RANKL/OPG Expression Induced By Metal (Cobalt,Chromium) Ions In Preosteoblastic Cells And The Molecular Mechanisms Of Signal Transduction

Objective:Through evaluation the effects of cobalt(Co²⁺) , chromium (Cr³⁺)ions on the expression levels of RANKL,OPG in osteoblast(MC3T3E1) and the role of ERK signaling pathway in this process, to investigate the relationship between metal ions and aseptic loosening and provide the theoretical basis to prevent the periprosthetic osteolysis.Methods:Osteoblasts were cultured in vitro and divided into 3 groups, According to the different solution. Group A: osteoblast was culture with normal saline ; Group B: osteoblast was cultured with cobalt(Co²⁺) and chromium (Cr³⁺)ions solution. Group C: osteoblast was cultured with Co²⁺,Cr³⁺ solution and inhibitor of ERK signaling (PD98059); cells viability and the expression of RANKL and OPG on the protein and gene levels were assessed by MTT, ELISA assays and RT-PCR at different time point.Results:MTT test demonstrated that Co²⁺ and Cr³⁺ ions can obviously decrease the osteoblast viability. ELISA revealed that at 24h and 48h after cultured , the expression of RANKL in Group B and C were significantly higher than that in Group A, the RANKL expressions were 61.6 times and 13.8 times as common in Group B as in Group A, and that were 44.4 times and 10.5 times as common in Group C as in Group A.the releasion of OPG in Group B and C had been increased respectively by 32.1% and 30.4% as compared with Group A at 24h and showing statistically significant difference(p <0.05),and 48 hours later, the secretion of OPG in Group B and C increased respectively by 17.8% and 19.8%(p <0.05). RT-PCR revealed that the gene expression of RANKL and OPG could found in three groups at 24 and 48 hours after cultured, Group B and C were higher than the Group A,especially the RANKL, and the difference has statistical significant (P < 0.05) ; the expression of RANKL mRNA in the Group C decreased obviously than those in Group B at 24 and 48 hours after cultured and the difference has statistical significant (P < 0.01),but the expression of OPG mRNA decreased slightly and the difference has statistical significant (P < 0.01).Compared to Group A,the ratio of RANKL/OPG in Group B and C elevates obviously, no matter in the protein level or the gene ,and which in Group C decreased obviously than that in Group B, but that was still higher than that in Group A.Conclusion:Co²⁺ and Cr³⁺ ions have a cytotoxic effect on osteoblasts. It can stimulate the expression of RANKL and OPG of the osteoblast on the protein and gene level and upregulate the the ratios of RANKL/OPG. inhibitor of ERK signaling(PD98059)could downregulate the expression of RANKL and the ratios of RANKL/OPG to a certain degree, so that may decrease the occurrence of the periprosthetic osteolysis.