A number of heart assist devices including the PediaFlowTM ventricular assist device (VAD), a magnetically levitated mixed flow rotary blood pump, and the Levitronix® PediVAS™, an extracorporeal magnetically levitated centrifugal blood pump are under development to address the urgent need for mechanical circulatory support suitable for children in heart failure. VADs are associated with a host of biological complications including bleeding, thromboembolism, and infection. The biocompatibility of these new devices must be characterized in a preclinical model (juvenile ovines) to ensure their safety and efficacy in children. However, biocompatibility studies in ovines are limited due to a lack of available assays. Flow cytometric assays were developed to detect ovine platelet activation and function. These assays were applied during in vitro assessment of potential biomimetic coatings for the blood contacting surfaces of pediatric VADs. These assays were then applied in vivo in 5 lambs undergoing a VAD sham surgical procedure for 30 days duration, in 20 lambs implanted with the Levitronix PediVAS for 30 days duration, and in 8 lambs implanted with the three design iterations of the PediaFlow VAD ranging from 6 - 72 days duration. The sham surgical procedure enabled characterization of the effects of the implant surgery on platelet activation. Platelet activation was reduced on surfaces that received a biomimetic coating compared to uncoated surfaces which was in agreement with platelet deposition results. Platelet activation levels rose post-operatively in the sham animals and returned to pre-operative levels at approximately two weeks. In PediaFlow and Levitronix implanted animals platelet activation also rose post-operatively and typically returned to baseline levels. In these implants platelet activation consistently rose following pump or animal complications. In a subset of studies platelet activation was elevated for the duration of the study and this high level of activation generally coincided with increased kidney infarcts or thrombus deposition in the cannulae at necropsy. Overall, the blood biocompatibility of the Levitronix PediVAS and the PediaFlow VAD as represented by a low level of platelet activation observed in the majority of studies is encouraging for the potential clinical use of these devices. The ability of the developed platelet activation assays to differentiate between surface coatings, and to discern trends with respect to pump complications and kidney infarcts following VAD implant demonstrate its utility in assessing the blood biocompatibility of pediatric heart assist devices.

All tissues in the human body continuously grow, remodel, and adapt to changes in their physiological environment, in order to maintain homeostasis or to retain it in case of pathologies. The growth and remodeling (G&R) process results in changes in structure and composition. To elucidate how observed changes in tissue components are related to altered tissue level mechanical behavior, structural constitutive models are required with physiologically relevant model parameters. Specifically what is required is a finite deformation constitutive model describing the tissue mechanical behavior, in combination with the appropriate kinematics for the multiple tissue components, and experimental data of a relevant tissue application. An excellent example is the urinary bladder wall (UBW), which undergoes profound remodeling in response to different pathologies, such as spinal cord injury (SCI). The overall objective of this dissertation was to develop a morphologically-driven, multi-phase constitutive model that would allow for separate investigation of the contribution of individual tissue components to the tissue-level remodeling process. As a first step, a constitutive model was developed of UBW extracellular matrix (ECM). Removing the smooth muscle cells from UBW tissue via decellularization allowed for the separate mechanical and structural investigation of UBW ECM. It was shown that the presence of de novo produced elastin in the UBW ECM post-SCI induced, indirectly, a distinct mechanical behavior with higher compliance, allowing for a higher overall extensibility of the post-SCI UBW and increased bladder storage capacity. The ECM constitutive model was extended and modified to be able to apply it to a multi-component tissue with individual model components existing in different reference states. Parameters were determined from biaxial mechanical data of decellularized and intact UBW tissue using a step-wise fitting approach implemented in MATLAB. As an initial step towards a theoretical G&R framework, a parametric analysis was performed to investigate if observed mechanical changes in post-SCI UBW were due to changes in morphology or intrinsic constituent properties. The developed model has the potential to explain underlying remodeling mechanisms of individual constituents in muscular tissues in several pathologies (e.g. UBW post-SCI), and predict remodeling events in a tissue engineering setting of muscular tissues.

Severe sepsis is defined as a systemic inflammation leading to organ failure and is characterized by the release of pro- and anti-inflammatory markers called cytokines. Current clinical techniques used to treat sepsis such as early goal-directed therapy, specific target drug therapies, and hemofiltration have had limited success and inconsistent outcomes. A newly investigated therapy, hemoadsorption, has proven to be nonselective and therefore broad-spectrum (i.e. restores homeostasis to the system as a whole); more efficient and cost-effective than affinity based removal; and is auto-regulating in that solutes at higher concentrations are removed more rapidly than those at lower, safer concentrations. The goal of this thesis was to characterize and compare adsorption profiles of several cytokines in our cytokine adsorption device (CAD) for use in the treatment of sepsis. To do this, we first characterized capture of our three main cytokines of interest in the original lot of CytoSorb resin for buffer and serum. This polymer is a highly porous polymer manufactured by Cytosorbents, Inc. (Monmouth Junction, NJ) and consists of a polystyrene divinylbenzene (PSDVB) copolymer covered in a biocompatible polyvinylpyrrolidone coating. We then detailed changes in adsorption profiles over a manufacturers lot change and re-established our baseline capture rates with the main cytokines of interest as well as a secondary group of cytokines in both buffer and serum. Further investigation into a lot of smaller diameter polymer followed and a re-design of our CAD ensued. Finally, we tested the polymer in three distinct red blood cell suspensions in order to methodically increase the degree of complexity of the capture suspension. With the information included in this thesis, further optimization and development to our CAD and the polymer will be done. Additionally, the characterization of the resin is crucial for our in-house mathematical model as well as the development and calibration of a systems model of sepsis. This model will be used to simulate the development and progression of sepsis in humans and the integration of a therapeutic CAD intervention protocol into the timecourse of sepsis to improve patient outcomes.

The anterior cruciate ligament (ACL) is the most important knee stabilizer and is frequently injured during sports and work related activities. Unfortunately, midsubstance ACL ruptures have a limited healing capacity. As such, surgical reconstruction using soft tissue autografts is often performed. However, long-term follow-up studies have revealed that 20-25% of patients had a less than satisfactory outcome. These negative results have renewed clinical interests in healing of a torn ACL by means of biological stimulation. Thus, there is a need for basic science studies in order to better understand such an approach and also to logically develop an effective functional tissue engineering (FTE) treatment for an injured ACL. The overall objective of this dissertation was to evaluate the positive impact of biological and mechanical augmentation on the healing of the ACL using a combined experimental and computational approach. The ability of an extracellular matrix (ECM) bioscaffold in combination with an ECM hydrogel to enhance ACL healing following suture repair was first demonstrated in the goat model. At 12 weeks of healing, ECM-treatment led to an increase in neo-tissue formation as well as improved biomechanical properties of the healing ACL compared to suture repair alone. Second, as the healing process of the ACL was relatively slow even with ECM treatment, mechanical augmentation to better restore initial joint stability was required. Therefore, a suture augmentation procedure was developed, and improved joint function was achieved versus suture repair alone at the time of surgery. Further, there was increased tissue formation and improved biomechanical properties of the healing ACL at 12 weeks of healing. Finally, as a step toward predicting long-term outcomes following these biological and mechanical augmentation procedures, a preliminary mathematical model was developed to describe the remodeling process of healing ligaments. The results of this work can now be used to guide future experiments using FTE treatments to enhance ACL healing. With a sound scientific basis, it is hoped that such exciting new technologies could then be translated into the clinical arena to improve patient outcome following ACL injuries.

Optical coherence tomography (OCT) provides real-time, objective, in-vivo, optical cross-sectional representations of the retina and optic nerve. Recent innovations in image acquisition, including the incorporation of Fourier/spectral-domain detection, have improved imaging speed, sensitivity and resolution. Still, there remain specific structures within ocular OCT images, such as retinal ganglion cells (RGCs), which are of clinical interest but consistently have low contrast. This makes it difficult to differentiate between surrounding layers and structures. The objectives of this project were: 1. To establish a reliable method for OCT imaging of the healthy and diseased mouse eye in order to provide a platform for testing the utility of OCT contrast agents for ocular imaging, 2. To develop antibody-conjugated gold nanoparticles suitable for targeting specific structures and enhancing OCT image contrast in the mouse eye, and 3. To examine the localized contrast-enhancing ability and biocompatibility of gold nanoparticle contrast agents in-vivo. Our organizing hypotheses were that nanoparticles could improve contrast by modulating the intensity of backscattered light detected by OCT and that they could be directed to structures of interest using antibodies specific to cellular markers. A reproducible method for imaging the mouse retina and quantifying retinal thickness was developed and this technique was then applied to a mouse model for retinal ganglion cell loss, optic nerve crush. Gold nanorods were designed specifically to augment the backscattering OCT signal at the same wavelengths of light used in current ophthalmic OCT imaging schemes (resonant wavelength Λ = 840 nm). Anti-CD90.2 (Thy1.2) antibodies were conjugated to the gold nanorods and a protocol for characterization of the success of antibody conjugation was developed. Upon injection, the gold nanorods were found to remain in the vitreous post-injection, with many consumed by an early inflammatory response and only very few reaching the internal limiting membrane and passing into the retina. Our findings suggest that, while gold nanorods are able to locally increase OCT signal intensity in the vitreous, their utility in the retina may be limited.

Despite the regenerative capacity of skeletal muscle, chronic myopathy and muscle trauma present significant clinical problems with limited therapeutic options. Myogenic cell therapies are being actively investigated to mitigate muscle degeneration that otherwise progresses to fibrosis and long-term loss of function. The rationale for this strategy is based on augmenting the native myogenic progenitor or stem cell reserve, populated by satellite cells, by transplanting myogenic cells into the injured muscle. However, engraftment efficiency in myogenic cell transplantation is impaired by rapid cell death, which represents a precipitous loss of cell viability within 48hrs of injection, therefore severely limiting engraftment and tissue regeneration. Upon transplantation, cells experience a host response of rapid inflammation; an environment of oxidative and inflammatory stress that may cause a dramatic loss in cell viability. This effect may determine the regeneration capacity irrespective of the cell's multilineage differentiation potential. Our lab has isolated and characterized multiple populations of myogenic progenitors from murine and human skeletal muscle, including the muscle derived stem cell (MDSC) isolated by a modified preplate technique. In our studies of MDSCs, we observed an increase in post-transplantation survival and skeletal muscle regeneration capacity over that of early preplate myoblasts. Furthermore, a strong correlation of improved survival and regeneration with inflammatory and oxidative stress resistance emerged. In this dissertation, we examined the role stress resistance and survival during myogenic differentiation of MDSCs. By treating the transplanted cells with a membrane permeable reactive oxygen species scavenger, XJB-5-131, prior to transplantation, the survival and myogenic differentiation capacity under stress conditions was significantly improved. Furthermore, we isolated a novel sub-population of muscle derived cells with elevated stress resistance from murine and human skeletal muscle by their enhanced aldehyde dehydrogenase activity. ALDHlo and ALDHhi cells were characterized in terms of their myogenic potential and stress tolerance in vitro and muscle regeneration capacity in vivo. These studies are aimed at understanding the importance of survival and stress tolerance of transplanted cells in skeletal muscle cell therapy and how this tolerance modifies the efficacy of cell therapy.

Falls threaten the quality of life of older adults and are associated with tremendous economic costs. Slips and trips are the two major causes of falls during locomotion and each requires a different postural response to prevent falling. However, a critical requirement in maintaining balance in either is the ability to generate proactive postural adjustments. Older adults have been shown to adopt proactive postural adjustments through repeated exposure to novel perturbations. However, the extent to which such learning abilities applied to perturbed and novel gait was unknown. This dissertation investigated reducing the incidence of falls in older adults through learning to better recover from perturbed gait based on a systems model theory. Potential associations between aging and anticipatory postural strategies when repeatedly exposed to forward slips were studied. Forward vs. backward walking slips were also compared to examine the impact of gait novelty on the ability to generate proactive adjustments. We also desired to know whether knowledge of the type of perturbation impacts the ability to generate proactive adjustments and whether such adjustments change with experience and when the nature of the perturbation is unknown. Subjects were exposed to multiple slip and trip perturbations to investigate these differences and to compare how young and older adults optimize their proactive adjustments. As anticipatory behavior improves perturbation recovery outcomes, changes in measures of severity with increased exposure were also analyzed. This study found young and older adults capable of adopting optimal proactive postural adjustments when repeatedly exposed to forward slips and their central nervous system was able to make internal representations applicable to a novel task. Awareness of a perturbation proved sufficient to induce proactive adaptations and with experience, adaptations became perturbation specific to reduce slip and trip risk in both age groups. Perturbation recovery improved with multiple exposures in both age groups as decreases in severity measures were observed. This study opens the door to studies evaluating the retention of postural control motor skills adapted through training and prior experiences and sheds light on the benefits of a systems model theory based fall intervention program for slips and trips.

Intervertebral disc degeneration is a leading cause of low back pain, a significant socioeconomic burden with a broad array of costly treatment options. Motion-based therapy has shown modest efficacy in treating LBP. Basic science research has begun to identify thresholds of beneficial and detrimental mechanical loading of the intervertebral disc. Ex-vivo mechanobiological systems are important experimental models for determining the effect of loading parameters on disc biology and matrix homeostasis. A novel experimental platform has been developed to facilitate in-situ loading of a rabbit functional spinal unit (FSU) with outcome measures relevant to disc matrix homeostasis and cell behavior. First, the system was designed for multi-axis motion outside of an incubator and validated for rigid fixation and stable, physiologic environmental conditions that maintained adequate cell viability. Following system development and validation, experimental testing on rabbit FSUs proceeded with cyclic compression and four-hour constant compression compared. Disc tissue was analyzed for cell viability using a colorimetric absorbance assay or relative gene expression. Conditioned media was assayed for matrix metalloproteinase activity, type-II collagen degradation fragments, prostaglandins, and an aggrecan epitope implicated in aggrecan synthesis. Cell viability remains high (>90%) regardless of loading. Relative gene expression shows small increases in anabolism and larger, variable increases in catabolic and inflammatory markers. These trends are more reliable in AF than NP. Interestingly, matrix metalloproteinase activity trends toward a decrease in media in loaded specimen culture. Although type-II collagen fragment concentrations do not correlate with loading, the aggrecan synthesis marker concentrations do. Results indicate increased catabolism and aggrecan turnover in response to loading, though the net effect on matrix homeostasis at later time points is unclear. Future work will explore applying other loading patterns, rotational loading, and coupling local inflammatory stimuli with loading. This novel experimental platform will explore the effect of physiologic motion simulations on disc homeostasis, helping to improve motion-based therapies.

Obesity is a condition that affects about 40% of US adults, and people with disabilities have a higher incidence of obesity than able-bodied individuals. Motor vehicle collisions (MVCs) are the number one cause of death in individuals under the age of 34 in the US, and people who ride in vehicles while seated in their wheelchairs are at increased risk of injury compared to people who ride in the automotive seat. Obese occupants appear to have a different risk of injury in MVCs than non-obese individuals. To reduce the risk of injury to obese occupants it is necessary to further understand the injury mechanisms to obese individuals in frontal MVCs. The purpose of this research was to investigate the mechanisms of injury and injury risk to obese occupants and obese wheelchair-seated occupants in frontal impact. Three full body occupant models were created to investigate the effects of increased mass, changes in obese torso mechanical response and geometry, and a combination of mass and torso changes on occupant injury risk. To investigate the effects of obesity on wheelchair-seated occupants a wheelchair/occupant model was created and validated. Parametric studies were used on all the models to investigate injury risk in frontal impact. The results show that increased mass is the most significant factor leading to injury for obese occupants. The differences in torso mechanical response and geometry as a result of increased adipose tissue in obese occupants, do not significantly affect the injury risk of obese occupants. Changes in the obese torso coupled with increased mass cause increased pelvis and chest excursion which results in increased risk of lower extremity injury. As BMI increases in wheelchair-seated occupants the risk of lower extremity injury increases, and obese wheelchair-seated occupants have a higher risk of injury to the lower extremities than obese non wheelchair-seated occupants. This research suggests that the reduction in injuries to certain body regions reported in the literature are not due to a cushion effect, but are more likely due to altered occupant kinematics that transfer load from the upper body to the lower extremities.

Muscle-derived stem cells (MDSCs) have been isolated from murine skeletal muscle and have the ability to differentiate into osteogenic, myogenic and chondrogenic lineages, among others. MDSCs may prove to be an attractive cell sources for orthopaedic tissue engineering therapies. However, MDSCs must prove successful in many patient populations before clinical translation becomes a reality. MDSCs have been characterized and studied extensively based on attributes of the donor animal. To date, little is known about the effect of the host animal or surrounding tissue environment on MDSC therapies. These studies were undertaken to determine the efficacy of MDSCs in bone formation and bone healing in different hosts, representing different patient groups. MDSCs are known to exhibit sexual dimorphism, by donor sex, of osteogenic differentiation. Therefore, MDSC-mediated ectopic bone formation and cranial bone defect healing were compared between male and female host animals. Castrated male and ovariectomized female hosts were also examined in MDSC-mediated ectopic bone formation and cranial bone defect healing studies, in order to determine the role of host sex hormones in MDSC osteogenic therapies. Moreover, castrated males and ovariectomized females represent aged hosts, as these animals exhibit some aging symptoms related to loss of sex hormones. Lastly, MDSCs have only previously been applied to freshly created defects, so a delayed application study was carried out to determine if MDSCs could heal established defects. MDSCs were applied to a cranial defect, which had been previously created and allowed to fill with scar-like fibrous tissue, to determine the efficacy of MDSCs in unfavorable bone defect environments or as treatment for non-unions. The results of these studies helped to elucidate the effect of host animal and tissue environment on MDSC-mediated bone tissue engineering therapies