Research On WNT/β-Catenin In Signaling Acting Synergistically On BMP9-Induced Osteo/Odontoblastic Differentiation Of Stem Cells From The Apical Papilla(SCAPs)

The dental caries, endodontium diseases and periapical diseases which have high incidence in children not only affect the children’s chewing function and pronunciation, endanger the normal growth of teeth and dentition, and seriously influence children’s physical and mental health. The children are in the growth and development period, so the tooth apical is mostly immature and the apical foramen is open. Be different from the adult treatment programs, the emphasis of children treatment focuses on promoting the normal development of the apical root hard tissue including dentin, cementum, alveolar bone, to restore normal function of the diseased tooth. Traditional drug therapy has some damage to the tooth surrounding tissue, so the more safe and effective clinical treatment should be needed to promote the periapical tissue regeneration. For the past few years, the study of stem cell technology and tooth tissue engineering provides a new thought for the development of children’s dental treatment.The Stem Cells from the Apical Papilla (SCAP) has the self-renewal ability and multilineage differentiation potential. Research shows that SCAP is the main source of odontoblast cells in the root development and the ideal seed cell of the apical tissue regeneration. But in view of the limited cell sources, lower quantity, easily differentiation, the immortalized stem cells from the apical papilla (iSCAP) are used in this project, which hold the sufficient number of cells and good biological properties.Because the stem cells can differentiate into multilineages, looking for the cell factors to promote osteogenic differentiation becomes a research hotspot. Wnt and Bone Morphogenetic Protein (BMP) signalling pathways play the important roles in embryonic development, organ formation and tissue regeneration, especially in the osteogenic differentiation. At present, more than 20 kinds of Wnt genes and more than 14 kinds of BMP genes have been isolated. We have made a comparative analysis that Wnt3 and Wnt3A could show the strongest transcription activation of P-catenin, BMP9 hade the strongest ability to induce the mesenchymal stem cells into osteogenic differentiation in vitro and in vivo. However, the role and mechanism of the crosstalk between Wnt and BMP is unclear to induce the SCAP cells into osteogenic differentiation.The purpose of this study is:(1) to analyze whether Wnt3A and BMP9 could induce iSCAP cells into osteoblast-like cells; (2) to investigate whether P-catenin plays a key role in the osteogenic differentiation of iSCAP induced by Wnt3A and BMP9; (3) to explore the synergistic effect and mechanism of Wnt3A and BMP9 in the osteogenic differentiation.The first part:Firstly the pMPB-simBC plasmid containing 3 interference fragments of silencing P-catenin was transfected into iSCAP cells (iSCAP-KD), the real-time PCR showed the expression of P-catenin mRNA was significantly reduced. The iSCAP-Ctrl and iSCAP-KD were infected with Ad-Wnt3A separately, pTOP-Luc luciferase reporter gene assay showed in iSCAP-KD group the transcriptional activity of β-catenin was significantly decreased; real-time PCR showed the expression of c-Myc mRNA--the downstream target genes of Wnt3A pathways decreased after silencing β-catenin; the immunofluorescence staining showed compared with iSCAP-Ctrl group, in iSCAP-KD group the intranuclear transferation of β-catenin was significantly reduced and the expression intensity in cytoplasm decreased. Next, the osteogenic differentiation was analyzed. Wnt3A could promote the ALP activity and expression which was the early osteogenic differentiation index in a dose-dependent manner, the ALP activity and expression in iSCAP-KD Group was significantly lower than the control group; the calcium deposition by Alizarin Red staining and expression of osteopontin (OPN) and osteocalcin (OCN) by Real-time PCR which were all the later osteogenic differentiation index were not significantly different between iSCAP-Ctrl and iSCAP-KD. The results showed Wnt3A could promote iSCAP into osteogenic differentiation in the early phase, but not in the late phase.The second part:Firstly the iSCAP-Ctrl and iSCAP-KD were infected with AdGFP or AdBMP9 separately to detect the osteogenic differentiation. BMP9 could promote the ALP activity and expression and the calcium deposition in a dose-dependent manner, but the level in iSCAP-KD group was significantly lower than the control group. Secondly, to verify the synergistic effect of inducing iSCAP into osteogenic differentiation mediated by Wnt3A and BMP9 in vitro and in vivo. The results showed, the ALP activity induced by Wnt3A and BMP9 combination was significantly increased than single infection in a dose-dependent manner, but silencing P-catenin could significantly weakened this synergistic osteogenesis effect.In vivo, the ectopic bone formation induced by Wnt3A and BMP9 combination showed the increased number of bone trabecula and good bone matrix continuity, Masson’s Trichrome staining showed the increased mature and mineralized bone matrix. Finally, AdWnt3A, AdBMP9 and Adβ-catenin infected separately the iSCAP-Ctrl and iSCAP-KD cells transfected with a luciferase reporter gene p6OSE2-Luc, showed the transcription activity of Runx2 all increased in the three kinds, however the β-catenin silence couldn’t activate Runx2.In summary, Wnt3A and BMP9 could induce iSCAP cells into osteogenic differentiation, their combination is more synergistic effect, silencing β-catenin gene significantly inhibit the osteogenic expression, and Runx2 activation could be the key intersection of promoting osteogenic differentiation synergistically mediated by Wnt3A and BMP9 signaling pathway.