Effect Of COL4A2 On The Tissue-specific Extracellular Matrix Involved In Osteogenic Differentiation Of Periodontal Ligament Stem Cells

Background: Periodontitis(PD)is a chronic disease characterized by irreversible and progressive degradation of periodontal tissue,which mainly causes tooth loss and alveolar bone defect.Periodontal ligament stem cells(PDLSCs),as seed cells of periodontal repair,have different ability to repair periodontal tissue defects in different microenvironments of periodontitis.However,existing clinical treatments,such as periodontal flap surgery,guided tissue regeneration,and adding growth factors,can only remove necrotic tissue and control inflammation,which has limited effect on periodontal tissue repair.Fortunately,in recent years,with the extensive application and research of tissue engineering in the regeneration and repair of damaged tissues,more and more attention has been paid to the research of stem cells and biomaterials in the biological and functional regeneration of periodontal composite tissues.There are many kinds of collagen in extracellular matrix,which can regulate the physiological activities of cells,thus affecting the repair ability of tissues.Objective: The purpose of this study was to investigate the effects of COL4A2 in specific extracellular matrix(ECM)on osteogenic differentiation and alveolar bone defect repair of PDLSCs.Contents: 1.To isolate the PDLCs and BMCs,and prepare periodontal ligament cell derived-ECM(P-ECM)and bone marrow cell derived-ECM(B-ECM)and decellurized ECM(d ECM).2.Decellurized P-ECM(P-d ECM)and decellurized B-ECM(B-d ECM)were analyzed by electron microscopy and protein mass spectrometry.3.Regulating COL4A2 in specific ECM,and investigating the effect of specific ECM on proliferation and differentiation of PDLSCs.4.Study on the mechanism of Wnt/β-catenin classical pathway on osteogenic differentiation of PDLSCs in specific ECM.5.Specific ECM combined with PDLSCs and Bio-Oss bone powder was implanted into subcutaneous tissue in immunocompromised mice and maxillary alveolar bone defect of SD rats.After 8 weeks,the specimens were detected by histological or immunohistochemical staining and Micro-CT to observe the growth of transplantation.Methods: The characteristics of PDLSCs with respect to surface markers and multiple-differentiation ability were determined.We prepared the periodontal ligament cell(PDLCs)-derived and bone marrow cell(BMCs)-derived ECM and the related decellularized ECM(d ECM).Transmission electron microscopy(TEM),scanning electron microscopy(SEM),atomic force microscopy(AFM),and protein mass spectrometry were used to distinguish the ECMs.The expression of Type IV collagen 2(COL4A2)in ECM was inhibited by si RNA or activated by lentiviral transfection on BMCs or PDLCs.The stemness,proliferation,and differentiation of PDLSCs were determined in vitro.Different d ECMs under the regulation of COL4A2 mixed with PDLSCs and Bio-Oss bone powder were subcutaneously implanted in immunocompromised mice or in the defects in rat alveolar bone.The repair effects were identified by histological or immunohistochemical staining and Micro-CT.Results: The d ECM derived from BMCs(B-d ECM)exhibited more compacted fibers than that derived from PDLCs(P-d ECM)as revealed by TEM,SEM,and AFM.Protein mass spectrometry showed that COL4A2 was significantly increased in B-d ECM than in P-d ECM.PDLSCs displayed a stronger proliferation,stemness,and osteogenic differentiation ability when cultured in B-d ECM than in P-d ECM.The upregulation of COL4A2 in the P-d ECM by lentiviral transfection increased the mineralization nodules and ALP activity of PDLSCs and the downregulation of COL4A2 in the B-d ECM by si RNA decreased the PDLSCs osteogenic capacity.At the same time,specific d ECM combined with bone powder and PDLSCs were implanted into immunocompromised mice and alveolar bone defects of SD rats.Up-regulation of COL4A2 enhanced the osteogenic differentiation ability of PDLSCs and improved the effect of repairing bone defects,while down-regulation of COL4A2 resulted in the opposite result.After up-regulation of COL4A2,the levels of nuclear and total β-catenin and p-GSK-3β were decreased and the levels of osteogenesis-related proteins were increased,while after down-regulation of COL4A2 resulted in the opposite result which confirmed that the classical Wnt/β-catenin pathway played an important role in the negative regulation of PDLSCs osteogenesis.Conclusion: Our study suggests that COL4A2 of the ECM promotes osteogenic differentiation of PDLSCs through negative regulation of Wnt/β-catenin pathway and can be used as a potential therapeutic strategy to repair the bone defect.