Turner, Joel; (2024) Understanding Bone Cell Interactions with Silicate Species Released from Bioactive Glasses. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Despite 50 years of silicate bioactive glass (SBG) research and commercial success, the effect of soluble silicate (Si) species on cellular responses remains poorly understood. This includes how Si is internalised, excreted, and localised within bone cells. With an aim to optimise SBG ion release rates for more precise control of cell behaviour, this thesis attempts to investigate silicate ion uptake dynamics in osteoblasts whilst examining some of the roles these ions may play in bone regeneration. By using a systematic analysis of previous in vitro literature, this thesis demonstrated, for the first time, a relationship between [Si] and cellular response. An [Si] range where an increased reporting of desirable cellular interaction was found (~30-40 ppm, P≤0.001), whilst the frequency of negative outcomes increased by ~3x above 50 ppm (1.8 mM) Si. This systematic approach also provided quantitative evidence of the variance in methodological practices to assess responses to bioactive glasses and the need for greater standardisation. Liquid state 29Si-NMR revealed that BGs release orthosilicate species up to 2 mM Si, whilst above this concentration both ortho and disilicate were identified. Sodium silicates (SS) were found to produce more and larger precipitates in water and cell culture media than BGs dissolution products (up to 3mM Si) up to 3 mM, suggesting that other ion may influence Si species and particle formation. Intracellular [Si] was observed to be lower in cells cultured in 45S5 BG dissolution products compared to SS, suggesting that Si species may be important in cellular uptake. An increase in intracellular [Si] was observed up to a maximum of 250 μM/cell after 4 days. Following the replacement of Si-conditioned media with media alone, intracellular [Si] decreased (P≤0.001), along with an increase in [Si] found in the supernatant suggesting the excretion of Si from cells. Si particles released from both BGs and SS were found to be localised within lysosomal vesicles (STEM-EDX and fluorescence imaging) in both osteoblast and osteoclast cells suggesting an endosomal uptake route. A clatherin-mediated endosomal inhibitor (Dynasore hydrate), however, did not reduce Si uptake suggesting aggregation post-uptake or a non-clatherin (e.g., caveolae) mediated uptake mechanism. This thesis suggests an [Si] range that may be more likely to produce, positive, negative and non-significant cell responses, whilst demonstrating how Si species, (in addition to other ions released from BGs) are important in influencing intracellular [Si] and Si localisation. These results are important in informing the composition of new BGs that release quantities of Si that produce desirable cell responses.

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