Scarless Wound Healing

Abstract

Wound healing is a complex, multiple-step mechanism and most lead to the development of scars, which may or may not affect the functional capability of the healed tissue. However, with the advanced healing techniques and our improved understanding of the wound-healing process, there has been some development towards limiting the scarification that develops as part of the process. This chapter will explore the major types of scar tissue as well as their development and complications arising from the same. With wound healing being a complex process, there have also been attempts towards modulating the wound environment to increase the rate of healing as well as limit the formation of scars. While there is no definitive procedure that can ascertain rapid, scar-free healing as yet, this chapter aims to explore both, the traditional and alternative techniques that are used (during or after the complete healing of the wound) to mitigate the development of scars.

1. Introduction

Scarless Wound healing is considered as the elusive Holy Grail of wound management [1]. While scars are considered as ‘badges of honour’ in some cultures and highly prized [2, 3]; in most scenarios, people prefer to avoid or minimise them as much as possible. And in some cases, formation of a ‘scar’ leads to functional impairment that hampers the quality of one’s life—such as scars from abrasions on the ocular surface that lead to corneal opacity and hamper vision [4, 5, 6, 7, 8].

Wound healing is composed of three overlapping stages involving cellular and molecular processes that generally culminate into a fibrotic patch to ‘repair’ the wound with [3]. These ‘scar tissues’ have no hair follicles or sweat glands, and are inflexible and weaker than regular skin. They also limit movement and do not easily adapt to temperature changes [9].

Wound healing is a complex series of reactions primarily involving three distinct stages: inflammation, proliferation and maturation or remodelling of the tissue [10]. Each stage involves a complex series of interactions among the involved cells and their mediators (Figure 1).

Figure 1.

Stages of wound healing- representative image indicating the four primary stages involved in wound healing since the inception of the injury (A), and the pathogens on the epidermal surface which may lead to opportunistic infections. The inflammation stage (B) when the scab is formed to staunch the bleeding and macrophages activated to combat pathogens. The injury leads to ‘signals’ that summon fibroblasts, macrophages, neutrophils and platelets to the site of the injury. The proliferation stage (C) when the arriving cells proliferate and re-structure the ECM. The maturation stage (D) when the wound contracts and leads to the development of a scar.

2. Steps involved in traditional wound healing

2.1 Inflammatory phase

The initial healing begins with the formation of a fibrin clot at the site of the wound that serves as a temporary extra-cellular matrix (ECM) and provides the necessary stimulus to summon inflammatory cells [11].

Characterised by haemostasis and inflammation, collagen exposure during injury leads to the activation of the intrinsic and extrinsic pathways of the clotting cascade, thereby initiating the inflammatory phase. Vasoconstrictors like thromboxane A2 and prostaglandin 2-α are immediately released, leading to the formation of a clot made of collagen, platelets, thrombin and fibronectin. The release of cytokines and growth factors initiates the inflammatory response and the fibrin clot serves as ECM for the arriving cascade of inflammatory cells (including neutrophils, monocytes, fibroblasts and endothelial cells) with the neutrophils being the first cells to arrive (Figure 1A and B). As these inflammatory regulators arrive, the signal released changes from vasoconstrictors (that aided in initial clot formation) to vasodilators to allow for the increased cellular traffic [10].

The presence of Interleukin (IL)-1, Tumour Necrosis factor (TNF)- α, Transforming Growth factor (TGF)-β, Platelet Factor-4 (PF-4) attract monocytes from nearby tissue and blood that convert into macrophages which in turn are essential for transitioning the wound from its initial inflammatory phase to the proliferative phase. Macrophage activation generally occurs between 2 and 4 days post injury and leads to angiogenesis (the formation of new blood vessels) and fibroplasia (growth of fibrous tissue). It also leads to the synthesis of nitric oxide that plays multiple roles including providing pain relief by serving as a partial agonist at opioid receptors, encouraging vasodilation and having anti-inflammatory effects [10]. Interestingly, nitric oxide is considered as a pro-inflammatory mediator that induces inflammation due to its overproduction in pathophysiological situations [10, 12].

2.2 Proliferation phase

The proliferation phase is another complex stage in wound healing that involve multiple simultaneous processes occurring at the site of the injury including (but not limited to) epithelialisation, angiogenesis, fibroplasia, granular tissue formation and collagen deposition (Figure 1C) [10, 11].

Epithelialisation, one of the early steps in wound repair occurs in one of two ways based on the severity of the wound:

1. Basement membrane intact—If the wound is shallow, leaving the basement membrane intact; epithelial cells migrate upwards in a normal pattern as epithelial progenitor cells remain undamaged. This type of injury allows the epidermis to be restored within 2–3 days.

2. Basement membrane damage—In case of deeper wounds where the basement membrane has been affected, the epithelial cells at the edges begin proliferation and sending out projections to aid in clot formation and re-establishment of protective barrier. This is followed by angiogenesis, which leads to endothelial cell migration and capillary formation, which allows granulation and tissue deposition due to the nutrient supply. The protective barrier formed by the epithelial cell projections from the edge of the wound plays crucial role in providing a protective role by preventing bacterial invasion and fluid loss.

Epithelialisation, while initially stimulated by inflammatory cytokines from the inflammatory phase of wound healing, leads to upregulation of keratinocyte growth factor (KGF) by the stimulation of IL-1 and TNF-α. KGF, in turn stimulates keratinocytes from wound-adjacent regions to migrate to the site of injury and proliferate as well as differentiate in the epidermis [10].

Granulation tissue formation is the final stage in the proliferation phase, characterised by activation of fibroblasts which in turn initialise collagen synthesis and turn into myofibroblasts that aid in wound contraction. Wound contraction, induced by the TGF-β1 that is secreted by macrophages, is primarily carried out by the fibroblasts present at the wound site (wound fibroblasts) that have less proliferative potential as compared to those at the periphery. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) are the primary signals that drive the attraction of fibroblasts and their activation [10, 11].

This in turn leads to the synthesis of a provisional matrix that is made up of collagen type-III, glycosaminoglycans and fibronectin [9, 10, 11].

2.3 Maturation phase

The maturation phase is often deemed as the most important phase from a clinical perspective as it is characterised by collagen deposition (Figure 1D). Any issue with collagen deposition and deviation in the orderly networked fashion it is meant to take can lead to compromised wound strength. Conversely, excessive collagen deposition leads to the development of a hypertrophic or keloid scar [8, 9].

As the wound matures, the collagen that is initially thinner than that produced over uninjured skin changes to become thicker and organised along the wound such that they are organised around the regions that are under greater stress than their surroundings. It must be noted that the collagen in these granulation tissues that develop in wound regions has greater hydroxylation and glycosylation of lysine residues, making it different from those that are found in uninjured areas. While the tissue strength rarely returns to its pre-injury state, wound strength gradually increases over time with the region needing a minimum of 90 days to regain 80% of its original strength (this may be compared with its strength during the first week of wound healing which is only 3%) [10, 11].

3. Scarless wound healing

While scars are revered in some cultures, with elaborate rituals centred around developing aesthetically pleasing, elaborate scars, they are more commonly categorised as a cosmetic concern. Apart from aesthetic concerns, scars can also lead to reduced functionality and limit mobility. Furthermore, unsightly scars, particularly those on the face or other visible areas of the body; can often lead to crippling self-esteem issues and affect the quality of life of the effected individual. The United States of America alone has a $12 million industry targeted to reducing or limiting scars [8, 10].

Scarless wound healing as it occurs in nature is a rare phenomenon that is limited to specific scenarios.

3.1 Foetal wound healing

While wounds in the early mammalian gestational period heal without the formation of scars, wounds beyond that period form scars. It has been speculated that the early foetal wound healing occurs by a process resembling regeneration. While the exact mechanism is elusive, it is speculated that the characteristics that differentiate foetal skin at the tissue and cellular levels plays a role such as the decreased tensile strength [11].

A fascinating study by Wong et al. [13] has connected mechano-transduction with fibrosis through the focal adhesion kinase (FAK) pathway. It was observed that the extra cellular-related kinase (ERK) initiated the secretion of monocyte chemo-attractant protein-1(MCP-1) in the FAK pathway is associated with multiple fibrotic disorders in humans. When components of the inflammatory FAK-ERK-MCP-1 pathways are inhibited, the development of scars is attenuated [13].

3.2 Oral mucosa

In the mammalian system, the oral mucosa demonstrates minimal scarring and is the closest model of regenerative healing. It is often described as a ‘protected environment’ for wound healing as the underlying mucosa in the oral cavity is protected from mechanical damage or infection from pathogens by hydrophilic viscoelastic gels that are formed by the salivary mucins [14]. Fascinatingly, they lose this property when transplanted [15].

Studies comparing the oral mucosa to the foetal wound healing environment have found similarities in the ECM with the mucosa revealing increased fibronectin, its splice variant ED-A and chondroitin sulphate as compared to the skin. Also, elastin levels were found to be higher in dermal cells, with those exhibiting greater differentiation property as opposed to the greater proliferative property demonstrated by the mucosa [16].

4. Difference between traditional and scarless wound healing

While not conclusive, studies have indicated that the mechanism of wound healing itself is different in the case of traditional wound healing and the scarless wound healing of early foetal cells (Figure 2). One of the primary differences being the niche and its composition itself. For instance, it has been observed that the early gestational cells modify their environmental, embryonic niche to promote regeneration during wound healing as a cell-intrinsic property [17]. However, late gestational cells are restricted to repetitive healing, in the sense that they are capable of modulating fibroblasts to increase proliferation and migration.

Figure 2.

Differences between scarless wound healing and traditional wound healing- representative image indicating the factors that influence traditional and scarless wound healing.

This is further confirmed by proteome analysis comparisons of foetal and adult fibroblasts which have revealed completely different patterns of protein expression such as the types of collagen that are more prevalent in scarless wound healing and wound healing with scar formation [11].

These findings were further supported by Siebert and his team [18], who had performed several histological and biochemical analyses on the healing of foetal wounds to determine how it differs from adult wound healing to lead to a scarless wound closure, and observed that the collagen (collagen type-I) that is identical to the one found in adult wound healing sites was minimal while it was abundant in a different type of collagen (collagen type-III). Additionally, the foetal wound matrix was also rich in hyaluronic acid that had earlier been associated with decreased scar-formation during post-natal wound closure [18]. This in turn, led to the development of a theory pertaining to a hyaluronic acid/collagen/protein complex with a highly efficient matrix reorganisation potential leading wound healing in the early foetal stage debunking the theory of ‘true regeneration’ wherein a completely new individual is formed from a small tissue/cell.

While the quantities of collagen deposited in foetal and in adult wounds are known to be different; it has been theorised that the collagen present in foetal wounds is more for ‘structural’ purposes than in the form of ‘scar tissue’ [18]. This is further supported by the deposition of glycosaminoglycans at the wound site which promotes the migration towards, differentiation and maturation of mesenchymal stem cells (MSCs) [18, 19].

Additionally, inflammation, that is the bedrock of adult wound healing, is absent in foetal wound healing. Thus, while epithelialisation occurs in foetal wound healing, the accompanying angiogenesis that is prevalent in adult injury repairing mechanisms is absent and, as already observed by Siebert and his team, has minimal, highly organised, deposition of (the same type of) collagen and is instead dominated by the presence of hyaluronic acid [18, 20, 21, 22, 23].

It has been speculated that altering the levels of growth factors and their inhibitors might aide in replicating the scarless wound healing mechanisms in the adult system. To that end, Bone morphogenetic protein-2, hypoxia-inducible factor 1α (HIF-1α), decorin (a TGF-β modulator), α- and β-fibroblast growth factors, IL-6, and IL-8; as well as Tenascin, which is a large, extracellular matrix glycoprotein synthesised by fibroblasts during embryogenesis are currently being explored by various teams. It has been theorised that Tenascin, which is present in early foetal wounds might be the factor responsible for initiating the rapid cell-migration and re-epithelialisation that is characteristic of foetal wound healing [24, 25].

5. Factors involved in wound healing

Physiological responses as well as cellular functions influence wound healing, and interruptions at any stage enhance the chances of scar development.

5.1 Local factors

5.1.1 Ischemia

Inadequate supply of blood to any part of the body is termed as ischemia, and since wounds require multiple elements such as energy (in the form of adenosine triphosphate or ATP), glucose and oxygen; that are all borne by the blood to the site of the wound, ischemia affects wound healing dramatically. The rate of wound healing is significantly lower in cases of Hypoxia, which in turn may be triggered by ischemia [26].

Hypoxia leads to vasodilation and stimulates fibrin deposition which increases pro-inflammatory activity, capillary leak and neovascularisation. Pro-inflammatory property is stimulated by Tumour Necrosis Factor (TNF)-α, leading to apoptosis and affecting the collagen organisation of the injured tissue [26, 27].

Similarly, fibroblasts exposed to long periods of hypoxia may not involve in extra cellular matrix, thereby delaying wound healing/closure [27].

5.1.2 Oedema (edema)

Swelling that arises due to excess fluid trapped in the body’s tissues is medically termed as an Oedema (/edema). This is most commonly associated with ischemia and severely delay wound healing as inflammatory response is delayed due to raised tissue pressure. This in turn compromises cellular function and lead to severe hypoxia or even cell death, thereby leading to necrosis and impairing wound healing [26].

5.1.3 Foreign bodies

Clinically, any object found in the wound, apart from its natural tissues is demarcated as a foreign body, the presence of which severely inhibits wound closure. This is primarily because foreign bodies prevent wound contraction and epithelialisation, thereby leading to the development of necrotic tissues. Unfortunately, necrotic tissues further prevent wound healing to the extent that all the necrotic tissue has to be removed before wound healing can commence [28].

‘Foreign bodies’, which may also indicate non-viable tissue; further complicate matters by serving as an asylum to bacteria and other pathogens.

5.1.4 Infection

Presence of bacteria or other pathogens at the site of the wound is termed as ‘wound infection’, and characterised by the one or more of the four cardinal signs of inflammation:

1. Rubor—the infected region might appear red,

2. Calor—the affected region might be warmer than the surrounding tissues,

3. Dolor—there might be increased pain and,

4. Tumour—or swelling.

A fascinating study by Robson and his team in 1997 [29] exhibited that bacterial counts exceeding 10⁵ organisms per gram of tissue prevent wound closure to the extent that skin-graft replacement or even primary sutures failed to heal the wound. Similarly, presence of beta-haemolytic streptococcus will also inhibit wound healing. This is because endotoxins present in bacteria stimulate phagocytosis and the release of the enzyme collagenase that contributes to collagen degradation and destruction of the priory normal tissue that surrounds the site of injury [29, 30].

5.2 Systemic factors

Certain factors such as obesity, cardiovascular and respiratory disease etc. might affect an individuals’ wound healing capacity and pre-dispose them to wound healing dysfunction [31, 32]. While wound healing is slower with age, it has been observed that those with co-morbidities also exhibit delayed healing which is indistinguishable from the delay caused due to the effects of age alone. This was confirmed with observations of improved healing ability in elderly female patients when they were prescribed/given topical oestrogen supplements [33, 34].

Although it is a prevalent belief that senior patients heal at a slower rate than their younger counterparts, it must also be acknowledged that older patients are more prone to co-morbidities which may interfere with wound healing. Fascinatingly, while longitudinal studies in animal models [35] support this theory, studies in humans have been inconclusive in the sense that dermal collagen deposition is equivalent in patients undergoing skin-grafting irrespective of age, however their re-epithelialisation rates are reduced [36]. While further research is needed to understand the consequences of age in wound repair, it has been speculated that it may be attributed to reduced availability of growth factors.

Some of the known chronic conditions that interfere with wound healing are listed below:

5.2.1 Diabetes mellitus

Increased serum glucose and hyperglycaemia related deleterious impact has been evidenced in cellular and molecular pathophysiology. For instance, sorbitol, a toxic by-product of glucose metabolism has been found to accumulate in tissues in case of hyperglycaemia and thereby involved in the renal, ocular as well as vascular complications commonly associated with diabetes. Additionally, collagen; which plays a vital role in wound healing mechanisms is more prevalent in its glycosylated form that is comparatively more resistant to enzymatic degradation and less soluble than normal protein product. These defects in collagen maturation led to decreased granulations and reduced collagen in granulation tissue, thereby hampering wound healing [37, 38, 39].

5.2.2 Hypothyroidism

Experimental studies have indicated that hypothyroidism leads to decreased collagen production and decreases tensile wound strength. These issues are further complicated by the co-morbidities that are often caused/present in hypothyroidism patients [40, 41].

5.3 Other factors influencing wound healing

5.3.1 Tissue perfusion

Tissue perfusion, which may be local due to external compression, or even systemic, when arising from alterations in circulatory volumes may lead to tissue hypoxia; which, as already noted earlier; interferes with wound healing [28].

5.3.2 Hypothermia

Hypothermia leads to peripheral vasoconstriction which in turn has an impact on cutaneous perfusion, thereby affecting wound healing [42, 43].

5.3.3 Debilitating pain

The pain stimuli may lead to the diffuse of an adrenergic discharge, which in turn is responsible for cutaneous vasoconstriction. Consequently, it has often been observed that proper pain control leads to improved wound healing [44, 45].

5.3.4 Major trauma

Severe trauma often results in hypo-volemic shock as a consequence of compromised cardiac functionality, thereby enhancing circulating cytokines as well as inflammatory mediators such as TNF- α and leading to anomalies in wound closure such as abnormalities in clotting [46, 47]. Thus, it is essential to maintain normo-volemic conditions as well as body temperature for proper wound management [31, 32, 46, 47, 48].

5.3.5 Septicaemia

Excessive secretion of pro-inflammatory mediators due to endotoxins is a critical part of systemic inflammation that leads to sepsis or septicaemia. Systemic inflammation has a hypo-inflammatory state characterised by monocyte deactivation and immunosuppression that compromises leukocytic activity and has inhibitory effect on wound healing.

Septicaemia also causes plasma and endothelial-related inflammatory modifications that impact coagulation and has been implicated in micro circulatory thrombosis that affects tissue perfusion and may even lead to fatalities due to associated multiple-organ failure [49, 50].

5.3.6 Nutrition

Wound healing is nutrition intensive as there is significant protein loss to maintain normoglycaemia, thus patients’ bodies consume body stores of fats and protein during this process and it is essential to maintain proper nutrition to ensure proper wound remediation. While glucose is the main fuel for wound healing, protein malnutrition, especially deficiencies of specific amino acids such as arginine and methionine have been associated with compromised wound repair due to prolonged inflammation stage, disruption of matrix deposition, cellular proliferation, and angiogenesis. Decreased rates of collagen deposition during dermal wound healing have also been associated with malnutrition [51, 52, 53].

5.3.7 Smoking

The detrimental impact of smoking on wound healing has been elegantly demonstrated nearly four decades ago by Mosely and Finseth in 1977 and re-assessed by Goldminz and Bennet in 1991 [54, 55].

The impact of smoking is multi-factorial: Nicotine has vasoconstriction properties that additionally decrease the proliferation of erythrocytes, macrophages, and fibroblasts - different cells that play indispensable role in wound-closure. Additionally, carbon monoxide severely limits the oxygen-carrying capacity of haemoglobin (thereby inducing tissue hypoxia) while simultaneously increasing platelet aggression and viscosity of blood and interfering with collagen deposition and prostacyclin formation [56].

5.3.8 Corticosteroids

Anti-inflammatory steroids are known to inhibit cell development. Additionally, they are known to decrease inflammatory response. Macrophage response to chemotactic signals as well as phagocytosis by polymorphonuclear neutrophils and macrophages is negatively affected by the stabilising impact of steroids on lysosomes. Epithelialisation, which is a critical step in wound healing is inhibited by glucocorticoids such as hydrocorticosterone [57].

Things are further complicated by the low-secretory state induced by corticosteroids due to their effect on the endoplasmic reticulum. Thus, risk of wound dehiscence is consequently enhanced due to the reduced deposition of collagen [58].

5.4 Genetic syndromes associated with abnormal wound healing

5.4.1 Cutis laxa

Defective elastin fibres as evidenced in cutis laxa- an acquired or congenital disorder, impacts wound healing. Characterised by skin that is loose (lax), wrinkled, sagging, and lacking elasticity (inelastic). The inelastic skin returns to place abnormally slowly when stretched. The skin around the face, arms and legs etc. are the predominantly affected parts and give patients a prematurely-aged appearance [59].

5.4.2 Ehlers-Danlos Syndrome

Characterised by deficit in collagen metabolism, it is a group of connective tissue abnormalities that lead to defects in the inherent strength, elasticity, integrity, and healing properties of the tissues [60, 61]. Even mild injury in Ehlers-Danlos patients may lead to severe bruising and the development of wide, ‘open’ wounds due to the delayed healing [27, 60, 61].

5.4.3 Osteogenesis imperfecta

An inheritable disorder of connective tissue, clinical features of this condition include bone fragility, neonatal dwarfism, deformities of the long bones, scoliosis, ligamentous laxity, blue sclerae, defective dentinogenesis, and deafness. While there are four major forms, mutations in the genes that encode type I collagen is a common trait that result in the formation of wide scars [27, 62].

6. Adjuncts to wound healing

6.1 Bioengineered skin

One of the best examples of Bioengineered skin is the Apligraf: a bi-layered bioengineered skin substitute composed of ‘bovine type I collagen matrix populated with human male neonatal fibroblasts and an epidermal sheet derived from male neonatal epidermal keratinocytes’, that has been approved by the US Food and Drug Administration (FDA) for cases where standard wound care has failed to treat ulcers. The mechanism that initiates wound healing by the use of these bioengineered products is still being researched but it has been revealed that these cells grow and proliferate, and produce growth factors, collagens, and extracellular matrix proteins, all of which are known to positively stimulate re-epithelialisation, formation of granulation tissue, angiogenesis, and neutrophil and monocyte chemotaxis. Thus, these have the potential to provide a potent cellular remedy and adaptable response in acute and chronic wounds [63].

6.2 Electrostimulation

While the first exploration of electrical current in skin was described as early as 1860 by DuBois-Reymond; the fact that wounds treated with electrostimulation had a positive potential compared with the surrounding skin was only confirmed in 1945 [64]. It is believed that electro-stimulations aides in wound healing by accelerate the wound-healing process by tissue increasing the migration of vital cells to the site of injury i.e., neutrophils, macrophages [64, 65, 66] and fibroblasts [67, 68, 69] by imitating the natural electrical current that occurs in skin when it is injured and thereby accelerates the healing process [70, 71, 72].

6.3 Hydrotherapy

One of the oldest adjunct treatment modalities that is still practiced today is the Whirlpool therapy that accelerates wound healing by wound debridation, warming the wounded tissue, and providing buoyancy and gentle limb resistance for physical therapy [73]. However, despite their prevalence, Whirlpool treatments have been subject to disapprobation in recent years because of the enhanced risk of nosocomial contamination and transmission of virulent infections associated with them [74, 75, 76, 77, 78]. These days, modified forms of the whirlpool therapy, such as the pulsed lavage and the VersaJet are more popular as they provide the benefits of hydrotherapy without the associated collateral trauma of traditional methods [78, 79].

6.4 Hyperbaric oxygen

Although hyperbaric oxygen treatment for narcotising soft-tissue infections has not always been successful, the treatment has been found to be beneficial for a variety of conditions including amputations, [80] osteoradionecrosis, [81, 82] surgical flaps, and skin grafts [81, 83, 84]. However, studies have failed to statistically corroborate significant outcomes regarding differences with respect to mortality rates and length of hospitalisation [82, 85, 86, 87, 88]. Benefits are suspected to primarily originate from the benefits of the increased nitric oxide levels that are developed by increasing oxygen pressure as nitric oxide is known to be vital for wound-healing [88].

Fascinatingly, in a study using an ischemic rabbit ear model, hyperbaric oxygen therapy was used in combination with PDGF or TGF-β1 and was found to have a synergistic effect that completely reversed the healing issues caused by ischemia [89].

7. Scars

A scar refers to a growth of tissue marking the spot where skin has healed after an injury. And while scars are used for body modification and ‘body art’ in some cultures, most take it to be a sign of someone surviving a catastrophic event or a debilitating disease [2, 3]. They are predominantly of two types:

1. Keloid scar: these are scars that have overgrown their boundaries with large collagen bundles in their midst and being limited in macrophage content but abundant in eosinophils, mast cells, plasma cells and lymphocytes [90, 91, 92].

2. Hypertrophic scar: Hypertrophic scars do not have collagen bundles, but nodules of α-smooth muscle actin–staining myofibroblasts that contain cells and collagen [93].

7.1 Treatment

While multiple treatments are promoted/suggested to minimise the development of scars, none have been proven to be completely effective. Keloid scars especially are difficult to treat because of their high recurrence rate.

Common scar-treatment modalities include:

1. Excision: While one of the more popular treatments, excision alone (especially in the case of keloid scars) has been proven to have a high recurrence rate ranging from 45 to 93 percent.

2. However, when coupled with other treatment practices, excision has been known to lower recurrence risk [90, 91, 92].

3. Laser excision: Lasers cause a range of specific thermal tissue reactions in a dry and bloodless environment and was initially utilised in the hopes of reducing scar formation, however they are not frequently used in the present date owing to their high recurrence levels [94, 95].

4. Radiation Therapy: Radiations have been used in the eradication of this benign lesion since the 1960s; however current concerns regarding the safety of patients - due to increased risks of developing skin cancer; have limited the perpetuity of this therapeutic modality [94].

5. Steroids: While steroids themselves are known to interfere with wound healing, they are used in initial treatment of scars as they suppress the inflammatory stage [95, 96].

6. Cryo-surgery: Traditionally, cryotherapy have been used for managing hypertrophic scars and keloids, with pre-treatment and post-treatment histological analyses indicating significant improvement in scar organisation after needle cryosurgery [93].

7. Interferon: IFN-α-2b normalises the collagen and glycosaminoglycan of the keloid, thereby interfering with the fibroblasts collagen synthesis [97]. Complications with IFN-α-2b include flu-like symptoms, headache, fever, and myalgias.

However, till date; prevention is the best keloid therapy.

8. Problems with current strategies to reduce scarring

Current strategies for non-surgical therapy for the treatment of keloids and hypertrophic scars include topical therapy and intra-lesional injections of corticosteroids. While literature has reported a success rate ranging between 50 to 100 percent; these methods have also been associated with hypopigmentation, dermal atrophy, telangiectasia, widening of the scar, and delayed wound healing [98].

8.1 Prospects being explored to promote scarless-wound healing

8.1.1 Dermal stem cells

While advances in genetic lineage-tracing technologies, cellular assays, and imaging techniques have revealed important stem and progenitor cell reservoirs in the inter- follicular epidermis, the eccrine sweat glands, and the hair follicle; given excisional wounds develop into areas lacking sweat and sebaceous glands as well as hair follicles, further understanding of stem and progenitor cells would aide in achieving the ultimate goal of scarless wound healing following injury [99].

8.1.2 Interfollicular epidermis

During wound healing of the interfollicular epidermis, a population of ‘slow-cycling cells’ is suspected to demonstrate autocrine regulation [100, 101] until mobilised by a wound healing signal. The cell division frequency of these slow-cycling cells increases following a wound, which provides excess daughter cells that help in repairing the damage. Further, injured epithelium has been known to demonstrate behavioural plasticity with progenitors capable of reverting to multipotent states and multipotent cells differentiating to fill unipotent roles, [102, 103]. Exploring these avenues may eventually allow us to discover the hitherto undiscovered processes that would allow regenerative wound healing with negligible scarring.

8.1.3 Sweat glands

Loss of sweat glands in burn patients remains an unsolved problem. However, recent studies of these epidermal appendages have identified gland-specific progenitors, [104] and indicated the role of these cells in development, homeostasis, and wound repair [104, 105, 106, 107].

8.1.4 Hair follicles

Mammalian hair follicles are known to house numerous progenitor cell populations that might play indispensable role in the repair of injured dermal tissue and exploring them might give us ground breaking insight towards achieving scarless wound healing [106, 108, 109].

8.2 Future prospects for scarless wound healing

8.2.1 Gene targets

Genes involved in the scarring response that influence fibrosis by regulating collagen production and degradation have been identified. If they can be safely down-regulated by emerging medical techniques, they may provide the answer to the mechanisms involved in the minimisation of scars [110].

8.2.2 Dermal substitutes

Wound coverage as well as a matrix to encourage engraftment and proliferation of endogenous cells and the function of transplanted cells is provided by dermal substitutes. These are typically acellular in nature, but biocompatible and morphologically similar to natural tissue structure with mechanical properties similar to host dermal tissue [111].

8.2.3 Mechanical offloading

Another alternative to limiting fibrosis in cutaneous injuries is mechanical offloading since mechanical tension plays a significant role in the development of fibrosis, activating numerous mechano-responsive signalling pathways such as the focal adhesion kinase (FAK) [112, 113].

8.3 Cellular targets for scarless wound healing

Identification of populations of cells contributing to scar formation allows us to explore options that reduce these specific cell populations during wound healing, thereby minimising scarification.

For instance, Dulauroy and team (2012) successfully distinguished a pro- inflammatory subset of perivascular cells that are activated upon acute injury in muscle and dermis by transient expression of a disintegrin and metalloprotease (ADAM12) [114]. Knocking down ADAM12 expression or ablating these cells was shown to decrease fibrosis and resulting scar formation.

This has been concordant with the findings of Rinkevich et al. [115] who found that fibroblasts originating from En1-lineages were the main culprits in the cutaneous scarring. These cells majorly contribute to scar formation in connective tissue.

Selective abrogation of the En1-fibroblast lineage with diprotin A by accompanying CD26 (also known as dipeptidyl peptidase-4, DPP4) surface marker has also been found to reduce cutaneous scarring without compromising the integrity of the healed tissue [115].

The ability of DPP4 inhibitors to curb the fibrogenic phenotypes of keloid-derived fibroblasts and normal fibroblasts have been further verified through various in vivo experiments. Observed decrease in collagen production and TGF-β1 expression have also been found to be enabled by underlying mechanisms involving the pro-fibrotic pp38 and pERK1/2 pathways [116].

Further, Myofibroblasts have been identified as the key players in the standard wound healing response, as they contribute to wound contraction and ECM production [117], making them ideal targets for reducing scar formation [118]. However, during the maturation phase of normal wound healing, the majority of these cells undergo apoptosis [119]. Thus, reversal of the myofibroblast phenotype might also help in decreasing this cell population [120, 121].

Unfortunately, like fibroblasts, myofibroblasts (or myofibroblast-like cells) also form a functionally heterogeneous population with potential precursors including fibroblasts, mesenchymal stem cells (MSCs), smooth muscle cells, endothelial cells, and fibrocytes [122]. Although it is still being determined whether fibroblasts and adipocytes share a common progenitor, Schmidt and Horsley [123] have demonstrated that dermal adipocytes are essential for fibroblast recruitment during wound healing mechanisms. Experimental interventions with direct and indirect targeting at both populations have been demonstrated to be responsible for scar formation.

Interestingly, Desai and his team [120] have found indications that the myofibroblast differentiation process is not terminal with basic fibroblast growth factor (bFGF) functioning as a phenotypic reversing agent as it led to diminishing expression of α-Smooth Muscle Actin (SMA), collagen I, and fibronectin, and a loss of focal adhesions and stress fibres being inversely co-related with tenascin-C and vimentin upregulation, in agreement with a more fibroblast-like phenotype [120]. These findings are in tune Rinella et al. [124] work that indicate that extracorporeal shockwaves (ESW), which cause myofibroblast precursors to differentiate into more fibroblast-like cells with lower contractility and higher migration potential, simultaneously reducing α-SMA and type I collagen expression may play a significant role in scar reduction.

8.4 Stem cells

Stem cells are known to modulate the wound environment and improve healing by reducing inflammation [125, 126, 127]. A recent study by Li et al. [128] has demonstrated the potential benefits of conditioned media from umbilical cord (UC)-MSC cultures wherein, dermal fibroblasts under the paracrine influence exhibit characteristics similar to those of foetal fibroblasts: low myofibroblast forming capacity, decreased TGF-β1/TGF-β3 ratio, as well as increased expression of enzymes (matrix metalloproteinases or MMPs) involved in ECM remodelling [128]. Other in vitro studies have indicated that human amniotic-fluid-derived MSC-conditioned media also has the potential to inhibit the pro-fibrotic actions of TGF-β1 and even reverse the myofibroblast phenotype to a fibroblast-like state. Conditioned media from ASCs produced similar results, but to a lesser extent [129]. However, this enhanced healing mechanism may not always translate to reduced scar-formation as these experiments do not have similar results when performed in vivo [130, 131]. Additionally, ethical, legal as well as practical barriers associated with stem-cell- based therapies might restrict their use in the exploration of scarless wound healing [132].

8.5 Wnt and regeneration

Various signalling pathways, such as the canonical Wnt/β-catenin have been implicated in the expression of foetal mouse keratinocytes and fibroblasts at embryonic day (E)16 and E18 time points, straddling the transition from scarless to scar-forming repair [117, 133]. While Wnt signalling is a key component of embryological development, it is also involved in various wound healing mechanisms with the canonical Wnt pathway being the most relevant [134]. In this pathway, the Wnt-ligand binding at the cell surface leads to cytoplasmic β-catenin accumulation, which subsequently translocates to the nucleus to exert its effects as a transcriptional co-activator [135].

Additionally, a link between TGF-β and the canonical Wnt/β-catenin pathway has been observed in the case of fibrosis with analysis of pathological scars (hypertrophic scars and keloids) revealing upregulated Wnt signalling secondary to TGF-β [136]. β-catenin levels have been shown to double during the proliferative phase in normal wound healing (with scarring) as well [137]. Keloids in humans also display Wnt-3a over-expression by inducing fibroblasts of endothelial origin to transition to mesenchymal cells that leads to collagen accumulation [138].

8.6 MicroRNA

MicroRNA (miRNA) gene therapies are a more recent avenue for potential therapeutic interventions as these molecules exert an inhibitory role on mRNA transcription within eukaryotic cells, effectively silencing genes at a post- transcriptional level [139]. Comparison of genome-wide miRNA expression between mid-gestational (E16) and late-gestational (E19) mouse skin discovered global repression of these molecules at the earlier time point where scarless healing is the norm [140]. miR-34 family have been established as potential candidates for scarless wound healing in human foetal keratinocytes, however; expression of these miRNAs was found to be significantly lower as gestation progressed [103].

Regenerative wound healing using miRNAs has been explored to reduce scarification [141, 142, 143]. For instance, miR-145 has been found at three times its normal levels in hypertrophic scars and pro-fibrotic TGF-β1-induced myofibroblasts. However, with the help of a commercial inhibitor of miR-145, Gras et al. (2015) were able to significantly decrease type I collagen expression, TGF-β1 secretion, and contractility in skin myofibroblasts [144].

Similarly, miRNAs have been combined with biomimetic scaffolds to enhance wound healing, and furthering their clinical potential [145]. Future in vivo studies will hopefully enumerate the clinical potential for other miRNA-based therapies.

9. Conclusions

Our current understanding of wound healing and cell subpopulations within the skin has allowed us to develop scar-reducing therapies, however, they are not as effective as needed to make them the mainstay of available wound healing modalities. While research regarding this elusive therapeutic modality is underway, successful scarless wound healing would require not only an understanding of signalling molecules and growth factors but also a thorough understanding of lineage-specific cellular origin and function during both foetal and adult stages.

Acknowledgments

I would sincerely like to thank Priya Ashrit and Dr. Shweta Sharma for their assistance and encouragement during the preparation of this chapter. I also acknowledge the kind aid of Mr. Subhasis Bhattacharjee in the design of the images that have been used in this chapter.

Conflict of interest

The author declares that there is no conflict of interest.

List of abbreviations/acronyms

ADAM

a disintegrin and metalloprotease

ASC

acellular stem cell (bioengineered cells)

ATP

adenosine triphosphate

ECM

extra-cellular matrix

ERK

extra-cellular related kinase

ESW

extracorporeal shockwaves

FAK

focal adhesion kinase

FDA

Food and Drug Administration (of the USA)

HIF

hypoxia inducible factor

IFN

interferon

IL

interleukin

KGF

keratinocyte growth factor

MCP

monocyte chemo-attractant protein

MMPs

matrix metalloproteinases

MSCs

mesenchymal stem cells

PDGF

platelet derived growth factor

PF

platelet factor

SMA

smooth muscle actin

TGF

transforming growth factor

TNF

tumour necrosis factor

bFGF

basic fibroblast growth factor