MG 63 cells were incubated with EMD protein in the presence or absence of SB203580, and were then cultured on DQ gelatin matrix. Consistent with results in Figure 1, EMD protein enhanced the deg radation of gelatin, as com pared with unstimulated MG 63 cells. When SB203580 was co incubated with EMD protein, Perifosine side effects degradation of gelatin was stopped, as demon strated by a significant decrease in specific fluorescent sig nals. These findings suggest that MMP 2 is a major gelatin degrading enzyme pro duced by EMD protein stimulated osteoblasts. These results also suggest that EMD protein stimulates matrix degradation involved in the catalytic activity of MMP 2. Furthermore, our study highlights the pivotal role for p38 MAP kinase pathways in inducing MMP 2 production from EMD protein stimulated osteoblasts.

Discussion In this study, we showed that EMD protein stimulates osteoblasts to degrade gelatin in vitro and that production of MMP 2 is up regulated in response to EMD protein stimulation. Our results also suggest that adhesion to gelatin enhances production of and activates pro MMP 2, which is spontaneously produced by osteoblasts. EMD protein stimulates p38 MAPK signaling pathways, which in turn, induce MMP 2 production by osteoblasts. Al though previous studies have shown that EMD protein applied to periodontal defects is absorbed into the denuded root dentin surface and induces periodontal tissue regener ation, the mechanisms of this action remain largely unknown. In this regard, our results show the pivotal role of MMP 2 produced from osteoblasts in response to EMD protein in facilitating periodontal connective regeneration.

MMP 2 plays a crucial role in bone remodeling and mineralization, and several studies have assessed the functional roles of specific MMPs. Our results further support the importance of MMPs produced by EMD protein stimulated osteoblasts in bone remodeling. The observations of residual degradation of ECM in the presence of TIMP 2 may be explained by the involve ment of other proteases such as serine protease. Indeed, recent studies have shown that osteoblasts express surface serine protease. However, the significant inhibition of gelatin degradation by TIMP 2 suggests that EMD protein enhances the production of MMPs on cell surfaces, which facilitates osteoblast mediated gelatinolysis.

In the present study, we demonstrated that MMP 2 is spontaneously pro duced from unstimulated osteoblasts, and that gelatinase is activated in the presence of EMD protein. In contrast, pre vious studies have shown that gelatinase MMP 9 was not produced by EMD protein activated osteoblasts. EMD protein contains both Brefeldin_A TGF B and BMP like growth factors, which contribute to the induction of biomineralization during periodontal regeneration.