Allogenic 3D scaffold-free tissue engineered product for deep thickness skin regeneration: in vitro characterization and in vivo biocompatibility

December 18, 2019

Valérie Lebrun1, Sophie Vériter1, Gaëtan Thirion1, Modarressi Ali2, Pierre-Yves Adnet1, Céline Caty1, Denis Dufrane1
1 Novadip Biosciences, Mont-Saint-Guibert, Belgium
2 Plastic, Reconstructive&Aesthetic Surgery department, University hospitals of Geneva, Switzerland

Introduction: Deep thickness skin wound remains a major challenge for reconstructive surgery. A biological substitute, based on an allogeneic adipose-derived 3D scaffold-free technology, was developed to restore the healing physiology.

Material and Methods: Adipose-derived stromal cells (ASCs) were isolated and processed to produce extracellular matrix (ECM). The ultrastructure and the mineralization of the graft were assessed by histology/microtomography/scanning electron microscopy. The protein/growth factors contents were determined by proteomic analysis (LC-MS/MS) and ELISA, respectively. The angiogenic ASCs properties were assessed by RNA sequencing (n=3),q-RT-PCR of a panel of angiogenic genes (n=5) and growth factors (VEGF,SDF1a) secretion (n=3) in view to compare ASCs in a single layer versus embedded in the extracellular matrix. The impact of the high glucose (4.5g/L) and low oxygen tension (1 % O2) on the ASCs bioactivity was evaluated in vitro by the quantification of growth factors content/secretion (n=3). The biocompatibility (immune response,biodegradation) was then assessed in vivo in nude/wistar rats up to 4 weeks (n=20).

Results: The 3D-graft is a translucid/malleable membrane easily handled with forceps to be place on the wound bed. Histomorphometric and SEM analysis showed a mean of 175±86 cells/mm² embedded 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 3D-graft versus ASCs alone. The VEGF and SDF1a contents (181±12 and 663±27 ng/g, respectively) were also improved by the embedding of ASCs in the ECM. The bioactivity of the 3D-graft was not affected by the in vitro conditions of high glucose and low oxygen tension. The biocompatibility of the 3D-graft was confirmed in vivo with a balance between the elicitation of the inflammatory reaction and the graft integration without evidence of negative impact by the xenorejection.

Conclusion: The allogenic scaffold-free 3D-graft (i) improves the ASCs bioactivity for the angiogenesis and (ii) the in vivo remodeling by the specific ECM-proteins of wound healing.