| Objective:Artificial composite skin is constructed by culturing adipose derived stem cells(ADSCs)combining with laser microporous burned denatured dermis matrix(LP-DDM)in vitro.To observe and analysis its biocompatibility,vascularization and other effects,in order to get a new composite skin.Methods:1.The perirenal fat of healthy male SD rats was taken to culture and pass ADSCs in vitro.Idantify ADSCs by adipogenic and osteogenic differentiation assay and detect ADSCs surface markers CD29,CD45,CD90 through flow cytometry.After constucting model of deep second degree burn on the SD rats' skin,its full-thickness skin takes acellular treatment and laser microporous technology treatment,being microporous denatured acellular demal matrix(LP-DDM),and transplanting the ADSCs on the surface of LP-DDM;2.Divided 16 healthy male SD into 4 groups randomly,group A: DDM,group B: LP-DDM,group C: DDM+ADSCs,group D: LP-DDM+ADSCs group,and each rat' s back was designed a flap(2cmí2cm).We implanted the new composite skin under subcutaneous,after 7 days,14 days,21 days and 28 days,we would observe the compatibility and the vascularization of the composite skin,and HE staining test.Results:1.The primary ADSCs was in star or polygon shape,passage of ADSCs showed long fusiform shape with fibroblast-like and swirl-like growth.From adipogenic differentiation experiment we observed big and round lipid droplet stained by oil red O after 4 weeks,osteogenic differentiation experiment showed that the precipitation of calcium can be stained with alizarin red after 4 weeks.Flow cytometer detected surface markers of ADSCs showed CD45 negative expression(0.70%),and at the same time 93.4% for the positive expression rate of CD29,CD90.ADSCs were co-cultured with LP-DDM found that ADSCs could live and grow well on the LP-DDM.After DAPI fluorescence staining,observing the blue fluorescence of ADSCs on the scaffolds by fluorescence microscropy.And the cells growth rate on the LP-DDM and DDM had no significant difference with that in control group(P>0.05).When added with CCK-8 and tested the OD value,LP-DDM and DDM also had no significant difference with that in control group(P>0.05),which explained that the material would not cause significant cytotoxicity and not harm the cells viability,proliferation and replication ability.2.Animal experiments:(1)General observation: 14 days after transplantation,in A,B,C,D four groups,a few peripheral vessels began to grow around the materials,28 days later,in Group D,there were a large number of red-like vascular tissue around the composite skin,which was much more than other groups,we also could see that color of some parts of the material had changed from creamy yellow to pink with red scattered dots on surface of the material.(2)Histology(HE): After 14 days,in group B and group D,there were a few vascular infiltration in materials' boundary and interior.But in group A and group C,there was few vascular infiltration.Inflammatory cells had been observed in each group.After 28 days,in group A,there were a small amount of vascular infiltration,and clear boundary between the composite skin and normal tissue was found.In group B,there were some vascular infiltration,even some adipose-like tissue.In group C,there was some vascular infiltration.In group D,there was a large number of vascular infiltration in the composite skin boundary and interior,and the adipose-like tissue was found.Microscopically,inflammatory cells were gradually reduced,the boundary of the composite skin was fused with normal tissue.Conclusion:The new artificial composite skin is found the good biocompatibility after grafting experiment,and there is no obvious immunological rejection.The composite skin can accelerate vascularization?differentiate to adipose-like tissue and reduce the inflammatory reaction.It will provide the experimental basis for the research of the composite skin in wound healing and clinical treatment of the wound in the future. |
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