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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: The organisation of cytoskeletal components in isolated chondrocytes cultured in agarose

Author: IDOWU, Bernadine

Year: 2001

Supervisor(s): Dan Bader, David Lee

The well characterised isolated chondrocyte/agarose model enables the investigation of the influence of cell deformation, independent of other possible mechanotransduction pathways. The organisation of the cytoskeleton is critical for normal cell metabolism and therefore plays an important role in maximising the elaboration of extracellular cartilaginous matrix.
The current study has optimised a series of methods to analyse and quantify the 3 main cytoskeletal components within isolated bovine articular chondrocytes from different zones seeded within 3% agarose gel, visualised using confocal laser scanning microscopy.

Four methods were developed namely, (i) a numerical scoring system, (ii) a visual ranking system, (iii) an intensity threshold method and (iv) an edge map method. The latter was selected as it provided an edge index (EI) value which described the organisation within the cell that satisfied the established criteria.

Results indicated that mechanical compression alters the organisation of the cytoskeleton within chondrocytes. This was assessed by comparing separate population of cells within unstrained and strained constructs. Using this method a consistent reduction in EI value for actin microfilament was revealed within cells in constructs subjected to static or dynamic strain when compared to unstrained control values. For example reductions of 15%, 10% and 20% were observed after culture periods of 1, 24 and 72 hours, respectively. A similar pattern of response was evident for both superficial and deep zone cells suggesting similar pathways are activated within both cell subpopulations. The results for the microtubules and vimentin intermediate filaments did not follow the same pattern.

This study has yielded an important step towards understanding the role of the cytoskeleton in response to both static and dynamic compression. It proposes upstream and downstream mechanisms by which the application of mechanical compression may induce alterations in cytoskeletal organisation via direct or indirect mechanisms, which may dictate alterations in cellular activity.