Mapping fluid flow in porous biomaterials for tissue engineering

Tissue engineering strategies seek to regenerate cartilage tissue in vitro using a combination of cells, scaffolds and cell stimulation via means including bioreactors, as an alternative treatment option to cartilage defects and injuries. There is great interest in capitalising on perfusion and the associated fluid induced forces as a means of providing mechanical stimulation to cells, to ultimately influence desired tissue formation. The use of perfusion bioreactors to introduce such mechanical stimulation has been shown to effectively encourage cartilage regeneration when applied to cells in 3D porous scaffolds. In tissue engineering, cell scaffold constructs are often matured in vitro for extended periods prior to in vivo use so it is vital that the culture environment facilitated by bioreactors enhances tissue formation. Nonetheless, application of excessive perfusion rates can be detrimental to cell attachment, and cause non-desirable changes to differentiation pathways. Therefore, it is imperative that perfusion flow is closely controlled to ensure it provides appropriate levels of mechanical stimulation to cells. However, the relationship between fluid flow and the cellular response to fluid flow in culture, in addition to how far these two influence one another over time needs to be elucidated. To investigate the effectiveness of perfusion bioreactors, research currently relies predominantly on computational models to predict behaviour, and post analysis of scaffolds after perfusion. Experimental real time data to understand how not only does a dynamic culture system change with culture time, but also how the porous architecture influences the fluid pathway would provide a great insight into how scaffold design and subsequent cell proliferation and differentiation effect the flow velocity and fluid induced forces. The optimisation of dynamic culture experiments, bioreactor design and scaffold porous architecture could all benefit from this level of insight. In this thesis, a technique for mapping fluid velocity in porous scaffolds using NMR and MRI is presented. This technique utilises the spin properties of nuclei in proton dense liquids to provide spatially resolved information about the location and physical properties of atom nuclei, and is able to distinguish between atoms with different physical properties, including those atoms experiencing different translational diffusion. All of this information can be obtained non-invasively and in real time making it an ideal tool to study perfusion in porous biomaterial scaffolds for tissue engineering. However, to date there has been very limited use of this technique with respect to tissue engineering, such that the studies in this will seek to validate NMR and MRI techniques in this field and further explore the extent of how it can be used. The impact of obtaining flow velocity profiles within porous scaffolds will undoubtedly inform decisions on scaffold design, bioreactor design and flow conditions. Small variations in flow distributions and velocities could impact cell responses in regards to proliferation, migration and differentiation. Small and unexpected variations in cell behaviour could lead to undesired and inhomogeneous tissue formation. Therefore understanding how variations in flow occur in culture and affect overall cell behaviour and tissue formation can be used to both mitigate for these factors, but also optimise experiments to ensure flow conditions constantly facilitate an environment desirable for tissue regeneration. Firstly, to examine the effects of scaffold porous architectures on fluid flow regimes, 3D printing techniques were used to fabricate cell free scaffolds with varied pore characteristics. 3D printing and computer aided design allow for a high level of control over pore architecture, which dependent on desired flow patterns, can be altered to facilitate such flow patterns. Results demonstrated the effects scaffold architecture has on flow can be mapped using NMR and MRI velocimetry. Secondly, this project further utilised the capabilities of MRI to investigate porous scaffold, which had in this instance been seeded with ihMSC cells. MR imaging was successful in visualisation of both cells that had been labelled with iron oxide nanoparticles, and unlabelled cells within the porous polymer matrix. This imaging method was non-destructive to the scaffold, and therefore could be used to monitor changes in cell densities and migration during cell culture. Finally, this project combined both velocimetry and cell visualisation techniques to link cell location and fluid field patterns. When compared with cell free scaffolds there was significant differences in velocity of fluid in cell-seeded scaffolds, which in some scenarios could be directly linked with cell location