An Experimental Study Of Rats' Wound Repair By HAAM Loaded With ADSCs

ObjectiveTo investigate the biocompatility of human acellular amniotic membrane (HAAM) with adipose-derived stem cells (ADSCs) and to explore the possibility of HAAM with ADSCs as a good engineered skin substitute.MethodsPrimary ADSCs were obtained from inguinal fat of healthy SD rats and cultured in vitro and purified. When primary ADSCs expansion to the 3rd passages, the cells were used to detect CD44,CD49d and CD34 by immunocytochemistry staining.After physical and trypsin preparation, the HAAM observated by HE staining and scanning electron microscope (SEM) respectively. ADSCs seeded on epithelial face of HAAM at the density of 2.0×105/cm2, and observated by SEM at different times. MTT test was used to detect viability of cells that seeded on HAAM.The 30 rats were random divided into A, B and C three groups. Full-thickness skin wound were performed in all groups, and A were repaired with HAAM loaded ADSCs, B group were only repaired with HAAM, C group were covered with gauze as control.The rats postoperative assessment of wound healing rate and the wound skin were observed by light microscope at the one week, two weeks and four weeks after surgery. ResultsADSCs could rapidly expand, CD44 and CD49d were showed positive on the cell while CD34 was negative. There were no residues of cells in the HAAM by HE staining. By SEM, we found there were different structures on the two sides of HAAM; One side of HAAM had compact reticular structure, the other side had fibrous structure. ADSCs growing was very good on HAAM for three days; The cells were closely packed onto the HAAM, they attached firmly and proliferated to confluence on the stromal surface of HAAM. MTT test showed that the cells on the HAAM growth well and proliferation vitality strongly; There was no significant difference between ADSCs cultured in the HAAM and control group.After graft, there were significant difference in wound healing rate between group A and group B, C (p<0.05). HE staining showed that the group A wound healed more faster than group B and group C; There are more cytokeratin 19 (CK19) positive cells in group A than in group B or in group C.ConclusionThe graft of HAAM with ADSCs played an effective role in promoting the repair of full-thickness skin wound.