The predominant approach for cartilage tissue engineering involves cell transplantation with or without cytokine delivery, biomaterial scaffolds, bioreactors, applied mechanical stimulation and altered oxygen tension. Despite its scientific merit, cell delivery faces drawbacks including scarce cell availability, donor site trauma, possible immune rejection and potential tumorigenesis. Tissue regeneration by cell homing is a novel concept and may offer the advantage of accelerated clinical translation. Promising results have been shown using a cell homing approach to engineer a number of tissue types including dental pulp, vascular tissue and bone. Various stem/progenitor cell populations are present in tissues adjacent to an articular cartilage defect including subsets of cells that have the ability to differentiate into cartilage-like tissue. Furthermore, several factors have been elucidated that stimulate stem/progenitor cell homing and selected cytokines have been discovered to be potent at inducing chondrogenic differentiation of stem/progenitor cells. Cell homing is an exciting area of regenerative medicine but many critical questions remain such as cell origin, homing distance, and effective chemotactic cues. In addition to currently studied cell homing cues, other cytokines present during inflammation that are not typically known for their homing abilities might be helpful in recruiting additional cells to the scaffold and improving the quality of cartilage tissue formation. The effect of concurrently exposing a cell population to multiple cytokine signals, similar to conditions that cells experience in vivo, has not been fully investigated. Determining which cytokine or groups of cytokines that induce high levels of chemotaxis would be critical for designing effective bioactive scaffolds for cell recruitment and chondrogenesis. This thesis develops novel systems to characterize stem/progenitor cell migration and uses the knowledge gained from these systems to develop new methods for inducing chondrogenesis by cytotactic homing. First, the concept of stem/progenitor cell homing for cartilage tissue regeneration is reviewed (Chapter 1). Next, a system was developed for the in vitro recruitment and chondrogenesis of Adipose Stem Cells (ASCs), Mesenchymal Stem Cells (MSCs) and Synovium Stem Cells (SSCs), all of which are natively located adjacent to a full-thickness articular cartilage defect (Chapter 2). Using microfluidic principles, novel assay systems were designed and built to characterize the process of stem cell migration in the presence of single and competing cytokine signals (Chapter 3). An in-depth study was conducted investigating the process of stem/progenitor cell migration in the presence of competing cytokine signals (Chapter 4). Lastly, the knowledge gained through extensive chemokine testing using these novel assay systems was used to develop a bioactive scaffold to induce cell homing and chondrogenesis for rhinoplasty augmentation in a rat model (Chapter 5). The novel migration devices developed herein offer a rare opportunity for screening of cell homing efficacy, potentially applicable to any stem cell population including embryonic, iPS, skeletal, muscular, neural, cardiac and adipose. A number of basic biological concepts have been examined by studies using these devices such as cell motility behavior and optimal migratory distances. The competitive cytotactic assay system provided new insight into stem cell behavior in response to gradients of multiple cytotactic cues, thus mimicking native in vivo conditions. By determining combinations of cytokines effective at maximizing cell homing, novel approaches for cartilage tissue engineering without the need for cell delivery, were developed for rhinoplasty augmentation. These systems for inducing chondrogenesis by chemotactic homing were shown to be an effective alternative to cell transplantation for cartilage tissue regeneration therapies.

The development of a multicellular organism from an embryo is one of the nature's most remarkable phenomena. Deciphering how this transformation occurs is a fundamental challenge in biology with profound biomedical implications. Insights into the molecular signals guiding developmental patterning may provide design strategies to promote multicellular structure formation in applications such as tissue engineering and regenerative medicine. In this thesis, we explored the applications of controllable gene expression techniques in combination with engineering strategies in regulation and patterning of cell differentiation and pluripotency, by pursuing three related research projects: 1. Reversible immortalization of cardiomyocytes to enable their proliferation necessary for obtaining large cell numbers 2. Patterning of the delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector, to mesenchymal stem cells cultured in a microfluidic 3. Patterning of the Nanog gene expression in embryonic stem cells, using a microfluidic device, to establish differentiation - pluripotency boundaries that mimic the developmental processes in vivo. In the first project, we developed a novel strategy for controlled expansion of non-proliferating primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then the reversal of the phenotype of expanded cells back to the cardiomyocytes state. Primary rat cardiomyocytes were transduced with simian virus 40 large T antigen (TAg), or with Bmi-1 followed by the human telomerase reverse transcriptase (hTERT) gene; the cells were expanded; and the transduced genes were removed by adenoviral vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than the Bmi-1/hTERT gene, based on the rate of cell proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, and reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (α-actin, connexin-43, and calcium transients). After the immortalization was reversed, cells returned to their differentiated state, as evidenced by molecular and functional properties inherent to terminally differentiated cardiomyocytes. This strategy for controlled expansion of primary cardiomyocytes by reversible gene transfer could provide large amounts of a patient's own cardiomyocytes for cell therapy, and enable controlled in vitro study of cardiogenesis. In the second project, we developed a novel patterning strategy by using inducible gene expression systems in conjunction with simple multi-laminar fluidic techniques, which can directly pattern the expression of particular gene at transcriptional level. Using osteogenic differentiation of human mesenchymal stem cells as a model, we describe a novel approach to spatially regulate the expression and secretion of bone morphogenetic protein (BMP-2) in a two-dimensional field of cultured cells, by flow patterning the modulators of inducible BMP-2 gene expression. We first demonstrated a control of gene expression, and control of osteogenic differentiation of the cell line with inducible expression of BMP-2. Then we designed laminar flow systems, with patterned delivery of Doxycycline (Dox), the expression modulator of inducible BMP-2 expression vector. The patterned concentration profiles were verified by computational simulation and dye separation experiments. Experiments conducted in the flow systems for a period of three weeks showed the Dox concentration dependent osteogenic differentiation, as evidenced by mineral deposition. This strategy combining inducible gene expression with laminar flow technologies provides an innovative way to engineer tissue interfaces. In the third project, we further developed the patterning strategy for gene expression to form boundaries of different gene expression domains in cultures of mouse embryonic stem cells. Using Nanog safeguarded embryonic pluripotency as a model; we demonstrated controlled Nanog expression, which lead to controlled early differentiation under the exposure or withdrawal of varied small molecules, as evidenced by alkaline phosphatase (AP) staining, immunofluorescent staining, and gene expression analysis. By patterning Nanog gene expression, as well as soluble factors in the laminar fluidic system, we successfully developed varied differentiation - pluripotency boundaries between Nanog expressing pluripotency zones and Nanog suppressed early differentiation zones from the same population of cells, which mimic the development process in vivo. Mechanistic insights can be gained on dissecting the signaling pathways that drive multicellular patterning during the natural processes of embryonic and adult development. In summary, we demonstrated that controlled expansion of non-proliferating primary cells can achieved by reversible genetic manipulation, and that varied continuous, graded pluripotency - differentiation boundaries can established by patterning the expression of target genes via a simple laminar fluidic system. Taken together, these approaches provide innovative models to modulate cell function at the transcriptional level. Additional cooperative research was conducted during my graduate training. The manuscript of this study "Micropatterned Mammalian Cells Exhibit Phenotype-Specific Left-Right Asymmetry" was submitted to Proc Natl Acad Sci U S A., and it is currently under review. We attached this manuscript in appendix.

The key to effective treatment of neurological diseases resides in the safe opening of the blood-brain barrier (BBB), a specialized structure that impedes the delivery of therapeutic agents to the parenchyma. Despite the fact that several approaches have been successful in overcoming the BBB impermeability, none of them can induce localized BBB opening noninvasively except for focused ultrasound (FUS) in conjunction with microbubbles. The physical mechanism behind the opening, however, has not been identified. Insight into the mechanism can be critical for delineating the safety profile for in both small and large animals alike. Therefore the purpose of this dissertation is to first determine the physical mechanism of FUS-induced BBB opening in mice and then translate this approach to non-human primates. To accomplish this goal, an in vivo transcranial cavitation detection system was developed and tested, built in phantoms and in vivo, to monitor the behavior of the microbubbles in the FUS bean, and to determine the type of cavitation, i.e., the activation of bubbles in an acoustic field, during BBB opening. We showed that the inertial cavitation (IC), a collapse of a bubble, which can vary from a fragmentation of the bubble to shock wave and liquid jets depending on the pressure, thereby damaging the endothelial cells of the brain capillaries, was not required to induce BBB opening in mice. With this system, the role of microbubble properties, including the diameter and shell components, in the BBB opening were determined. When the BBB opens with stable cavitation (SC), i.e., relatively moderate amplitude changes in the bubble size, the bubble diameter is similar to the capillary diameter (i.e., at 4-5, 6-8 µm) while with inertial cavitation it is not (i.e., at 1-2 µm). The bubble may thus have to be in closer proximity to the capillary wall to induce BBB opening without IC. The BBB opening properties, such as volume and permeability, however, were not affected by the shell component of the microbubbles in mice. The connection between the physical and physiological mechanism was then investigated to identify the lowest peak rarefactional pressure BBB opening threshold at 1.5 MHz (0.18 MPa). A sufficiently long pulse (pulse length = 0.5 ms) was required for the SC to induce BBB opening at the lowest pressure. However, the tight junctions, the main formation of the BBB, were found not to be disrupted after sonication at both low (0.18 MPa) and high (0.45 MPa) pressures. Therefore, the transcellular pathway may be the main route of the FUS-induced BBB opening. Finally, the cavitation-guided BBB opening system was used to induce reversible BBB opening in non-human primates. This is a major step towards clinical feasibility. In conclusion, a transcranial cavitation detection system was developed, in order to characterize the physical mechanism, the role of the microbubbles, and the corresponding physiological response of the FUS-induced BBB opening.

Cardiovascular disease is a rising epidemic worldwide, and curative therapies remain elusive. Heart and vascular disease remain some of the hardest to cure due to the limited capacity of the heart to repair itself, necessitating a cell or organ based therapy to cure the inevitable descent into heart failure. Tissue engineering is uniquely poised to significantly alter this disease burden though the fabrication of cardiac and vascular tissues in vitro. However, the challenges for achieving these aims are significant - for cardiac tissues, the therapy must adhere to strict requirements of adequate perfusion and functional integration with the damaged heart. Vascular tissues are required to be amenable to surgical anastomosis while at the same time provide nutrient transport on the cellular level. Recently, a new set of technologies based from the semiconductor industry, have enabled micron level control over the cellular environment and cells themselves and may enable novel approaches to fulfill these tissue engineering requirements. In this dissertation, these microtechnologies will be leveraged to address some of the current obstacles that limit the use of tissue engineering approaches for functional therapy. Specifically, microtechnologies were used to screen the effect of electrical stimulation on the function and maturation of human embryonic stem cell derived cardiomyocytes, which resulted in the ability to program specific individual beating frequencies of the cells while improving contractile function and led to the identification of a channel specific effect for frequency modulation. These technologies were also used to distinguish the vasculogenic potential of different mesenchymal stem cell sources for nascent vessel stabilization, and enabled the development of a powerful hydrogel docking platform with the novel capability to spatially pattern any number of cells, cytokines or drugs on the microscale, while permitting scale up for larger tissue generation without the loss of precision. Finally, these technologies were used to create vascular networks with hierarchical branching patterns that could be implanted and used in vivo fulfilling a major criterion of vascular tissue function - surgical compatibility with microscale architecture for tissue perfusion. Therefore, these microtechnologies support novel interrogation of cell function and enable new methods to engineer cardiovascular tissues.

The importance of atherosclerotic disease in coronary artery has been a subject of study for many researchers in the past decade. In brief, the aim is to understand progression of such a disease, detect plaques at risks (vulnerable plaques), and treat them selectively to prevent mortality and immobility. Consequently, several imaging modalities have been developed and among them intravascular ultrasound (IVUS) has been of particular interest since it provides useful information about tissues microstructures and images with sufficient penetration as well as resolution. In general, the ultimate goal is to provide interventional cardiologists with reliable clinical tools so they can identify vulnerable plaques, make decisions confidently, choose the most appropriate drugs or implant devices (i.e. stent), and stabilize them during catheterization procedures with minimal risk. In this work, we review existing atherosclerotic tissue characterization algorithms including the state-of-the-art virtual histology (VH) framework, which has been implemented in the Volcano (Rancho Cordova, CA) IVUS clinical scanners using 64-elements 20 MHz phased-array transducer. Initially, we intended to extend this technique for data acquired with 40 MHz single-element transducers. For this reason, we started acquiring in vitro IVUS data and studied involved challenges from specimen preparation toward classification. We observed inconsistency among extracted features along with transducer's spectral parameters (i.e. bandwidth, center frequency). This, in addition to infeasibility of construction of reliable training dataset due to heterogeneity of atherosclerotic tissues motivated us to develop an unsupervised texture-based atherosclerotic tissue characterization algorithm. We proposed a two-dimensional multiscale wavelet-based algorithm to expand IVUS backscattered signals and/or grayscale images onto orthogonal symmetric quadrature mirror filters (QMF) such as Lemarie-Battle. At the bottom of decomposition tree, we employed ISODATA to cluster enveloped detected features in an unsupervised fashion and classify atherosclerotic plaque constitutes into fibrotic, lipidic, calcified, and no tissues. For the first time, we studied numbers of factors that were necessary for extension of in vitro derived classifier for in vivo applications such as reliability of classified tissues behind arc of calcified plaques and effects of pressure changes as well as flowing blood on constructed tissue color maps, called prognosis histology (PH) images. The second half of this dissertation is devoted to automatic detection of lumen borders in IVUS grayscale images acquired with high frequency (40 MHz up) transducers where more scattering exhibited within lumen area that makes the problem of interest more challenging. We established our framework on three-dimensional expansion of IVUS sub-volumes onto orthonormal brushlet basis function. The rational behind our framework was presence of incoherent (i.e. blood) versus coherent (i.e. plaque, surrounding fat) textural patterns along pullback direction, which was motivated by what an interventional cardiologist does to locate the lumen border visually by going back and forth among IVUS frames. We studied the feasibility of brushlet analysis through filtering blood speckles and supervised classification of blood versus non-blood regions. Our preliminary study confirmed that the most informative features reside in the innermost cubes, representing low-frequency components in transformed domain. Finally, we explored that tissue responses to IVUS signals are proportionally preserved in brushlet coefficients and it enabled us to classify blood regions in complex brushlet space. Subsequently, we employed surface function actives (SFA) to estimate the lumen borders after regularization. In a comparison study, we quantified our results with two of existing algorithms, employing IVUS grayscale images acquired with 40 MHz and 45 MHz single-element transducers. Overall, our proposed algorithm outperformed and the resulting automated detected borders showed good correlation with manually traced borders by an expert.

580,350 (35%) of 1,660,290 cancer patients are estimated to die in the US in 2013. Routine monitoring by X-Rays and CT scans are hazardous and evaluating this disease is time consuming. Magnetic Resonance Spectroscopy (MRS) has changed this mal-routine significantly in the past few years. MRS can help with better understanding of tumor pathology, study of tumor vascularization and progress, and having a predicting value for the treatment response and disease-free survival of the patients even before they start their treatment. Unfortunately, MRS is still not a common practice among the medical community because of three main reasons: First and far most is the fact that MRS acquisition is usually very time consuming. For a classic 1H 3D MRS with a spatial matrix of 20x18x10 with TR = 1000 ms, the scan time is about 1 hour which is "practically" impossible to acquire on a patient. Second, MR time is extremely expensive. Depending on the site, specific procedure, and strength of the magnet a simple MR study can cost somewhere between 1000 to 3500 US dollars. Finally, non-standardized MRS acquisitions and analysis protocols could create havoc in interpretation and usefulness of the technique. MRS scan parameters such as spatial resolution and echo times have been used non-uniformly in variety of different combinations in research and clinical studies. These parameters must be chosen with utmost care as they have direct impact on signal to noise ratio, quantification of the metabolites, and an overall interpretation of the results. For the reasons said, having a method that could shorten the length of an MRS scan, reduce the cost, and potentially become a sensible routine in clinical practice is of a huge value. 3D Zero J-modulation Echo Planar Chemical Shift Imaging (3D ZJ-EPSI) is a fast MRS technique that can not only achieve all that was mentioned above, it can also provide additional detailed anatomical/pathological information due to its 3D nature. 3D ZJ-EPSI technique acquires proton magnetic resonance spectroscopy with time to acquisition (TE') of less than 1.7 ms and zero J-modulation effects. 3D ZJ-EPSI consisted of a slab excitation, followed by two phase encoding gradients and an echo planar switching readout gradient. The Free induction decay (FID) acquisition occurred during the plateaus of the switching gradient. The lipid suppression was achieved via ten Regional Saturation Technique (REST) pulses placed prior to the main slab excitation RF. The water suppression technique was a chemical shift selective (CHESS) pulse with RF-80º-80º-160º that was placed prior to lipid suppression pulses. The sequence was tested on a brain metabolite phantom with spatial resolution of 15×15×6 mm3 in 4:04 min, yielding spectra with comparable quality to the spectra obtained using conventional chemical shift imaging (CSI) technique taking 56:34 min. The sequence was also tested on human subjects with spatial resolution of 15×15×6 mm3 and 7.5×7.5×6 mm3 and the metabolic ratios were calculated and compared to literature values. Signals of coupled resonances were improved due to near zero TE' and zero J-modulation effects, while the macromolecules were more pronounced in the spectra. With non-water suppressed sequence, variations of waterline shape of different tissues in three spatial dimensions could be studied. The 3D ZJ-EPSI technique addresses the need for a fast MRS method that allows for a better quantification capability by acquiring proton spectra with zero J-modulation. The short acquisition time and near zero TE' make this methodology suitable for uniform quantification of metabolites in clinical studies.

Nonlinear microscopy refers to a range of laser scanning microscopy techniques that are based on nonlinear optical processes such as two-photon excited fluorescence and second harmonic generation. Nonlinear microscopy techniques are powerful because they enable the visualization of highly scattering biological samples with subcellular resolution. This capability is especially valuable for in vivo and live tissue imaging since it can provide both structural and functional information about tissues in their native environment. With the use of a range of exogenous dyes and intrinsic contrast, in vivo nonlinear microscopy can be used to characterize and measure dynamic processes of tissues in their normal environment. These advances have been particularly relevant in neuroscience, where truly understanding the function of the brain requires that its neural and vascular networks be observed while undisturbed. Despite these advantages, in vivo nonlinear microscopy still faces several major challenges. First, observing dynamics that occur in large areas over short time scales, such as neuronal signaling and blood flow, is challenging because nonlinear microscopy generally requires scanning to create an image. This limits the study of dynamic behavior to either a single plane or to a small subset of regions within a volume. Second, applications that rely on the use of exogenous dyes can be limited by the need to stain tissues before imaging, the availability of dyes, and specificity that can be achieved. Usually considered a nuisance, endogenous tissue contrast from autofluorescence or structures exhibiting second harmonic generation can produce stunning images for visualizing subcellular morphology. Imaging endogenous contrast can also provide valuable information about the chemical makeup and metabolic state of the tissue. Few methods have been developed to carefully and quantitatively examine endogenous fluorescence in living tissues. In this thesis, these two challenges in nonlinear microscopy are addressed. The development of a novel hyperspectral two-photon microscopy method to acquire spectroscopic data from tissues and increase the information available from endogenous contrast is presented. This system was applied to visualize and identify sources of endogenous contrast in gastrointestinal tissues, providing robust references for the assessment of normal and diseased tissues. Secondly, three methods for high speed volumetric imaging using laser scanning nonlinear microscopy were developed to address the need for improved high-speed imaging in living tissues. A spectrally-encoded high-speed imaging method that can provide simultaneous imaging of multiple regions of the living brain in parallel is presented and used to study spontaneous changes in vascular tone in the brain. This technique is then extended for use with second harmonic generation microscopy, which has the potential to greatly increase the degree of multiplexing. Finally, a complete system design capable of volumetric scan rates >1Hz is shown, offering improved performance and versatility to image brain activity.

Molecular motors perform diverse functions in cells, ranging from muscle contraction, cell division, DNA/RNA replication, protein degradation, and vesicle transport. The majority of molecular motors use energy from the ATP hydrolysis cycle, converting chemical energy into mechanical work in cells. All ATP-dependent molecular motors have a similar ATP binding site, although the functions can be drastically different. Myosins comprise a large group of ATP-dependent molecule motors. The structure-function relationship governing different functions for different myosin families remains elusive. Hypothesizing that members of each family possess conserved residues for their consensus functions and residues distinctive from those of other families to differentiate their functions from functions of other myosin families, we developed an algorithm for comparative sequence analysis in a phylogenic hierarchy to identify family-specific residues for 38 myosin families/subfamilies that comprise human myosin members. We found a number of family-specific residues that have been reported, such as residues in β-cardiac myosin associated with hypertrophic cardiomyopathy and residues in myosin 7A associated with hereditary deafness. We also identified distinct features among myosin families that have never been reported, including a unique signature of the SH1 domain in each of the myosin families, residues differentiating α- and β-cardiac myosins, and a unique converter domain of myosin VI. We further examined myosin VI to understand why it moves toward the (-)-end of actin filaments, opposite to the direction of all other myosins and to shed light on their links to prostate cancer and ovarian cancer, where myosin VI is over-expressed. We found that many of myosin VI specific residues locate in or adjacent to the converter domain, including a cluster of unique residues at the interface between the motor domain and the converter. Using molecular dynamics (MD) simulation, we found mutations of M701 on the SH1 helix and F763 on a helix of the converter caused the separation of the motor domain and the converter, indicating their important roles in linking the converter and the motor domain in the pre-power stroke state structure, potentially critical for positioning of lever arm. Using the location of the unique residues at the interface of the motor domain and the converter as the site of drug docking, we identified a set of candidate small molecules binding to this unique binding site selectively, potentially blocking the converter rotation of myosin VI. A benzoic acid (C15H17N3O3) was found to have the best score in docking, binding to both the converter and motor domain stably in a 200 ns MD simulation run. This molecule can be a good lead to be optimized to inhibit myosin VI functions in cancer patients. We have also applied our algorithm to other ATP-dependent molecular motors, including hepatitis C virus NS3 helicase and DEAD box helicase Mss116. We found an important residue, T324, in NS3 helicase connecting domains 1 and 2 acting as a flexible hinge for opening of the ATP-binding cleft and an atomic interaction cascade from T324 to residues in domains 1 and 2 controls the flexibility of the ATP-binding cleft in NS3 helicase. We also found a conserved flexible linker for Mss116, and the tight interactions between the Mss116-specific flexible linker and the two RecA-like domains are mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

Diabetes Mellitus is one of the most common diseases in the world. Cardiovascular diseases account for ~80% of deaths amongst diabetic patients, primarily through coronary artery disease (CAD). However, a new clinical entry, termed Diabetic Cardiomyopathy (DC), may lead to heart failure in diabetic patients independently of CAD or hypertension. In DC, hyperglycemia and hyperlipidemia associated with diabetes produce structural and biochemical alterations at the cardiac cell level. Early stage cell alterations include hypertrophy, calcium mishandling, cell apoptosis, excessive ROS production, and increased collagen production by fibroblasts. Eventually, major structural and functional changes can appear in myocardial tissue, characterized by diastolic and systolic dysfunction, and eventually heart failure. While specific changes associated with DC are well characterized, the mechanism underlying disease development and progression as a whole remain to be elucidated. The ability of researchers to develop general treatment options for this disease is thus limited. Currently, a majority of DC studies focus on either in vitro molecular pathways, or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease of changes in structural and functional properties, these studies can not precisely quantify what conditions (such as hyperglycemia and hyperlipidemia) are producing specific biomechanical changes such as increased myocardial stiffness or diastolic dysfunction. To address this, we developed an in vitro approach, based on culturing cardiac cells in elevated glucose and fatty acid, to examine how structural and functional properties may change as a result of a diabetic environment. Increased myocardial stiffness is associated with increased collagen production in the heart. However, diastolic dysfunction is found to occur in DC prior to significant collagen accumulation. We hypothesized that increased cardiac cell stiffness could contribute to early stage diastolic dysfunction. To test this hypothesis, we developed and used contemporary biomedical engineering tools to characterize the biomechanical properties of cardiac myocytes and fibroblasts under a variety of hyperglycemic and hyperlipidemic conditions. We showed that our in vitro model of DC exhibits increased stiffness in myocytes, but not fibroblasts. We then developed an assay to measure cardiac myocyte contractile force, as well as assess systolic and diastolic function. This assay was then used to determine the role of N-acetyl-cysteine (NAC), towards regulating reactive oxygen species (ROS) and reversing cellular-level changes associated with our DC model. We found that DC model cardiac myocytes exhibited greater incidences of diastolic, but not systolic, dysfunction, and that treatment with NAC reduced dysfunction to a normal level. In terms of structural properties, we additionally determined that treatment with NAC attenuated increases in myocyte stiffness found in our DC model, and that NAC reduced myocyte hypertrophy for certain diabetic conditions. Overall, treatment with NAC attenuates the maladaptive mechanical and functional changes found in our DC model.

The menisci are fibrocartilaginous tissues of the knee that specialize in load-bearing and stabilization of the joint. Though once believed to be "the functionless remains of leg muscles," and therefore routinely removed after injury, it is now understood that loss of meniscus integrity leads directly to degenerative changes in the knee, inspiring new pursuits to overcome the intrinsic limitations to meniscus repair. However, many components of the meniscus environment and their roles in regulating the cellular response of injured tissue remain unclear. This thesis presents novel strategies to enhance integrative repair of the meniscus, via control of the cell environment from the micro- to macro-scale. The unifying hypothesis of this dissertation was that the application of chemical, physical, and environmental factors can significantly influence the meniscus cell environment and contribute to healing. Specifically, we studied the direct role of vasculature on the meniscus, and identified angiogenic factors that regulate cell migration and tissue repair between the inner and outer regions. We analyzed the effects of pulsatile direct current electrical stimulation on meniscus cell migration and repair, and describe the mechanisms of electrotransduction in the meniscus. Finally, we developed a decellularized meniscus extracellular matrix hydrogel to deliver and dictate the behavior of stem cells for meniscal repair. By rigorous study of each element at the micro-scale and integration at the macro-scale, novel therapies for meniscus repair will emerge towards clinical applications.