Cartilage is a robust connective tissue found throughout the body and, due to its properties and complex composition, is extremely difficult to repair when damaged. The main cells found in cartilage are chondrocytes, which play a special role in the development, maintenance and repair of the extracellular matrix. Chondrocytes were the focus of our research. The aim of this thesis is to determine the influence of different growth media and inflammation on the expression of cartilage-related genes in primary chondrocytes. The expression of the investigated genes was measured in 43 primary chondrocyte samples obtained from knee cartilage of 5 different patients. The tissue samples were obtained during total or partial knee arthroplasty performed at the Department of Orthopaedic Surgery of the University Clinical Centre Maribor and Celje General Hospital. Primary chondrocytes were isolated from the samples and cultured in different growth media (S1-S6), the composition of which is confidential due to the patent application process. In some cells, an inflammatory environment was created by the addition of tumor necrosis factor α (TNF-α) in the culture medium. After culture, cell lysis was performed and total RNA was isolated. This was then reverse transcribed into cDNA, which was then used to determine the expression of cartilage-related genes by quantitative polymerase chain reaction (qPCR). The expression of chondrocyte genes (SOX-9), cartilage genes (COL1A1, COL1A2, COL2A1, COL11A2, COL9A2, COL3A1, ACAN1, COMP, VCAN, DCN, FMOD, BGN, SNORC, RUNX2), MAPK signalling pathway genes (MAP3K1, MAPK1, MAP2K1, MAPK3), and other genes (BAX, ALP, SPP1, and PPARG) was determined. We hypothesised three hypotheses: the first, which assumes that the expression of the genes under investigation differs between chondrocytes grown in different growth media under physiologic conditions; the second, which assumes that the expression of the genes under investigation differs between chondrocytes grown in different growth media under inflammatory conditions; and the third, which assumes that the expression of the genes under investigation differs between chondrocytes grown in the same growth media under inflammatory and physiologic conditions. All three hypotheses were partially accepted. Under physiologic conditions, we found differences in the expression of 4 genes between the media, i.e. BGN (between S4 and S5), DCN (between S1 and S4), MAP3K1 (between S4 and S5 and S4 and S6) and ALP (between combinations of all 6 media). Under inflammatory conditions, we observed an interesting difference in BAX gene expression between growth media S1, S3 and S4. When examining the differences between inflammatory and physiologic conditions, we found a statistically significant difference in BAX gene expression in growth medium S1. Our results thus demonstrate a differential impact of the investigated growth media on primary chondrocytes at the molecular level. Further studies of the investigated molecules at the protein level would be necessary to confirm the differences observed. However, functional studies on primary chondrocytes would be necessary to determine the potential of the investigated growth media for cartilage regeneration.
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