The Role Of RANKL/RANK/OPG System In Periprosthetic Osteolysis With Septic Loosening

Imbalanced expression of RANKL/RANK/OPG system is closely related to periprosthetic osteolysis with aseptic loosening after Total Hip Athroplasty(THA), which has been confirmed by the extensive literature. However, the pathogenesis of periprosthetic osteolysis with septic loosening is rarely reported. The purpose of this study was to investigate whether the RANKL/RANK/OPG system is associated with the incidence of periprosthetic osteolysis with septic loosening, by detecting the expression of the RANKL/RANK/OPG signaling pathway in vitro and interface membrane.Part Ⅰ The Expression of RANK in MacrophagesObjective To investigate differences of RANK mRNA expression in macrophages with UHMWPE particles or thrombin treated.Methods U937monocytes were induced to macrophages with PMA treated, cell morphology observation, immunological marker examination and phagocytic function detection were used to identify whether the induction was succeeded. The diameter, nature and endotoxin content of UHMWPE particles were detected after grinded by ball miller. Macrophages were co-culture with thrombin or prepared UHMWPE particles, MTT assay was used to detect optimum concentration of thrombin and UHMWPE particles for macrophages. The macrophages were divided into macrophages+thrombin group, macrophages+UHMWPE particles group and simple macrophages group (control group), real-time PCR technology was used to detect differences of RANK mRNA expression in three groups.Results U937monocytes can induce macrophages successfully after PMA treated, which had the morphological characteristics of macrophages, the expression of specific differentiation antigen-CD14and good phagocytosis. Average diameter of UHMWPE particles after grinding was about8.46μm±6.30pm and endotoxin content was0.053EU/ml, which meets the experiment requirements. The optimum concentration of thrombin and UHMWPE particles for macrophages detected by MTT assay was50U/ml and0.1mg/ml respectively. RANK mRNA expression level in macrophages with particles treated was significantly higher than the other two groups, P<0.01, while the level of RANK mRNA expression in thrombin-treated group was also significantly higher than control group, P<0.01.Conclusion U937monocytes can successfully induce mature macrophages with PMA treated. The RANK mRNA expression level in macrophages with thrombin treated was significantly increased, but elevated level of RANK mRNA was less significant than that with UHMWPE particles treated.Part Ⅱ The Expression of RANKL and OPG in hFOB1.19OsteoblastsObjective To investigate differences of RANKL and OPG mRNA expression in hFOB1.19osteoblasts with UHMWPE particles or thrombin treated.Methods hFOB1.19cells were co-culture with thrombin or prepared UHMWPE particles, MTT assay was used to detect optimum concentration of thrombin and UHMWPE particles for hFOB1.19cells. The hFOB1.19cells were divided into cells+thrombin group, cells+UHMWPE particles group and control group, real-time PCR technology was used to detect differences of RANKL and OPG mRNA expression in three groups.Results The optimum concentration of thrombin and UHMWPE particles for hFOB1.19cells detected by MTT assay was50U/ml and0.1mg/ml respectively. RANKL and OPG mRNA expression level in macrophages with particles treated was significantly higher than the other two groups, P<0.01, while the level of RANKL and OPG mRNA expression in thrombin-treated group was also significantly higher than control group, P<0.01. The RANKL/OPG ratio in particle-treated group was higher than the other two groups, but no significant differences between the thrombin-treated group and the control group. Conclusion RANKL, OPG mRNA expression and RANKL/OPG ratio was significantly increased in hFOB1.19cells with UHMWPE particles treated. RANKL and OPG mRNA was also increased in hFOB1.19cells with thrombin treated, however the active effect was less significant than UHMWPE particles treated.Part Ⅲ The Expression of RANKL/RANK/OPG System in septic interface membranesObjective To investigate expression differences of RANKL/RANK/OPG system in normal synovium, septic and aseptic interface membranes.Methods17cases of septic interface membranes (SIM),26cases of aseptic interface membranes (AIM) and12cases of normal synoviums were collected. Scanning electron microscope (SEM) and transmission electron microscope (TEM) were used to observe microscopic morphology of three different types of tissues. Real-time PCR technology was used to detect difference of RANKL, RANK and OPG mRNA expression; Immunohistochemistry and western blot technology was used to detect difference of RANKL, RANK and OPG protein expression in three different kinds of tissues.Results Microscopic morphology of three types of tissues had significant differences, SEM showed wear debris widely distributed on AIM surface; TEM showed bacillus was exited in SIM. RANKL, RANK and OPG mRNA expression in AIM was significantly higher than the other two tissues, P<0.01. RANKL mRNA expression in SIM was higher than normal synoviums, P<0.05. However, RANK and OPG mRNA expression in these two tissues was not significant difference, P>0.05. The RANKL/OPG ratio both in AIM group (P<0.01) and SIM group (P <0.05) were higher than normal synoviums groups. RANKL, RANK expression in AIM was significatly higher than the other two groups, P <0.01. RANKL protein expression in SIM was higher than normal synoviums, P<0.05; however OPG protein expression in these two tissues was not significant difference, P>0.05.Conclusion RANKL expression and RANKL/OPG ratio was significantly increased in SIM. Imbalance RANKL/RANK/OPG system was related to periprosthetic osteolysis with septic loosening, but it was not the only pathogenesis.