Development of a dermal matrix from glycerol preserved allogeneic skin.

Dermal substitutes can be used to improve the wound healing of deep burns when placed underneath expanded, thin autologous skin grafts. Such dermal matrix material can be derived from xenogeneic or human tissue. Antigenic structures, such as cells and hairs must be removed to avoid adverse inflammatory response after implantation. In this study, a cost-effective method using low concentrations of NaOH for the de-cellularization of human donor skin preserved in 85% glycerol is described. The donor skin was incubated into NaOH for different time periods; 2, 4, 6 or 8 weeks. These dermal matrix prototypes were analyzed using standard histology techniques. Functional tests were performed in a rat subcutaneous implant model and in a porcine transplantation model; the prototypes were placed in full thickness excision wounds covered with autologous skin grafts.An incubation period of 6 weeks was most optimal, longer periods caused damage to the collagen fibers. Elastin fibers were well preserved. All prototypes showed intact biocompatibility in the rat model by the presence of ingrowing blood vessels and fibroblasts at 4 weeks after implantation. An inflammatory response was observed in the prototypes that were treated for only 2 or 4 weeks with NaOH. The prototypes treated with 6 or 8 weeks NaOH were capable to reduce wound contraction in the porcine model. In neo-dermis of these wounds, elastin fibers derived from the prototype could be observed at 8 weeks after operation, surrounded by more random orientated collagen fibers. Thus, using this effective low cost method, a dermal matrix can be obtained from human donor skin. Further clinical studies will be performed to test this material for dermal substitution in deep (burn) wounds.

Immunology of skin transplantation.

Untreated viable allogeneic skin is highly immunogenic. Epidermal Langerhans migrate after transplantation out of the donor skin into the lymph node of the recipient where they can activate T cells capable to mediate rejection. Allogeneic skin is used as a temporary coverage of burn wounds, often in combination with autologous skin grafts. Several methods to pretreat the allogeneic skin have been used to delay the rejection process. Processing of allogeneic skin in 85% glycerol results in a non-viable skin with a well-preserved structure. Experiments in a full thickness porcine wound model showed that rejection of glycerol treated allogeneic skin grafts was up to six days delayed. Viable, untreated allogeneic skin grafts were rejected predominantly by CD8 positive T cells whereas in the glycerol treated grafts the influx of host cells was lower and the majority of the cells were macrophages. The outgrowth of the autologous skin grafts underneath glycerol treated allogeneic skin was three days earlier completed when compared to grafts in combination with untreated allogeneic skin. Thus, by processing the allogeneic skin into 85% glycerol, the direct route to induce graft rejection is blocked since the Langerhans cells are non-viable. The glycerol-preserved skin grafts are finally rejected via an indirect route mediated by macrophages; this process is less disturbing for the outgrowth of autologous cells.

Rat monocyte-derived dendritic cells function and migrate in the same way as isolated tissue dendritic cells.

Dendritic cells (DC) are the most potent antigen-presenting cells and are therefore useful to induce immune responses against tumor cells in patients. DC can be generated in vitro from monocytes using GM-CSF and IL-4, the so-called monocyte-derived DC (MoDC). To achieve antitumor responses, MoDC must be able to migrate to the draining lymph nodes after injection to induce cytotoxic T cells. Therefore, we studied migration of MoDC in a rat model. Functional rat MoDC were generated from PVG-RT7B rats and injected subcutaneously into PVG rats. These rat strains differ only at one epitope of the leukocyte-common antigen, which can be recognized by the antibody His 41. The advantage is that migrated cells can be detected in the draining lymph nodes by staining sections with His 41+; thus, migration is not influenced by labeling procedures. Rat MoDC migrated to the T-cell areas of the draining lymph nodes, just as isolated Langerhans cells or spleen DC do. In contrast, monocytes also migrated to the B-cell areas and the medulla.