Tissue engineering of the annulus fibrosus (AF) is challenging due to its complex lamellar structure. Polyurethane scaffolds have shown promise in AF tissue engineering. The current study examines whether matrix protein coatings (collagen type I, fibronectin, or vitronectin) would enhance cell attachment and promote cell and collagen orientation that more closely mimics native AF. The results demonstrate that the greatest cell attachment occurred with fibronectin (Fn)-coated scaffolds. Cells on Fn-coated scaffolds were also aligned parallel to scaffold fibers, a process that involved α5β1 integrin, determined by integrin-specific blocking antibodies. The inhibition of this integrin reduced AF cell spreading and alignment and the changes in cell shape were regulated by the actin cytoskeleton, demonstrated using cytochalasin D inhibitor. Cells on Fn-coated scaffolds formed fibrillar Fn, synthesized significantly more collagen, and showed alignment of type I collagen that more closely mimics native AF therefore facilitating the development of the tissue in vitro.

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Description based on contents viewed Feb. 5, 2008; title from title screen. Includes bibliographical references (p. 65-68).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed Sept. 2, 2009). Additional advisors: Uday Vaidya, Burton Patterson, Susan Bellis, Mark Weaver, Vinoy Thomas. Includes bibliographical references.

Metallic materials have gained much attention recently from the areas of medical devices and orthopaedics. Artificial organs, dental implants, prostheses and implants that replace damaged or malfunctioning parts in the body are, or contain, metal components. Our ageing society poses an increased demand to provide devices and implants that can demonstrate better performance than those presented by traditional solutions. Matching the mechanical properties (i.e. stiffness and strength) of the device to those of the host tissue is a major challenge for the design and manufacture of engineered metal materials for biomedical applications. Failure in doing so provokes implant loosening, patient discomfort and repeated surgeries. Therefore, tailoring physical properties and biocompatibility of those materials is the main final aim of this research programme. This PhD study has focused on the tailoring of the mechanical properties of titanium-based materials and titanium-based alloys. Titanium inertness and the selection of biocompatible alloying elements were set as the baseline. Two approaches were employed to decrease stiffness (i.e. Young s modulus): one, by introducing porosity in a titanium matrix and therefore, reduce its Young s modulus, and two, by designing and manufacturing beta-titanium-based alloys with a reduced Young s modulus. Titanium scaffolds were manufactured using powder metallurgy with space holder technique and a sintering process. Different space holder sizes were used in four different categories to study the effect of pore size and porosity on the mechanical properties of the porosity engineered Ti scaffolds. Ti-based alloys were manufactured using manufacturing techniques such as sintering and arc-melting. The effect of different fabrication processes and the addition of beta-stabilising elements were studied and investigated. The obtained results of mechanical properties for pore size and porosity were within the values that match bone properties. This means these materials are suitable for biomedical application and the beta-Ti alloys results show that the mechanical properties can be decreased via tailoring the crystal structures. The characterisation of the Ti-based alloys helps to develop this material for its use in biomedical application.

Bone has the capacity to regenerate itself, however for challenging defects such as non-uniform factures, repair can be problematic. A similar challenge is presented in the repair of osteoporotic bone. Osteoporotic bone becomes increasingly porous and brittle and the risk of fracture is greatly increased. A drug mimic, poly(vinyl phosphonic acid – co – acrylic acid)(PVPA), has been incorporated into FDA approved poly(ε-caprolactone)(PCL), and aims to mimic the action of bisphosphonates to reduce the activity of osteoclasts. The PVPA polymer contains pendant phosphonic acid groups which are hypothesised to mimic the P-C-P backbone found in bisphosphonates. The PCL/PVPA scaffold has been found to have sufficient mechanical strength in order to be used as a bone void filler as well as providing a hydrophilic surface for superior cell attachment. The substrate has been found to significantly enhance the deposition of collagen, alkaline phosphatase activity and the expression of osteocalcin. Alizarin red staining revealed an increase in the rate of mineralisation in the presence of the drug mimic. The PCL/PVPA substrates have been suggested to induce osteoblast cells from a proliferative phase to a mineralisation stage. This is believed to be due to the presence of phosphorous within the scaffold which could lead to the critical concentration required for the initiation of mineralisation being reached more rapidly and effectively. The PVPA polymer has been found to mimic the action of bisphosphonates by inducing osteoclast apoptosis in vitro, and its actions of osteoclast apoptosis are comparable to that of Alendronate, a commonly administered bisphosphonate. A critical size defect model has demonstrated that the PVPA polymer has the ability to heal critical size defects; the healing potential was two fold greater than the control PCL substrate. Initial in vivo studies using a subcutaneous model demonstrated an improvement in mineralisation in the presence of PVPA. Untreated PCL/PVPA substrates displayed a high level of branched blood vessel formation, essential for healthy bone formation. However PVPA samples pre-treated with VEGF, hindered blood vessel formation and the infiltration of cells. This suggests that the PVPA alone is capable of inducing neovascularisation without the addition of VEGF. The findings suggest that the PCL/PVPA system could be used to treat challenging bone defects such as non-unions and osteoporotic fractures.

Hydrogels aim to mimic the natural living environment by entrapping large amount of water or biological fluids in their polymeric network. There has been growing interest in the development of peptide and protein hydrogels, due to their improved biocompatibility, biodegradability and biological properties in comparison to purely synthetic polymer hydrogels. Under the appropriate conditions, biomacromolecular protein hydrogels can self-assemble into ordered meso- to macroscopic supramolecules with better resulting networks that promote tissue development. The work presented here mainly focuses on producing protein hydrogels with controlled physical properties useful for tissue regeneration process and drug delivery applications. Hen egg white lysozyme (HEWL) hydrogels were studied in the presence of water and different reducing agents forming three HEWL systems including HEWL/water, HEWL/DTT and HEWL/TCEP gels. Strong, self-supporting HEWL gels were successfully prepared in the range of pH 2 to 7, using a temperature of 85°C. At pH 2, the protein denaturation in water was relatively slow resulting in a high percentage of turn structure (~50%) that promotes HEWL gelation after 3 days of heating. No lysozyme gelation in water was observed at pH 3, 4 and 7 even after 21 days of heating. A small quantity of DTT (~20 mM) was added to encourage lysozyme unfolding and HEWL/DTT samples formed gels at higher pH including at physiological pH. The pH 2 HEWL/water gel was found to be stronger but more brittle than pH 7 HEWL/DTT gel. It was observed there were some irregularities in the distribution of pH 2 fibrils (~7µm in length) that form large pore sizes within the network. The pH 7 sample contained shorter and stiff fibrils with repetitive polygon-shaped mesh network. The use of TCEP, which is a stronger reductant than DTT, led to the formation of self-supporting HEWL gels between pH 3.5 and 5.5. The highest storage modulus was observed at pH 5, which is related to the high β-sheet content of the sample (~45%). In addition, a promising strategy has been devised to form thermoresponsive HEWL hydrogels by synthesising and incorporating a small fraction of lysozyme-PNIPAAm bioconjugates into the major protein matrix. Results show the thermoresponsive nature of PNIPAAm was conferred to HEWL protein that exhibits higher storage stability in response to changing temperature.

This thesis is focused on the development and evaluation of different hybrid scaffolds for the treatment of injuries in cartilage or bone. These hybrid materials were three-dimensional polycaprolactone macroporous scaffolds obtained through freeze extraction and particle leaching combined method and modified with hyaluronic acid or mineral particles. In order to facilitate the description of the obtained results, the thesis is divided in two sections dedicated to bone and cartilage tissue engineering respectively. In the case of bone tissue engineering we addressed the treatment of disorders associated with the spine that require spinal immobilization. This Thesis proposes the development of a synthetic macroporous support for intervertebral fusion as an alternative to commercial bone substitutes. Macroporous scaffolds were developed with bare polycaprolactone or its blends with polylactic acid in order to increase its mechanical properties and degradation rate. Furthermore, the scaffolds obtained were reinforced with hydroxyapatite or Bioglass®45S5 to improve their mechanical properties and turn them in bioactive scaffolds. The supports were characterized physicochemically and biologically to determine if they met the requirements of the project. Finally, materials were tested in vivo in a bone critical size defect preformed in a rabbit model against a commercial support. Cartilage engineering has been extensively studied in the last years due to the inherent limited self repair ability of this tissue. The second part of the thesis was focused in developing a construct composed by in vitro differentiated chondrocyte like cells in a hybrid scaffold for cartilage tissue engineering. Polycaprolactone hybrid substrates coated with hyaluronic acid scaffold were developed obtaining a substrate with positive influence over the development of chondrocyte phenotype in culture and able to protect the cells from excessive mechanical loading in the joint. Cell-scaffolds constructs were obtained combining hybrid scaffolds with mesenchymal stem cells and differentiating them to chondrocytes using chondrogenic culture medium combined with hypoxia, mechanical stimulus or co-culture. Finally the cellularized scaffolds were mechanically, biochemically and histologically characterized to determine the production of extracellular matrix and expression of chondrogenic markers. / Ródenas Rochina, J. (2015). Scaffold surface modifications and culture conditions as key parameters to develop cartilage and bone tissue engineering implants [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48526 / TESIS

Bioactive glasses exhibit unique characteristics as a material for bone tissue engineering. Unfortunately, their extensive application for the repair of load-bearing bone defects is still limited by low mechanical strength and fracture toughness. The main aim of this work was two-fold: the reinforcement of brittle Bioglass®-based porous scaffolds and the production of bulk Bioglass® samples exhibiting enhanced mechanical properties. For the first task, scaffolds were coated by composite coating constituted by polyvinyl alcohol (PVA) and microfibrillated cellulose (MFC). The addition of PVA/MFC coating led to a 10 fold increase of compressive strength and a 20 fold increase of tensile strength in comparison with non-coated scaffolds. SEM observations of broken struts surfaces proved the reinforcing and toughening mechanism of the composite coating which was ascribed to crack bridging and fracture of cellulose fibrils. The mechanical properties of the coating material were investigated by tensile testing of PVA/MFC stand–alone specimens. The stirring time of the PVA/MFC solution came out as a crucial parameter in order to achieve a more homogeneous dispersion of the fibres and consequently enhanced strength and stiffness. Numerical simulation of a PVA coated Bioglass® strut revealed the infiltration depth of the coating until the crack tip as the most effective criterion for the struts strengthening. Contact angle and linear viscosity measurements of PVA/MFC solutions showed that MFC causes a reduction in contact angle and a drastic increase in viscosity, indicating that a balance between these opposing effects must be achieved. Concerning the production of bulk samples, conventional furnace and spark plasma sintering technique was used. Spark plasma sintering performed without the assistance of mechanical pressure and at heating rates ranging from 100 to 300°C /min led to a material having density close to theoretical one and fracture toughness nearly 4 times higher in comparison with conventional sintering. Fractographic analysis revealed the crack deflection as the main toughening mechanisms acting in the bulk Bioglass®. Time–dependent crack healing process was also observed. The further investigation on the non-equilibrium phases crystallized is required. All obtained results are discussed in detail and general recommendations for scaffolds with enhanced mechanical resistance are served.

No description available.

Titania (Titanium dioxide, TiO2) has been researched as a promising biomaterial due to its excellent biocompatibility. However, the main limitation of titania is its poor mechanical properties which limit its use in many load-bearing applications. In this thesis report, the properties of titania were improved by doping with small quantities of MgO, ZnO and SiO2 as sintering additives. Nanocrystalline powder was selected, as it possesses outstanding properties over conventional coarse-grained powders due to reduced grain size. Nanocrystalline anatase powder of size 5-15 nm was synthesized via a simple sol-gel technique. Small quantities of dopants were introduced into pure titania powder, through homogeneous mixing. The doped powder compositions were compacted uniaxially and sintered at 1300°C and 1500°C, separately, in air. The effects of sintering cycle and temperature on the microstructure, densification and mechanical properties of the sintered structures were studied. Mg doped structures recorded maximum sintered density of 3.87 g.cm-3. Phase analysis was carried out using powder XRD technique using Cu K[alpha] radiation. Microstructural analysis was performed using Scanning electron microscopy. The mechanical properties were assessed by evaluating hardness and biaxial flexural strength (ASTM F-394) of the structures. Results showed 12% increase in hardness and 18% increase in biaxial flexural strength in structures doped with ZnO and SiO2, respectively. Further, simulated body fluid maintained at 36.5°C was used to study the bioactivity and degradation behavior of the structures. The second part of the work aimed in the processing of porous titania scaffolds using polyethylene glycol as the pore-former. The green structures were sintered at 1400°C and 1500°C, separately in air and their properties have been studied. Microstructural analysis was carried out using Scanning electron microscope (SEM). Porosity was evaluated using the immersion technique. Vickers hardness and biaxial flexural tests were used to carry out the mechanical characterization. Further, the biomechanical/biodegradation behavior of the structures was assessed in simulated body fluid (SBF). Biodegradation and change in biomechanical properties as a function of time were studied in terms of weight change, change in Vickers hardness and biaxial flexural strength. The mechanical properties of porous titania scaffolds doped separately with MgO and ZnO have also been studied to investigate the influence of these additives on the properties of porous structures. The Vickers hardness and biaxial flexural strength were seen to improve with the addition of these sintering additives.