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Joint disease is detrimental to basic quality of life. Articular cartilage is responsible for reducing friction and distributing loads in joints as they undergo large, repetitive load cycles each day, but damaged tissue has very limited intrinsic regenerative ability. Osteoarthritis (OA), the most common joint disease, affects over 500 million people worldwide, contributes more than $27 billion dollars in annual healthcare expenditures, and has increased in prevalence by nearly 50% since 1990 with our aging population. In spite of all this, OA remains a chronic degenerative condition lacking in effective treatment strategies. For cartilage repair in late-stage disease, synthetic joint replacements carry risk of altered loading and metal hypersensitivity, while clinically approved autografts or autologous chondrocyte implantation procedures suffer from lack of donor tissue and donor site morbidities. Prior to surgical intervention, OA management is focused on analgesia rather than preventing or slowing early-stage disease. Disease-modifying OA drugs are yet to successfully complete clinical trials, in part due to the widespread use of animal models for therapeutic discovery rather than high-fidelity human models. Alleviating the burden of cartilage damage will require improvements in both early-stage therapeutic interventions and late-stage repair. Tissue engineering has the potential to offer more biologically faithful cartilage derived with minimal invasiveness, but the resulting cartilage currently lacks the organization or maturity of native tissue. Thus, the central concept of my thesis work was to introduce biologically inspired spatiotemporal cues to guide engineered cartilage formation, establishing novel methods for cartilage tissue engineering that would provide (i) cartilage-bone grafts for regenerative implantation and (ii) advanced in vitro models for studying osteochondral disease. United by the central theme of cartilage, this dissertation spanned three complementary and interacting areas of tissue engineering: regenerative medicine in Aim 1, tools and technological development in Aim 2, and organs on a chip in Aim 3. In Aim 1, we created patient-specific cartilage-bone constructs with native-like features at a clinical scale, using decellularized bone matrix, autologous adipose-derived stem/stromal cells, and dual-chamber perfusion bioreactors to recapitulate the anatomy and zonal organization of the temporomandibular ramus-condyle unit with its fibrocartilage. We validated key tissue engineering strategies for achieving in vivo cartilage regeneration, with the cartilage-bone grafts serving as templates for remodeling and regeneration, rather than providing direct replacements for the native tissue. To enable precise in vitro manipulation of TGF-β signaling, a key pathway in cartilage development, in Aim 2 we developed an optogenetic system in human induced pluripotent stem cells and used light-activated TGF-β signaling to direct differentiation into smooth muscle, tenogenic, and chondrogenic lineages. This optogenetic platform served as a versatile tool for selectively activating TGF-β signaling with precise spatiotemporal control. Using optogenetic recapitulation of physiological spatiotemporal gradients of TGF-β signaling in Aim 3, we formed stratified human cartilage integrated with subchondral bone substrate, towards in vitro engineering of native-like, zonally organized articular cartilage. Collectively, these studies established novel cartilage tissue engineering approaches which can be leveraged to alleviate the burden of joint disease.

The purpose of this study was to examine the impact of brain injury upon the family of the brain-injured patient. Although some research into the psychosocial sequelae following head injury and the direct and indirect effects of severe head-injury upon the family have been conducted, these studies have largely been of a quantitative nature. For this reason, and with a view to expanding upon the existing findings, the present study was qualitative in nature in order to gain an in-depth understanding of relatives' experience of living with and caring for a severely head-injured family member. In addition, ways in which family members coped with the impact of the brain injury were elicited. Participants were caregivers to 11 severely head-injured patients who were representative of a range of socio-economic groupings and racial classifications. Severity of head injury was controlled for and participants were interviewed two or more years after the head injury had occurred. Demographic and injury related data were elicited by means of a questionnaire. Thereafter, data was collected by means of in-depth semi-structured interviews. A number of indices were developed based on a qualitative and descriptive analysis of the data. The study found that the mam source of distress experienced by caregivers related to emotional rather than physical symptomatology. All types of family relationships were profoundly affected by the patient's brain injury, although the caregiver's relationship with the patient appeared most vulnerable. Formal sources of support were considered inadequate and most caregivers relied heavily on emotional support, particularly from an intimate source. Coping responses were dependent upon coping resources available. Families of brain-injured patients who were racially and socio-economically disadvantaged were subjected to additional stress related to the unavailability of services and the randomness with which services that were available were dispensed. Rehabilitation services appeared to be failing to meet the needs of "brain-injured families" (Brooks, 1984) as they concentrated on the patient's physical handicap rather than emotional sequelae of the head injury.

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The human bone marrow (BM) is one of the most complex and critical tissues in the adult, functioning as the site for blood and immune cell production in homeostasis, injury, and disease. The marrow acts as an incredibly diverse stem cell niche, containing stromal and blood cells that help support the maintenance and differentiation capacity of hematopoietic stem and progenitor cells (HSPCs). The cell-cell and cell-matrix interactions within the niche help alter the marrow to trigger blood cell production in response to injury, as well as harbor downstream changes that may persist in the hematopoietic system during disease, such as in cancer metastasis or leukemias of the BM. As the development of engineered human tissue models including organs-on-a-chip (OoC) have emerged over the past decade, there has been an increased relevance of using human BM models to study human- and patient-specific immune interactions in vitro. In this dissertation, we have developed patient-specific bioengineering technologies to model the BM, as well as those to study multi-organ interactions, for a host of translational applications of injury and disease. In Chapters 1 and 2, we introduce a number of concepts in bioengineering and stem cell biology for studying human organ functions outside of the body. In Chapter 3, we describe the tools that are critical for modeling individual organ functions (healthy human BM) and immune cells, as well as when combining multiple OoC systems together. In Chapter 4, we apply these tools for disease modeling, in studying the complex interactions in either acute leukemia development or metastatic colonization of the BM. In Chapter 5, we use our human-specific engineered models for studies of acute and systemic injury, including the effects of cosmic radiation on human tissue function. To tie together the tissue engineered tools developed in this thesis, we described in Chapter 6 the utility of scientific outreach and social media in the widespread dissemination of tissue engineering and stem cell principles to the broader scientific community and general public. Collectively, this dissertation provides a unique look at the use of engineered human tissue systems to model human blood and immune interactions using bioengineering tools, with applications in disease modeling of primary and metastatic cancers, as well as in acute and systemic injuries.

Blast-induced traumatic brain injury (bTBI) has been a health concern in both military and civilian populations due to recent military and geopolitical conflicts. Military service members are frequently exposed to single and repeated blasts throughout their training and deployment. As a result of blast exposures, military personnel report symptoms of various neurological and neurosensory deficits. Our group has previously reported decreased long term potentiation (LTP) following either single or repeated bTBI in a rat organotypic hippocampal slice culture (OHSC) model. LTP is a neuronal correlate for learning and memory and is a neurological metric that can be used to evaluate blast injury severity and the efficacy of therapeutic interventions. In the first aim of this thesis, we characterized LTP deficits following repeated bTBI to develop tolerance criteria for blast exposures. We did so by varying the blast injury severity, the inter-blast interval between blasts, and the recovery period following blast exposure. We determined that LTP deficits were compounded as a result of repeated mild bTBI. LTP deficits were attenuated with increasing inter-blast intervals and with increasing recovery periods after injury. Even after three repeated mild bTBIs, LTP spontaneously recovered after 6 days. In the second aim, we investigated the pathological changes in OHSCs following repeated blast exposures. Following injury, we observed robust microglial activation, evidenced by increased expression of the pro-inflammatory marker, CD-68, and decreased expression of the anti-inflammatory marker, CD-206. We also observed increased expression of MIP-1α, IL-1β, MCP-1, IP-10, and RANTES and decreased expression of IL-10 in the acute period after both single and repeated bTBI. Following partial depletion of microglia prior to injury, injury induced LTP deficits were significantly reduced. Lastly, treatment with a novel drug, MW-189, immediately after a repeated bTBI prevented LTP deficits. In the third aim, we investigated changes in inflammatory markers like cyclooxygenase (COX) and tested the efficacy of COX or prostaglandin receptor (EP3R) inhibitors in attenuating LTP deficits. We observed that expression of COX-2 increased 48 hours following repeated blast injury; however, COX-1 expression was unchanged. Following repeated bTBI, EP3R expression was upregulated and cyclic adenosine monophosphate (cAMP) concentration was decreased. Treatment of blast injured OHSCs with a COX-1 specific inhibitor, SC-560, a COX-2 specific inhibitor, rofecoxib, a pan-COX inhibitor, ibuprofen, or an EP3R inhibitor, L-798,106 improved LTP deficits. Delayed treatment with L-798,106 and ibuprofen also improved LTP deficits. Our data suggests that bTBI induced neuroinflammation may be partially responsible for the functional deficits that we have observed in blast-injured OHSCs. Additionally, we also conclude that COX and EP3R inhibition may be viable therapeutic strategies to reduce bTBI induced neurophysiological deficits. In the final aim, we investigated bTBI induced changes to the electrophysiological network of OHSCs. Following blast exposure, sham and injured OHSCs were administered increasing concentrations of bicuculline, a GABAA receptor antagonist. Doing so revealed an increase in connectivity and clustering coefficients in sham slices compared to injured slices. This suggested that the underlying neuronal network of injured slices was dysfunctional. Biologically, this dysfunction could be explained by the decreased expression of GABAA receptor α1 and α5 subunits. A loss of GABAA receptor expression or function may explain the electrophysiological network disruptions that we observed. More work will be required to determine how blast exposure decreases the expression of GABAA receptors and how these receptors may contribute to network deficits. This thesis has expanded upon the tolerance criteria for repeated blast exposures. These studies have also further characterized the pathological changes in microglial activation and explored promising therapeutic pathways that could be used to attenuate functional deficits. Lastly, this thesis has also provided novel ways to interrogate neuronal networks following blast injury, revealing subtle deficits that will need to be explored in more detail.

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