Construction Of Artificial Composite Skin Based On Mesenchymal Stem Cells Derived From Induced Pluripotent Stem Cells Combined With Microporous Dermal Matrix

Objective:To construct a new artificial complex skin by mesenchymal stem cells(MSCs)derived from induced pluripotent stem cells(iPSCs)reprogrammed from human adult fibroblast cells as seed cells combined with microporous acellular dermal matrix(MADM),and to verify the feasibility of iPSC-MSCs as the seeding cells of skin tissue engineering for the repair of skin defect.Methods:1?The iPSC-MSCs reprogrammed from human fibroblast were resuscitated,and the cell growth curve was recorded in the passage culture.The osteogenic and adipogenic differentiation of the cells was verified by the lipid induced differentiation and the osteogenic differentiation experiment.ADM was isolated from rat dorsal skin by enzyme digestion combined with detergent,and MADM was obtained after laser drilling.The iPSC-MSCs were inoculated on ADM to prepare the artificial compound skin.The cells were stained and labeled by DAPI dye,and the cell growth was observed under the microscope.The enzyme labeling instrument was used to detect the cytotoxicity of ADM at the wavelength of 450 mm.2.Twelve SD rats were fed for several days before the experiment.Routine abdominal anesthesia was used to make a U shaped flap in the middle of the back.The length of the flap was 2 centimeters long.The whole layer of skin was completely removed,the subcutaneous fascia was separated,the hot wire was used to stop bleeding and the skin was scalded.Twelve SD rats were randomly divided into two groups: group A(group MSCs+MADM),group B(single MADM).The materials were grafted on the defect wounds of the corresponding groups respectively.Vaseline gauze was covered,and suture was fixed after packing.The appearance of the wound and the tissue growth of the skin graft were observed at first,second,third,and fourth weeks after implantation,and the tissue growth was identified by histological HE staining.The survival of composite skin cells in the experimental group was observed under fluorescence microscope.The complete recovery time of each group of rats was recorded and analyzedstatistically.Result:1.iPSC-MSCs in vitro culture and generation: after eighth generation of resuscitation cells,the cells were round,and most of the cells were adhered to the wall after 24 hours.The adherent growth cells gradually showed the spindle type and round type.After 6-8 days,the cells were mostly spindle shaped,and fibroblast like and whirlpool like growth.The incubation period was 10-12 days.Oil red O staining showed large round lipid droplets,and osteogenic differentiation was confirmed by calcium precipitation after alizarin red staining.It indicates that iPSC-MSCs has very similar growth characteristics with other types of MSCs.The detection of the enzyme labeling instrument showed that cell viability has no difference between the combined culture cells and the cultured cells,which showed that the prepared MADM had good performance and no cytotoxicity.It could be used in the preparation of compound skin and animal experiments.2.Gross observation of the wound: the average healing time of the A group was 17.33 days.A large number of new reticulated blood vessels around the artificial composite skin were wrapped around the artificial compound skin.The color was red,and the texture of the skin was soft.The skin was close with the subcutaneous tissue,and the boundary was blurred.In group B,the average healing time of wound healing in single MADM transplantation group was 21.83 days,and there was more neovascularization around the skin graft,but it was obviously less than that of the experimental group A,and the fusion of skin slices and subcutaneous tissue was general,and the boundary was clearer.The healing time of the two groups was statistically significant(P<0.05).It is suggested that artificial composite skin made from new seed cells can promote wound healing better.3.Histology staining and fluorescence microscope observation: In the early transplantation,the experimental group A and the control group B around the skin graft showed mild inflammatory response,HE staining showed inflammatory cell infiltration.After two weeks of transplantation,the observation showed that the vascular network structure in the group A was more than that of the control group B,and the papillary structure of the A group penetrated into the compound skin;and wound tissue was interlaced with the compound skin,and a little fat tissue appeared.There was a little ring neovascularization around the control group,and the skin slices and subcutaneous tissue were clearly demarcated.Under the fluorescence microscope,there were more blue fluorescent points in the first week of transplantation,and the fluorescence gradually decreased with time,and the fluorescence points were reduced and disappeared,which indicated the normal cell apoptosis.Conclusion:The new seed cells(iPSC-MSCs)is similar to common MSCs and has high differentiation potential.Artificial preparation of MADM cells is not toxic,and is a good choice for composite skin scaffold materials.The use of new seed cells iPSCMSCs cells combined with MADM to make artificial composite skin can better accelerate the process of wound repair and increase the filling of tissue,and hope to become a widely used seed cell.