[An experimental study on repairing full-thickness skin wound by human acellular amniotic membrane loaded with adipose-derived stem cells in rats]

Abstract

Objective: Human acellular amniotic membrane (HAAM) contains collagens, glycoproteins, protein-polysaccharide, integrin, and lamellar, which can supply rich nutrition to cell proliferation and differentiation. To explore the possibility of HAAM with adipose-derived stem cells (ADSCs) as a good engineered skin substitute for repairing skin defect.

Methods: Primary ADSCs were obtained from inguinal fat of 30 healthy 4-month-old SD rats, male or female, weighing 250-300 g, and cultured in vitro and purified. The 3rd passage ADSCs were used to detect CD44, CD49d and CD34 by immunocytochemistry staining. After physical and trypsin preparation, the HAAM was observed by HE staining and scanning electron microscope(SEM) respectively. ADSCs were seeded on epithelial side of HAAM at the density of 2 x 10(5)/cm2, cocultured, and observed by SEM at different time. MTT test was used to detect viability of cells that seeded on HAAM, the group without HAAM was used as control. Thirty SD rats were made models of full-thickness skin wound and randomly divided into three groups (A, B, and C). Wound was repaired with HAAM/ADSCs composites in group A, with HAAM in group B, and with gauze as control in group C. The rats underwent postoperative assessment of wound healing rate and histological observation at the 1st, 2nd, and 4th weeks.

Results: HE staining showed that the 3rd passage ADSCs was spindle-shaped with an ovoid nucleus which located in the middle of cell; the immunocytochemistry staining showed positive result for CD44 and CD49d and negative result for CD34. There were no residues of cells in the HAAM by HE staining. SEM showed that there were different structures at the two sides of HAAM: one side had compact reticular structure and the other side had fibrous structure. After 3 days of co-culture, ADSCs showed good growth on HAAM; the cells were closely packed onto the HAAM, attached firmly and proliferated to confluence on the stromal surface of HAAM. MTT test showed that the cells on the HAAM grew well and had strong proliferation vitality. There was no significant difference between ADSCs cultured in the HAAM and control group (P > 0.05). One, 2, 4 weeks after graft, there were significant differences in wound healing rate between group A and groups B, C (P < 0.05), between group B and group C (P < 0.05). HE staining showed that wound healed faster in group A than in groups B, C. Cytokeratin 19 (CK19) immunohistochemical staining showed that there were more CK19 positive cells in group A than in groups B, C.

Conclusion: The graft of HAAM with ADSCs plays an effective role in promoting the repair of full-thickness skin wound.