Changes in cytoskeleton and glycosaminoglycan synthesis in passaged articular chondrocytes / 中华创伤骨科杂志

ABSTRACT

Objective To observe the changes in cytoskeleton (CSK) and glycosaminoglycan (GAG) synthesis following passage culture of articular chondrocytes and the correlation between CSK and GAG.Methods Eight male New Zealand White rabbits (8-month-old) were sacrificed by air embolism.After the chondrocytes from their knee joints were isolated by enzymolysis method,monolayer culture was performed.The chondrocytes of primary passage (P0) and passages 1 & 2 (P1,P2) were inoculated into 24-well plates with round cover slips put at the bottoms.Cell climbing slices were fixed after attachment of chondrocytes.The CSK proteins,actin,vimentin,tubulin and vinculin were stained by immuuofluorescence antibody on P0,P1 and P2 cell climbing slices,respectively.The CSK morphology was observed by laser confocal scanning microscopy and the fluorescence intensities of CSK proteins were detected by the fluorescence intensity software.The medium was changed for each generation after cell fusion and the GAG concentrations in the supernatants were measured at 24,36,48,60 h after medium change by alcian blue method.Results The intermediate filament networks became loosen and the dense distributions surrounding the nucleus decreased;more microtubule processes formed at the cell periphery with passage.The fluorescence intensity of actin of P1 chondrocytes was significantly increased than that of P0 (P ＜ 0.05),but there were no such significant differences between P0 and P2 or between P1 and P2 (P ＞ 0.05).The fluorescence intensities of vimentin and tubulin were significantly decreased with passage respectively,and there were such significant differences between any two of P0,P1 and P2 (P ＜ 0.05).The GAG concentrations in the supernatants were significantly decreased with passage at each time point,and there were such significant differences between any two of P0,P1 and P2 (P ＜ 0.05).Conclusions Passage culture of articular chondrocytes may lead to changes in morphology and protein expression intensity of the main components of CSK,and accordingly to decreased synthesis amount of GAG,one of the extracellular matrix of chondrocytes,indicating the changed characteristics of chondrocytes after passage and a certain correlation between CSK and GAG.