Experiment Studies On The Effects Of MMP-2 Expression Induced By Titanium Particles In MC-3T3-E1 Cells In Vitro

Background: Periprosthetic osteolysis poses a significant complication in patients managed with joint replacements surgeries. It has been generally accepted that wear debris contributes to osteolysis which was critical reason of implant loosening after total joint arthroplasty. However, the molecular mechanism underlying osteolysis still remains to be described. Matrix metalloproteinases 2(MMP-2) was expressed in osteoblasts. It play an important role in bone metabolism, since they are most likely involved in bone remodeling in which bone resorption is balanced with bone formation. But we have little information about the MMP-2 expression in osteoblasts in response to wear-particles, and whether it contributes to periprosthetic osteolysis is largely unknown. In the present study, we investigated the role of the titanium particles in the production of MMP-2, and regulation mechanism in murine calvarial preosteoblastic cells.Objective: To study effects of MMP-2 expression and regulation mechanism in osteoblasts after exposed to titanium particles. The purpose is to better understand the molecular mechanism of periprosthetic osteolysis induced by wear-debris during aseptic loosening.Methods: Firstly, cultured MC3T3-E1 murine calvarial preosteoblastic cells were incubated with endotoxin-free titanium particles with different particles loading. The specimens were collected on the 2nd,4th,6thand 8th days respectively, and the mRNA levels of MMP-2 were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR).Secondly, MC3T3-E1 cells were challenged with titanium particles with 0.1mg/ml concentration in the presence and absence of specific inhibitors of p38 (SB203580). Activation of the mitogen activated protein kinase (MAPK) signal-regulated protein kinase p38 was determined over a time course from 0 to 3 days by Western-blot analysis. It was also tested using immunofluorescence staining. The mRNA level of MMP-2 was analyzed by RT-PCR as well.Results: Titanium particles with endotoxin-free increased the mRNA level of MMP-2 through p38MAPK signaling pathway in MC3T3-E1 cells, suggesting that titanium particles induced MMP-2 gene expression at least at the transcriptional level. Titanium particles induced MMP-2 mRNA expression in the cells reaching a maximum on 2 day, and maintained for 8 day. There was significant difference between every time at each group (F=23.757, P=0.000). The increase in MMP-2 production with titanium particles was also dose-dependent (F=295.097, P=0.000). Active p38 showed an elevated protein expression by Western-blot analysis which was also shown increase in immunofluorescence staining following titanium particles (0.1mg/ml) stimulation. Titanium particles exposure causes an increase in p38 protein expression by 1.5-fold on 1 day and by 1.25-fold on 3 day. Moreover inhibition studies showed that a specific p38 inhibitor, SB203580, completely blocked the increase in active p38 expression induced by titanium particles, meanwhile, the expression of MMP-2 mRNA was inhibited in the presence of SB203580.Conclusions: This study demonstrates that titanium particles increase MMP-2 expression in osteoblasts, and p38 signaling pathway is required for production of MMP-2 in response to titanium particles in vitro. Thus, p38 signaling pathway could play a significant role in contributing to the development of periprosthetic osteolysis.