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Queen Mary University of LondonQueen Mary University of London
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School of Engineering and Materials Science
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PhD Thesis: Calcium mediated signalling pathways for chondrocytes in 3D constructs subjected to cyclic loading

Author: PINGGUAN, B

Year: 2006

Supervisor(s): Dan Bader, Martin Knight

This thesis presents the development and validation of a novel computer-controlled mechanical system to load bovine chondrocytes seeded in an agarose construct with porous glass endplates. A single wavelength Ca2+ indicator, Fluo 4AM, was selected to monitor intracellular Ca2+ response. Within unloaded controls a sub-population of 40-50% of chondrocytes exhibited characteristic spontaneous Ca2+ transients over a 300 second imaging period. The response could be categorised into those cells which exhibited one to three transients and those exhibiting four or more, so called oscillatory transients. Static compression did not cause any upregulation of Ca2+ response from control values. By contrast, cyclic compression for only 1 cycle significantly upregulated the percentage of cells exhibiting Ca2+ transients. A similar effect was noted after 10 or 100 cycles. Furthermore, the reduced response following 300 cycles indicates a possible receptor desensitisation mechanism. In addition it was shown that Ca2+ signalling was, to some degree, modulated by strain rate and frequency of the dynamic loading. A further set of experiments were designed to investigate the autocrine-paracrine signalling mechanisms. These involved treatment with specific chemical modulators such as apyrase, significantly reduced the percentage of cells responding during cyclic loading, suggesting the role of ATP in the mechanotransduction pathway. Incubation with other modulators indicated a role of mechanosensitive ATP release channels during cyclic loading and highlighted the interplay between extracellular and intracellular Ca2+ in mechanically-induced Ca2+ signalling.

These findings should considered when designing biomechanical conditioning regimes for tissue engineering repair of articular cartilage.