A Comparative Study of Inhibitory Effects on Peri-implant Osteolysis between Anti-tnf- Alpha Antibody and Bisphosphonate in a Rat Model

Introduction; Osteolysis around joint prostheses is reported to be caused by osteoclastic bone resorption, which stimulated by the release of bone-resorbing cytokines from macrophages phagocytosed wear debris such as polyethylene particles. Previous studies have demonstrated that a rat osteolysis model with continuous infusion of polyethylene particles is useful for observing osteolysis over a short period. Anti-tumor necrosis factor alpha (TNF-α) antibody have been reported to inhibit bone resorption in a rat adjuvant arthritis. TRK-530 is a novel synthetic bisphophonate to have a direct effect on osteoclastic bone resorption as well as suppressive effects on bone resorbing cytokines from macrophages. This study compared the inhibitory effects of anti-TNF-α antibody and a new bisphosphonate (TRK-530) on peri-implant oseteolysis in a rat model with continuous infusion of polyethylene particles. Materials and Methods; Eighty Wister rats were randomized to four groups (n=20 each). In each rat, a Kirshner wire (Kwire) was inserted into the femur and polyethylene particles (HDPE, mean size; 2 microns) were continuously infused into the knee joint using an osmotic pump (6 million particles per week). The animals were subcutaneously injected with saline (control group) or 1 mg/kg of TRK (TRK group) or intraperitoneally injected with 100 μg of rat monoclonal anti-TNF (TNF-α 100 group) or with 500 μg of rat monoclonal antiTNF (TNF-α 500 group) every second day after surgery until 8 weeks. At 4 weeks or 8 weeks after surgery, rats were sacrificed. Radiographs were evaluated for the presence of endosteal erosion, focal osteolysis and trabecular bone loss. The presense of osteolysis was defined on the basis of these findings. Radiographs were compared for osteolysis by three independent observers who were blind to the animal group. Grarnulation tissues were stored for reverse transcription polymerase chain reaction (RT-PCR) analysis for interleukin-1-α (IL-1-α) mRNA as well as TNF-α mRNA. After removal of the K-wire distal femurs were suspended in 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.067 mol/l cacodylic acid buffer, and demineralized for 2 weeks in 10% ethylenediaminetetraacetic acid solution at 4 deree celsius. Dehydrated specimens were embedded in paraffin, cut into mid-sagital sections, and stained with hematoxylin and eosin. To obtain the average membrane thickness, the circumference of the K-wire was measured by light microscopy on sagital sections, and the total membrane area was obtained using computer-image analysis. Then the average membrane thickness was calculated for each group. Other thin sections from the same specimens were prepared for TRAP (tartrateresistent acid phosphatase) stainings. The total number of osteoclast-like cells around the membrane in each section was determined by counting in 10 fields per section under light microscopy at a magnification of 200x. Then the mean number of osteoclast-like cells was calculated by averaging the total number of osteoclast-like cells in three sections. Histomorphometric analysis composed of the average membrane thickness and the average number of TRAP positive multinucleated cells (osteoclast-like cells) in the interface membrane around K-wire in each group. RT-PCR for detection of IL-α mRNA and TNF-α mRNA was performed as previously reported using electrophoresis. Statistical analysis was performed by the chi-square test for radiographic findings and Student’s t-test was used for histomorphometric analysis. Results; The results of radiographic and histomorphometric analysis are shown in Table 1. At 4 weeks the radiographs of TRK group as well as TNF-α 500 group showed significantly less osteolysis compared to the other three groups (p<0.01). The histologic findings showed that the average membrane thickness in TNF-α 500 group and TRK group was significantly less than the other two groups (p<0.01), while the average number of osteoclasts was significantly less in TRK group compared to the other three groups (p<0.01). At 8 weeks, radiographs of TRK group showed significantly less osteolysis compared to the other three groups (p<0.01). The histologic findings showed that the average membrane thickness as well as the average number of osteoclasts in TRK group was significantly less than the other three groups (p<0.01). RT-PCR study showed that the expression of IL-α mRNA and TNF-α mRNA tended to be suppressed in the TRK group at 8 weeks after surgery while the significant suppression of those mRNA was not observed in the other groups. Discussion; The present study demonstrates that cumulative effects of a novel bisphosphonate (TRK-530), such as the suppression of bone resorbing cytokines as well as direct suppression of osteoclasts, reduce the polyethylene induced peri-implant osteolysis more effectively when compared to single anti-cytokine therapy in our model.. Future studies are needed to investigate the mechanism by which TRK inhibits peri-implant osteolysis in a clinically relevant model, such as a weight-bearing canine model using a real prosthesis with continuous infusion of polyethylene particles.