The Role Of The Canonical Wnt Signaling In The Odontogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells In Vitro

At present tooth loss is still a very common dental disease, traditional treatments has been gradually inconsistent with the principles of modern medicine because of the characteristics of damage to adjacent structures. Stem-cell-based tooth tissue engineering has been a much discussed subject, because stem-cell-based therapy for the regeneration of tissue is considered as a desirable approach of future medicine. The cell source for dental regeneration can be divided into two types: dental-derived stem cells and non-dental-derived stem cells. Dental-derived stem cells has a good potential to regenerate tooth tissue, but sometimes the difficult access limits its applications, so that the research of non-dental-derived stem cells have a practical significance. There are many similarities in biological characteristics between bone marrow-derived mesenchymal stem cells and dental-derived stem cells.In this study, we cultured bone marrow-derived mesenchymal stem cells in tooth germ cells conditioned medium, to explore its capacity of the odontoblast-like differentiation. In the same time, the bone marrow-derived mesenchymal stem cells were pretreated by Wnt3a or Dkk-1, to detect the role of the canonical Wnt signaling pathway in the odontoblast-like differentiation of bone marrow-derived mesenchymal stem cells in vitro.The experiment is divided into two parts:PartⅠ: The odontogenic differentiation of bone marrow mesenchymal stem cells in vitro.Objective: To explore the possibility of bone marrow mesenchymal stem cells to differentiate into odontoblast-like cells. Methods: Bone marrow-derived mesenchymal stem cells were isolated from two-week Sprague-Dawley rats and cultured in tooth germ cells conditioned medium. The odontogenic differentiation was observed by immunofluorescence staining and RT-PCR. Results: The immunofluorescence staining showed many of BMSCs expressed dentin sialoprotein (DSP) and dentin matrix protein 1 (DMP-1),which were treated by TGC-CM for 7days; RT-PCR showed BMSCs became to express dentin sialophosphoprotein (DSPP) and DMP-1 mRNA after being induced in TGC-CM. Conclusion: The rat BMSCs induced by the tooth germ cell conditioned medium could differentiate into odontoblast-like cells.PartⅡ: The role of the canonical wnt signaling in the odontogenic differentiationObjective: To study the role of the canonical Wnt signaling pathway in the odontoblast-like differentiation of bone marrow mesenchymal stem cells in vitro. Methods: Bone marrow-derived mesenchymal stem cells were pretreated by Wnt3a or Dkk1 for 48h, then Western blot was done to detect theβ-catenin levels in nucleus; 7 days after being cultured in tooth germ cell conditioned medium, the expression of the odontoblast-specific marker DSPP and DMP-1 was observed by immunofluorescence staining and real-time quantitative PCR. Results: Western blot showed that theβ-catenin level in nucleus of bone marrow-derived mesenchymal stem cells was significantly up/down after treated by Wnt3a/Dkk1; Immunofluorescence staining and real-time quantitative PCR showed that the DSPP and DMP-1 expression was significantly down/up in the group which was pretreated by Wnt3a/Dkk1. Conclusion: The canonical Wnt signaling pathway plays a negative role in the regulation in the process of bone marrow mesenchymal stem cells differentiate into odontoblast-like cells.