| Objective: To study human acellular amniotic membrane as the scaffold materials when loaded the human amniotic mesenchymal stem cells to construct tissue engineered“biomimetic amniotic Membrane”,as well as survival,proliferation and cytokines secretion in vitro.Methods:Preparation and selection of HAAM scaffolds: The fresh human amniotic membranes were treated as follows: Group A: no treatment(control group);Group B:1%Triton X-100+0.25% trypsin-0.02% EDTA-2Na(Triton+ trypsin);Group C: 3m M Na Cl solution +0.25% trypsin-0.02% EDTA-2Na(hypertonic + trypsin);Group D: 0.25% trypsin-0.02% EDTA-2Na + scraping(trypsin + scraping).Comparing the preparation of HAAM scaffolds with different methods,and identified acellular condition,composition of residual extracellular matrix and integrity of basement membrane by HE staining,scanning electron microscopy,thickness and mechanical properties.So as to select HAAM scaffolds with good biocompatibility and mechanical properties.Isolation and identification of h AMSCs:h AMSCs were separated by trypsase and collagenase Ⅱ,purified and cultured by differential adhesion method.The cell morphology was observed by inverted phase contrast microscope and then cultured to the third generation,and then identified by cell morphology and flow cytometry for subsequent composite culture experiments with HAAM scaffolds.Detection of the preparation and characterization of “Bionic Amniotic Membrane”: The cells were divided into two groups: two-dimension with no scaffold(the control group)and threedimension with scaffold(the experimental group).The h AMSCs were devided to seeded on the ordinary cell culture plate and the HAAM scaffold.The inverted microscope and scanning electron microscope are used to observe the growth and proliferation of h AMSCs cells after 1d,3d and 7d.Live/Dead and CCK-8 assays were used to detect the survival and proliferation of h AMSC.The cytokines secretion were detected by ELISA.Results: The preparation of HAAM scaffold by Triton+ trypsin is better than the method by hyperosmolar + trypsin and trypsin + scraping.After HE staining and scanning electron microscopy,it shows that all the cell components in the upper cortex and matrix,prepared the HAAM scaffold by Triton+trypsin method were removed.The basement membrane layer was continuous and intact,and collagen components still existed.Compared with fresh human amniotic membrane,the thickness and mechanical properties of human fresh amniotic membrane were not significantly weakened.The h AMSCs were separated by collagenase Ⅱ and trypsase,adherent-like,spindles and spirals,with high expression of CD44,CD73,CD90 and CD105,but low expression of CD34,CD19,CD45,CD11 b and HLA-DR.The h AMSCs survived well in the designed HAAM scaffold,cells adhered to the three-dimensional space network and the number of cells on the scaffold increased gradually with the extension of culture time by inverted phase contrast microscope and scanning electron microscopy.The results of Live/Dead and CCK-8 showed that the survival rate and proliferation activity of HAAM scaffold group were higher than those in control group.The expression levels of VEGF,b FGF and collagen I of h AMSCs in experimental group were significantly higher than control group(P < 0.05),while the expression levels of TGF-β1and α-SMA were lower than that control group(P < 0.05).Conclusion:(1)The HAAM scaffold preparing with Triton+ trypsin is ideal scaffolds,it has good cytocompatibility and biomechanical propertiesand,and no cell residue at the same time.(2)The h AMSCs were successfully separated by collagenase Ⅱ and trypsase.(3)The h AMSCs loaded with HAAM scaffold successfully constructed “biomimetic amniotic membrane”,and the HAAM scaffold has good cytocompatibility,which can simulate extracellular matrix in vitro and promote the adhesion,proliferation and secretion of factors related to endometrial repair of h AMSCs. |
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