**2. Skin wounds and healing process**

Generally, wounds are classified on the basis of location, depth and tissue loss into three categories: superficial wounds where damage affects the epidermis only; partial thickness wounds when both the epidermis and dermis are involved; and full thickness wounds which involve the dermis, subcutaneous fats and sometimes, bones. However, depending on normal healing trajectory, there are two principal categories of skin wounds: acute and chronic wounds [26, 27].

### **2.1. Acute wounds**

**1. Introduction**

100 Wound Healing - New insights into Ancient Challenges

non‐healing wounds [13].

In the United Kingdom, around 200,000 patients experience a chronic wound of varying type, ranging between ulcerations, scars, trauma and burns. Unfortunately, patient morbidity and in some cases mortality may result from such injuries for which chronic ulceration is a major factor [1–3]. One of these impacts is the reduced contribution to society by the individuals suffering from these chronic wounds including their inability to work [3]. In addition, healthcare treatment and hospitalisation for chronic wounds are costly [2] involving lengthy treatment and nursing care. In 2005 and 2006, the care of patients with a chronic wound costs the UK NHS approximately £2.3bn–£3.1bn each year, with £6.08 million in England lone being attributed to nursing care [4]. In the United States, approximately 6.5 million patients suffered from chronic wounds with expenses for wound care management exceeding US \$25 billion in 2009 [5]. Furthermore, infection is inevitable, which not only negatively affect wound healing but can also be life threatening, requiring more hospitalisation and increased healthcare expenditure [6] and repetitive treatment [7]. Consequently, both the society and the health sector are negatively affected by the burden of chronic wounds. Moreover, despite great progress in wound treatment including the implementation of growth factors and biological engineering of skin equivalents, present treatment options for burns and non‐healing chronic wounds are restricted and not always effective [8]. Engineered skin to aid the development of novel wound care strategies is limited by their construction from substances that are hard to be degraded, and do not always result in complete replication into normal uninjured skin. Furthermore, complete renewal of this model requires the alteration of immune responses to reduce fibrotic reactions in order to diminish scar production [9]. Gene therapy may also be limited by insufficient selection of target cells, the identification of the factors which may affect the introduction of genes or the inability to produce stable prolonged specific gene product which is the main problem with systemic gene therapy [10]. Additionally, biofilms give rise to hypoxic conditions. Therefore, there is an urgent need for new therapies for wounds with delayed healing [8]. Specific extracellular matrix (ECM) proteins equivalent to the skin, specific growth factors, mesenchymal stem cells (MSCs), fibroblasts or viable epithelial cells may, however, aid the wound‐healing process, and their addition to potential wound‐healing treatments may improve the efficacy of current therapeutic strategies [11]. The availability of MSCs in normal human skin [12], and their vital function in wound healing suggests that the exogenous application of such cells may represent a promising solution for the treatment of

Mesenchymal stem cells (MSCs) are generally defined as self‐renewable, multi‐potent pro‐ genitor adult stem cells present in peripheral blood. *In vivo*, they have the ability to differentiate widely into many mesenchymal lineages such as cartilage, bone, muscle and adipose tissues [14]. Furthermore, MSCs have the ability to migrate from the bone marrow to an injured site and differentiate into functional skin cells [15]. *In vitro* they can be defined as fibroblast‐like cells capable of self‐renewal with the ability to adhere to plastic and subsequently differentiate into adipose, bone, cartilage tissue [16] as well as a multi‐layered epidermis‐like structure [17]. Paracrine factors secreted by MSCs are considered the principle factors with therapeutic potential for tissue wound healing [18] including growth factors, cytokines and chemokines Acute wounds arise either as a result of surgical incision or following traumatic accidents including abrasions, superficial burns and partial thickness injuries with significant loss of tissues. Irrespective to their causes, the healing process of acute wounds is complex and utilises different types of cells and cytokines [26].

### **2.2. Chronic skin wounds**

Wounds are defined as chronic when they fail to heal during one or all of the phases of the healing process causing an injury that cannot be repaired within the expected time period of normal wound repairs [11]. Chronic wounds mainly accompany disorders such as pressure ulcers, diabetes, burns, vascular insufficiency and vasculitis [5]. The chronic state of non‐ healing wounds is exacerbated by many factors including tissue hypoxia, microbial infection, necrosis, exudates and an elevated ratio of inflammatory cytokines during the different healing stages [28]. Neutrophils also contribute by releasing excessive amounts of collagenase which leads to break down the ECM [29] and enzyme elastase destroying important healing factors such as PDGF and transforming growth factor‐beta (TGF‐β). Chronic wounds do not respond to therapeutic methods unless the prolonged inflammation is targeted [11]. Consequently, human skin with its limited abilities will fail to heal itself in cases of wounds penetrating the epidermis [30] due to the deficiency in growth factors and cytokines which are depleted during the healing process [31, 32]

### **2.3. Phases of wound‐healing process**

Each wound undergoes a series of successive events for repairing and healing. These processes take from several minutes such as coagulation, several days such as inflammation to several months or years such as remodelling and can be divided into three, four or five overlapping phases and stages. Monaco and Lawrence [26] state the wound‐healing process consists of five distinct phases: (a) haemostasis, (b) inflammation, (c) cellular migration and proliferation, (d) protein synthesis and wound contraction and (e) remodelling, while Gosain and DiPietro [33] and Zhou et al. [34] describe the healing process as consisting of four highly integrated and overlapping phases: (a) haemostasis, (b) inflammation, (c) proliferation and (d) tissue remod‐ elling or resolution [7]. A normal wound‐healing mechanism is a dynamic and complex process involving a series of coordinated events, including (a) bleeding and coagulation, (b) acute inflammation, (c) cell migration, (d) proliferation, (e) differentiation, (f) angiogenesis, re‐ epithelialisation and (g) synthesis and remodelling of ECM. Conversely, Maxson et al. [11] report that the healing process is a complex event occurring in three overlapping phases: (a) inflammatory, (b) proliferative and (c) remodelling. These phases and their biophysiological functions must occur in the proper sequence, at a specific time, and continue for a specific duration and intensity [35]. There are many factors that can affect wound healing which interfere with one or more phases in this process, thus causing improper or impaired tissue repair [28]. All in all, a successful healing process cannot be accomplished without any one of these processes; haemostasis, inflammation, angiogenesis, proliferation, contraction, re‐ epithelialisation and remodelling [36]. To better understand the healing process, we will discuss the five phases and how they overlap.

### *2.3.1. Haemostasis phase (coagulation)*

During blood circulation in an intact blood vessel, endothelial cells of the blood vessel secrete coagulation and aggregation inhibitors, that is they release heparin‐like molecules to prevent blood coagulation and thrombomodulin to prevent platelet aggregation. Prostacyclin and nitric oxide are also involved in this process [37]. In contrast, the endothelial cells of broken blood vessels replace the secretions of clot inhibitors with a blood glycoprotein called von Willebrand factor (vWF) which initiates haemostasis [37, 38].

Haemostasis is the first phase of wound healing and consists of three successive steps: vasoconstriction, blockage the wound by platelet aggregation and blood coagulation. When skin is injured, a blood extravasation begins to fill the injured site. Immediately after the skin injury and bleeding, the blood vessel contracts and reduces the blood flow to the wounded site thereby keeping the blood within the damaged vessel and causing bleeding to stop [38, 39]. Not only do vessel contractions stop haemorrhage, but also blood changing from a liquid phase to a gel phase forming a blood clot (coagulation) and platelet aggregation generates a haemostatic buffer (plasma) which is rich in fibrin, thereby stopping the haemorrhage and restoring a barrier protecting the wound from infection by invading microorganisms. This process constitutes a matrix what encourages cell migration [40, 41]. In this phase, the role of platelets is not only restricted to blocking the damaged area and in clot formation, but also in the formation of a transient extracellular matrix by secreting adhesion molecules such as fibronectin and thrombospondin, as well as growth factors such as epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), transforming growth factor‐alpha (TGF‐α) and beta (TGF‐β), and vascular endothelial growth factor (VEGF) [42]. This matrix serves as a reservoir for growth factors and cytokines critical to subsequent healing phases [41]. Collec‐ tively, the matrix, activated cascade coagulation and parenchymatous cells make the injured vessel a chemotactic environment to attract inflammatory cells at the wound site and initiate the start of inflammatory phase [43].

### *2.3.2. Inflammation phase*

**2.3. Phases of wound‐healing process**

102 Wound Healing - New insights into Ancient Challenges

discuss the five phases and how they overlap.

Willebrand factor (vWF) which initiates haemostasis [37, 38].

*2.3.1. Haemostasis phase (coagulation)*

Each wound undergoes a series of successive events for repairing and healing. These processes take from several minutes such as coagulation, several days such as inflammation to several months or years such as remodelling and can be divided into three, four or five overlapping phases and stages. Monaco and Lawrence [26] state the wound‐healing process consists of five distinct phases: (a) haemostasis, (b) inflammation, (c) cellular migration and proliferation, (d) protein synthesis and wound contraction and (e) remodelling, while Gosain and DiPietro [33] and Zhou et al. [34] describe the healing process as consisting of four highly integrated and overlapping phases: (a) haemostasis, (b) inflammation, (c) proliferation and (d) tissue remod‐ elling or resolution [7]. A normal wound‐healing mechanism is a dynamic and complex process involving a series of coordinated events, including (a) bleeding and coagulation, (b) acute inflammation, (c) cell migration, (d) proliferation, (e) differentiation, (f) angiogenesis, re‐ epithelialisation and (g) synthesis and remodelling of ECM. Conversely, Maxson et al. [11] report that the healing process is a complex event occurring in three overlapping phases: (a) inflammatory, (b) proliferative and (c) remodelling. These phases and their biophysiological functions must occur in the proper sequence, at a specific time, and continue for a specific duration and intensity [35]. There are many factors that can affect wound healing which interfere with one or more phases in this process, thus causing improper or impaired tissue repair [28]. All in all, a successful healing process cannot be accomplished without any one of these processes; haemostasis, inflammation, angiogenesis, proliferation, contraction, re‐ epithelialisation and remodelling [36]. To better understand the healing process, we will

During blood circulation in an intact blood vessel, endothelial cells of the blood vessel secrete coagulation and aggregation inhibitors, that is they release heparin‐like molecules to prevent blood coagulation and thrombomodulin to prevent platelet aggregation. Prostacyclin and nitric oxide are also involved in this process [37]. In contrast, the endothelial cells of broken blood vessels replace the secretions of clot inhibitors with a blood glycoprotein called von

Haemostasis is the first phase of wound healing and consists of three successive steps: vasoconstriction, blockage the wound by platelet aggregation and blood coagulation. When skin is injured, a blood extravasation begins to fill the injured site. Immediately after the skin injury and bleeding, the blood vessel contracts and reduces the blood flow to the wounded site thereby keeping the blood within the damaged vessel and causing bleeding to stop [38, 39]. Not only do vessel contractions stop haemorrhage, but also blood changing from a liquid phase to a gel phase forming a blood clot (coagulation) and platelet aggregation generates a haemostatic buffer (plasma) which is rich in fibrin, thereby stopping the haemorrhage and restoring a barrier protecting the wound from infection by invading microorganisms. This process constitutes a matrix what encourages cell migration [40, 41]. In this phase, the role of platelets is not only restricted to blocking the damaged area and in clot formation, but also in the formation of a transient extracellular matrix by secreting adhesion molecules such as An inflammatory reaction begins soon after the haemorrhage stops at the site of injury. This reaction promotes mobility of various cells toward the injured tissue giving rise to a multitude of complicated and successive series of reactions ending with rebuilding of a tissue‐like structure [44]. The main advantages of this phase are isolating the injured tissues from the surrounding contaminated environment, cleaning out cell debris and damaged tissues and the initiation of the healing process [45]. The main reactivity observed in this phase is an increased migration of inflammatory cells from intravascular tissue towards the extracellular wound site due to increased vascular permeability. This permeability increases due to vasodilation when both fibrin and thrombin are activated by the coagulation cascade. Meanwhile, clot formation and their stimuli are dissipated and plasminogen converted to plasmin [46]. Three main cell types are involved in the inflammatory phase: neutrophils, macrophages and lymphocytes whose activity is initiated within hours of injury [44, 45]. Neutrophils seem to be the most dominant cell type during the first 48 hours, cleaning the wound site from bacteria, cell debris and damaged tissue by releasing free radicals; however, they are not essential for the healing process [40, 41, 47]. Approximately 48 hours following the injury, stimuli for neutrophils no longer persist and neutrophil numbers cease when macrophages (monocyte‐derived macro‐ phage) then penetrate the wound site via the blood and become the dominant cellular component of the inflammatory phase by phagocytosing cell debris and bacteria including expended neutrophils. Macrophages also secrete collagenases and elastases to break down the damaged tissues [48]. In contrast to neutrophils, the role of macrophages is not restricted to cleaning of the tissues as they also play a crucial role in the healing process by secreting prostaglandins, which act as vasodilators increasing microvessel permeability and attracting other inflammatory cells into the wounded site [41, 49, 50]. In addition, macrophages secrete fibroblast growth factor (FGF), PDGF, TGF‐α and VEGF which are important for proliferation and migration of fibroblasts as well as cytokines, which attract endothelial cells to the injury site promoting their proliferation and the development of a new tissue [48, 49, 51]. Within three days of the inflammatory phase, T lymphocytes home to the injury site by the activity of interlukin‐1 and secret lymphokines such heparin‐binding epidermal growth factor (HB‐EGF) and basic fibroblast growth factor (bFGF), promoting fibroblast proliferation [52].

### *2.3.3. Proliferation phase (epithelialisation)*

The proliferation phase (epithelial proliferation phase) represents the main phase responsible for actual wound closure. In the case of skin wounds, endothelial non‐inflammatory cells such as keratinocytes and fibroblasts start to proliferate and migrate towards the edges of the wound‐producing collagen for the development of new tissues [53–55]. Within a few hours (between 6 and 24 hours) of injury, TGF‐β and EGF act as mitogenic and chemotactic stimu‐ lators attracting keratinocytes which migrate towards the wound and start epithelialisation [54]. Fibroblasts are activated and start to differentiate into myofibroblasts which participate in reducing the wound size by contracting and secreting extracellular matrix (ECM) proteins giving rise to healing of the connective tissue [56, 57]. Meanwhile, angiogenesis progresses, coordinating the transfer of nutrients and oxygen from newly formed capillaries to the wound site enhancing metabolic activity [58]. Epithelisation, fibroplasia and angiogenesis collectively comprise granulation tissue which covers the damaged tissues within four days of injury [55].

### *2.3.4. Contraction phase*

Wound contraction could be defined as mobility of wound margins towards the wound core to facilitate closure; this phase begins when fibroblasts stop proliferating and undergo apoptosis within 5–15 days post‐injury which occurs concurrently with collagen synthesis [59, 60]. The rate of movement of wound edges depends on tissue laxity and wound shape; for instance, the looser tissues tend to contract more rapidly than the compact tissues and squared wounds contract more quickly than rounded wounds. The contraction rate also depends on the availability of myofibroblast and their proliferation and connection to the surrounding extracellular matrix [61].

### *2.3.5. Remodelling phase (resolution)*

Remodelling or resolution is the last phase of the wound‐healing process. The biological processes observed in this step involve gradual resolution of the inflammatory phase, collagen deposition, complete coverage of the injured site by the new tissues and formation of scar tissue [62]. Successful remodelling requires stable collagen content; therefore, the important step in this phase is controlling collagen remodelling [34]. Although collagen synthesis is continuing during this phase, its level is restricted due to the activity of collagenases and metalloproteinases which aid in removing the excess collagen [63, 64]. For optimal remodel‐ ling, collagen levels need to be balanced by the activity of metalloproteinases inhibitors secreted by tissue arresting the collagenolytic enzymes and balancing the production of new collagen with that of the removed old collagen [64]. The outcome of this process is that collagen type III is replaced by collagen type I, hence replacing both hyaluronic acid and glycosami‐ noglycans by proteoglycans and the disappearance of fibronectin as well as resorbing water from scar tissues. These events start approximately 3 weeks after the injury and may last indefinitely as collagen fibres stack closer to each other decreasing scar thickness and increas‐ ing wound bursting strength 'resistance to rupture' [65].

As described above, the main issues in the wound‐healing process are how cells are attracted to the site of injury site and how to enhance their proliferation and differentiate at the wounded region. These cells include inflammatory cells (neutrophils, macrophages and lymphocytes) and epithelial cells (fibroblasts and keratinocytes). All these activities are mainly regulated by growth factors and cytokines. In many cases, these cells fail to migrate, proliferate and differentiate due to deficiency in growth factors and cytokines; consequently, the healing process will be impaired and chronic wounds will arise [54]. Therefore, in order to improve wound healing, there is a need for an alternative source of healing cytokines and growth factors to enrich the injury site. MSC‐CM acts as a rich source of 36 growth factors, cytokines and chemokines which collected from MSC *in vitro* under good manufacturing practice could be used as therapy for wounds in the future [66]. The main events and phases of the wound‐ healing process are summarised in **Table 1**.

as keratinocytes and fibroblasts start to proliferate and migrate towards the edges of the wound‐producing collagen for the development of new tissues [53–55]. Within a few hours (between 6 and 24 hours) of injury, TGF‐β and EGF act as mitogenic and chemotactic stimu‐ lators attracting keratinocytes which migrate towards the wound and start epithelialisation [54]. Fibroblasts are activated and start to differentiate into myofibroblasts which participate in reducing the wound size by contracting and secreting extracellular matrix (ECM) proteins giving rise to healing of the connective tissue [56, 57]. Meanwhile, angiogenesis progresses, coordinating the transfer of nutrients and oxygen from newly formed capillaries to the wound site enhancing metabolic activity [58]. Epithelisation, fibroplasia and angiogenesis collectively comprise granulation tissue which covers the damaged tissues within four days of injury [55].

Wound contraction could be defined as mobility of wound margins towards the wound core to facilitate closure; this phase begins when fibroblasts stop proliferating and undergo apoptosis within 5–15 days post‐injury which occurs concurrently with collagen synthesis [59, 60]. The rate of movement of wound edges depends on tissue laxity and wound shape; for instance, the looser tissues tend to contract more rapidly than the compact tissues and squared wounds contract more quickly than rounded wounds. The contraction rate also depends on the availability of myofibroblast and their proliferation and connection to the surrounding

Remodelling or resolution is the last phase of the wound‐healing process. The biological processes observed in this step involve gradual resolution of the inflammatory phase, collagen deposition, complete coverage of the injured site by the new tissues and formation of scar tissue [62]. Successful remodelling requires stable collagen content; therefore, the important step in this phase is controlling collagen remodelling [34]. Although collagen synthesis is continuing during this phase, its level is restricted due to the activity of collagenases and metalloproteinases which aid in removing the excess collagen [63, 64]. For optimal remodel‐ ling, collagen levels need to be balanced by the activity of metalloproteinases inhibitors secreted by tissue arresting the collagenolytic enzymes and balancing the production of new collagen with that of the removed old collagen [64]. The outcome of this process is that collagen type III is replaced by collagen type I, hence replacing both hyaluronic acid and glycosami‐ noglycans by proteoglycans and the disappearance of fibronectin as well as resorbing water from scar tissues. These events start approximately 3 weeks after the injury and may last indefinitely as collagen fibres stack closer to each other decreasing scar thickness and increas‐

As described above, the main issues in the wound‐healing process are how cells are attracted to the site of injury site and how to enhance their proliferation and differentiate at the wounded region. These cells include inflammatory cells (neutrophils, macrophages and lymphocytes) and epithelial cells (fibroblasts and keratinocytes). All these activities are mainly regulated by growth factors and cytokines. In many cases, these cells fail to migrate, proliferate and

*2.3.4. Contraction phase*

104 Wound Healing - New insights into Ancient Challenges

extracellular matrix [61].

*2.3.5. Remodelling phase (resolution)*

ing wound bursting strength 'resistance to rupture' [65].


This table shows the main phases and events of the wound‐healing process which are divided into five overlapping phases. For example, the phase (a) indicates that the wound is not healed and there is a possibility to reach a chronic state if the coagulation phase failed. The phase (b) indicates that the wound is still not healed, but it is progressing towards healing; however, if the inflammation is not terminated, a chronic condition has a chance to be initiated. The phase (c) indicates that the active healing process has been initiated. The phase (d) represents further development of the healing process with less chance of progression to a chronic condition. The phase (e) represents the complete healing and remodelling.

**Table 1.** The main phases and events of the wound‐healing process.
