Synthetic Graft Materials Single Laminar Grafts

In order to provide a simple method of closure, a number of simple, single-layer, polymer wound dressings have been developed. The Ivalon sponge was developed in 1962, and had the same problems as many of the simple coverage schemes. Bacteria present under the graft wound spread into deep structures when the sponge was used on third-degree burns. Also, the brittle nature of the material led to small pieces breaking off when the graft was removed, leading to foreign body reactions within the ultimately healed wound.

Spray-on materials, such as hydroxyvinylchloride-acetate and sebacic acid copolymer, produce the same problems of suppurative bacterial spread when placed over open third-degree wounds. If the film is nonpermeable to moisture, accumulation of serous exudate and suppurative material will result in uneven adhesion of the film to the wound. Other polymer materials have been tried over the years with similar results.

A spray-on silicone membrane, Hydron (Hydron Laboratories, Inc., New Brunswick, NJ), consists of poly (hydroxyethyl methacrylate) powder and liquid polyethylene glycol sprayed onto the wound surface, resulting in a thin film. The membrane is easily removed from the wound bed by contact with bed clothing, and infection can expand beneath its surface. These dressings would be best for treatment of donor sites from split thickness skin grafts and possibly clean superficial second-degree burns, but their high cost limits their use over simpler dressings.

A liquid gel made from agar and acrylamide copolymer has been introduced, This material is again best used as a temporary cover only because of its lack of adherence to the wound and its fragile nature. Some advantages of the gel are that it allows fluid to freely drain from the wound without accumulating and because the material is highly translucent, it allows easy inspection of the wound. As with Hydron, this should be used in clean wounds expected to heal rapidly, like donor sites and superficial second-degree burns.

Collagen (as discussed earlier) is an excellent material owing to many of its unique properties. A fairly pure form can be extracted in large amounts from many commercial sources. It is inherently low in antigenicity and exerts a hemostatic effect on the fragile and vascular wounds. Collagen can be formulated in many different ways from gels to films, depending on the properties desired and methods for application. Cross-linking the collagen fibers increases the tensile strength, but unfortunately it also tends to make the fibers stiff and brittle. The permeability of collagen materials may be controlled by their thickness, but mechanical properties must be considered if the material becomes too thick. Another property of collagen is that it naturally adheres to the wound initially because of its binding with fibrin. The particular form of collagen is important for success because various forms of collagen have been tried unsuccessfully over the years. Sponges of collagen dry slowly and behave as a serous crust or scab which prevents effective ingrowth of fibroblastic tissue from the bound bed; collagenases present both naturally in the wound bed and from wound bacteria result in the eventual shedding of these collagen sponges.

MiittUaminar Grafts

In order ro both control the ingrowth of fibroblasts and long-term adhesion to the wound, a large pore size must be maintained. On the other hand, the requirements of water permeability necessary to prevent the wound from drying out require a smaller pore diameter. The best approach to date has been to create bilaminar membranes. This approach makes designing the wound dressings simpler because differences in physical strength, adhesion, pliability, and other properties can be separated between the two layers of the device.

Biobrane is a synthetic bilaminate material developed by Woodroof Laboratories, Santa Ana, CA. This material consists of a finely knit nylon and hydrophilic type I porcine collagen which is covalently bound to the hydrophobic inert nylon. The outer layer of Biobrane is a silicone rubber which, through its porosity, controls the water permeability of the graft. The pore size of the outer layer is maintained small enough to provide a barrier for bacteria while being large enough to be permeable to topically applied antibiotics. If nylon mesh is placed against a clean wound (low bacterial count), then the Biobrane may-adhere to the wound surface, and through its loose pore structure, permit ingrowth of cells from the wound bed below.

Biobrane is flexible enough to deform, maintaining adequate contact with the contours of the wound while still having the mechanical stability necessary for ease of handling and durability as a dressing. Biobrane is not biodegradable and serves at best only as a temporary closure material, lasting up to a month. Biobrane must eventually be removed either manually or by epithelial growth from below. Care must be taken not to prevent reepithelialization when Biobrane is used as a wound cover, as when the graft is placed over a finely meshed autograft. If Biobrane is left on for more than 6 days, it prevents epithelialization in the interstices of the graft (Lin et al., 1982). The problems of wounds with significant bacterial contamination have become apparent and development of Biobrane with the incorporation of antibiotics is under way. As with any inert skin substitute, Biobrane must eventually be removed and the wound then covered with autograft for those wounds without epidermal elements. Biobrane has been successfully used only in clean superficial second-degree burns and donor sites.

Levine etal. have developed a completely inert wound cover consisting of six layers of a nylon stocking fabric, covered with a 1-mm poly(tetrafluoroethylene) membrane with 0,1-ix.m pores. This dressing allowed fibroblasts to grow into the mesh while providing a semipermeable moisture and bacterial barrier. As there is no inherent inhibition of bacteria beneath the graft, the material was first soaked in an antimicrobial solution. This material is used more as a temporary wound dressing than as a skin replacement because these dressings must be changed every 48 to 72 hr to provide local debridement. Eventually they are replaced with a more definitive wound closure when it becomes available.

Yannas and Burke have developed a bilaminar replacement membrane consisting of an inner layer of collagen—chondroitin 6-sulfate, and an outer layer of silastic (Figs. 7 and 8). The collagen binds to the wound bed and is invaded by neovascula-ture and fibroblasts. The collagen chondroitin 6-sulfate very significantly inhibits or prevents wound contracture, and with time the collagen is rresorbed by collagenase and is completely replaced by remodeled dermis. Pore size of the collagen—GAG was found to be optimized at 50 /wm, which supported active ingrowth of tissues from below. Smaller pores excluded fibroblasts and mesenchymal tissue, promoting a fibrouslike formation around the openings of the pores. The silastic layer is 0.1 mm thick and provides the bacterial barrier needed while maintaining the proper water flux through the membrane. In fact, the flux of 1 to 10 mg/cm2/hr is similar to that of normal epidermal tissue. The silastic layer also provides the mechanical

Epithelial cells

Biodegradable protein layer

FIG. 7. Schematic w

Fibroblasts, Endothelial Cells diagram of s hi laminar artificial skin-

rigidity needed to suture the graft in place, preventing movement of the material during wound healing.

The GAG contenr of the collagen controls such things as the porosity and elasticity of the material, as explained in more detail in the section on materials. The collagen-chondroitm 6-sulfate adheres to the wound within minutes (in contrast to collagen sponge), and neovascularization can be observed within 3—5 days. The strong attachment of the collagen-GAG to the wound is evident by measuring the peel strength of the graft, which is 9 N/m at 24 hr and increases to 45 N/m by 10 days. Successful adherence of the artificial skm to the wound rresults in biological wound closure and its beneficial effects. Slow resorption of the collagen from native collagenase results in remodeling of the dermis. The three-dimensional structure furnished by the collagen—GAG material provides a scaffolding or suptastrucrure which results in controlled ordered dermal formation without the resulting scarring seen with simpler materials. When autografted epidermis (0,1 mm thick) becomes available from regenerating donor sites, the silastic layer can be removed from 3—4 weeks and the epidermal layer then can be applied directly to the developing neodermis with 95 to

FIG. 8. Diagram of Burke and Tannas artificial ikin (from Hombach et at., 1988).

100% successful grafting rates. If the graft wrinkles or fails to conform adequately for adherence to the wound, it must be trimmed to prevenr seromas or hematomas from forming beneath it.

Histological cross sections of wounds closed with this artificial skin show complete replacement of bovine collagen at 7 weeks with remodeled human dermis. No evidence of hypertrophic scar formation has been noted, and the grafted areas are more supple than those areas simply covered with meshed autograft.

Yannas and Burke have extended their development by seeding dermal portions of a graft with autologous basal skin cells (stage II membranes). A small sample of cells is removed from the patient and the top layer of the epidermis is discarded. The basilar cells are then dissociated from one another with trypsin, and suspended in media. The basilar cells are introduced into the matrix, either by direct injection or by centrifugal force (this entire procedure can be accomplished in the guinea pig mode) in under 4 hr). The keratinocytes rapidly proliferate and form sheets of keratinized cells. Functional skin replacement has been achieved in guinea pigs in under 4 weeks (Yannas et al., 1982a); preliminary studies in humans have been encouraging.

Because of the unique ability of this artificial skin to participate in the natural healing process and to allow remodeling of the dermis, infection rates are low and in fact are similar to those seen with autografted tissues. Cosmetic results are dramatic and result in a smooth, supple, homogeneous surface similar to that of normal skin. This artificial skin is the only long-term skin replacement with a large human experience. More than 100 patients have been treated with this artificial skin at Massachusetts General Hospital and the Boston Unit of the Shriner's Burns Instinite, in addition. 106 patients have had this treatment in a U.S. Food and Drug Administration Phase II trial. Recent use of the artificial skin in 43 adults admitted to Massachusetts General Hospital showed a significant increase in survival, particularly in those patients over 40 years of age (64% survival of those receiving artificial skin, versus 22% of those receiving standard treatments only). This is particularly significant because those receiving the artificial skin generally had a larger percent of body surface area involved m their burn than those not treated (Tompkins et al., 1989). This material received FDA approval recently.

The use of artificial skin allows for ptompt debridement and closure of the largest of bum wounds, limiting wound contracture and providing permanent coverage with a readily available, easily stored skin replacement material. When seeded stage II material begins its routine clinical use, the advantages of cultured epidermis without the limiting delay of several weeks and the use of exogenous multiple mutagenic components will also be realized. A bilaminant artificial skin has also been developed by Boyce and Hansbrough (1987).

Culture Grafts

Grafts composed of epidermal cells grown from small samples taken from a patient have the theoretical advantage of significantly amplifying the donor sites available for epidermal cells. Studies have been conducted in which cells were grown on collagen film by seeding the film with basal cells. The sheets

DispOsablaSilaslic (Epidermis)

FIG. 8. Diagram of Burke and Tannas artificial ikin (from Hombach et at., 1988).

Artificial Oarmis Collagen Fiber (chlndrollln-6-Sulla(o)

DispOsablaSilaslic (Epidermis)

Artificial Oarmis Collagen Fiber (chlndrollln-6-Sulla(o)

were grown by placing the film in the subcutaneous tissue or subcapsular region of the kidney. When a confluent layer of epithelial cells appeared, the sheets were applied to the wounds and an occlusive dressing was placed to immobilize the sheets. In those grafts that took, the epithelialization seemed complete after 1 week. Contraction was noted in the wounds and whether the cells were replaced by the animal's own cells was not established. Extensive experience in humans with long-term experience has not been reported with these methods.

Results in short-term human studies show histological sections with a mildly hyperkeratotic epidermal layer with no glandular structures. A mild chronic inflammatory response was noted in the underlying connective tissue, similar to that seen with scar formation. No stratum corneum or rete pegs were present, making the epidermal cells susceptible to trauma. Moist petroleum drressings were kept on the wounds for 4 weeks to prevent graft desiccation. A limitation to this technique is that the period required for cellular growth is 2-3 weeks; thus immediate closure of the burn wound must be provided by other means.

Green has extended the ability to generate cultured epidermal keratinocytes by coculturing them with lethally irradiated 3T3 cells (an immortal transformed mouse cell line) in the presence of epithelial growth factor (EGF), and cholera toxin—a nonlethal agent that increases intracellular cyclic AMP (cAMP) (Green et al., 1979). Using these techniques, the keratinocytes plated at a density of 5000 cells/cm2, displaced the 3T3 cells, and grew to confluence in approximately 14 days. This technique improved yield and provided the ability to quickly culture cells, producing a monolayer of epidermal cells which could then be grafted over a healing wound site. When a modification of this technique (without the 3T3 coculture) was used on human patients, significant amplification of the patients' unburned epidermal areas could be achieved. Of course these grafted sites show no evidence of any stratum corneum because only the epidermal cell layer is being replaced, thus making them susceptible to desiccation.

Bell has developed a "living skin equivalent" consisting of a collagen fibrilar lattice seeded with autologous fibroblasts; epidermal cells can be cultured on its surface (Bell et al., 1981). This technology is being tested by Organogenesis, Inc. (Cambridge, MA). In rodent studies, rapidly neovascularization of the graft allowed the fibroblasts to proliferate. It was suggested that the graft prevented contracture of the wound edges by as much as 75 to 80%. In animal studies using small grafted areas, the dermal layer is remodeled after the graft is placed and over the next 10 weeks, the collagen layers thin to half the thickness of the surrounding dermis. Epithelial growth can be seen at the edges of the wound. There are no published human studies available to evaluate this approach.

More recently, another approach for an artificial dermal matrix has been taken in which human fibroblasts are grown on surgical mesh materials. The artificial dermal matrix is expected to provide a dermis for the interstices of meshed skin. In athymic mice studies (Hansbrough et al., 1992a), polygly-colic acid (PGA) and polyglactin-910 (PGL) mesh containing confluent, cultured human fibroblasts were applied to full-

thickness wounds. Expanded mesh, human, split-thickness skin grafts were placed over the artificial dermal matrix graft. During a 99-day period after graft placement, the PGA/PGL-fibro-blast grafts were incorporated into the wound and epithelialization from the skin bridges proceeded rapidly across the surface of the PGA/PGL-fibroblast grafts. Basement membrane formation at the dermal-epidermal junction of the epithelialized interstices was seen and minimal inflammatory reaction to the PGA/PGL-fibroblast grafts was noted.

In a controlled clinical study with this approach, Hansbrough et al. (1992b) evaluated Dermagraft (Advanced Tissue Sciences, La Jolla, CA), which is composed of human neonatal fibroblasts grown on a PGA Vicryl mesh. The study tested the ability of Dermagraft to function as a dermal replacement material when placed beneath meshed, expanded, split-thickness skin grafts. Full-thickness burn wounds in 17 patients with burns (mean age, 31 years; range: 6—69 years; mean burn size, 23.8% total body surface area) were excised to subcutaneous fat (nine patients), to fascia (three patients), or to a combination of deep dermis and fat (five patients). The results showed that "take" of skin grafts on control sites was slightly better than take on the Dermagraft; however, the differences were not statistically significant. Mesh interstices epithelialized over the surface of the full-thickness wound in a fashion comparable to the surface of Dermagraft. No evidence of rejection of the allogeneic fibroblasts and minimal inflammatory reaction to the Vicryl fibers were seen. The Vicryl was hydro-lyzed within the wound over 2—4 weeks although expulsion of the fibers was noted as the healing epithelium advanced to close the interstices. Further clinical trials with Dermagraft are in progress. Comparisons of currently available skin replacements are shown in Table 1.

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