Novel Pre-vascularized Tissue-engineered Dermis Based On Stem Cell Sheet Technique Used For Dermis-defect Healing

Objective:To solve insufficient donor dermis and the shortage of three-dimensional vascular networks,a pre-vascularized tissue-engineered dermis was constructed by using cell sheet technology,and then implanted in the nude mice dorsal dermis-defect wound.To explore the feasibility of this composite for clinical use,the wound healing effect was quantified using the methods of histochemistry and immunohistochemistry.Methods:1)Construction and characterization of pre-vascularized tissue-engineered dermis?PTED?:Rabbit tibial bone marrow mesenchymal stem cells?BMSCs?were isolated by adherent method;The growth curve of BMSCs was measured by MTT method,and BMSCs in P2 generation were used in subsequent experiments;Surface markers of BMSCs were identified;Multidirectional differentiation of BMSCs was detected;Fibroblasts?Fb?and endothelial cells?ECs?were incubated;BMSCs and Fb were incubated with high density and cultured into the cell sheets before ECs were seeded.After a total of 5 days of culture,the pre-vascularized bone marrow mesenchymal stem cells?PBMCS?and pre-vascularized fibroblast membranes?PFCS?were formed.Finally,PTED was constructed by superposition or folding.PBMCS and PFCS were observed and detected by CD31.2)PTED?PBMCS?PFCS groups on repairing dermis-defect in the back of nude mice:The dermis-defect model was constructed in the back of nude mice;The constructed PTED was then implanted into the dermis-defect of the back of nude mice.And the wound healing effect at days 1,7 and 14th was observed respectively and evaluated through histochemistry and immunohistochemistry methods.Results:1)BMSCs showed a long spindle shape under an inverted optical microscope,and the MTT experiment showed an"S"growth curve of BMSCs in P2 generation.Immunohistochemical identification of BMSCs indicated that BMSCs showed positive expression of surface markers CD29,CD44 and CD90.Alizarin red,alcian blue and oil red O staining showed that BMSCs had the potential to differentiate into adipogenic,osteogenic and chondroblastic cells.Immunofluorescence staining verified that ECs were migrated on bone marrow Mesenchymal stem cell sheet?BMCS?and fibroblast cell sheet?FCS?,benefiting to the generation of pre-vascularized structures in PFCS.2)The constructed PTED?PBMCS?PFCS were respectively implanted in nude mice dorsal dermis-defect wound and wound healing effect was quantified at days 1,7 and 14 using the methods of histochemistry and immunohistochemistry.The results showed that PTED could rapidly promote the wound closure,especially on 14^th day.The wound healing rate of 3-layers PTED could reach 97.2%?p<0.01?,which was faster than the blank control group?89.1%?,PBMCS?92.4%?,PFCS?93.8%?and6-layers PTED?92.3%?.Moreover,the vessel density in the PTED group was higher than the other groups on the 14th day.Conclusions:Taken together,the results of this study showed that the PTED,especially3-layers PTED,is more conducive to the full-thickness dermis-defect repair and construction of the three-dimensional vascular networks,indicating its potential application in dermis-defect repair.