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Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Boyan, Barbara; Committee Member: Bonassar, Lawrence; Committee Member: Sambanis, Anthanassios; Committee Member: Schwartz, Zvi; Committee Member: Wick, Timothy.

Joint disorders significantly affect quality of life and present unique challenges for tissue engineering. In the craniofacial space, and especially for the temporomandibular joint (TMJ), there is an unmet need for anatomically precise and mechanically robust cartilage and bone tissues to recapitulate native function. Current surgical reconstruction methods, whether using autologous or synthetic options, suffer from imprecision, comorbidities, complications, and frequently require subsequent operations. Furthermore, many craniofacial graft efforts have focused on improving bone without addressing cartilage, which is essential to proper TMJ function. Thus, there is a compelling need to engineer a human-sized, biologically and anatomically matched cartilage-bone TMJ replacement. This dissertation demonstrates the ability to generate such a graft with native-like properties in a human-sized large animal model by focusing on two aims: (i) establish methods to fabricate and culture anatomically specific, autologous cartilage-bone grafts (Aim 1), and (ii) show improvement of graft performance after six months implantation in vivo compared to previous methods, controls, and native tissue (Aim 2). Using Yucatan mini-pigs as a human-sized model, the ramus-condyle unit (RCU), a geometrically intricate portion of the mandible and primary load bearing section of the TMJ, was targeted for reconstruction. Scaffolds were created using computer tomography (CT) image-guided micromilling of decellularized bone matrix, then infused with autologous adipose-derived chondrogenic and osteogenic progenitors. These biological constructs were then cultured in vitro in a novel dual-perfusion bioreactor before in vivo implantation. Similar in vitro culture of representative constructs done in parallel demonstrated cell attachment and some differentiation. After six months implantation, the dual cartilage-bone RCU grafts maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a significantly greater extent than either bone-only grafts or acellular scaffolds, and showed remarkable similarity to native tissue. Furthermore, tracking of implanted cells enabled additional insights into the progression of cartilage and bone regeneration. The methods and results established in this dissertation form a promising basis for the next evolution in engineering full-sized, patient-specific, and biologically and mechanically robust TMJ replacements.

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.

Articular cartilage underwent serious joint injuries seldom repair spontaneously and might progress to post-traumatic osteoarthritis. This is majorly because articular cartilage’s unique properties that lack blood and nerve supply intrinsically. This peculiar structure, in addition, generates an unfavorable environment for certain phagocytes (macrophages, monocytes, neutrophils, etc) to infiltrate to cartilage to scavenge debris from cartilage matrix and cell caused from joint injuries. Therefore, physiological and functional regeneration of damaged cartilage is urgently needed and several clinical techniques have been developed, including microfracture, autograft transplantation, autologous chondrocytes implantation. We previously identified highly migratory cells emerged and repopulated in cartilage damaged surface after ~10 days of artificial cartilage injury. These cells were later named chondrogenic progenitor cells (CPCs) due to their enhanced potential of chondrogenic differentiation. However, this important finding contrasts the conventional theory that cartilage harbors only one cell type, chondrocytes. Here we hypothesize that CPCs are a distinct cell type in cartilage, and more importantly, one of CPCs’ crucial natures is to phagocytose debris more effectively than chondrocytes. To test these, we first harvested CPCs from cartilage surfaces, chondrocytes, synovial cells (synoviocytes and synovial fluid cells) for microarray assay to evaluate the closeness among these joint cells on whole gene expression level. Quantitative PCR were then conducted to verify gene expression of certain functional interests. Moreover, debris from cell and extracellular matrix were generated and incubated with CPCs and chondrocytes to compare their phagocytic capacity via multiple experimental assessments. In confocal microscopy examination, the emergence of CPCs could be clearly observed after cartilage injury. Aside from their distinguishable morphology compared to chondrocyte, CPCs possess several vital properties including highly migratory, chemotactic, clonogenic. Microarray data revealed that CPCs, from gene expression profile, are distinctively isolated from chondrocytes and are more akin to synovial cells. Additionally, the series of phagocytosis related experiments showed that CPCs are dramatically superior to chondrocytes in engulfing debris, along with enhanced lysosomal activities indicating the following debris degradation. Taken all these data together, CPCs, activated by cartilage injury, emerged and migrated to damaged sites. They are a distinct cell type residing in cartilage apart from chondrocytes. Their enhanced capacity to sustainably phagocytose and clear debris provides a novel insight for cartilage regeneration and prevention of osteoarthritis.

This thesis demonstrated the effects of exogenous stimuli on engineered articular cartilage constructs and elucidated mechanisms underlying the responses to these agents. In particular, a series of studies detailed the effects of chondroitinase-ABC (C-ABC), hyaluronic acid (HA), and TGF-β1 on the biochemical and biomechanical properties of self-assembled articular cartilage. Work with C-ABC showed that this catabolic agent can be employed to improve the tensile properties of constructs. When constructs were cultured for 6 weeks, treating with C-ABC at 2 weeks enhanced the tensile stiffness. Furthermore, treating at 2 and 4 weeks synergistically increased tensile properties and allowed compressive stiffness to recover to control levels. Another study showed that combining C-ABC and TGF-β1 synergistically enhanced the biochemical and biomechanical properties of neotissue. Microarray analysis demonstrated that TGF-β1 increased MAPK signaling in self-assembled neocartilage whereas C-ABC had minimal effects on gene expression. SEM analysis showed that C-ABC increased collagen fibril diameter and fibril density, indicating that C-ABC potentially acts via a biophysical mechanism. Constructs treated with C-ABC and TGF-β1 also showed stability and maturation in vivo , exhibiting a tensile stiffness of 3.15±0.47 MPa compared to a pre-implantation stiffness of 1.95±0.62 MPa. To assess the response to HA application, studies were conducted to optimize HA administration and examine its effects in conjunction with TGF-β1. Applying HA increased the compressive stiffness 1-fold and increased GAG content by 35%, with these improvements depending on HA molecular weight, application commencement time, and concentration. Microarray and PCR analyses showed that HA also influenced genetic signaling, up-regulating multiple genes associated with the TGF-β1 pathway. In addition to genetic effects, the enhanced GAG retention due to HA treatment could increase the fixed charge density of the matrix and thereby increase resistance to compressive loading. Additive effects were observed when HA was applied in conjunction with TGF-β1, with the combined treatment increasing compressive stiffness and GAG content by 150% and 65%, respectively. In general, results demonstrated mechanisms underlying C-ABC, HA, and TGF-β1 treatments and showed how these agents can be applied to improve cartilage regeneration efforts.

Focal damage to cartilage sustained in serious joint injuries typically goes unrepaired and may progress to post-traumatic osteoarthritis. However, in a bovine explant model we found that cartilage damage provoked the emergence of highly migratory cells that homed to the site of injury and appeared to re-populate dead zones. We hypothesized that the migrating population were chondrogenic progenitor cells engaged in cartilage repair. The surfaces of bovine osteochondral explants injured by blunt impact were serially imaged to follow cell migration. Migrating cells harvested from cartilage surfaces were tested for clonogenic, side population, chemotactic activities and multipotency in in vitro assays. Gene expression in migrating cells was evaluated by microarray and their potential for spontaneous cartilage regeneration was assessed in a chondral defect model. Migrating cells emerged from superficial zone cartilage and efficiently repopulated areas where chondrocyte death had occurred. In confocal examination with high magnification, we could clearly observe the morphology of elongated progenitor cells which were migrating toward cartilage defect area and these cells were distinguishable from round chondrocytes. The cells were also activated to migrate in cartilage defect model. Most migrated cells in fibrin were morphologically elongated and a few cells were differentiating to chondrocyte-like cells with the deposit of proteoglycans. These cells proved to be highly clonogenic and capable of chondrogenesis and osteogenesis, but not adipogenesis. They were more active in chemotaxis assays than chondrocytes, showed a significantly larger side population, and over-expressed progenitor cell markers and genes involved in migration, chemotaxis, and proliferation. To active migration of chondrogenic progenitor cells (CPCs) short-term enzymatic method was used around edge of cartilage defect. Surprisingly, CPCs migrated into fibrin defect and were differentiating into chondrocytes with abundant deposit of proteoglycans. This result strongly supports that progenitor cells are activated in traumatic cartilage injury and have great potential for cartilage repair. In conclusion, migrating cells on injured explant surfaces are chondrogenic progenitors from the superficial zone that were activated by cartilage damage to attempt repair. Facilitating this endogenous process could allow repair of focal defects that would otherwise progress to post-traumatic osteoarthritis.

Osteoarthritis is a degenerative joint disease characterized by painful, progressive articular cartilage lesions that deteriorate joint function. It remains leading cause of disability in the United States, affecting nearly 30 million Americans with increasing prevalence in the aging population, which has resulted in an annual economic burden of $128 billion. Symptomatic, full thickness cartilage injuries often require surgical intervention, because the tissue is predominantly avascular and thus has a limited self-healing capacity. However, clinical management strategies including matrix-induced autologous chondrocyte implantation and osteochondral grafting are inadequate in the long-term due to poor integration of cartilage grafts with surrounding host cartilage and subchondral bone. In addition to physical congruence between graft and host cartilage, a structural or chemically functional barrier that limits osseous invasion into the cartilage compartment is critical in order to maintain the integrity of repaired cartilage. Given these significant clinical challenges, the objective of this thesis is to establish design principles for homotypic and heterotypic tissue integration via a cup-shaped fibrous scaffold system that encapsulates cartilage grafts (autologous or engineered), and integrates them simultaneously with host cartilage and bone at their respective interfaces. Additionally, to facilitate clinical translation of the scaffold cup, an innovative “green electrospinning” method is developed using FDA Q3C Class 3 solvents with minimal manufacturing impact on the environment. It is hypothesized that, to fuse cartilage grafts with host cartilage, the walls of the envisioned cup can direct cell migration directly to the graft-host cartilage interface via chemotactic agent delivery, where scaffold electroactivity will encourage cells to deposit a structurally contiguous neocartilage matrix. At the boundary between the graft and underlying bone, the scaffold cup base will mimic the topography and ceramic chemistry of the native osteochondral interface while preventing bone vasculature from growing upwards into the cartilage, guided by the hypothesis that this will enable the formation of a calcified cartilage interface layer that merges the graft and subchondral bone. To test these hypotheses, this thesis began with green electrospinning the scaffold cup walls incorporated with insulin-like growth factor 1 (IGF-1), a well-established chondrocyte chemoattractant that induced cell migration from cartilage autografts towards resulting fibers. Additionally, the walls contained an optimized dose of graphite nanoparticles to impart electroactivity to the fibers. Mimicking the fixed charge density of cartilage in this way promoted chondrocyte proliferation and biosynthesis of a hyaline cartilage-like matrix in vitro, with selective regulation of proteoglycans (biglycan and decorin) and downregulation of collagen type I compared to a graphite-free fiber control. Moreover, the graphite fibers sequestered IGF-1, sustaining release of the growth factor and improving functional graft-cartilage shear integration strength in vitro. In a full thickness defect osteochondral construct repaired with the scaffold cup and implanted subcutaneously in rat dorsi, localized IGF-1 delivery promoted graft-host cartilage interface matrix elaboration with significantly greater integration strength measured with graphite in the cup walls. For integration with subchondral bone, design criteria for the scaffold cup base were set by quantitatively mapping the compositional and morphometric characteristics of healthy and osteoarthritic human osteochondral tissues, and evaluating FEBio simulations of calcified cartilage and polymer-ceramic composite fibers in silico. These analyses established the need for an interdigitating mesh topography and ceramic particle incorporation, which minimize shear and distribute loading across the fibers, respectively, recapitulating the osteochondral interface’s force gradient from cartilage to bone in order to functionally integrate the tissues. Thus, the dose of calcium deficient apatite (CDA) nanoparticles, which capture the high calcium-phosphate ratio and semi-crystalline atomic structure of native bone mineral, was optimized to promote deep zone chondrocyte growth and biosynthesis of a calcified cartilage matrix in vitro. Moreover, CDA enhanced remodeling of the interface in vivo, with undulating fibers preventing osseous upgrowth. Taken together, these findings delineate the importance of strategic biomimicry in scaffold design, specifically with regards to interface regeneration and cartilage integration. The proposed approach is unique in that it utilized cell homing and an electroactive substrate to mimic properties of the cartilage matrix, with a strategy for simultaneous graft integration with host cartilage and bone. Moreover, the cup design is readily adaptable to current cartilage repair techniques including press-fit autografting and cell-based graft implantation, as well as emerging tissue engineered grafting strategies. Beyond cartilage repair, the scaffold design criteria established in this thesis are broadly applicable to integrating other complex tissue systems, and may inform the regeneration of critical soft-soft (muscle-tendon) and soft-hard (tendon- or ligament-bone) interfaces in the musculoskeletal system.

Osteoarthritis (OA) is a debilitating, degenerative joint disease that affects over 32.5 million adults in the United States and nearly 595 million people globally. OA is a major cause of pain and disability and is among the most expensive conditions to treat, carrying an annual healthcare cost of over $16.5 billion. The disease has classically been characterized by the degradation of articular cartilage and subchondral bone; however, changes to the synovium have recently garnered appreciation as synovitis has been linked to OA symptoms and progression. While the importance of the synovium in diarthrodial joint health and pathology is now widely accepted, quantitative structure-function data remains sparse. There is a need to investigate synovium structure-function relationships to better understand the synovium’s role in joint homeostasis and disease. The role of sex-based differences in OA has gained attention as epidemiological studies reveal that the incidence and prevalence of OA is higher in women than in men. Sex as a variable has rarely been considered in preclinical animal studies and in vitro laboratory experiments that explore the mechanisms of OA development and progression. Furthermore, therapeutic approaches for the treatment of OA have not adequately considered sex-based differences. As the population of those at risk for OA grows, the influence of sex-based differences in OA warrants more attention, particularly in the regenerative strategies for cartilage repair. This dissertation seeks to address persistent questions regarding OA etiology and the mechanisms underlying disease progression, as well as strategies to enhance cartilage tissue engineering therapies. The objectives of this dissertations are three-fold: (1) to further the understanding of synovium tribology (2) to develop a tissue-engineered (TE) human synovium to facilitate the study of synovium structure-function relationships and (3) to elucidate sex-based differences in cartilage regenerative medicine strategies. In Chapter 2, we assess the hypothesis that tissue glycosaminoglycan (GAG) content contributes to the low friction properties of the synovium. Bovine and human synovium tribological properties were evaluated using a custom friction testing device. Following proteoglycan depletion, synovium friction coefficients increased while GAG content decreased. In a second study, synovium samples were treated with interleukin-1 (IL) to observe inflammatory-induced structural changes. IL treatment elevated GAG concentration and decreased friction coefficients. No changes to collagen content were observed following IL treatment. For the first time, a relationship between synovium friction coefficient and GAG concentration was demonstrated. The study of synovium tribology is necessary to fully understand the mechanical environment of the healthy and diseased joint. Chapter 3 documents the development of a human TE synovium and its ability to recapitulate native tissue properties and responses to chemical stimuli. A mixed donor population of primary human fibroblast-like synoviocytes was combined with a commercially available extracellular protein mixture to fabricate TE synovium constructs. At baseline, mature TE synovium exhibited characteristics of native synovium such as the formation of an intimal lining and the expression of critical proteins like lubricin, cadherin-11, and collagen type IV. In response to IL and dexamethasone treatment, TE synovium underwent biochemical changes that mimicked the changes observed in human explants. In addition, solute transport measurements were performed to highlight the relationship between synovium extracellular matrix (ECM) composition and its functional properties, resulting in a proposed link between tissue GAG content and diffusion coefficient. A human TE synovium enables the investigation of synovium structure-function relationships in a controlled manner and can serve as a platform for disease modeling and drug screening, which may accelerate the development of new treatments for maintaining joint health that specifically target the synovium. In Chapter 4, sex-based differences in the ECM properties of canine engineered cartilage and in its degradative response to IL insult are evaluated. Isolated chondrocytes from male or female cartilage donors were encapsulated in agarose to create cylindrical cartilage constructs. Mechanical and biochemical measurements demonstrated that the sex of the donor chondrocytes did not influence intrinsic, de novo tissue formation after 42 days of tissue maturation. Following IL treatment, the mechanical, biochemical, and media analyses revealed that the sex of the donor cells did not influence the engineered cartilage’s response to IL insult. By understanding how sexual dimorphism impacts cartilage growth and susceptibility to proinflammatory cytokine insult, we may better direct cell-based strategies for cartilage repair that are personalized to account for patient sex.

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