Bi-layered living cellular construct resulted in greater healing in an alloxan-induced diabetic porcine model.

Tissue-engineered skin constructs, including bi-layered living cellular constructs (BLCC) used in the treatment of chronic wounds, are structurally/functionally complex. While some work has been performed to understand their mechanisms, the totality of how BLCC may function in wound healing remains unknown. To this end, we have developed a delayed wound healing model to test BLCC cellular and molecular mechanisms of action. Diabetes was chemically-induced using alloxan in Yucatan miniature pigs, and full-thickness wounds were generated on their dorsum. These wounds were either allowed to heal by secondary intention alone (control) or treated with a single or multiple treatments of a porcine autologous BLCC. Results indicated a single treatment with porcine BLCC resulted in statistically significant wound healing at day 17, while four treatments resulted in statistically significant healing on days 10, 13, and 17 compared to control. Statistically accelerated wound closure was driven by re-epithelialisation rather than contraction or granulation. This porcine diabetic model and the use of a porcine BLCC allowed evaluation of healing responses in vivo without the complications typically seen with either xenogenic responses of human/animal systems or the use of immune compromised animals, expanding the knowledge base around how BLCC may impact chronic wounds.

Survival of Allogeneic Self-Assembled Cultured Skin.

BACKGROUND: Deficiency of autologous skin for reconstruction of severe wounds is a major problem in plastic surgery. Autologous substitutes can provide additional coverage, but due to the duration of production, treatment is significantly delayed. The allogeneic approach offers a potential of having an off-the-shelf solution for the immediate application. METHODS: In this study, we assess the engraftment and immunogenicity of allogeneic bilayered bioengineered skin prepared by a self-assembly method. Bioengineered skin has the potential immunological advantage of lacking passenger leukocytes including antigen-presenting cells. The skin constructs were transplanted across major histocompatibility complex (MHC) barriers in a porcine animal model. Animals received a second grafting of the same skin construct 7 weeks after the first set of grafts together with MHC-matched constructs to assess for clinical sensitization. RESULTS: All alloconstructs successfully engrafted with histologic evidence of neovascularization by day 4. Complete cellular rejection and tissue loss occurred by day 8 for most grafts. After the second application, accelerated rejection (<4 days) took place with the development of swine MHC-specific cytotoxic alloantibody. CONCLUSIONS: These data demonstrate preclinically that self-assembled allogeneic constructs engraft and reject similar to allogeneic skin despite the absence of professional donor antigen-presenting cells.

Bioengineered Self-assembled Skin as an Alternative to Skin Grafts.

For patients with extensive burns or donor site scarring, the limited availability of autologous and the inevitable rejection of allogeneic skin drive the need for new alternatives. Existing engineered biologic and synthetic skin analogs serve as temporary coverage until sufficient autologous skin is available. Here we report successful engraftment of a self-assembled bilayered skin construct derived from autologous skin punch biopsies in a porcine model. Dermal fibroblasts were stimulated to produce an extracellular matrix and were then seeded with epidermal progenitor cells to generate an epidermis. Autologous constructs were grafted onto partial- and full-thickness wounds. By gross examination and histology, skin construct vascularization and healing were comparable to autologous skin grafts and were superior to an autologous bilayered living cellular construct fabricated with fibroblasts cast in bovine collagen. This is the first demonstration of spontaneous vascularization and permanent engraftment of a self-assembled bilayered bioengineered skin that could supplement existing methods of reconstruction.