Successful gene therapy for breast and ovarian cancer will likely require that anti-cancer genes be delivered specifically to primary and metastatic tumor sites while avoiding normal tissues. Adenoviral vectors are attractive for cancer gene therapy, since they can deliver transgenes to many different tumors. While adenovirus is quite potent at gene delivery, it is also non-specific and delivers genes into tumor and non-tumor cells in vivo. For effective gene therapy, the natural tropism of adenovirus must be removed and the virus re-targeted to tumor cells using cancer-specific ligands. To identify new cell binding ligand, peptide presenting phage libraries were selected against human breast cancer cell lines. Displayed on phage, these peptides bound specifically to their selection target, cross-reacted to varying degrees on other breast cancer cell lines, and did not bind to normal breast epithelial cells. The binding properties of these peptides were compared with those of commercially available antibodies such as Herceptin and binding proteins such as EGF to determine viable candidates for vector targeting. Viral targeting methods developed in our laboratory show promise in both ablating the natural tropism of adenovirus and retargeting the virus. The targeting ligands were complexed to biotinylated adenovirus through avidin bridges and chemically cross-linked to adenovirus using bifunctional PEG molecules. The viral complexes were tested in vitro before delivery was evaluated in the in vivo xenograft tumor models. Fluorescent and luminescent reporter genes were used to determine the location of vector delivery through gene expression in vivo. Targeted adenovirus had reduced background transduction and somewhat increased breast and ovarian cancer transduction. Finally to better evaluate ligand performance, real time, dynamic imaging was used to track ligand distribution and kinetics in vivo in tumor models. Fluorescent conditions were first evaluated in mouse models, demonstrating that imaging in the near infrared had superior signal to noise profiles over fluorescence in the visual range. Therefore ligands were labeled with the near infrared dye IR800 and their distribution was tracked in real time. To evaluate the feasibility of tracking virus (not transgene products), adenovirus was labeled with IR800, given to mice and virion trafficking was successfully imaged.

An estimated 20-25% of Americans suffer from the symptoms of temporomandibular joint disorder, or TMD. The National Institutes of Health have reported between 14.4 and 43.2 million Americans having symptoms associated with TMJ disorders, with peak prevalence in young adults. TMD can be caused by many different factors and can have complex symptoms which can include but are not limited to deviation in opening and closing, clicking, locking, joint and muscle pain, and limited opening. A characterization of the temporomandibular joint as it pertains to kinematics is required in order to understand the pathology. A new method for defining quantifiable jaw motion parameters is developed and used to describe the motion characteristics in a normal population, as well as to compare between normal and pathological subjects. Additionally, a motion prediction algorithm is developed which is used to accurately predict the mandibular motion of two subjects. A subject specific finite element model is created to examine the stress profile of the temporomandibular joint disc during normal jaw motion in vivo. Finally, a muscle activation prediction algorithm is created and used to create a kinematic model for the investigation of mechanical changes in the temporomandibular joint.

The integration of bioactive and biomimetic signals into materials for drug delivery and tissue engineering serves to improve cellular responses and therefore healing by more closely resembling the natural cellular microenvironment. The materials developed in this thesis show promise in delivering therapeutic doses of nitric oxide (NO) to physiological systems and provide novel surfaces for the study of cell adhesion and spatial organization. NO has several biological functions that make it an ideal candidate therapeutic agent for the prevention of the occlusive scarring of blood vessels following treatment of coronary artery disease through procedures such as balloon angioplasty and bypass grafting. The present work incorporates NO donors into polymeric biomaterials, resulting in copolymers that release NO over controllable time frames depending on material design. These NO-generating polymers have proven effective in significantly reducing platelet adhesion and smooth muscle cell proliferation in vitro. Endothelial cells exposed to these materials displayed enhanced proliferation, which is essential in restoring vessel function. Local, sustained release of NO from perivascularly-applied hydrogels reduced unwanted neointimal formation by approximately 90% in an experimental balloon angioplasty model. Novel NO releasing dendrimers have been synthesized to establish the potential for injectible NO therapy and can be targeted to sites of active vascular disease. NO-releasing polyurethane has been synthesized as a candidate material for vascular grafts. The superior mechanical properties of polyurethane combined with the inhibition of platelet adhesion by NO promise increased patency in small diameter vascular prostheses. Bioactive poly(ethylene glycol) (PEG) hydrogels have also been synthesized with covalently bound cell adhesion moieties to elucidate the mechanisms of immune cell adhesion to the vascular wall under shear. Leukocytes perfused over the surfaces of these hydrogels in a parallel plate flow chamber display rolling and adhesion properties like those seen on vascular endothelium in vivo. This work also presents a system of patterning bioactive regions onto hydrogels using transparency masks. This system allows the formation of complex patterns of cell-adhesive regions that closely mimic in vivo cellular arrangement. The intrinsic biocompatibility of PEG and the decreased thrombogenicity that NO affords make these materials ideal for incorporation into blood contacting devices.

Novel polymer networks based on poly(propylene fumarate) (PPF) and the crosslinking agent poly(propylene fumarate)-diacrylate (PPF-DA) were investigated as a material for a biodegradable interbody fusion cage. The aim of this work was to establish the effect of the macromolecular network structure on the physical properties in order to tailor the material to demonstrate high strength, controllable degradation, and suitable biocompatibility for this implant. The PPF/PPF-DA network structure was characterized with a newly developed technique in which the networks were degraded into simpler linear constituents that provided insight to the macromolecular structure. The double bond conversion and crosslinking density of the polymer networks was controlled by the concentrations of PPF and PPF-DA in the network, as dictated by the double bond ratio of fumarate groups in the PPF backbone to acrylate groups in the PPF-DA crosslinker. Lower double bond ratios yielded higher conversions and a more densely crosslinked network. The network structure was further influenced by the free radical initiator system. The mechanical properties of the PPF/PPF-DA networks increased with decreasing double bond ratios as a result of higher crosslinking densities. Photo-crosslinking produced a stronger material and also facilitated processing of PPF/PPF-DA networks because there is greater control over the crosslinking reaction. Examination of the in vitro degradation behavior of PPF/PPF-DA networks in simulated body fluids showed that the degradation rate was faster for networks with lower crosslinking densities. The biocompatibility of the material was also controlled by the macromolecular structure as PPF/PPF-DA networks with higher double bond conversions and crosslinking densities exhibited no adverse cytotoxicity and enabled fibroblast attachment. A prototype PPF/PPF-DA interbody fusion cage was fabricated by photo-crosslinking the polymers in transparent silicone molds. The PPF/PPF-DA implant demonstrated similar mechanical properties as a clinical approved allograft spacer and suggested that the device can provide sufficient support for interbody fusion. This work demonstrated that PPF/PPF-DA networks are a suitable material for a biodegradable interbody fusion device as well as other load bearing orthopaedic implants.

A composite material consisting of a thermally-sensitive hydrogel and optically active nanoparticles would possess the temperature sensitive characteristics of the NA copolymer as well as the absorption spectrum of the nanoshells. The combination of these two properties in a single material opens up several very interesting opportunities in a number of applications. Composite hydrogels consisting of either Au-Au2S or SiO2-Au nanoshells or gold colloid embedded in a N-isopropylacrylamide-co-acrylamide copolymer collapse upon exposure to light that matches the peak extinction wavelength of the nanoparticles. These composite materials have successfully delivered a wide range of molecular weight compounds, from methylene blue (MW 345) to bovine serum albumin (MW 66,000), in a pulsatile fashion in vitro and in vivo. Furthermore, insulin that has been released from the composite hydrogels in this manner retains its activity. Due to the ability of the nanoshell-composite to provide on-demand release of a therapeutic agent, such a system may prove to be beneficial in the treatment of diseases that require a flexible therapeutic regimen, such as insulin dependent diabetes mellitus. The collapse of the composite hydrogels has also been investigated for use as valves or gates in microfluidic devices. Hydrogels containing different nanoparticles exhibit independent optical addressibility, and were polymerized in situ within existing microfluidic chambers. Composite materials of optically active nanoparticles and thermally sensitive hydrogels should prove useful in a wide range of applications where remote optical activation of a mechanical device, such as a gate or switch, is desired, in particular where large relative displacements of such a structure are desirable.

Bone regeneration through transplantation of genetically modified cells provides an opportunity to combine the fields of tissue engineering and gene therapy. The ex vivo modification of cells for overproduction of proteins can be used for therapeutic and/or diagnostic applications in tissue regeneration. The aim of this work was to develop genetically modified cells for enhanced osteogenic potential and for non-destructive diagnostic evaluation of cell behavior on biomaterials. Prior to modification of cells, a luminescent based assay was developed for the evaluation of osteodifferentiation of progenitor cells. A chemiluminescent substrate was used for detection of the early osteodifferentiation marker, alkaline phosphatase (AP). The results of this work demonstrated a faster, simpler, and more sensitive method of monitoring changes in AP levels during osteodifferentiation using the chemiluminescent substrate relative to the traditional colorimetric substrate. The ability of primary rat marrow stromal cells (MSCs) to be genetically modified by adenoviral, retroviral, and cationic lipid gene therapy vectors was investigated. All vectors delivered the reporter transgene luciferase to MSCs. We also explored the influence of MSC differentiation induced by different culture conditions on the vectors' gene delivery efficiency. Only adenovirus demonstrated a substantial increase in transgene expression when cells were cultured in the presence of osteogenic supplements. Moreover, it was determined that increased transgene expression was specific to the supplement concentration of dexamethasone in the culture medium. For therapeutic bone regeneration the osteogenic transgene bone morphogenetic protein 2 (BMP-2) was delivered to MSCs. Adenovirus, retrovirus, and cationic lipids delivering this transgene were used and the osteogenic potential of the MSCs was evaluated. Results both in vitro and in vivo indicated that MSCs genetically modified with adenovirus had an enhanced osteogenic response compared to unmodified control MSCs and MSCs modified by the other vectors. For diagnostic non-destructive evaluation of cells on biomaterials, two fibroblastic cell lines were developed to stably express the fluorescent transgene enhanced green fluorescent protein (EGFP) and the luminescent transgene luciferase. Cells were able to be visualized both microscopically and macroscopically on two separate biomaterials. In addition, quantitative growth of cells on the materials was assessed non-destructively both in vitro and in vivo.

The meniscus, a fibrocartilagenous tissue found between the femur and tibia, is responsible for shock absorption, load transmission, and stability within the knee joint. Damage to this tissue can lead to osteoarthritic changes, suggesting that the meniscus protects the knee joint from degenerative joint disease. Historically, repair techniques consisted of excision or suturing of the damaged tissue. Unfortunately, neither of these techniques successfully repairs the damaged tissue; tissue engineering is one possible solution. When attempting to tissue engineer a tissue, it is ideal to start in a small animal model, such as a rabbit, before attempting the repair process in a larger animal model. The objective of this work was to characterize the medial rabbit meniscus ultrastructurally, biomechanically, biochemically, and cellularly and to perform biomechanical characterization on larger animal models for future scale up efforts. The medial rabbit meniscus was found to have a higher hydration level, greater amount of sulfated glycosaminoglycans, and lower level of hydroxyproline at the inner 1/3 of the tissue, which confirmed the more chondrocytic nature of this region. It was also found that the anterior portion of the tissue, particularly in the inner 1/3, had a higher hydration level, sulfated glycosaminoglycan level, aggregate modulus, permeability, shear modulus, and a lower hydroxyproline level than the central and posterior locations. It is believed that this topographical variation is due to the bent-knee resting stance of the rabbit and its propensity to jump. It was also determined that significant variations exist in the compressive creep properties, both intraspecies and interspecies, in a variety of animal models, indicating caution when comparing animal models and determining which animal model to use in future tissue engineering efforts. The characterization in this study can serve as a "gold standard" reference for future meniscal tissue engineering efforts and be used as a baseline for future large animal tissue engineering efforts.

The temporomandibular joint (TMJ) disc is a specialized fibrocartilaginous tissue located between the mandibular condyle and the glenoid fossa-articular eminence of the temporal bone. It has been observed that up to 70% of patients with temporomandibular joint disorders (TMDs) suffer from displacement of the disc. When the displaced disc becomes an obstacle to movement and degeneration is severe, surgeons have no choice but to replace the disc with various allopastic or biological materials. Tissue engineering may provide a better alternative for discectomy patients. Toward this end, the work described in this thesis provides a systematic approach for tissue engineering the TMJ disc. Initial studies first characterized the biochemical composition of the porcine TMJ disc. It was determined that the majority of the porcine TMJ disc matrix is collagen, while glycosaminoglycans are present in small quantities. Once this biochemical standard was established, an appropriate scaffold composed of poly(glycolic) acid (PGA) non-woven meshes for TMJ disc tissue engineering was identified. Dynamic spinner flask seeding was determined to be the most effective method for seeding TMJ disc cells on PGA, based on an increased production of collagen. The addition of two growth factors, whether insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), or transforming growth factor-beta1 (TGF-beta1), at high concentrations of 100 ng/ml for IGF-I and bFGF and 30 ng/ml for TGF-beta1, improved the cellularity of constructs after six weeks in culture, but did not improve matrix production. Ascorbic acid concentration was found to be an important factor affecting attachment of passaged TMJ disc cells onto PGA. A concentration of 25 mug/ml of medium was observed to be more beneficial than no ascorbic acid and 50 mug/ml of medium. A high cell seeding density of 75 million cells per ml of construct produced twice more collagen than previous attempted seeding densities. In the last portion of this work, the effects of hydrostatic pressure were examined as part of an in vitro tissue engineering approach. A constant hydrostatic pressure regimen of 10 MPa for 4 hrs was shown to increase collagen expression in 2D and 3D, and increase collagen production. Surprisingly and perhaps counter-intuitively, an intermittent exposure of the hydrostatic pressure regimen at 1 Hz was observed to be detrimental to TMJ disc cells. The results of this work established general criteria, both in terms of biochemical and biomechanical factors, toward addressing the complex problem of regeneration of the TMJ disc.

Adenoviral (Ad) and adeno-associated viral (AAV) vectors have great promise as gene delivery vehicles for gene therapy and genetic immunization. However, these vectors can non-specifically target tissues and cell types in vivo. Redirected targeting of these vectors by the addition of cell-specific ligands would improve the therapeutic efficacy and safety of these vectors by reducing the effective dosage needed for gene therapy. Phage display technology has been exploited to discover novel cell-specific ligands for vector targeting. However, these ligands are selected in the context of phage and translation of the ligands back into the viral capsid can ablate viral assembly and function or inactivate the targeting function of the ligand itself. To circumvent this ligand-vector compatibility problem, a novel approach to identify cell-specific ligands is described. We have introduced structural "context" onto filamentous bacteriophage and generated random peptide libraries within these contexts for use in ligand selection. The HI loop of the adenoviral capsid was displayed on phage and a random peptide library was generated within this scaffold and used to identify cell-specific ligands against mouse skeletal muscle in vitro. A cell-specific peptide ligand, designated 12.51, was incorporated back into Ad capsid and the redirected Ad vector improved targeting in vitro, suggesting the viability of this approach for ligand discovery. This "context"-based approach was extended towards generating random peptide libraries within streptavidin protein for ligand selection. In addition, a system for conjugation of targeting ligands to the AAV capsid based on the streptavidin-biotin interaction, has been developed. A biotin acceptor peptide was engineered into the AAV capsid and resulted in the development of vectors that are metabolically biotinylated during production in cell lines. This avidin-biotin technology was previously utilized for construction of metabolically biotinylated Ad vectors. However, Ad vectors are extremely immunogenic compared to AAV and may not be suitable for in vivo applications. We constructed metabolically biotinylated AAV vectors and demonstrated proof-of-principle targeting in vitro using various biotinylated ligands. Eventually, streptavidin-context ligands can be conjugated to biotinylated vectors for targeted delivery.

Cardiovascular disease is a major cause of death in the U.S., and atherosclerosis, the principle cause of myocardial and cerebral infarctions, accounts for most of these deaths. In current clinical practice, cholesterol status, or atherosclerotic risk, is evaluated by fasting serum lipoprotein measurements. However, disease variation lies in the biologic responses of circulating blood cells and cells in the arterial wall, in the presence of given levels of plasma cholesterol. Lipid-laden monocytes or circulating macrophage 'foam cells' may be useful markers of atherosclerotic disease. Macrophage 'foam cells' can be identified by their massive accumulation of an atherogenic lipoprotein, oxidatively modified low-density lipoprotein (LDL). Oxidized LDL possesses unique fluorescence spectral characteristics that distinguish it from native (non-oxidized) LDL. Thus, fluorescence spectroscopy was chosen as a tool for identifying oxidized LDL accumulations in monocyte/macrophages, and may be useful for identifying a novel risk factor in the assessment of atherosclerosis. In cell suspension autofluorescence analysis, spectral characteristics of oxidized LDL were shown in cultured macrophages incubated with oxidized LDL preparations. Additionally, it was demonstrated that isolated peripheral blood monocytes may acquire spectroscopic signatures characteristic of oxidatively modified LDL. Identifying the percentage of oxidized LDL filled monocytes in a patient will be important in determining any correlation with atherosclerosis incidence or risk of atherosclerotic complications. Thus, more sophisticated analysis of the distribution of intracellular oxidized LDL content within a population of monocyte/macrophages required the development of instrumentation for spectroscopy at the microscopic level. A microspectrofluorometer was designed and constructed to detect intracellular oxidized LDL concentrations, based on autofluorescence signals, in individual cells smeared onto microscope slides. Good characterization of system losses improved the capability to quantify measurements in terms of absolute concentrations, a problem inherent in most fluorescence instruments. Using autofluorescence microspectrofluorometry, intracellular oxidized LDL concentrations could be identified within individual monocyte/macrophages incubated with oxidized LDL and calibrated with respect to oxidized LDL standards. Histologic comparisons using oil red O staining and transmission electron microscopy verified cultured macrophages were accumulating oxidized LDL. Increases in lipid droplets, cholesterol clefts, and secondary lysosomes correlated with increasing autofluorescence signals.