To create a more efficient and effective method of osteogenic drug delivery in vivo, drugs were modified with high calcium affinity moieties including pamidronate, poly(aspartic acid), and poly(glutamic acid). To test the initial hypothesis that modified drugs can demonstrate the same bone binding capabilities of pamidronate, poly(aspartic acid), and poly(glutamic acid), these motifs were conjugated to model peptides and exhibited high affinity to hydroxyapatite (HA). An in vitro controlled release experiment was conducted for native and modified TP508. Native and modified TP508 drugs were loaded in PLGA-PEG microparticles. Porous PPF scaffolds were injected with these drug-loaded particles, and in some instances with HA microparticles (20-50 or 50-100 mum). Less mineral surface resulted in less binding of drugs after release from the PLGA-PEG carriers and therefore a greater release than with the large HA particles. A final study was performed in the presence of 383 ng/mL collagenase, which cleaved the TP508 from the bone-binding domains at the point of the degradable peptide linker sequence. The dose effect of TP508 was established by delivering 0, 25, 50, and 100 mug TP508 loaded into PPF scaffolds and implanted in a sized rat cranial defect. After 4 weeks, microCT analysis of the skulls revealed a statistically significant increase in bone formation for the 50 mug dose compared to controls and the 25 mug dose. Based on these findings, an equivalent of 50 mug TP508 or modified drugs were delivered from PLGA-PEG microspheres in the presence of 20-50 mum HA microparticles in the PPF scaffolds' pore network, which revealed no significant differences between drug groups. These results were promising in that this strategy of drug modification had no apparent negative effect on the bioactivity of TP508. Another finding of this work was that the incorporation of HA into PPF composites resulted in significantly greater bone formation, even after subtraction of the initial amount of HA. The addition of this osteoconductive material stimulated an increase in new bone over 4 weeks for both the control and 50 mug TP508 groups.

The clinical need for bone graft substitutes has led tissue engineering strategies to investigate strategies to create osteoconductive, osteoinductive, and osteogenic constructs for bone repair and regeneration. The strategy in this research work is to use a cell-secreted extracellular matrix (ECM) to influence the osteoblastic differentiation of marrow stromal cells (MSCs). The effects of an in vitro generated ECM on MSC osteoblastic gene expression under static culture indicated that MSCs differentiated down the osteoblastic lineage evidenced by significant increases in expression of the osteoblastic markers, such as collagen type I and osteocalcin. When cultured on an ECM, osteoblastic differentiation was accelerated and enhanced through (1) earlier upregulation of osteopontin and osteocalcin; and (2) maintenance of a high level of expression of these and other osteoblast-specific genes. The upregulation in osteoblastic gene expression leading to significant increases in calcium deposition is likely mediated through the interactions between the growth factors and other matrix molecules found in the in vitro generated ECM since the expression of insulin-like growth factor, vascular endothelial growth factor, fibromodulin, and dentin matrix protein was found to display significant peaks in expression level. To further modulate MSC differentiation, the cellular constructs were cultured in the presence of fluid shear stresses. The effects of fluid shear stress and the bioactivity of the in vitro generated ECM acted synergistically to enhance mineralized matrix deposition. However, this synergy occurred only when the constructs were cultured in the presence of dexamethasone. The spatial distribution of cells and ECM was markedly improved compared to static culture. The osteoinductive and angiogenic potential of ECM constructs generated in a flow perfusion bioreactor for different peroids of time were tested in an ectopic site in a rodent model. No osteoinduction was observed although an increase in the number of blood vessels within the implants with increasing ECM was noted. This study emphasized the challenges in creating ideal conditions for de novo bone formation and underscored the need for optimization of this strategy for in vivo applications. These studies led to the transition from titanium to polymeric fiber scaffolds generated via electrospinning. The system was used to fabricate scaffolds with varying fiber diameters and pore sizes as well as multi-layered scaffolds. It was demonstrated that nanofibers could inhibit the spatial distribution of cells and ECM, despite culture in the bioreactor. These scaffolds hold tremendous potential as tissue engineering scaffolds and will be investigated further as part of the cell-secreted ECM-based scaffold strategy.

Differently loaded regions of the mitral valve contain distinct amounts and proportions of glycosaminoglycans (GAGs) and proteoglycans (PGs); these GAG/PG profiles are altered in abnormal loading conditions such as myxomatous degeneration. However, the role of mechanical stimulation on GAG and PG synthesis by valvular interstitial cells (VICs) is still unclear. This research analyzed first the PGs in differently loaded regions of mitral valve (leaflet and chordae) and then the effects of mechanical strains on GAG and PG synthesis by VICs using an in vitro 3-dimensional tissue-engineering model to develop a deeper understanding of valve mechanobiology. This original research investigated the specific PGs present in human mitral valves and found that the regions in compression (leaflets) are rich in versican and regions in tension (chordae) are rich in decorin and biglycan; these PGs were also detected in the engineered tissues seeded with VICs. Applying constraint increased the synthesis of decorin, biglycan and 4-sulfated GAGs. Constraint also increased versican secretion but reduced its retention within the engineered tissues. The application of constraint was found to be more influential than the directionality (biaxial vs. uniaxial) of strain. Constrained collagen gels containing leaflet cells retained more decorin and biglycan than did those containing chordal cells. The application of cyclic strains decreased the total GAG synthesis, increased the proportions of 4-sulfated GAGs, and reduced the proportions of hyaluronan. Synthesis of the PG versican was increased by leaflet cells and decreased by chordal cells in response to cyclic strain. Chordal cells were found to be more responsive to cyclic strains than leaflet cells, which has implications in the dramatic remodeling of myxomatous chordae tendineae. Synthesis of total GAGs, 4-sulfated GAGs and decorin was found to be strain dependent, whereas synthesis of versican and decorin was frequency dependent. In general, VICs within collagen gels synthesize GAG in proportions and amounts close to that of native valve tissue. This research is the first to show that strains can modulate GAG/PG synthesis by valve cells. These results provide insight into valve mechanobiology and pathology and have implications for understanding the remodeling process of many soft tissues.

Successful translation of experimental therapeutics to the clinical setting will require development of challenging in vivo models which mimic oral and craniofacial wound healing environments, and can accurately assess a construct's angiogenic and osteogenic performance. With the intent of developing an easily accessible and reproducible, non-healing alveolar bone defect in the rabbit, 10-mm diameter partial- and full thickness cylindrical defects were created in the premolar/molar region of the mandible. Microcomputed tomography (micro-CT) and histological analysis of the partial thickness defect demonstrated significant bone formation at 8 weeks, and complete union and contour regeneration at 16 weeks. In contrast, the full thickness defect was never able to bridge itself and only exhibited partial bone regeneration by 16 weeks, demonstrating the potential of the mandibular full thickness defect as a test bed for tissue engineering constructs. A subsequent study examined the use of contrast-enhanced micro-CT to characterize neovascularization in the rabbit alveolar bone defect model. Scaffold-implanted groups were found to have differences in vessel network morphology versus empty defects. These results suggest the rabbit alveolar bone defect model in conjunction with micro-CT imaging is a robust system for evaluating the angiogenic and osteogenic potential of tissue engineering constructs. Lastly, the scale-up to larger human applications will require rapid and adequate vascularization throughout implanted scaffolds, perhaps necessitating simultaneous delivery of angiogenic and osteogenic growth factors with specific release kinetics and dosages for effective tissue regeneration. A final study investigated the dose effect of simultaneous delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) for bone regeneration in a critical size rat cranial defect at 12 weeks. A dose-dependent decrease in percent bone formation was observed as BMP-2 alone decreased from 2 microg to 0.5-1 microg. The addition of VEGF in amounts of 6-12 microg was unable to reverse this decrease in bone formation, although improvements in bony bridging were seen in some of the dual release groups. Thus, further optimization of the growth factor doses and release kinetics may be required to observe long-term benefits over single growth factor release in this particular animal model.

Mammalian hearing exhibits exquisite sensitivity and frequency selectivity attributed to the unique properties of cochlear outer hair cells (OHCs). These sensory epithelial cells are electro-mechanical transducers, capable of converting sound-induced electrical signals into mechanical forces which provide feedback via a mechanism known as the cochlear amplifier. In a process aptly termed electromotility, electro-mechanical transduction manifests as whole-cell axial length changes in OHCs that occur in response to changes in the transmembrane potential. The polytopic motor protein prestin functions as the voltage sensor and molecular motor, both in OHCs and when expressed in heterologous systems. As the molecular mechanism(s) of electromotility remain unknown, examining the structure and function of prestin is a major focus of ongoing research. Since changes in membrane composition and biophysical properties affect protein function and organization, we are particularly interested in membrane-protein interactions. Recent studies suggest that manipulations in membrane cholesterol levels reversibly shift the membrane microdomain distribution of prestin, modulate prestin oligomerization states, and alter prestin function, thus regulating electromotility through membrane-protein interactions. Measurements of protein and lipid lateral mobility provide a powerful tool to dynamically examine such interactions. We hypothesize that OHC plasma membrane cholesterol levels affect electromotility either through microdomain-mediated mechanisms that cluster or segregate prestin molecules or via alterations in the material properties of the membrane, which in turn affect the resident proteins. Using fluorescence recovery after photobleaching (FRAP), we evaluated the lateral mobility of both protein and lipid components of the OHC. Then we showed that the diffusion of both prestin in HEK cells and lipids in OHCs is altered in response to changes in membrane cholesterol concentration. Cumulatively, this work demonstrates the complexity of prestin-membrane interactions and highlights the importance of their inclusion in current models of prestin function and electromotility.

Temporomandibular joint (TMJ) disorders arise from disease or trauma and may result in degeneration of the soft tissues. Tissue engineering may provide a solution to disorders of the TMJ without the side effects seen with artificial materials, such as improper incorporation with the surrounding tissues or immunological rejection of the artificial replacement. Several experiments were completed toward the goal of creating a functional TMJ disc replacement using a cell-based approach; the cell types that were primarily examined in this work were TMJ disc cells and costal chondrocytes. Attempts were made to improve the properties of scaffolds seeded with TMJ disc cells, and while proliferation was increased for the monolayer expansion phase of the approach, improvements were not seen in the properties of the three-dimensional constructs by adding L-proline to the culture. Due to the limited success of the TMJ disc cell constructs and the donor scarcity of this cell type, alternative cell sources were investigated in a scaffoldless tissue engineering method to improve the functionality and translatability of the engineered constructs. Chief among the cell types investigated, costal chondrocytes (CCs) consistently produced constructs with considerable amounts of extracellular matrix that were relevant to regenerating TMJ disc fibrocartilage. From this initial success, other aspects in using CCs for TMJ disc tissue engineering were investigated, specifically, passaging the CCs and adding exogenous stimuli. Examining passaged costal and articular chondrocytes showed that while the process of passaging and expanding chondrocytes caused an increase in collagen type I over type II, constructs made from passaged chondrocytes had higher collagen content and tensile properties than primary chondrocyte constructs. The observation that passaged cells were just as, if not more, capable of producing functional constructs also enhanced the translatability of this method by addressing the issue of donor tissue scarcity. Therefore, CCs at a variety of passages were examined in construct culture. Passaged CC constructs consistently produced more glycosaminoglycans per wet weight than primary cell constructs. Passaged CC constructs were then examined in the presence of exogenous stimuli to further improve their properties. At the regimens examined, hydrostatic pressure did not affect the constructs. In contrast, insulin-like growth factor-I improved construct properties over the no growth factor control. Overall, this thesis presents considerable support for the use of passaged costal chondrocytes for the purposes of improving functionality and clinical translatability of constructs for TMJ disc tissue engineering.

Almost 25 years ago, the unique electromotile activity of mammalian outer hair cells (OHCs) was discovered. This cylindrical neuro-epithelial cell is capable of transducing changes in transmembrane potential into whole-cell axial deformations. The resulting contractions and elongations feedback into the sound-induced vibrations of the basilar membrane, enhancing hearing sensitivity and frequency detection capabilities. Although the complete mechanism of electromotility is not presently understood, the discovery and localization of the putative motor protein, prestin, to the OHC plasma membrane promotes the importance of the membrane in both OHC and cochlear behavior. A thorough understanding of the constituents of the OHC membrane, both lipids and proteins, as well as their interactions, is then crucial to current and future analyses of OHC function. In this work, we describe the use of quantitative optical microscopy to investigate protein-protein and membrane-protein interactions. For the former, we employ fluorescence resonance energy transfer (FRET) microscopy to assess prestin-prestin interactions in a heterologous expression system. For the latter, we have developed a specific implementation of fluorescence polarization microscopy (FPM) to measure the orientation of the fluorescent lipid analog di-8-ANEPPS in OHCs. Using both of these techniques, we can investigate the effects of treatments known to affect prestin function and non-linear capacitance in a membrane-dependent manner. The results undeniably highlight the importance of the plasma membrane in regulating prestin and OHC electromotile function.

Respiratory patient motion often causes image blur and inaccurate quantitation results in positron emission tomography (PET) imaging. Traditional gated acquisition approach corrects motion artifacts by generating a snapshot sequence. Each snapshot, however, is of poor image quality due to insufficient photon counts. This dissertation addresses the problem of how to correct motion artifacts without sacrificing image quality or extending scan duration. To this end, a motion-incorporated model of the PET imaging system has been developed which enables utilization of data acquired during the entire scanning period in an image reconstruction process, thereby improving photon statistics while eliminating motion artifacts. Three related approaches within this framework are presented. In the first approach, motion information is first derived from a corresponding four-dimensional computed tomography image sequence, and then incorporated into the PET system model to get a motion-free image. The second approach develops a region of interest extension to the motion-incorporated system model to improve efficiency and accuracy of motion estimation. Finally, the motion-incorporated system model is integrated with an anatomy based image reconstruction algorithm, thereby enabling utilization of mismatched anatomical information during the image reconstruction process to improve image quality and quantitation performance.

This research introduces a delivery system for the release of an angiogenic and an osteogenic growth factor to address the inability of large bone defects to sufficiently heal. These critical size defects (CSDs) affect approximately one million patients annually. Current treatments rely on the use of bone grafts or permanent orthopedic implants; alternatively, tissue engineering strategies utilize a combination of biomaterials for the delivery of osteoprogenitor cells and osteogenic growth factors. However, few studies address the importance of angiogenesis for bone regeneration, particularly in CSDs which require the presence of an underlying vasculature to recruit and support osteoprogenitor cells within the large defect. We hypothesized that controlled delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), and an osteogenic growth factor, bone morphogenetic protein-2 (BMP-2), would demonstrate a compounded effect to induce the closure of CSDs. Both growth factors were delivered from gelatin microparticles incorporated within a porous polymer scaffold. In vitro studies showed that VEGF and BMP-2 release kinetics were affected by the extent of gelatin crosslinking, but growth factor dose was found to affect release only minimally for the doses investigated. Additionally, gelatin type was also found to affect the release profiles of BMP-2. Early delivery of VEGF and minimal burst release and sustained delivery of BMP-2 were attained in vitro and in vivo by using acidic and basic gelatin, and low and high gelatin crosslinking, respectively. A critical size rat cranial defect was also used to evaluate the regenerative potential of this dual delivery system in vivo. At 4 weeks, there was no difference in blood vessel formation between groups, but dual release groups exhibited significantly higher bone formation at 4 weeks along with increased bony bridging at 12 weeks. Bone formation at 12 weeks in the dual release and BMP-2-only groups were significantly higher than in VEGF-only groups or blank scaffolds. These results suggest a synergistic effect for dual release at early time periods and indicate faster healing times at later periods. The studies presented here demonstrate the potential of this unique dual release system for use in strategies for bone regeneration.

Tissue engineered scaffolds are often considered "black boxes." Post implantation, they are solely expected to provide temporary mechanical support and foster tissue ingrowth while de novo tissue forms around its matrix. This is rarely the case however, as the post implantation interaction between this foreign body and the host biological system is largely uncontrolled. A growing body of concrete results is overwriting previous holistic knowledge to provide firm and hierarchical guidelines for successful scaffold design. Two areas have recently demonstrated fertile ground for progress: (1) the mechanical strength of architecture and (2) the fluid flow properties of that architecture, both of which act on different void phases. Mechanical properties are controlled by the solid phase of the matrix, while the void space determines fluid flow characteristics. The objective of this dissertation was to demonstrate the benefits of an analysis of the structural properties of tissue engineered scaffolds combined with the specific design potentials of computer-aided tissue engineering (CATE) for orthopaedic applications. Two overarching goals directed this research. The first was focused on antipodal properties and addressed solutions which included an interplay between opposing poles while matching biological properties and secondly, to apply that knowledge towards the design of patient specific implants. Two antipodal properties were studied; (1) modification of the solid phase was addressed with respect to structural mechanical properties and (2) modification of the void phase was studied to determine fluid flow characteristics of porous architectures. These concepts were then applied in real applications using CATE towards the goal of tissue engineered scaffolds for bone repair and drug regimen.