Biocompatibility Of Human Amniotic Mesenchymal Stem Cells And Human Decellularized Amniotic Membrane Scaffold

Objective: To investigate if human amniotic mesenchymal stem cells can survive when seeded in human amniotic membrane scaffolds.Methods: 1)After taking placenta from different parturient women,the human decellularized amniotic membrane scaffold was prepared by detergent and enzyme digestion method.The prepared HAAM was stained with HE,and the morphological characteristics and cell removal of HAAM and HAM were observed by inverted phase contrast microscope.Immunofluorescence staining was used to further observe the removal of HAAM cells and the damage of Extracellular matrix(ECM)on the inverted fluorescence microscope.2)The amniotic membrane was obtained with the same method,and h AMSCs was obtained by enzyme digestion.The proliferative activity and phenotypic molecules of h AMSCs were detected by CCK-8 and flow cytometry.The alizarin red,toluidine blue and oil red O staining were used to evaluate the differentiation effect of the h AMSCs into osteocytes,chondrocytes and lipocytes like.The expression of vimentin and CK-19 in the third generation of h AMSCs was detected by immunofluorescence in the purity identification of h AMSCs.3)HAAM and 3rd h AMSCs were cultured respectively in the culture of 14 d and21d,which should be observed in the inverted microscope and scanning electron;in addition,the 3rd generation h AMSCs cell membrane after staining with HAAM to the same method after co culture observed under inverted fluorescence microscope;The h AMSCs are divided into HAAM group and negative control group,which were treated with HAAM extracts and basal medium,then the profileration ability was recorded by CCK-8 assay.Results: 1)The human decellularized amniotic membrane was digested by detergent and enzyme.After staining with HE,microscopic observation showed that the amnioticepithelium of normal people was intact,and there was a large number of hAMSCs nuclei on the deep basement membrane.The human acellular amniotic epithelial layer is completely detached,h AMSCs has been completely removed,and the basement membrane is not destroyed.Immunofluorescence staining showed that type I collagen and type III collagen were positive in fresh human amniotic membrane and human acellular amniotic membrane.DAPI staining showed that human acellular amniotic membrane was negative,and human amniotic membrane was positive.2)T A full amount of h AMSCs primary cells can be extracted by enzyme digestion.After subculture,the cell morphology gradually changed into a long spindle shape.The growth pattern is whirlpool and adhered to the wall.The results of flow cytometry identification showed that h AMSCs could express the MSCs phenotype.The expression of vimentin in the third generation of h AMSCs was positive,and the expression of CK-19 was negative.The Toluidine Blue staining showed positive results after chondrocytes induction.After adipogenic differentiation,strong refraction of lipid droplets were observed in cytoplasm of the cells.The Oil red O staining showed positive results.3)The HAAM and the third generation hAMSCs were cultured after 7d in the process of cultivation,the growth activity was detected by CCK-8 assay,the results showed that HAAM extract group and ordinary medium cells were both growing well,and there were significant differences in cell proliferation level(P<0.05)during the 3rd-7th day.The obverstion of h AMSCs-HAAM complex at 14 d and 21 d by Inverted microscope and scanning electron microscope and by the inverted fluorescence microscope showed that h AMSCs owned good adhesion on HAAM,widely distributed in HAAM,and still showed long fusiform,neat.Conclusion: 1)HAAM with detergent enzymatic digestion method can completely remove cell components of HAM,and will not damage its ECM structure.2)h AMSCs can differentiate into osteoblasts,chondrocytes and adipocytes,and contain proliferatation with MSCs characteristics.Accordingly it can be used as seed cells in tissue engineering.3)HAAM can effectively promote the adhesion of h AMSCs,and there are no rejection and toxicity is found.HAAM can be used as a carrier of the load cell to provide a new type of scaffold for tissue engineering research.It lays the preliminary experimental foundation for the construction of tissue engineering ligaments and the healing of tendon bone healing in the later period.