Frozen bean curd-inspired xenogeneic acellular dermal matrix with triple pretreatment approach of freeze–thaw, laser drilling and ADSCs pre-culture for promoting early vascularization and integration

Xenogeneic acellular dermal matrix (ADM) is widely used in clinical practice given its good biocompatibility and biomechanical properties. Yet, its dense structure remains a hindrance. Incorporation of laser drilling and pre-culture with Adipose-derived stem cells (ADSCs) have been attempted to promote early vascularization and integration, but the results were not ideal. Inspired by the manufacturing procedure of frozen bean curd, we proposed a freeze–thaw treatment to enhance the porosity of ADM. We found that the ADM treated with −80°C 3R+−30°C 3R had the largest disorder of stratified plane arrangement (deviation angle 28.6%) and the largest porosity (96%), making it an optimal approach. Human umbilical vein endothelial cells on freeze–thaw treated ADM demonstrated increased expression in Tie-2 and CD105 genes, proliferation, and tube formation in vitro compared with those on ADM. Combining freeze–thaw with laser drilling and pre-culture with ADSCs, such tri-treatment improved the gene expression of pro-angiogenic factors including IGF-1, EGF and vascular endothelial growth factor, promoted tube formation, increased cell infiltration and accelerated vascularization soon after implantation. Overall, freeze–thaw is an effective method for optimizing the internal structure of ADM, and tri-treatments may yield clinical significance by promoting early cell infiltration, vascularization and integration with surrounding tissues.