Preliminary Investigation Of BMP-2 Influence On Osteogenic And Cementogenic Differentiation Of Human Periodontal Ligament Cells

Objective:The influence of different concentrations of BMP-2 on osteogenic and cementogenic differentiation of human periodontal ligament cells is experimentally investigated in vitro, which aims to clarify its mechanism. Meanwhile, the relationship between their reaction time and cell differentiation is also discussed to explore whether there is an optimal period. It could provide theoretical support to the clinical application of BMP-2 in periodontal tissue regeneration therapy. Methods:1. Cultivation of h PDLCs:Tissue cultivation is utilized to cultivate h PDLCs, followed by observation of cell growth and curve plot.2. Identification of h PDLCs:Cell phenotype is analyzed by flow cytometry(FCM) and cell sources are identified by immunocytochemistry of vimentin and cytokeratin.3. Proliferation of h PDLCs:Periodic variation of cells is analyzed by flow cytometry(FCM).4. Cementogenic Differentiation of h PDLCs:Variation of m RNA and protein expression of related gene in h PDLCs, treated by BMP-2 treatment, is measured. CAP and m RNA expressions are analyzed by quantitative PCR. While CAP and CP-23 protein changes are investigated by Western blot.5. Osteogenic Differentiation of h PDLCs:Variation of m RNA and protein expression of related gene in h PDLCs, treated by BMP-2 treatment, is measured. RUNX2 and COL-1 m RNA expressions are analyzed by quantitative PCR. While EGFR and COL-1 protein changes are investigated by Western blot.Results:1. h PDLCs cultivated have a good ability of cell dryness, high proliferation and active growth.2. The influence of BMP-2 on h PDLCs proliferation: according to the result obtained by flow cytometry, the majority of the cells(92.31%)were in the stationary stage and the early one of DNA synthesis(G0/G1 stages).3. The influence of BMP-2 on cementogenic differentiation:1) The results obtained by quantitative PCR show that under the condition of 20ng/ml BMP-2, expression of CAP m RNA increases a lot and reaches the peak on the 5th day, which is statistically valid(p<0.05).2) The results obtained by western blot show that under the condition of 20ng/ml and 100ng/ml BMP-2, CAP and CP-23 in h PDLCs expresses more, which is also statistically valid(p<0.05).4. The influence of BMP-2 on osteogenic differentiation:1) The results obtained by quantitative PCR show that under the condition of 80ng/ml BMP-2, expression of COL-1 increases a lot and reaches the peak on the 7th day, which is statistically valid(p<0.05); under the condition of 100ng/ml BMP-2, expression of RUNX2 m RNA enhances with time. It stands statistically(p<0.05).2) The results obtained by western blot show that under the condition of 100ng/ml BMP-2, EGFR and COL-1 expresses more, which is also statistically valid(p<0.05). Conclusion:1. BMP-2 has a positive influence to osteogenic and cementogenic differentiation of periodontal ligament cells. At the low concentration(20ng/ml), it can enhance the cementogenic differentiation while at high centration(100ng/ml), it motivates the osteogenic one;2. The stimulation time of BMP-2 on h PDLCs is valid statistically(p<0.05) and an optimal period exists. Cementogenic differentiation reaches the peak on the 5th day and osteogenic differentiation reaches the peak on the 7th day. This is important to BMP-2 clinical treatment.