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

Abstract

INTRODUCTION: This study was performed to evaluate the viability, persistence and immunogenicity of a novel self-assembled porcine bilayered cellular construct (SA-PBCC) 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 SLAcc and SLAdd 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 SLAdd SA-PBCC were grafted on split thickness wounds in SLAcc (n=5) and SLAdd (autologous control) animals. Engraftment and/or rejection were evaluated by gross examination and biopsy histology. Mixed lymphocyte reactions, alloantibody flow cytometry, and complement-dependent cytotoxicity assays were used to assess immunogenicity of the grafts.3 Animals received a second grafting of full MHC mismatched SA-PBCC 7 weeks following the first set of grafts together with MHC matched SA-PBCC to assess for clinical sensitization. RESULTS: Autologous SA-PBCC engrafted and persisted similarly to autologous skin grafts. All fully mismatched SA-PBCC successfully engrafted with histologic evidence for neovascularization of the fibroblastic component by day 4 (Figures 1 and 2). By day 6, perivascular inflammatory 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-PBCC (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.Figure 1: MHC mismatched SA-PBCC on POD4.Figure 2: Histology of the MHC mismatched SA-PBCC on POD4 (HE, 20X).CONCLUSION: These data demonstrate that SA-PBCC engraft, undergo vasculogenesis, and mature into structures resembling normal skin. Despite the lack of donor hematopoietic-derived cells, MHC-mismatched SA-PBCC are rejected in a similar time course to allogeneic skin and sensitize across MHC barriers. Since the absence of professional donor antigen presenting cells does not appear to prolong graft survival, sensitization to minor histocompatibility antigen likely occurred through indirect antigen presentation following vascularization and host cell infiltration into the construct. The fact that autologous SA-PBCC permanently engrafts could have important implications for wound treatment and may someday complement the use of tissue expanders in reconstructive surgery.