Skin Substitutes and Wound Healing: Current Status and Challenges

David Eisenbud, MD, CWS; Ngan F. Huang, BS; Sunny Luke, DSc; Melvin Silberklang, PhD


Wounds. 2004;16(1) 

In This Article

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.[32,33,34] Later, it was shown that replacing the connective tissue may increase mechanical strength of healed wounds and reduce ultimate scarring,[35,36,37] 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.

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. TranscyteTM (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 medium[72] (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.


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