Experimental Preparation And Transplantation Of Living Dermal Substitue

Objective:To prepare living chitosan-Collagen Laser Micropore Porcine acellular composite dermal matrix(CCLMPADM)with CCLMPADM and fibroblasts;Transplantation was carried out in full thickness defect of skin model;this work will be a basis for future researching.Methods:1.Chitosan-Collagen Membranes(CCM)in various rations of chitosan(1mg/ml)and collagen(3g/L)were prepared by freeze drying or dry heating method,and their physical performance and biocompatibility have been detected to determine the perfect preparative method.2.Further perfected the skin cells bank in vitro;to studied fibroblasts culture method and regulatory effect of Ca²⁺on HKC;To prepared full-chickness tissue engineering skin for future study.3.To prepared living CCLMPADM with CCLMPADM and fibroblasts.Full thickness defect wounds were created on the back of 36 SD rats, the rats were divided into 3 groups randomly,with 12 rats in each group:â‘ PADM group:the wounds were grafted with PADM with micropores,and covered with split-thickness autograft;â‘¡CCLMPADM group:CCLMPADM grafted,and covered with split-chickness autograft;â‘¢living CCLMPADM group:living CCLMPADM + split-thickness autograft.The gross observation of wound healing and histological observation were performed after surgery.Results:1.The CCMs prepared by dry heating method were smooth transparent sheets,their mechanical strength were higher than the CCMs prepared by freeze drying.The latter were ivory white semitransparent sheets, they had better water absorption ratio and water retaining rate.the mechanical strength of CCM reached the max when the proporation of chitosan and collagen was 8/2.With the increment of collagen concentration,the water absorption ratio and water retaining rate increased.The biocompatibility tests showed that there was no significant differences in HKCs proliferation rate among CCMs which were prepared by different drying methods,but fibroblasts proliferation rate in CCMs which were prepared by freeze drying were more higher than that in CCMs prepared by dry heating methods.With increment of collagen concentration,HKCs and fibroblasts proliferation rate were increased too.we also founded that CCMs had good biocompatibility,and the biodegradation of CCMs speeded up as the collagen proporation increased.2. Successfully established fetus skin cells line;Improved tissue mass method had short cultured cycle and little cell damagement.Ca²⁺had critically effects on the proliferation and differentiation of HKC;successfully prepared full-thickness tissue engineering skin.3.Successfully prepared living CCLMPADM;the healing results of living CCLMPADM group were better than PADM, CCLMPADM group.Conclusions:1.The 5/5 and 3/7 maybe the ideal proportion of chitosan and collagen in prepare tissue engineering materials.The freeze drying is good method to reconstruct dermal substitute.2.Improved tissue mass method is ideal method to culture fibroblast for tissue engineering.Ca²⁺have critically effect on the proliferation and differentiation of HKC.Using collagen,technology of three-dimentional and air-liquid culture can build up simple model of full thickness tissue engineering skin.3.The adding of chitosan and collagen makes cells more easily to be added to PADM,and the living CCLMPADM is an ideal dermal substitute.