**4. Oxidative stress**

300 Recent Advances in Arthroplasty

is principle factor in this process. Abundant evidence show that the macrophages play a key role in wear debris induced periprosthetic osteolysis (Brooks et al., 2002, Park et al., 2005, Purdue et al., 2006, Sabokbar et al., 1997, Wang et al., 2002). Phagocytosis of wear particles induces secretion of various proinflammatory cytokines such as tumor necrosis factor alpha (TNF-), IL-1 and PGE2 (Goldring, Lam et al., 2000, Yao et al., 2008). This inflammatory response is modulated by various factors including chemical composition, size, shape, and volume of the particles (Sieber et al., 1999, Yang et al., 2002). The prostheses-bone interface could be also influenced by other factors such as endotoxins (Kido et al., 2004), matrix metaloproreinases secreted from activated macrophages that directly resorb bone (Brooks et al., 2002), and mechanical factors such as high fluid pressure (Aspenberg & Van der Vis, 1998, Van der Vis et al., 1998). Although the inflammatory response to wear debris is central to the process of aseptic loosening, the detailed nature of the local response may vary based upon several parameters, including prosthetic type and material, patterns of wear, and

Similar to polyethylene debris, metallic debris migrate in the periprosthetic tissues, and because of its smaller size is easily phagocytosed by histiocytes (Doorn et al., 1999). Metal ions can be involved in various cellular, local and systemic biological reactions. However, the mechanism of metal toxicity is not fully understood today. It is well known fact that metals are involved in production of reactive oxygen species (ROS), such as superoxide ions (O2**·–**), hydrogen peroxide (H2O2), hydroxyl radical (OH**·**), and nitrogen oxide (NO**·**) via Fenton/Haber-Weiss chemistry (Sawyer, 1990). Free radicals may damage purine and pyrimidine bases of DNA (Valko et al., 2006). Moreover, direct binding of Cr to DNA is reported (Wolf et al., 1989) that may inhibit the process of DNA repair (Witkiewicz-Kucharczyk & Bal, 2006). These gene modifications and eventual mutations can lead to carcinogenesis. Metal ions have been also associated with a delayed immune reaction (hypersensitivity) (Hallab et al., 2001). It was suggested that a vicious circle of particle phagocytosis, cellular lysis and subsequent release of particles by the involved cells may play role in delayed hypersensitivity reaction mediated by T-lymphocytes (Hallab et al., 2005). In addition to systemic and cellular reactions various local adverse reactions such as extensive necrosis (Ollivere et al., 2009), periprosthetic osteolysis (Amstutz et al., 2011) and pseudotumour reactions (either cystic or solid) (De Haan et al., 2008, Wynn-Jones et al., 2011) have been reported with MoM bearings. Perivascular accumulation of activated macrophages and T-lymphocytes has also been associated with periprosthetic osteolysis (Park et al., 2005). In addition, direct toxicity may also be involved in bone loss (Fleury et al.,

Cells in synovial membrane of the artificial hip joint generate synovial fluid that is called pseudosynovial fluid and secrete the mediators of inflammation into it. Schmalzried et al. hypothesized that wear debris is dispersed into the joint fluid (Schmalzried et al., 1992). Access to the joint fluid for the wear particles is dependent on the contact between implant and bone. Wear debris activates macrophages, which activate osteoclasts or become osteoclasts themselves and initiate bone resorption (Sabokbar et al., 1997). The resulting bone loss will enlarge the interface and ease the flow of joint fluid, resulting in higher transportation capacity of the debris and gradual loosening of the implant. This concept is in concordance with the high pressure theory that was suggested by Aspenberg and Van der

patient-related factors (Purdue et al., 2006).

2006, McKay et al., 1996).

Vis (Aspenberg & Van der Vis, 1998).

Oxidative stress is a condition when the balance of formation of oxidants exceeds the rate of metabolism and the ability of antioxidant systems to remove ROS. High levels of ROS can damage proteins, lipids, and DNA, and eventually cause cell death.

Alternatively, oxidative stress can trigger activation of specific physiologic signaling pathways (Rached et al., 2010). In several studies, reactive oxygen species have been demonstrated as one of the key factors in inflammation (Kamikawa et al., 2001, Wang et al., 2002, Tsaryk, 2009). Because of the inflammatory nature of aseptic loosening of total hip replacement, arthroplasty, it is likely that free radicals play a major role in this condition as well. In the periprosthetic tissues, detection of reactive oxygen species provides evidence for the formation and activity of free radicals (Windhager et al., 1998, Kinov et al., 2006). Under physiological conditions, ROS are part of normal regulatory circuits, and the cellular redox state is tightly controlled by antioxidants. However, increased concentrations of ROS and loss of cellular redox homeostasis following extensive particulate challenge can lead to upregulation of inflammatory processes in the interface membrane.

The interface between implant and bone is rich of transitional metals from the alloy of the implant. Transition elements like vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), molybdenum (Mo), so-called d-block elements, show variable valence, which allow them to undergo changes in oxidation state involving one electron. If free radicals have a causative role in aseptic loosening than transition metals would have a strong promotional effect (Windhager et al., 1998). Via the Fenton reaction they would greatly stimulate inflammation and loosening. Iron i.e., exerts its toxicity through a series of reactions with reactive oxygen species called modified Haber-Weiss or Fenton reaction (Fe2+ + H2O2 → Fe3+ [H2O2-] → .OH + -OH), generating the highly toxic hydroxyl radical (.OH) (Lubec, 1996).

The generation of hydroxyl radicals via Fenton chemistry represents one of the most important mechanisms in various pathologic conditions. Hydroxyl radicals can lead to DNA and protein damage and impairment of normal DNA and protein synthesis and cell proliferation and thus has been thought to be casually involved in the multistep process of loosening (Wang et al., 2002). Furthermore, ferrous/ferric ion has a decisive function in lipid peroxidation process by direct reaction with unsaturated fatty acids or reaction with preformed lipid hydroperoxides to form chain-carrying alkoxyl and peroxyl radicals, leading to severe damage of cellular integrity (Lin & Girotti, 1993, Minotti & Aust, 1992, Schaich, 1992).

The effects of the metal wear particles on oxidative stress are not augmented by polyethylene and cement wear debris, originating from materials used for implant fixation. In an in vitro study, Petit et al. compared the effects of different wear products from hip prostheses on the nitration of proteins (an evidence for oxidative damage) in macrophages (Petit et al., 2005). The effect of both Co(2+) and Cr(3+) ions was inhibited by glutathione monoethyl-ester that provides protection against oxidative stress. However, ultra-high molecular weight-polyethylene and alumina ceramic particles had no significant effect on the nitration of proteins.

Because of their excellent mechanical properties, titanium and titanium alloys are widely used in orthopedic implants. Moreover, superb corrosion resistance and biocompatibility are characteristic for titanium and are the main reasons for its wide use in various biomaterials. However, as a result of wear and corrosion, titanium ions are released in the periprosthetic tissues and can be found in systemic circulation. Titanium may be directly involved in ROS production interacting with H2O2 leading to formation of hydroxyl radicals

Evidence Linking Elevated Oxidative Stress and Aseptic Loosening of Hip Arthroplasty 303

mean interval between primary THA and revision was 97 months (range, 14–157 months) (p=0.405). As a control group, 16 samples of fascia lata were obtained from 16 patients

In a second study, periprosthetic tissues and pseudosynovial fluid were obtained at revision of 18 consecutive primary THA performed at the Department of Orthopedics and Traumatology, Medical University of Sofia. The eight men and 10 women in the series had mean age 63.2 years (range, 52 to 78 years) at the time of revision. The mean interval between primary THA and revision was 10.8 years (range, 2.1 to 22.3 years). The pseudosynovial fluid was immediately deep frozen at –80°C until analysis. The periprosthetic samples were fixed in 10% formalin until being processed. Patients with multiple revisions and infections were excluded from the studies. As a control group, 18

Prostheses fixation was graded according to the criteria of Engh et al. (Engh et al., 1989) for the cementless and Harris & Penenberg (Harris & Penenberg, 1987) for the cemented components. Osteolysis was graded according to Paprosky (Paprosky & Burnett, 2002). Annual polyethylene wear was measured as described by Livermore et al. (Livermore et al.,

A portion of each specimen was embedded in paraffin, processed with xylene, cut into 5 mm thick sections, and stained with hematoxylin and eosin. All sections were studied blindly at a maximum magnification of 600x and were graded in a semiquantitative fashion for cellular constituents and particulate debris according to Mirra et al. (Mirra et al., 1976).

Evaluation of the ultrastructure of collagen was obtained by examination of ten representative cases with electron microscopy. These samples were fixed in glutharaldehyde

Reduced glutathione and oxidized glutathione was measured by spectrophotometry (Beckman Instruments, Fullerton, CA) according to the method of Tietze (Tietze, 1969). Measurements with GSH concentrations bellow 200 μmol/g wet weight were excluded from calculation because of high possibility for error. Results were then weighted for hydroxyproline content and expressed as μmol/mg. Samples were run in duplicates and

Tissue malondialdehyde levels were determined by Khoschsorur's method (Khoschsorur et al., 2000). The samples were chromatographed on a high performance liquid chromatographer (HPLC) (Spectrochrom, Brackley, UK) interfaced to a LiChrosorb RP18 column. Fluorometric detection was performed with excitation at 527 nm and emission at 551 nm.

run according to supplier's instructions (OxisResearch, Portland, OR).

**5.6.1 Malondialdehyde determination in periprosthetic tissue** 

samples of joint fluid were obtained from 18 patients during primary TKA.

during primary THA.

**5.2 Radiographic analysis** 

1990) and corrected for magnification.

Tissue necrosis was recorded as present or absent.

**5.4 Electron microscopic examination** 

**5.5 GSH and GSSG determination** 

**5.6 Malondialdehyde determination** 

and were processed with standard techniques.

**5.3 Histological examination** 

(Lee et al., 2005). ROS production may exceed physiological protection mechanisms and can thus be referred to as oxidative stress (Tsaryk, 2009).

CoCr alloys have higher corrosion rate compared to titanium and titanium alloys and release toxic Co and Cr ions. Furthermore, Co ions mediate oxidative stress and could increase up to eight times oxidative stress in the cell (Limbach et al., 2007).

It proves that elevated oxidative stress in the setting of aseptic loosening is a local phenomenon. Recent studies showed that increased levels of Co and Cr ions are not connected with elevation of the level of oxidative stress in the blood of patients (Antoniou et al., 2008, Tkaczyk et al., 2010).

### **4.1 Response to oxidative stress - oxidative stress and bone**

Excessive amounts of ROS are toxic to the organism and cells have specific protection mechanisms against oxidative stress. In ROS deactivation, superoxide dismutase (SOD), catalase and gluthatione (GSH-GSSG) system play central role. In one of the most important systems, glutathione peroxidases detoxifies peroxides with GSH acting as an electron donor in the reduction reaction, producing GSSG as an end product (Townsend et al., 2003). Hence, the balance between reduced (GSH) and oxidized gluthatione (GSSG) is very important for protection against oxidative stress. A deficiency of GSH puts the cell at risk for oxidative damage. It is not surprising that an imbalance of protection mechanisms against oxidative stress is observed in wide range of pathologies including inflammatory and degenerative disorders with tissue fibrosis.

Elevated oxidative stress was associated with low bone mineral density (Ozgocmen et al., 2007, Basu et al., 2001) and gene polymorphisms in antioxidant enzymes were also associated with low bone mineral density (Mlakar et al., 2010). Further research elucidated oxidative stress as a potential modulator of osteogenesis in different skeletal diseases (Liu et al., 2010). ROS have been involved in osteoporosis by causing cellular death and by inhibiting osteoblast proliferation and stimulating osteoclast differentiation (Hamel et al., 2008, Weitzmann & Pacifici, 2006). It was proven that H2O2 inhibits osteoblast proliferation time- and dose-dependently (Li et al., 2009) and that decreasing oxidative stress normalizes bone formation and bone mass in mice (Rached et al., 2010). Although extensively studied (Bai et al., 2005, Basu et al., 2001, Mody et al., 2001, Rached et al., 2010)., the mechanisms of action of ROS on bone formation are not completely understood. (Bai et al., 2005, Basu et al., 2001, Mody et al., 2001, Rached et al., 2010).
