Artificial embryonic stem cell niches were made from murine embryonic stem cells (ESCs) and SAOS-2 osteoblast-like cells (a human osteosarcoma cell line) by constructing aggregates with well-defined architectures with dielectrophoresis between the castellations of interdigitated oppositely castellated electrodes. This combination of the cells was chosen to mimic the bone marrow endosteal niche that harbours haematopoietic stem cells in a quiescent stage, with the aim of transforming the embryonic stem cells into hematopoietic precursor cells. Within aggregates made with dielectrophoresis cells are in very close contact, allowing strong cell-cell interactions to occur. Puramatrix gel was used to immobilize the cells; a concentration of 25% Puramatrix was found to be optimal. Aggregates consisting of only ESCs formed embryoid bodies upon aggregation with dielectrophoresis within 24 hours. The size of the electrodes determines the size of embryoid bodies. Embryonic bodies formed at electrodes with a characteristic size larger than 100 μm tended to split; electrodes smaller than 75 μm gave embryonic bodies which tended to merge. 75 to 100 μm was optimal. When aggregates were made containing both SAOS-2 and ESCs, the reorganization of the two cell types after their aggregation was found to be controlled by the different adhesive-cohesive properties of the two cell types and their initial position. Optimum cell-cell interaction was obtained in an aggregate with a layered architecture with the osteoblasts initially in bottom position, and the ESCs in top position. The study of differentiation in ESCs was made by conducting experiments with Bry ESCs, which mark the onset of differentiation along mesenchymal lineage with the production of GFP. The results indicated that aggregation with dielectrophoresis causes the ESCs to take the first steps towards differentiation along the mesenchymal lineage, and that the differentiation is stronger in aggregates formed at electrodes of 75 μm than at electrodes of 100 and 50 μm. Co-culture with SAOS-2 cells did not lead to differentiation along the mesenchymal lineage. Lastly it was shown that optical tweezers could be combined with dielectrophoresis to move individual cells between niches.

The main aim of this work is to develop methods of assessing soft biological tissue quality using structure-property relationships. “Tissue quality” is here taken to mean the condition of the biological tissue structure which can be quantified in terms of features at a range of scales, including histological features. The purpose is to serve as a diagnostic aid in clinics and provide quantitative information that might replace the more traditional palpation. It is recognised that any structure-property relationship may be local to particular areas and may be differently expressed at a range of scales of probe size. In order to arrive at some generic outcomes, two model systems were chosen, one a biological mimic (gelatin-oil mixtures) which had the advantage that certain aspects of the structure could be varied. The biological system was chosen to be ovine connective tissue (collagen-lipid mixture) because it was expected to be similar in its components to the mimic, although with a more natural variation in structure. Static and dynamic indentation tests were carried out on all of the material and the static and dynamic elastic and viscoelastic properties were determined. Two experimental rigs were developed, a macro-scale one with a 0.5mm radius indenter and micro-scale one with a, pyramidal indenter of base side 4µm, height 6µm and opening angle . The macro-scale rig was purpose-built with indentation distance being controlled using software specifically configured for the work. The macro-scale rig was a modified atomic force microscope with a piezo-ceramic actuator being used to vibrate the specimen stage. Experiments were carried out under displacement control, with the mean indentation distance in the range from 1-4% of the specimen height, a frequency range of 1-10Hz and an indentation displacement amplitude of 30 and 90µm. Nine different oil in gelatine formulations were used and the ovine biological tissue was classified as; collagen (from the ligamentum nuchae), fat (from the tail) and a collagen-fat mixture (from the abdomen). The dynamic tests were carried out over a range of frequency and the variation of the loss and storage modulus with frequency was used to arrive at an appropriate visco-elastic model for each of gelatine-oil and biological tissue systems. Finally, the elastic and viscous moduli were correlated with tissue quality (structure) to obtain the structure-property relationship. Of the various three-parameter visco-elastic models tested, a Maxwell model in parallel with a spring was found to be the most suitable for the biological tissues whereas a Kelvin model in series with a spring was best for the gelatine-oil mixtures. Using the resulting visco-elastic moduli, it was found that a structure-property relationship exists for biological tissues and gelatine phantoms at both scales. Furthermore, the results suggest that the scale of the probe affects the dynamic mechanical properties for the biological tissues but not for the gelatine phantoms.

The PhD thesis concerns the development of scaffolds inspired by biology for musculoskeletal tissue engineering. This was accomplished by chemical modification of poly- DLy-glutamic acid (y-PGA) to reduce its water solubility for use in regenerative medicine applications. A series of water-insoluble modified y-PGA polymers were synthesised, yielding materials with varying hydrophilicity and dissolution rates. All polymers were fully characterised using techniques such as differential scanning calorimetry, size-exclusion chromatography, wide-angle x-ray scattering, thermogravimetric analysis, Fourier transform infrared spectroscopy, and contact angle determination. Following characterisation, esterified y-PGA polymers were oriented to different degrees above their glass transition temperatures using tensile deformation. This drawing induced crystallisation, and thus orientation, on a molecular scale, leading to increased mechanical properties that were calculated from tensile tests. Synthesised polymers were found to be non-cytotoxic using the pre-clinical ISO 10993:5 test, and supported human fibroblast cell growth, as determined by LIVE/DEAD® staining. Oriented, modified y-PGA polymers were produced with a wide range of mechanical properties, displaying tailorability for specific tissue engineering applications.

Background: Nanoparticles (NPs) of different material composition (silica, gold) have gained increasing attention in drug therapeutics and diagnosis of disease. In particular, Gold nanoparticles (AuNPs) demonstrate potential for cell tracking and imaging diagnostics, however, their impact on cellular and vascular function remains uncertain. The AuNPs emission characteristics can vary when they are suspended in different physiological fluids due to their aggregation; hence, to overcome this effect, organic polymer composite coatings have been applied. The aim of the present study is to investigate the effects AuNPs and their surface modifiers on endothelial cell and murine aortic vascular function, ex vivo. Methods: A number of nanoparticles were fabricated (silica, gold), but AuNPs were selected because they were simple to synthesise and characterise. AuNPs (12±3nm) were synthesised according to the Turkervich method. They were then surface modified with the polymers polyvinylpyrrolidone (PVP) and mercapto-polyethylene glycol - (mPEG) and characterised by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDAX) and UV-Vis spectroscopy. Silica NPs (SiNPs, 100 and 200 nm) were synthesised using a modification of the Stöber method. They were characterised using TEM and the Malvern Zeta Sizer. Cellular uptake of AuNPs by cultured bovine aortic endothelial cells (BAECs) was visualized using transmission electron microscopy (TEM). The effects of AuNPs on BAEC proliferation, cell viability and apoptosis were determined using the automated cell counter and flow cytometry for cell number counting, exclusion dye propidium iodide (PI) and Annexin V/PI detection, respectively. Aortic vessel rings from male Wistar rats were mounted between two fine steel wires in an organ bath system and constantly superfused in oxygenated physiological salt solution (PSS) at 37oC. Cumulative doses of the endothelial dependent agonist acetylcholine (ACh; 0.01-100μM) and endothelial independent sodium nitroprusside (SNP; 0.1nm-10μM) were added to KCl preconstricted vessels, before and 30 minutes after incubation with modified and non-modified AuNPs. Results: Maximum cellular uptake of AuNPs was observed 24 hours and 48 hours, after incubation in non-modified and modified AuNPs, respectively. Both non-modified and modified AuNPs significantly decreased cell viability and proliferation and increased apoptosis up to 24 hours after incubation, whereas no inhibitory effect was observed after 48 hours of incubation. Both modified and non-modified AuNPs influenced cellular signalling and reduced Erk and Akt phosphorylation. Non-modified AuNPs had no overall effect on ACh responses, but significantly reduced responses to SNP. PVP modified AuNPs (AuPVP), but not mPEG modified AuNPs (AumPEG) lead to a significant attenuation in ACh responses (ACh concentration 0.01-100μM; p<0.05). AumPEG at 2.9μg/mL had no overall significant effect on either ACh or SNP responses; however, at higher concentration (5.8μg/mL) AumPEG NPs led to a significant reduction in ACh dilator response at most ACh concentrations. PVP alone, at the minimal concentration required to stabilise the AuNPs, reduced ACh dilator responses. Conclusions: We demonstrate that surface modification of AuNPs using polymers enhances their stability in physiological solutions and culture media, and also reduces cellular uptake by BAECs. Furthermore, we show that cellular and vascular effects of AuNPs depends on the type and concentration of polymer modification used. mPEG modified AuNPs show greater biocompatibility and are less detrimental to vasodilator function, than PVP modified AuNPs, thus showing greater potential use as agents for diagnostic imaging and therapeutics.

The world's population is aging and cases of debilitating degenerative diseases are increasing. Bone is the second most transplanted tissue after blood but natural bone grafts are in short supply. Bioglass, which is a particular composition of bioactive glass, stimulates more bone repair than other synthetic bone grafts. However, it is brittle so cannot be used in cyclically loaded sites. A promising solution is the use of hybrid materials that can potentially combine the toughness of polymers with the stiffness and bioactivity of the glass through interpenetrating inorganic-organic networks. Hybrids have the unique feature of tuneable mechanical properties and degradation rates. In this thesis, two very different polymers were investigated as the organic component of hybrids; chitosan and poly(2-hydroxyethyl methacrylate-co-(3-trimethoxysilane)propyl methacrylate). The natural polymer chitosan was incorporated into the silica sol-gel process to produce hybrids and scaffolds were fabricated using freeze drying and foaming techniques. The chemical, morphological, mechanical and degradation properties of the scaffolds were studied. In order to covalently bond the organic and inorganic components, the chitosan was functionalised with an alkoxysilane crosslinker, 3-glycidoxypropyl trimethoxysilane. Using NMR and FTIR, the functionalisation reaction and side-reactions were characterised, discovering that the reaction was only 20% efficient at all pH values. To avoid the inefficient functionalisation reactions and concerns over the reproducibility of natural polymers, the synthetic co-polymer poly(2-hydroxyethyl methacrylate-co-(3-trimethoxysilane)propyl methacrylate) was synthesised by controlled polymerisation techniques (ATRP and ARGET ATRP) and typical free radical polymerisation (FRP). ATRP gave good control over molecular weight distributions, but the copper catalyst had serious implications on the chemical and architectural structure of the polymers. An NMR kinetics study was used to identify alternative polymerisation routes that could avoid the problems associated with the copper catalyst. The polymers were introduced into the sol-gel process to produce entirely synthetic hybrids with non-brittle (tough) behaviour and dissolution rates controlled by the polymer composition. The hybrids also exhibited hydroxyapatite precipitation in simulated body fluid, indicative of potential bioactivity in vivo. Hence, the aim of producing non-brittle, bioactive materials with controllable degradation rates was achieved.

Biomolecules such as bone growth factors, extracellular matrix proteins, bioactive factor-derived peptides, and other mineralisation-promoting proteins are gaining increasing attention in bone tissue engineering due to the fact that they can play an important role in cell attachment, growth, differentiation, and migration, and therefore be used to enhance bone formation and develop bone substitutes. This study is focused on the development of engineered biomolecules with improved functionality, and their combination with supporting matrix materials for bone tissue engineering applications. Two types of biomolecules are studied: The first are short peptides derived from the growth factor bone morphogenetic protein 7 (BMP-7), which induces bone regeneration, and regulates bone formation. The second is an engineered recombinant metalloenzyme E. coli alkaline phosphatase (E. coli ALP), which hydrolyses organic phosphate and generates the inorganic phosphate required for the mineralisation of hard tissue. Titanium (Ti) implants and polycaprolactone (PCL) scaffolds were used to incorporate these engineered biomolecules to verify the improvement of their functionality and biological efficiency. Results indicate that the BMP-7-derived peptides have a limited function and are not promising candidates for bone tissue engineering applications; while the mutant E. coli ALP shows superior activity compare to the wild-type ALP (nearly 25 times) and bovine ALP (more than 9 times), and is shown to be a promising factor to enhance bone formation. The activity of E. coli ALP can be affected by many factors including NaCl, lyophilisation, histidine, and imidazole. When the ALP is combined with either a Ti implant or an ALP/PCL scaffold, it demonstrates outstanding mineral-promoting ability, making it a potential candidate for bone tissue engineering applications. In conclusion, this study demonstrates the potential of engineering biomolecules with improved functionalities for bone tissue engineering applications.

Scaffolds suitable for trabecular bone regeneration were produced via the gel-cast foaming process using the melt-derived glass composition SBP-3. A problem common to most bioactive glass compositions that undergo sintering in order to achieve scaffold form is that they crystallise, lowering bioactivity as well as incurring mechanical instability of the structure via unpredictable local degradation. The scaffolds did not exhibit significant bulk crystallisation upon sintering and the presence of potassium sodium sulphate crystals were not detected on the surface, which were formed with a previously studied composition ICIE16. The process was then optimised further and up-scaled to produce scaffolds in sufficient quantities for an in vivo ovine condyle defect study (undertaken at University College London by Prof. Allen Goodship and Prof. Gordon Blunn). Despite possessing a relatively high network connectivity - 2.31 - and low bioactivity from SBF studies, results from the in-vivo study concluded that the SBP-3 scaffolds performed better than the original 45S5 Bioglass granules, displaying 99 % bone aposition over a period of 91 days, in addition to the new bone more closely resembling the trabecular bone structure. Work was then carried out to simplify the process, switching from a chemical polymerisation process to a thermal gelation process, thus allowing for greater optimisation of the process.

Hybrid materials have been found to be possessed with unique and novel properties, by superimposing the advantages of each component to provide material properties far superior to the individual constituents alone. These types of material are used throughout the field of bioengineering, and can facilitate biological interactions on both a molecular and cellular level, responding to biological and mechanical queues. With these inherent unique properties these materials provide novel platforms to help repair, replace and regenerate body tissues and function. This thesis explores the synthesis of new 'smart' hybrid hydrogels and their properties. Through the conjugation of self-assembling β-sheet peptide sequences to a naturally occurring poly (γ-glutamic acid) polymer backbone, peptide-polymer hybrid hydrogels were formed. These hybrid hydrogels were attributed with robust and tunable mechanical properties. Through the reassembly of the physical β-sheet crosslinks the hydrogels can respond to their mechanical environment, exhibiting 'self-healing' capabilities and a resistance to cyclic strain. Also, being composed entirely of natural peptide bonds they have excellent biocompatibility and are a promising platform for future tissue engineering scaffolds and biomedical applications. These hybrid hydrogels were further functionalised to detect for the activity of enzymatic biomarkers. A simple assay based on Förster resonance energy transfer was incorporated in to the hydrogel platform through the immobilisation of quantum dots modified with peptide substrates. The activity of matrix metalloproteinase-7 was targeted specifically, a marker for inflammation and immunity. This hydrogel sensing platform provides a basis for the in vivo sensing of enzymes and also, the potential to be used as a powerful tool to investigate biological processes in vitro.

This thesis is concerned with the nature of biomaterial interactions with compromised host tissue sites. Both ocular and dermal tissues can be wounded, following injury, disease or surgery, and consequently require the use of a biomaterial. Clear analogies exist between the cornea/tear film/contact lens and the dermal wound bed/wound fluid/skin adhesive wound dressing. The work described in this thesis builds upon established biochemistry to examine specific aspects of the interaction of biomaterials with compromised ocular and dermal tissue sites, with a particular focus on the role of vitronectin. Vitronectin is a prominent cell adhesion glycoprotein present in both tear fluid and wound fluid, and has a role in the regulation and upregulation of plasmin. The interaction of contact lenses with the cornea was assessed by a novel on-lens cell-based vitronectin assay technique. Vitronectin mapping showed that vitronectin-mediated cell adhesion to contact lens surfaces was due to the contact lens-corneal mechanical interaction rather than deposition out of the tear film. This deposition is associated predominantly with the peripheral region of the posterior contact lens surface. The locus of vitronectin deposition on the contact lens surface, which is affected by material modulus, is potentially an important factor in the generation of plasmin in the posterior tear film. Use of the vitronectin mapping technique on ex vivo bandage contact lenses revealed greater vitronectin-mediated cell adhesion to the contact lens surfaces in comparison to lenses worn in the healthy eye. The results suggest that vitronectin is more readily deposited from the impaired corneal tissue bed than the intact healthy tissue bed. Significantly, subjects with a deficient tear film were found to deposit high vitronectin-mediated cell adhesion levels to the BCL surface, thus highlighting the influence of the contact lens-tissue interaction upon deposition. Biomimetic principles imply that adhesive materials for wound applications, including hydrogels and hydrocolloids, should closely match the surface energy parameters of skin. The surface properties of hydrocolloid adhesives were found to be easily modified by contact with siliconised plastic release liners. In contrast, paper release liners did not significantly affect the adhesive surface properties. In order to characterise such materials in the actual wound environment, which is an extremely challenging task, preliminary considerations for the design of an artificial wound fluid model from an animal serum base were addressed.

This thesis is concerned with the design and synthesis of a novel, injectable proteoglycan analogue for tissue repair. This is of particular relevance to the restoration of disc height to a degraded nucleus pulposus of the intervertebral disc. The focus is on the use of sulfonate monomers as proteoglycan analogues, in particular sodium 2-acrylamido-2-methylpropane sulfonic acid and the potassium salt of 3-sulfopropyl acrylate. For most biomedical applications, synthetic hydrogels need to show dimensional stability to changes in pH, osmolarity, and temperature. This is readily achieved by neutral structures however ionic sulfonate containing hydrogels are responsive to environmental change which renders them difficult to manage in most tissue replacement applications. In this case osmotic responsiveness rather than stability is desirable. Therefore sulfonate based materials possess advantageous properties. This is a result of the sulfonate becoming an ideal surrogate for the sulfate group present within the structure of natural proteoglycans. This thesis reports polymerisation studies based on the production of a redox initiated copolymer system capable of polymerising in situ within a timescale of circa. 5-7 minutes. The rheological properties, osmotic drive, and residual monomer content of successful compositions is analysed. Properties are adapted to mimic those of the target natural tissue. The adaptation of the material for use as an injectable intra-ocular lens, with hyaluronic acid as an interpenetrate is reported. The synthesis of a radiopaque macromer to allow visibility of the repair system once in situ is investigated and discussed. The results presented in this thesis describe a suitable proteoglycan tissue analogue which is injectable, biomimetic, osmotically responsive and mechanically stable in its desired application.