Objective: To investigate the influence of tubulin disassembly on the in vitro metabolism of articular chondrocytes.
Methods: Eight New Zealand rabbits aged 2 months were sacrificed by air embolism. The full-thickness cartilages were harvested from both knees under sterile conditions. Then 0.4% pronase and 0.025% II collagenase were used to digest for primary chondrocytes. The cells were cultured for 3 days after attachment and then divided into the control and experimental groups. The control group continued culturing with primary medium and the experimental group with colchicine, a tubulin destructive agent, at a final concentration of 0.1 µmol/L. At Days 1 & 2, the early apoptosis of two cell groups was assayed with phosphatidylserine (Annexin V). At Day 6, the morphological changes of cells were observed by hematoxylin and eosin staining. At Days 3, 6 & 9, the expression levels of type II collagen, proteoglycan and MMP (matrix metalloproteinase)-13 mRNA were measured by real time quantitative fluorescent reverse transcription-polymerase chain reaction (real time RT-PCR). Meanwhile at Days 3, 6 & 9, the cultured supernatant of each group was obtained to detect the contents of type II collagen and proteoglycan by the methods of ELISA (enzyme-linked immunosorbent assay) and Alcian blue.
Results: At Day 2, the early cell apoptosis rate of the experimental group was higher than that of the control group (P < 0.05). At Day 6 post-dosing, the experimental cell morphology was found to be irregular and polygonal. Hyperchromatic nucleus and division phase increased while matrix cell decreased. At Days 3, 6 & 9, the expressions of collagen and proteoglycan mRNA in the experimental group decreased versus those in the control group (P < 0.05). The level of MMP-13 mRNA showed no significant changes in the control group at Day 3. And it significantly increased at Days 6 & 9 (P < 0.01). Meanwhile, type II collagen and glycosaminoglycan were significantly lower in the experimental group at Days 3, 6 & 9 than those in the control group (P < 0.05).
Conclusion: A disassembly of tubulin can induce early apoptosis of chondrocytes. The synthesis and secretion of type II collagen and proteoglycan decrease while there is an elevated expression of MMP-13. The degeneration of cartilage cells and their disrupted synthesis and catabolism result in a serious decline in biological functions.