Understanding the contribution of individual mechanical stimuli to cardiovascular pathogenesis is critically important for understanding and treating cardiovascular disease, and microfluidic bioreactors are a useful tool for these studies. Cardiovascular bioreactors are uniquely complex because they require the simultaneous application of fluid shear stress and dynamic strain. One of the key shortcomings of current mechanotransduction bioreactors that we wanted to address in this thesis was the effect of dynamic strain on the fluid shear conditions in a bioreactor system. In this thesis, we designed, developed and validated a microfluidic bioreactor system which can accurately recapitulate the major hemodynamic mechanical parameters of shear stress and strain. We characterized the system using computational modeling and validated computational models by developing novel three dimensional particle velocimetry (PIV) techniques. Computational modeling of the microfluidic bioreactor during dynamic strain demonstrated that shear stress experience by cells in a bioreactor is altered by applying dynamic strain. The PIV methods developed allowed us to visualize the three dimensional fluid velocity profile in the bioreactor during dynamic strain and allowed us to measure and validate the computational models of bioreactor dynamics. In addition, we applied this system to investigate the effects of mechanical stimuli on omental mesothelium.

Purpose: This study evaluates a new magnetic resonance imaging method for detecting calcium deposits, using their characteristic susceptibility effects, in practical conditions to provide insight into its clinical value for detecting breast microcalcifications at high field (7T). Methods: Signatures of calcium deposits in phase images were detected via cross-correlation between the images and a library of templates containing simulated phase signatures of deposits. The influences of deposit position, signal-to-noise ratio, spatial resolution, high-pass filtering, and fat suppression on the method were determined and used to optimize the method for detecting simulated microcalcifications inserted in silico into breast MRI of healthy controls. Results: In images acquired with a clinical scanner and acquisition times below 12 minutes, simulated microcalcifications with sizes of 0.8 1.0 mm were detected in images with voxel sizes of (0.4 mm)3 and (0.6 mm)3 with sensitivity and specificity of 75-87% and 54-87%, respectively; smaller microcalcifications with sizes of 0.6 0.7 mm were detected better in images with voxel size of (0.4 mm)3, with sensitivity and specificity of 87% and 54%, respectively, than in images with voxel size of (0.6 mm)3, with sensitivity and specificity of 56-78% and 44-47%, respectively. Conclusions: The new method is promising for detecting large microcalcifications (approximately 0.8 1.0 mm in longest dimension) within the breast at 7T. Detection of smaller deposits may be possible in images with higher spatial resolution; unfortunately, these images take too long to acquire using current MR methods and therefore are clinically impractical. Although mammography can detect smaller microcalcifications with sensitivity between 74-95%, and specificity between 89-99%, this alternative MRI method does not expose breasts to ionizing radiation, is not affected by breast density, and can be combined with other quantitative MRI exams to increase the diagnostic specificity of breast MRI.

Articular cartilage has a poor intrinsic healing response, so tissue engineering provides a promising approach for cartilage regeneration. The major objective of this proposal was to enhance the self-assembling process, used in articular cartilage tissue engineering, by investigating the effects of construct confinement, hydrostatic pressure application, and growth factor addition. First, the effects of construct confinement in different directions and at different times were investigated. It was demonstrated that construct confinement resulted in enhanced biomechanical properties in the direction orthogonal to the confinement surface, either by enhancing collagen organization or by increasing collagen production. Next, the effects of hydrostatic pressure at different timepoints, magnitudes, and frequencies on the biomechanical and biochemical properties of self-assembled constructs were determined. It was demonstrated that the application of static hydrostatic pressure, at 10 MPa, for 1 h/day, from days 10-14 days led to significant increases in compressive and tensile properties, accompanied by significant increases in GAG and collagen content, respectively. To our knowledge, this was the first study to demonstrate increases in the biomechanical properties of tissue from pure HP application. Furthermore, the effects of exogenous application of growth factors, at varying concentrations, dosages, and combinations, with and without hydrostatic pressure, were assessed on the biochemical and biomechanical properties of engineered constructs. A systematic approach was used to determine the effects of BMP-2, IGF-I, and TGF-beta1, alone and in combination, on the functional properties of engineered constructs. This was the first study to demonstrate significant increases in both compressive and tensile biomechanical properties as a result of growth factor treatment. Also, for the first time, synergistic and additive effects on construct biomechanical and biochemical properties were found when combining growth factor treatment with hydrostatic pressure application. Finally, the effects of various decellularization treatments were examined, and it was determined that it was possible to remove cells while maintaining construct functional properties. The results presented in this thesis are exciting, as they have allowed for a better understanding of the self-assembling process, and have allowed the self-assembled constructs to mature into functional articular cartilage, as evidenced by biomechanical and biochemical properties spanning native tissue values.

Optical imaging has the potential to improve early detection of oral cancer. Reflectance and fluorescence based optical devices have demonstrated improved sensitivity and specificity compared to conventional visual oral examination. Although these devices are increasingly used as clinical tools in developed countries, they are a less practical solution in low-resource settings as the cost of these devices is relatively high, their portability is limited, and results from them are often subjective. This dissertation focuses on development of optical imaging platforms that specifically addresses these challenges and can be used to aid in screening and detection of oral pre-cancers. The first part of this dissertation describes the construction and evaluation of a macroscopic imaging system with multi-modal imaging capability. This system can be used to screen the surface area of oral tissue at risk to detect abnormal sites. It is low-cost, portable and battery powered, which is ideal for screening and detection of oral cancer in high-risk populations in low-resource and remote settings. The system was evaluated in a clinical study in India and results from the study were used to develop a computational algorithm for objective interpretation of images from the system. In addition, this system was used to characterize optical properties of pathological conditions that are population specific. Results from this trial system are promising and show that normal oral sites can be differentiated from high-risk and cancerous sites with high sensitivity and specificity. Although results show that sites with low-risk can be differentiated from normal tissue using this system, the sample size of the low-risk measurements is relatively small and they can not be differentiated from high-risk and cancerous tissue. The second part of this dissertation involves developing a low-cost and simple microscopic system that is capable of high-resolution imaging for early detection of cancer. It was demonstrated that the 'optical-sectioning' concept of structured illumination can be integrated with optically active exogenous contrast agents for high-resolution molecular-specific imaging of pre-cancer. Finally, this dissertation also incorporates evaluation of a multi-modal miniature microscope (4M) device developed based on structured illumination. Results from the system show that the device is capable of high-resolution imaging and can be used with molecular-specific contrast agents for detection of cancer and its precursors.

Despite the accessibility of the oral cavity to inspection, patients with oral cancer most often present at a late stage, leading to high morbidity and mortality. Multispectral widefield optical imaging has emerged as a promising technology to aid clinicians in screening and resection of oral neoplasia, but current approaches rely on subjective interpretation. This work focuses on the design, construction, and clinical testing of a novel multispectral widefield optical imaging device for objective screening and delineation of oral neoplasia. The Multispectral Digital Microscope (MDM) acquires in vivo images of oral tissue in autofluorescence, narrow band reflectance, and orthogonal polarized reflectance modes that the diagnostic value of each modality may be qualitatively and quantitatively evaluated alone and in combination. Using in vivo imaging data collected from 56 patients and 11 normal volunteers, combined with computer aided diagnostics, a sensitivity of 100% and a specificity of 91.4% was achieved for discriminating oral dysplasia and cancer from normal tissue in an independent validation set. A single feature calculated from the autofluorescence images at 405 nm excitation was used to achieve this performance. Disease probability maps were constructed using this feature to help identify areas with a high probability of abnormality. Autofluorescence imaging at 405 nm excitation also provided the greatest image contrast which was significantly higher than that using standard white-light illumination. Features extracted from other imaging types did not appear to aid in diagnosis. Ex vivo image data from the MDM was combined with image data from a high-resolution microendoscope (HRME) in order to determine if a synergistic relationship existed between these devices. The ability to objectively diagnose oral lesions substantially increased when using both devices in combination compared to using either alone. This combination of devices provides a practical means of screening the entire mucosal surface for suspicious regions, using the MDM, and then using the HRME for confirmation of diagnosis. This work has demonstrated that widefield autofluorescence imaging at 405 nm excitation can be highly effective for the objective discrimination of oral lesions.

Dysplasia and cancer of epithelial tissues, including the oral cavity and esophagus, typically have much higher survival rates if diagnosed at an early stage. Unfortunately, the clinical appearance of lesions in these tissues can be highly variable. To achieve a definitive diagnosis of a suspected lesion at these sites, an excisional biopsy must be examined at high-resolution. These procedures can be costly and timeconsuming, and in the case of Barrett's esophagus, surveillance biopsy strategies may not be entirely effective. Optical imaging modalities have the potential to yield qualitative and quantitative high-resolution data at low cost, enabling clinicians to improve early detection rate. This dissertation presents a low-cost high-resolution microendoscopy system based on a fiber optic bundle image guide. In combination with a topical fluorescent dye, the fiber bundle can be placed into contact with the tissue to be observed. A high-resolution image is then projected onto a CCD camera and stored on a PC. A pilot study was performed on both resected esophageal tissue containing intestinal metaplasia (a condition known as Barrett's esophagus, which can transform to esophageal adenocarcinoma) and resected oral tissue following surgical removal of cancer. Qualitative image analysis demonstrated similar features were visible in both microendoscope images and standard histology images, and quantitative image processing and analysis yielded an objective classification algorithm. The classification algorithm was developed to discriminate between neoplastic and non-neoplastic imaging sites. The performance of this algorithm was monitored by comparing the predicted results to the pathology diagnosis at each measurement site. In the oral cancer pilot study, the classifier achieved 85% sensitivity and 78% specificity with 141 independent measurement sites. In the Barrett's metaplasia pilot study, 87% sensitivity and 85% specificity were achieved with 128 independent measurement sites. The work presented in this dissertation outlines the design, testing, and initial validation of the high-resolution microendoscope system. This microendoscope system has demonstrated potential utility over a wide range of modalities, including small animal imaging, molecular-specific imaging, ex vivo and ultimately in vivo imaging.

This thesis describes the development of photothermal nanoshell therapy activated by proteases. Hirsch et al. previously showed that the optical spectrum of crosslinked nanoshells within the UV-visible range reveals a broader, red-shifted, and lower peak absorbance as compared to disperse nanoshells. As described in this thesis, studies showed this decrease in absorbance corresponds with a lower temperature change upon laser irradiation of the nanoshells. Near infrared (NIR)-absorbing silica-gold nanoshells were crosslinked with a proteolytically degradable linker, resulting in a broadening, red-shifting, and decrease of the peak absorbance. After collagenase was added to the crosslinked nanoshells, the peak absorbance increased, suggesting degradation of the linker and subsequent dispersion of the nanoshells. The results described here suggest that this may be applied to protease-activation in vivo within the tumor in an effort to increase tumor specificity and to protect surrounding normal tissue.

This body of research explored mitral valve remodeling due to changes in the mechanical and chemical environments of the tissue in order to better predict patient response to medical device and drug therapies. This work was novel both in its characterization of the mitral valve and in the consideration of the impacts of the biological environment on the multilayered valve structure. Left ventricular dilation due to congestive heart failure (CHF) changes the mechanical forces experienced by the mitral valve. Collagen, cellular, glycosaminoglycan, and proteoglycan compositions of valve tissues were therefore compared between CHF patients treated with and without the left ventricular assist device (LVAD). Mechanical properties of these tissues were also tested to assess effects of matrix changes on valve function. Since mechanical stimulation was determined to be required to maintain valve structure and function, a novel splashing bioreactor was then designed to maintain the valve mechanical environment in vitro in preparation for studies altering the chemical environment of the tissue. Alter validation, this bioreactor was used to study initial changes to valve structure alter two weeks of organ culture with media containing the serotonin receptor agonists serotonin and norfenfluramine (fen-phen) as well as a receptor antagonist. In the mechanical stimulation study, CHF valve tissue exhibited fibrotic remodeling compared to normal tissue, impeding normal mitral valve function. LVAD treatment, while it tended to restore functionality of the mitral valve chordae, encouraged compensatory remodeling that did not improve leaflet structure or mechanical behavior. Conversely, the gentle stretching provided by bioreactor was deemed sufficient to maintain matrix composition after two weeks of culture. Serotonin and norfenfluramine exposure appeared to upregulate different proteoglycans within the mitral valve structure, and exposure to a serotonin receptor antagonist had varying levels in success in blocking these drug effects. The results from these studies showed that changes to the mechanical (CHF and LVAD) and chemical (serotonin and norfenfluramine) environments altered mitral valve tissue structure and function. These effects of device use and drug therapies should be considered when assessing treatment for pathologies, such as CHF and obesity, which may not appear to directly impact the mitral valve.

Injuries to avascular regions of menisci do not heal and result in significant discomfort to patients. Current treatments, such as partial meniscectomy, alleviate the symptoms, but lead to premature osteoarthritis due to reduced stability and changes in knee biomechanics. An alternative treatment to overcome these problems involves functional tissue engineering. This thesis examined several exogenous factors to enhance the capability of meniscus cells (MCs) to synthesize relevant ECM markers and improve the functionality of constructs in vitro. First, the effect of passage on the phenotype of MCs in monolayer was investigated, and rapid changes were observed in collagen I, collagen II, and COMP expression. Collagen I and aggrecan protein coatings assisted in reversing expression levels of certain ECM markers; however, collagen II expression could not be reversed. Next, 3D tissue engineering studies were conducted using a cell-scaffold approach with MCs seeded on PLLA meshes. Anabolic stimuli that aided in meniscus regeneration included (1) hypoxia and bFGF, which resulted in synergistic increases in the total glycosaminoglycan content and compressive properties of constructs; (2) 10 MPa static hydrostatic pressure (HP), which resulted in increases in collagen content and the relaxation modulus of constructs; and (3) 10 MPa static HP and TGF-beta1, which resulted in additive increases in collagen content, and synergistic increases in the compressive moduli of constructs. Finally, a self-assembly, scaffoldless approach was employed for meniscus regeneration using co-cultures of MCs and articular chondrocytes (ACs). A high density of cells were seeded on non-adherent agarose molds and allowed to coalesce into a construct without a scaffold. Different co-culture ratios of MCs and ACs resulted in a spectrum of fibrocartilages that recapitulated some biochemical and biomechanical properties of the rabbit meniscus. Cell culturing conditions were optimized with the identification of a smooth 1% agarose mold that resulted in geometrically-mimetic meniscus constructs. In conclusion, this thesis quantified phenotypic changes in MCs over passage, and used scaffold-based and scaffoldless approaches to regenerate constructs with biochemical and biomechanical properties in the range of native tissue values. Successful replacement of a damaged meniscus will improve the quality of patient life and reduce the risk of osteoarthritis.

Damage to or loss of craniofacial tissues, often resulting from neoplasm, trauma, or congenital defects, can have devastating physical and psychosocial effects. The presence of many specialized tissue types integrated within a relatively small volume leads to difficulty in achieving complete functional and aesthetic repair. Tissue engineering offers a promising alternative to conventional therapies by potentially enabling the regeneration of normal native tissues. Initially, a stimulus responsive biomaterial designed for injectable cell delivery applications was investigated with the goal of providing a substrate for osteogenic differentiation of delivered cells. In order to enable faster clinical translation, later efforts focused on novel combinations of regulated materials. Most common approaches using cell delivery for bone tissue engineering involve the harvest and ex vivo expansion of progenitor cell populations over multiple weeks and cell passages. The effect of aging and passage on proliferation and differentiation were analyzed using murine mesenchymal stem cells as a model. These cells lose their ability to proliferate and differentiate with increases in donor age and passages during cell culture. Delivery of uncultured bone marrow mononuclear cells was then investigated, and it was determined that when delivered to porous scaffolds these cells, which can be harvested, isolated, and returned to the body within the setting of a single operation, significantly increased bone regeneration in vivo. Finally, because these techniques of scaffold implantation and cell delivery would likely fail if delivered to an exposed or infected wound, a method of space maintenance was investigated. Space maintainers made of poly(methyl methacrylate) and having tunable porosity and pore interconnectivity were evaluated within a clean/contaminated mandibular defect. Low porosity space maintainers were found to prevent soft tissue collapse or contracture into the bony defect and allowed surrounding soft tissues to penetrate the pores of the implant, enabling healing over 12 weeks. The tissue response and wound healing characteristics of these implant was favorable when compared to solid or high porosity implants. Although optimization and further investigation of these techniques is necessary, in combination these approaches demonstrate one possible and translatable approach towards craniofacial tissue regeneration.