Isolation, culture and biological characteristics of high-purity orofacial-bone-derived mesenchymal stem cells of the rats / 中国组织工程研究

ABSTRACT

BACKGROUND: It is widely accepted to recruit mesenchymal stem cells from the jaws to repair the defects of the jaws. However, there are relatively few researches on orofacial-bone-derived mesenchymal stem cells, mainly due to the difficulty in separating mesenchymal stem cells from the jaws. OBJECTIVE: To establish the methods of in vitro isolation and culture of rat orofacial-bone-derived mesenchymal stem cells and observe and study its related biological characteristics. METHODS: The mandibles of rats were dissected. The attached muscles were stripped and cut into pieces. Cortical bone was loosened by digesting with collagenase II. The migration and adherent growth ability of mesenchymal stem cells was used to isolate orofacial-bone-derived mesenchymal stem cells. Cell morphology was observed by inverted microscope. Surface markers of the cell were detected by flow cytometry. Cell proliferation was detected by MTT assay and cell growth curve was drawn. Fibroblast colony forming rate was calculated by colony formation. Osteogenic and lipogenic induction experiments were conducted to study the multi-directional differentiation potential of cells. RESULTS AND CONCLUSION: Cells isolated by collagenase digestion and bone slice culture were positive for CD29, CD44 and Sca-1, and negative for CD31, CD34 and CD45. Cell proliferation test showed that the growth curves of orofacial-bone-derived mesenchymal stem cells exhibited incubation period, logarithmic phase and platform period. In addition, the cells had a strong ability of proliferation, and the cell clone formation rate was 20% and the cells in DNA synthesis stage accounted for 52.5%. Alizarin red and oil red O staining showed positive reaction after osteogenic and lipogenic induction, indicating that the cells have the potential of multi-directional differentiation. It is concluded that the method of bone fragment culture after digestion with collagenase II could separate orofacial-bone-derived mesenchymal stem cells sufficiently and purely. Besides, the orofacial-bone-derived mesenchymal stem cells show strong proliferative and osteogenic differentiation capacities. Thus, it provides abundant source of seed cells for bone tissue engineering of maxillofacial represented by bone defects repairing of implants.