## 4 Discussion
### Oxidative Stress-Induced Ultrastructual Chondrocyte Damages  
Four types of death are described for chondrocytes: apoptosis (type I), autophagic chondrocytes death (type II), necrosis (type III), and chondoptosis, the last involving both apoptotic and autophagic components [68]. In our study we found that H2O2 exposure led to variable morphological changes in primary human chondrocytes, and the ultrastructural damages were correlated with OS increase. The main ultrastructural changes in chondrocytes exposed to OS were found in mitochondria and RER. Mitochondria displayed a more condensed matrix, and RER was expanded and in large quantities in most viable chondrocytes. In almost all cells we identified a large amount of electron-dense fibrillar material. All these ultrastructural changes found in chondrocytes exposed to H\({}_{2}\)O\({}_{2}\) were recognised as markers for cellular OS. Condensed matrix of mitochondria was reported as consequence of mitochondrial membrane potential (mMP) damage induced by OS [69]. Previous studies communicated that RER exposure to OS induced accumulation of unfolded proteins, RER expanding, and endoplasmic reticulum stress (ERS) occurrence [70]. Accordingly, in our study the large amount of expanded RER in most viable chondrocytes was the proof of ERS induced by OS. At the same time, the abundant fibrillar material in chondrocyte exposed to OS was a marker for chondrocyte injury. Fibrillar material was also found in ear chondrocytes exposed to papain, an enzyme with proteolytic effect on cartilage [71], and in rabbit OA chondrocytes [72]. Ultrastructural Damages in Chondrocytes Exposed to OS Were Correlated with MDA and TNF-\(\alpha\) Increase  
In our study MDA and TNF-\(\alpha\) levels increased in chondrocytes exposed to H\({}_{2}\)O\({}_{2}\) and were associated with chondrocytes necrosis, mitochondria condensed matrix, and a large amount of expanded RER. Previous studies also demonstrated elevated MDA and TNF-\(\alpha\) levels in rat chondrocyte cultures exposed to H\({}_{2}\)O\({}_{2}\)[73; 74]. MDA is a stable product of the reaction between high-energy radicals and lipids [45] and the most used indicator of OS in clinical and experimental studies. In previous studies it was demonstrated that MDA was involved in collagen oxidation [75], reacted with histidine and lysine to form stable adducts, and might induce DNA cross-linking in cartilage [76]. The chondrocytes necrosis, ERS, and mitochondrial damages were found to also be correlated with MDA increase in previously published studies. In [PERSON] disease MDA enhancing was correlated with chondrocytes necrosis in the deep-zone of cartilage [77]. In rat monosodium iodoacetate OA, a MDA increase was associated with elevated levels of TNF-\(\alpha\) and biochemical markers of ERS [78]. In human OA chondrocytes higher MDA values coexist with lower respiratory capacity and higher proton leak in mitochondria correlated with structural mitochondria damage [79]. In our experimental model, cell necrosis, damaged mitochondria matrix, and expanded RER occurrence were associated with a significant TNF-\(\alpha\) increase in in chondrocytes exposed to H\({}_{2}\)O\({}_{2}\). In accordance with our results, in a previous study the H\({}_{2}\)O\({}_{2}\) capacity to induce TNF-\(\alpha\) synthesis was proven [80]. TNF-\(\alpha\) was also found to stimulate NF-\(\kappa\)B mediated inflammatory response and ERS in human chondrocytes. [81] In another study [PERSON] communicated that in TNF-\(\alpha\)-treated chondrocytes mitochondria cristae and ATP decreased as consequence of the mitochondria injury [82]. Previous studies demonstrated that TNF-\(\alpha\) can induce both apoptosis and necrosis, depending on cell types and the experimental model [83; 84]. Although most studies have associated TNF-\(\alpha\) synthesis with apoptosis, our study showed that most chondrocytes died from necrosis.