Cytotoxicity of divalent cobalt ions and their effects on cell proliferation (in vitro)  Cells were cultured with Co2+ of different concentrations for 72 hours. MTT cell proliferation assay showed that the experimental groups which included 125, 250, 500 μmol/L Co2+ inhibited the proliferation of MC3T3-E1 cells in varying degrees (P < 0.05; Figure 2). LDH assay showed significant toxicity when Co2+ reached 1 000 μmol/L and Co2+ showed slight toxicity at 500 μmol/L (P < 0.05; Figure 3).  "

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ALP activity (in vitro) Figure 4 showed the different effects of two concentrations of Co2+ (62 μmol/L and 250 μmol/L) on ALP expression in MC3T3-E1 cells, respectively. When Co2+ was 250 μmol/L, it showed significant inhibition of ALP expression compared with the control group (P < 0.05)."

Gene expression in preosteoblasts (in vitro)  RT-PCR was used to detect the expression of a variety of genes to further investigate the molecular biological mechanisms underlying the effects of different concentrations of Co2+                   (62 μmol/L and 500 μmol/L) on MC3T3-E1 cells. The data showed that Co2+ significantly promoted MCP-1 gene expression at 500 μmol/L compared with the control group (P < 0.05). Both groups significantly promoted the expression of TNF-α and IL-6, and they also promoted the expression of RANKL. When Co2+ was at 500 μmol/L, the expression of RANKL was significantly increased compared with that of 62 μmol/L group. The expression of NFATc1 stimulated by Co2+ was significantly enhanced at 500 μmol/L compared with the control group (P < 0.05). The expression levels of OPG and OSX were inhibited in both experimental groups. When Co2+ was at 500 μmol/L, there were significantly differences with the control group; thus, the RANKL/OPG ratio was much higher than the control value (P < 0.05). Same trend with Lrp-5 and Runx2, both of them promoted the expression of Co2+ at 62 μmol/L and inhibited the expression at 500 μmol/L compared with the control group (P < 0.05; Figure 5). "

General situation and surgical results of experimental mice (in vivo) The mice in each group grew and moved well, and generally responded well. No mice died in each group. The uneven distribution of cobalt and chromium particles around the prosthesis and the knee joint was observed after the knee joint was severed, and no significant anatomical changes were observed in the gross morphology of the knee joint (Figure 6)."

MicroCT evaluation results (in vivo) MicroCT scans showed that mice in the stable control group had good fixation and no displacement, bone ingrowth around the titanium nail could be seen obviously. There was visible bone resorption around the titanium nail in the cobalt ion group (Figure 7), and the percentage of changes of bone mineral density before and after treatment was significantly lower than that in the loose control group and the stable control group   (P < 0.05; Figure 8)."

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Stability evaluation of titanium nail prosthesis-nail pulling test (in vivo) The blade holding the distal tibia of the mice which were fixed well by bone cement was able to withstand at least 100 Newtons (N) tension. In the stable control group, the maximum shear force needed at the moment of pullout was (11.58±2.47) N and (10.92±2.44) N in the loose control group, while in the cobalt ion group, the pullout force of the titanium nail was further reduced to (5.93±1.91) N. There were significant differences between the experimental group and the control group (P < 0.05; Figure 9)."

Observation of histomorphology (in vivo) Hematoxylin-eosin staining showed that there was obvious bone ingrowth and few inflammatory cells infiltration around the titanium nail in the stable control group. There was obvious inflammatory reaction membrane formation and a large number of inflammatory cell infiltration around the prosthesis in the cobalt ion group (Figure 10A-C). The comparison of periprosthetic membrane thickness between the above groups was calculated by computer image analysis software. The thickness of inflammatory reaction membrane around prosthesis in cobalt ion group was significantly thicker than that other two control groups (P < 0.05; Figure 10D). Differentiation and quantity of osteoclasts at the titanium nail prosthesis-bone interface were evaluated by TRAP staining, which showed that the degree of them in the surrounding tissues of the cobalt ion group was significantly higher than that of the control group (P < 0.05; Figure 11)."

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Immunohistochemical assessments (in vivo) The expression of the positive-staining of TNF-α among groups is shown in the Figure 12A-C. There was almost no expression of TNF-α in the stable control group, but the cobalt ion group was the most obvious. All staining conditions were summarized, and it was concluded that the cobalt ion group and loosening control group were significantly higher than the stable control group in staining (P < 0.05; Figure 12D)."