| ObjectiveAseptic loosening is the major factor that limits the longevity of prosthetic arthroplasty. Retrieval studies have revealed cement, metallic, and polyethylene wear debris within macrophages at the interface of loose prostheses. Bone resorption in aseptic loosening is theorized to be an inflammatory response to wear debris at the bone-prosthesis interface. We examined the effects of these particles on macrophages, as we believe that macrophages play a central role in the response to wear particles. Macrophages are known to be producers of a large number of potent inflammatory mediator, many of which stimulate bone resorption. The principal aim of this study was to eximine the differences in the release of interleuk in- 1, tumor necrosis factor, in the effect on macrophages by different particles. We also determined the patterns of mediator release in response to increased concentration or size of these wear particles. In the second part, the ability of macrophage conditioned medium to induce bone resorption was determined using radiolabeled rat calvaria in organ culture. Additionally, the therapeutic effect of non-steroidal and steroidal drugs, biphosphonates on process of bone resorption was investigated.MethodAna- 1 macrophage cell line was isolated from C57BL/6 murine fresh bone marrow by a V-raf/V-myc recombinant retrovirus. 1.5*106/mi Ana1 macrophage cells were suspended in 1 .Sml of RPMI/1 640 medium supplemented with SOug/ml glutamine gentamycin, 10% fetal bovine serum and containing Sug/ml penicillin and 50U/ml streptomycin and placed in each well of a 16-mm flat bottomed 24 well tray. After the cells were incubated at 37oC in 5%C02 for 1 hour, the nonadherent cells were removed by washing with PBS solution. These cells were incubated inVAT~tliJf~2ml of the RPMI medium containing particles (it抯 size and concentrations were according to the study ). After 48 hours, the supernatants were sampled and centrifuged to remove any particles, or cell debris. The levels of IL-i f3 ,TNF- a were measured using commercially available immunoassay kits. For the bone resorption assay, Sprague-Dawley rats at approximately 1 to 3 days were injected subcutaneously with 1.5 uCi of 45Ca. After 4 days these rats were beheaded . Calvaria were dissected free of soft tissue leaving the periosteum intact, and a 4-mm biopsy was taken from each parietal bone. Each bone disk was incubated with macrophages and particles for 48 hours. Duplicate 50 ul aliquots of medium were taken from each well and counted for 45Ca in a scintillation counter. Bone resorption was expressed as a ratio between the counts in the experimental well and the counts in the control well.Results1.TNF- a , IL-i P release was stimulated by all six types of particles at l.5*l0l0particles/ml. There were significant statistical difference in the levels of cytokines release when the different particles were compared(p<0.0 1). The results were as follows:No Particles UHMWPPMMACoCrMoTiAIVHAPEEKEILl-P41.67?.24901.67?748.33?490.00?210.00?122.67140.80?(pg/mI)12.9611.308.504.28?.250.78TNF-a45.83?1.92821.67?730.83?391.67?238.33?115.00145.00?(pg/mI)2132.8917.1812.88?.583.862.Macrophages exposed to PE particles released TN7F- a and IL-ifB in a dose-dependent manner. But, there was no statistically significantdifference between unexposed macrophages and those exposed to 106particles /ml per well(P>0.05)3.Macrophages exposed to PE particles released TNF- a and IL-iviP in a size-dependent manner.4. Incubation of radiolabeled calvaria with medium from macrophages exposed to PE particles and biphosphonates did not lead to an increased release of 45Ca . The ratios of bone resorption were as follows (n8,P |
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