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Cellular senescence is a program activated during diverse situations of cell stress. Chondrocytes differ from other somatic cells as articular cartilage is an avascular tissue. The effects of oxidative stress on chondrocytes are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of human osteoarthritic chondrocytes, subjected to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by RT-PCR. Sub-lethal doses of oxidative stress induced cell-cycle arrest, senescentmorphological features and senescence-associated b-galactosidase positivity. Prolonged oxidative treatment had no effects on cell proliferation or morphology. Sub-lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. The effects of sublethal oxidative stress regarding proliferation and telomere biology were more distinct in senescent cells. Acute oxidant insult caused upregulation of p21 expression to levels comparable to senescent cells. TRF2 protects telomere ends and showed elevated expression levels. SIRT1 and XRCC5 enable cells to cope with unfavorable growing conditions. Both were up-regulated after oxidant insult, but expression levels decreased in aging cells. Taken Keywords: cellular senescence; oxidative stress; telomeres; DNA damage; human chondrocytes Cellular aging or senescence refers to metabolically active somatic cells having entered a state of permanent growth arrest. Replicative senescence has been associated with telomere shortening. Telomeres consist of noncoding repetitive DNA sequences and associated proteins located at the end of linear chromosomes. Human telomeres form a 2,000-30,000 base pair (bp) array of duplex DNA strand of TTAGGG repeats, ending in a 100-200 bp nucleotide 3 0 single strand overhang invading the duplex array to form a lariat-like loop structure (t-loop). Telomeric repeats ensure the complete replication of encoding DNA. Chromosome ends, uncapped by telomeres, are susceptive to degradation and fusion and can activate DNA damage checkpoints. 1 A six-protein complex-known as telosome or shelterin complex-functions to form and maintain the t-loop structure. TRF1 and TRF2 are part of the core telomeric proteins. Various signaling pathways coordinating telomere function and cell cycle regulation start off the telosome complex. TRF1 modulates the length of telomeres. TRF2 is important for stabilizing the t-loop structure. 2,3 XRCC5 and SIRT1 are associated with the telomerenucleo-protein complex and link telomere biology and DNA damage checkpoints with genes involved in other cellular stress responses. XRCC5 is involved in repairing DNA double-strand breaks. 4,5 SIRT1 is a negative regulator of p53 and prevents growth arrest, senescence, and apoptosis. 6 Telomere uncapping, DNA damage foci at telomeres 7 and oxidative insults cause p21 elevation. The p21...

Decreasing replicative potential and dedifferentiation of articular chondrocytes during expansion in cell culture are essential limitations for tissue engineering and cell therapy approaches. Telomeres and telomerase play a key role in cell development, aging, and tumorigenesis. There is evidence that growth factors are involved in regulating telomerase activity. Therefore, the objective was to evaluate the effect of selected growth factors on telomere biology of serially passaged chondrocytes. Human articular chondrocytes were isolated from cartilage of three patients undergoing total knee arthroplasty. The chondrocytes were cultured in monolayer with the growth factors PDGF-BB, TGF-b1, and bFGF. Telomere length was measured by telomere restriction fragment length assay, and telomerase activity was determined by quantifying the gene expression of its catalytic subunit hTERT by rtPCR. Chondrocytes cultured with PDGF-BB and TGF-b1 showed a significantly higher proliferation rate than control cells. None of the growth factor cultures revealed an accelerated rate of telomere shortening. Telomerase was not expressed in significant amounts in any of the chondrocyte cultures. Growth factor treatment of chondrocyte cell cultures for cell therapy purposes can be regarded as safe in terms of telomere biology. ß

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