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The principal aim of this thesis was to investigate the ability of surface topography in inducing bone cell differentiation for biomedical purposes. In orthopedic research, regeneration of bone defects can be performed in vitro using biomaterials. Third generation biomaterials aim not only to support tissue (first generation) and not only to be ‘bioactive’ (second generation), but to stimulate specific, known and desirable responses at the molecular level. Nanoscale topography offers a possible route to the development of third generation biomaterials. Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) is a new method for assessing protein expression strategies and here, a micro-grooved topography was used as a model for protocol optimization. The protocol was successfully developed and proved that 2D DIGE can be used as a powerful tool in the evaluation of biomaterial can direct cell behavior and cell fate. Next, the refined protocol was applied to the evaluation of the novel nanotopographic features; near-square nanopits (120 nm diameter, 100 nm depth with the pitch between the pits was set to an average of 300 nm with a ± 50 nm error). Protein expression profiles indicated that ERK1/2 might play part in cell proliferation and cell differentiation. However, to make a clear conclusion about molecular signalling, the study of sub-cellular proteome is needed in the future work. Additionally, the use of another comparative proteomic technique; dimethyl labelling, implicated the possibility of sub-population differentiation, i.e. the formation of multiple cell types that could be advantageous in tissue engineering of complex organs. Furthermore, the application of fluid-flow bioreactors was shown to enhance the growth rate and possibly increased differentiation of cells cultured on nanotopographical features.

Silicon substituted hydroxyapatite (SiHA) has been reported to produce faster bone in- growth in vitro compared to hydroxyapatite (HA). The mechanism by which silicate ions in these materials trigger bone growth and differentiation remains unclear. In vitro models were used in this thesis to investigate human osteoblast cell responses on exposure to silicon containing materials and silicate ion solutions. The amounts of serum protein bound to SiHA was significantly higher than that in HA (p<O.OOl). Culture of both primary human osteoblast (HOB) cells and an osteosarcoma cell line (MG-63 cells) showed that SiHA discs were biocompatible to the cells; flat cell morphologies, higher degree of cellular processes, and a covering with minuscule bone mineral-like crystals were observed. To elucidate the effects of silicate ions alone on osteoblast functions, a 1000 ppm standard silicon solution was supplemented into cell culture medium to produce silicate ion concentration of 20 and 500 ~M; it was found that the former had little effect on both cell types. Significant increases in levels of total DNA (p<0.001), protein (p<0.001), and collagen (p<0.001) were observed in HOB cells, but not in MG-63 cells, in cultures with 500 ~M silicate ions. Likewise, expression of COL-J al (p<0.001), BMP-2 (p<0.05), PHOSPHO-J (p<0.001) genes were up-regulated in both cells types cultured with 500 ~M silicate ions. Further studies proposed that the activation of cell proliferation by this silicate ion-containing medium, observed as increases in total DNA, involved TGF~1 and/or IGF-I receptors. In trying to understand this, it was latterly identified that the pH changes of the serum- supplemented culture medium that occurred during supplementation with the alkali silicate ion solution and subsequent neutralisation with HCI were the actual cause of the marked enhancement in HOB cell proliferation. Silicate ions did still appear to have a direct effect on some HOB cell responses, due to observing comparable effects of 20 and 500 ~M silicate ions on e.g. TNAP and PHOSPHO-J gene expression, compared to silicate ion-free controls.

Phosphate glass with additions of sodium, magnesium and/or calcium were investigated for their potential to be used as the reinforcing phase in a completely degradable long fibre composite. Glasses were prepared from phosphate salts as opposed to oxides and melted under air in platinum/gold crucibles. The effect of cation addition on the material properties and biocompatibility was investigated. Glasses were characterised using a number of complimentary techniques, including: XRD, XPS, DSC, IR and EDX. The findings from these techniques were used to explain the observed thermal and dissolution properties. The thermal and dissolution properties were found to be dependant on both the thermal history and composition of the phosphate glass. For a phosphate glass with low cation content, the temperature and length of time held at that temperature increased the Tg by 10 C for sodium phosphate glass and slightly improved the durability of sodium phosphate glass containing 10 mol.% MgHPO4, as phosphate chain growth was greater under those conditions. Addition of divalent cations increased the Tg of phosphate glasses from 295 C for sodium phosphate glass by up to 150 C with the addition of 50 mol.% MgHPO4. The dissolution rate was decreased exponentially with the addition of calcium phosphate or magnesium phosphate to sodium phosphate glass. Rates as low as 1x10-7 g/(cm2.h) were achieved with the addition of 50 mol.% divalent cation phosphates. The divalent cations inhibited phosphate chain growth but formed a new network based upon divalent cation/non bridging oxygen cross-links. These cross-links were found to exert greater influence over the material properties then the phosphate chain length. Cell culture assays were used to establish the biocompatibility of phosphate glasses with different compositions. Preliminary tests were conducted with craniofacial derived osteoblast like cells cultured on glass surfaces. Initial assays performed showed that the most durable glasses sustained the greatest amount of proliferation and differentiation over a seven day period. The most promising glass compositions, 40 Ca, 40 Mg, 30 Mg/20 Ca and 20 Mg/30 Ca, and were selected for longer term osteoblast culture and short term macrophage culture. Long term osteoblast culture showed that cells were able to attach, spread and proliferate throughout the 28 day duration of the study. Assays performed on the culture showed that cells were differentiating, producing specific osteoblast markers for each of the three differentiation phases of proliferation, matrix maturation and mineralisation. ECM production and mineralisation was confirmed on all surfaces tested via type I collagen staining and alizarin red staining respectively. Over the 28 day period, it was found that the composition did not have a significant effect on the production of the osteoblast markers, namely alkaline phosphatase, collagen, osteocalcin and mineral deposition. Immunological studies show that macrophages are not activated by the presence of phosphate glass. This demonstrated that phosphate glass has shown potential for use a biomaterial.

Bone cement is required for the majority of implant procedures. The mechanical integrity of cemented implants may be compromised by fatigue failure of the bone cement, mainly due to internal defects or debonding at the implant interfaces; improvements in the mechanical properties of bone cement may therefore be valuable if the implant lifetime of cemented arthroplasties are to be increased and revision rates decreased. The present study investigated the use of synchrotron X-ray microtomography for the observation of internal defects and failure processes that occur during fatigue loading. Initial assessments of fatigue damage processes in in-vitro fatigue test specimens demonstrated the uncertain nature of locating fatigue cracks and other defects, identifying the need for a synthesis of high resolution tomographic imaging with complementary prior damage monitoring methods. This was achieved via a novel amalgamation of acoustic emission, ultrasound and/or microfocus computed tomography scans prior to testing. Location of cracks/defects prior to high resolution tomographic imaging increased the probability of capturing crack initiation, furthering the underlying understanding of crack formation and propagation. Experiments performed at the European Synchrotron Research Facility have shown that the microstructural features of a commercial bone cement are readily imaged using microtomography of short exposure times. Furthermore, interactions (for example crack deflection and ligament formation) have been clearly identified between failure processes and both the cement defect population and internal microstructure. Early stages of crack initiation have also been captured: a new mechanism of crack initiation is proposed where porosity and local BaSO4 distribution are seen to act together to cause resultant crack initiation in the cement matrix rather than directly from pore surfaces. An opportunity for cement enhancement has been identified in the use of carbon nanotubes (CNTs); improved mechanical and physical properties of acrylic bone cement reinforced with CNTs are reported in the literature, although current methods utilised for CNT dispersion in polymers do not immediately lend themselves to surgical deployment. Adding CNTs to bone cement may further provide bio-active and sensing capabilities, beyond the conventional fixation and load-bearing rôle. The present study confirms that CNT-reinforcement (using shear mixing techniques) enhances the fatigue performance of a PMMA matrix and additional acoustic emission parameter based analyses confirm that the presence of CNTs alters the associated failure mechanisms. An insight into the potential capabilities of CNT reinforced cements, using relatively simple preparation techniques suitable for surgical deployment, is provided. These results suggest that enhanced fatigue performance may be achieved by means of CNT reinforcement of the matrix leading to crack shielding mechanisms such as crack bridging. Biologically, the presence of CNTs may reduce local thermal necrosis in the tissue surrounding the cemented construct through a reduction of the peak exothermic polymerisation temperature.

Hydroxyapatite (HAP) coatings are used in orthopaedic surgery for bone regeneration. Current methods of phase quantification of HAP coatings suffer from drawbacks. A novel methodology of quantitative phase analysis of HAP coatings has been devised and validated. This method, based on whole pattern fitting with a fundamental parameters approach, incorporates amorphous calcium phosphate (ACP) and apatite phases into structural refinements. A comparison of the structural and chemical properties of plasma sprayed (PS) and novel electrodeposited (ED) HAP coatings has been conducted. ED coatings contained less ACP and more preferred orientation than the PS coatings, although the stoichiometry was similar. In vitro investigations of PS and ED coatings in simulated body fluid and foetal calf serum revealed that both are bioactive. A carbonated apatite layer produced on the ED coatings was -0.7μm thick with a stoichiometry and chemical constituents similar to that of natural bone apatite. PS coatings produced a nanocrystalline carbonated apatite layer (-4μm). For the first time it has been possible to model crystalline HAP and nanocrystalline apatite as independent phases and obtain accurate lattice parameters for each. A positive linear correlation has been made between microstrain and the solubility of HAP and carbonated apatites. Dissolution studies have shown that the behaviour of HAP and carbonated apatite is dominated by crystallite size at low undersaturation and by crystallite size and microstrain at high undersaturation for crystallites between -30OA- 1000A. Metastable equilibrium occurred for crystallites <_400A at low undersaturation. Carbonate content did not affect the solubility or dissolution behaviour. A novel technology for coating polymeric tape with HAP for potential use in anterior cruciate ligament reconstruction has been devised. Mechanical tests have demonstrated that no adverse properties are induced by the coating technology. Cell culture studies have shown that the HAP layer is capable of enhanced attachment, proliferation and differentiation of osteoblast cells compared to uncoated tape.