Objective To investigate the results of human amniotic membrane(HAM) which are loaded with marrow mesenchymal stem cells(MSCs) and epidermis cells in treating fullthickness skin defect combined with radiation injury. Methods Eight minipigs were used in this study. Three round fullthickness wounds（Ф3.67cm）, which combined with radiation injury, were created on the dorsum of each side close to the vertebral column in each animal. Among 48 wounds, 24 left side wounds were treated with HAM loaded with MSCs and epidermis cells as experimental group (group A), 16 right side wounds with simple HAM (HAM group, group B) and 8 right side wounds with oil gauze as control (group C). The granulation tissue, reepithelization and wound area were observed after 1,2 and 3 weeks. Immunohistochemistry was performed using vWF as a marker for blood vessels.Image analysis was employed to test new area of wound at different interval time and healing rate of wound.Results The healing time of group A was 6 to 7 days faster than that of group C and 5 to 6 days faster than that of group B. After 15-17 days of graft, there were significant differences in new area of wound and healing rate between group A and groups B,C(Plt;001). New epidermis fully covered whole wound surface in group A, and their granulation tissue, which contained a lot of vWF, fibroblasts, capillaries and collagen, grew well. Many inflammatory cells still were seen in groups B and C, and their contents of vWF, fibroblasts, capillaries and collagen in granulation tissue were smaller than that in group A.Conclusion The graft of HAM loaded with MSCs and epidermis cells played an effective role in promoting healing of wound combined radiation injury with high quality.
Objective Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×106 cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×106 cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation. Conclusion The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.