Optimisation and validation of a tri-axial bioreactor for nucleus pulposus tissue engineering

Mechanical stimulation, in combination with biochemical factors, is likely to be essential to the appropriate function of stem cells and the development of tissue engineered constructs for orthopaedic and other uses. A multi-axial bioreactor was designed and built by Bose ElectroForce to simulate physiologically relevant loading conditions of the intervertebral disc (IVD), including axial compression, hydrostatic pressure and perfusion flow to multiple constructs under the control of a software program. This research optimises the design and configuration of the perfusion system of the bioreactor and presents results of preliminary experimental work on the combined effects of axial compression and perfusion on the viability of mesenchymal stem cells encapsulated in alginate hydrogels and the ability of the cells to produce extracellular matrix (ECM). The results of this thesis illustrated the power of a design of experiments (DOE) approach as a troubleshooting quality tool. With a modest amount of effort, we have gained a better understanding of the perfusion process of the tri-axial bioreactor, improved operational procedures and reduced variation in the process. Furthermore, removing unnecessary tubing lengths, equipment and fittings has made cost savings. The steady flow energy equation (SFEE) was used to develop a numerical analysis framework that provides an insight into the balance between velocity, elevation and friction in the flow system. The pressure predictions agreed well with experimental data, thus validating the SFEE for fluid analysis in the bioreactor system. The numerical predictions can be used to estimate the pressures around the three-dimensional constructs with a given arrangement of the tubing and components of the bioreactor. The system can potentially support long-term cultures of cell-seeded constructs in controlled environmental conditions found in vivo to study the mechanobiology of nucleus pulposus tissue engineering and the aetiology of IVD degeneration. However, dynamic compression and perfusion with associated hydrostatic pressurization of culture medium resulted in significant loss of cell viability compared to the unstimulated controls. Due to a large number of factors affecting cell behaviour in the tri-axial bioreactor system, it is difficult to identify the exact parameters influencing the observed cell response. A strategy that could help to distinguish the effects of mechanical stimuli and specific physiochemical factors should combine experiments with mathematical modelling approaches, and use the sensing incorporated in the bioreactor design and process-control systems to monitor and control specific culture parameters. Optimisation of the cell passage and cell seeding density were identified as key areas to improve the production of GAG in future studies; since the production of ECM was not observed in both static and dynamic cultures. Further studies could also attempt to use other hydrogel scaffolds, such as agarose, which has been widely used in cartilage tissue engineering studies and hyaluronic acid - a component of the nucleus pulposus ECM