Abstract: This paper reviews the physiology of wound healing and properties of the “ideal” dressing and also reviews advanced wound therapeutics and dressings, such as growth factors and biological skin substitutes.

Introduction

       Treatment of diabetic foot ulceration is much more complex than simply putting a dressing over a wound. Diabetic foot ulceration is a significant cause of morbidity and is the most common reason for hospital admission in diabetic patients. Annually, two to three percent of diabetic patients1,2 will develop foot ulcers, and up to 15 percent of diabetic patients will develop chronic ulcers during their lifetimes.3 In those who require lower-limb amputation, 70 to 90 percent will be preceded by a foot ulceration.

Physiology of Wound Healing

       The three general phases involved in wound healing are the acute inflammatory phase, the proliferative phase, and the maturation phase. The initiation and transition of these phases have no clear-cut boundaries but are descriptors on a continuum of events. The initial phase, inflammation, involves transient vasoconstriction of local arterioles and capillaries followed by an influx of inflammatory cells and plasma proteins to mediate the repair process. The next phase is proliferation, where fibroblastic activity and angiogenesis by the endothelial cells begin. The maturation phase may last for up to two years and involves collagen synthesis and breakdown.

Developments in Physiological Aspects of Wound Healing

       Chronic diabetic foot ulcers have been shown to result from a number of causes, one of which involves faulty wound healing. The normal wound healing process entails a complex interplay between connective tissue formation, cellular activity, and growth factor activation. All three of these physiologic processes are altered in the diabetic state and contribute to the poor healing of diabetic foot ulcers. More specifically, the chronic diabetic foot ulcer is stalled in the inflammation phase of the normal wound healing process.4 During this delay, there is a cessation of epidermal growth and migration over the wound surface.5,6 Analyses of fluid from chronic wounds have demonstrated elevated levels of matrix metalloproteinases (MMPs) directly resulting in increased proteolytic activity and inactivation of the growth factors that are necessary for proper wound healing. A number of recent studies have investigated these alterations in an attempt to better understand the wound healing abnormalities in diabetes and to target therapy specifically aimed at correcting these deficiencies, as described below.

       Collagen. Collagen, the most abundant protein in connective tissue, is an integral component of dermis, bones, tendons, and ligaments. Collagen synthesis and degradation in wound repair are complex processes that continue at the wound site long after the injury. The resulting scar tissue following wound repair never fully regains the tensile strength of the original intact skin. Instead, scar collagen retains only 70- to 80-percent tensile strength of the original collagen.7 The balance between collagen synthesis and degradation in wound repair is tenuous, and disease states, such as diabetes, can shift the balance to one side, disrupting the wound healing process.

       In diabetes, collagen synthesis is markedly decreased, resulting in chronic connective tissue complications. The defect in collagen metabolism in diabetes is present at both the collagen peptide production level as well as the posttranslational modification of collagen degradation. The resultant collagen production deficits can be observed in several systems, including thickening of the vascular basement membrane, limited joint mobility, and poor wound healing.

       Cellular activity. The inflammatory stage of wound repair involves an orchestrated interaction of resident cells, such as epithelial cells, fibroblasts, dendritic cells, and endothelial cells, with biochemical activity. In addition to these resident cells, platelets, neutrophils, T-cells, natural killer cells, and macrophages are recruited to the wound site. These cells migrate to the injury site to mediate the inflammation, coagulation, and angiogenesis processes occurring in the wound healing process.

       In diabetes, the resultant hyperglycemia can potentially mitigate the cellular activity in the inflammatory process. More specifically, the morphological characteristics of macrophages have been observed to be transformed in such a manner as to impair their function.8 Morphological changes have also been noted in skin keratinocytes. Additionally, inhibition of keratinocyte proliferation in the presence of increased cellular differentiation leads to an imbalance in keratinocyte production, an essential step in the wound healing process.9 Consequently, impairment of the cellular mediators during the wound healing process in diabetes is implicated as a cause of faulty wound healing.

       Growth factors. Growth factors influence the wound healing process through inhibitory or stimulatory effect on the local wound environment. Growth factors, such as platelet-derived growth factor, basic fibroblast growth factor, vascular endothelial growth factor, and nitric oxide, have all been found in wound fluid. These growth factors are known to be integral in the chemotaxis, migration, stimulation, and proliferation of cells and matrix substances necessary for wound healing. Therefore, the altered secretion or absence of these growth factors in diabetic foot ulcers can potentially impair wound healing. Recent investigation of the role these growth factors play in wound healing appears to support this hypothesis.

       Becaplermin. Recombinant human platelet-derived growth factor-BB (rhPDGF-BB)(becaplermin) is the only growth factor to date approved by the US Food and Drug Administration for the treatment of diabetic foot ulcers. Levels of PDGF have been shown to be lower in chronic wounds. Therefore, studies have investigated the results of topical augmentation of PDGF in these chronic wounds.10 Administered in a gel formulation concurrent with a standardized regimen of good wound care, becaplermin gel increased both the incidence of complete wound closure and decreased the time to achieve complete wound healing.11 Becaplermin is believed to enhance wound healing through the expression of PDGF-B by macrophages, endothelial cells, and platelets. PDGF-B is known to be a potent mitogen and chemotactic agent for connective tissue and stromal cells and may act to increase the wound vascularization by stimulating endothelial cell proliferation, movement, and tube formation.

       Basic fibroblast growth factor (bFGF). bFGF provides the initial stimulation of endothelial cell migration and proliferation in wound repair. It is also a potent mitogen for all cell types and found predominantly in the early proliferative phase of the wound. The importance of bFGF in wound repair has been demonstrated with studies using neutralizing antibodies against bFGF, which resulted in marked decrease in production of granulation tissue.12

       Topical application of bFGF has demonstrated faster granulation tissue formation and epidermal regeneration in the context of burn injuries. However, topical application of bFGF had no discernible advantage over placebo for healing chronic diabetic foot ulcers.13 Application of bFGF in a 5mg/mL saline spray that was performed daily for six weeks showed no increase in healing rate to that of saline gauze dressings. While bFGF has been reported in other studies to enhance wound healing slightly, this enhancement was not deemed significant.14 The failure of topically applied bFGF to promote wound healing may be due perhaps to inadequate dosage of the growth factor or the necessity of a “cocktail” of several growth factors to effectively influence wound stimulation.

       Vascular endothelial growth factor (VEGF). VEGF also induces endothelial cell proliferation and migration in the wound healing process. Secretion of VEGF can be performed by a number of cell types, including keratinocytes, macrophages, fibroblasts, and endothelial cells, generally in a hypoxic environment.15,16 The concentration of VEGF reaches maximal levels at day seven following wound development and appears to be the major stimuli for induction of angiogenesis. In the presence of diabetes, the production of VEGF has been shown to decrease.8 Furthermore, the histological appearance of the wounds were found to have diminished cellularity, decreased angiogenesis, and delayed granulation tissue formation and reepithelialization. Current research is aimed at the possible benefits of VEGF gene therapy on promotion of diabetic wound healing.

       Nitric oxide (NO). NO is normally secreted by macrophages and fibroblasts during the wound healing process. However, variation in the NO production in the chronic wound has been found to parallel the reparative collagen formation.17 When fibroblasts were genetically modified to prevent synthesis of NO, there was diminished fibroblast proliferation, decreased collagen synthesis, and delayed contraction of collagen lattices.18 Replacement of the NO restored collagen synthesis to normal levels. These observations demonstrate the integral role NO production plays in the wound healing process and how therapy aimed at maintaining optimal levels may facilitate healing of the chronic diabetic foot ulcer.

Wound Care Dressings

       The effective use of dressings is essential to ensure the optimal management of diabetic foot ulcers. In recent years, the concept of a clean, moist, wound-healing environment has been widely accepted. Benefits to this approach include prevention of tissue dehydration and cell death, acceleration of angiogenesis, and facilitation of the interaction of growth factors with the target cells.19 Additionally, patients have reported less discomfort with moist wound dressings. The notion that a moist wound environment increases the risk of developing an infection appears to be unfounded.

       Properties of the “ideal” dressing. Modern dressings are designed to promote and maintain a moist wound environment in the different phases of wound healing. While no perfect wound dressing exists for the diabetic foot ulcer, the properties sought should include:
• A moist wound environment
• Promotion of wound healing
• Provision of mechanical protection
• Nonadherence to the wound
• Allowance for removal without pain or trauma
• Capability of absorbing excess exudate
• Allowance of gaseous exchange
• Impermeance to microorganisms
• Acceptability to the patient
• Ease to use
• Cost effectiveness.

       Types of dressings. Wound dressings may be described as passive, active, or interactive.20 While passive dressings simply serve a protective function, both active and interactive dressings create a moist environment at the wound/dressing interface. Furthermore, interactive dressings are believed to also be capable of modifying the physiology of the wound environment by modulating and stimulating cellular activity and growth factor release. Interactive dressings include the hydrocolloids, alginates, hydrogels, iodine dressings, and, though not technically dressings, living skin equivalents. These dressings additionally promote debridement and may enhance granulation and reepithelialization (Table 1).20–22

Table 1

       Hydrocolloid dressings. As some of the first interactive dressings to be developed, the hydrocolloids are occlusive dressings designed to create and maintain a moist wound environment.23 They are capable of absorbing a moderate amount of wound exudate, resulting in a moist gel formation on the wound surface. However, oversaturation of the dressing may lead to leakage of the gelatinous substance, causing maceration of the surrounding skin. Therefore, hydrocolloids should be avoided on plantar ulcers of the foot, as the periwound skin is susceptible to maceration. Additionally, hydrocolloids have been shown to retain growth factors under the dressing as well as promote granulation and epithelialization.24

       Alginate dressings. Alginates are naturally occurring polysaccharides composed of the sugars mannuronic and guluronic acids. They are found primarily as the sodium salt in the brown algae, Phaeophyceae. Alginate dressings are composed entirely of calcium alginate or a combination of calcium and sodium alginate. Alginates can absorb between 15 to 20 times their own weight in fluid.25 The absorptive capacity of alginates makes them eminently suited for the treatment of heavily draining wounds. However, caution should be used as the pooling exudate from alginate dressings may cause maceration of the surrounding tissues.

       Hydrogel dressings. Composed of insoluble polymers, hydrogels contain hydrophilic sites that enable interaction with aqueous solutions and absorb and retain significant volumes of wound exudate.26 Hydrogels facilitate autolysis and rehydrate the wound. Hydrogels may be appropriate in diabetic ulcers that require debridement when surgical debridement is not an option. However, hydrogels are generally considered inappropriate for ischemic and gangrenous ulcers.

       Iodine dressings. The use of antisepsis in the treatment of ulcers is controversial, as antiseptics have been found to be harmful to healing wounds. Iodine, considered an antiseptic, has been found to be toxic to human cells as well as bacteria and fungi at high doses.27,28 However, in a diluted form, the antimicrobial effects are still present, but the toxicity to human cells is diminished. Therefore, iodine dressings may be useful in the diabetic ulcer at risk for infection.

       Promogran. Promogran is a combination collagen and oxidized regenerated cellulose (ORC) topical wound dressing. The importance of collagen synthesis and growth factor activation to normal wound healing has been demonstrated in a number of studies showing increased proteolytic activity and inactivation of growth factors in chronic foot ulcers.29,30 It is believed that the combined use of collagen and ORC can potentially inhibit the proteolytic activity while allowing continued growth factor activity.

       In a prospective, randomized, multicenter trial, 37 percent of promogran-treated patients had complete wound closure compared to 28.3 percent of gauze-treated patients. While this was not statistically significant, there was a statistical significance in wounds of less than six months duration. In wounds of less than six months duration, 32.6 percent of promogran-treated patients healed compared to 20.4 percent of gauze-treated patients. The results indicate that promogran may be employed as an alternative to moistened gauze for the treatment of diabetic foot ulcers with a duration of less than six months.

Developments in Biological Skin Substitutes

       Biological skin substitutes, also known as living skin equivalents (LSEs), are commercially offered in both autograft and allogeneic form. The allogeneic LSEs are produced from neonatal fibroblasts and keratinocytes using tissue-engineering technology. Available for epidermal, dermal, and composite (epidermal and dermal) wounds, LSEs offer distinct advantages over traditional skin grafting because LSEs are noninvasive, do not require anesthesia, can be performed in an outpatient setting, and avoid potential donor-site complications, such as infection and scarring.31 Furthermore, more rapid wound coverage of chronic diabetic foot ulcers with the use of LSEs can provide both social and economic advantages by reducing the number of office visits and hospital stays and preventing serious wound complications that often lead to amputation.

       Epidermal grafts. Epidermal grafts, available as autografts, are produced through a technique allowing in-vitro cultivation and serial subculture of epidermal cells, resulting in sheets of viable keratinocytes. Commercially available since 1988 (Epicel®, Genzyme Tissue Repair Corporation, Cambridge, Massachusetts), epidermal grafts can provide coverage of large skin defects with acceptable cosmetic results and are currently indicated for burns and leg ulcers. The disadvantages associated with autologous epidermal grafts include a necessary biopsy specimen, product fragility (making handling of the product very difficult), and the two- to three-week delay for the cultivation procedure.

       In response to the delay in product procurement, cultured allogeneic keratinocyte grafts were developed. Cultured from neonatal foreskin, keratinocyte allografts can be cryopreserved, allowing for a longer shelf life and increased availability.32,33 While availability is improved and biopsy specimens made unnecessary, allogeneic epidermal grafts, like autogeneic epidermal grafts, are fragile and difficult to handle due to a lack of a backing material or dermal layer. Additionally, they are unsuitable for deep wounds, as they provide only temporary coverage of the wound for eventual replacement by host epithelium. Blistering has also occurred following grafting onto burn wounds.34–37 Epidermal replacements are most successful when placed on a dermal bed, suggesting that the dermal elements play a key role in the stability and durability of the graft.

       Dermal grafts. Dermal replacements are allogeneic in nature and possess the advantage of immediate wound coverage. It is believed that the elements in the dermal layer exert positive effects on epithelial migration, differentiation, attachment, and growth in the wound healing process providing a good base for future epidermal grafting.38 Initial dermal replacements were harvested from cadaver skin for use in temporary wound coverage of full-thickness burns. While successful for this specific function, cadaveric allograft has limited availability, and safety concerns have restricted its use. The cadaver allografts can be treated chemically to eradicate antigenic components, thus leaving an immunologically inert acellular dermal matrix with an intact basement membrane (Alloderm®, Life Cell Corporation, Woodlands, Texas). While encouraging results have been found in pilot studies for burn wounds, procurement of the cadaver skin and virus screening still pose problems, limiting its use.39,40

       As an alternative to cadaver skin, a dermal graft consisting of a polymer of bovine collagen and chondroitin-6 sulfate with an overlying silastic sheet of human keratinocytes and fibroblasts has been developed (Integra®, Life Sciences Corporation, Plainsboro New Jersey). It is indicated for burns, as a surgical space filler for deep tissue defects, and for cosmetic procedures. However, it cannot be generated in large quantities, is susceptible to infection, and is not indicated for use in diabetic foot ulcers.

       Recently approved by the FDA for use in diabetic foot ulcers, Dermagraft® (Advanced Tissue Sciences Inc., La Jolla, California, distributed by Smith & Nephew Inc., Largo, Florida) consists of neonatal dermal fibroblasts cultured in vitro onto a bioabsorbable polyglactin mesh, producing a living, metabolically active tissue containing the normal dermal matrix proteins and cytokines. Dermagraft has been shown to incorporate quickly into the wound with good vascularization and with no adverse side effects.41–43

       In a prospective, randomized, multicenter study, Dermagraft-treated ulcers were associated with more complete and rapid healing with the added benefit of a reduction in the ulcer recurrence rate compared to conventional therapy (Figure 1).44 There was a dose relationship found in which multiple applications of Dermagraft applied to the wound led to more rapid and complete healing. Additionally, more weekly applications of Dermagraft also exhibited greater improvement compared to application of the Dermagraft every two weeks.

Figure 1

Figure 1. Dermagraft study. Results of randomized, prospective, multicenter Dermagraft study for diabetic foot ulcers. Of 50 patients with diabetic foot ulcers, 32 percent of the control (CT) achieved wound healing compared to 39 percent of patients who received Dermagraft (DG-All) treatment (p = 0.14). Patients who received metabolically active products at the first two applications and the majority of the following application (DG-TR) achieved 51-percent healing rate by week 12 (p = 0.006). Patients who received metabolically active products at all times (DG-E) achieved 54-percent healing rate (p = 0.007).

       While the precise mechanism of action of Dermagraft is not completely understood, it is hypothesized that the matrix components and cytokines produced by the fibroblasts may provide a stimulus for more rapid and complete healing. Because collagen production and growth factor secretion in diabetic skin have been found to be abnormal, the engrafting of normal matrix components by Dermagraft may respond physiologically to the recipient host tissues to contribute to improved rate and quality of wound healing.44

       Composite grafts. Composite grafts are bilayered skin equivalents consisting of both epidermal and dermal components. One such composite graft, Apligraf® (Organogenesis, Inc., Canton, Massachusetts, distributed by Novartis Pharmaceutical Corp., East Hanover, New Jersey) contains an outer layer of allogeneic human keratinocytes on an inner dermal layer consisting of human fibroblasts on type 1 collagen dispersed in a protein matrix. While Apligraf looks and feels like human skin, it does not contain blood vessels, hair follicles, or sweat glands. Interestingly, Apligraf acts like human skin, producing all the cytokines and growth factors produced by normal skin during the wound healing process (Table 2).45

Table 2

       In diabetic foot ulcers, Apligraf was shown to significantly increase the wound healing rate as well as decrease the median time to complete wound closure (Figure 2).46,47 Ulcer recurrence rate was similar in both Apligraf-treated ulcers and standard treatment groups.47 Additionally, no adverse events or immunological effects have been associated with the use of Apligraf. While it is available immediately, Apligraf does have a limited shelf life of only five days; therefore, it requires patient scheduling within that time frame.

Figure 2

Figure 2. Apligraf study. Frequency of complete wound closure. Results of randomized, prospective, multicenter Apligraf study for diabetic foot ulcers. The difference between the control and Apligraf treatment is apparent early in the study. At week 4, three percent of the patients who received control treatment had wound healing compared to 20 percent of patients who received Apligraf once a week for the first four weeks. At week eight, 25 percent of control patients achieved wound healing compared to 45 percent of the Apligraf-treated patients. By 12 weeks, 39 percent of the control patients achieved complete wound healing compared to 56 percent of the patients who received Apligraf treatment (p = 0.0026).

       Similar to Dermagraft, the exact mechanism of action for Apligraf is not fully understood. It is believed that rapid wound healing by Apligraf is due to filling of the wound with extracellular matrix proteins and with the subsequent induction and expression of growth factors and cytokines necessary for wound healing. Additionally, the matrix components may further facilitate the recruitment of cells to the wound, improving wound repair.

Conclusions

       Intensive work over the last twenty years has considerably increased our understanding of the physiology and pathophysiology of wound healing. This has resulted in the development of new wound healing treatments, such as living skin equivalents and wound care products, directed at increasing the bioavailability of growth factors in the chronic wound environment. Clinical trials conducted over the last five years have shown very promising results with several products recently approved for clinical use in the diabetic foot ulcer. The variety of advanced wound care techniques and products will aid the clinician in developing appropriate algorithms for the diabetic foot wound as well as potentially increasing wound healing overall.