Dental caries and traumatic impact are two major causes of destruction of dental soft and mineralized tissues that affect a large segment of the population and pose major public health concerns. Conventional treatment strategies rely on mere replacement with bioinert filling materials. Hence, a critical need exists for biology-based therapeutic approaches to restore damaged dental tissues to their original form and function. Recent developments in tissue engineering, material sciences and stem cell research offer considerable potential to impact dental therapies. A customized scaffolding system laden with bioactive factors could deliver dental stem cells to the site of injury. An applicable scaffold should be biocompatible and biodegradable, accommodate cells, incorporate growth and differentiation factors, and allow for injection into small defects. Synthetic peptide hydrogels are particularly interesting in all these aspects. Our pilot study demonstrated their compatibility with two dental stem cell lines. In Specific Aim 1, peptide sequences were developed to further optimize the system for cell proliferation and spreading. In Specific Aim 2, the gels were modified to incorporate bioactive molecules and growth factors for cell differentiation and vasculogenesis. Release profiles were established, and cell culture studies demonstrated the induction of cellular differentiation. For Specific Aim 3, the generated material was utilized in an animal model, where constructs of cell- and growth-factor-laden gels in standardized dentin cylinders were transplanted into immunocompromised mice. Soft connective tissue formation and new blood vessel formation could be observed, along with localized collagen deposits, indicating beginning dentin formation. In summary, the objective of this research was to modify and optimize peptide-based hydrogels in order to develop a novel tissue engineering approach for the regeneration of dental tissues.

Disorders of the temporomandibular joint (TMJ) are widespread, afflicting millions of people. The majority of these cases involve displacement or injury to the TMJ disc. Current treatments do not fully address severe cases of TMJ dysfunction; therefore, efforts to engineer functional tissues for repair or replacement are warranted. While previous studies have laid the groundwork for these efforts, significant challenges remain, including (1) identification of appropriate animal models, (2) development of methodologies for in vitro TMJ tissue engineering, and (3) refinement of tissue culture procedures for clinically relevant cells sources. This thesis contributes to overcoming these challenges by (1) exploring topographical and interspecies variation in functional properties of the TMJ disc, (2) developing an in vitro tissue engineering strategy capable of recapitulating native tissue characteristics, and (3) enhancing protocols for chondrogenesis of dermis-derived cells. The first aim of this thesis characterized the biomechanical and biochemical properties of human TMJ disc in relation to several animal models. Significant regional and interspecies variations were indentified, though certain characteristics were observed across all species. While the human disc displayed properties distinct from the other species, the pig was the most similar and was therefore identified as the most appropriate animal model. The second aim applied these findings as design criteria in the development of an in vitro tissue engineering strategy. Scaffoldless constructs derived from co-cultures of chondrocytes and fibrochondrocytes were enhanced through optimization of growth factor and serum supplementation, such that they recapitulated many characteristics of native TMJ cartilage. Finally, the third aim refined the differentiation process for chondroinduction of dermis-derived cells. Using an optimized, low-cost surface coating, chondrogenesis was significantly enhanced through incorporation of hypoxia during culture. These experiments address several aspects of fibrocartilage tissue engineering and represent a significant step towards in vivo application of these technologies.

This work investigated an injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) as a cell and growth factor carrier for osteochondral tissue engineering applications. An in vitro study first investigated chondrogenic differentiation of rabbit marrow mesenchymal stem cells (MSCs) encapsulated in single-layer hydrogel composites of different swelling ratios with or without transforming growth factor-beta1 (TGF-beta1). The results showed that hydrogel composites containing TGF-beta1-loaded MPs and of higher swelling ratios supported MSC chondrogenic differentiation. When implanted in a rabbit osteochondral defect, these hydrogel composites containing MSCs facilitated subchondral bone formation in the presence of TGF-beta1. However, the delivery of MSCs either with or without TGF-beta1 did not improve cartilage morphology. Accordingly, a bilayered OPF/MP hydrogel composite consisting of a chondrogenic layer and an osteogenic layer was fabricated. In vitro culture of the construct demonstrated that MSCs encapsulated in the chondrogenic layer differentiated into chondrocyte-like cells in the presence of TGF-beta1-loaded MPs. In the osteogenic layer, osteogenically precultured MSCs maintained their osteoblastic phenotype, and synergistically enhanced chondrogenic differentiation of the MSCs in the chondrogenic layer with TGF-beta1. In a following study investigating similar hydrogel composites, TGF-beta3-loaded MPs in the chondrogenic layer showed a more effectively stimulatory effect on MSC chondrogenic differentiation than TGF-beta1-loaded MPs. Furthermore, encapsulated cells of different degrees of osteogenic differentiation in the osteogenic layer were found to significantly influence the chondrogenic gene expression of co-cultured MSCs in both the presence and absence of TGF-beta3. Overall, this study demonstrated the fabrication of hydrogel composites that mimic the structure and function of osteochondral tissue, along with the application of these composites as cell and growth factor carriers for osteochondral tissue engineering.

Injuries to the inner-portion of the meniscus, common with today's active lifestyles, have little ability for intrinsic repair due to the lack of vascularity. Current treatments only alleviate the symptoms of meniscal damage and do nothing to prevent the eventual osteoarthritic changes to the articular surfaces of the knee joint. To prevent these changes by restoring the structure and functionality of the meniscus, the generation of biochemically and biomechanically robust tissue engineered constructs for tissue replacement is desirable. This thesis investigated methods to engineer and enhance a self-assembled meniscal replacement using both a leporine and bovine cell source. First, the leporine cell source was considered as it represents the potential for future small animal, allogenic, in vivo studies. The use of a chondrogenically-tuned expansion procedure, involving a chemically defined medium and high density monolayer culture, was employed to expand leporine articular chondrocytes (ACs). Not only did this protocol outperform traditional expansion in terms of promotion of a cartilaginous phenotype, but constructs formed with expanded ACs had higher GAG/WW and collagen 2/collagen 1 than constructs formed with primary ACs. To further enhance cartilaginous quality and potential clinical translatability, the effects of passage number, cryopreservation, and redifferentiation culture prior to self-assembly were studied for both leporine ACs and meniscus cells (MCs). This study found that by increasing the passage number to obtain more cells from the same amount of starting material, the biochemical and biomechanical properties of constructs were not detrimentally affected. Cryopreservation and aggregate pre-culture redifferentiation were found to enhance biomechanical properties of AC and MC self-assembled constructs. The remaining tissue engineering studies in this thesis employed immature bovine ACs and MCs because these cells have been successfully applied in the self-assembly process to create constructs of complex shapes. In addition, a study was performed to assess the immunogenicity of xenogenic, bovine and allogenic, leporine ACs and MCs when co-cultured with leporine peripheral blood mononuclear cells (PBMCs). The mixed lymphocyte reaction assay showed that an immune response was not elicited by either bovine or leporine cells. This result suggests that the use of bovine cells for leporine meniscal replacement may be a feasible option. Studies assessing chemical and mechanical stimulation of anatomically-shaped meniscus constructs formed from bovine ACs and MCs followed. First, effects of temporally coordinated chemical stimuli, chondroitinase ABC (C-ABC) and transforming growth factor p1 (TGF-beta1), were studied on anatomically-shaped meniscal constructs. A stimulation regimen, consisting of TGF-beta1 applied continuously and C-ABC applied after 1 wk of culture, was found to synergistically enhance the radial tensile modulus and compressive relaxation modulus; in addition, this regimen additively increased the compressive instantaneous modulus and collagen/WW. Next, the effects of combining the previously determined chemical stimulation regimen with physiologic mechanical stimulation were studied. The shape of the construct and compression stimulator allowed for application of simultaneous compression and tension stimulation which mimicked the types of forces experienced by native menisci. This study found that the application of mechanical stimulation from days 10-15 resulted in significant enhancement of all measured biochemical and biomechanical properties. Further, combined chemical and mechanical stimulation resulted in additive increases to collagen/WW and all biomechanical properties. Finally, the effects of self-assembly well topography and compliance were studied. This study indicated that a smooth topography and higher compliance resulted in constructs possessing higher GAG/WW, collagen/WW, and tensile modulus. In conclusion, this thesis identified (1) expansion, cryopreservation, and pre-self-assembly redifferentiation as factors able to enhance the cartilage-forming capability of leporine ACs and MCs, (2) determined that the use of bovine ACs and MCs in leporine meniscal engineering could be feasible due to lack of immunogenicity, and (3) discovered chemical and mechanical stimulation treatments that were able to enhance the functional properties of bovine AC and MC meniscus constructs to values in the range of native tissue. In the future, the translation of these techniques to clinical usage could reduce the risk of osteoarthritis following meniscus injuries by providing functional replacement tissue.

Optical techniques targeting biomarkers of cancer have the potential to aid in the early detection of this disease. Molecular and biochemical changes in neoplastic tissue could be interrogated, potentially providing an objective assessment and identification of suspect tissue. A method of interrogating such changes involves the development of optical based probes that provide a molecular-specific source of signal. This dissertation aims to develop an effective optical strategy to target the epidermal growth factor for in vivo detection. The optical probe design consists of an optically active component (quantum dot, fluorescent dye) and a molecular specific probe (antibody, peptide). The optically active components investigated were cadmium telluride quantum dots (QDs) and Alexa Fluor 647 dye. Epidermal growth factor receptor (EGFR) was chosen as a molecular target because it is over-expressed in a large number of epithelial cancers. Both peptides and antibody to EGFr were investigated as molecular specific probes. The work presented in this dissertation provides a systematic approach for developing optically active probes targeting EGFR. The ability to synthesize, passivate and functionalize red-emitting QDs was investigated. It was demonstrated that in order for QDs to be properly passivated for biological imaging applications, a crosslinked amphiphilic-based polymer should be used. Such polymers afforded the best surface protection of all the coating strategies tested. Unfortunately, such systems are generally very large (>35nm) and therefore might be hindered in in vivo delivery. Due to the large size of QD-based optical probes, further research in this dissertation utilized the commercially available dye, Alexa Fluor 647. Alexa 647 was tethered individually to three different proteins: anti-EGFR antibody; mouse epidermal growth factor peptide (mEGF); and human epidermal growth factor peptide (hEGF). The specificity of each Alexa 647-protein conjugate was verified in vitro. Each protein-dye conjugate was further investigated to determine the specificity and efficiency of intravenous delivery to EGFR positive and EGFR negative tumors. These investigations revealed the importance of both the mean fluorescent signal (normalized for dye:protein ratio) as well as distinctive, recognizable patterns of contrast agents for in vivo applications. Such criteria yield a contrast agent with the best case scenario for being detected and recognized. The use of a systematic approach to design and test contrast agents for in vivo applications was demonstrated through this work. This work stressed a comprehensive investigation including in vitro, ex vivo and in vivo testing. Additionally, it was shown that care must be taken in designing in vitro experiments to interpret in vivo results as the two do not necessarily correlate. This dissertation lays the groundwork for the development of contrast agents targeting specific molecular markers of cancer.

The objective of this dissertation is to investigate the accuracy of point and surface based image space to physical space registration performed using a spatially tracked A-mode ultrasound transducer to localize features and to determine the applicability of these techniques for use in interactive, image-guided surgery. The accuracy of subcutaneous marker localization has been demonstrated using a phantom. An spatially tracked A-mode ultrasonic localization system was constructed. The system was used to examine the accuracy of transcutaneous localization of bone implanted fiducial marker analogs. The relationship between the number of signals used to localize the fiducial markers and localization accuracy was determined. Validation was performed by comparison to an optical system. The accuracy of surface registrations based on matching the outer surface of the skull as identified by ultrasound and in CT images has been estimated in a phantom. The ultrasonic localization system was modified for use in localizing the outer surface of the skull. The effect of changes in the image model parameters and image slice thickness on registration error were examined. The effect of variations in the speed of sound was also examined. The surface registration results were validated by comparison to a fiducial marker registration. A preliminary study on the accuracy of surface registrations in twelve human patients has been performed. The ultrasonic localization system was enhanced to synchronize the acquisition of ultrasonic and optical information. Patient motion in the CT images was compensated for using the Nbar system of the CRW stereotactic frame. The surface registration results were evaluated for three different speed of sound values corresponding to the speed of sound in the tissue components of human scalp. The correlation between the number of ultrasonic points used in the surface registration algorithm and the surface registration error was evaluated. Fiducial markers attached to the CRW stereotactic frame were used to validate the surface registration results. These experiments have demonstrated a spatially tracked A-mode ultrasound transducer capable of localizing both point and surface features that can be used in registration processes for interactive, image-guided procedures.

<p> Sudden cardiac death is the primary cause of mortality in the industrialized world. Ventricular tachycardia and lethal arrhythmias such as ventricular fibrillation are believed to be the result of reentrant electrical activity, i.e., self-sustained electrical activity which continues to re-excite regions of cardiac tissue independently of the natural pacemaker rhythm. The mechanisms behind fibrillation initiation, maintenance, and termination by a defibrillatory shock are largely unknown. </p> <p> Cardiac fibrillation is characterized by a complex spatial interaction of non-stationary spiral waves; however, the nature of this interaction is an ongoing topic of investigation. The organizing center of reentry is a topological defect called a phase singularity in two dimensions, a filament in three dimensions; an understanding of fibrillation behavior may be obtained by localizing and tracking these defects. Experimentally, the electrodynamic behavior is typically investigated via optical mapping using voltage-sensitive fluorescent dyes. </p> <p> In this thesis, a technique to detect phase singularities based upon topological charge was applied to nonlinear time-series and phase portrait analysis of optical signals, and later extended to filament detection; this procedure was shown to be both efficient and mathematically robust. An alternate method to reconstruct the phase portrait was also explored and shown to overcome some of the limitations of the time-series method as well as permitting singularity detection closer to initiation than previously allowed. Numerically examining the interaction dynamics of a simple filament configuration paralleling that seen in experimental preparations indicated that a critical bifurcation in filament life-time exists between attractive and repulsive behavior along with annihilation by mutual collision and collapse by shrinkage; which could be represented by a difference of Yukawa potentials by treating the filaments as a pair of point charges. The inclusion of optical depth effects into a numerical model of three-dimensional filament activity was studied, and suggested that these effects have a significant impact on observed epicardial activity. Finally, a three-dimensional geometric reconstruction of an isolated, perfused heart with the fluorescence information as a texture map, previously developed as a proof-of-concept, was shown to be a viable tool for whole-heart singularity visualization.</p>

The objective of this research was to design and develop a system capable of displaying cortical brain function during image-guided neurosurgery. Brain function was determined using a cortical stimulator, classified according to function type, and displayed along with pre-operative tomographic and rendered images of the brain. In addition to displaying brain function acquired from the patient undergoing surgery, a probabilistic map of functional information acquired from a database or previous patients may also be displayed. This information is stored in an atlas coordinate system and can be mapped to the patient's coordinate system for display during surgery. The entire system was tested and evaluated during three human neurosurgery procedures. Functional information corresponding to speech, motor, and sensory regions was identified and displayed during surgery. This data was then mapped to a common reference database using a non-linear registration algorithm to evaluate the feasibility of using this system to create a functional atlas of the human brain.

Raman Spectroscopy has been shown to have the potential for providing differential diagnosis in the cervix with high sensitivity and specificity in previous in vitro and in vivo studies. Two clinical studies further evaluated the potential of near infrared Raman spectroscopy to detect cervical dysplasia in a clinical setting. In the first study, the Raman spectral features of the different pathologies found in the cervix were characterized and mathematical algorithms were developed to classify the spectra according to pathology. The second study examined and quantified the sources of spectral variability within a given pathology. Experiments using organotypic raft cultures examined the biochemical and cellular basis for the spectral differences seen between normal and dysplastic tissue. These studies have laid the foundation for the development of Raman spectroscopy as a non-invasive, real-time diagnostic tool for cervical dysplasia.

The correlation between the stiffness and health of tissue is an accepted form of organ disease assessment. As a result, there has been a significant amount of interest in developing methods to image elasticity parameters (i.e. elastography). This work presents a technique that frames the elastography imaging problem within a non-rigid iterative registration approach. Through the use of finite element modeling and image comparison methods, material properties are varied in order to optimize the registration between a post-compressed image and a model-generated compressed image. The results shown here demonstrate the strong connection between image similarity and appropriate tissue parameters and the algorithm's ability to detect contrast in tissue stiffness. Simulations demonstrate that the method is effective over a wide range of scenarios. Also, we were successfully able to localize regions of stiffness within phantom data taken in both CT and MRI. By casting elasticity image reconstruction within the context of image similarity, the method is generalized to all forms of medical imaging.