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Experimental Research of Differentiation of BMMSCs into Odontoblast-like Cells Induced by Natural Dentin

Author: LeiGang
Tutor: ZhangGuangDong；YuJinHua
School: Nanjing Medical University
Course: Clinical Stomatology
Keywords: Bone marrow mesenchymal stem cells Dentin Pulp Odontoblast
CLC: R781
Type: Master's thesis
Year: 2011
Downloads: 23
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Abstract

Objective: use of natural dentin to odontoblast-like cell differentiation induced bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMMSCs), looking for the source of seed cells for new dental tissue regeneration and endodontic from the traditional physical restorative treatment to provide a theoretical basis to the changes in the biological treatment. Methods: whole bone marrow adherent method to obtain rat bone marrow mesenchymal stem cells and their tissue of origin and differentiation capacity preliminary identification, and take 3 ~ 5 cells were used for subsequent experiments. In vitro experiment: Take 1 × 104 BMMSCs cells dentin pieces were cultured in 24-well plates, culture fixed after 2 w, demineralization, embedded serial sections by hematoxylin - eosin (hematoxylin-eosin, HE) Masson tri-color method and immunohistochemical staining (immunohistochemistry, IHC) cells around the dental film growth conditions, expression of the histological changes and dentin sialoprotein (dentin sialoprotein DSP). Transplanted into rats in vivo: Collect 5 106 BMMSCs cells and dentin chip composite renal capsule, 6 w after drawn, fixed, demineralization, embedding, serial sections by HE, Masson tri-color method and IHC staining cells was detected in the dental films around the morphological changes and the expression of DSP. In addition, the extracted sample dentin chip soft tissue total RNA and total protein were subjected to reverse transcription-PCR (reverse transcription-polymerase chain reaction, RT-PCR) and Western blot (Western blot) detection dentin sialophosphoprotein protein gene (dentin sialophosphoprotein, DSPP) and dentin sialoprotein (DSP) expression. Experimental results: in BMMSCs cultured rat cells adherent growth of fibroblast-like cells, the cytokeratin negative waveform silk protein positive staining proved mesenchymal source of cells, and induction microenvironment multi-differentiation capacity successfully established a culture system of rat BMMSCs. The BMMSCs dentin slices in vitro co-culture after 2 w observation, found that cells in the growth of dentin film surface, some cells appear polar growth and cell processes can be seen inside the dentinal tubules, and anti-DSP antibody staining were positive. The BMMSCs dentin slices in vivo transplantation 6 W dentin sheet around a typical layer of high columnar polarity arranged cells, some cells extend protrusions into the dentinal tubules, here cells DSP staining was positive expression. Dentin slices or the BMMSCs cell group after transplantation alone were not found in similar cells. Detected by RT-PCR and Western Blot also found the experimental group dental film the soft tissue Dspp and DSP expression was significantly higher. Conclusion: The established rat BMMSCs training system; natural dentin can be induced by in vivo and in vitro co-culture form BMMSCs to odontoblast-like cell differentiation and expression of specific markers DSPP / DSP.

Experimental Research of Differentiation of BMMSCs into Odontoblast-like Cells Induced by Natural Dentin

Abstract

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