An allogenic 3D scaffold-free tissue engineered product for deep thickness skin regeneration: in vitro development to in vivo proof of concept

Abstract

Background & Aim Deep thickness skin wound remains a major challenge for reconstructive surgery. A novel approach of tissue engineering, based on an allogeneic adipose-derived 3D scaffold-free technology, was proposed. Methods, Results & Conclusion Material and Methods Adipose-derived stromal cells (ASCs) were isolated from human adipose tissue to constitute the 3D-scaffold free graft by the production of the extracellular matrix (ECM, n=9). The ultrastructure of the graft was assessed by microtomography/SEM. The protein and growth factors contents were determined by proteomic analysis (LC-MS/MS) and ELISA, respectively. The in vivo biocompatibility (inflammatory reaction, biodegradation) was assessed in nude and Wistar rats up to 4 weeks (n=20) as well as the safety in terms of tumorigenicity/toxicity/biodistribution. The efficacy was then evaluated in a xenogenic (human to rat) model of ischemic (vs. non-ischemic) wound in hyperglycemic Wistar rats (n=42, 3D grafts vs. sham/Ctrl+). Results The 3D-graft is a translucid and malleable membrane with a mean of 175±86 cells/mm2 found to be embed in the ECM with a low level of mineralization (0.30±0.31%v/v). The proteomic and genes analysis revealed the stimulation of the biological pathways involved in early wound healing and the over-expression of pro-angiogenic genes (ANG, ANGPT1, EPHB4, VEGFA, VEGFB, VEGFC, EDN1, THBS1, PTGS1, LEP) in the graft (in comparison to ASCs alone), respectively. The VEGF and SDF1a contents (181±12 and 663±27 ng/g, respectively) were also improved in the scaffold-free implant. The biocompatibility and the safety of the 3D-graft were confirmed at 4 and up to 24 weeks post-implantation, respectively. The 3D-graft was easily handled and applied by a simple bandage on the ischemic/hyperglycemic wounds (on the leg) and promoted an earlier irreversible wound closure (27 vs. 34 days for sham, respectively) associated with angiogenesis, dermis/epidermis reconstruction, transient and reversible increase of aSMA, lymphocytes/macrophages recruitment at 10-15 days. Conclusion The scaffold-free approach with allogenic 3D-graft (derived from ASCs) demonstrated the safety and efficacy in stringent xenogenic model of hyperglycemic and ischemic deep-thickness wound.