C. D. Richters

Skin replacement in burn wounds.

Historically, the main goal in burn management was increasing the survival of severely burned patients by rapid debridement and early closure of burn wounds, consequently reducing the infection risk.1–4 However, in the last decennia, surgical emphasis has shifted from survival to “quality of survival,” especially by improving the residual scars and preventing contractures. Traditionally, surgeons divide burns into deep burns requiring surgical therapy, and superficial burns which heal spontaneous by re-epithelialization with minimal scarring. Nevertheless, there is a gray zone between those two groups in which therapeutic decision making is difficult. The final decision for surgery generally remains case and surgeon dependent, and will mainly depend on the total burned surface area.5 Wound closure can be obtained by diverse therapeutic modalities depending on the depth and healing potential of the burn wound.5 In this article, the main focus is on the surgical treatment of deep dermal and full thickness burns. We endeavor to give a comprehensive overview of the developments in skin substitutes, which is impossible without mentioning some alternative treatments. The current golden standard for deep burns is surgical debridement and closure with autologous split thickness skin grafts or “STG” (epidermis plus a thin layer of dermis). Nevertheless, donor areas are limited in extended burns, and the residual scars remain unsatisfactory due to the lack of dermis. A more aesthetical reconstruction can be obtained with full thickness skin grafts (epidermis and whole dermis), which are limited in dimension and can only be harvested in a few areas (groin, lower abdomen, etc.). Deep defects with exposed bone or neurovascular structures are currently treated with flap surgery, which gives an optimal aesthetical and functional result. Nevertheless, the severe donor-site morbidity, the technical difficulty, and sometimes severe complications limit its use mostly to secondary reconstructions. Consequently, alternative conservative and surgical treatments were developed to improve the healing and the quality of the residual scars.6 Several mechanisms are supposed to enhance healing: (i) providing the ideal wound environment (wound dressings, etc.), (ii) by assisting the intrinsic healing capacities (growth factors, cytokines, etc.), or (iii) by surgically replacing the damaged skin (“skin substitutes”), which also should reduce scarring in full thickness defects. A permanent skin substitute is a surgically fixated “long lasting” skin replacement, consisting of naturally occurring skin elements which become incorporated in the normal skin. The main issue of this definition is the longevity of a skin substitute, which seems to be mostly of commercial importance, where terms such as biological dressings, and permanent and temporary skin substitutes are used without a clear distinction. This literature review showed that technically similar products are commercialized as “permanent” by one company and as biological dressing by another. Therefore, we chose to divide all these products in the following categories, depending on the skin layer which is (temporary) replaced: epidermal, dermal and combined skin substitutes (or composite grafts; Fig. 1). In the future, a fourth group might need to be added: the combined skin substitute with a subcutaneous adipose layer.7 However, the difference between skin replacements and some wound dressings can be small. Wound dressings are intended for coverage instead of replacement, to optimize wound healing. Wound dressings can roughly be divided in dressings containing natural elements (such as honey ointments), synthetic dressings (such as silver-impregnated dressings), and biological dressings containing mammalian cells or cell-derived substances like collagens and growth factors (human donor skin). Synthetically manufactured, naturally occurring elements, such as cellulose membranes, are also synthetic dressings. Wound dressings are not considered as (permanent) skin substitutes because they are not incorporated in the healing wound. Some authors previously named some of these products “skin substitutes” (without mentioning “permanent”) but this only lead to confusing terminology. The most important biological dressing, used since the 1940s, is human donor skin or “cadaver skin.”8 It contains several beneficial factors (growth factors, cytokines, etc.), and it provides the ideal environment for healing. Because of better preservation techniques (glycerol or cryopreserved), the risk of infection transmission is minimized, and its rejection will be delayed up to 3 weeks to 5 weeks.8,9 One of the Submitted for publication May 18, 2009. Accepted for publication November 6, 2009. Copyright

Morphology of glycerol-preserved human cadaver skin.

Donor allograft skin preserved in 85 per cent glycerol has been used successfully as a temporary coverage for large burn wounds. The glycerol preservation is a method with low costs and has practical advantages such as antibacterial and virucidal effects. This report shows that the glycerol treatment did not affect the fundamental structural integrity of the skin. Intact keratinocytes and Langerhans cells with their characteristic Birbeck granules were still present in the glycerol-treated skin. After treatment with glycerol, the cells in the prepared epidermal cell suspensions were non-viable. MHC class II positive and CD1a positive cells could still be identified in situ and in the suspension.

Isolation and characterization of migratory human skin dendritic cells

A method is described to isolate and characterize human skin dendritic cells (DC). This method is based on the migratory capacities of these cells. The cells migrated “spontaneously” out of split‐skin explants into the medium during a 24‐h culture period and contained up to 75% CD1a+ cells. After removal of co‐migrated T cells and macrophages, the highly enriched (>95% CD1a*) DC showed potent allo‐antigen‐presenting capacities. About 25% of the CD1a+ cells were also positive for the dermal DC marker CD1b, whereas only 15‐20% of the cells contained Birbeck granules, the characteristic cell organelle of the epidermal Langerhans cell. Before culture. CD1a* DC were observed on cryostat sections not only in the epidermis but also in the dermis. After culture, the number of CD1a+ cells in both epidermis and dermis had decreased. Not all the cells had migrated during the culture period; some CD1a+ cells could still be detected in the epidermis and dermis after culture. Thus, using this method, potent allo‐stimulating CD1a+ cells, migrating from both epidermis and dermis can be obtained without the use of enzymes.

Immunogenicity of Glycerol-Preserved Human Cadaver Skin In Vitro

Donor allograft skin preserved in 85% glycerol is used as a temporary coverage for large burn wounds. Glycerol treatment does not affect the structural integrity of the skin; cells are well preserved but dead. However, cells expressing major histocompatibility class II molecules can still be observed. In this study we investigated the mechanism underlying the clinical observation that glycerol-treated alloskin will be destroyed but after a prolonged period. We compared the in vitro immunogenicity of untreated and 85% glycerol-treated human skin cells. Human purified blood T cells did not proliferate when cultured with allogeneic treated skin cells, whereas untreated cells induced a distinct response. A moderate response was measured after adding T cells and viable antigen presenting cells, such as monocytes, to the allogeneic treated skin cells. However, the response on untreated skin cells was much higher. These results favor the suggestion that after transplantation of glycerol preserved skin is performed, an inflammatory process mediated by infiltrating host monocytes occurs rather than a rejection process mediated by T cells.

Effect of low dose UVB irradiation on the migratory properties and functional capacities of human skin dendritic cells

We recently described the ‘spontaneous’ migration of skin dendritic cells out of human split skin during culture. Since newly infiltrating cells from the circulation are excluded, this in vitro model is very suitable for studying the effect of UVB irradiation on the migratory properties, phenotype and functional capacities of skin cells. In the present study, we show that UVB irradiation of the skin before the culture period results in a significantly lower number of migrated cells that could be obtained compared with untreated skin. Relatively more dendritic cells of the population that migrated from UVB‐irradiated skin were of dermal origin, as indicated by a higher percentage of CD1b+ cells. These data imply that UVB irradiation inhibits migration, especially of the epidermal Langerhans cells. Ultrastructural analysis of the irradiated skin revealed that the UVB dose used did not cause any directly visible damage to the cells. However, the cell population that had migrated from UVB‐irradiated skin showed a significantly lower capacity to stimulate allogeneic T cells. This was not due to a lower expression of MHC class II on these cells. The percentage of cells expressing B7.1, B7.2 and LFA‐3 was decreased in the population migrated from irradiated skin. The possible mechanism underlying the UVB‐induced suppression is discussed.

Migration of rat skin dendritic cells.

This study examines the in vivo migration of rat skin dendritic cells (including Langerhans cells) after skin transplantation. As donor animals, PVG‐RT7b rats were used. The leukocytes of these rats bear an epitope of the leukocyte common antigen that can be recognized by use of the antibody His 41. The cells of allogeneic (ACI) recipient strains do not label with this antibody. Four days after transplantation of PVG‐RT7b skin on allogeneic recipients, His 41+ cells showing a dendritic morphology were present in the T cell area of the draining lymph nodes. During culture of rat skin explants, dendritic cells migrated spontaneously into the medium. These in vitro migrated cells showed a high capacity to stimulate allogeneic T cells. When these cells, obtained from PVG‐RT7b skin, were injected into the hind footpads of allogeneic recipients, they migrated to the same compartments of the draining lymph node. These data indicate that the cells that migrate from a transplanted allogeneic skin graft are the same cells that migrate in vitro from explants. Most probably, they initiate graft rejection in the draining lymph nodes of the recipient. J. Leukoc. Biol. 60: 317–322; 1996.

Glyaderm® dermal substitute: Clinical application and long-term results in 55 patients

INTRODUCTION Glycerol preserved acellular dermis (Glyaderm(®)) consists of collagen and elastin fibers and is the first non-profit dermal substitute derived from glycerol-preserved, human allogeneic skin. It is indicated for bi-layered skin reconstruction of full thickness wounds. METHODS A protocol for clinical application and optimal interval before autografting with split thickness skin graft (STSG) was developed in a pilot study. A phase III randomized, controlled, paired, intra-individual study compared full thickness defects engrafted with Glyaderm(®) and STSG versus STSG alone. Outcome measures included percentage of Glyaderm(®) take, STSG take, and scar quality assessment. RESULTS Pilot study (27 patients): Mean take rates equaled 91.55% for Glyaderm(®) and 96.67% for STSG. The optimal autografting interval was 6 days (±1 day). Randomized trial (28 patients): Mean Glyaderm(®) take rate was 88.17%. STSG take rates were comparable for both research groups (p=0.588). One year after wound closure, Glyaderm(®)+STSG was significantly more elastic (p=0.003) than STSG alone. Blinded observers scored Glyaderm(®) treated wounds better in terms of scar quality. DISCUSSION The efficacy of Glyaderm(®) as a suitable dermal substitute for full thickness wounds is attested. Currently a procedure for simultaneous application of Glyaderm(®) and STSG is adopted, allowing for further widespread use of Glyaderm(®).

Administration of prednisolone phosphate–liposomes reduces wound contraction in a rat partial-thickness wound model

Macrophages play an important role in the inflammatory phase of wound healing and their activity regulates fibroblasts and keratinocytes. Modulation of macrophage function may result in improvement of the wound healing process. Prednisolone phosphate (PLP) encapsulated into liposomes was administered to partial‐thickness wounds in rats. A single dose of 75 μg/kg, applied directly after wounding, resulted in up to a 30% reduction of wound contraction at 28 days after wounding. This effect could not be achieved in the group that was administered free PLP or liposomes containing phosphate‐buffered saline to the wound. The number of myofibroblasts was up to 50% lower in wounds treated with the liposomal PLP at 4 days after wounding. The number of macrophages present in the wounds was not statistically different between groups. Most probably, the production of cytokines and growth factors by macrophages is altered after phagocytosing the liposomes, resulting in reduced wound contraction.

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts.

Evaluation of Acellular Dermis for Closure of Abdominal Wall Defects in a Rat Model

Background: Abdominal wall repair can be performed with synthetic or biological materials. Biological materials may reduce the risk of infections and fibrosis. The aim of this study was to evaluate two acellular human dermis products. Materials and Methods: A rat model was used to compare the two materials. One was prepared using low concentrations of NaOH; the other material was SureDerm™, which is commercially available. Full thickness defects were prepared in the abdominal wall and closed with the materials. Rats were sacrificed at 1 or 4 months after operation and the numbers of adhesions to the bowels were scored. Samples were tak- en for histological analysis and to measure the breaking strength. Results: In both groups a good functional integration of the implants with the abdominal wall was observed. There was no adhesion formation with the bowels in the group with the NaOH prototype. In the SureDerm group, 4 out of 7 rats showed only small adhesions at 4 months after operation. Breaking strength of the healed tissue was significantly higher in the NaOH prototype group at 4 months after operation (p < 0.0026). Conclusions: The results indicate that both human acellular dermis products may be used in clinical trials for closure of abdominal wall defects.