Effects Of BMP2 In Autologous Teeth On Proliferation And Osteogenesis Of Human Periodontal Ligament Cells

Objective: To establish a human periodontal ligament cell line expressing human bone morphogenetic protein 2（BMP2）gene stably by lentiviral method to explore the effect of BMP2 on the proliferative and osteogenic ability of human periodontal ligament cells,and to explore the changes of endoplasmic reticulum stress level during BMP2-induced osteogenesis of human periodontal ligament cells,which provides a theoretical basis for the application of autologous bone in the study of periodontal tissue regeneration and the repair of maxillofacial defects.Methods: 1.In vitro culture of primary human periodontal ligament cells for passage and identification;2.The full-length BMP2 gene was amplified by PCR,and the recombinant lentiviral plasmid p LV-BMP2 was constructed.After packaging the lentiviral particles,human periodontal ligament cells were infected to construct a stable periodontal ligament cell line overexpressing BMP2;3.The effect of BMP2 on the proliferation of periodontal ligament cells was detected by CCK-8;4.The osteogenic induction of periodontal ligament cells was carried out on 7,14,and 21 days,and determined by alizarin red staining and ALP activity detection.What’s more,RT-q PCR was used to detect the m RNA expression levels of osteogenesis-related genes to detect the effect of BMP2 on its osteogenic ability;5.The expression levels of endoplasmic reticulum stress-related proteins were detected by Western Blot and RT-q PCR to explore the changes of endoplasmic reticulum stress levels in the process of BMP2-promoted periodontal ligament cells osteogenesis.Results: 1.Primary periodontal ligament cells were successfully cultured and identified;2.The homologous recombinant lentiviral plasmid p LV-BMP2 was successfully constructed and transfected into periodontal ligament cells,which were morphologically similar to normal periodontal ligament cells,with typical radial arrangement;3.The results of CCK-8 showed that compared with h PDLCs group and p LV-puro group,the proliferation ability of periodontal ligament cells in p LV-BMP2 group was enhanced（P<0.05）;4.The results of alizarin red staining and ALP activity detection showed that the osteogenic ability of periodontal ligament cells in p LV-BMP2 group was significantly enhanced compared with h PDLCs group and p LV-puro group（P<0.01）.And the results of real-time quantitative PCR showed that osteogenesis-related genes were highly expressed in periodontal ligament cells in p LV-BMP2 group（P<0.01）;5.The results of Western Blot showed that in the process of BMP2 promoting osteogenesis of periodontal ligament cells,the expression level of endoplasmic reticulum stress-related protein PERK was significantly increased.Conclusion: The stable periodontal ligament cell line overexpressing BMP2 was successfully established by lentiviral method,which has a strong ability to promote the proliferation and osteogenesis of periodontal ligament cells.And the endoplasmic reticulum stress level were increased during the process of BMP2 promoting periodontal ligament cell osteogenesis,which provides an experimental basis for the study of periodontal tissue regeneration,and also helps autologous teeth,as a new type of bone repair material,provides a certain theoretical basis for periodontal treatment and maxillofacial bone defect repair.