CCN3Coordinates Proliferation And Odontoblastic Differentiation Of Dental Pulp Stem Cells Via Modulating Notch And BMP Signaling Pathway During Reparative Dentinogenesis

Backgroud:Dentinogenesis occurs with terminal odontoblastic differentiation during embryonicdevelopment and continues throughout the whole tooth life. Primary dentinogenesis isformed by the active secretory odontoblasts during the crown and roots formation.Secondary dentinogenesis follows the completion of root formation, and the secretoryactivity of odontoblasts is obviously reduced to near quiescent. When tooth development iscomplete, dentin homeostatic and self-protective mechanisms are maintained by thedentin-pulp complex, which is involved in the reparative dentin formation (tertiarydentinogeneis) after dentin injury to protect dental pulp from external damage. Thepost-natal dental pulps contain stem cells that are responsible for the repair of damageddentin and pulp tissues under the cavities and trauma stimulation. Dental pulp stem cells(DPSCs), with clonogenic ability, high reproductive activity and multiple differentiationpotentials, have been demonstrated to be an extremely crucial cell element for dental tissuerestoration. Upon dental tissue damage, DPSCs recruit, migrate, proliferate and differentiateinto odontoblasts, which then synthesize matrix to form the tertiary dentin at the damagedsites.Reparative dentinogenesis is orchestrated by many signaling factors. Recent years, newmethods of dentin regeneration treatments have been based on the understanding of themolecular and cellular mechanisms required for the dentinogenesis during dentalregeneration and their potential for clinical exploitation. During tooth morphogenesis oftooth development, numerous signal molecules have been associated with the reciprocalepithelial-mesenchymal interactions. Notch receptors as well as its ligands were alsoexpressed both in epithelial and mesenchymal tissues in the stages of tooth morphogenesis. Notch signaling has been indicated to be associated with the fate determination of dentalepithelial cells leading to the terminal differentiation during odontogenesis. Notch receptorswere weekly expressed in normal adult dental tissues, whereas their expression was stronglyupregulated after dentin injury. Notch1was expressed in the stem cell compartment ofdental pulp, and the expression of Notch2was observed in subodontoblasts andodontoblasts during denting repair. Bone morphogenetic protein (BMPs) were identified tobe crucial signal in the transmission of inductive interactions between the dental epitheliumand mesenchyme. BMP2transcripts have been demonstrated to be confined in dentalpapillae, and then increased remarkably in odontoblastic differentiation process.Inflammatory reaction, as a result of dentin injury, was inherently linked to the dentinregenerative process. The inflammatory products during wound repair and healing havebeen observed to interact with BMPs and affect their biologic potential. However, thepivotal activation factors of Notch and BMP signaling that stimulate the proliferation andodontoblastic differentiation of DPSCs for dentin repair still remain unknown.Nephroblastoma overexpressed (NOV, or CCN3) was firstly characterized as a promoterof progenitor activity of human hematopoietic stem cells, as knockdown of CCN3canabrogate the function of primitive progenitors. Later studies showed that CCN3is alsoactively involved in the process of wound healing. CCN3is highly expressed in granulationtissues of cutaneous wounds and capable of inducing synthetic responses of fibroblasts. Wehypothesized that CCN3may actively participate in the modulation of DPSCs functions andplay an important role in regulating dental tissue regeneration.Methods:To study tertiary dentinogenesis in response to injury, an exposed dentin cavities werecreated in rat incisor by diamond cylindrical burs under aseptic conditions. Specificalexpression of CCN3was profiled in the potential population of cells in dental pulp. HumanDPSCs was infected by lenti-virus vector for ectopic expression of CCN3(DPSCs/CCN3).The effects of over-expression of CCN3on the proliferation were detected by cell cycle assay,CCK-8analysis and PCNA expression determination. Impacts of ectopic expression of CCN3on odontoblastic differentiation of DPSCs were examed by alkaline phosphatase activity,Alizarin red S staining and calcium quantification. Expression of Notch signaling pathway and Runx2were detected by qRT-PCR and Western-blot. Expression of BMP2as a knownligand responsible for odontoblastic differentiation of DPSCs was evaluated by qRT-PCR andWestern-blot. DPSCs/CCN3and DPSCs/vector were mixed at a ratio of1:4and co-culturedin odontoblastic differentiation medium, and the odontoblastic differentiation of DPSCs wereassayed by alkaline phosphatase activity, Alizarin red S staining and calcium quantification.DPSCs/CCN3and DPSCs/vector were seeded together into porous PLGA scaffolds andinduced the cell-scaffold constructs in vitro for2weeks and subcutaneously transplanted theconstructs into nude mice and the mineralization was evaluated by von kossa staing. FinallyDPSCs were trated by BMP2(3.5nM). Expression of CCN3was detected by qRT-PCR andWestern-blot.Results:Transient expression of CCN3is observed during reparative dentinogenesis.Over-expression of CCN3in human DPSCs promoted proliferation of human DPSCs throughelevated Notch signaling and the over-expression of CCN3inhibited odotoblasticdifferentiation of DPSCs through elevated Notch signaling. Then Over-expression of CCN3was found to up-regulate BMP2signaling through increased BMP2secretion. DPSCs/CCN3promoted odontoblastic differentiation through BMP signaling in non cell autonomousmanner. DPSC/CCN3was capable of enhancing mineralization in vivo model. BMP2inhibited CCN3expression and Notch signaling.Conclusion:1. This study indicated that CCN3specifically express in dentin stem cells and wassignificantly up regulated during recovery process.2. Over-expression of CCN3leads to elevated proliferation and attenuated odontoblasticdifferentiation of DPSCs through elevated Notch signaling pathway.3. CCN3promoted BMP2expression of DPSCs. And the elevated Notch signaling wasable to suppress the effect of BMP signaling, to prevent odontoblastic differentiation.4. Elevated BMP2secretion mediated by over-expression of CCN3is able to promoteDPSCs differentiation through non cell autonomous manner.5. Over-expression of CCN3can promote dentin regeneration in a co-culture mode (containing DPSCs with over-expressed CCN3and DPSCs negative control at1:4ratioseeded into PLGA scaffolds) in vivo.