4D kwantificatie van massa transport in skeletale weefsel engineering draagstructuren

Molecular transport phenomena underlie many of the processes that regulate cell behaviour during skeletal growth and development. This fundamental knowledge has largely originated from advances in imaging technologies that enabled the iterative interaction between experiments and theoretical models of mass transport. The demand for quantitative data that can be derived from imaging observations has hereby strongly increased, finding its application in rational experimental design. Quantitative descriptions of the mass transport environment have also become an obvious need in biomimetic tissue engineering approaches, aiming for the repair of skeletal tissue damage through recapitulation of embryonic tissue development. Gradually the tools for mass transport quantification are growing in this field, but the need for novel quantitative imaging technologies is still high. The research presented in this dissertation has combined quantitative luminescence techniques, microbead technology, and mathematical modelling with the aim of better understanding mass transport phenomena in tissue engineering constructs, with a central focus on the role of oxygen transport.To this end, photoluminescent oxygen sensitive microbeads were developed for the localized, in situ measurement of oxygen concentration. The microbead oxygen sensors were successfully calibrated in normal and turbid media, indicating that the ratiometric measurement principle in combination with a suitable intensity peak fitting algorithm could be applied to monitor oxygen concentrations in a wide variety of experimental setups. Oxygen transport within these setups was shown not to be influenced by the microscale dimensions of the oxygen sensors. Integration of the oxygen sensors into cell aggregates, mimics of skeletal cell condensations, was achieved by a binding method that relied on the non-covalent interactions between biotin and streptavidin. This binding method enabled the targeted and robust incorporation of microbeads into a specific environment. The non-invasive and non-interfering application of this method and sensor integration was corroborated experimentally.The use of fluorescence recovery after photobleaching was explored to provide a quantitative description of solute mobility within tissue engineering constructs. Tracer diffusion rates were shown to be significantly hindered by interactions with the bulk volume of a hydrogel and by the presence of cells. These results and use of the photobleaching technique were successfully validated by comparison with other methods for mass transport quantification. Oxygen transport within engineered constructs, and more specifically in cell aggregates, was quantified from microbead oxygen sensor measurements. Values for the oxygen consumption rate and induced oxygen concentration profiles were derived which could be correlated to observed changes in cell proliferation and differentiation behaviour.Finally, the influence of mass transport phenomena on bioluminescent reporter cell activity within a tissue engineering construct was explored. Oxygen concentration gradients were measured within the constructs that clearly interfered with the emitted bioluminescence signal. Using a model-based approach, oxygen-independent bioluminescence intensities could be obtained that were shown to be a more accurate and reliable representation of the active reporter cell population within the construct. These results revealed a dual role for the study of mass transport phenomena, as these transport phenomena do not only control many of the cellular and molecular processes during skeletal development, but they also are a basic requirement for the use of luminescent assays based on biochemical reactions.nrpages: 192status: publishe