Observations on the effect of BMP-2 on rat bone marrow cells cultured on titanium substrates of different roughness

Abstract

The objective of this study was to examine the osteoinductive capacity of different concentrations of BMP-2 on bone marrow stromal cells in vitro. Further, we intended to determine whether titanium provided with an increased surface roughness is more efficient in osteoblast differentiation than machined titanium. Therefore, 20,000 cells/ml were seeded and cultured on machined and grit-blasted titanium discs for 4, 8 and 16 days. Different concentrations of rhBMP-2 (0, 10, 100, 1000 ng/ml) were supplemented to the medium for 8 days of culturing. To evaluate cellular proliferation and differentiation, specimens were examined for DNA, alkaline phosphatase activity, and calcium content. Morphological appearance of the specimens at 8 and 16 days of incubation was evaluated using scanning electron microscopy. Two separate experimental runs were performed. Evaluation of the DNA and alkaline phosphatase data revealed that a significant difference existed for these data between both experimental runs. Further analysis of the DNA figures learned that roughening of the titanium surface and addition of BMP-2 had no effect on cell proliferation. The alkaline phosphatase analysis and calcium measurements revealed that BMP-2 stimulated the early differentiation of osteogenic cells on machined titanium substrates in a dose-dependent manner. After 16 days of culture, no significant differences in calcium content could be observed anymore between machined and roughened titanium surfaces. Further, the data revealed that the machined surfaces showed a significant increase in calcium deposition when 100 and 1000 ng/ml BMP-2 were supplemented to the medium. However, the roughened surfaces showed this significant enhancement in calcium content only with 1000 ng/ml BMP-2. In addition, SEM evaluation revealed a dose-dependent response to BMP-2. Increasing BMP-2 concentrations resulted in more calcified globular accretions on bone surfaces than when no BMP-2 was added. On the basis of our results, we conclude that (1) due to the heterogeneous nature of bone marrow, experimental results with primary rat bone marrow cells are difficult to reproduce from one experiment to the other, and (2) addition of rhBMP-2 in the medium stimulates the early differentiation and matrix mineralization of osteogenic cells on machined titanium surfaces in a dose-responsive manner. Further, we concluded that our roughened titanium surfaces had no effect on proliferation and differentiation of primary derived rate bone marrow cells.