Rezaei, Azadeh; (2019) The role of HIF pathway in bone regeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bone regeneration is initiated (in part) by cells sensing a drop in oxygen pressure (hypoxia), followed by a number of steps that include; inflammation, angiogenesis, soft and hard callus formation and remodelling. The oxygen sensing hypoxia inducible factor- 1α (HIF-1a) transcription factor has an important role in many of the stages of fracture repair including; angiogenesis, inflammation, stem cell recruitment, soft callous formation and bone remodelling. Furthermore, diabetic and elderly patients who have an increased risk of impaired bone healing, have a reduced ability to respond to hypoxia. Prolonged hypoxia has, however, also been shown to inhibit bone formation both in vivo and in vitro. Considering the importance of hypoxia in bone repair this thesis investigates the role of hypoxia (1% O2) and two HIF stabilisers (chemicals that inhibit the degradation of HIF- 1a), CoCl2 and DMOG, on bone formation in vitro, in both normal and hyperglycaemic conditions. This will enable us to determine if artificial stabilisation of the HIF pathway in patients with impaired bone healing could be a new target for regenerative medicine. To explore the controlled delivery of these HIF mimetics, cobalt was also incorporated into the glass network of silicate bioactive glasses with the intention of improving upon the existing bone regenerative properties of these materials, with controlled cobalt release. As there is limited information on the role of Si and Si-bioactive glasses on bone nodule formation in vitro, this was also studied. A multidisciplinary characterisation approach that included the biological (angiogenic, proliferation, and differentiation), biochemical (Raman spectroscopy), ultrastructural (TEM) and microstructural quantitative techniques (interferometry) for bone nodule formation was used to compare between treatments and with native bone. Hypoxia (1% O2) completely inhibited bone nodule formation, whilst DMOG and cobalt showed a concentration dependant inhibition, bone nodules were still formed within the ranges of CoCl2 [12.5μM-25μM] and DMOG [250μM-500μM]. CoCl2 [12.5μM] did not inhibit bone nodule formation. Si [0.5mM] and Si-based bioactive glasses conditioned media [0.5mM Si] enhanced bone regeneration (as determined by average nodule height P ≤ 0.05) but a concentration of 2mM Si and above inhibited bone nodule formation. Cobalt releasing bioactive glasses were successfully manufactured with controlled release of cobalt. Cobalt bioactive glasses conditioned media with 50μM cobalt (in a similar manner to CoCl2) inhibited bone nodule formation but not at lower cobalt concentrations (12.5μM). A high glucose environment completely inhibited bone nodule formation, while cobalt and DMOG treatment restored bone nodule formation. HIF stabilisation through the controlled release of HIF stabilisers, together with understanding the cellular mechanisms of these ions, may allow for the development of new materials with patient specific ion release profiles tailored to underlying disease. Moreover, this research highlighted the importance of multi-modal characterisation together with quantification for comparing bone tissue engineering approaches in vitro.

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