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.

Damage to articular cartilage, whether through degeneration (i.e. osteoarthritis) or acute injury, is particularly debilitating due to the tissue's limited regenerative capacity. These impairments are common: nearly 27 million Americans suffer from osteoarthritis and 36% of athletes suffer from traumatic cartilage defects. Allografts are the preferred treatment for large cartilage defects, but demand for these tissues outweighs their supply. To generate additional replacement tissues, tissue engineering strategies have been studied as a cell-based alternative therapy. Our laboratory has had great success repeatedly achieving native or near-native mechanical and biochemical properties in grafts engineered from chondrocyte-seeded agarose hydrogels. The most common iteration of this technique yields a disk of ~4 mm diameter and ~2.3 mm thickness. However, much work is still needed to increase the potential for clinical translation of this product. Our laboratory operates under the premise that in vivo success is predicated on replicating native graft properties in vitro. Compared to these engineered grafts, native grafts are larger in size. They consist of cartilage, which has properties varying in a depth-specific manner, anchored to a porous subchondral bone base. They are able to be stored between harvest and transplantation. This dissertation presents strategies to address a subset of the remaining challenges of reproducing these aspects in engineered grafts. First, graft macrostructure was addressed by incorporating a porous base to generate biomimetic osteochondral grafts. Previous studies have shown advantages to using porous metals as the bony base. Likewise, we confirmed that osteochondral constructs can be cultured to robust tissue properties using porous titanium bases. The titanium manufacturing method, selective laser melting, offers precise control, allowing for tailoring of base shape and pore geometry for optimal cartilage growth and osteointegration. In addition to viability studies, we investigated the influence of the porous base on the measured mechanical properties of the construct's gel region. Through measurements and correlation analysis, we linked a decrease in measured mechanical properties to pore area. We promote characterization of such parameters as an important consideration for the generation of functional grafts using any porous base. Next, we investigated a high intensity focused ultrasound (HIFU) denaturation of gel-incorporated albumin as a strategy for inducing local tissue property changes in constructs during in vitro growth. HIFU is a low cost, non-contact, non-invasive ultrasound modality that is used clinically and in the laboratory for such applications as ablation of uterine fibroids and soft tissue tumors. Denaturing such proteins has been shown to increase local stiffness. We displayed the ability incorporate albumin into tissue engineering relevant hydrogels, alter transport properties, and visualize treatment with its denaturation. HIFU treatment is aided by the porous metal base, allowing for augmented heating. Though heating cartilage is used in the clinic, it is associated with cell death. We investigated this effect, finding that the associated loss of viability remains localized to the treatment zone over time. This promotes the option of balancing desired changes in tissue properties against the concomitant cell viability loss. In order to match clinically utilized allografts, engineered constructs must be scaled up in size. This process is limited by diffusional transport of nutrients and other chemical factors, leading to preferential extracellular matrix deposition in the construct periphery. Many methods are being investigated for overcoming this limitation in fixed-size constructs. In this chapter, we investigated a novel strategy in which small constructs are cultured for future assembly. This modular assembly offers coverage of variable sized defects with more consistent growth with more uniform distribution of biochemical constituents than large constructs cultured on their own. Physiologic failure testing showed that integration of these tissues may be strengthened by increased subunit strength or assembled culture. It is expected that bioadhesive caulking and/or the incorporation of osteochondral bases would further increase integration of the assembled large graft. Finally, we sought to provide a preservation/storage protocol for engineered cartilage constructs. Such a technique is critical for clinical translation, providing the engineered graft with a “shelf-life.” We adopted and evaluated the Missouri Osteochondral Allograft Preservation System (MOPS), which had been shown to maintain cell viability in native grafts for at least 63 days at room temperature without serum or growth factors. Within the current clinical of 28 days, MOPS maintained chondrocyte viability and 75% of the pre-preservation Young's modulus without significant decline in biochemical content, however it did not extend the clinical window as it had with native grafts. Refrigeration with MOPS did not show any benefit at day 28, but proved better with longer preservation times. These results are the first evaluating engineered cartilage storage. Further optimization is necessary to extend storage tissue property maintenance in storage. Overall, this dissertation presents four strategies for increasing the translation potential of engineered articular cartilage grafts by better matching the clinically utilized native allograft system. Combining these techniques, one could ideally engineer small, interlocking ostechondral constructs with HIFU modified interface properties, which could be stored from maturity to implantation. Future optimization is required to better understand and utilize these methods to engineer fully functional, clinically relevant grafts.

Osteoarthritis is a debilitating joint disease characterized by the degradation of articular cartilage due to long term wear or acute injury. OA can lead to pain, limited mobility, and stiffness in the joint, and current treatment options often require invasive surgery or are limited to corrective attempts at mitigating pain. Due in part to the complexity of the disease and lack of holistic understanding of its advancement, there is no known treatment to halt or reverse the effects of OA progression in the joint. In order to address this need, the underlying mechanisms that drive the mechanical degradation of cartilage structure in its progression must be determined. The objective of this dissertation is to (1) investigate the mechanical breakdown of cartilage through fatigue failure in physiologically relevant models and (2) to introduce a minimally invasive method for increasing the mechanical integrity of cartilage in an effort to reverse the effects of OA. In order to classify the mechanical mediation of wear in OA disease pathology, wear progression in human articular cartilage must be fully characterized. Human articular cartilage exhibits a remarkable resilience to wear during frictional sliding, making it difficult to induce damage in the tissue in experimental models. Previous work established reciprocal compressive stresses, and not frictional stresses, as the primary initiator of delamination fatigue wear in immature bovine cartilage. In Chapter 2, we tested the hypothesis that reciprocal compressive stresses could induce fatigue wear in human articular cartilage and thus establish a reproducible and characterizable model of wear induction in human tissue. Human articular cartilage was subjected to 24 hours of frictional sliding in two contact configurations: stationary contact area (SCA), and migrating contact area (MCA). Five samples were tested in the SCA configuration, which induces frictional stresses, and five were tested in the MCA configuration, which induces reciprocal compressive stresses and frictional stresses. The SCA samples showed no conclusive damage after 24 hours of sliding, and recovered 99.3% ± 2.34% of their original thickness after testing. Three out of five MCA samples showed conclusive signs of damage, one in the form of tissue splitting, one in the form of blister formation, and one in the form of complete tissue tearing. The average friction coefficient in the SCA group (μ_SCA= 0.090 ± 0.008) was higher than the average friction coefficient in the MCA group (μ_MCA= 0.066±0.020; p=0.03). Although conducted as two separate studies, the results in Chapter 2 provide a preliminary data set to suggest that reciprocal compressive stresses are responsible for fatigue failure in human tissue, coherent with the results in the immature bovine model. Additionally, results of Chapter 2 establish a reproducible and physiologically relevant protocol for damage induction in human tissue. Future work will investigate this hypothesis with directly paired SCA and MCA human articular cartilage tissue samples of similar OA grade. To further understand cartilage damage mechanics in physiologically relevant conditions, Chapter 3 and 4 investigate the role of synovial fluid in fatigue failure of immature bovine cartilage. Synovial fluid is often incorrectly identified as the source of low friction in cartilage sliding. In fact, it has an effect on the friction coefficient that is far secondary to interstitial fluid load support. Further, reciprocal compressive stresses, not frictional stresses, have been shown to be responsible for fatigue failure. It is imperative to understand the function of synovial fluid in wear mechanics. We tested the hypothesis that synovial fluid reduces the rate of fatigue failure in immature bovine articular cartilage due to the protective effects of its molecular constituents. Eight paired medial and lateral tibial plateaus were tested in MCA sliding in phosphate buffer saline (n=8) or synovial fluid (n=8) to directly compare fatigue rate in synovial fluid versus phosphate buffer saline. An additional study evaluated the effect of molecular constituents on wear rate by testing medial and lateral tibial plateaus in 50% (n=8) and 25% (n=8) synovial fluid diluted with phosphate buffer saline. All eight samples tested in phosphate buffer saline damaged after 24 hours of reciprocal sliding, and none of the samples tested in pure synovial fluid became damaged over the same duration. After an additional two days of sliding, two of eight samples tested in pure synovial fluid got damaged. In the samples tested in 50% and 25% synovial fluid-phosphate buffer saline dilutions, one sample and five samples got damaged after 72 hours of sliding respectively. The results of this study confirmed the hypothesis that synovial provides a protective effect against fatigue failure. The study also suggests that dilution of the synovial below a critical value reduces the concentration of molecular constituents available to protect the cartilage against damage. Chapter 4 investigates the mechanism of synovial fluid’s protective effect further, by examining its potential to extend the duration of elevated fluid load support under compression and thereby reduce cartilage susceptibility to fatigue. The results of Chapter 4 illustrated that synovial fluid had no effect on the stress relaxation response of the cartilage to unconfined compression, disproving the presented hypothesis. Therefore, future work will investigate the function of synovial fluid in reducing the rate of fatigue, independent of its effect on friction. The final two studies of this dissertation present a novel treatment modality to induce collagen crosslinks that enhance the cartilage equilibrium modulus. The technique introduced is presented as a minimally invasive alternative to current surgical interventions and proposes to increase the integrity of early-OA tissue. In Chapter 5, we investigate the hypothesis that low-level femtosecond laser treatment of cartilage can increase the stiffness of the equilibrium modulus without damaging tissue integrity or cell viability. In the first experiment, six immature bovine cartilage samples were treated with the laser and the equilibrium modulus was found to increase in stiffness (p<10⁻³). The technique was also applied to human articular cartilage tissue with “low” and “high” OA, and tissue was found to have an increase in equilibrium modulus (p=0.003 and p=0.03, respectively). Cell viability was preserved under these treatment conditions. Chapter 6 further outlines a safe envelope of laser treatment parameters through evaluation of the effect of thermal heating on the equilibrium modulus of cartilage samples. The results of this study found that temperatures above 65 ℃ (p<10⁻³) increase the tissue modulus, but no change in modulus occurs below 65 ℃ (p=1.00). The results of Chapter 6 provide an insight to the mechanical effect of thermal exposure, and informed the laser treatment parameters presented in Chapter 5, which were confirmed to produce thermal heating far below temperatures that result in thermal stiffening. Through the results presented in Chapter 5 and 6, preliminary data is provided to introduce a novel method for crosslink induction in the superficial zone of articular cartilage. In future work, this technique can be applied as a potential strategy to increase fatigue wear resistance, and to reduce the progression of OA in diseased tissue. The work presented in this dissertation seeks to contribute to the understanding of fatigue wear in articular cartilage under physiologically relevant conditions, as well as introduce a method for enhancing cartilage tissue properties with laser treatment. In the first half of the dissertation, the effect of reciprocal compressive stresses was evaluated in human articular cartilage tissue and in immature bovine cartilage immersed in synovial fluid in an effort to understand the mechanism of delamination fatigue failure in OA progression. In the second half, a laser treatment modality was shown to increase tissue equilibrium modulus stiffness without compromising tissue viability.

Post-traumatic osteoarthritis (PTOA) of the knee is a complication resulting from direct injury to the joint, such as anterior cruciate ligament and meniscus tears, and accounts for approximately 12% of all OA cases. The economic and clinical impact of PTOA is also greater than idiopathic OA, as patients are younger and often more active, requiring treatments for symptomatic OA over a greater fraction of their lifetime. A common strategy to manage pain and inflammation associated with PTOA is the intraarticular administration of corticosteroids. However, these injections are limited due to the requirement of high-doses imposed by synovial joint clearance rates and their resulting systemic side effects. In addition, currently used broad-spectrum corticosteroids are palliative and not curative, stemming from incomplete knowledge of specific mechanisms that drive cartilage degeneration and other joint pathologies. Thus, most patients with PTOA eventually undergo surgical procedures such as osteochondral graft transplantation for focal defects and in more severe cases, total knee arthroplasty. As such, the studies presented in this dissertation (i) offer specific insights into mechanisms by which traumatic injury can drive joint degeneration and (ii) present novel strategies to modulate joint responses to pathological factors by leveraging sustained drug-delivery platforms. In Part I, mechanistic assessments of human cartilage and synovium responses to insults are conducted to identify novel pathways that may lead to impaired joint homeostasis. First, a direct consequence of traumatic injury, hemarthrosis, is explored as a potential contributor to the development of PTOA specifically through contributions by red blood cells. We demonstrate for the first time the differential roles of erythrocytes in their intact and lysed states through measures of oxidative stress and changes to metabolomic profiles in the context of ferroptosis. Furthermore, we demonstrate the therapeutic potential of Ferrostatin-1, a lipophilic radical scavenger in inhibiting pathological changes to cartilage and its crosstalk with the neighboring synovium in an in vitro model of hemophilic arthropathy. Second, a strategy to prevent an indirect consequence of traumatic injury, arthrofibrosis, is presented in an in vitro model of joint contraction. Fibrosis and the presence of hyperplastic synovium are implicated in the progression of OA through pathological shifts in tissue composition as well as secreted factors that promote cartilage degeneration and the maintenance of a pro-inflammatory joint environment. A type I transforming growth factor beta-1 receptor inhibitor, SB-431542, is encapsulated in polymeric microspheres for the prophylactic treatment of arthrofibrosis through sustained low-dose drug delivery to circumvent the challenges associated with resident joint clearance rates. Utilizing human-based in vitro models of cartilage and synovium pathology, we present novel mechanisms and therapeutic strategies to prevent pathological changes following traumatic joint injury that may contribute to the development of PTOA. In Part II, the sustained delivery platform introduced in Part I is extended to the treatment of PTOA. Osteochondral graft transplantation is currently the clinical gold standard for large focal cartilage lesions. However, allograft procedures are limited due to the lack of available donor tissues and autografts are associated with complications due to donor-site morbidity. In both cases, grafts are subject to failure, potentially in part due to the continual presence of pro-inflammatory factors following surgical procedure. In this section, we present cellular agarose hydrogels embedded with dexamethasone-releasing microspheres that are integrated with a titanium base as a functional tissue-engineered alternative to native osteochondral allografts. These allogenic tissue-engineered grafts were assessed in an in vivo preclinical canine model in their ability to maintain clinical function and to modulate the inflammatory response over the course of 12 months. We successfully demonstrated the feasibility of using engineered grafts by comparing clinical measures of range of motion, function, lameness, and pain, as well as modified cartilage graft scores, against native osteochondral allograft controls. In addition, improvements in the histopathological scoring of neighboring synovial and meniscal tissues indicate the therapeutic capacity of dexamethasone released from within the joint to modulate the inflammatory response up to one-year post-implantation. Taken together, the studies presented in this dissertation identify novel mechanisms behind pathological changes to the cartilage and synovium that may contribute to the development of PTOA following injury. Potential therapeutic targets, inhibitory compounds, and delivery strategies are also assessed using human-based in vitro models of disease and further validated in an in vivo canine model through a clinically relevant timeframe. Ultimately, we demonstrate for the first time, the use of dual-function tissue-engineered grafts in a weight-bearing region of the knee joint to circumvent limitations associated with the clinical gold standard for the treatment of large focal cartilage defects.