## 4 Discussion  
Increasing evidence in both experimental and clinical studies suggests that OS is one of the main causal factors in the OA pathogenesis [3]. An increased production of ROS and a deficient antioxidant protection coexist in OA. In chondrocytes the primarily formed radical is superoxide (O2\({}^{\bullet-}\)), which is converted to H2O2 by superoxide dismutase (SOD). Usually, catalase (CAT) and glutathione peroxidase (GPx) neutralise H2O2, but in OA these enzymes are deficient and allow H2O2 accumulation. In late-stage OA, SOD synthesis declines, and a O2\({}^{\bullet-}\) neutralization deficit appears.  
H2O2 and O2\({}^{\bullet-}\) impairs mitochondrial membrane permeability, leads to cytochrome c release into the cytoplasm [49], and may cause chondrocytes apoptosis [50,51]. H2O2 also induces the expression of some proinflammatory cytokines, such as IL-1\(\beta\) and TNF-\(\alpha\), that exert stimulating effects on the expression of MMPs and contribute to the degradation of ECM [52].  
Due to the analgesic and anti-inflammatory effects, US therapy has many applications, both in human and veterinary pathology, even when classical non-steroidal therapy is contraindicated [53, 54, 55, 56, 57, 58, 59]. A large number of clinical and experimental studies that support US use in OA treatment have been published over the last years. Nevertheless US influences on OS in OA are unclear. Some studies performed in cancer therapy have demonstrated that low-intensity US (LIUS) may induce apoptosis in human hepatocarcinoma cells in vitro, by mitochondrial dysfunction and OS [60]. This effect was also reported in human leukemic cells [61]. On the other hand, some studies reported US antioxidant effects. According to [PERSON], LIUS therapy diminished OS during reperfusion in ischemia-reperfusion experimental animal models [62]. In our previous studies we demonstrated that US therapy improved plasma antioxidant capacity in OA patients [63], and recently antioxidant and anti-inflammatory effects of US have been reported in experimental model of muscle injury in rats [64]. Moreover, [PERSON] and coworkers communicated that LIUS, at a lower intensity (<3 W/cm2), induced\"more of antioxidant effects in non-cancer cells compared to cancer cells\" [65].  
Since US effects depend on the dose and the irradiated tissues, it is not surprising that the results of studies regarding the influences of US on OS are contradictory.  
In our study we applied US at the frequency and the intensity used in OA therapy to assess their effects on chondrocytes exposed to OS.  
Because the chondrocytes cultures are more vulnerable to stress than the in vivo chondrocytes, we chose to use US at low therapeutical intensity.  
In our study H2O2 was used for inducing OS in chondrocyte cultures because H2O2 is an important factor in OA pathogenesis [66]. It was also used to reproduce oxidative environment in chondrocytes cultures.  
In a previous study, H2O2 demonstrated its capacity to increase the level of ROS and mRNA levels for pro-inflammatory and prooxidative genes (cyclooxygenase-2 and inducible nitric oxide synthase) and to induce a significant increase in caspase 3-like activity and chondrocyte death [67].  
In our study H2O2 added to chondrocyte cultures increased MDA and TNF-\(\alpha\) synthesis and induced the death of some chondrocytes by necrosis and ultrastructural damage for most viable chondrocytes. After H2O2 exposure only a small number of chondrocytes remained quite normal in appearance. Pre-exposure to US-protected chondrocytes from MDA and TNF-\(\alpha\) increased as did ultrastructural damages.