Hydrogels of oligo(poly(ethylene glycol) fumarate) (OPF) were investigated toward the controlled release of therapeutic plasmid DNA for tissue engineering applications. The release of DNA from OPF hydrogels and the swelling characteristics of the hydrogels were characterized in vitro. The results demonstrated that the nominal molecular weight of the poly(ethylene glycol) from which the OPF was synthesized affects the hydrogel swelling and the DNA release kinetics. Further, these studies indicated that the degradation of the OPF dominates the control of DNA release. The retention of DNA bioactivity over the course of release was demonstrated through bacterial transformations. Subsequent studies characterized the release of plasmid DNA from composites of OPF and cationized gelatin microspheres (CGMS) in vivo, as well as the degradation kinetics of CGMS in these composites. Comparisons between the composite groups and material control groups indicated that the bioavailability of DNA can be extended through release from CGMS encapsulated within OPF, relative to CGMS or DNA solution injection alone, although no difference was observed between the composites and OPF. A related study characterized the release of DNA from the composites of OPF and CGMS in vitro. The results demonstrated that plasmid DNA can be released in a sustained fashion over the course of 49 to 149 days, with the release kinetics depending upon the material composition and the method of DNA loading. Released DNA retained viable structure over the course of release. A final study investigated the release of plasmid DNA encoding an osteogenic protein from composites of OPF and CGMS toward enhancing bone regeneration in a rat calvarial defect model. No enhancement in new bone formation was observed with release of DNA, relative to material controls. However, the reason for the absence of enhancement could not be elucidated in the study. Thus, hydrogels of OPF and composites of OPF and CGMS demonstrate potential as tissue engineering scaffolds for the controlled release of plasmid DNA, yet further investigation is warranted to assess transfection efficiency and gene expression with these systems.

The growing interest in using exogenous agents to enhance the subtle differences between optical signatures of normal and cancerous tissue, has spurred the development of novel nanoparticulate agents that exhibit desirable optical responses and at the same time, used to target biomolecular markers of diseases. Gold nanoshells are a class of core-shell nanoparticle, exhibiting an extremely agile peak optical resonance, ranging from the near-ultraviolet to the mid-infrared wavelengths. Although optical properties of gold nanoshells in transmission have already been well documented, the reflectance characteristics have not been elucidated. Yet, in order to use gold nanoshells as a contrast agent for scattering-based optical diagnostic tools, it is critical to study the reflectance behavior. Using a combination of experimental observations and Monte Carlo models, we investigated gold nanoshell reflectance characteristics and its effect on tissue phantoms. Gold nanoshells were shown to significantly alter reflectance signatures of tissue phantoms, both experimentally and in computer models. Monte Carlo simulations of gold nanoshell reflectance demonstrated the efficacy of using such methods to model diffuse reflectance and also reaffirm the experimental observations. Our studies suggest that gold nanoshells are an excellent candidate as an optical contrast agent and Monte Carlo methods can be a useful tool for optimizing nanoshells best suited for scattering-based optical methods to enhance the detection and imaging of cancers.

Vertebroplasty has the potential to be a highly effective vertebral fracture prevention treatment, but the procedure must first be optimized for maximum benefit and minimal risk of safety to the patient. The procedure involves the percutaneous injection of a liquid bone cement into the vertebral body, which upon hardening provides instantaneous structural reinforcement. This research characterizes the effects of bone cement volume, material properties and distribution patterns on the global and internal vertebral biomechanics after prophylactic vertebroplasty in order to optimize these cement properties based on biomechanical efficacy and risk of complications which pose a threat to patient safety. In light of the many factors affecting the biomechanical outcome, a computational approach was employed since multiple analyses can be repeated on the same specimen. The accuracy of the models is assured by using realistic, image-based finite element models of human vertebral bodies that are specimen-specific, anatomically detailed and calibrated to experimental results. Prophylactic vertebroplasty was simulated on these models under various cement configurations and their biomechanical efficacies were evaluated based on the criteria for biomechanical success developed in this research---maximum mechanical reinforcement to reach low fracture risk levels with minimal amount of cement and maintenance of intravertebral mechanical compatibility to retain the normal dynamics of the weight-bearing spine. The biomechanically optimal bone cement is determined as one that results in a spatially dispersed distribution when injected into the vertebral body. The higher vertebral reinforcements achieved with a dispersed cement fill may lower the risk of complications due to cement leakages since smaller cement volume would be just as biomechanically effective. Furthermore, the disperse fill results in minimal intravertebral stress concentrations that may reduce the risk of subsequent fractures in the adjacent untreated vertebrae. Now that the bone cements with spatially dispersed fill patterns is known to produce optimum biomechanical effects, the biochemistry of materials with this unique characteristic can be specifically tailored to include biodegradability and drug release capabilities for various applications that require the merger of fracture prevention, tissue engineering and drug delivery innovations into one without any concerns for adverse biomechanical affects.

Composite materials based on the synthetic polymer poly(propylene fumarate) (PPF) were developed and characterized for use as porous controlled release scaffolds in bone tissue engineering. Through the use of poly(DL-lactic-co-glycolic acid) (PLGA) microparticles, the osteogenic peptide TP508 (Chrysalin RTM) was incorporated into the polymer phase of PPF-based scaffolds, creating a composite material that could act as a scaffold for guided tissue ingrowth as well as a vehicle for targeted drug delivery. Alteration of formulation parameters such as the TP508 loading of the microparticles, the microparticle to PPF ratio, and the initial leachable porogen content lead to variation in the release kinetics of the incorporated peptide in vitro. Inclusion of the microparticles into the scaffolds as well as changes in the scaffold formulation parameters did not alter the scaffolds in vitro degradation profile through 26 weeks. Using results from the in vitro studies, two distinct release kinetic profiles were selected for further evaluation in vivo . Composite formulations exhibiting either a large initial burst release or a minimal initial burst release of 200 mug TP508 were implanted in 15.0 mm segmental defects in rabbit radii. Radiography, micro-computed tomography, and histomorphometry were used to elucidate the effect of varied release kinetics on bone formation at 12 weeks post-operative. Results showed that composite scaffolds exhibiting a large burst release of TP508 resulted in the greatest amount of bone. Analysis showed that bone formation was characterized by growth both into the pores of the scaffold as well as guided across the defect along the surface of the implant. Further investigation revealed minimal degradation of the polymer after 18 weeks in vivo. The studies presented here demonstrate the potential of PPF/PLGA composite materials for use in bone tissue engineering. These composite scaffolds offer controlled, targeted delivery of bioactive molecules as well as structural support for cellular infiltration and bone formation within osseous defects. Additional work was conducted in the area of controlled release of polysaccharide oligomers. Initial experiments established that hyaluronan oligomers could be incorporated into PLGA microparticles and that parameters including PLGA molecular weight, hyaluronan molecular weight, and hyaluronan loading influenced the oligomer release kinetics.

Tissue-engineering scaffolds were designed to mimic several features of the extracellular matrix using a combination of the synthetic polymer, PEG diacrylate, and bioactive factors. Scaffolds were formed by exposing aqueous solutions of PEG diacrylate and bioactive factors modified with PEG monoacrylate to ultraviolet or visible light in the presence of a suitable photoinitiator. Light exposure generated free radicals that targeted acrylate groups in the monomer and in PEG conjugated bioactive factors resulting in crosslinked hydrogel scaffolds with bioactive factors covalently incorporated. This study extended the capability for directing cell behavior using PEG-based hydrogels to include control over the spatial distribution of bioactive factors and the presentation of the growth factor, bFGF. Additionally, PEG hydrogels were modified with a degradable peptide sequence to enable cells to remodel the scaffold by secreting matrix metalloproteinases (MMPs). A continuous linear gradient was formed by simultaneously using a gradient maker to combine hydrogel precursor solutions with photopolymerization, which locks the gradient in place. Coomassie blue staining confirmed the formation of protein gradients. Fibroblast cells responded to covalently immobilized gradients of the adhesive peptide, RGD, by changing their morphology to align in the direction of increasing RGD concentration and by migrating differentially on RGD-gradient hydrogels compared to control hydrogels. Next, bFGF was covalently immobilized to hydrogels with retention of its mitogenic and chemotactic effects on smooth muscle cells (SMCs). A covalently immobilized bFGF gradient was also formed using the gradient maker and shown to increase linearly along the hydrogel's length by silver staining. SMCs responded to these bFGF-gradient hydrogels by aligning in the direction of increasing bFGF concentration and by migrating differentially, up the concentration gradient, compared to migration on control hydrogels. Finally, the MMP-sensitive peptide sequence, GPQGILGQ, was inserted into the main polymer chain's backbone to allow targeted degradation by cell-secreted proteases. Cells were observed to change their morphology and migrate when seeded within these degradable hydrogel scaffolds, but not in scaffolds lacking this degradable peptide sequence. This hydrogel system is expected to be useful for studying tissue formation leading eventually to an improved understanding of the factors needed to form engineered tissues.

This dissertation determines the feasibility of photopolymerizable hydrogels as novel tissue engineering scaffolds and constructs a pulsatile flow bioreactor for the development of tissue engineered vascular grafts (TEVGs). The large number of small diameter bypass surgeries performed each year coupled with the shortage of suitable, patent vascular grafts has spurred the development of tissue engineered vascular substitutes. This investigation characterizes the mechanical properties of polyvinyl alcohol (PVA) and polyethylene glycol (PEG) hydrogels and evaluates their ability to support cell viability, proliferation, and extracellular matrix protein production for use as a tissue engineering scaffold. The elasticity and tensile strength of PVA and PEG hydrogels may be tailored by changing the polymer concentration, number of crosslinkable groups per PVA chain, PEG molecular weight, or using blends of high and low PEG molecular weights to transmit cyclic strain and still maintain structural integrity in a pulsatile flow bioreactor. At least 75% of cells cultured over two weeks inside PVA hydrogels and for four weeks in PEG hydrogels remained viable, with no differences in viability across the thickness of the hydrogel. Once seeded inside hydrogels, cells continue to function; following two weeks in culture, cells produced hydroxyproline in both PVA and PEG hydrogels. After determining that these hydrogels were suitable materials for scaffolds, a pulsatile flow bioreactor, mimicking transmural strain encountered in vivo, was constructed to culture tubular hydrogel-cell constructs. PEG hydrogels placed in the bioreactor exhibited strains at 2 Hz and between 5.9--15.9%, depending on the material elasticity, with pressures around 70/20 mmHg. Furthermore, smooth muscle cells seeded in PEG hydrogels and cultured in the bioreactor for one week showed similar DNA content to static gels, indicating that the bioreactor does not hinder cell viability. These results suggest that PVA and PEG hydrogels are appropriate materials for TEVG scaffolds and that the bioreactor generates conditions suitable for tissue formation and organization. In the future, this system will require optimization to incorporate the right combination of bioactive molecules, cell types, and bioreactor parameters to achieve a TEVG with composition, organization, and mechanical properties resembling those of native blood vessel.

In this thesis, a novel injectable copolymer hydrogel was developed that may be suitable for cardiovascular applications. This material consists of a triblock copolymer of poly(propylene fumarate) and poly(ethylene glycol). The first part of this work involved characterizing the kinetics of the poly(propylene fumarate) (PPF) reaction using zinc chloride as a catalyst. The reaction kinetics were shown to be dependent on the reaction temperature. The second part of the work involved characterizing various properties of the hydrogel by varying components of the water soluble crosslinking system and the copolymer structure. The properties examined included equilibrium water content, sol fraction, mechanical strength, onset of gelation, molecular weight between crosslinks, and endothelial cell adhesion. In addition, these hydrogels were modified with bioactive peptides to test for their feasibility as a future promoter of endothelial cell adhesion and migration. The results from all these studies show that this novel injectable hydrogel may be suitable for cardiovascular applications and other tissue engineering applications.

Near infrared tuned gold nanoshells have been developed to enhance the contrast of optical coherence tomography (OCT) images, and we have completed a systematic study which quantifies and optimizes the specifications of nanoshells that provide improved efficacy of OCT imaging and photothermal ablation of cancer. The optical properties of gold nanoshells, such as scattering, absorption, and asymmetry values were calculated with Mie scattering theory. For comparison and experimental quantifications, scattering coefficients were extracted from OCT images using Extended Huygens-Fresnel (EHF) principle based algorithms. With the addition of Her2 conjugated nanoshells, ex vivo OCT images of human breast cancer tissue, which express signatures of Her2/neu, provide significant contrast in comparison to the normal and malignant controls. As an extended study of dual NIR absorbing/scattering nanoshells for integrated cancer imaging and therapy in vitro, combined OCT imaging and photothermal tumor ablation was performed in vivo. Results showed that gold nanoshells selectively accumulated in the tumorous regions and enabled clear differentiation of tumor. Tumor regression by the photothermal ablation using NIR tuned nanoshells was also reported. Our studies have demonstrated that nanoshells can be designed specifically for diagnostic and therapeutic purposes.

Cell migration is an essential step during processes such as embryonic development, wound healing, angiogenesis, and cancer metastasis. Migration is a complex integration of cellular adhesion to its substratum, reorganization of the cytoskeleton, proteolysis and remodeling of surrounding extracellular matrix (ECM), and activation and regulation of chemical signaling by growth factors and other mitogenic cues. These cooperative mechanisms enable a cell to move to its target to perform its function, whether to repair injured tissue, fight infections, or build new blood vessels. The main objective of this research is to study mechanisms of cell migration within a biomimetic hydrogel system. Because of the complexity of the ECM, studying cell migration in ECM derivatives or even single components of the ECM can make it difficult to decipher the importance of each factor involved. In addition, the ECM imposes a spatial barrier to cells. For migration, the cells must not only interact with matrix adhesive ligands for force generation, but also develop strategies to overcome biomechanical resistance imposed by the matrix. Thus, the biomimetic hydrogel system developed can provide the mechanical support, adhesion ligands, degradation sequences, and other signals, so that a cell can migrate. This system will provide tight control over many experimental parameters and minimize nonspecific cell-material interactions. Hence the aim of the hydrogel system is to stimulate an active interaction between the synthetic polymer and the biological environment. The biomimetic hydrogels are photopolymerizable hydrogels based on acrylated derivates of polyethylene glycol. This material contains proteolytically degradable peptide sequences, targeted for specific enzymes involved in cell migration, in the polymer backbone. Cell adhesion peptides are also grafted into the hydrogels during photopolymerization to promote interaction with specific cell surface receptors. Other bioactive signals, such as growth factors, can also be grafted into the network during photopolymerization. Thus a single hydrogel material can contain several different proteolytically sensitive segments, many cell adhesion ligands and various growth factors, allowing for one to mimic many properties of the ECM. This hydrogel system is used to assess cell migration mechanisms by controlling the identity and availability of bioactive signals presented to the cells and studying their affects.

Synthetic hydrogel materials based on oligo(poly(ethylene glycol) fumarate) (OPF) were developed and characterized as injectable cell carriers for orthopaedic tissue engineering. Through alteration of the poly(ethylene glycol) molecular weight used in the synthesis of the OPF macromer, swelling and mechanical properties of the resulting crosslinked hydrogels could be controlled. These hydrogels were characterized, in both the swollen and dry states, leading to calculation of their mesh sizes, which varied depending on the OPF type used in crosslinking. In addition, it was found that these gels could be laminated during crosslinking, with each layer having distinct mechanical properties. Before their use as injectable cell carriers, the cytotoxicity of all OPF hydrogel precursor molecules, including radical initiators and their derivatives, was evaluated using rat marrow stromal cells as a model cell type. Results indicated that the overall pH of the formulation, as well as length of exposure to the components, had significant effects on cell viability. Using this information, an initiator was identified which remained near neutral pH in cell culture media and resulted in crosslinking of two types of OPF hydrogels in 8 min at 37°C. The optimized OPF formulations were then used to investigate effects of changes in hydrogel swelling properties and media supplements on osteogenic differentiation of encapsulated rat marrow stromal cells. After 28 days of in vitro culture, evidence of cellular differentiation was found in all sample types, indicating that the encapsulation procedure did not have a detrimental effect on the ability of the marrow stromal cells to form bone-like tissue. In the presence of osteogenic supplements, OPF hydrogels with greater swelling promoted embedded MSC differentiation over those that swelled less. In all specimens examined, areas of mineralized matrix were obvious many microns away from the cells, indicating that the hydrogel mesh size was large enough to allow diffusion of matrix components throughout the material. These results demonstrate the great potential of OPF hydrogels as injectable carriers for delivery of cells to a variety of complex orthopaedic defects.