Monosodium Urate and Tumor Necrosis Factor-α Increase Apoptosis in Human Chondrocyte Cultures.

Monosodium urate and tumor necrosis factor-α, are two potent mediators of separate inflammatory response pathways in arthritic joints where inflammation may be accompanied by the loss of chondrocyte vitality via apoptosis. To address this possibility in vitro, chondrocyte cultures were employed to determine the extent to which monosodium urate and recombinant TNF-α altered the frequency of apoptotic chondrocytes. Apoptosis as a function of the activation of p38 kinase, C-Jun-terminal kinase, signal transducer and activator of transcription-3 and/or the activity of xanthine oxidase was also studied. Using normal human chondrocytes, monosodium urate or recombinant tumor necrosis factor-α increased the frequency of apoptosis and activity of xanthine oxidase. However, the xanthine oxidase-specific inhibitor, febuxostat, failed to blunt this response. Monosodium urate, tumor necrosis factor-α or the Janus kinase inhibitor, AG-490, increased the frequency of apoptotic nuclei in macroaggregate pellet cultures initiated from juvenile human chondrocytes, but not in pellet cultures derived from mesenchymal stem cells. In OA chondrocytes, activation of p38, C-Jun-NH2-kinase and signal transducer and activator of transcription-3 preceded apoptosis. Activation of signal transducer and activator of transcription-3 also was seen in pellet cultures initiated from juvenile chondrocytes and MSCs incubated with MSU, recombinant tumor necrosis factor-α or febuxostat, but apoptosis was increased only in the pellet cultures derived from juvenile chondrocytes. Although AG-490 or the combination of AG-490 and febuxostat inhibited signal transducer and activator of transcription-3 activation, apoptosis was unaffected. These results showed that recombinant tumor necrosis factor-α, monosodium urate and AG-490 increased apoptosis in normal human chondrocytes, OA chondrocytes and human juvenile chondrocyte pellet cultures, but not in chondrocyte pellet cultures initiated from MSCs. The increased frequency of apoptotic chondrocytes in response to recombinant tumor necrosis factor-α or monosodium urate was not dependent on either activation of STAT3 or the activity of XO.

Low oxygen tension during incubation periods of chondrocyte expansion is sufficient to enhance postexpansion chondrogenesis.

To determine whether low oxygen (O(2)) tension during expansion affects the matrix density, as well as quantity, of cartilage formed, and to determine whether application of low O(2) tension during incubation periods alone is sufficient to modulate chondrogenic expression, rabbit chondrocytes expanded at either 21% O(2) or 5% O(2) were analyzed for glycosaminoglycan (GAG) and DNA content, total collagen, and gene expression during expansion and postexpansion aggregate cultures. When cultured as aggregates at 21% O(2), chondrocytes expanded at 5% O(2) produced cartilage aggregates that contained more total GAG, GAG per wet weight, GAG per DNA, and total collagen than chondrocytes expanded at 21% O(2). Less of an effect on GAG and collagen content was observed when aggregate culture was performed at 5% O(2). Real-time polymerase chain reaction analysis of COL2A1 expression showed upregulated levels of type IIA (an early marker) and IIB (a late marker) during expansion and elevated levels of type IIB during aggregate culture in chondrocytes expanded in low O(2). The application of low O(2) tension during incubation periods of chondrocyte expansion enhances the ultimate cartilage matrix density and quantity, and this enhancement can be achieved through the use of an O(2) control incubator.