| Objective: To investigate the ability of amniotic membrane homogenate to promote the proliferation and osteogenic differentiation of periodontal ligament stem cells,and select the most appropriate concentration of amniotic membrane homogenate.Iron oxide nanoparticles were loaded with AMH to co-culture periodontal ligament stem cells,to observe whether it can promote the osteogenic differentiation of periodontal ligament stem cells in vitro.Methods:(1)Preparation,culture and identification of periodontal ligament stem cells:healthy periodontal ligament stem cells were cultured by enzyme digestion method,then we cultured cells by limited dilution cloning method and passaged routinely,and periodontal ligament stem cells were identified by flow cytometry.(2)Preparation and concentration determination of amniotic membrane homogenate: the supernatant of amniotic membrane homogenate was obtained by homogenizer and centrifuge,and its concentration was determined by BCA method.(3)Co-culture of amniotic membrane homogenate and periodontal ligament cells: CCK-8method and clone formation were used to detect the effects of incremental concentration gradients of 0mg/L,4mg/L,8mg/L,16mg/L and 32mg/L AMH on the proliferation of periodontal ligament stem cells,and ALP and ARS staining were used to detect the the osteogenic differentiation of periodontal ligament stem cells cultured on incremental concentration gradients of AMH.(4)Co-culture of periodontal ligament stem cells with iron oxide nanoparticles loaded with amniotic membrane homogenate: The third-passage periodontal ligament stem cells were co-cultured with IONPs-loaded AMH to detect their osteogenic differentiation potential.(Control group: OS group;IONPs group;AMH group;IONPs+AMH group)four groups were the experimental subjects,ALP and ARS staining was used to detect its effect on the osteogenic differentiation of periodontal ligament stem cells,and RT-PCR and WB were used to further detect the expression of osteogenesis-related genes and proteins.Results:(1)Periodontal ligament stem cells were cultured by enzymatic digestion and limiting dilution cloning method,and the cells were long spindle and polygonal.Flow cytometry identified the phenotype of periodontal ligament stem cells with high expression of mesenchymal stem cells.(2)Under aseptic operation,the clear supernatant of amniotic membrane homogenate was obtained by homogenizer and centrifuge,and its concentration was determined to be588.7±141.20mg/L by BCA method.(3)CCK-8 assay showed that the final concentration of AMH was less than 16mg/L,the cell proliferation was not affected,and the ability of cell proliferation was the strongest in16mg/L(P<0.05).The results of colony formation showed that the cell proliferation ability increased after adding amniotic membrane homogenate,and the proliferation ability was the strongest in 16mg/LAMH group(P<0.05).ALP and ARS staining showed that 16mg/L AMH group had the deepest staining and the most calcified nodules.(4)The results of ALP staining showed that the staining of IONPs+AMH group was the deepest,the IONPs group and AMH group was deeper respectively,and the control group was the lightest.The results of ARS staining were consistent with the results of ALP staining.The expression of osteogenesis-related genes ALP and RUNX2 detected by RT-RCR showed an upward trend in control group,IONPs group,AMH group and IONPs+AMH group(P<0.05).Osteogenesis-related proteins ALP and RUNX2 were further verified by WB.Conclusion: Amniotic membrane homogenate can promote the proliferation and osteogenic differentiation ability of periodontal ligament stem cells at optimal concentration,and iron oxide nanoparticles loaded amniotic membrane homogenate can promote their osteogenic differentiation ability. |
