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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21019号 / 医博第4365号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 安達 泰治, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Articular cartilage degeneration, called osteoarthritis, in the hip joint is a serious condition that affects millions of individuals yearly, with limited clinical solutions available to prevent or slow progression of damage. Additionally, the effects of high-risk factors (e.g. obesity, soft and hard tissue injuries, abnormal joint alignment, amputations) on the progression of osteoarthritis are not fully understood. Therefore, the objective of this thesis is to generate a finite element model for predicting osteochondral tissue stress and strain in the human hip joint during gait, with a future goal of using this model in clinically relevant studies aimed at prevention, treatment, and rehabilitation of OC injuries. A subject specific finite element model (FEM) was developed from computerized tomography images, using rigid bones and linear elastic isotropic material properties for cartilage as a first step in model development. Peak contact pressures of 8.0 to 10.6 MPa and contact areas of 576 to 1010 mm2 were predicted by this FEM during the stance phase of gait. This model was validated with in vitro measurements and found to be in good agreement with experimentally measured contact pressures, and fair agreement with measured contact areas.

Mechanical properties of articular cartilage, including stiffness, biolubrication, and wear-resistance, undergo deterioration during progression of diseases such as osteoarthritis. When the tissue becomes softened and wear-prone, resulting from biochemical alterations within the cartilage matrix, osteoarthritis patients experience painful joint degeneration and erosion of the bone-protective cartilage. Moreover, the synovial fluid bathing the cartilage also experiences a reduction in lubricating capacity as osteoarthritis advances, further hastening wear. An existing treatment paradigm known as viscosupplementation, designed to restore a viscous and lubricating nature to the synovial fluid, involves intraarticular injection of hyaluronic acid into affected joints. While this technique relieves pain for some individuals, the majority of patients experience neither pain relief nor protection of the cartilage from further damage. To address the unmet need of patients requiring chondroprotective thera-pies, this dissertation describes two potential intraarticular strategies based on the application of polymer chemistry principles to bodily tissues and interfaces. One strategy involves the synthesis of a non-hyaluronic-acid synovial fluid sup-plement, based on a phosphorylcholine-containing polyacrylate network, de-signed to functionally mimic the lubricity of the glycoprotein lubricin, phospho-lipid macromolecular assemblies, and high molecular weight hyaluronic acid. The second strategy involves the in situ photopolymerization of a related polyacrylate within cartilage bulk tissue to strengthen, prevent wear, and in-crease the proportion of compressive load supported by the tissue’s interstitial fluid rather than solid matrix. In this strategy, the branched polymer network functionally mimics the glycosaminoglycans that are found in healthy cartilage but depleted in osteoarthritic cartilage. For both potential therapies, chemical and physical properties of the respective fluid and tissue are analyzed, and ex vivo cartilage mechanical testing involving axial and shear deformation reveal the biotribological and compressive reinforcement conferred by the zwitterionic polymer. The synovial fluid supplement significantly decreases cartilage friction through a variety of lubrication mechanisms depending upon tissue fluid flow state and articulation conditions, and the cartilage-reinforcing supplement pro-tects cartilage during accelerated wear testing while also improving synovial flu-id’s ability to lubricate polymer-impregnated cartilage. The fundamental tissue—biomaterial tribological interactions investigated in this dissertation will inform the rational design of therapeutic, friction-reducing polymers for diverse applications. / 2019-04-04T00:00:00Z

<p>  </p> <p>Articular cartilage is a strong but flexible connective tissue that covers and protects the ends of long bones. Osteoarthritis (OA) is a degenerative joint disease which is the most prevalent type of arthritis. The progression and development of OA involves changes in cartilage composition and tissue degradation. As a result, the biomechanical and biotribological properties of the joint may be affected. It has not been determined how cartilage composition and mechanical properties affect its wear and friction, or if there are feasible strategies to improve cartilage performance.</p> <p>Photochemical crosslinking is one method to enhance the modulus and strength of collagenous tissues and improve their resistance to enzymatic degradation. In chapter 2, the effect of photochemical crosslinking on viscoelastic properties of cartilage using an indentation test were investigated. Results of the study indicated that chloro-aluminum pthalocyanine tetrasulfonic acid (CASPc) photo-initiator and 670 nm light increases the modulus of articular cartilage, though this effect is likely limited to the tissue surface.</p> <p>The objective of research described in chapter 3 was to assess correlations between tissue composition and modulus with friction and wear properties in healthy cartilage specimens. Viscoelastic properties of cartilage were obtained via indentation and then the coefficient of friction was measured during an accelerated <em>in vitro</em> wear test. The composition of adjacent cartilage tissue including collagen, glycosaminoglycans, and pyridinoline crosslinks were obtained by biochemical assays. Correlation analysis suggested that stiffer cartilage with higher glycosaminoglycans (GAGs) and collagen content leads to higher wear resistance of the cartilage. Enzymatic collagen crosslinks in type II collagen, pyridinoline (PYD), also enhances the wear resistance of the collagen network. The three parameters of wear, composition, and mechanical properties of cartilage were interrelated and were all correlated with one another. However, friction was independent of these in healthy cartilage tissue. </p> <p>In chapter 4, the hypothesis that mechanical wear is exacerbated in degraded cartilage tissue was tested. Fresh osteochondral specimens were treated with interleukin-1β, with chondroitinase ABC (ChABC) to specifically remove GAGs, or with collagenase to degrade the collagen network during culture. Viscoelastic properties of the tissues were characterized followed by an accelerated <em>in vitro</em> wear test. Results of this study suggest that although the degradation of cartilage was observed with exposure to IL-1β, ChABC and collagenase, wear was not uniform between the three. All three treatments lost GAGs across their superficial zone, and tissue loss due to wear appeared to be confined to the superficial zone. The passive loss of GAGs did not induce increased wear of the tissue. However, an increase in wear was observed with degradation of the collagen network. As the COF was not affected by the degradative treatments, the changes in wear were attributed to alterations in tissue structure and composition</p> <p>Finally, in chapter 5, conclusions, and summary of all main three chapters were stated and directions for future studies were presented.  </p>

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13518号 / 論医科博第10号 / 新制||医科||10(附属図書館) / (主査)教授 金子 新, 教授 松田 秀一, 教授 山中 伸弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Osteoarthritis is a highly prevalent, debilitating disease characterized by the wear and degradation of articular cartilage. While many surgical interventions exist, few are consistently effective and those that are effective are not necessarily suitable for all patients. The objective of this dissertation is to improve patient care through the development of a new surgical technique and through basic science studies which seek to better understand articular cartilage wear initiation. Four studies, which address this objective are summarized below. Osteochondral allograft transplantation provides a safe and effective treatment option for large cartilage defects, but its use is limited partly due to the difficulty of matching articular surface curvature between donor and recipient. We hypothesize that bendable osteochondral allografts may provide better curvature matching for patella transplants in the patellofemoral joint. The finite element study presented in Chapter 2 investigates patellofemoral joint congruence for unbent and bendable osteochondral allografts, at various flexion angles. Finite element models were created for 12 femur-patella osteochondral allograft pairings. Two grooves were cut into the bony substrate of each allograft, allowing the articular layer to bend. Patellofemoral joints with either unbent (OCA) or permanently bent (BOCA) allografts were articulated from 40 to 70 degrees flexion and contact area was calculated. OCAs and BOCAs were then shifted 6 mm distally toward the tibia (S-OCA, S-BOCA) to investigate the influence of proximal-distal alignment on congruence. On average, no significant difference in contact area was found between native patellofemoral joints and either OCAs or BOCAs (p > 0.25), indicating that both types of allografts restored native congruence. This result provides biomechanical support in favor of an emerging surgical procedure. S-BOCAs resulted in a significant increase in contact area relative to the remaining groups (p < 0.02). The fact that bendable osteochondral allografts produced equally good results implies that these bendable allografts may prove useful in future surgical procedures, with the possibility of transplanting them with a small distal shift. Surgeons who are reluctant to use osteochondral allografts for resurfacing patellae based on curvature matching capabilities may be more amenable to adopting bendable osteochondral allografts. The recent development of bendable osteochondral allografts provides the potential for improved osteoarthritis treatment for joints whose current treatment is unsatisfactory. One such joint is the carpometacarpal joint in the thumb. While the current standard of care for carpometacarpal osteoarthritis, ligament reconstruction and tendon interposition, can reduce pain in the joint, it does not restore full joint function and mobility. A proposed alternative includes using an osteochondral allograft harvested from the femoral trochlea in a donor knee, machining grooves in the bone to allow the allograft to bend, and replacing the trapezium with this bent osteochondral allograft [1,2]. Chapter 3 of this dissertation discusses adjustments to the original design of the bendable allograft and the design of a custom surgical tool to perform the proposed surgery. Specification changes of the allograft included an overall size reduction in order to better fit within the carpometacarpal joint, minimum bone thickness requirements to avoid bone cracking during the surgical procedure, and a reduction from three grooves to two grooves, which provided sufficient bending yet avoided fracture of the allograft. The surgical tool was designed to be a custom forceps device, whose primary features included (1) jaws with an angled face to match the angle of allograft bending and (2) insertion holes for the Kirschner wire and compression screws used to anchor the allograft in the bent position. These customizations allow the tool to be used to bend the allograft, fix it in the bent configuration, and place the allograft in its proper position in the hand during anchoring of the bent allograft to the native trapezium. The final two studies presented in this dissertation focus on furthering our current understanding of wear and structure-function relationships of articular cartilage. We hypothesize that cartilage wears due to fatigue failure in reciprocating compression instead of reciprocating friction. Chapter 4 compares reciprocating sliding of immature bovine articular cartilage against glass in two testing configurations: (1) a stationary contact area configuration (SCA), which results in static compression, interstitial fluid depressurization and increasing friction coefficient during reciprocating sliding, and (2) a migrating contact area configuration (MCA), which maintains fluid pressurization and low friction while producing reciprocating compressive loading during reciprocating sliding. Contact stress, sliding duration, and sliding distance were controlled to be similar between test groups. SCA tests exhibited an average friction coefficient of μ=0.084±0.032, while MCA tests exhibited a lower average friction coefficient of μ=0.020±0.008 (p<10^(-4)). Despite the lower friction, MCA cartilage samples exhibited clear surface damage with a significantly greater average surface deviation from a fitted plane after wear testing (R_q=0.125±0.095 mm) than cartilage samples slid in a SCA configuration (R_q=0.044±0.017 mm, p=0.002), which showed minimal signs of wear. Polarized light microscopy confirmed that delamination damage occurred between the superficial and middle zones of the articular cartilage in MCA samples. The greatest wear was observed in the group with lowest friction coefficient, subjected to cyclical instead of static compression, implying that friction is not the primary driver of cartilage wear. Delamination between superficial and middle zones imply the main mode of wear is fatigue failure under cyclical compression, not fatigue or abrasion due to reciprocating frictional sliding. The final study of this dissertation, presented in Chapter 5, investigates the importance of collagen fibril distribution in articular cartilage computational models. Finite element models were created to approximate a bovine humeral head and replicate previous experimental loading conditions [3]. Five different finite element analyses were run, each using a different fibril distribution model. Three of the models used two, four, or eight discrete fibril bundles, while two models used continuous fibril distributions with either isotropic or depth-dependent ellipsoidal distributions. Two primary findings arose from this investigation. The first was the discovery that as the fibril distribution became more isotropic, the strain throughout the tissue decreased, even though the contact area between the articular surface and rigid platen remained relatively equal across distribution models. This suggests that computational models which approximate the collagen fibrils with an isotropic distribution may be underestimating the strain through the depth of the tissue. The second primary finding was that in the discrete distribution model with two fibril bundles, which followed the classically described Benninghoff structure [4], the greatest magnitude of shear strain during compressive loading was observed in the middle zone. However, the highest magnitude of shear strain observed in the isotropic fibril distribution model occurred in the deep zone near the subchondral surface. The observed results suggest that the type of fibril distribution used to model collagen in articular cartilage plays a role in depth-dependent strain magnitude and strain distribution.

Osteoarthritis (OA) is a prevalent joint disease without a cure and leading cause of disability worldwide. Knee OA is the most common and expected to increase with a growing, aging population. OA is clinically diagnosed by the measure of joint space narrowing on a radiograph and pain scores, though OA is now known to be a disease of the whole joint and characterized by cartilage degradation, subchondral bone sclerosis, synovitis, meniscal erosion and inflammation. There are currently no disease-modifying therapies because the pathogenesis and progression of these events are unknown. Recent studies have demonstrated the importance of subchondral bone in OA initiation. Using an advanced imaging technique called individual trabecula segmentation (ITS) to decompose the trabecular network into individual plates and rods, subtle microstructural changes were identified beneath both intact and damaged cartilage in human OA. These findings were also observed during OA initiation before cartilage changes in a model of guinea pig OA. The aims of this study were to further investigate these bone microstructural changes and investigate how they affect the mechanical properties of trabecular bone and overlying articular cartilage. In the first aim, the Dunkin-Hartley guinea pig model of spontaneous OA was used to quantify ITS-based trabecular microstructural changes in the knee joint during OA initiation in both a longitudinal and cross-sectional analysis. In the longitudinal study, microstructural changes in the subchondral bone were quantified from μCT images and voxel-based modeling and remodeling. In the cross-sectional study, structural, biomechanical and biochemical properties of articular cartilage, subchondral bone plate and trabecular bone were analyzed. In both the longitudinal and cross-sectional analyses, there was a trend towards increased thickness, a significant decrease in porosity, and increased mineralization in the subchondral bone plate. There was an increase in the plate-to-rod (PR) ratio due to a loss of trabecular rods, an increase of trabecular plates, and thickening of trabecular plates before any visible histological changes in the cartilage. Voxel-based bone modeling and remodeling analysis confirmed that there was a loss of rods and not merely that the rods were turning into plates. This confirmed our hypothesis that trabecular rod loss precedes cartilage damage and could be a potential therapeutic target. There was a trend towards an increase in the apparent elastic modulus of bone followed by a reduction in the modulus, with the stiffest and most drastic reduction in the medial tibial plateau, which coincided with cartilage fibrillations and a trend towards reduction in the cartilage aggregate modulus. The tissue-level mechanical properties of the trabecular bone are due to both microstructural changes in the trabecular network and material changes in the tissue modulus. Micro-indentation of the trabecular bone revealed a trend towards an increase in the tissue modulus in the medial tibial plateau, followed by a reduction in the tissue modulus. This suggested that during OA progression, there is rapid formation of lower-quality bone with a reduced capacity to mechanically support the joint. In summary, there were structural and mechanical bone changes observed before histological, mechanical or biochemical changes in the articular cartilage. In the second aim, a mechanically-driven subchondral bone computational model was developed. Under equilibrium conditions to simulate aging, there was no change in bone volume fraction and there was a shift from plate-like to rod-like trabecula. There was a slight decrease in the apparent elastic modulus of the bone. Under increased applied strain to simulate the effects of obesity, there was an increase in bone volume fraction, due to both a rod loss and plate thickening. This change came from rod loss, rods thickening and becoming plates, as well as rods and plates merging together. These microstructural changes caused an increase in the apparent elastic modulus of bone. This study demonstrated that the microstructural changes observed in OA can be simulated by increasing the applied strain. Taken together, these studies demonstrate how bone microstructural changes in OA initiation affect the mechanical integrity of the subchondral bone and could cause abnormal stress distributions in the overlying cartilage and promote cartilage degradation. Therapies that prevent bone loss, like bisphosphonates, could be investigated to prevent this initial rod loss as a means of potentially slowing or reversing OA progression.

No description available.

Articular cartilage lesions are often caused by joint trauma and can progress to osteoarthritis (OA) if left untreated. Cartilage tissue engineering is a promising approach for chondral lesion repair, involving the cultivation of cell-seeded scaffolds to generate neocartilage tissues recapitulating composition, structure, and function of native cartilage. Transforming growth factor beta (TGF-β) is widely utilized in cartilage tissue engineering for its ability to promote chondrogenesis and extracellular matrix (ECM) biosynthesis. Conventionally, TGF-β is supplemented in culture medium at supraphysiologic doses (10-100 ng/mL) during in vitro cultivation to regenerate neocartilage with native-matched sGAG content and mechanical properties. However, these doses are 10-1000-fold higher than the physiologic range, promoting undesirable tissue features that are detrimental to the functional behavior of hyaline cartilage. Additionally, TGF-β gradients from media supplementation can induce pronounced heterogeneities in ECM distribution, potentially compromising the survival of engineered cartilage under physiologic loading. The dissertation aims to enhance cartilage regeneration quality using bio-inspired latent TGF-β (LTGF-β) conjugated scaffolds. We hypothesize that LTGF-β scaffolds can achieve uniform delivery of moderated, near-physiologic doses of TGF-β through cell-mediated activation, inducing homogeneous and more hyaline cartilage-like tissue growth. We first evaluated the impact of physiologic TGF-β doses on tissue growth. To address issues related to TGF-β concentration gradients and tissue heterogeneities, we employed a reduced-size construct model. Our findings demonstrate that physiologic doses of TGF-β promote significant enhancements in tissue properties for reduced-size tissues, while also mitigating undesirable outcomes associated with excessive TGF-β. Subsequently, we developed bio-inspired LTGF-β-conjugated scaffolds to deliver physiologic doses of TGF-β. We established a quantification platform based on TGF-β autoinduction to accurately measure the bioactivity level of delivered TGF-β, confirming conjugated LTGF-β can be activated in physiologic range. Further, this quantification platform exhibits versatility for applications in native tissue studies and other TE platforms. Lastly, we determined that LTGF-β conjugation led to enhancements in tissue functional properties comparable to native tissue, while mitigating the abnormal features of neocartilage associated with TGF-β excesses. Moreover, LTGF-β conjugation significantly improves tissue spatial homogeneities in composition and mechanical properties, offering promising implications for enhancing clinical regeneration outcomes.