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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: Chondrocyte metabolism in alginate constructs for tissue engineering

Author: HEYWOOD, Hannah

Year: 2004

Supervisor(s): David Lee, Dan Bader

The work involved a model system incorporating primary bovine chondrocytes within a 4 mm thick alginate construct. A viability profiling protocol, in conjunction with histology and biochemistry, permitted a spatial description of cellular activity. Subsequently, the cellular utilisation of glucose and oxygen was assessed and data evaluated by a numerical model. In addition, the novel application of Fluorescence Recovery After Photobleaching to the quantitative and non-invasive analysis of developing matrix distribution was examined.

A general paucity of matrix and reduced chondrocyte viability were observed in the centre of the alginate constructs. However, the study revealed that cellular utilisation, as opposed to scaffold concentration, is the dominant factor that defines neo-cartilage development. Increasing the medium volume to at least 6.4 mL.10-6 cells permitted homogeneous tissue development in constructs seeded at high cell densities (40x106 cells.mL-1). Glucose supplementation maintained the cellularity of constructs cultured in volumes less than 1.6 mL.10-6 cells, but did not account for the continued augmentation of GAG biosynthesis in enhanced volumes, from 1.6-6.4 mL.10-6 cells. The metabolism of the chondrocytes was characterised by a high conversion of glucose to lactate and low oxygen consumption rate, equivalent to 9.6x10-16 mol.cell-1.hr-1. However, primary chondrocytes are capable of double their oxygen consumption rate when glucose is lower than 2.7 mM. Under these conditions the oxygen consumption by the chondrocytes reduced the oxygen tension beneath 2 mm thick constructs to values less than 10 mM. Accordingly, knowledge of how chondrocyte nutrient utilisation is regulated will contribute to intelligent bioreactor design.