**7. Rationale of oxidative stress and aseptic loosening**

Three different mechanisms are mainly responsible for osteolysis and loosening: exacerbated inflammation caused by ROS production in the periprosthetic tissue; cascade of cellular and molecular interactions ultimately resulting in osteoclasts activation; and, compromised bone formation resulting from increased cytotoxicity on mesenchymal osteoprogenitors.

Wear debris such as polyethylene, PMMA and metal particles, metal degradation products and ions may be exposed to the ROS produced by the inflammatory cells in the periprosthetic tissues. Mediators such as H2O2, NO and ONOO− are released by the macrophages and the inflammatory cells. Metal debris and metal degradation products could react with the free radicals resulting in elevation of oxidative stress. H2O2 in the cells can also undergo the Fenton reaction in the presence of metal ions with subsequent formation of highly toxic hydroxyl radical (Lubec, 1996, Sawyer, 1990). Various studies have reported that ROS are connected with tissue damage and fibrosis (Park et al., 2001, Riedle & Kerjaschki, 1997, Wang et al., 2002, Windhager et al., 1998). On the other hand, the effect of submicron wear debris on macrophage production of reactive oxygen species is largely unexplored. Thus, these facts led to the hypothesis that ROS play a role in aseptic loosening and formation of fibrous pseudocapsule around hip implants. Our results demonstrated in vivo elevated oxidative stress in periprosthetic tissues and pseudosynovial fluid from loose hip prostheses and hip implants with high rate of wear. This added further insight into the mechanism of aseptic loosening of hip arthroplasty.

When the cells are exposed to a large amount of oxidants, the capacity of the regulatory mechanisms of cellular response to oxidative stress may be exceeded by the rate of ROS production, resulting in a condition of "oxidative stress". In the present study, we found increased levels of markers of oxidative stress in periprosthetic tissues and pseudosynovial fluid from loose THAs. Our results are difficult to compare as the available studies on the possible role of oxidative stress in aseptic loosening of THA are in vitro experiments. We believe that, in the early stage of aseptic loosening, the exposure to wear debris could be responsible for the increase in the values of markers of oxidative stress in total hip arthroplasties with high rate of wear and osteolysis. Moreover, free radicals may be involved in sustaining the foreign-body reaction to wear debris. Later on, chronic exposure would result in a triggering of compensatory mechanisms leading to progressive increase in antioxidants and low oxidative stress as observed in the beginning of loosening (Table 1). This is consistent with the conception of oxidative stress regulation. However, other mechanisms may play role, too.

Evidence Linking Elevated Oxidative Stress and Aseptic Loosening of Hip Arthroplasty 311

contact is not uniform along the entire surface there are gaps between the surfaces. During the healing phase, these gaps are filled with callus tissue which may mature in different directions, i.e. by bony healing or osseointegration, where there is no fibrous lining between the implant and the bone, or by soft tissue encapsulation with formation of synovial-like membrane (Albrektsson, 1990). In osseointegration of the prosthesis the interface can resist shearing as well as tensile loads whereas the fibrous tissue interface can withstand compressive and, to lesser extent, shear loads, but fails with tensile loads. In the case of soft tissue anchorage when the prosthesis is loaded there will be movements. As far as they are micro-movements the implant will be stable. Some bone resorption will occur, but it is usually compensated for by new bone formation to maintain equilibrium between applied load and strength of tissue. But even in the physiological range of movement there is a constant risk of overloading the device which will result in macro-movements and implant failure. Improving initial implant fixation with bisphosphonates may represent a promising strategy for improving initial fixation and clinical outcome after THA (Friedl et al., 2009,

Successful inhibition of osteoclast activity in postmenopausal osteoporosis and increase of bone mineral density with use of bisphosphonates turn research efforts in this direction. Osteoclast is the ultimate cell in the cascade of events that lead to periprosthetic osteolysis. Various in vitro and animal studies have shown encouraging results (Horowitz et al., 1996, Millett et al., 2002, Shanbhag et al., 1997, von Knoch et al., 2005). However currently, convincing results from clinical studies supporting the use of bisphosphonates are still lacking. The local inflammatory response to wear debris particles with cascade of cellular and molecular interactions ultimately results in periprosthetic osteolysis and aseptic loosening. Biological approach to prevention of this deleterious complication of THA seems promising. In-depth research into bone biology and molecular mechanisms of bone metabolism has led to the identification of novel possible strategies. Receptor activator of nuclear factor kappa-B ligand (RANKL) plays a key role in osteoclastogenesis which makes this cytokine an attractive target in efforts for prevention of osteolysis. Clinical studies have demonstrated its effectiveness in decreasing bone turnover and fracture prevention (Bekker et al., 2004, Cummings et al., 2009). However, its potent effect on osteoclast function has not been

Other promising novel therapeutical targets include: osteoclast protease cathepsin K; sclerostin and dickkopf-1, two endogenous inhibitors of bone formation; the osteoclast ATPase proton pump, vitronectin receptor, and src tyrosine kinase, all of which are required

Such approaches would be expected to decrease bone loss, but not influence inflammation. It was proven that tumor necrosis factor alpha (TNF-) is capable of inducing osteoclastogenesis in presence of small amounts of RANKL (Lam et al., 2000) whereas interleukin-1 (IL-1) is directly involved into osteoclast differentiation (Yao et al., 2008). This suggests that effective inhibition of osteolysis includes blockade of proinflammatory mediators. In support of this direct evidence that cyclo-oxygenase 2 (COX-2) inhibits wear induced osteolysis in an animal model were provided (Zhang et al., 2001). However, these treatment possibilities should be approached with caution as signaling molecules are involved in variety of processes and their

The local inflammatory response of the organism to wear debris is the main cause for osteolysis and prostheses loosening. However, current knowledge of wear-induced

Kinov et al., 2006).

evaluated in periprosthetic osteolysis.

for resorption (Purdue et al., 2006).

inconsiderate manipulation may have deleterious effects.

Lipids of cell membranes are a prominent target for free radicals generated in a complex series of oxygen-dependant reactions via Fenton's chemistry (Sawyer, 1990). Iron-induced lipid peroxidation has been demonstrated in various studies (Marmunti et al., 2004, Lim & Vaziri, 2004). However, we could not establish correlation between levels of MDA and iron. As metal-catalyzed lipid peroxidation is dependent on metal as a catalyst, there might be little influence of metal concentrations, and many other factors might also contribute to MDA formation. On the opposite, we found elevated MDA level in hips with prostheses made of titanium alloy. This finding might be explained by higher rate of wear of titanium implants compared to Co-Cr implants (Table 2) (Wang et al., 2002). We hypothesized that the process of wear debris-mediated loosening leads to elevation of oxidative stress. In support of this, Wang et al. found that exposure to particles stimulates superoxide production by macrophages and osteoclasts (Wang et al., 2002). Furthermore, it was observed that the increase of free radicals on polyethylene correlated with the degree of inflammation of synovial cells in culture (Fiorito et al., 2001). The correlation between markers of oxidative stress and hydroxyproline levels suggest, first of all, that the increase of collagen in periprosthetic tissues in the presence of wear debris is due to elevated oxidative stress. Connective tissue metabolism is normally characterized by equilibrium between degradation and synthesis of extracellular matrix. Deviation from the equilibrium may lead to the replacement of extracellular matrix by fibrous tissue (Park et al., 2001, Riedle & Kerjaschki, 1997, Wang et al., 2002, Windhager et al., 1998). The fibrous pseudocapsule formed is probably related to high intraarticular pressure and expansion of the effective joint space as well as production of inflammatory substances and subsequent loosening of the implant (El-Warrak et al., 2004, Van der Vis et al., 1998).

Most of the studies elucidating the mechanism of loosening of hip arthroplasty are performed on animal models or are in vitro studies. However, the results from in vitro or animal studies could not be easily translated to humans. The findings of our in vivo studies provide further insight into the possible mechanisms of aseptic loosening. The number of cases in the studies was relatively small but the differences in measurements of markers of oxidative stress between patients and controls were high which compensated for small sample size.
