Experimental Research Of Differentiation Of BMMSCs Into Odontoblast-like Cells Induced By Natural Dentin

Objective: To induce bone marrow mesenchymal stem cells (BMMSCs) to differentiate into odontoblast-like cells by use of natural dentin. And then, provide the theoretic basis for finding new seed cell sources in tooth regeneration and change of the pulp treatment approaches from traditional obturation therapy to biological treatment.Methods: BMMSCs were harvested by the whole marrow method and preliminary identified according to the tissue origin and multiple differentiation potential. Cells at the 3rd ~ 5rd passages were collected for subsequent experiments. For in vitro studies, 1×104 cells were seeded on the surface of the dentin slices and co-cultured for 2 weeks in the 24-well plates, then fixed, decalcifid, embedden in paraffin and prepared the serial sections for subsequent experiments. Hematoxylin-eosin (HE) and Masson trichrome staining were used to evaluate the growth and morphology of BMMSCs on the dentin slices. The protein expression of dentin sialoprotein (DSP) in co-cultured BMMSCs was detected by immunohistochemistry (IHC) staining. For in vivo studies, 5×106 cells were collected as cell pellets, combined with dentin slices and then incubated in renal capsules for 6 weeks. Retrieved implants were fixed, decalcified, embedded in paraffin and prepared for serial sections. Morphology and DSP expression of these retrieved tissues were detected by HE, Masson trichrome and IHC staining. Total RNA and protein were extracted from some recovered tissue. Dspp/DSP expression in the retrieved samples was respectively investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Results: BMMSCs separated from rat bone marrow were fibroblast-like cells, in which Vimentin was expressed, while Keration was not expressed, indicating these stem cells were of mesenchymal orgin. Moreover, these stem cells had the multi differentiation potential in different induction medium. These data implied that rat BMMSCs culture system was successfully established. After 2 weeks co-culture with dentin slices, BMMSCs adhered and grew well on the dentin surface, some of them were polarized and extended their cytoplasmic processes into dentine tubules. Besides, co-cultured BMMSCs were positive with DSP. Recombination of BMMSCs with dentin slices showed similar morphological changes and DSP expression in the cells near dentin slices. The findings of PT-PCR and Western Blot also demonstrated that the expression of Dspp/DSP in the co-cultured BMMSCs groups were obvious higher than the control groups.Conclusion: The culture system of rat BMMSCs was successfully established. Natural dentin can induce BMMSCs to differentiate along the odontoblast-like cell lineages and express odontoblast-specific Dspp/DSP in vivo and in vitro.