The Experimental And Mechanism Study Of In Vivo Construction Of Dermis-fat Composte:Optimizing Heterogeneous Acellular Dermal Matrix With Laser Micropore Technique And RADSCs Pre-culture In Vitro

Objectives:Skin flap transplantation is an important clinical repairment and reconstruction technique,while it can impair the donor site.Tissue Engineering is a promising technique to solve this dilemma.Since dermis-fat composite is the main component of skin flap,we try to construct the composite using PADM in vivo.However,the PADM has such a compact structure that it prevents host cells to infiltrate into the material,resulting in delayed neovascularization.Transplantation also triggers a certain degree of inflammatory response.Both these points bring difficulties to the construction.The study used two in vitro pretreatments to optimize the PADM.The first is Laser Micropore Technique?LMT?.The other is pre-culture with rADSCs.The aim of this study was to investigate whether PADM before and after laser microporation can provide a microenvironment that is beneficial for adipose-derived stem cells to exert their paracrine function,and to verify whether optimization in vitro can promote vascularization of PADM after implantation and reduce the intensity of inflammatory response,thereby constructing dermis-fat composite with considerable mechanical strength.Methods:First,the PADM was processed by a CO₂ drilling machine to produce micropores with 1 mm of both pore diameter and pore pitch.rADSCs were then seeded at a density of 1×10⁵/well in PADM,LPADM and mircoplate to detect the expression of genes and proteins involved in angiogenesis and inflammation regulation in rADSCs cultured in different microenvironments.Then PADM and laser microporous treated PADM?LPADM?were embedded in the back of the rats,respectively.After 4 weeks,the effect of LMT on the vascularization of PADM was evaluated by gross and histological detection.rADSCs were seeded on a 24-well plate and PADM/LPADM with the same pore size at a density of 1×10⁵/well to detect the expression of genes and proteins involved in angiogenesis and inflammatory regulation in rADSCs cultured on different systems.2×10⁵ rADSCs were co-cultured with PADM for one week in vitro,and embedded in the back of the rats together with PADM without co-culture.After 4weeks,the effects of rADSCs pre-culture on the vascularization of PADM were observed.Finally,the pretreated and unpretreated PADMs were subcutaneously implanted into the back of the rat,with autologous fat implanted two weeks later.At 4weeks after surgery,the in vivo products were harvested for gross observation,histology and biomechanical testing.Results:Laser microporation and pre-culture of rADSCs significantly increased the angiogenesis rate after PADM implatation,and the inflammatory response of PADM co-cultured with rADSCs was significantly relieved.PADM with and without LMT can both provide a beneficial microenvironment for rADSCs,demonstrating by up-leveled expression of pro-angiogenic genes?VEGF,bFGF,HGF?,inflammation regulatory genes?TGF-?,TSG-6,COX-2,iNOS?and stemness gene?SOX-2?.The results of in vivo composite tissue construction showed that the PADM treated by LMT and rADSCs pre-cultured in vitro,compared with untreated PADM,the inflammatory reaction induced by the implant was significantly weakened,localized,and rapid vascularization achieved.The fat survival of the second stage transplantation increased.The dermis-fat composite tissue finally formed in the in vitro pretreatment group possess better biomechanical strength.Conclustions:By LMT treatment,the cell infiltration and neo-vascularization of PADM were efficiently enhanced.Pre-culture with rADSCs in vitro promotes neovascularization and alleviates inflammatory response after PADM implantation.PADM/LPADM can both provide a microenvironment that facilitated the paracrine function of rADSCs,while the paracrine products of rADSCs can promote angiogenesis and regulate the inflammatory response of the PADM after implantation,which made the material and seed cells promote each other.The PADM,pretreated with LMT and rADSCs,and autologous fat were respectively implanted into the back of the rat,which can construct a dermis-fat composite with sufficient mechanical strength.This study laid the foundation for the next step of constructing skin flap with epidermis-dermis-fat full layer structure.