Peticone, C; (2017) Microscale tissue engineering: a modular approach for vascularized bone regeneration. Doctoral thesis , UCL (University College London).

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Abstract

Four million surgeries involving bone grafting or bone substitutes for the treatment of bone defects are performed yearly worldwide. However, limited donor tissue availability, pain and the risk of infection and immune rejection, have led to the development of alternative strategies for bone repair. Tissue engineering represents an alternative to current treatments as it consists of using a biomaterial scaffold alone or in combination with proteins, genes or cells, as a bioactive implant to stimulate bone repair. Microspherical scaffolds have been proposed as a potential modular unit for bone tissue engineering applications as their shape could facilitate filling of irregular shaped defects. Furthermore, microspheres could be used as a support for ex vivo expansion of adherent cells as well as a carrier to directly deliver cells to the defect site. In this study, the use of phosphate glass microcarriers for bone tissue engineering applications was investigated. As this material is completely soluble and non-toxic, it can be implanted in the patient together with cells. Furthermore, the tuneable glass composition can be easily engineered to induce specific structural and biological properties. Here, the effect of culturing MG-63 and hBM-MSCs on titanium-doped phosphate glass microspheres containing increasing concentration of cobalt (0, 2 and 5%) was investigated, as these ions have been shown to induce osteogenesis and angiogenesis, respectively. Furthermore, as part of this study a novel perfusion microfluidic bioreactor was fabricated to culture cells on microspheres under perfusion and to enable parallel screening of multiple culture variables. Cells proliferation on the microspheres as well as secretion of ECM proteins in response to the substrate was observed over time, thus confirming the biocompatibility of all compositions tested. Upregulation of osteogenic markers by MSCs also occurred in response to the microspheres in the absence of exogenous supplements. However, this effect was suppressed when cobalt was added to the glass composition. On the other hand, while cobalt doping was found to induce key angiogenic responses (i.e. VEGF secretion), this did not translate into improved functional vascularization in comparison to the cobalt-free microspheres. Successful MSCs culture on the microspheres within the microfluidic reactor was achieved and it was possible to efficiently quantify functional outputs, such as the expression of ECM proteins as a function of microspheres substrates and nutrient feeds under perfusion. In conclusion, titanium-doped phosphate glass microspheres were identified as a potential substrate for bone tissue engineering applications in terms of MSCs expansion and differentiation, as well as to support endothelial cells migration towards the scaffold and vessel formation, while additional doping with cobalt was not found to improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled to identify optimal parameters for perfused cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing.

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