Objective Human acellular amniotic membrane (HAAM) contains collagens, glucoproteins, proteinpolysaccharide,integrin, and lamellar, which can supply rich nutrition to cell prol iferation and differentiation. To explore the possibil ity of HAAM with adi pose-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 epithel ial side of HAAM at the density of 2 × 105/cm2, cocultured, and observed by SEM at different time. MTT test was used to detect viabil ity 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 heal ing 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 prol iferated to confluence on the stromal surface of HAAM. MTT test showed that the cells on the HAAM grew well and had b prol iferation vital ity. There was no significant difference between ADSCs cultured in the HAAM and control group (P gt; 0.05). One, 2, 4 weeks after graft, there were significant differences in wound heal ing rate between group A and groups B, C (P lt; 0.05), between group B and group C (P lt; 0.05). HE staining showed that wound healed faster in group A than in groups B, C. Cytokeratin 19 (CK19) immunohistochemical statining 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
To investigate the immunoreaction, histological reaction and turnover by comparing the xenotransplantation of fresh human amniotic membrane (HAM) with that of preserved HAM, and to analyze the cl inical appl ication value of different kinds of HAM preparations. Methods Subcutaneous implant models were establ ished in 150 BALB/C mice, which were randomized into 5 groups of 30 mice each, based on different implants: fresh amniotic membrane (FAM), double fresh amniotic membrane (DFAM), glycerin preserved amniotic membrane (GPAM), chorion (positive control) or merely operation (negative control). The tissue samples from grafted area were observed with SABC and HE staining, and the inflammatory cells were calculated with l ight microscopy. 1, 2, 4, 8 and 12 weeks after surgery. Results The mice in all of groups were normal in eating and moving, and the wound surface healed well. In all of AM groups, the expression of MHC Ⅱ and the calculation of inflammatory cells were much less than those in chorion groups, showing significant differences (P lt; 0.01). At 1, 8 and 12 weeks after surgery, there were no significant differences in the expression of MHC Ⅱ and the calculation of inflammatory cells in all of AM groups, compared with other groups (P gt; 0.05). From 2 weeks to 4 weeks after surgery, there were no significant differences in the expression of MHC Ⅱ and the calculation of inflammatory cells between FAM and DFAM groups (P gt; 0.05), but they were both more than those in GPAM groups, showing significant differences (P lt; 0.05). At the 4th week after surgery, in all of AM groups, the expression of MHC Ⅱ and the calculation of inflammatory cells were less than those at the 2nd week, showing significant difference (P lt; 0.01).The amniotic epithel ium was still al ive in fresh AM groups until 4 weeks after transplantation. Early after surgery, fibroblasts infiltrated AM from the substantia basilaris layer while made fibrous capsule around the epithel ium. After 12 weeks, the amnion absorbed. Conclusion As a kind of heterologous biomaterial, whether fresh or preserved, HAM can be seemedof ideal immunocompatibil ity and histocompatibil ity. Fresh HAM with al ive epithel ium may be more successful in area ofrepair and reconstruction.
Objective To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane(HAAM).Methods To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The realtime PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simplycultured osteoblasts were used as the control group (n=20).Results The expression of Cbfa1 was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day(P＜0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results(P＜0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference(P＞0.05). Conclusion Cbfa1 and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.
Objective To prepare human acellular amniotic membrane(HAAM) and to measure its cytocompatibility and biocompatibility. Methods HAAM were preparedby chemical detergent-enzymatic extraction. Fresh human amnion was crosslinkedwith glutaradehyde, shaken in 0.5% SDS for 24 hours, and then treated with 0.25%trypsin for 4 hours. The production were freeze-drying and sterilized using ethylene oxide. Human fibroblasts were isolated from embryo and expanded in vitro. The fibroblasts were seeded in HAAM. HAAM and specimen were stained with HE and Mallory, and observed grossly, under light microscopy and scanning electron microscopy. The HAAM were implanted in the back of SD rats. Results There wereno residues of cells in the HAAM (HE, Mallory staining). One side of HAAM had reticular and porous structure, the other side had compact fibrous structure.Pore size was from 10 to 80 nm. The HAAM could be seeded with expanded fibroblasts in vitro,and fibroblasts had the potential of spread and proliferation. The SD rat in the implant test had no death, convulsions and other abnormal response. Conclusion The detergent-enzymatic extraction process can remove cellsand solvable components effectively and preserve the tissue matrix well and keep the reticular structure. The HAAM can be used as an ideal scaffold of biological membrane for tissue engineering.
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.
Application research on human amniotic membrane has been carried out for nearly a hundred years and people found that there were more than dozens of kinds bioactive substances in the amniotic membrane. It has been proved that the amniotic membrane has a lot of functions, such as anti-inflammatory, anti-bacterial, anti-virus, anti-angiogenic and promoting cell apoptosis, and so on. As effective treatments, amniotic membrane has been used for adjunctive therapy of burns, trauma, ophthalmic damage, dermatopathya. Recent advances of amniotic membrane and amniotic membrane-derived cells research have led to enormous progress in skin tissue engineering, vascular tissue engineering, biological scaffold material, and biological sustained-release materials. Amniotic membrane and amniotic membrane derived cells have a significant advantage and unique charm in medical field. Therefore, they have higher research value and broad prospects in the applications.
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.