Abstract: Bioengineered skin substitutes have emerged over the past 20 years as the most carefully studied and proven of the advanced wound management technologies. While the initial impetus for their development was to replace autograft, allograft, and xenograft in burn applications, they have found even wider application in the treatment of chronic venous and chronic diabetic ulcers. The current review addresses the history of skin substitutes, surveys the landscape of existing Food and Drug Administration-approved products and other promising innovations that appear close to market, and discusses the reasoning and controversies associated with design of these products. While acellular biologic constructs are discussed, the authors focus on products that include autologous or allogeneic cells. The various clinical trials supporting the use of skin substitutes for different wound healing indications are reviewed. The preponderance of literature supports the cost effectiveness of using skin substitutes in healing burn, autograft donor site, diabetic, and venous wounds. In addition, better methods for early identification of diabetic and venous ulcers that may not heal well with standard treatment should improve the process of triaging candidates for skin substitute therapy. In the future, attaining a more detailed understanding of the mechanisms by which skin substitutes induce accelerated healing, including a better appreciation of the roles of cytokines and cell scaffolds, may lead to product enhancements that increase efficacy. Ongoing progress toward overcoming issues, such as abbreviated shelf life and distribution difficulties, as well as high manufacturing costs, should enable broader implementation of skin substitutes in acute and chronic wound therapy.

Part 1 of 2

Introduction

In the 1980s, the emergence of tissue engineered skin substitutes and cultured skin cells (“cell-based wound therapy”) for use as experimental wound healing therapies was motivated primarily by the critical need for early coverage of extensive burn injuries in patients with insufficient sources of autologous skin for grafting. Since then, skin substitutes have been widely used to address the prevalent problem of chronic wounds associated with non-burn etiologies. The treatment of such hard-to-heal, chronic, open wounds has assumed increasing importance as the aged segment of the population in the United States and the rest of the industrialized world has increased and as the incidence of comorbid states, such as diabetes mellitus and atherosclerosis, has increased. To put these wound healing indications into quantitative perspective, the annual incidence of serious burns in the United States is estimated at 70,000;[1] the prevalence of venous leg ulcers is between 600,000 and 1,500,000;[2] and 15 to 20 percent of people with diabetes eventually suffer a chronic foot wound.[3] The direct cost, in the US, of dressings alone for all these conditions has been estimated at over $5 billion per year.

Concurrent with the increasing socioeconomic importance of wound management, an infusion of scientific understanding of the cellular and biochemical steps involved in wound repair has spawned multiple new advanced technologies that may be applied to treat nonhealing wounds. The old paradigm for wound management was essentially passive: to remove impediments to wound healing (e.g., uncontrolled diabetes mellitus, infection, and ischemia) and allow nature to take its course. The contemporary approach, while not ignoring these basics, seeks to more actively intervene and improve upon natural healing using such modalities as hyperbaric oxygen, electrical stimulation, skin surface negative pressure, exogenous growth factors and bioengineered skin substitutes. Thus, a “one-size-fits-all” approach, whereby a clinician would use his favorite dressing on most wounds, has given way to individualization of therapy based upon the physiology of each particular patient and wound.[4] The current challenge for clinicians is to understand the relative merits and roles of each available technology. One objective of this review will be to compare and contrast various commercial or nearly-commercial skin substitutes and cell-based wound therapies, and illustrate their roles in wound management.

A variety of acellular matrices that are used to promote regenerative skin wound healing, such as Alloderm® (LifeCell Corporation, Branchburg, New Jersey), Oasis® (Healthpoint Ltd., Fort Worth, Texas), Integra® Dermal Regeneration Template (Integra Life Sciences Holding Co., South Plainfield, New Jersey) and Biobrane® (Bertek Pharmaceuticals, Sugarland, Texas) are generally included in the skin substitute category; these have been reviewed elsewhere[5–7] and will be mentioned briefly here, but will not be the focus of this discussion. Cultured epithelial autografts,[8,9] acellular dermal matrices,[10] as well as composite dermal/epidermal cultured autografts,[1,11] can act as autograft alternatives to provide life-saving skin replacement. Many recent reviews have summarized the history and current status of skin substitutes made with acellular matrices or with autologous cells.[1,5] Other authors have provided encyclopedic overviews of the landscape of biological skin substitutes.[12] The authors of this review aim not to repeat a detailed summary of all available skin substitute and cell-based wound therapy devices, but rather to consider current advances and challenges within this field and to use particular products to exemplify various concepts. Also, while many centers have reported limited experience with “home-grown” cell-based wound therapies, most of which are technical variations on the platform of cultured autologous keratinocytes, alone or in combination with a dermal layer,[13,14] the authors will focus primarily on cellular, mostly allogeneic, commercial devices that already have achieved or appear to be nearing Food and Drug Administration (FDA) approval.

Rationale for the Use of Cell-Based Skin Substitutes in Chronic Wound Therapy

While autologous tissue transfers can be highly effective in securing wound healing, such procedures (e.g., grafts and flaps) are invasive, painful, and expensive, and are not within the purview of many wound care practitioners. In practical terms, skin substitutes represent artificial, off-the-shelf alternatives to skin grafts that avoid the pain and potential complications of harvesting, are always available in any quantity needed, and can be applied in an office setting. The ideal skin substitute would adhere quickly to the wound and would, during its residence in the wound bed, mimic the physiology and some of the mechanics of normal skin. It would be inexpensive, not subject to immune rejection by the host, and would be highly effective in accelerating tissue regeneration and wound repair.

Unfortunately, this ideal skin substitute has not yet been achieved, and expectations for such a product may be unrealistic. Nevertheless, some very useful products have been developed that do provide many of the desired clinical characteristics. In order to be available off-the-shelf, these products rely on the use of donor allogeneic cells. Allogeneic skin substitutes do not provide cells that persist on the recipient site long term. Deoxyribonucleic acid (DNA) analyses of biopsies of healed wounds after application of a living skin equivalent (LSE) (Apligraf®, Organogenesis Inc., Canton, Massachusetts), for example, have demonstrated one-month persistence of allogeneic cells in only a minority of venous ulcer patients, and the complete disappearance of these cells by two months post-application.[15] In the case of chronic wounds, therefore, the goals of skin substitute therapy have evolved away from providing an immediate new skin towards the more reasonable goal of providing a temporary biologic dressing that accelerates skin tissue regeneration and wound healing by stimulating the recipient’s own wound bed-derived skin cells.[16]

The mechanisms by which bioengineered skin substitutes aid wound repair are not completely understood, but may range from maintenance of a biochemically balanced moist wound environment to structural support for tissue regeneration and/or the provision of beneficial cytokines and growth factors to the wound bed. The FDA has regulated skin substitute products as medical devices and has not required extensive elucidation of their therapeutic mechanisms. As knowledge of the myriad cellular events involved in the wound healing cascade increases, dozens of molecular mediators (growth factors) have been found to be involved in wound repair.[17,18] One hope implicit in the use of any cell-based wound therapy, as opposed to exogenous growth factor therapy, is that cells applied to the wound surface are “smart” and will bathe the wound bed with balanced cocktails of such mediators appropriate for the particular physiology of the wound environment that is sensed. For example, incubation of cells derived from biopsies of venous ulcers in conditioned medium supernatant from human fibroblast cell culture induces a highly significant increase in skin cell proliferation, and this effect has been correlated with levels of several cytokines.[19] There is mounting evidence that a bioengineered skin substitute is indeed acting as an interactive “drug” delivery system.[20] Mansbridge, et al., for example, have reported that the viability and metabolic activity of the cellular component of a cellular skin substitute is essential for therapeutic efficacy and have proposed that this is due to the need for ongoing cytokine expression in the wound bed following application.[21] Osborne and Schmid[22] have used measurements of matrix metalloproteinases (MMPs) secreted by the epidermis and dermis of LSE to show that the individual layers of bioengineered skin sense their environment and secrete MMPs in different quantities and ratios depending upon their environment.

While the end point of wound closure is the most intensely studied, there is also increasing focus upon the quality of the healed wound. Cell-based wound therapies, such as cellular skin substitutes, have the potential to reduce wound contraction and to influence the nature of the final healed tissue; indeed, there are reports to indicate an additional benefit of skin substitute therapy in leading to a healed wound with properties that more closely resemble those of normal uninjured skin.[23]

History of Skin Transplantation

Autologous skin grafts have been used for more than two thousand years, apparently beginning in India; the first scientific reports come from nineteenth century Europe.[24,25] Cadaver skin has also been used extensively, especially over the past thirty years, mostly in treating extensive burns.[26] Prompt wound coverage is thereby achieved in patients with limited sites for harvesting autologous skin grafts. Typically the allograft is harvested within a day after death and is cryopreserved at liquid nitrogen temperature. While allograft skin will ultimately require excision and autologous regrafting, it may adhere well to the wound for up to several weeks before clinically obvious rejection occurs. Nevertheless, although results with cadaver skin for burn coverage are good, problems with availability, expense, potential disease transmission and questions about the detrimental effects of the cryopreservation process have all limited its use. In answer to the limited availability of cadaver skin, xenograft[27,28] (most often porcine or bovine) skin has also been tested extensively over the years. Results are similar to those with allograft in that initial take is often observed when grafted onto a clean, debrided wound, and angiogenesis is encouraged, albeit with ultimate rejection and the requirement for removal within a few days to a few weeks.

Grafting of cultured cells as skin replacements did not become a clinical reality until the work of O’Connor, et al., who were the first to deliver autologous keratinocyte sheets to burn patients in 1981.[9] The major technological advancement which presaged O’Connor’s clinical success was the development of an in-vitro keratinocyte expansion technique by Rheinwald and Green in 1975[8] that paved the way for culturing sheets of autologous keratinocytes.[29] Other researchers have used both autologous fibroblasts and keratinocytes to create composite autologous skin substitutes for burn wound closure.[30] Such successes notwithstanding, the advantage of off-the-shelf availability and the greater commercial potential has led to much more effort in the industrial sector toward the use of allogeneic donor cells to create tissue engineered skin substitutes.

Design and Application of Cultured Skin Substitutes

Tissue engineering of cultured skin substitutes is largely based on the strategy that the following three components are important in a tissue-engineered construct:[31] 1) cell source, 2) tissue-differentiation-inducing substance, and 3) matrix. A variety of cells, soluble mediators, and biopolymers have been tested in various combinations to engineer cultured skin substitutes. Epidermal sheets of cultured keratinocytes have been applied to wounds as allografts or autografts.[32–34] Later, it was shown that replacing the connective tissue may increase mechanical strength of healed wounds and reduce ultimate scarring,[35–37] so fibroblasts have been included in some artificial skin substitutes.[30,38,39] Others have used matrix-cultured dermal fibroblasts alone as a wound healing device.[40] Most commercial bioengineered skin devices consist of sheets of biomaterial matrix containing allogeneic cells, which are typically derived from neonatal foreskin, a convenient tissue source with the added advantages of having a higher content of putative keratinocyte stem cells,[41] vigorous cell growth and metabolic activity, and minimal antigenicity.[42] The steps in creating and combining the components of bioengineered skin have been comprehensively discussed by Boyce and Warden.[1]

A listing of commercial skin substitutes is provided in Table 1. Epicel® (Genzyme Biosurgery, Cambridge, Massachusetts) the original cultured epidermal autograft (CEA), conceptually represents one of the simplest of the bioengineered skin substitutes,[43] in that it consists of a single cell type (keratinocytes) delivered on a petrolatum gauze backing (no matrix.) The autologous keratinocytes are cultured with irradiated murine fibroblast feeder layers to form stratified keratinocyte sheets 2 to 8 cell layers thick. Based upon the seminal work of Rheinwald and Green,[8] who developed an optimized method for growing and differentiating keratinocytes, these cells are derived from a small full-thickness biopsy of skin from the wounded individual. By manufacturing CEA sheets that are customized and unique for each patient, immune rejection issues are eliminated and the cells have the potential for permanent engraftment. The product has a 24-hour shelf life at room temperature. In a study of six extensively burned children, the average initial and final engraftment rates of this CEA were 79 percent and 84 percent, respectively.[44] Biopsies post-engraftment demonstrated that an organized epidermis with rete ridges and anchoring fibrils and a mature vascularized dermis regenerated over 6 to 12 months after CEA application. A retrospective review of 30 patients with burns of a mean 78 percent of total body surface area found an extraordinarily high survival rate (90%) using CEA to reduce the need for autologous harvesting by about half; sixty-nine percent of the CEA adhered permanently to the patient.[45] The primary difficulties associated with CEA use relate to high cost and logistics. Treatment with Epicel requires taking a skin biopsy, sending it to the manufacturer, and waiting about three weeks to receive the finished product. For these reasons, this product has been used primarily in life-threatening high body surface area burns, but has not been used extensively in the chronic wound arena.

Table 1

Another CEA product is Laserskin® (Fidia Advanced Biopolymers Srl, Italy), which is indicated both for deep second-degree burns and for chronic ulcers. This CEA product is based on a biodegradable matrix composed of a benzyl esterified hyaluronic acid derivative with ordered laser-perforated microholes for the in-growth and proliferation of autologous keratinocytes.[46] Autologous keratinocytes are isolated from a skin biopsy and cultured directly on the matrix. The graft can be removed from culture without disturbing the arrangement of basement membrane proteins. Currently, this CEA is commercially available only in Europe. To investigate its efficacy for treatment of diabetic foot ulcers, a pilot noncontrolled study was conducted in which this CEA was applied to 14 patients with type 2 diabetes mellitus who suffered nonhealing foot lesions.[47] It was found that 11 of the 14 lesions completely healed with an average healing time of 41 days. These results suggest that this CEA may be an effective treatment for the healing of diabetic foot wounds, and other data suggests its potential to reduce hospital length-of-stay. Product advantages include the immunological safety of using autologous cells, and disadvantages include cost, short shelf life, fragility, and the need for custom preparation. Controlled clinical trial data have not yet been obtained.

In parallel with the development of customized, autologous products, a variety of allogeneic keratinocyte sheets have been produced by many groups and used to heal burn and venous wounds successfully since the 1980s. Commercialization of cultured epithelial allografts has been difficult, although Celadon Science, LLC (Brookline, Massachusetts) may have surmounted at least some of the problems in creating Celaderm®, which contains metabolically active foreskin-derived allogeneic keratinocytes that are not capable of proliferating. For treatment of chronic wounds, this epithelial allograft treatment has the potential to reduce the expense and inconvenience associated with Epicel, as manufacturing costs for an allogeneic product are inherently somewhat lower, and the cryopreserved product can be stored in an ordinary freezer for six months and applied without elaborate thawing or rinsing procedures. This epithelial allograft treatment and other epithelial allografts made using a similar technique have been tested in several single-site pilot human studies. Seven of eleven venous ulcer patients with wounds recalcitrant to standard therapy were healed after an average of 4.14 applications of the product.[48] Alvarez-Diaz and colleagues found in a one-center pilot group of 11 patients that matched-pair partial-thickness burns healed at least 44 percent more quickly with epithelial allograft treatment than with control dressings (no wounds were autografted).[49] The product was also tested in a pilot group of ten patients with chronic ulcers of many etiologies, including some cases that presented with tendon exposure but managed to heal completely.[50] More extensive controlled clinical studies will be needed to fully evaluate the safety and effectiveness of epithelial allograft treatment.

Dermagraft® (Smith & Nephew, Inc., Largo, Florida) is a cryopreserved human fibroblast-derived dermal substitute that is designed for treatment of diabetic foot ulcers of greater than six weeks’ duration, including full-thickness wounds. In the manufacturing process, fibroblasts are isolated and expanded from human neonatal foreskin. The cells are then cultured on a bioabsorbable polyglactin mesh for approximately three weeks. During this time, the cells secrete matrix proteins, including human dermal collagen, and soluble factors to create a human protein-containing three-dimensional matrix that can be used as a dermal replacement.[51] The product is sterility tested and is supplied in a cryopreserved form that requires thawing and rinsing before use. Initial difficulties with maintaining cell viability of the product impeded regulatory approval,[52] but the product has since been approved based upon subsequent studies; it has also been approved (Humanitarian Device Exemption) for treatment of ulcers secondary to epidermolysis bullosa. The product, though fragile, handles moderately well, but a significant preliminary thawing and rinsing procedure is required prior to its use. Transcyte™ (formerly Dermagraft-TC, Smith & Nephew), another fibroblast-containing construct containing non-viable cells, uses a silicone covered nylon mesh for cellular support, and is indicated for temporary coverage of surgically excised burn wounds (i.e., as an alternative to cadaver skin.)[53,54]

LSE is a composite cultured skin substitute indicated primarily for treatment of diabetic foot ulcers and venous leg ulcers.[55] Like other allogeneic products, it has limited persistence in vivo, and cannot therefore be considered a skin replacement. The design and manufacture of LSE have been well reviewed by several authors.[56] This construct, containing human foreskin-derived keratinocytes and fibroblasts in a bovine collagen gel matrix, was the first FDA-approved bilayered cell-based wound therapy indicated to treat chronic wounds. It is composed of one layer of human fibroblasts grown in bovine collagen gel and a second overlying layer of stratified, differentiated human keratinocytes with a well-formed stratum corneum. In the manufacturing process, fibroblasts and keratinocytes are first isolated from neonatal foreskin and expanded in vitro to establish a cell bank. The donor and cell bank are extensively safety tested. Fibroblasts are mixed with purified acid-soluble bovine type I atelo-collagen and cast into a gel form. The fibroblasts contract the gel matrix to form a dermal equivalent after four to six days. Keratinocytes are then seeded onto the dermal matrix and cultured for two days, after which the cultures are exposed to the air-liquid interface to allow the epidermal layer to differentiate and stratify. The final LSE mimics some of the biochemical and histological properties of skin.5 The advantages of LSE include its ability to mimic some aspects (insofar as it is absent minority skin cell types, such as melanocytes, Langerhans cells, and endothelial cells) of the structure and function of skin, as well as the capability of clinical application in an outpatient procedure. The main disadvantages are somewhat awkward handling and the short shelf life of five days at room temperature.

OrCel® (Ortec International, Inc., New York, New York) is another allogeneic bilayered cellular matrix (BCM) containing neonatal foreskin-derived cultured keratinocytes and fibroblasts, but it is constructed on a porous cross-linked collagen (bovine type I) sponge matrix, rather than a gel. Foreskin donors (through screening and blood tests on the donor mother) and cells are tested extensively for absence of transmissible diseases, tumorigenicity, or cytogenetic abnormalities to assure complete safety. The sponge matrix is asymmetric, in that one side is coated with a thin film of acid-soluble atelo-collagen gel to close the macroscopic pores. Dermal fibroblasts are cultured on and within the porous sponge side of the collagen matrix while keratinocytes, from the same donor, are cultured on the gel-coated, non-porous side of the collagen matrix. Cell-seeded matrix cultures are maintained submerged so as to inhibit keratinocyte differentiation and stratification (Figure 1). The fresh form of this product has been FDA-approved for two indications: treatment of hand reconstructions in patients suffering from recessive dystrophic epidermolysis bullosa and healing of autograft donor sites in burn patients. Recently, a cryopreserved form of the product, with prolonged shelf life, has also been tested clinically in an FDA-approved pivotal trial for the treatment of venous leg ulcers. The timing and ratio of fibroblast and keratinocyte cell seeding and other aspects of the bilayered cellular matrix culture production process are designed to control cell density and in-vitro cytokine expression of the final product. It has been shown, in fact, that co-cultured keratinocytes and fibroblasts exhibit synergistic (as opposed to additive) expression levels of some cytokines and growth factors.[57,58] Figure 2 compares daily levels of in-vitro expression of two key cytokines—keratinocyte growth factor (KGF-I) and granulocyte-macrophage colony stimulating factor (GMCSF)—from transwell cultures of equivalent sized pieces of freshly thawed units of cryopreserved BCM, as compared to the more highly differentiated form of skin substitute represented by LSE. It is worth noting that the net cytokine/growth factor balance expressed by such co-cultures is not unlike that of normal human acute wound fluid (Prajapati R and Silberklang M, manuscript in preparation).

Figure 1

Shown here is a photomicrograph illustrating cross-section of BCM (OrCel®): K, keratinocytes; F, fibroblasts; G, collagen gel laminate layer; S, collagen sponge.

Figure 2

This graph illustrates in-vitro cytokine expression by BCM (OrCel®) vs. LSE (Apligraf®): Cultures of equivalent-sized pieces of BCM (freshly thawed and rinsed in saline solution) and LSE (freshly removed from its packaging at room temperature) in BCM growth medium72 (Ortec International) were incubated for 48 hours, and media were sampled daily; assay of KGF-I and GMCSF were by commercial ELISA kits (Quantikine, R&D Systems) following manufacturer’s recommendations. Results are expressed as picograms (pg) of cytokine secreted per square centimeter area of tissue per day.

Fenestration or meshing of more dense skin substitutes, such as LSE, enables exudate to escape without disrupting the apposition of the product to the wound bed. More porous products, such as the BCM product, allow exudate permeation without meshing. Nevertheless, meshing can be used, in some cases, to expand the matrix to allow greater wound surface area to be covered. The effect of meshing on bilayered cellular matrix has been reported by Martin and Kirsner,[59] who noted that even as great as 6:1 expansion was associated with acceptable product handling characteristics and apparent patient benefit. Most clinicians have gravitated away from suturing or stapling bioengineered skin products onto the wound bed in favor of fixing the cells in place using steristrips along the wound edge or by a using a bulky secondary compression dressing.

Article continued under same article title, PART 2.