Articular cartilage disorders are a leading cause of human disability in many countries around the world. In this work, new techniques and strategies were developed to improve the quality of cartilage produced in vitro by methods of tissue engineering. Chondrocytes were isolated from the hip and knee joints of aborted human foetuses. The cells were expanded and seeded into scaffolds and the seeded scaffolds were cultured in perfusion bioreactors. The quality of the final cartilage constructs was assessed biochemically by measuring their content of glycosaminoglycan (GAG), total collagen and collagen type II and histologically by staining cross-sections of the constructs for GAG, collagen type I and collagen type II. The amount of proteoglycan released in the culture medium was also measured at regular intervals. Proteoglycans from tissue-engineered cartilage and spent culture medium were compared and analysed for degradation and capability of aggregation. During monolayer expansion, the chondrocyte differentiation indices decreased, the cell size increased and the percentage of cells present in G2/S??M phase decreased with the greatest changes occurring during the first passage. Expanding chondrocytes in PGA or PGA??alginate scaffolds produced cells with a higher level of differentiation than monolayer-expanded cells. However, PGA and PGA??alginate could not be justified as suitable systems for the routine expansion of chondrocytes mainly because of the relatively low cell proliferation obtained. Two new methods for seeding of cells into scaffolds were investigated using PGA and PGA??alginate as scaffold materials. Both methods produced high seeding efficiencies and homogeneous distribution of cells. When seeded PGA??alginate scaffolds were cultured in perfusion bioreactors, they produced good quality constructs with higher concentrations of extracellular matrix (ECM) components compared with previously described methods. However, when seeded PGA scaffolds were cultured in perfusion bioreactors, they produced small constructs of poor quality. Investigation of the effect of medium flow rate on the PGA scaffolds showed that a low flow rate was needed at the beginning of the culture to enable the cells to form a framework onto which other synthesised elements could deposit. Applying a gradual increase in medium flow rate to PGA scaffolds cultured in perfusion bioreactors solved the shrinkage problem and produced constructs with quality similar to those produced using PGA??alginate scaffolds. A novel compression bioreactor that mimicked the physiological stimulation of cartilage by joint movement was constructed. Using this bioreactor, compressed constructs showed significantly higher wet weight and higher concentrations of GAG, total collagen and collagen type II compared with non-compressed constructs.
| Identifer | oai:union.ndltd.org:ADTP/222265 |
| Date | January 2008 |
| Creators | Shahin, Kifah, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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