**1. Introduction**

A wound may be defined as a disruption to the physiological arrangement of the skin cells and a disturbance to its function in connecting and protecting underlying tissues and organs. It may be primary caused by accidental cut, tear, scratch, pressure, extreme temperatures, chemicals, and electrical current, or secondary to surgical intervention or disease (i.e., diabetes, ulcers, or carcinomas) [1]. It ranges from superficial (affecting the epidermis) to partialthickness (affecting both epidermis and parts of the dermis) and full-thickness (including subcutaneous fat and bones) wounds [2]. Wound healing is a physiological process, by which the living body repairs tissue damages, restores its anatomical integrity, and regains the functionality of the injured parts. A wound can be closed by primary intention or left to heal by secondary intention, and in both ways the healing process occurs through a series of overlapping events and is influenced by a number of intrinsic and extrinsic factors [3].

### **1.1. Acute wounds**

Acute wounds can heal within a limited amount of time, usually show no complications, and are characterized by the loss of skin integrity (injury) that occurs suddenly. The injured tissue heals in a predictable manner where platelets, keratinocytes, immune surveillance cells, microvascular cells, and fibroblasts play major roles in the restoration of tissue integrity [4]. These wounds are either surgical or traumatic [5].

### **1.2. Chronic wounds**

Chronic wounds are wounds that do not heal within normal period and are associated with predisposing factors that weaken the integrity of dermal and epidermal tissues. Those factors either disrupt the balance between wound bioburden and the patient's immune system or impair the wound healing cycle. In terms of duration, if the wound fails to heal or shows no sign of recovery within 12 weeks, it is considered a chronic wound. Predisposing factors may affect the tissue perfusion causing chronic wounds such as vascular ulcers, associated with metabolic disorders such as diabetes causing diabetic foot ulcers [6]. They can be identified by criteria such as delayed healing and friable granulation tissue, prolonged inflammatory phase, persistent infection, and presence of resistant microorganisms [7–10].

### **1.3. Wound healing**

The repair (wound healing) process involves four overlapping biochemical, physiological, and molecular phases.

### I. Hemostasis

This stage is characterized by microvascular injury and release of blood components at the wound site. Platelets come into contact with and adhere to the wall of the injured blood vessels. This adherence activates the platelets to release cytokines, growth factors, and numerous pro-inflammatory mediators, resulting in platelet aggregation and triggering the intrinsic and extrinsic coagulation pathways to form a fibrin clot which limits further blood loss. Growth factors produced by the platelets initiate the healing cascade [11, 12].

II. Inflammatory phase

**1. Introduction**

374 Wound Healing - New insights into Ancient Challenges

**1.1. Acute wounds**

**1.2. Chronic wounds**

**1.3. Wound healing**

molecular phases.

I. Hemostasis

These wounds are either surgical or traumatic [5].

persistent infection, and presence of resistant microorganisms [7–10].

A wound may be defined as a disruption to the physiological arrangement of the skin cells and a disturbance to its function in connecting and protecting underlying tissues and organs. It may be primary caused by accidental cut, tear, scratch, pressure, extreme temperatures, chemicals, and electrical current, or secondary to surgical intervention or disease (i.e., diabetes, ulcers, or carcinomas) [1]. It ranges from superficial (affecting the epidermis) to partialthickness (affecting both epidermis and parts of the dermis) and full-thickness (including subcutaneous fat and bones) wounds [2]. Wound healing is a physiological process, by which the living body repairs tissue damages, restores its anatomical integrity, and regains the functionality of the injured parts. A wound can be closed by primary intention or left to heal by secondary intention, and in both ways the healing process occurs through a series of overlapping events and is influenced by a number of intrinsic and extrinsic factors [3].

Acute wounds can heal within a limited amount of time, usually show no complications, and are characterized by the loss of skin integrity (injury) that occurs suddenly. The injured tissue heals in a predictable manner where platelets, keratinocytes, immune surveillance cells, microvascular cells, and fibroblasts play major roles in the restoration of tissue integrity [4].

Chronic wounds are wounds that do not heal within normal period and are associated with predisposing factors that weaken the integrity of dermal and epidermal tissues. Those factors either disrupt the balance between wound bioburden and the patient's immune system or impair the wound healing cycle. In terms of duration, if the wound fails to heal or shows no sign of recovery within 12 weeks, it is considered a chronic wound. Predisposing factors may affect the tissue perfusion causing chronic wounds such as vascular ulcers, associated with metabolic disorders such as diabetes causing diabetic foot ulcers [6]. They can be identified by criteria such as delayed healing and friable granulation tissue, prolonged inflammatory phase,

The repair (wound healing) process involves four overlapping biochemical, physiological, and

This stage is characterized by microvascular injury and release of blood components at the wound site. Platelets come into contact with and adhere to the wall of the injured blood vessels. This adherence activates the platelets to release cytokines, growth factors, and numerous pro-inflammatory mediators, resulting in platelet aggregation and triggering the intrinsic and extrinsic coagulation pathThe inflammatory phase starts at the same time as hemostasis sometime between a few minutes after injury up to 24 h and lasts for about 3 days. Aggregated platelets store vasoactive amines such as prostaglandins and histamine while other amines from granules released by mast cells, in response to injury, result in increased microvascular permeability and vasodilation, leading to exudation of fluid into the extravascular space [13]. This allows the migration of monocytes and protein-rich exudate into the wound and surrounding tissue, resulting in edema. These are typical signs of the inflammation process, and patients start complaining about pain at the site of injury within 24 h.

### III. Proliferative phase

This phase commences after the inflammatory phase wanes. The remaining inflammatory cells produce growth factors to initiate angiogenesis, which is important to keep adequate blood supply within the wound bed [14]. Newly formed blood vessels will contribute to granulation tissue (composed of collagen and extracellular matrix) formation and provide the required nutrients.

### IV. Maturation phase

This commences when the wound is superficially sealed. It involves the reepithelialization and remodeling of newly formed tissues in the proliferative phase and restoration of epidermal integrity [15]. It also involves transferring collagen III to collagen I.

### **1.4. Factors affecting wound healing**

Multiple factors affect wound healing and lead to the impairment of healing classified into local and systemic factors [16].

### *1.4.1. Oxygenation*

Oxygen is crucial to wound healing and for resistance to infection, and used for cellular energy production by adenosine triphosphate [17]. It acts on different levels of wound healing by inducing angiogenesis, keratinocytes differentiation, migration, re-epithelialization, fibroblast proliferation, and collagen synthesis, and promotes wound contraction [18]. When injury occurs, temporary hypoxia and oxygen are useful to trigger wound healing by inducing the production of cytokines and growth factors from macrophages, keratinocytes, and fibroblasts [16]. Chronic wounds are generally hypoxic with oxygen tissue tension of 5–20 mm Hg compared to normal levels of 30–50 mm Hg [19]. Factors predisposing chronic wounds such as advancing age and diabetes can induce poor oxygenation through impaired vascular flow. Interventional revascularization therapies have been used to reverse hypoxic conditions in diabetic foot ulcers [20]. However, it has also been reported that such procedures can cause adverse effects to diabetic patients [21]. Recently, some topical foam dressings containing dissolved oxygen were developed to increase oxygen perfusion into the chronic wound area [22]. Results showed that dissolved oxygen from topical foam dressing penetrates into skin layers compared to topical gaseous oxygen.

### *1.4.2. Wound bioburden and infection*

### *1.4.2.1. Bioburden*

The intact skin acts to control the microbial population on the skin surface itself [23]. Once the integrity is lost through injury, the subcutaneous tissue becomes exposed, providing an environment for colonization and growth of microbes. However, this does not necessarily lead to an infection as there is a balance between the wound bioburden and the immune system [24].

### *1.4.2.2. Wound infection*

Skin microflora is present to about 105 colonies without any clinical problems [25]. However, if the balance is disrupted, microorganisms will proliferate and start a microbiological chain of events by invading tissues resulting in an inflammatory response which may lead to tissue damage and delayed healing [7]. Once it causes damage to the host tissue, infection will arise. One of the consequences of infection is the prolonged inflammation due to prolonged elevation of pro-inflammatory cytokines, which causes the wound to enter the chronic stage and fail to heal within the expected 8–12 weeks [26]. This prolonged inflammation is also associated with increased levels of matrix metalloproteases which are capable of degrading the extracellular matrix which is the key component of proliferative phase of wound healing [9]. This increase in protease levels happens at the expense of the naturally occurring protease inhibitor levels that are decreased. From a microbiological perspective, wound infection is described as the presence of replicating microorganisms at the wound site overwhelming the host's immune system. It delays wound healing due to the release of toxins and exhibits active signs and symptoms of infections.

### *1.4.2.3. Common bacterial species present in chronic wounds*

Generally, most infected wounds are polymicrobial and are commonly contaminated by pathogens found in the immediate environment, the endogenous microbes living in the mucous membranes, and the microflora on adjacent skin. Bacteria are the main cause of wound infection among other microorganisms present in the skin, though other microorganisms such as fungi have been implicated in certain mixed infections. In the initial stages of chronic wound formation, Gram-positive organisms such as *Staphylococcus aureus* and *Escherichia coli* are predominant [9]. In the later stages, Gram-negative *Pseudomonas* species are common and tend to invade deeper layers in the wound causing significant tissue damage [27]. Other aerobes implicated include *Staphylococci* and *Streptococci* species as well as anaerobic bacteria and are estimated in 50% of chronic wounds [28, 29].

### *1.4.3. Chronic wounds and biofilm*

adverse effects to diabetic patients [21]. Recently, some topical foam dressings containing dissolved oxygen were developed to increase oxygen perfusion into the chronic wound area [22]. Results showed that dissolved oxygen from topical foam dressing penetrates into skin

The intact skin acts to control the microbial population on the skin surface itself [23]. Once the integrity is lost through injury, the subcutaneous tissue becomes exposed, providing an environment for colonization and growth of microbes. However, this does not necessarily lead to an infection as there is a balance between the wound bioburden and the immune system [24].

if the balance is disrupted, microorganisms will proliferate and start a microbiological chain of events by invading tissues resulting in an inflammatory response which may lead to tissue damage and delayed healing [7]. Once it causes damage to the host tissue, infection will arise. One of the consequences of infection is the prolonged inflammation due to prolonged elevation of pro-inflammatory cytokines, which causes the wound to enter the chronic stage and fail to heal within the expected 8–12 weeks [26]. This prolonged inflammation is also associated with increased levels of matrix metalloproteases which are capable of degrading the extracellular matrix which is the key component of proliferative phase of wound healing [9]. This increase in protease levels happens at the expense of the naturally occurring protease inhibitor levels that are decreased. From a microbiological perspective, wound infection is described as the presence of replicating microorganisms at the wound site overwhelming the host's immune system. It delays wound healing due to the release of toxins and exhibits active signs and

Generally, most infected wounds are polymicrobial and are commonly contaminated by pathogens found in the immediate environment, the endogenous microbes living in the mucous membranes, and the microflora on adjacent skin. Bacteria are the main cause of wound infection among other microorganisms present in the skin, though other microorganisms such as fungi have been implicated in certain mixed infections. In the initial stages of chronic wound formation, Gram-positive organisms such as *Staphylococcus aureus* and *Escherichia coli* are predominant [9]. In the later stages, Gram-negative *Pseudomonas* species are common and tend to invade deeper layers in the wound causing significant tissue damage [27]. Other aerobes implicated include *Staphylococci* and *Streptococci* species as well as anaerobic bacteria and are

colonies without any clinical problems [25]. However,

layers compared to topical gaseous oxygen.

*1.4.2. Wound bioburden and infection*

376 Wound Healing - New insights into Ancient Challenges

Skin microflora is present to about 105

*1.4.2.1. Bioburden*

*1.4.2.2. Wound infection*

symptoms of infections.

*1.4.2.3. Common bacterial species present in chronic wounds*

estimated in 50% of chronic wounds [28, 29].

Biofilm is defined as "a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a secreted matrix of extracellular polymeric substances (EPSs), and exhibit an altered phenotype with respect to growth rate and gene transcription" [30]. Firstly, conditioning film forms and is composed of proteins and polysaccharide molecules adsorbed onto the solid surface. This makes the surface ready to receive the first cells of the insipient biofilm. Secondly, bacteria will start to approach and attach onto the surface by forces such as van der Waals forces and the negative electrostatic charges of bacterial surface [31]. The attached bacteria become encased in a polymeric matrix called extracellular polymeric substance (EPS). This bacterial attachment induces a phenomenon called quorum sensing, which is responsible for "the regulation of gene expression in response to fluctuations in cell population density" [32]. This causes the bacteria within biofilm to alter their phenotypes resulting in the production of more virulent factors in response to signals from other bacteria within biofilm. These factors with barrier made from EPS contribute to the increased resistance to antibiotics. It has been suggested that EPS can interact with antibiotics spontaneously thereby preventing them reaching the bacteria to exert their antimicrobial activity [33]. The biofilm also protects the bacteria from host defenses by the covering of glycocalyx while bacteria secrete products within the film which makes phagocytic penetration poor [34].

This understanding is of great importance for intervention modalities in chronic wounds especially the use of antimicrobial wound dressing. For example macrolides can have inhibitory effect on the film formation or induce phagocytic invasion into biofilms [35]. Furthermore, in clinical wound management, it is always essential to promptly clean the wound and remove necrotic tissue and foreign material (e.g. bacteria and biofilms) from areas around the wound to improve the chances of enhanced wound healing, and this is known as debridement [1]. This is important because the presence of necrotic tissue increases the risk of infection and sepsis, which prolongs the inflammatory phase. Several approaches are employed including surgical removal, wound irrigation (e.g. saline and antiseptics such as chlorhexidine), autolytic rehydration using hydrogel dressings, applying enzymes such as collagenases or streptokinase preparations as well as using maggots to selectively dissolve necrotic and infected tissue (including biofilms) without destroying healthy or newly formed tissue [1].
