Browsing by Subject "Cartilage"

Human joints are able to withstand millions of loading cycles with loads regularly more than 3 times an individual's body weight in large part due to the unique bearing properties of articular cartilage, a strong, slippery tissue that covers the ends of long bones. PRG4 is a boundary lubricating glycoprotein present on the cartilage surface and in the synovial fluid surrounding it. While evidence that PRG4 lubricates and preserves normal joint function is strong, little is known of its effect on cartilage surface properties, the mechanism by which it lubricates, or its postulated role of preventing wear on joints. The effect of PRG4 on cartilage friction, wear, structure, morphology, and the mechanisms by which it mediates these factors are studied here. Methods to study these parameters at the microscale using atomic force microscopy are also developed.

Cartilage of mice with the Prg4 gene (which expresses PRG4) deleted is shown to be different in a number of ways from wild type cartilage. The uppermost layer is thicker and less uniform and the surface is rougher and softer. There is also a loss of proteoglycans, structural components of cartilage, from the underlying superficial tissue, and apparent tissue damage in some cases. Wear in the presence of PRG4 in shown to be significantly lower than in its absence, a finding which may have direct implications for prevention and treatment of osteoarthritis. It appears that PRG4 needs to be present in solution, not merely on the cartilage surface to have this effect, indicating that adsorption properties are important for wear prevention.

The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain.

Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.

Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation.

Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.

In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Articular cartilage is a connective tissue that lines the surfaces of diarthrodial joints; and functions to support and distribute loads as wells as facilitate smooth joint articulation. Unfortunately, cartilage possesses a limited capacity to self-repair. Once damaged, cartilage continues to degenerate until widespread cartilage loss results in the debilitating and painful disease of osteoarthritis. Current treatment options are limited to palliative interventions that seek to mitigate pain, and fail to recapitulate the native function. Cartilage tissue engineering offers a novel treatment option for the repair of focal defects as well as the complete resurfacing of osteoarthritic joints. Tissue engineering combines cells, growth factors, and biomaterials in order to synthesize new cartilage tissue that recapitulates the native structure, mechanical properties, and function of the native tissue. In this endeavor, there has been a growing interest in the use of scaffolds derived from the native extracellular matrix of cartilage. These cartilage-derived matrix (CDM) scaffolds have been show to recapitulate the native epitopes for cell-matrix interactions as well as provide entrapped growth factors; and have been shown to stimulate chondrogenic differentiation of a variety of cell types. Despite the potent chondroinductive properties of CDM scaffolds, they possess very weak mechanical properties that are several orders of magnitude lower than the native tissue. These poor mechanical properties lead to CDM scaffolds succumbing to cell-mediated contraction, which dramatically and unpredictably alters the size and shape of CDM constructs. Cell-mediated contraction not only prevents the fabrication of CDM constructs with specific, pre-determined dimensions, but also limits cellular proliferation and metabolic synthesis of cartilage proteins. This dissertation utilized collagen crosslinking techniques as well as ice-templating in order to enhance the mechanical properties of CDM scaffolds and prevent cell-mediated contraction. Furthermore, the decellularization of CDM was investigated in order to remove possible sources of immunogenicity. This work found that both physical and chemical crosslinking techniques were capable of preventing cell-mediated contraction in CDM scaffolds; however, the crosslinking techniques produced distinct effects on the chondroinductive capacity of CDM. Furthermore, the mechanical properties of CDM scaffolds were able to be enhanced by increasing the CDM concentration; however, this led to a concomitant decrease in pore size, which limited cellular infiltration. The pore size was able to be rescued through the use of an ice-templating technique that led to the formation of large aligned grooves, which enabled cellular infiltration. Additionally, a decellularization protocol was developed that successfully removed foreign DNA to the same order of magnitude as clinically approved materials, while preserving the native GAG content of the CDM, which has been shown to be critical in preserving the mechanical properties of the CDM. Altogether, this body of work demonstrated that dehydrothermal crosslinking was best suited for maintaining the chondroinductive capacity of the CDM, and given the appropriate scaffold fabrication parameters, such as CDM concentration and ice-templating technique, dehydrothermal treatment was able to confer mechanical properties that prevented cell-mediated contraction. To emphasize this finding, this work culminated in the fabrication of an anatomically-relevant hemispherical scaffold entirely from CDM alone. The CDM hemispheres not only supported chondrogenic differentiation, but also retained the original scaffold dimensions and shape throughout chondrogenic culture. These findings illustrate that CDM is a promising material for the fabrication of tailor-made scaffolds for cartilage tissue engineering.

Articular cartilage is a highly specialized avascular tissue and consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. The chondrocyte morphology and extracellular matrix properties vary with the depth of cartilage. Some past studies have defined the zonal distribution of a broad range of cartilage proteins in different layers. Based on the variations within each layer, the extracellular matrix can be further distinguished to pericellular, territorial and interterritorial regions. However, most of these studies used guanidine-HCl extraction that leaves an unextracted residual with a substantial amount of collagen. The high abundance of anionic polysaccharide molecules from cartilage adversely affects the chromatographic separation. Scatter oriented chondrocytes only account for the small proportion of the whole tissue protein extraction. However, the density of the cell varies with depth of cartilage as well. Moreover, the physiological status may also altered the extracellular matrix properties. Therefore, a comprehensive strategy to solve all these difficulties are necessary to elucidate the molecular structure of cartilage.

In this study, we used quantitative and qualitative proteomic analysis to investigate various cartilage tissue processing protocols. We established a method for removing chondrocytes from cartilage sections that minimized matrix protein loss. Quantitative and qualitative proteomic analyses were used to evaluate different cartilage extraction methodologies. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. The different extraction methods yielded different protein profiles. For instance, an overwhelming number of collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction.

Subsequently we applied these methods to extract cartilage sections from different cartilage layers (superficial, intermediate and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic). We also utilized lase capture microscopy (LCM) to harvest cartilage sample from individual subregions (territorial and interterritorial regions). The results suggested that there is more unique proteins existed in the superficial layer. By removing the chondrocytes, we were able to identify more extracellular matrix proteins. The phenotyping of cartilage subregions provided the chance to precisely localize the protein distribution, such as clusterin protein. We observed that the guanidine-HCl extractability (guanidine-HCl/ guanidine-HCl + in situ digestion extracts) of cartilage proteins. Proteoglycans showed high extractability while collagen and non-collagenous proteins had lower extractability. We also observed that the extractability might differ with depth of cartilage and also disease states might alter the characters as well.

Laser capture microscopy provides us the access to the cartilage subregions in which only few studies have investigated because of the difficulties to separate them. We established the proteomic analysis compatible-protocol to prepare the cartilage section for LCM application. The results showed that most of the proteoglycans and other proteins were enriched in the interterritorial regions. Type III and VI collagens, and fibrillin-1 were enriched in the territorial regions. We demonstrated that this distribution difference also varied with depth of cartilage. The difference of protein abundance between subregions might be altered because of disease states.

Last we were looking for the post-transliational modification existed in these subregions of cartilage. Deamidation is one of the modification without the enzyme involved. Previous studies have showed that deamidation may accumulated in the tissue with low turnover rate. Our proteomic analysis results suggests that abundance of deamidated peptides also varied in different layers and subregions of cartilage.

We have developed the monoclonal antibody based immunoassay to quantify the deamidated cartilage oligomeric matrix protein within cartilage tissue from different joints (hip and knee) and disease states (healthy, para-lesion, and remote lesion). The results suggests that the highest concentration of deamidated COMP was identified in arthritic hip cartilage.

The results of this study generated several reliable protocols to perform cartilage matrix proteomic analysis and provided data on the cartilage matrix proteome, without confounding by intracellular proteins and an overwhelming abundance of collagens. The discovery results elucidated the molecular architecture of cartilage tissue at different joint sites and disease states. The similarities among these cartilages suggested a constitutive role of some proteins such as collagen, prolargin, biglycan and decorin. Differences in abundance or distribution patterns, for other proteins such as for cartilage oligomaric matrix protein, aggrecan and hyaluronan and proteoglycan link protein, point to intriguing biological difference by joint site and disease state. Decellularization and a combination of extraction methodologies provides a holistic approach in characterizing the cartilage extracellular matrix. Guanidine-HCl extractability is an important marker to characterize the statue of cartilage; however it has not been fully understand. The protein distributions in matrix subregions may also serve as an index to characterize the metabolic status of cartilage in different disease states. A large sample cohort will be necessary to elucidate these characters.

Osteoarthritis (OA) is a common joint disorder, affecting over 27 million Americans. OA is characterized by the degeneration of cartilage tissue, and presents clinically with joint pain, stiffness, and limited range of motion. As such, it is a leading cause of disability in the United States. Current treatment options for OA focus on relieving pain (either pharmacologically or through surgical joint replacement), but do not treat or reverse cartilage degeneration. A main reason for this is that the diagnosis of OA depends on pain and radiographic findings, which are not present until advanced stages of the disease. Development of therapies focused on treating or reversing OA degeneration would therefore be enhanced if OA pathology was detectable at earlier stages of the disease. Because changes in mechanical properties (i.e. the stiffness and permeability) occur in OA cartilage before pain and radiographic features are visible, measurement of cartilage mechanics may be used for earlier assessment of OA degeneration. As cartilage mechanics are traditionally measured in the ex vivo environment, the goal of this dissertation was to develop a noninvasive methodology for measuring cartilage mechanical properties in vivo.

Specifically, the methodology consists of a combination of noninvasive magnetic resonance imaging (MRI) techniques to quantify in vivo cartilage composition and mechanical response, as well as a statistical model predicting cartilage stiffness based on these MRI measurements. Porcine knee joint cartilage was used to develop the statistical model, where stiffness was quantified in the traditional manner using ex vivo mechanical testing. The statistical model was then applied to in vivo data from a cohort of healthy human volunteers, for whom the noninvasive MRI techniques were used to measure the composition and mechanical response of their tibial cartilage. Thus, human tibial cartilage stiffness in vivo was quantified.

Overall, the in vivo estimates of healthy human tibial cartilage stiffness (ranging from 0.39 ± 0.05 MPa to 1.06 ± 0.24 MPa) compare well with ex vivo measurements of human cadaveric tibial cartilage stiffness (ranging from 0.45 ± 0.28 MPa to 0.65 ± 0.25 MPa). This finding supports the validity of the methodology developed in this dissertation. Future work using this in vivo methodology for measuring cartilage mechanical properties has diverse applications regarding cartilage health. For instance, this technique may be used clinically to provide earlier detection of OA pathology, or it may be used in future biomechanics research to evaluate the efficacy of different therapeutic approaches toward ameliorating OA pathology and restoring healthy cartilage mechanics. Therefore, the methodology for measuring cartilage mechanical properties in vivo developed here represents an important contribution to the fields of biomechanics and OA research.

It is possible to investigate in vivo musculoskeletal biomechanics using multimodal medical imaging techniques; however, the analysis of medical image sets is often time-prohibitive. In this dissertation, I outline various projects that utilize magnetic resonance imaging (MRI) scans acquired before and after exercise to quantify cartilage thickness changes incurred by the loading activity. A better understanding of cartilage mechanics is crucial for prediction and prevention efforts related to osteoarthritis, patellofemoral pain, and other musculoskeletal conditions. While this cartilage "stress test'' protocol has been used in the past to investigate knee, ankle, and spine mechanics, this work expands the methodology to the shoulder and hip joints and further addresses the impact of various exercises on the knee joint in different subject populations. For instance, I outline how patellofemoral cartilage deforms after a series of single-legged hops in anterior cruciate ligament-deficient and intact knees, how body mass index impacts patellofemoral cartilage strain and T1rho relaxation times in the context of walking, how tibial cartilage T1rho relaxation times change over the course of the day due to activities of daily living, and how pushups affect glenohumeral cartilage. I also discuss the development and validation of a semi-automated technique to isolate bones from MRIs, which reduces the time required for manual segmentation by approximately 75% and thus significantly improves research efficiency. As an expansion of the semi-automatic segmentation work, I will cover how I developed a technique to assess the minimum moment of inertia along the femoral neck from clinical computed tomography (CT) scans, with the goal of understanding relative fracture risks between individuals with and without diabetes. Finally, I quantify running-induced changes in knee cartilage thickness and composition (as measured by T1rho relaxation times), as well as changes in hip joint bone-to-bone distances and hip cartilage T1rho relaxation times. Running is a known activity linked to patellofemoral pain, yet the underlying etiology of this condition is unknown. As both knee and hip kinematics have been linked to patellofemoral pain, the goal was to assess how running influences these joints biomechanically and biochemically to better understand why people suffer from patellofemoral pain.

Osteoarthritis (OA) is a degenerative disease affecting articular cartilage, leading to loss of its structure and function. Early stage OA is characterized by changes in the extracellular matrix (ECM), including a reduction in proteoglycans (PG) concentration, increased water content within the tissue, and increased synthesis and degradation of matrix molecules with disorganization of collagen network [1, 2]. The ability to noninvasively quantify PG changes in cartilage would therefore be useful for early OA diagnosis, monitoring cartilage response to therapies, and assessing efficacy of cartilage repair procedures [3]. T1rho and T2 weighted magnetic resonance (MR) imaging techniques have been shown to have potential in tracking early biochemical compositional changes within cartilage associated with degeneration [3, 4]. Additionally, this method has the potential to be a powerful tool to better understand how cartilage responds to different loading environments both acutely and over time. The main objective of this work is to validate T1rho and T2 relaxation times as non-invasive measures for the assessment of biochemical and biomechanical properties of cartilage.

The first two studies presented in this work focus on the validation of this T1rho and T2 imaging for non-invasive cartilage assessment. The first study examines both normal and osteoarthritic cartilage containing both OA defect regions and healthy appearing areas. This study aims to comprehensively assess the relationship between OA cartilage composition, biochemical, and biomechanical properties with T1rho and T2 relaxation times in order to validate this technique as an in vivo diagnostic method for early stage OA. The second study utilizes targeted enzymatic depletion of both glycosaminoglycan (GAG) and collagen to determine the specific effect of each ECM component on T1rho and T2 relaxation times. A repeated measures design examines the effect of targeted enzymatic cartilage degradation (to isolate changes in cartilage biochemical composition, mechanical properties, and histology) on the T1rho and T2 relaxation times. These studies utilize confined compression for biomechanical analysis of cartilage, biochemical assays for the determination of S-GAG and collagen content, and histology for visualization of cartilage structure and composition. These measures are compared to the associated T1rho and T2 relaxation times. The results of these studies indicate that increases in T1rho relaxation times are correlated with S-GAG depletion, increased percent extractable collagen, decreases in mechanical strength of cartilage, and areas of OA defects (within which the previously mentioned biomechanical and biochemical conditions exist). Together, the results of these two studies validate T1rho and T2 quantitative imaging techniques for the in vivo diagnosis of early OA and the non-invasive assessment of cartilage biomechanical and biochemical properties.

Altered patterns of mechanical loading can result in morphological and compositional changes to cartilage that lead to cartilage degeneration. Quantitative MR imaging is a unique tool with the potential to provide insight into the relationship between biomechanics and the biophysical environment of cartilage, which is vital to better understanding the development of OA and degeneration of cartilage. The third study presented utilizes T1rho as a method for assessing localized changes to cartilage with dynamic activity. Sagittal MR images were obtained before and immediately after subjects completed a single legged hopping activity to dynamically load cartilage. A system of equally spaced grid points were registered to 3D surface mesh models of the tibial and femoral cartilage surfaces constructed from the MR images. T1rho relaxation times were then determined at each grid point to examine site-specific changes before and after exercise. A significant decrease in relaxation times was found after exercise in both the tibial plateau and the femoral condyle, with a greater decrease observed in the lateral femoral cartilage than in the medial femoral cartilage. No significant correlation between location and exercise was found. At each grid point, T1rho cartilage maps were also divided into superficial and deep regions of cartilage to determine where the greatest changes occurred. Ongoing analysis of the layer specific results will provide insight into where in the cartilage thickness these changes are most localized. The decrease in relaxation times after loading is likely due to the relative increase in PG content that results from the exudation of water from the cartilage ECM due to loading. This study demonstrates how T1rho may be used to non-invasively provide insight into the biophysical environment of cartilage with loading.

T1rho and T2 imaging represent a very powerful tool for the non-invasive assessment of articular cartilage. This work is significant in that it validates this method for the assessment of cartilage biomechanical and biochemical properties. Additionally, these methods can be used in future work to better understand how various risk factors contribute to OA development and to give valuable insight into the connection between biomechanical factors, biochemical composition, and the development of cartilage degeneration.

Articular cartilage is a smooth connective tissue that covers the ends of bones and protects joints from wear. Cartilage has a poor healing capacity, and the lack of treatment options motivates the development of tissue engineering strategies. The widespread cartilage degeneration associated with osteoarthritis (OA) is dramatically accelerated by joint injury, but the defined initiating event presents a therapeutic window for preventive treatments. In vitro model systems allow investigation of OA risk factors and screening of potential therapeutics. This dissertation develops stem-cell based strategies to 1) treat cartilage injury and OA using tissue-engineered cartilage, 2) prevent the development of OA by delivering stem cells to the joint after injury, and 3) study cartilage by establishing systems to model genetic and environmental contributors to OA.

Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are promising human adult cell sources for cartilage tissue engineering, but require distinct chondrogenic conditions. As compared to ASCs, MSCs demonstrated enhanced chondrogenesis in both alginate beads and cartilage-derived matrix scaffolds.

We hypothesized that MSC therapy would prevent post-traumatic arthritis (PTA) by altering the balance of inflammation and regeneration. Highly purified MSCs (CD45-TER119-PDGFRα+Sca-1+) rapidly expanded under hypoxic conditions. Unexpectedly, MSCs from control C57BL/6 (B6) mice proliferated and differentiated more than MSCs from MRL/MpJ (MRL) "superhealer" mice. We injected B6 or MRL MSCs into mouse knees immediately after fracture, and MSCs of either strain were sufficient to prevent PTA.

Genetically reprogramming adult cells into induced pluripotent stem cells (iPSCs) generates large numbers of patient-matched cells with chondrogenic potential for therapy and cartilage modeling. We produced murine iPSC-derived cartilage constructs with a multi-phase approach involving micromass culture with bone morphogenetic protein-4, flow cytometry cell sorting of chondrocyte-like cells, monolayer expansion, and pellet culture with transforming growth factor-beta 3. Successful differentiation was confirmed by increased chondrogenic gene expression, robust synthesis of glycosaminoglycans and type II collagen, and the repair of an in vitro cartilage defect.

The diverse applications pursued in this research illustrate the power of stem cells to deepen the understanding of cartilage and guide the development of therapies to prevent and treat cartilage injury and OA.

Knee pain is a common complaint among older Americans, nearly half of whom have developed or will develop painful osteoarthritis. Osteoarthritis is primarily a disease of articular cartilage, the low-friction, shock-absorbing connective tissue that lines long bones at their articulating surfaces. Within these joint tissues and within arthritis, the minor protein collagen VI plays an uncertain role, although it has been implicated in several muscle and ligament disorders. Determination of the collagen VI role in bone and cartilage of the knee is the focus of this dissertation.

Within articular cartilage, collagen VI exclusively localizes to and delimits the pericellular matrix (PCM), which differs from the extracellular matrix (ECM) in composition and structure. To interact with the cell, a molecule must first pass through the PCM. Fluorescent dextran diffusivities were quantified in the cartilage PCM using a newly developed model of scanning microphotolysis (SCAMP), a line photobleaching technique. Diffusion was slower in the PCM than in the ECM, although not in early-stage arthritic tissue. These results support the hypothesis that diffusivity is lower in the PCM than in the ECM of healthy articular cartilage, presumably due to differences in proteoglycan content.

Arthritic degradation is partly mediated by interleukin-1 (IL-1), a catabolic cytokine that affects the mechanical properties of articular cartilage and preferentially binds to cell-surface receptors in the surface zone. Since cells are the cartilage metabolic units, matrix degradation is hypothesized to influence molecular transport in the PCM before the ECM. Cartilage was cultured with or without IL-1, soaked in FITC-ovalbumin, and photobleached using SCAMP to measure diffusivity. Over 7 days of culture, IL-1 doubled the diffusivity in both zones (surface, middle) and matrices (PCM, ECM) of the cartilage. Diffusivity within the PCM was slightly lower than within the ECM. No increase in PCM diffusivity relative to ECM diffusivity was detected within either zone, suggesting that PCM-localized degradation either cannot be distinguished at these time points or cannot be detected by measures of ovalbumin diffusion.

To determine the effects of collagen VI absence on the morphometry and physical properties of the joint, knees of 2-, 9-, and 15-month-old Col6a1+/+ and Col6a1-/- mice were studied. Bone morphometry was evaluated using micro-computed tomography (microCT). Subchondral bone thickness, joint-capsule thickness, and cartilage degradation were assessed by histology. Cartilage elastic modulus, roughness, and coefficient of friction were measured by atomic force microscopy (AFM). Diffusion through the cartilage ECM was determined by SCAMP. Overall, collagen VI absence had profound effects on the morphometry of the proximal tibia and the overall histological structures of the mouse knee, yet minimal effects on the friction, roughness, elastic modulus, and diffusional properties of the articular cartilage. Musculoskeletal abnormalities at the knee do result from collagen VI absence.

There are approximately 900,000 people in the US suffering from damage to the articular cartilage, with the knee being most commonly affected. Articular cartilage lacks a vasculature and has a limited ability to heal. A variety of surgical treatments have been developed to repair cartilage lesions. Current strategies for cartilage repair include microfracture, autologous chondrocyte implantation (ACI) and osteochondral allograft transfer (OAT). These strategies suffer from high failure rates (25-50% at 10 years), long rehabilitation times (more than 12 months) and decreasing efficacy in patients older than 40-50 years. Focal joint resurfacing with traditional orthopedic materials is being explored as an alternative strategy, but due to their high stiffness and coefficient of friction relative to cartilage, these implants may ultimately contribute to joint degeneration through abnormal stress and wear. A focal joint resurfacing method that is widely available, allows immediate weight bearing, has short recovery times and has low long-term failure rates remains an unmet need.This thesis explores a strategy to address this need. There are two major criteria within this strategy: 1) develop a material that mimics the properties of cartilage and 2) attach this material to an orthopedic base to enable integration with bone. I developed the first hydrogel to achieve the strength and modulus of cartilage in both tension and compression properties. This hydrogel also exhibits cartilage-equivalent tensile fatigue at 100,000 cycles. The hydrogel was created by infiltrating a PVA-PAMPS double-network hydrogel into a bacterial cellulose (BC) nanofiber network. The BC fibers provide tensile strength in a manner analogous to collagen in cartilage. The PAMPS provides a fixed negative charge and osmotic restoring force similar to the role of aggrecan in cartilage. Subsequently, I further improved and developed the hydrogel to reach a strength that exceeds that of cartilage. The high strength was achieved through reinforcement of crystallized PVA with BC. Experimental results show that reinforcement of annealed PVA with BC leads to a 3.2-fold improvement in the tensile strength (from 15.6 to 50.5 MPa) and a 1.7-fold increase in the compressive strength (from 56.7 to 95.4 MPa). The BC-reinforced PVA was also 3 times more wear resistant than cartilage over 1 million cycles and exhibited the same coefficient of friction. These properties make the BC-reinforced BC hydrogel an excellent candidate material for replacement of damaged cartilage. Current strategies for adhering hydrogel to a surface are 10 times weaker than the shear strength with which cartilage is attached to bone. The osteochondral junction is characterized by mineralized collagen nanofibers anchoring cartilage to bone. I sought to mimic this strategy by bonding freeze-dried BC to porous titanium with a hydroxyapatite-forming cement. The cement penetrates about 10 microns into the bacterial cellulose, forming a nanofiber-reinforced zone of adhesion. The PVA-PAMPS hydrogel is then infiltrated into the bonded bacterial cellulose. This strategy achieved a shear strength of attachment three times greater than the state of the art. I soon proposed an important enhancement of the attaching strategy by introducing shape memory alloy ring to change the direction of shear load bearing. It is the first method for attaching a hydrogel to metal with the same shear strength as the cartilage-bone interface. The average shear strength of the junction between 1.2-mm-thick hydrogel and metal made in this manner exceeded the shear strength of porcine cartilage-bone interface. The shear strength of attachment increased with the number of bacterial cellulose layers and with the addition of cement between the bacterial cellulose layers. Such improved strategies for attaching hydrogels to a metal surface with sufficient strength to allow for weight-bearing can enable the creation of hydrogel-capped titanium implants for cartilage repair.