Effect Of Titanium Particles And Inflammatory Cytokines On The Activity Of MMP-2in Human Knee Joint Synovial Cells And Explor Prevention And Cure Of Aseptic Loosening

Total joint replacement is still the most common and highly successfultreatment for severe degenerative or other arthritic joint diseases such asend-stage osteoarthritis or rheumatoid arthritis, However, the asepticloosening, a major factor resulting in the late failure of total jointreplacement, limits the longevity of prosthetic arthroplasty. The mechanismof aseptic loosening is not clear, but it is accepted that osteolysis is the mainreason. In the failed total joint arthroplasties, one of the pathologicalcharacteristics of the osteolysis is the formation of a membranous tissuewhich contain fibroblasts, macrophages and foreign body giant cells.Previous data inferred that these cells phagocytized the particles from theinterface of the bone-prosthetic materials, which led to the inflammation.The inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α)and interleukin-1beta (IL-1β), could activate the osteoclasts and induce acascade of the osteolytic events. MMP-2is a most important member of thematrix metalloproteinases (MMPs) which play an essential role in the extracellular matrix (ECM) degradation. The activity of MMP-2weremodulated by Ti particles and some cytokines in human macrophages,fibroblasts. Synovial cells present in or around the peri-prosthetic membraneare the important targets of wear debris during osteolysis. Few study focuson wear particle and inflammatory cytokines and investigate their relationin membranous.Objective1. To study the effect of different diameter of titanium particles on theMMP-2activity in synovial cells of human knee joint, and sift the size oftitanium particle in this study；2. To study the effect of different concentration of TNF-α and IL-1β on theMMP-2activity in synovial cells of human knee joint, and sift the suitableof concentration of TNF-α and IL-1β in this study；3. To study the effect of titanium particle and inflammatory cytokines onthe MMP-2activity in synovial cells of human knee joint；4. To study the effect of special inhibitors on the MMP-2activity insynovial cells of human knee joint；By means of those studies，the probable mechanism of asepticloosening after MOM arthroplasty at the level of molecular and cell wouldbe discussed. And explore prevention and cure of aseptic looseningpreliminarily. MethodsHuman knee joint synovial cells were cultured in vitro. The first partstudy the different diameter of titanium particles (≤20μm,0.1wt%）, it wascomposed of the following four groups:(1) Control: mono-culture ofsynovial cells；(2) group I：diameter of titanium particles:≥10μm；(3)group II：diameter of titanium particles：5μm~10μm;(4)groupIII：diameter of titanium particles:≤5μm. The second part study thedifferent concentration of TNF-α and IL-1β, it was composed of thefollowing eight groups:(1)Control: mono-culture of synovial cells;(2)TNF-α treatment (1、10、20ng/m);(3)IL-1β treatment (1、5、10ng/m);(4)TNF-α+IL-1βtreatment (TNF-α：10ng/m；IL-1β5ng/m). Thesecond part study the effect of titanium particle and inflammatory cytokines,it was composed of the following five groups:(1)Control: mono-culture ofsynovial cells;(2)titanium particles treatment;(3)Ti and TNF-α treatment;(4)Ti and IL-1β treatment;(5)Ti、 TNF-α and IL-1β treatment. The fourthpart study the effect of inhibitors, it was composed of the following ninegroups:(1)Control: mono-culture of synovial cells;(2)titanium particlestreatment;(3)Ti and KT5720;(4)Ti and Curcumin;(5)Ti and Bay11-7082;(6)Ti and Bay11-7085;(7)Ti and SP600125;(8)Ti and PD98059;(9)Ti andSB203580. The conditioned media were collected at12,24,48and72h inthose experiments, and MMP-2activity was determined by zymography.Results1、The activity of MMP-2were increased in the synovial cells after Ti particle treatment. Moreover，At72h, the high activity of MMP-2is up to1.66folds in group I,2.12folds in group II and3.95folds in group III.2、Both factors(TNF-α and IL-1β) could up-regulate the activity of MMP-2and showed a concentration and time dependent-manner. Furthermore, theIL-1β had a greater influence on MMP-2activity than TNF-α. At72h, thecombination induced MMP-2up to8.94folds compared to normal control;in comparison, IL-1β up to4.28folds (5ng/ml) and TNF-α up to3.32folds(10ng/ml).3、Combination of Ti particles and TNF-α induced a higher activity thaneach factor did at72h (up to5.48folds vs control). The combination of Tiparticles and IL-1β had an even higher activity at48h (up to7.76folds vs.control). The combination of Ti particles, TNF-α and IL-1β induced bothpro-and active-enzyme of MMP-2to a most high level (up to9.08folds vs.control,72h).4、Those inhibitors (KT5720,SP600125, PD98059,SB203580,curcumin,bay11-7082, bay11-7085) could reduce the activity of MMP-2，but over thecontrol level. Bay11-7082and Bay11-7085could significantly reduce theactivitys of MMP-2, at72h, down-regulation the MMP-2activity to29.4%、30.5%(vs. titanium particles treatment).Conclusion1. The activity of MMP-2were increased in the synovial cells afterdifferent Ti particles treatment, Group III (diameter≤5μm) had the most effect.2. Both factors (TNF-α and IL-1β) could up-regulate the activity of MMP-2and showed a concentration and time dependent-manner. IL-1β had agreater effect and showed a synergistic effect. The concentration of TNF-αand IL-1β appropriate in our study was10ng/ml5ng/ml.3. Combination of Ti particles and inflammatory factor had a synergisticeffect, Ti particles and IL-1β had a greater effect. The combined effect(high activity of MMP-2) of Ti particles, TNF-α and IL-1β maybe oneprobable mechanism of osteolysis.4、Those inhibitors could reduce the activity of MMP-2，but over thecontrol level. Bay11-7082and Bay11-7085could significantly reduce theactivity of MMP-2, there was no difference in the two group. Inhibitor ofNF-κB pathway may one possible target to prevent aseptic loosening.