The Investigation Of P2X7 Receptor In The Osteogenic Differentiation Of Periodontal Ligament Stem Cells Promoted By ECM

Periodontitis is a common infectious diseases happens in periodontal tissue. The main symptoms of Periodontitis is gingival atrophy, loss of attachment, damage of cementum, periodontal ligament. The continuous progress of inflammation will eventually lead to the absorption of alveolar bone and tooth loss. Although the clinical treatment of periodontitis can largely prevent the deterioration of periodontitis, the damaged periodontal tissue cannot be restored due to limited self-repair capacity. Therefore, how to effectively achieve the regeneration of periodontal tissue integrity is an important research direction of periodontal tissue engineering.Periodontal tissue engineering research mainly includes seed cells, biological scaffold carrier and micro environment. The periodontal ligament stem cells(h PDLSCS) are the biological basis of periodontal tissue regeneration while the extracellular matrix(ECM) micro environment is a key factor to maintain stemness of h PDLSCS. Therefore, the ECM originating from different tissues may influence the differentiation of h PDLSCS in different manners. On the other hand, various recent studies have found that P2X7 receptors play an important role in the differentiation of mesenchymal stem cells into osteoblasts. This implies that the P2X7 may be the key point during the osteogenic differentiation mediated by ECM micro environment. So in this study, the ECM of periodontal ligament cells and bone marrow cells were prepared in vitro to simulate different microenvironment for periodontal ligament stem cells differentiation. Afterwards, the P2X7 expression profile during the osteogenic differentiation of h PDLSCS was determined to validate their relationship. Finally, the role of P2X7 in the osteogenic differentiation of HPDLSCS was checked on bone marrow ECM micro environment, in order to confirm the interaction of ECM and h PDLSCS osteogenic differentiation.Research contents: 1. Effect of tissue specific ECM on osteogenic differentiation of periodontal ligament stem cells 2. Role of P2X7 receptor in osteogenic differentiation of periodontal ligament stem cells. 3. Role of P2X7 receptor in ECM promoting osteogenic differentiation of periodontal ligament stem cellsMethods: 1.Tissue specific ECM was prepared from human bone marrow cells and periodontal ligament cells in vitro. The human periodontal ligament cells were seeded in the h PDLCs-ECM, h BMCs-ECM, Fibronectin and blank 6-well plates respectively, and then induced by osteogenic induction. We used WESTERN-BLOT, RT-PCR, and alizarin red staining to detect the Osteogenesis effect of the four groups. 2.Human periodontal ligament stem cells were isolated and divided into four groups based on different media, e. g. ?-MEM, osteogenic medium(10-7 M dexamethasone, 10 m M ?-glycerophosphate and 50 mg/L ascorbic acid), osteogenic medium+blocker(100 n M KN-62) and osteogenic medium+activator(100 n M ATP). After induction for 7 days, the P2X7 expression was measured by q RT-PCR and western blot. The alizarin red staining was performed after 21 days induction. 3.The periodontal ligament stem cells were seeded in 6-well plates and h BMCs-ECM to perform the osteogenic induction. After induction for 7 days, the immunofluorescence, RT-PCR and western blot were used to check the expression of OCN, RUNX2 and P2X7. After induction for 21 days, the alizarin red staining was used to detect the mineralization. In the in vivo study, the h PDLSCS sheet was prepared on both normal tissue culture plate surface and ECM surface. The sheets were embedded in combination with HA/TCP granules. After 60 days healing, the samples were isolated, fixed and decalcified to perform histological evaluation.Result: 1.In all the four groups, h BMCs-ECM group after osteogenic induction, the osteogenic related gene and protein was significantly higher than the other three groups, and the expression of P2X7 receptors was also higher than the other three groups 2. Exogenous ATP significantly enhance the osteogenic effect of periodontal ligament stem cells in first 7 days. ATP can activate periodontal ligament stem cells with P2X7 receptor expression. The P2X7 receptor expression and osteogenic differentiation of HPDLSCS were positive correlated. 3.In the in vitro study, the expression of P2X7 receptor, OCN and RUNX2 on ECM surface were higher than on tissue culture plate surface. The alizarin red staining indicated that the mineralization on ECM surface was higher than culture plate. In the in vivo study, the HE staining suggested that the new bone formation in ECM group was significantly promoted.Conclusion: 1.Tissue specific ECM can effectively maintain the osteogenic differentiation potential of periodontal ligament stem cells, and h BMCs-ECM has the best effect, and ECM can increase the expression of P2X7 receptor on the surface of periodontal ligament stem cells. 2. P2X7 receptors are involved in the process of osteogenic differentiation of periodontal ligament stem cells. 3. P2X7 receptor plays an important role in promoting the osteogenic differentiation of periodontal ligament stem cells in tissue specific ECM.