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℃3Rt-30℃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 proangiogenic 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.