL‐10 prevents the deposition of excessive collagen and inhibits the invasion of neutrophils into the wound and their release of reactive oxygen species (ROS) factors, which collectively participate in the prevention of scar tissue formation [30].

### *3.6.2. Migration and engraftment capacity*

Various studies have reported the capability of MSCs to selectively migrate to and engraft into the wound site and exert local functional effects on inflammatory reactions regardless of tissue type [30, 73]. In this context, murine studies have shown that MSCs can home to the lung, adopting phenotypic characteristics of epithelium‐like cells and reducing inflammation in response to injury [131]. Another study in mdx mice, a strain of mice arising from a spontaneous mutation (mdx) in inbred C57BL mice showed that MSCs may migrate to muscle tissues [132]. MSC migration has been shown to be regulated by a multitude of signals [133] ranging from growth factors such as PDGF and IGF‐1, cytokine such as SDF‐1, chemokine such as CCL5 and chemokine receptors, including CCR2, CCR3 and CCR4 [73, 134].

### *3.6.3. Wound closure acceleration*

MSCs play a role not only in wound healing but also in accelerating the healing process by increasing the strength of wound and by reducing scaring [23]. The effects of MSCs during wound healing include acceleration of epithelialisation, an increase in angiogenesis and the formation of granulation tissue [11]. These activities are attributed to the ability of MSCs to produce biologically active substances capable of accelerating the regeneration process [135] including IL‐8 and CXCL1, responsible for stimulating the migration of epithelial cells and accelerating wound closure [111].

### *3.6.4. Antimicrobial activity*

response. They have the potential to reduce inflammation and enhance repair wounds [122]. The exact mechanism by which MSCs modulate the immune system is not fully understood. The potential mechanism includes cell‐to‐cell direct contact and by the secretion of immune suppressive factors, interacting with other immune cells such as T lymphocytes, B lympho‐ cytes, dendritic cells (DC) and natural killer cells (NKCs) [123]. In 2013, Patel et al. [74] reported that MSCs suppress both the activation and proliferation of lymphocytes in response to allogeneic antigens as well as enhancing the development of CD8+ regulatory T cells (Treg) in the suppression of an allogeneic lymphocyte response. Additional immune suppressive activities of MSC include inhibition of differentiation of peripheral blood monocyte progenitor cells and CD34+ haematopoietic progenitor cells (HPCs) into antigen‐presenting cells (APCs) [124]. MSCs also inhibit the proliferation of NK cells mediated by IL‐2 or IL‐15 [125]. MSCs have been shown to exert other immunomodulatory activities including altering the prolifer‐ ation and activation of B cells, IgG production, antibody secretion, chemoattractant behaviour, and reducing the expression of CD40, and CD86 and major histocompatibility complex class

The ability of MSCs to modulate T cell and their proliferation, participation and activity [127] and suppress the proliferation of B cells [128] and NK cells [125] is well documented. By attenuating the function of these cells, MSCs reduce the pro‐fibrotic process [129]. Importantly, by the secretion of Prostaglandin E2 (PGE2) [102, 103] and 1L‐10 [11, 40, 106], MSCs regulate macrophage and lymphocyte function [30]. For instance, PGE2 attenuates mitogenesis and proliferation of T cells in the wound [124] acting as co‐parameter in regulating the transition from Th1 cells into Th2 cells [130]. On the other hand, IL‐10 prevents the deposition of excessive collagen and inhibits the invasion of neutrophils into the wound and their release of reactive oxygen species (ROS) factors, which collectively participate in the prevention of scar tissue

Various studies have reported the capability of MSCs to selectively migrate to and engraft into the wound site and exert local functional effects on inflammatory reactions regardless of tissue type [30, 73]. In this context, murine studies have shown that MSCs can home to the lung, adopting phenotypic characteristics of epithelium‐like cells and reducing inflammation in response to injury [131]. Another study in mdx mice, a strain of mice arising from a spontaneous mutation (mdx) in inbred C57BL mice showed that MSCs may migrate to muscle tissues [132]. MSC migration has been shown to be regulated by a multitude of signals [133] ranging from growth factors such as PDGF and IGF‐1, cytokine such as SDF‐1, chemokine such

MSCs play a role not only in wound healing but also in accelerating the healing process by increasing the strength of wound and by reducing scaring [23]. The effects of MSCs during wound healing include acceleration of epithelialisation, an increase in angiogenesis and the formation of granulation tissue [11]. These activities are attributed to the ability of MSCs to

as CCL5 and chemokine receptors, including CCR2, CCR3 and CCR4 [73, 134].

II (MHC‐II) [126].

formation [30].

*3.6.2. Migration and engraftment capacity*

114 Wound Healing - New insights into Ancient Challenges

*3.6.3. Wound closure acceleration*

Conditioned medium of hBM‐MSCs is capable of inhibiting bacterial growth directly indicat‐ ing the ability of MSCs to produce and release substantial amounts of antibacterial substances known as human cathelicidin peptide‐18 (hCAP‐18) or LL‐37 peptide which are characterised by their ability to retard *in vitro* growth of *Pseudomonas aeruginosa* and *Escherichia coli* [24], thus avoiding wound contamination and infections complications which exacerbate the healing process [28].
