Dynamic culture of osteogenic mesenchymal progenitor cells in a 3D porous scaffold

There is an increasing need to treat bone defects that arise from disease or trauma and bone tissue engineering offers an alternative solution to the limitations that are present in current treatments. Until now, regenerative medicine strategies rely on the static culture of human mesenchymal stem cells (hMSC) on a three-dimensional (3D) scaffold in the presence of biochemical cues in order to stimulate cell differentiation. However, this approach neglects the importance of mechanical stimuli in the homeostasis of bone tissue. Perfusion bioreactors have been designed to improve the nutrient supply and induce a mechanical stress in in vitro cell culture. Within a bioreactor system, many important parameters including flow type, insertion of rest periods, duration, frequency and magnitude of shear stress can create different biomechanical and cellular microenvironments which can accelerate the formation of bone matrix. Therefore, the ultimate aim of this project was to study the effects of a perfusion bioreactor (with particular emphasis on flow parameters) to achieve progenitor cell commitment towards an osteogenic lineage and accelerate the biological process of bone formation. In order to achieve this, hES-MP cells were seeded on to a novel glass scaffold and subjected to oscillatory and unidirectional flow. It was shown that direct perfusion in combination with oscillatory flow improved cell growth and enhanced genes associated with osteogenic differentiation of hES-MPs in comparison to static cultures. The methods developed were also used to study the cell’s response to a combination of peptide coated scaffolds and bioreactor culture. Scaffolds were received as coated by Orla Protein Technologies with peptides from Tenascin C, Osteopontin and BMP-2. It was shown that the combination of peptide coated scaffolds and oscillatory flow resulted in an improved cell distribution and an upregulation in early and late markers of bone formation. Finally, the 3D model was used to investigate the role of the primary cilia as a mechanosensory organelle. It was demonstrated that MLO-A5 cells were less responsive and synthesized less matrix in response to fluid shear stress in the absence of the primary cilium. Suggesting that presence of intact primary cilia is essential for load sensing and absence of the cilium (or changes in its morphology), inhibit the ability of cells to respond to fluid flow in a 3D scaffold. The work in this thesis indicated that short bouts of oscillatory fluid flow may be sufficient to stimulate bone differentiation and maintain cell viability in an open pored scaffold such as used here. Initial data suggested that a combination of coating the scaffolds with peptides from ECM proteins or osteogenic growth factors can act synergistically with fluid flow. Finally it was demonstrated for the first time that the primary cilia of bone cells can be a mechanosensor in a 3D porous scaffold culture system. This work contributes to the ongoing work in the field of bone tissue engineering to optimise in vitro culture conditions for the creation of 3D bone matrix that could be used for future tissue engineering strategies.