An understanding of the interactions between orthopaedic and dental implant surfaces with the surrounding host tissue is critical in the design of next generation implants to improve osseointegration and clinical success rates. Critical to the process of osseointegration is the rapid establishment of a patent neovasculature in the peri-implant space to allow for the delivery of oxygen, nutrients, and progenitor cells. The central aim of this thesis is to understand how biomaterials regulate cellular and host tissue response to elicit a pro-angiogenic microenvironment at the implant/tissue interface. To address this question, the studies performed in this thesis aim to 1) determine whether biomaterial surface properties can modulate the production and secretion of pro-angiogenic growth factors by cells, 2) determine the role of integrin and VEGF-A signaling in the angiogenic response of cells to implant surface features, and 3) to determine whether neovascularization in response to an implanted biomaterial can be modulated in vivo. The results demonstrate that biomaterial surface microtopography and surface energy can increase the production of pro-angiogenic growth factors by osteoblasts and that these growth factors stimulate the differentiation of endothelial cells in a paracrine manner and the results suggest that signaling through specific integrin receptors affects the production of angiogenic growth factors by osteoblast-like cells. Further, using a novel in vivo model, the results demonstrate that a combination of a rough surface microtopography and high surface energy can improve bone-to-implant contact and neovascularization. The results of these studies also suggest that VEGF-A produced by osteoblast-like cells has both an autocrine and paracrine effect. VEGF-A silenced cells exhibited reduced production of both pro-angiogenic and osteogenic growth factors in response to surface microtopgraphy and surface energy, and conditioned media from VEGF-A silenced osteoblast-like cell cultures failed to stimulate endothelial cell differentiation in an in vitro model. Finally, the results show that by combining angiogenic and osteogenic biomaterials, new bone formation and neovascularization can be enhanced. Taken together, this research helps to provide a better understanding of the role of material properties in cell and host tissue response and will aid in the improvement of the design of new implants.

<p>Today, the majority of materials used for single-use packaging are petroleum-based synthetic polymers. With increased concern about the environmental protection, efforts have been made to develop alternative biodegradable materials from renewable resources. Starch offers an attractive alternative since it is of low cost and abundant. However, the starch material is brittle without plasticizer and the mechanical properties of starch materials are highly sensitive to moisture.</p><p>In nature, the plant cell walls combine mechanical stiffness, strength and toughness despite a highly hydrated state. This interesting combination of properties is attributed to a network based on cellulose microfibrils. Inspired by this, microfibrillated cellulose (MFC) reinforced starch-based nanocomposites films and foams were prepared. Films with a viscous matrix and MFC contents from 10 to 70wt% were successfully obtained by solvent casting. The films were characterized by DSC, DMA, FE-SEM, XRD, mercury density measurements, and dynamic water vapor sorption (DVS). At 70wt% MFC content a high tensile strength together with high modulus and high work of fracture was observed. This was due to the nanofiber and matrix properties, favourable nanofiber-matrix interaction, a good dispersion of nanofibers and the MFC network.</p><p>Novel nanocomposite foams were obtained by freeze-drying aquagels prepared from 8wt% solutions of amylopectin starch and MFC. The MFC content was varied from 10 to 70wt%. For composite foam with MFC contents up to 40wt%, improved mechanical properties were observed in compression. The mechanical properties depended both on the cell wall properties and the cell-structure of the foam. The effect of moisture (20-80% RH) on the dynamical properties of composite foam with 40wt% MFC was also investigated and compared to those of neat starch foam. Improved storage modulus was noted with MFC content, which was a result of the nanofiber network in the cell-wall. In addition, the moisture content decreased with MFC content, due to the less hydrophilic nature of MFC.</p>

<p>Biomaterials used for stabilization of compressed vertebraes due to osteoporosis, have mainly been based on resin materials, like PMMA (polymethyl methacrylate), but have recently expanded to consist of injectable ceramics, such as calcium-aluminate. In this in vitro study human osteoblast-like cells, MG-63, were cultured on three different injectable biomaterials based on: Ca-aluminate, Bis-GMA (bisphenol A-glycidylmethacrylate) and PMMA, to investigate the cellular response elicited by these materials. Cell proliferation was measured by the NucleoCounter® system, cell viability was investigated by LDH (lactate dehydrogenase) analysis, cell differentiation and mineralization was evaluated by mRNA gene expression of the osteoblastic markers: ALP (alkaline phosphatase), OC (osteocalcin) and COLL-I (collagen type I) by qPCR (quantitative polymerase chain reaction) analysis. Two control materials were used: TCP (tissue culture polystyrene, negative control) and PVC (polyvinyl chloride, positive control). The results showed that all the bone cement materials were non-toxic and biocompatible, i.e. they provided good cell viability and proliferation of the MG-63 cells. They are specific for bone cells, since they expressed high values of the osteoblast-specific differentiation markers, and are thus promising as injectable bone cement materials. Among the bone cements, Xeraspine appears to be the most biocompatible material for bone cells. It is followed by Cortoss and then Vertebroplastic.</p>

Chitosan is widely investigated for biomedical applications due to its excellent properties, such as biocompatibility, biodegradability, bioadhesivity, antibacterial, etc. In the field of neural engineering, it has been extensively studied in forms of film and hydrogel, and has been used as scaffolds for nerve regeneration in the peripheral nervous system and spinal cord. One of the main issues in neural engineering is the incapability of neuron to attach on biomaterials. The present study, from a new aspect, aims to take advantage of the bio-adhesive property of chitosan to develop chitosan-based materials for neural engineering, specifically in the fields of brain repair and neuroprosthetics. Neuronal responses to the developed biomaterials will also be investigated and discussed.In the first part of this study (Chapter II), chitosan was blended with a well-studied hydrogel material (agarose) to form a simply prepared hydrogel system. The stiffness of the agarose gel was maintained despite the inclusion of chitosan. The structure of the blended hydrogels was characterized by light microscopy and scanning electron microscopy. In vitro cell studies revealed the capability of chitosan to promote neuron adhesion. The concentration of chitosan in the hydrogel had great influence on neurite extension. An optimum range of chitosan concentration in agarose hydrogel, to enhance neuron attachment and neurite extension, was identified based on the results. A “steric hindrance” effect of chitosan was proposed, which explains the origin of the morphological differences of neurons in the blended gels as well as the influence of the physical environment on neuron adhesion and neurite outgrowth. This chitosan-agarose (C-A) hydrogel system and its multi-functionality allow for applications of simply prepared agarose-based hydrogels for brain tissue repair.In the second part of this study (Chapter III), chitosan was blended with graphene to form a series of graphene-chitosan (G-C) nanocomposites for potential neural interface applications. Both substrate-supported coatings and free standing films could be prepared by air evaporation of precursor solutions. The electrical conductivity of graphene was maintained after the addition of chitosan, which is non-conductive. The surface characteristic of the films was sensitively dependent on film composition, and in turn, influenced neuron adhesion and neurite extension. Biological studies showed good cytocompatibility of graphene for both fibroblast and neuron. Good cell-substrate interactions between neurons and G-C nanocomposites were found on samples with appropriate compositions. The results suggest this unique nanocomposite system may be a promising substrate material used for the fabrication of implantable neural electrodes. Overall, these studies confirmed the bio-adhesive property of chitosan. More importantly, the developed chitosan-based materials also have great potential in the fields of neural tissue engineering and neuroprosthetics.

Sustainable waste treatment and lignin development strategies targeted for biorefineries will benefit industry, consumers, and the environment. This dissertation demonstrates the feasibility of a novel biochemical pathway capable of converting sugars and lignin sourced from biorefinery waste streams into microbial oils suitable for biodiesel, cosmetic, and biopharmaceutical applications. This biochemical pathway also presents interesting avenues for the commercial production of higher-value intermediate metabolites such as catechol, protocatechuate, pyruvate, and succinate. Alternatively, this dissertation also demonstrates a unique polymerization strategy for lignin that can be adopted towards the production of green polymeric biomaterials. Overall, these strategies jointly present intriguing routes for lignin valorization.

This dissertation has aimed to fabricate polypeptide based biomaterial and characterize physical properties. Electrospinning is used as a tool for the sample fabrication. Project focused on determining the feasibility of electrospinning of certain synthetic polypeptides and certain elastin-like peptides from aqueous feedstocks and to characterize physical properties of polymer aqueous solution, cast film and spun fibers and fiber mats. The research involves peptide design, polymer electrospinning, fibers crosslinking, determining the extent of crosslinking, fibers protease degradation study, fibers stability and self-organization analysis, structure and composition determination by various spectroscopy and microscopy techniques and characterization of mechanical properties of individual suspended fibers. Fiber mats of a synthetic cationic polypeptide poly(L-ornithine) (PLO) and an anionic co-polypeptide of L-glutamic acid and L-tyrosine (PLEY) of defined composition have been produced by electrospinning. Fibers were obtained from polymer aqueous solution at concentrations of 20-45% (w/v) in PLO and at concentrations of 20-60% (w/v) in PLEY. Applied voltage and spinneret-collector distance were also found to influence polymer spinnability and fibers morphology. Oriented fibers were obtained by parallel electrodes geometry. Fiber diameter and morphology was analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). PLO fibers exposed on glutaraldehyde (GTA) vapor rendered fiber mats water-insoluble. A common chemical reagent, carbodiimide was used to crosslink PLEY fibers. Fiber solubility in aqueous solution varied as a function of crosslinking time and crosslinker concentration. Crosslink density has been quantified by a visible-wavelength dye-based method. Degradation of crosslinked fibers by different proteases has been demonstrated. Investigation of crosslinked PLEY fibers has provided insight into the mechanisms of stability at different pH values. Variations in fiber morphology, elemental composition and stability have been studied by microscopy and energy-dispersive X-ray spectroscopy (EDX), following the treatment of samples at different pH values in the 2-12 range. Fiber stability has been interpreted with reference to the pH dependence of the UV absorbance and fluorescence of PLEY chains in solution. The data show that fiber stability is crucially dependent on the extent of side chain ionization, even after crosslinking. Self-organization kinetics of electrospun PLO and PLEY fibers during solvent annealing has been studied. After being crosslinked in situ, fibers were annealed in water at 22 °C. Analysis by Fourier transform infrared spectroscopy (FTIR) has revealed that annealing involved fiber restructuring with an overall time constant of 29 min for PLO and 63 min for PLEY, and that changes in the distribution of polymer conformations occurred during the first 13 min of annealing. There was a substantial decrease in the amount of Na+ bound to PLEY fibers during annealing. Kinetic modeling has indicated that two parallel pathways better account for the annealing trajectory than a single pathway with multiple transition states. Bacteria have been engineered to make novel 250-mer elastin-like polypeptides (ELPs). Each was predicted to have an absolute net charge of less than 0.05 electron charges per amino acid residue in aqueous solution at neutral pH. Polymer structure in solution has been assessed by Circular dichroism spectroscopy (CD) and dynamic light scattering (DLS). Suitability for materials manufacture has been tested by electrospinning. Here, we have also tested the hypothesis that blending polypeptides of radically different amino acid composition will enable the realization of novel and potentially advantageous material properties. Aqueous polymer feedstock solutions consisted of pure ELP or ELP blended with a synthetic polypeptide, PLEY, which is highly ionized at neutral pH and spinnable. Morphology analysis of blended fibers by SEM has revealed the formation of a surprising variety of structures that are not seen in fibers of ELP or PLEY alone, for example, hollow beads. Analysis of blended fibers by fluorescence microscopy showed that there was little or no phase separation, despite the large difference in electrical properties between ELP and the synthetic polymers. Structure and composition of PLO, PLEY, ELPs and blends and electrospun fibers made of these polymers have been determined and compared. CD and FTIR have been utilized to obtain structural information on these polymers in aqueous solution, cast films and fibers. Fiber composition has been analyzed by EDX. Protein adsorption has been analyzed by quantitative fluorescence microscopy. The polymers adopted random coil-like conformations in aqueous feedstocks at neutral pH and in dehydrated cast films and fibers on glass, and the fibers comprised numerous counterions, according to spectral analysis. Adsorption of model proteins and serum proteins onto hydrated and crosslinked fibers depended on the electrical charge of the proteins and the fibers. The surface charge density of the fibers will be comparable to, but less than, the charge density on the outer leaflet of the plasma membrane of usual eukaryotic cells. Mechanical properties of a series of as-spun and crosslinked PLO and PLEY nanofibers with various diameters have been analyzed by using the pure bending mode and AFM technique. Aligned nanofibers were deposited on top of a microsized groove etched on a glass substrate. AFM tip was used as a probe, which could apply a measurable deflection and force onto the suspended nanofiber at a force calibration mode, so that the Young's modulus of a single nanofiber can be calculated based on the basic beam bending theories. The Young's moduli of the studied peptide nanofibers increased significantly with decreased fiber diameters. This study has also demonstrated that crosslinked electrospun PLO and PLEY fibers have a higher Young's modulus compared with their as-spun counterparts. Taken together, the results will advance the rational design of polypeptides for peptide-based materials, especially materials prepared by electrospinning. It is believed that this research will increase basic knowledge of polymer electrospinning and advance the development of electrospun materials, especially in medicine and biotechnology. The study has yielded two advances on previous work in the area: avoidance of an animal source of peptides and avoidance of inorganic solvent. The present results thus advance the growing field of peptide-based materials. Non-woven electrospun fiber mats made of polypeptides are increasingly considered attractive for basic research and technology development in biotechnology, medicine and other areas.

The work presented in this thesis focuses on the development of strategies and smart bioactive materials for the treatment of osteoporosis. High and low molecular weight soluble hyaluronic acid-bisphosphonate (HA-BP) derivatives were investigated for their ability to inhibit osteoclasts. Low molecular weight HA-BP (L-HA-BP) was most effective in inhibiting active resorption of both murine and human osteoclasts (without affecting osteoblasts) compared to free bisphosphonate (BP). Precursor monocytes were unaffected, suggesting the specificity of HA-BP towards osteoclasts. This new class of functionalized hyaluronic acid could lead to rapid development of tailor-made pro-drugs for targeted treatment of osteoporosis. Polyphosphoesters (PEP) have been widely studied for their pro-osteoblast effects, primarily due to their involvement in cellular energy production pathway leading to the formation of inorganic phosphates that contribute to mineralized bone. Given that the effect of PEP on human osteoclasts is little studied, this work on poly(ethylene sodium phosphate) (PEP.Na) explores the potential to use PEP.Na as an inhibitor of osteoclast activity for the first time. PEP.Na exposure led to a dose-dependent toxicity of osteoclasts with reduction in their capacity to form resorption pits over 24h. Currently, there is a dearth of in vitro cell-culture systems that can study osteoclast-related resorption and osteoblast-related mineralization in a single co-culture system, and to simultaneously quantify the effects of soluble factors on these processes. Described here, is the development of a novel and simple two-sided co-culture system that can overcome these limitations with reliable and quantifiable readouts. In comparison with traditional one-sided co-culture systems, the two-sided co-culture was able to generate similar readouts for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) markers. There is also the advantage of distinctly separate and quantifiable readouts for mineralization and resorption, which has been demonstrated using Pamidronate. Finally, HA-BP was synthesized with pre-determined amounts of BP groups. The BP groups attached to HA allowed the tunable incorporation of BMP-2 in hydrogels. The charge-based affinity of BMP-2 and BP allowed stable incorporation of specific amounts of BMP-2, which could be tuned by the ratio of BP groups. 125I-labelled BMP-2 was loaded into hydrogels and their release was studied. Radioactive measurements revealed the tunable sequestration and controlled release of protein over time. This result was corroborated by ALP measurements of cells exposed to released BMP-2. ALP production was found to be almost 5-fold higher in HA-BP hydrogels loaded with BMP-2 which suggested that the sequestered BMP-2 is not only available to cells but also remains highly potent, even in entrapped form, The release of BMP-2 is dependent upon the rate of diffusion, swelling in hydrogels and degradation pattern of the gels and may assist in the long-term and rapid regeneration of osteoblasts in vitro.

Many important therapeutic agents are associated with significant undesired side effects which often limit treatment duration and dosing. Specifically, most major classes of antitumor chemotherapeutics have deleterious effects on cell division and DNA synthesis throughout the body due to systemic biodistribution. Engineering systems for controlled drug delivery allows for improved quality of life during treatment; as well as higher localized therapeutic concentrations by isolating toxic drugs used in many diseases to specific physiological compartments. An important drug delivery strategy for controlled release of therapeutics is based on responsive polymer matrices, which undergo swelling transitions in response to environmental stimuli. Biologically relevant factors which may trigger the release of therapeutics from responsive polymers include pH, ionic strength, and temperature. Temperature responsive polymers integrated into a composite system with metal nanoparticles allow for on demand drug release via an externally-applied optical or magnetic energy source. The intent of this work was to develop a temperature-responsive drug delivery platform for controlled therapeutic release, as well to expand the toolbox for rational design of responsive hydrogel nanoparticles intended for therapeutic delivery. Temperature-responsive hydrogels were synthesized and examined in the form of nanoparticles and bulk polymer networks. These materials are based on interpenetrating polymer networks (IPNs) of polyacrylamide (PAAm) and poly(acrylic acid) (PAA), which exhibit a positive volume swelling response with respect to temperature. Since this system responds to pH, ionic strength, and temperature, these IPNs were characterized over a wide range of solution conditions. Critical synthesis parameters needed to optimize thermal responses for specific solution conditions were identified, as were the specific effects of pH and ionic strength on network swelling and stability. The reverse emulsion process used to synthesize IPN nanoparticles was characterized to determine how particle growth proceeds during preparation. To enhance biocompatibility, IPN nanoparticles were surface-modified with a corona of poly(ethylene glycol) to reduce protein adsorption, a common strategy to improve in vivo performance. Due to the large amounts of surfactants employed in the preparation of IPN nanoparticles, purification methods needed to improve safety of IPN nanoparticles were optimized, and studied in vitro to ensure cellular compatibility. / text

Myocardial Infarction (MI) is one of the most prevalent and deadliest diseases in the United States. Since the host myocardium becomes irreversibly damaged following MI, current research is focused on identification of novel, less invasive, and more effective treatment options for patients. Cellular cardiomyopathy, in which viable cells are transplanted into the necrotic tissue, has the potential to regenerate and integrate with the host myocardium. Stem cells, specifically pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC), are ideal candidates for this procedure because they are pluripotent; however, ESCs must be predifferentiated to avoid teratoma formation in vivo. In this dissertation, our goal was improve upon current protocols to direct differentiation of ESCs into cardiomyocytes using an embryoid body (EB) model. We immobilized pro-cardiomyogenic proteins, specifically Sonic Hedgehog (SHH) and Bone Morphogenetic Protein 4 (BMP4) to paramagnetic beads and delivered them in the interior of the EB. While lineage commitment was indiscriminate, the presence of the beads alone appeared to guide differentiation into cardiomyocytes: there were significantly more contracting areas in EBs containing beads than in the presence of SHH or BMP4. To take advantage of this result, we immobilized Arginine-Glycine-Aspartic Acid (RGD) peptides to the beads and magnetized them following incorporation into the EB. Magnetically mediated strain increased the expression of mechanochemical markers, and in combination with BMP4 increased the percentage of cardiomyocytes. Finally, PEGylated fibrin gels were used to investigate the effect of seeding method and fibrinogen concentration on cardiomyocyte behavior and maturation. Cells seeded on top of compliant hydrogels had the most contracting regions compared to stiffer PEGylated fibrin gels, whereas cardiomyocytes seeded within the hydrogels could not remodel the matrix or maintain contractility. As an alternative to 3D culture, we seeded cardiomyocytes within gel layers, which maintained viability as well as contractile activity. We observed that PEGylated fibrin gels can maintain ESC-derived cardiomyocytes; however, the ratio of cardiomyocytes and non-cardiomyocytes should be optimized to maintain contractile phenotypes. Therefore, this dissertation presents novel methods to differentiate ESCs into cardiomyocytes, and subsequently promote their maturation in vitro, for the treatment of MI. / text

A multitude of biophysical signals, including chemical, mechanical, and contact guidance cues, are embedded within the extracellular matrix (ECM) to dictate cell behavior and determine cell fate. To understand the complexity of the cell-matrix interaction and how changes to the ECM contribute to the development of tissues or diseases, three-dimensional (3D), culture systems that can decouple the effects of these cues on cell behavior are required. This dissertation describes the development and characterization of approaches based on multiphoton excitation (MPE) to control the chemical, mechanical, and topographical presentation of micro-3D-printed (μ-3DP) protein hydrogels independently. Protein hydrogels were chemically functionalized via the MPE-induced conjugation of benzophenone-biotin without altering the physical properties of the matrix. Complex, immobilized patterns and chemical gradients were generated within protein hydrogels with a high degree of spatial resolution in all axes. Hydrogel surfaces were also labeled with adhesive moieties to promote localized Schwann cell adhesion and polarization. Laser shrinking, a method based on MPE to manipulate the topographical and mechanical presentation of protein hydrogels after fabrication, is also presented. Topographical features on an originally flat substrate are created with depths approaching 6 μm. The Young’s modulus of protein hydrogels can also be increased by 6-fold (~15 – ~90 kPa) using laser shrinking, and parameters can be adjusted to create continuous gradient profiles for studying durotaxis. At determined scan conditions, the two properties can be adjusted independently of each other. Most importantly, the physical properties of the hydrogels can be manipulated in situ to study the effects of dynamic changes to the substrates on cells. As a potential tool to monitor cellular responses to presented cues, fluorescent probes that detect nitric oxide are characterized. Collectively, these technologies represent a key advance in hydrogel tunability, as the platforms presented offer independent, dynamic, and spatiotemporal control of the chemical, mechanical, and topographical features of protein hydrogels. The introduced technologies expand the possibilities of protein hydrogels to clarify underlying factors of cell-matrix interactions that drive morphogenesis and pathogenesis, and are broadly applicable to a multitude of physiological systems. / text