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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: Mechanotransduction pathways associated with intracellular calcium in chondrocytes within 3D constructs.

Author: ROBERTS, S.R.

Year: 2001

Supervisor(s): Dan Bader, David Lee

Calcium (Ca2+) signalling forms part of a possible mechanotransduction pathway by which chondrocytes may alter their metabolism in response to mechanical loading. This thesis presents the development of a series of techniques used to quantify intracellular Ca2+ in isolated adult bovine articular chondrocytes seeded within agarose constructs. A ratiometric Ca2+ indicator, Indo-1, was selected to monitor intracellular Ca2+ dynamics. A standard operating procedure was developed for the staining of chondrocytes with Indo-1, with minimal artefacts associated with the AM ester dye. Intracellular Ca2+ levels were expressed using a Ca2+ ratio.

Basal Ca2+ levels remained constant in cell seeded constructs cultured up to day 6 and following exposures to varying concentration of extracellular Ca2+ up to 20 mM. However, exposure to either Tg or EGTA significantly altered the basal levels. results suggest that chondrocytes within agarose have highly efficient systems for both Ca2+ buffering and ion transport. ATP-induced Ca2+ signalling was observed in cells in both monolayer and agarose cultures. The observed response in agarose was significantly reduced in the presence of both Tg and EGTA, suggesting release from intracellular stores and a Ca2+ influx across the membrane.

A technique was adapted to apply unconfined compressive strain to chondrocyte/agarose constructs in a repeatable manner. This allowed the same cell to be tracked prior to and following the application of 20% strain. In association with a change in morphology, isolated chondrocytes seeded in compressed agarose constructs demonstrated intracellular Ca2+ signalling on days 1 and 3 of culture. Following the application of compression a delay, with a mean time of 200 seconds, was observed prior to the onset of the Ca2+ response. This delay suggested the activation of upstream signalling events prior to Ca2+ mobilisation. The Ca2+ response following compression was significantly reduced in the presence of either EGTA or Gd3+.