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Currently used composites for tooth and bone repair share a similar composition. A major issue with dental composites is polymerisation shrinkage leading to bond damage and increased risk of bacterial microleakage. Concerns with bone composites for vertebral fracture repair (vertebroplasty) include low monomer conversion, high stiffness, and lack of antibacterial agent release. The aim of this study was to develop novel dental composites and injectable bone composites to overcome these limitations. The effects of components on various properties of the materials were also examined. The main components of experimental composites consisted of dimethacrylate monomers mixed with dental glass, mono calcium phosphate monohydrate (MCPM), tristrontium phosphate (TSrP), and polylysine (PLS). The experimental dental composites exhibited higher monomer conversion than a commercial material. The addition of MCPM with TSrP and PLS promoted hygroscopic expansion, apatite precipitation, and early polylysine release. These properties are expected to reduce bacterial microleakage. The incorporation of these additives reduced the monomer conversion and strength of the composites but these were still within an acceptable range. To produce bone composites, the dental composites were modified by replacing a light activated initiator with a chemically activated initiator and decreasing powder to liquid ratio. The pre-cured bone composites exhibited viscoelastic properties and shear- thinning behaviour which are desirable for injectable materials. The use of high molecular weight diluent monomer (polypropylene glycol dimethacrylate, PPGDMA) increased monomer conversion and shelf life of the bone composites. The addition of MCPM and PPGDMA increased strontium release, which is known to promote in vivo bone formation. The use of small glass fillers and fibres improved mechanical properties of the composites. Furthermore, the composites showed fatigue properties that compared favourably with commercially available materials. Modulus of elasticity of the experimental bone composites was, however, too high compared with that of cancellous bone. This could potentially lead to increased adjacent vertebral fracture risk. An attempt was made to decrease the modulus by raising the level of PPGDMA, phosphates, and polylysine. Increasing of PPGDMA improved monomer conversion and reduced the injection force required for the composites. Furthermore, the increase of PPGDMA and phosphates enhanced surface apatite precipitation which is known to enable in vivo bone bonding. The increase of these components also increased polylysine release. This may reduce postoperative infection, which is a life-threatening complication of vertebroplasty. Increasing PPGDMA and phosphates, however, reduced metabolic activity of mesenchymal stem cells limiting optimal levels.

The energy-regulatory network of the hypothalamus controls the everyday balance of food intake and energy expenditure. The close proximity of this region to the leaky blood brain barrier (BBB) enables many factors such as hormones to regulate energy homeostasis. This thesis aimed to determine the role of glucocorticoids (Gcs) within the hypothalamus in a model of Gc excess. Recent research has shown that high-fat diet (HFD) induces hypothalamic inflammation, which is detrimental to the energy-regulatory network. Over a series of time-points, the hypothalamic expression of Pomc, Npy, and Agrp were quantified and a decrease in orexigenic neuropeptide expression was observed after 4 and 20 weeks. In contrast to predictions, Pomc was also decreased after 20 weeks. At all time-points, the mRNA expression of inflammatory markers was too low to detect, however after 20 weeks, microglia morphology indicated activation. Mice gained weight throughout the study, but it is unclear whether the very low hypothalamic inflammation contributed to the development of obesity. The pleiotropic actions of Gcs enable them to be used as a treatment for a wide number of conditions, including inflammatory diseases, such as rheumatoid arthritis. Long-term Gc treatment is recognised to cause adverse metabolic side- effects including increased body weight gain and hyperphagia. In order to establish the role of hypothalamic Gcs in the development of Gc-induced metabolic syndrome, mice were treated with either corticosterone (Cort) or vehicle (1% EtOH) in the drinking water alongside a HFD or chow for 4 weeks. Exogenous Cort treatment induced a metabolic phenotype as food intake, body weight, and fed glucose increased during the study. Cort treatment also increased hypothalamic corticosterone levels after 24 hours, which remained elevated after the four week treatment period. HFD+Cort treatment exacerbated this effect, despite HFD alone not altering hypothalamic corticosterone levels. To determine the role of Gcs within the mediobasal hypothalamus (MBH) in the development of Gc-induced obesity and hyperphagia, Cre-lox technology was used to knockdown the glucocorticoid receptor (GR) in the MBH. To ensure that the Cre recombinase enzyme was functional, Cre and YFP expression were co- located within the MBH of AAV-Cre injected ROSA26-EYFP mice. Although the successful injection of AAV-Cre in GR flox mice should reduce GR expression, the low sensitivity of qRT-PCR in micro-punch and laser capture micro-dissections and dual immunofluorescence prevented confirmation of GR knockdown. After 3 weeks Cort treatment, a number of GR flox mice injected with AAV-Cre had a reduced gain in body weight and food intake. Furthermore, AAV-Cre injected GR flox mice were phenotypically monitored over a 6 month period without Cort treatment. These mice did not gain as much weight over time, suggesting that central Gc actions are important in the control of every day energy homeostasis. Collectively, this study has shown that Gcs acting within the hypothalamus have a significant role in the regulation of energy homeostasis. Further investigations will enable the development of co-therapies or ‘drug holidays’ to prevent the negative metabolic sequelae associated with Gc therapies.

This thesis examined the metabolic effects of acute and intermediate hypoxic exposure in humans, specifically, physiological mechanisms associated with weight loss. Namely; increased metabolic rate, changes in substrate oxidation, altered lipid metabolism and changes in taste. Study one assessed the validity and reproducibility of an online gas analyser in normobaric hypoxia [Fraction of inspired oxygen: 0.12 (FiO2:0.12) equivalent to approximately 4,500m] (n=nine; two females, seven males). The MetaMax3x demonstrates good reproducibility between repeated trials. Differences exist between the system and the gold standard Douglas Bag method for measures of oxygen uptake (percent differences of V̇O2; 21%), carbon dioxide production (V̇CO2; 10%) and minute ventilation (V̇E; 5%). The second study investigated the free fatty acid (FFA) and triglyceride (TAG) response to an acute (45 minutes) hypoxic exposure (FiO2: 0.12) (n=10; five females, five males). A greater resting metabolic rate (RMR) (+28 ± 6 kcal.hr-1 ) was observed, through increased carbohydrate (CHO) and fat oxidation. Increased plasma FFA (+54%) and TAG (+26%) were observed, highlighting metabolic perturbations from acute exposure. Study three investigated the metabolic responses to an acute (60 minute) hypoxic exposure (FiO2: 0.12) at rest and a subsequent bout of moderate exercise in normoxia following a high fat meal (n=eight males). Experimental trials included a lipid ingestion prior to a rest period at hypoxia or normoxia followed by moderate intensity exercise (60% heart rate reserve). Control trials consisted of the same protocol without lipid ingestion. Acute, severe hypoxia increased energy expenditure (EE), (+22 ± 11 kcal.hr-1 ) CHO and fat oxidation following exposure. A prior acute bout of severe hypoxia did not alter EE and substrate use during subsequent moderate intensity exercise. An exercise bout, postlipid ingestion, resulted in lower triglyceride concentration. No changes in Meteorin-like were observed throughout trials. These findings suggest that an increase in RMR occurs following a single resting hypoxic exposure and independently to Meteorin-like protein. The fourth study observed reductions in body mass (-2.36 ± 1.41 kg) and increases in CHO oxidation during an altitude stay in Peru (18 days, 3400 m) (n=10; five females, five males). The reduction in body mass (-1.89 ± 1.31 kg) was sustained four weeks post-return to sea-level. Salt, sweet and bitter taste sensations were reduced at 3,400 m compared to sea-level. No changes in self-reported appetite were observed throughout the testing period. Furthermore no changes in circulating Meteorin-like protein were observed upon return to sea-level at one and four weeks post-altitude stay. Study five investigated the blood lipid response to a high lipid meal consumed one and four weeks post-return to sea-level following an altitude stay (18 days, 3400 m) (n=10; five females, five males). No lasting postprandial effects were observed. It is likely that a time dependent effect of hypoxia exists with regards to postprandial blood lipid responses. Taken together acute and intermediate exposure to hypoxic conditions alter substrate oxidation with the potential to induce losses in body mass, independently to changes in Meteorin-like protein and self-reported appetite. Specifically, prolonged stay at moderate altitude results in a greater dependency on CHO use. Increases in RMR were observed during an acute severe bout of hypoxia, although this was not a consistent effect throughout prolonged exposure and should be further investigated. Altered taste during an altitude stay may influence food preferences, energy intake and subsequent changes in body mass and should be considered an area of future investigation. Higher circulating levels of FFA and TAG, demonstrates a metabolic perturbation from a single, acute severe hypoxic exposure.

This thesis concerns the development of a new and facile sol-gel synthesis route for production of phosphate-based glasses for biomedical applications including; tissue engineering, imaging contrast agents and drug delivery systems. The structure of the prepared samples was probed by XRD, 31P MAS-NMR, EDX and FTIR spectroscopy that confirmed successful synthesis and production of phosphate based glasses via the sol-gel method. In this study, for the first time, quaternary phosphate-based sol-gel derived glasses in the P2O5-CaO-Na2O-TiO2 system with a high TiO2 content of up to 30 mol% were synthesised. While incorporating a high percentage of titanium into the phosphate network is non-trivial via traditional melt quench methods. Investigation of quaternary glasses with the general formula of (P2O5)55-(CaO)25-(Na2O)(20-x)-(TiO2)x, where X = 0, 5, 10 or 15 revealed, substituting titanium in place of sodium significantly improves the stability and prolongs the degradation of these glasses, which opens up a number of potential biomedical applications. Cell studies on titanium-stabilised glasses suggested glasses containing 5 or 10 mol% TiO2 have optimal potential for bone tissue engineering applications. Electrospraying was used to prepare (P2O5)55-(CaO)30- (Na2O)15 glass nanospheres with a diameter size range of 200-500 nm. These glass nano spheres were used as a transient contrast agent for ultrasound imaging to label mesenchymal stem cells and it was determined in vitro and in vivo that these nanospheres had a detection limit of 5 and 9 μg.mL-1, respectively. Cell counts down to 4000 could be measured with ultrasound imaging with no cytotoxicity at doses required for imaging. Glass nanospheres were also used as a carrier for drug delivery applications with a linear release of tetracycline hydrochloride molecules within the first 4 hours of the study period. Importantly, ion release studies confirmed these glass nanospheres biodegrade into an aqueous medium with degradation products that can be easily metabolised in the body. To the knowledge of the author, this is the first report of sol-gel synthesis and electrospraying to prepare glass nanospheres at low processing temperature and the first use of such a system for both diagnostic and therapeutic purposes.

AIMS: To investigate the influence of canal design (closed, open), irrigant concentration, agitation, canal complexity, and biofilm type on the efficacy of sodium hypochlorite to remove biofilm. To examine the extent of biofilm destruction following irrigation protocols. METHODOLOGY: Standardized in vitro models were developed (Endo-Vu block, flow cell, and 3D printing root canal models). The canal consisted of two halves of an 18 mm length, size 30 and taper 0.06, with or without a lateral canal of 3 mm length, and 0.3 mm diameter. Biofilms were grown for 10 days, and stained using crystal violet. The model was attached to an apparatus and observed under a fluorescent microscope. Following 60 s of 9 mL NaOCl irrigation using syringe and needle, the irrigant was either left stagnant or agitated using gutta-percha, sonic and ultrasonic methods for 30 s. Images were captured every second using an external camera. The residual biofilm percentages were measured using image analysis software. The SPSS software was used for statistical analysis. The residual biofilms were observed using confocal laser scanning, scanning electron, and transmission electron microscope. RESULTS: The removal of biofilm by NaOCl was more extensive in the open than in the closed canal. The concentration and extent of the needle had an influence on the amount of the residual biofilm. Ultrasonic agitation increased the biofilm removal from the main canal (90.13%) and lateral canal (66.76%). Extensive destruction of residual biofilm was observed in the ultrasonic groups. More residual multi-species biofilm than single species biofilm was identified (p = 0.001). CONCLUSION: The 3D-printing model provides a reliable method to investigate irrigation procedure. The closed canal adversely affect the efficacy of NaOCl. Concentration and position of the needle affect the efficacy of NaOCl. The results recommend the ultrasonic method for NaOCl agitation. The multi-species biofilm was more resistant than the single species biofilm.