Innovative approaches in wound healing: trajectory and advances

Abstract

Wound is one of the oldest suffering associated with the mankind and its history is as old as humanity. Advances in the field of medical sciences created a pile of knowledge and paved the path for the development of a separate branch specifically devoted for wound healing. The understanding and treatment strategies for wound healing have gone through a great revolution. This article reviews all the aspects of wound healing including the pathway, types and recent advances made in the wound care management in particular moist wound dressings using natural polymers, skin grafts, debridement, growth factor and drug delivery.

Keywords::

• acute
• wounds
• chronic wounds
• dressings
• skin grafts
• wound
• wound healing

Introduction

Wound healing is a complex and dynamic cascade of events initiated by injury (Masre et al. 2012). This response to injury is a phylogenetically primitive, yet essential, innate host immune response for the restoration of tissue integrity (Martin 1997, Singer and Clark 1999). The process of wound healing is divided into sequential phases (Lawrence 1998, Phillips 2000, Orgill and Demling 1988). These phases overlap in terms of time, physiology, and cell type, with each phase not entirely completed before the next begins. Wounds have historically been treated by bizarre methods. Lizard’s dung, pigeon’s blood, cobwebs, boiling oil and hot irons are few of the odd treatments (Orgill and Demling 1988). The recent developments and discoveries made in the last decade expanded the knowledge base and technologies related wound sciences. Today, there is an increase in the scientific approach to healing as ‘old’ treatments like larvae therapy, sugar, leeches, tea tree oil, etc. are now rationalized and shown to be effective. The paradigm shift from dry dressings based on woven fabrics towards a moist environment revolutionized developments in this field. Nevertheless, the pathological cases of chronic wounds are still difficult to handle (Reiss et al. 2009). Even with the moist concept they require patient and persistent treatment. Continuous advances made in the study of the wound microenvironment, pathophysiology of wounds, and improved techniques in monitoring the response of healing and treatment of chronic wounds offered new hopes for patients suffering from acute and chronic wounds. This article reviews recent advances made in wound care management including stem cell technique, growth factor delivery and modern dressings.

Historical developments

Hippocrates is among pioneers credited with one of the earliest mentions of chronic wounds as he apparently recognized some relationship between leg ulceration and venous disorders (Hippocrates 1849). As early as the tenth century, there was widespread belief that curing an ulcer prevented the efflux of dangerous humors which was widely upheld until the eighteenth century (Avicenna et al. 1783). In a recently discovered early fifteenth- century manuscript, chronic wounds, regardless of cause, were divided into seven classes of ulceration based on a Latin nomenclature. The classes include corrosium—a wound that slowly progresses to form a shallow cavity prior to healing followed by virulentum—an old wound in which exudate is liquid and plentiful and difficillis consolidationis—an old wound that is difficult to heal, but not a cancer or an inflamed sore.

In 1775, the physician John Hunter reported that the sores of poor people can be healed in a speedy manner by rest in a horizontal position, fresh provisions and warmth in hospitals (Hunter 1837). Another significant contribution was made by John Gay in 1867, when he not only described clot formation and postthrombotic recanalization, but also recorded that ulceration could occur in the absence of varicose veins and introduced the term “venous ulcer”(Gay 1868). Perhaps the most significant advances in both the understanding of chronic wound pathophysiology and the introduction of modern wound dressings have taken place within the last 40–50 years. Table I highlights the historical aspects of developments made in wound treatments.

Table I. Historical development in wound treatments.

S.No.	Year	Treatment	References
	1600 B.C.	Grease, honey, lint, castor oil—Egyptians—Smith papyrus	(Reed and Clark 1985)
1	A.D. 1–200	Plaster, linen bandages—Celus/Galen–initial concept of “laudable pus”	(Galen 1976)
2	1000–1700	General belief maintained throughout this period: “to cure an ulcer was to prevent the efflux of dangerous humors	(Avicenna 1783)
3	1000–1100	Promotion of per secundum intentionem by promoting the production of pus	(Scholz 1993)
4	1200–1300	Bandages soaked in wine—per priman intentionem, with out infection	(Scholz 1993)
5	1400–1500	Practice of wound cautery, i.e., burning oil/hot irons-Paracelcus	(Scholz 1993)
6	1500	Ligation introduced by Parc; general use of ointments, bandages, and cotton lints	(Scholz 1993)
7	1600	Blood cupping = release of blood through the skin—Schropfkopf + cutting glass	(Scholz 1993)
8	1676	First description of a laced stocking	(Wiseman 1676)
9	1700	Introduction of compression bandages for old sores and edema; bandages were usually made out of linen, wool, silk or parchment	(Theden 1782)
10	1870s	Development of rubber bandages—Martin	(Scholz 1993)
11	1920s	Elastoplast released into the market	(Scholz 1993)
12	1960s	Concept of moist wound healing developed—Winter studies → UK registration of Opsite first occulsive dressing	(Winter 1962) (Seymour et al. 1972)
13	1980s	Development of Hydrocolloids (e.g., Granuflex), Foams (e.g., Alleyn), and Hydrogels (e.g., Intrasite)	(Friess 1988)
14	1990s	Development of Alginates (e.g., Sorbsan, Hydropolymers (e.g., Tielle), and Hydrofibers (e.g., Aquacel)	(Friess 1988)
15	2000+	Development of biopolymer materials, e.g., Hyalofil, an esterified hyaluronic, acid biopolymer, and therapeutic agents, e.g., drugs—Regranex	(Eaglstein 2001)
16	More recent	Possible development of biomaterials and use of tissue engineering	(Ruszczak and Friess 2003)

Types of wounds

Wounds can be classified as acute or chronic (Table II). Acute wounds can be defined as any interruption in the continuity of any tissue of the body (Wysocki 1999). This is in contradistinction to a chronic wound, which can be defined as an intervention in the continuity of any tissue that requires a prolonged time to heal, does not heal, or reoccurs (Wysocki 1999). Acute wounds heal as anticipated and proceed through a normal, orderly, and timely reparative process that results in sustained restoration of anatomic and functional integrity. Chronic wounds do not heal as anticipated and fail to proceed through this process or proceed through the process but fail to establish a sustained anatomic and functional result (Lazarus et al. 1994). Chronic wounds may get stuck in any of the phases of wound healing for greater than 6 week (Collier 2006).

Table II. Types of wounds.

S.No.	Type of Wounds	Important Features	References
1.	Acute wounds	Simple dividing of tissue caused by a simple shearing force (e.g., from a knife or glass)	(Provencher et al. 2006)
a)	Puncture wounds	•Includes 3% to 5% of all traumatic injuries• Caused by nails but can also occur via envenomation, human and animal bites, iatrogenic causes, and foreign bodies such as wood, metal, glass, and plastic.	(Baldwin and Colbourne 1999)
b)	Bite wounds	• Caused by dogs, cats, and humans and often young children• Cause crushing, tearing, or avulsion of tissue with significant bacterial contamination.	(Stefanopoulos and Tarantzopoulou 2005)
c)	Abrasions	• Caused by a tangential force to the epidermis and dermis• Varying thicknesses of epidermis and or dermis can be lost, and deeper abrasions can occur	(Trott 1997) (McCarthy 2006)
d)	Burns	•Damage to the skin and underlying structures caused by excessive heat or caustic chemicals	(De Santi 2005)
2.	Chronic wounds	Arise from acute wounding due to inappropriate treatment and predominance of factors known to delay healing	(Wysocki 1999)
a)	Pressure ulcers	• Disruption of the normal anatomic structure and function of skin that results from an external force associated with a bony prominence• Does not heal in an orderly and timely fashion	(Margolis 1995)
b)	Diabetic foot ulcers	• Occur on the planar area of the foot prone to excessive pressure•Present as symmetric, round, puncture-appearing wound cavities with a clean wound bed and callous around the wound• Diabetic patients typically manifest lesions in the distal popliteal and tibioperoneal arteries, with usual sparing of the dorsal foot	(Margolis 1995)
c)	Venous insufficiency ulcer	•Partial-thickness wounds resulting from chronic venous insufficiency	(Bruncardi 2005)
d)	Ischemic wounds	• Generally atrophic•Wounds are shallow, painful, have a pale base•Caused by a decrement in blood supply to the affected body part•Lead to necrosis of the affected part	(Bruncardi 2005)

Trajectory wound healing

Stages in wound healing

Wound healing involves a highly orchestered sequence of events that is triggered by tissue injury and ends by either partial or complete regeneration or by repair. Healing process can be divided into the five overlapping stages of hemostasis, inflammation, proliferation, contraction, and remodeling involving various effector cells (Figure 1).

Figure 1. Overlapping stages of wound healing.

Hemostasis

The first step in wound healing involves vasoconstriction to decrease blood loss and stimulation of Hageman Factor (XII) to initiate the clotting cascade (Figure 2) (Monaco and Lawrence 2003, Ryan and Manjo 1987). Wound causes exposure of collagen which is the major protein present in all tissues of the body leading to stimulation of the alternate complement pathway as well as platelet adherence and degranulation (Monaco and Lawrence 2003, Wakefield et al. 2001). Degranulation causes release of numerous cytokines, such as PDGF (platelet derived growth factor) (Clark et al. 1982, Herndon et al. 1993). The hemostatic stage prepares for and influences the onset of the next stage of healing- inflammation.

Figure 2. Cascade of events following tissue injury.

Inflammation

After hemostasis, inflammation is initiated in a few hours after injury. Chemotactic signals attract neutrophils and monocytes to wound sites (Reibman et al. 1991). Neutrophils normally begin arriving at the wound site within minutes of injury to provide protection against contaminating bacteria and also activate local fibroblasts and keratinocytes (Hübner et al. 1996). The neutrophil infiltration ceases after a few days, and neutrophils are themselves phagocytosed by tissue macrophages. Macrophages continue to accumulate at the wound site by recruitment of blood-borne monocytes and are essential for effective wound healing (Martin 1997). Once activated, macrophages also release a series of growth factors and cytokines at the wound site, thus amplifying the earlier wound signals released by degranulating platelets and neutrophils (Martin 1997).

Migration and proliferation

The next phase is classified as migration and proliferation phase. This phase of wound healing is characterized mainly by anabolic reactions, i.e., angiogenesis, epithelization, and fibroplasia. The formation of blood vessels is known as angiogenesis, which starts with an endothelial cell bud formed by existing intact vessels (Fisher et al. 1995). Soon, endothelial cells migrate through the resulting gap in the direction of the wound following the oxygen gradient. They divide and form tubular structures that connect with other buds. As a result, during the maturation process a new basal membrane develops from the extracellular matrix components. The newly formed vascular loops then connect with intact vessels and differentiate accordingly into capillaries, arterioles, and venules (Martin 1997).

For epithelization, proceeding in parallel to angiogenesis, keratinocytes migrate across the wound and as a result reconstitute epidermal covering from the wound margin and hair follicle remnants (Rochat et al. 1994). Fibroblasts are the predominant cell type especially at wound edges to form the new loose extracellular matrix consisting of proteoglycans as well as the water-soluble collagen fibers essential for tissue stability (Figure 2) (Garlick and Taichman 1994). Collagen is a fibrous protein crucial to the process of wound healing and identified as the most abundant connective tissue protein.

In healthy tissue, the collagen fibers are aligned in basketweave patterns. This organized structure is not achieved in wound healing as the collagen fibers at the wound site fashion themselves in an alignment parallel to the stress lines of the wound. Type I and type III are the collagens most commonly found in healing wounds, although at least 19 different types of collagen have been identified and characterized (Friess 1988).

Contraction

In this step wound essentially shrinks by recruiting adjacent tissue and pulling it into the wound. This process is intimate with the phases of proliferation and remodeling, because the key effector cell is the fibroblast (Figure 2). More specifically, the cell involved is the myofibroblast as first described by Diegelmann and Evans (2004). While motor function is present in all fibroblasts as well as in other cells such as leukocytes, these cells are modified in a way that moves the edges of the wound towards the center, rather than just being motile and moving themselves. Collagen in the extracellular matrix aids in locking the cells in place, thus augmenting the contraction process (Gilbertson et al. 2001, Davie et al. 1975).

Matrix remodeling

Matrix remodeling, cell maturation, and cell apoptosis create the final phase of wound repair, which overlaps with tissue formation. Once the wound is filled with granulation tissue and covered with a neoepidermis, fibroblasts transform into myofibroblasts, which contract the wound, and epidermal cell differentiate to reestablish the permeability barriers. Endothelial cells appear to be the first cell type to undergo apoptosis, followed by the myofibroblasts, leading gradually to a rather acellular scar (Clark 1996). During the proliferation phase of wound healing (Figures 1 and 2), fibroblasts assume a myofibroblast phenotype characterized by large bundles of actin-containing microfilaments disposed along the cytoplasmic face of the plasma membrane of the cells and by cell-cell and cell-matrix linkages (Welch et al. 1990, Desmouli re and Gabbiani 1996). The contraction probably requires stimulation by TGF-β1 or TGF-β2 and PDGF, attachment of fibroblasts to the collagen matrix through integrin receptors, and cross-links between individual bundles of collagen (Schiro et al. 1991, Clark et al. 1989, Montesano and Orci 1988, Woodley et al. 1991). The overall collagen content of the wound diminishes, while tensile strength increases as a result of structural modification of the newly deposited collagen. The degradation of collagen in the wound is controlled by several proteolytic enzymes termed matrix metalloproteinases (Mignatti et al. 1996).

During the granulation tissue formation wounds gain only about 20% of their final strength (Viljanto 1964). During this time, fibrillar collagen accumulates relatively rapidly and remodeled by contraction of the wound (Bailey et al. 1975). The complete healing can be compared to a synchronized chronological task of various parts of machinery to produce the desired product (Table III).

Table III. Chronological events involved in wound healing.

Postinjury Time	Inflammatory stage	Debridement stage		Repair stage			Maturation stage
5–10 minutes	Vasoconstriction, coagulation and scab formation				Epithelialization starts immediately in a sutured wound	During the formation of granulation tissue and epithelialization, wound contraction occurrs at a rate of 0.6 to 0.7 mm/day by centripetal movement of whole-thickness skin surrounding the wound via the actions of myofibroblasts
3 hours	Lag phase of healing Entry of inflammatory mediators at the scab site and persist at the site of injury for the first 3–5 days		May start as early as 3 hours postinjury
6–12 hours		PMNs enter site at 6 hours, Monocytes by 12 hours.	Appearance of fibroblasts
1 day 2 days 3 days		Monocytes transform into macrophages in 24–48 hours
		Monocytes attract fibroblasts into the wound via migration along fibrin strands ◊ stimulate antiogenesis.	Most fibroblasts usually arrive in a wound on day 3 and can remain as long as 14–21 days. Fibroblasts secrete ground substance (water, ions, and polysaccharides) to set up for collagen synthesis ◊ secretion of collagen by fibroblasts. Fibroblasts that come into contact with new epithelium secrete a collagenase. The fibroblastic phase of healing lasts 2–4 weeks	Orientation of wound fibers is perpendicular to wound
6 days				Orientation of wound fibers becomes parallel to wound	Epithelialization starts on day 4–5 if granulation tissue form
2 + weeks							After a sufficient amount of collagen has been placed in the wound bed (17–20 days), remodelling and reorientation of collagen fibers takes place, increasing the strength of the wound.

Factors affecting wound healing

Skin is body’s first point of contact with the outside world. It provides protection for the body and serves many complex functions. These functions can be put at risk if the skin’s integrity is not maintained. The wound healing process is influenced by numerous factors which include intrinsic factors and extrinsic fectors.

Intrinsic factors

Drugs

Drugs such as inotropes, steroids, and nonsteroidal anti-inflammatory drugs can affect wound healing. Steroid therapy can produce paper-thin skin which can be easily damaged. Chemotherapy can lower immunological resistance leading to higher rates of infection and kill new cells forming within the body, thereby destroying new cells within the wound bed and delaying healing.

Diseases

Diabetes. Diabetes can delay healing as viscous blood, functional impairment of polymorphonuclear neutrophils, and low delivery of nutrients and oxygen to the wound increases the risk of clinical infection and slows the healing process. Hyperglycaemia impairs leucocyte chemotaxis. Pathological changes in diabetes and nonenzymatic glycation is the mechanism by which proteins, such as collagen, are subject to chronic attack from glucose (Majno and Joris 1996).

Rheumatoid arthritis. Rheumatoid arthritis delay healing by affecting the microcirculation within the tissues, lowering the supply of nutrients and oxygen to the wound bed.

Shock

In acute shock, particularly following hemorrhage, the blood supply becomes inadequate to the tissues and leucocytes become sticky and trapped. The number of leucocytes available is greatly reduced and, therefore, the inflammatory response is depressed. Further complications are vasodilation and lowered blood supply, and these factors open the channels for bacterial invasion and potential for septic shock (Majno and Joris 1996). Stress causes vasoconstriction and the lowered blood delivery can affect the required nutrient supply to the wound bed (Kiecolt-Glaser et al. 1995). Pain causes a stress reaction within the body with resultant vasoconstriction.

Age

The ageing process has a detrimental effect on the skin and immunological system and affects the healing process with age related differences in wound healing. Aging skin recognizes numerous structural changes which include loss of melanocytes and loss of natural protection against ultraviolet rays, decrease in vascularity, decreased innervation with lowered tissue responses, elastic fiber changes, decrease in number and function of sweat glands, sebaceous gland hyperplasia and drying of the tissues, thinning of the epidermis, reduction in production of mast cells, and reduction in fibroblast proliferation leading to a decrease in wound contraction and slower wound healing (Desia 1997).

Free radicals

Free radicals are molecules without the full complement of electrons making the molecule unstable and reactive. The instability can cause the molecule to react with other molecules in a chain reaction sometimes causing the radicals to attack DNA, which in turn can make the cell division unstable, with the potential for forming cancer. Free oxygen radicals have also been reported to cause chronic damage to newly formed tissue in chronic injuries (Niwa 1987).

Oedema

Human body has a fine fluid balance between potassium and sodium exchange. Serum albumin maintains a pressure within the venous system that balances the fluid between the extracellular and venous systems.

Several conditions produce oedema such as low serum albumin, venous obstruction and lymphatic obstruction (Majno and Joris 1996).

Oxygen

Oxygen is vital for the repair of the tissues and, therefore, wound healing is impaired if hypoxia persists (Stadelmann et al. 1998). Chronic nonhealing wounds are frequently hypoxic as a consequence of poor blood perfusion, and host and microbial cell metabolism contribute further to lowering the pO2 (Bowler et al. 2001). Wound hypoxia predisposes the wound to infection as neutrophils require large amounts of oxygen for phagocytosis and, without oxygen, bacteria could proliferate unchecked. Lack of oxygen reduces mitotic and leucocyte activity, thereby increasing the potential for clinical infection. This condition can be checked by oxygen saturation through administration of fluids to increase blood volume and decrease viscosity. There is also a potential for the use of hyperbaric oxygen although the research undertaken by Winter and Perins demonstrated faster wound epithelialization when patients were placed in a hyperbaric oxygen chamber (Albert 2008).

Extrinsic factors

Pressure

Unrelieved pressure should be avoided in pressure ulcers.

Temperature

Cold delays wound healing as any drop in temperature of more than two degrees delay mitotic activity for up to four hours. The reduction in heat also delays entry of macrophages and leucocytes to the wound bed by up to 12 hours. This increases the risk of clinical infection. Wound temperature of greater than 30°C reduces the tensile strength of wounds through decreased perfusion from vasoconstriction.

Application

Poorly applied dressings, too tight compression, no compression when required, dry dressings, dressings that adhere to the wound, and antiseptics all delay wound healing (Leaper 1987).

Exudates

Chronic wound exudate delays wound healing by inhibiting the growth of fibroblasts (Phillips et al. 1998). Furthermore the presence of microbial antigens perpetuates the inflammatory process leading to further tissue damage (Thompson 1998).

Approaches for wound healing

Many older but obsolete methods are still in use in clinical practice. As a first treatment the wound is debrided. After that, under a moist dressing depending on the wound type the healing process is allowed to proceed in moist environment. Infection control treatment is crucial. For further support of the healing process skin substitutes are available as well as vacuum treatment devices. There are numerous wound healing strategies employed (Figure 3, Table III).

Figure 3. Therapy and target for wound healing.

Debridement

Debridement refers to the removal of devitalized tissue. Accelerating this process makes healing more efficient as devitalized tissue in the wound bed supports bacterial growth and is a physical barrier to healing. Devitalized tissue may also cause excessive amounts of proteases to be released.

The methods of debridement in today’s clinical practice are surgical, enzymatic, autolytic, mechanical, and biologic.

a. Surgical—Sharp surgical debridement is a very fast and efficient way to remove necrotic tissue from the wound bed. It is performed where there is an extensive amount of necrotic tissue or if there is a widespread infection requiring infected material to be removed.

b. Enzymatic debridement means the use of manufactured proteolytic enzymes, i.e., collagenases, papain, and serratiopeptidase. These support naturally occurring enzymes to degrade necrotic tissue (Reiss et al 2009). Autolytic debridement is a process performed by phagocytic cells and proteolytic enzymes in the wound site liquefying and separating necrotic tissue from healthy tissue. Wound dressings, which maintain a moist wound bed, can provide an optimal environment for debridement, as they allow migration of the phagocytic cells. Our group has been working on debriding enzyme, particularly serratiopeptidase, which is highly acid labile. We have developed serratiopeptidase loaded lipospheres and PLGA microspheres for safe administration of enzyme to dissolve the necrotic tissue at wound site and to improve the co administration of antimicrobial agents (Singh et al. 2009, Singh and Singh 2011).

We have also developed multiphase hydrogel of gentamicin (GM) and serratiopeptidase (STP) using PVA-Gelatin for effective and complete wound healing (Singh and Singh 2012).

c. Mechanical debridement is a method that physically removes debris from the wound. Examples include conventional dressings causing mechanical separation of necrotic tissue from the wound bed once the dressing is removed and wound irrigation using a pressurized stream of water to remove necrotic tissue.

Biologic larval therapy is an alternative method using sterile maggots that break down, liquefy and remove dead tissue secreting powerful proteolytic enzymes followed by eating of the digested tissue (Saap and Falanga 2002).

Infection control in wounds

Bacterial bioburden can cause a delayed or impaired healing. Bacterial burden present in the wound is divided into several degrees depending on the extent of microbial infestation and necessary treatment.

a. Contamination is defined as the presence of nonreplicating bacteria. This is a normal condition in chronic wounds and does not contribute to impair healing.

b. Colonization is defined as the presence of replicating bacteria without a host reaction. Replicating bacteria colonize and contaminate all chronic wounds. Bacterial colonization does not contribute to impair healing.

c. Critical colonization is defined as the presence of replicating microorganisms, which are beginning to cause local tissue damage. There may be subtle local indications that a change in the equilibrium, or increasing bioburden, could be contributing to delay healing (Falanga et al. 1994).

d. Infection occurs when bacteria have invaded tissue leading to impaired healing where microbes are multiplying causing a host reaction.

Although bacteria are present in all chronic wounds, generally, only critical colonization and infection indicate an antimicrobial treatment. The most frequently used topical antimicrobials in modern wound care practice include octenidine, iodine, and silver containing products. Chlorhexidine, hydrogen peroxide, and honey as well are in discussion but seem to be used more rarely. In the past, acetic acid, sodium hypochlorite, potassium permanganate, and proflavine have been used. Iodine as element was used in treating wounds mainly in the nineteenth century. Due to its heavy adverse effects it is obsolete today. Therefore, the safer formulations povidone iodine and cadexomer iodine have been developed. Povidone iodine is a polyvinylpyrrolidoneiodine complex; cadexomer iodine is composed of beads of dextrin and epichlorhydrin that carry iodine (Zhou et al. 2002). Silver also has a long history as an antimicrobial agent, especially since the late nineteenth century (Klasen 2000). Metallic silver is not active, but in aqueous environments silver ions are released and antimicrobial activity depends on the intracellular accumulation of low concentrations of silver ions. These bind to negatively charged components in proteins and nucleic acids, thereby effecting structural changes in bacterial cell walls, membranes, and nucleic acids that affect viability (Lansdown and Silver 2002a). Skin discoloration and irritation associated with the use of silver nitrate is responsible for its limited use (Lansdown and Silver 2002b). Our group recently demonstrated the safe and controlled delivery of antimicrobial agents through microsphere system of natural and synthetic polymers like Chitosan, Eudragit RS100 and PLGA for effective wound healing (Singh et al. 2008, Singh et al. 2010a, Singh et al. 2010b, Singh et al. 2010c, Singh et al. 2011).

Skin substitutes for wound healing

Dermal grafts are cellular or acellular, allogenic in nature and, hence, available for immediate use. Tissue engineering has added several skin substitutes to the variety of dressings available for wound treatment (Table IV). These products for example consist of fibroblasts and keratinocytes grown on collagen matrices. Bioengineered skin, a bilayered living skin construct has been approved for venous and diabetic ulcers. A living dermal skin equivalent was also approved for diabetic neuropathic ulcers (Cha and Falanga 2007). In addition to standard and more advanced treatments, other less commonly used and unusual therapeutic products, not necessarily new, are being used by some clinicians (Lanza 2004). For example, cadaver skin is such an alternative; it is a true allograft and is always eventually rejected by the recipient. Skin that lacks a dermis is less able to resist trauma and is prone to contraction, resulting in a poor functional and cosmetic outcome (Cha and Falanga 2007). All currently available examples of artificial dermis lack a vascular plexus for the nourishment of the epidermis and require host vasculogenesis into the dermis graft to supply nourishment to the grafted epidermis (Lanza 2004). Other strategies for treatment of chronic nonhealing cutaneous wounds include temporary substitutes such as porcine xenografts, synthetic membranes, and autologous and allogeneic epidermal substitutes (Sheridan and Tompkins 1990). Recent artificial dermal substitutes are structurally optimized to incorporate the surrounding tissue and to allow cell invasion by fibroblasts and capillaries for subsequent dermal remodeling (Sheridan and Tompkins 1990). There are a number of representative skin substitutes and in one such clinical trial the application of Apligraf® has been shown to accelerate wound closure compared to control (Falanga and Sabolinski 1999). But, in spite of advances in the treatment of chronic wounds with bioengineered skin, there remain almost 50% of patients who do not heal when their ulcers have been previously resistant to conventional therapy.

Table IV. List of skin substitutes with their composition.

S.No.	Name	Components	Company
1.	Biobrane	acellular dressing, collagen bound to nylon fabric	Bertek Pharmaceuticals; Morgantown, WV
2.	Epicel	autologous epidermal graft	Genzyme; Cambridge, MA
3.	AlloDerm	accellular, allogeneic dermal graft	LifeCell Corporation in Palo Alto, CA
4.	Integra Artificial Skin	bovine collagen and chondroitin-6-sulfate	Integra Lifesciences; Plainsboro, NJ
5.	Oasis	pig intestinal mucosa	Cook Biotech, Inc. West Lafayette, IN
6.	TranCyte	devitalized fibroblasts on nylon mesh	Advanced Tissue Sciences; La Jolla, CA
7.	Dermagraft	Human fibroblasts in an absorbable matrix (approved in United States for diabetic ulcers)	Smith & Nephew, La Jolla, CA
8.	Orcel	Human fibroblasts and keratinocytes in a bovine collagen sponge	Ortec International, Inc. New York, NY
9.	Apligraf	human fibroblasts and keratinocytes in a bovine collagen matrix (approved in United States for venous and diabetic ulcers)	[Novartis; East Hanover, NJ]:

Growth factors

There are several growth factors that are being evaluated in clinical trials (Gharaee-Kermani and Phan 2001). These growth factors play major roles in local inflammation, reepithelialization, granulation, tissue formation, neovascularization, and extracellular matrix production from various cell sources and through diverse mechanisms. There have been extensive investigations into wound healing by the exogenous application. Based on the diverse results, the type of the individual wound is an essential criterion for the choices of growth factors. Therefore, the approval of Regranex® only for diabetic foot ulcers is feasible. Richard et al. conducted a trial with b-FGF on diabetic foot ulcers and observed no advantage of verum over the placebo control (Richard et al. 1995). In another trial, EGF was exogenously applied to patients with diabetic foot ulcers and a significant enhancement of healing and a reduction of healing time were reported (Tsang et al. 2003). Effect of KGF-2 or repifermin on chronic venous ulcers was evaluated during clinical trial and a significant acceleration of wound closure was observed (Robson et al. 2001).

For PDGF-BB (platelet-derived growth factor consisting of BB-homodimer) or becaplermin, several clinical trials finally leading to the approval of Regranex®in 1999 for the treatment of diabetic foot ulcers have also been published. Efficacy and safety in diabetic foot ulcers have been proofed (Embil et al. 2000). Similar trials, e.g., concerning pressure ulcers, acute, and open surgical wounds have also been conducted with promising results but not yet leading to an approval (Cohen and Eaglstein 2001, Shackelford et al. 2002). In Regranex® PDGF is formulated in an aqueous carboxymethyl cellulose hydrogel. Further, the formulation contains an acetate buffer, lysine hydrochloride, and sodium chloride. Another new technology for augmenting levels of growth factors in wounds is by gene transfer. Andree et al. used particle-mediated and micro seeding gene transfer to deliver human EGF to porcine wounds (Andree et al. 1994). A high expression of EGF, as well as a significant acceleration of healing, was shown in the transfected wounds.

To overcome this problem and to make allowance to the thought of growth factors acting in concert, methods of autologous growth factor application have been developed (Andree et al. 1994).

Moist wound treatment

In 1962, George Winter, from Smith & Nephew Inc. demonstrated that wounds epithelialized more rapidly under occlusive dressings as it maintained a moist wound surface (Winter 1962). Numerous studies followed which demonstrated that wound occlusion and moisture increased all phases of healing (Mertz and Eaglstein 1984).

Advantages of moist wound treatment (Eaglstein 2001):

• During moist treatment the likelihood of scarring is reduced because there is no scab formation.

• Moisture is essentially required for the activity of growth factors and proteolytic enzymes. It is as well necessary for surface oxygen delivery and an efficient nutrient delivery. As a result, moisture improves the processes of the migration and proliferation phase by providing the cells with the ability to migrate across the wound surface leading to increased rate of epithelization and angiogenesis.

• With a moist environment the nerve endings are cushioned and protected which gives relief from pain.

Moist wound treatment is done by various means including moisture-retentive dressings like occlusive films, hydrogels, and hydrocolloids; absorbent dressings like foams and alginates.

• Film dressings—occlusive films are semipermeable polyurethane dressings that are coated with an adhesive. They are used for minor exudating wounds. Their purpose is to prevent bacterial infection by shielding, to absorb low amounts of exudate and to maintain a moist wound environment for fresh epithelial tissue. The dressings insure a gaseous exchange for vaporizing superfluous liquid.

• Hydrogels—Hydrogels are used to treat wounds with low exudate levels. They contain high amounts of water. Most products contain sodium carboxymethyl cellulose or polyacrylates swollen to an amorphous gel in a propylene glycol water mixture. Hydrogel dressings are used to hydrate necrotic tissue, facilitating autolytic debridement, while being able to absorb exudates (Singh and Singh 2011).

• Hydrocolloids—used for moderate exudation. They contain a layer of hydrocolloid. This is defined as liquid absorbing particles in an elastic, self-adhesive mass. The particles mostly consist of sodium carboxymethyl cellulose, calcium alginate, pectine, and gelatin. The elastic mass contains different synthetic polymers. The wound exudate binds to the absorbing particles of the hydrocolloid matrix to form a cohesive gel maintaining a moist wound environment. Most products are covered on the upper side by a semipermeable polyurethane film.

• Foams—Double-layer dressings consist of a polyurethane film carrier and polyurethane foam layer on the wound side. They are used for moderate to heavily exuding wounds. Specialty absorbent dressings can be used as secondary dressings.

• Alginates—Alginate dressings are used to cover heavily exuding wounds. They mostly contain a combination of calcium and sodium alginate fibers. Alginates support healing by binding bacteria and debris inside the gel structure and by providing a moist environment (Eaglstein 2001).

Gene therapy

This technique involves the introduction of the gene rather than product (growth factor) that is thought to be cheaper and more efficient for treating nonhealing wounds. Technology to introduce genes through physical or biological vectors has existed for some time. The limited success in clinical trials of topical delivery of pluripotent growth factors attracted the notice of scientists toward gene delivery systems. An approach in which genetically modified cells synthesize and deliver the desired growth factor in regulated fashion has been used to overcome the limitations associated with the (topical) application of recombinant growth factor proteins. Preliminary promising results have given hope for treatment of complicated wounds (Sabine et al. 2007). This is a new area of research and new trials are underway.

Stem cell therapy

The development in stem cell therapy has opened the path for treatment of wound using this technology. There is potential that stem cells may reconstitute dermal, vascular, and other elements required for optimum wound healing. Though the technique is still in its infancy, Badiavas et al. (2003) have shown that direct application of autologous bone marrow and its cultured cells may accelerate the healing process (Badiavas et al. 2003). Because of the relative ease with which they are cultured, bone marrow-derived mesenchymal stem cells have been tested in pilot studies as a promising approach for introducing them into the wound. It might turn out, however, that other types of stem cells will be more effective, including those derived from hair follicles, or perhaps subsets of bone marrow-derived cultured cells (Cha and Falanga 2007). Still, proper wound care and adherence to basic principles cannot be bypassed, even by the most sophisticated approaches.

Miscellaneous

Recent approaches to wound healing includes the use of metals like gold and silver for complete wound healing. Hendi, 2011 has synthesized and evaluated silver nanoparticle for its wound healing activity and found that rapid healing and improved cosmetic appearance occurred within 15 days (Hendi 2011). Leu et al 2012 studied wound healing activity of topically applied nanoparticles of gold with epigallocatechin gallate (AuEA) and concluded that AuEA significantly accelerated cutaneous wound healing (Leu et al. 2012).

Conclusion

Wounds are one of the major causes of deformity and death in the world. The extensive research in the area of wound healing has greatly enhanced the knowledge regarding treatment strategies for a variety of wounds. It is the major cause of financial burden on the healthcare system in general and on patients in particular. No single treatment is completely successful for wound healing as complete wound healing depends on the multiple molecular and cellular mechanisms. The evolution of wound healing as specialty division of medicine is selfexplanatory to emphasize the importance of wound healing. This has led to revolutionary advances in wound healing knowledge and treatment including tissue engineering, growth factors, animal fetal cell research, stem cell research, gene therapy, human skin substitutes, and dressings. These developments in therapy has opened new hopes for the patients suffering from complicated wounds, but still there is a sturdy requirement for improved methods of therapy that should involve the principle of minimizing harm and augment the basic principles of wound care.

Acknowledgement

The authors are thankful to the Director, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G.), India for providing all necessary facilities for carrying out this work and University Grants Commission (UGC/MRP 39-169/2010 (SR)) and (UGC-RA 70-371/2012) for financial assistance.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.