The Regulatory Mechanisms Of β-catenin In Odontoblastic Differentiation Of Dental Pulp Cells

Dental pulps have regenerative capacity to form reparative dentin in cases of tooth injury. Intense stimuli, such as cavity preparation and advanced dental caries, may cause death of odontoblasts and stimulate odontoblastic differentiation of the stem/progenitor cell populations of dental pulp cells (DPCs), which replace the necrotic odontoblasts; this is followed by reparative dentin formation. The odontoblastic differentiation of DPCs in response to tooth injury is essential to the reparative dentinogenesis of DPCs. Previous studies suggested that dentin-like structures lined with odontoblast-like cells could be generated by isolated DPCs. Therefore, delineation the mechanism of odontoblastic differentiation of DPCs will be helpful for developing more biologically based strategies to treat dental tissue injury in clinics.Wnt/β-catenin signaling pathway plays crucial roles in the development of many self-renewing organs such as bone, gut, and skin and is required for the maintenance of homeostasis in these organs, β-catenin, as the central component of the Wnt/β-catenin pathway, is the bottleneck through which all signals pass. Especially, β-catenin has also been found to have a central role in tooth development. For instance, inactivation of β-catenin in mesenchyme of developing tooth results in arrested tooth developmental at the bud stage, while forced P-catenin activation in embryogenesis or post-natal life causes ectopic tooth formation. There are studies also showing that tooth development and dental repair share some common molecular mechanisms. Additionally, robust studies have demonstrated that osteoblast differentiation, chondrocyte differentiation and adipocyte differentiation of stem/progenitor cells can be regulated by β-catenin. Given these previous findings, we hypothesize that P-catenin may participate in odontoblastic differentiation during reparative dentin formation.β-catenin regulates a number of genes in various biological processes. Among those, Runx2is a transcriptional factor and known master regulator in controlling osteoblast and odontoblast differentiation. It has been shown that expression of genes that are required for osteoblastic or odontoblastic differentiation is regulated by Runx2. For instance, Runx2activates the transcription of the DSPP gene, which encodes two major dentin specific proteins DSP and DPP. Additionally, we and others have shown that Runx2was upregulated during odontoblastic differentiation. Although the important role of Runx2in osteoblastic and odontoblastic differentiation has been well appreciated, how Runx2itself in these processes is regulated remains unclear. Because β-catenin binds the Runx2promoter and control its transcription, in this study, we aim to define the role of β-catenin in odontoblastic differentiation during reparative dentin formation and determine if such a role is fulfilled through activation of Runx2by P-catenin.The present study, divided into three parts, was performed to define the regulatory mechanisms of β-catenin in the odontoblastic differentiation of dental pulp cells during reparative dentin formation.Part one: Expression pattern of β-catenin during reparative dentin formationObjective: The purpose of this part of our study was to identify the expression pattern of β-catenin during the reparative dentin formation.Methods:24male Wistar rats (9weeks old, weighing200-250g) were intraperitoneally anaesthetized with urethane. The maxillary teeth were cleaned and disinfected, then class V cavities with1mm diameter were prepared on the mesial surfaces of the maxillary first molars. The cavities were then slightly perforated. The bleeding was slight and stopped in several seconds by the pressure of a sterile cotton pellet. Mineral trioxide aggregate (MTA) was placed on the perforation sites and then the cavity was sealed with glass ionomer cement. Eight rats of each group were sacrificed on day0,7and14after tooth preparation. The maxillary first and second molars were dissected immediately after sacrifice and then fixed with4%paraformaldehyde. The samples were then demineralized, dehydrated, and embedded. Sagittal sections were obtained for hematoxylin-eosin (H&E) staining, immunohistochemical staining.Results:Immediately after tooth preparation (day0), the odontoblast layer with weak β-catenin expression was detached from the dentin surface and the cells beneath the perforation site were irregularly arranged. At7days post-preparation, reparative dentin was formed beneath the perforation site and a few odontoblast-like cells were surrounding the reparative dentin. At this stage, β-catenin was expressed not only in odontoblasts but also highly expressed in odontoblast-like cells and DPCs underneath the perforation. Two weeks after tooth preparation, there was formation of dentine bridge-like calcified tissue underneath the perforation site. We also found that some tall columnar odontoblast-like cells were lined along the inner surface of the calcified tissue, with some being entrapped in the calcified tissue. At this stage of reparation, β-catenin was found abundantly expressed in odontoblast-like cells and pulp cells beneath the perforation site. Additional, β-catenin was translocated into the nuclei of the odontoblast-like cells lining the reparative dentin.Conclusion:These data suggest that P-catenin participates in the odontoblastic differentiation of the DPCs during reparative dentin formation.Part two:The role of β-catenin on the odontoblastic differentiation of human dental pulp cellsObjective: The purpose of this part of our study was to explore the role of β-catenin on the odontoblastic differentiation of human dental pulp cells.Methods:The human DPCs were isolated and treated with odontoblastic induction medium. Alizarin Red staining, alkaline phosphatase (ALPase) activity, Quantitative real-time PCR (Q-PCR) and Western Blot were performed to evaluate the in vitro odontoblastic differentiation. Knockdown of β-catenin by lentivirus-mediated shRNA and accumulation of P-catenin by lithium chloride (LiCl) treatment were performed to determine the function of β-catenin in odontoblastic differentiation.Results:Odontoblastic differentiation of human DPCs was confirmed by mineralization nodules formation and increased ALP activity in differentiated cells. The expression of β-catenin was significantly upregulated during odontoblastic differentiation of DPCs. Knockdown of β-catenin inhibits odontoblastic differentiation of DPCs. β-catenin accumulation induced by LiCl treatment promotes odontoblastic differentiation of DPCs.Conclusion:β-catenin can promote the odontoblastic differentiation of human DPCs.Part three:β-catenin enhances odontoblastic differentiation of DPCs through activation of Runx2Objective: The purpose of this part of our study was to make sure whether β-catenin promote odontoblastic differentiation of human dental pulp cells through activation of Runx2.Methods:The expression pattern of Runx2was determined by immunohistochemical staining and immunofluorescence staining during reparative dentin formation. The expression of Runx2was also identified by Q-PCR and Western Blot during odontoblastic differentiation of human DPCs and during odontoblastic differentiation in company with β-catenin knockdown or β-catenin accumulation. Chromatin immunoprecipitation was performed to detect the changes in binding of β-catenin to the promoter of Runx2.Results: The expression pattern of Runx2was similar to that of β-catenin. Immunofluorescence staining indicated that Runx2was also expressed in β-catenin-positive odontoblast-like cells and DPCs during reparative dentin formation. β-catenin knockdown resulted in a reduction of β-catenin binding to Runx2promoter region and reduced expression of Runx2. Consistently, accumulation of β-catenin produced opposite effect to that caused by β-catenin knockdown.Conclusion:It was reported in this study for the first time that β-catenin can enhance the odontoblastic differentiation of DPCs through activation of Runx2, which might be the mechanism involved in odontoblastic differentiation during reparative dentin formation.