The Study Of Osteogenesis Of Dental Pulp Stem Cells On Micro-arc Oxidation Titanium

Objectives:To explore the osteogenic differentiation potential of rat dental pulp stem cells (DPSCs) on micro-arc oxidation (MAO) titanium surface, and compare osteogenesis with rat bone marrow stromal cells (BMSCs), to provide the theory basis for DPSCs being the new seed cells in implant and tissue engineering combined application.Methods:1. The separation, culturing, identification and inducing differentiation of rat DPSCs. DPSCs were cultured by explants combined with enzyme digestion technique. Single cell line of DPSCs was isolated by limited dilution of cultured cells and identified by immunofluorescence. Inducing differentiation of DPSCs to osteoblast and adipocyte.2. The study of biological activity of DPSCs on MAO titanium surface. Groups are: group DPSCs- MAO, group DPSCs- bone condition induction and group DPSCs- no induction. At different time points during cells cultured, scanning electronic microscopy (SEM), biochemical tests and real-time PCR to observe differentiation potential of DPSCs.3. The osteogenic differentiation potential comparison between DPSCs and BMSCs on MAO titanium surface. Groups are:group DPSCs- MAO, group BMSCs- MAO and DPSCs- smooth. At different time points during cells cultured, SEM, energy dispersive spectroscope (EDS), biochemical tests and real-time PCR to observe the difference osteogenic differentiation potential between DPSCs and BMSCs.Results:1. DPSCs showed a high proliferation rate and arranged closely to form colony, besides the cells expanded as spindle-shape. Immunofluorescence analysis showed that DPSCs expressed CD44, STRO-1 and collagen I while CD34 was absent. DPSCs had the ability to differentiate into osteoblasts and adipocytes.2. SEM observation showed that the surface of MAO film was rough and porous, uniform distribution of 5-12μm microporous. And DPSCs were prone to grow toward micropore. ALP activity of DPSCs- MAO and DPSCs- bone condition induction were no significant difference (P>0.05), but there were significant difference with DPSCs-no induction (P<0.05). Cells cultured 21 days, the expression of Runx2, Osterix, DSPP and DMP-1 were increased between DPSCs- MAO and DPSCs- no induction, as the same between DPSCs- bone condition induction and DPSCs- no induction (P< 0.05); the expression of genes in DPSCs- MAO and DPSCs- bone condition induction were similar (P>0.05). Mineralization ability of DPSCs- MAO and DPSCs- bone condition induction, the calcium nodule area was no significant difference (P>0.05) and DPSCs- no induction was negative.3. MTT results showed that the cell viability of DPSCs- MAO was significantly enhanced compared to the DPSCs- smooth (P<0.05). The cell viability of DPSCs- MAO and BMSCs- MAO was no significance difference (P>0.05). Cells cultured 7 days, SEM showed both cells expressed the trend to differentiation on DPSCs- MAO and BMSCs-MAO. EDS showed that Ca was raised and P was declined because of cells cultured. The results showed higher ALP activity in DPSCs- MAO than in DPSCs- smooth (P< 0.05). Compared DPSCs- MAO and BMSCs- MAO, there was no significance difference except at the 5th and 13th day (P>0.05). The alizarin red positive mineralized area in DPSCs- MAO and BMSCs- MAO were no significant difference (P>0.05). The RT- PCR results showed that the expression levels of Runx-2, Osterix, DSPP and DMP-1 were similar. The expression levels of genes showed no significant difference between DPSCs- MAO and BMSCs- MAO (P>0.05).Conclusion:Rat DPSCs have good biocompatibility and biological activity on MAO titanium which was rough, porous and rich in calcium and phosphorus. The DPSCs differentiation may be regulated by MAO titanium surface. The osteogenesis potential of DPSCs on the MAO titanium like the BMSCs did.