Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells in vitro

Abstract

To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cells in vitro. Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10 -8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10 -8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance ( A) value at 562 nm by alizarin red staining at 21 days after culture. Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D ( P<0.05), in group C than group A ( P<0.05), and in group A than group B ( P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D ( P<0.05), and in group C than groups A and B ( P<0.05), but no significant difference was found between groups A and B ( P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D ( P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days ( P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D ( P<0.05), and in groups A and B than group D ( P<0.05), but there was no significant difference between groups A and B ( P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. The A values of mineralized nodules showed significant difference between groups ( P<0.05). BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.