Effects Of Human Acellular Amniotic Membrane On The Biological Function And Osteogenic Differentiation Of Periodontal Ligament Stem Cells

Objective: The aim is to explore the effect of human acellular amniotic membrane on the biological function and osteogenic differentiation of periodontal ligament stem cells in vitroMethods: In this experiment,we separated and cultured periodontal ligament stem cells by tissue block improved culture method,identified periodontal ligament stem cells by flow cytometry,prepared acellular amniotic matrix by mechanical / enzyme digestion method,and identified acellular condition and main components by HE staining and immunohistochemistry;observed the acellular amniotic membrane surface structure by inverted microscope and scanning electron microscope to evaluate the potential of acellular amniotic membrane matrix as a carrier of cell delivery.The cells were divided into two groups: acellular amniotic matrix group and TCP group(cell culture plate group).The apoptosis rate of Annexin V-FITC/PI,CCK8,cell adhesion and Transwell migration test were used to detect the effect of acellular amniotic membrane on the biological function of periodontal membrane stem cells.TCP(-),TCP(O)(fine Methods: ALP staining,alizarin red staining,ALP activity and alizarin red staining were used for 7,14,21 days,RT-PCR and WB were used to detect the osteogenic related proteins in 7,14,21 days,COL1A1,ALP and OPN were used to observe the osteogenic differentiation of different groups).Results: The separation and extraction of periodontal ligament stem cells and the preparation of acellular amniotic membrane matrix were successful.The main collagen structure of the acellular amniotic matrix was type I and type III collagen.The periodontal ligament stem cells could grow normally on the acellular amniotic membrane,and the cell arrangement was guided by the structure of the acellular amniotic membrane fiber.There was no significant difference in apoptosis rate and cell cycle between the cells loaded with acellular amniotic membrane and those cultured with cell culture plate(P > 0.05);and the acellular amniotic membrane could promote the proliferation,adhesion and migration of periodontal ligament stem cells compared with the cell culture plate;The results of ALP staining,alizarin red staining,quantitative analysis of ALP and alizarin red,RT-PCR and WB confirmed each other at 7,14 and 21 days.The results showed that the acellular amniotic membrane can promote the early osteogenic differentiation of periodontal ligament stem cells,and maintain the maximum osteogenic differentiation ability in the medium and late stage of the acellular amniotic membrane induction solution group,while the osteogenic differentiation ability of the pure acellular amniotic membrane compared with the cell culture plate has also been strengthened.Conclusion: It is speculated that acellular amnion matrix can be used as a suitable cell transfer carrier,mimicking natural extracellular matrix to positively affect the proliferation,adhesion and migration of periodontal ligament stem cells and promote the osteogenic differentiation of periodontal ligament stem cells.