## 4 Discussion
### US Therapy Induced Autophagosomes Occurence  
The most important ultrastructural change in the US-OS group was the large number of lysosomes and the occurrence of autophagosomes. A variety of essential cellular pathways are associated with lysosomal function, including the clearance of substrates, autophagy, exocytosis, and cell membrane repair. Cells with a larger number of lysosomes have an enhanced degradative capacity against lysosomal substrates such as glycosaminoglycans and autophagy substrates such as protein aggregates [90]. The autophagosomes occurrence is a characteristic feature for macroautophagy, a stress-induced form of autophagy that allows removal and continuous replacement of nonfunctional proteins and organelles. After fusion of lysosomes with autophagosomes to form autophagolysosomes, and after degradation of their content, a secondary lysosome may result [29]. This phenomenon explains how the number of lysosomes may remain higher under the autophagy process. In our study autophagosomes' presence in the US-OS group confirms that the autophagy process is stimulated by US. Autophagy is a cellular mechanism that supports adaptation and survival of cells in OS and other stress conditions [91, 92]. It has been shown that human age-related OA and surgically induced OA in mice were associated with a reduction in key regulators of autophagy and were accompanied by an increased rate of apoptosis [2]. In rat chondrocytes cultures, blocking autophagy also accelerated apoptosis [93]. The senescence--defective autophagy--OA axis was illustrated by [PERSON], who demonstrated that early changes occurring in aging cartilage were due to an autophagy defect. Thus, he hypothesized that \"agents preventing aging-related loss of autophagy might promote chondrocyte survival and cartilage biosynthetic capacity\" [94]. Autophagy defects cause an accumulation of dysfunctional mitochondria in chondrocytes [32]. This indicates that mitochondria function and autophagy are interdependent. In our study US preserved mitochondria ultrastructure most probably by autophagosomes occurrence. Recently, pre-treatment with vitamin D was found to be able to protect endothelial cells from mitochondrial lesions induced by H2O2 and to prevent endothelial cell death by reducing the apoptosis-related gene expression and activation of pro-autophagic molecule Beclin 1 [95]. In previous studies it was found that stimulation of autophagy attenuated mitochondrial injuries and ERS in chondrocytes exposed to OS [96] and ERS in mice hepatocytes exposed to a high-fructose diet [97]. In our US-OS group mitochondria had a normal aspect, and RER was swollen in most chondrocytes, but in a small amount. It could mean: firstly--US inhibited ROS production and ROS induced mitochondrial damages, secondly--autophagocytosis rapidly removed damaged cell organelles, or thirdly--both previous mechanisms were involved. In accordance with previous research, two molecular pathways could explain the US-mediated autophagy increase: MAPK pathways and phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway. In his study [PERSON] demonstrated that US can improve viability of chondrocytes exposed to H2O2 by modulation of p38 MAPK autophagy inhibitory pathways [34; 98]. In another study [PERSON] showed that US induced autophagy in chemotherapy-resistant prostate cancer cells via the PI3K/AKT/mTOR signaling pathway [99]. When mTOR is inhibited, the activity of PI3K/Akt pathway increases, and the autophagy is activated to protect chondrocytes [100]. Recently [PERSON] summarised in a narrative review the effects of the PI3K/AKT/mTOR signaling pathway in OA showing that PI3K/AKT could prevent chondrocyte death by autophagy stimulation [101]. To date only few studies demonstrated US capacity to induce autophagocytosis, and, to our knowledge, none of them proved this effect in chondrocytes.