A Novel Tissue Engineered Construct for Wound Treatment and Skin Immunity Research

Purpose: This study was performed to evaluate the viability, persistence and immunogenicity of a novel self-assembled bilayered living cellular construct (SA-BLCC) when transplanted across MHC barriers in a preclinical swine model^1,2.

Methods: Fibroblast and keratinocyte cell banks were generated from skin punches of neonatal SLA ^cc and SLA^dd MGH MHC-defined miniature swine.  A dermal matrix was prepared from fibroblasts stimulated to generate extracellular matrix, and then seeded with keratinocytes to form the epidermal layer.   The resulting SLA^dd SA-BLCC were grafted on split thickness wounds in SLA^cc (n=5) and SLA^dd (autologous control) animals. Engraftment and/or rejection were evaluated by gross examination and by histology of biopsies. Mixed lymphocyte reactions, alloantibody flow cytometry, and complement-dependent cytotoxicity assays were used to assess immunogenicity of the grafts³. Animals received a second grafting of full MHC mismatched SA-BLCC 7 weeks following the first set of grafts together with MHC matched SA-BLCC to assess for clinical sensitization.

Results:  Autologous SA-BLCC engrafted and persisted similarly to autologous skin grafts. All fully mismatched SA-BLCC successfully engrafted with histologic evidence for neovascularization of the fibroblastic component by day 4.  By day 6, perivascular infiltrates were present in the fibroblastic layer; some with extension to the epidermal component (grades 1-2 by Banff classification of skin-containing composite tissue allograft pathology). Complete cellular rejection and tissue loss occurred by day 8 for most grafts. Following the second application, both SA-BLCC (full MHC mismatched and MHC matched) underwent accelerated rejection (<4 days). MHC-specific cytotoxic alloantibody could be detected within 10-18 days post first grafting, with increased titer post re-grafting.

Summary and Conclusions: These data demonstrate that SA-BLCC engrafted, with vasculogenesis into the dermal equivalent of the SA-BLCC.  They are then rejected in a similar time course as allogeneic skin and sensitize across MHC barriers, despite the lack of donor hematopoietic-derived cells. Thus, the absence of professional donor antigen presenting cells does not appear to prolong graft survival, implying that sensitization likely occurred through indirect antigen presentation with sensitization to minor histocompatibility antigens following vascularization and host cell infiltration into the construct. The fact that autologous SA-BLCC permanently engrafts could have important implications for wound treatment.

Figure 1. Autologous SA-BLCC, POD12

Figure 2. Autologous SA-BLCC, POD12, HE