Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Includes bibliographical references (leaves 63-70). Also issued on the Internet. Also available on the Internet.

Osteoarthritis (OA) affects an estimated 250 million people worldwide, representing an enormous economic and social burden across demographic groups. While classically attributed to ‘wear and tear’ of the articular cartilage, there is a growing appreciation that OA is a whole-joint disease with a complex etiology involving the synovium and surrounding tissues. The synovium is a specialized connective tissue membrane that envelops the diarthrodial joint and maintains the synovial fluid environment through molecular secretion as well as bi-directional filtration of these constituents, nutrients, and cellular waste products. Moreover, synovium-derived cells have been directly implicated in both the native repair response as well as degradation of articular cartilage. Much of the existing research of synovium has been conducted in the context of rheumatoid arthritis (RA). And while synovitis is a key feature of both RA and OA, clinical reports have described OA synovium as distinct in its cellular and structural composition, molecular secretion, and chronic onset. However, literature studies have not adequately addressed the mechanisms by which alterations in synovium structure-function affect joint and cartilage health, particularly the contribution of different cell types within the synovium to solute transport and lubrication. The work described in this dissertation addresses these knowledge gaps in the context of existing and emerging OA therapies, namely glucocorticoids and electrical stimulation. We anticipate that a more comprehensive characterization of changes to the synovium composition, secretion of key metabolic mediators, lubrication properties, as well as its ability to regulate solute transport in and out of the joint space will not only contribute to our basic science understanding of the synovium but also the development and modification of therapeutic strategies aimed at restoring and maintaining joint health. This characterization will be facilitated by our laboratory’s expertise in tissue engineering and explant culture, IL-1 and DEX stimulation, and electrical stimulation of joint tissues. The approach of using an engineered synovium model is attractive in that quantitative high throughput in vitro mechanistic studies can be performed on tissues that are fabricated from cells derived from normal and OA synovium of patients and corresponding immune cells at defined density and cell type ratios. It also facilitates isolating effects of certain cell types or starting composition that are found in explant specimens. Intra-articular glucocorticoid injections are commonly administered to patients in an effort to control inflammation and pain. And while these high dose injections are known to have significant detrimental local and systemic effects, comparatively low doses of dexamethasone (DEX), a synthetic glucocorticoid, are known to have pro-anabolic and anti-catabolic effects on cartilage cultures. Our laboratory has published extensively on the benefits of DEX stimulation in growth and maintenance of engineered and explanted cartilage as well as chondroprotection from pro-inflammatory cytokines (e.g interleukin-1; IL-1), both in juvenile bovine basic science and adult canine preclinical systems. However, the concomitant effects of DEX on synovium structure-function have not been elucidated. In Part I, we describe a functional tissue engineered synovium model that was validated against explant behavior. We were able to recapitulate many of the unique structural and functional characteristics of synovium, including protein expression, intimal lining formation, solute transport, and friction coefficient. Additionally, changes in engineered synovium structure-function mirrored that of explants when treated with IL-1 or DEX. The engineered synovium model was then expanded to include resident macrophage-like synoviocytes (MLS), demonstrating the key role that these cells play in structural reorganization of synovium. The model was also translated to human cells, showing the potential of the system for personalized medicine. Finally, motivated by insights into solute transport in the synovium as well as its strong anti-inflammatory response to DEX, we developed a sustained low-dose DEX delivery platform for mitigating synovial inflammation while simultaneously stimulating cartilage growth. Utilizing a preclinical adult canine model, we showed that extended intra-articular delivery of DEX improved functional outcomes and cartilage tissue quality. In Part II, we evaluated synovium behavior and cartilage repair in response to modes of electrical stimulation. Electrical stimulation of cells and tissues has been a topic of interest for decades, owed in part to the knowledge that endogenous electric field (EF) gradients guide cell behavior during embryogenesis and wound healing. Pulsed electromagnetic fields (PEMFs) have been used in a clinical setting to stimulate bone repair and alleviate pain, however their use for OA and cartilage repair is controversial. Culture studied of PEMFs have shown anti-catabolic and pro-anabolic effects on isolated FLS and cartilage, respectively. And previous work in our laboratory demonstrated directed 2D migration of synoviocytes and chondrocytes in response to direct current (DC) EF stimulation. These modes of electrical stimulation have not been explored in synovium explants, so it is unclear to what extent the observed phenomena translate to the 3D tissue environment. For the first time, we characterized the biological response of both healthy bovine and OA human synovium explants, showing distinct anti-inflammatory behavior in bovine tissues and a highly variable response in arthritic human tissues, likely due to different inflammatory cell content. Motivated by the potent anti-inflammatory effect seen in normal tissue and previous work showing a pro-anabolic effect on cartilage, the PEMF system was then adapted for use with a preclinical adult canine model of engineered cartilage repair. In this model, PEMFs significantly enhanced functional outcomes and cartilage tissue quality. Finally, we investigated the potential for direct synovial cell-mediated cartilage repair via induced migration with DC EFs. By developing and validating a novel tissue-scale bioreactor capable of applying DC EFs in sterile culture conditions to three-dimensional constructs, we showed increased recruitment of synovial repair cells to the site of a cartilage wound. Taken together, the sum of the work builds on existing therapeutic strategies by developing models to understand the contribution of the synovium to joint maintenance and repair. By modeling dexamethasone- and electrical- induced changes to composition and function of synovium and cartilage, via complementary explant and engineered approaches, valuable mechanistic insights into osteoarthritis pathogenesis and cartilage repair were gathered. These findings lay the groundwork for more complex and personalized in vitro models of OA and motivate future work to capitalize on knowledge of the functional plasticity of the synovium to develop synovium-targeted strategies for OA treatment and prevention.

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.

Besides the molecular mechanisms regulating activation of FLS mentioned above, we also investigated anti-inflammatory activities of Chinese herbal medicine sinomenine and Liang Miao San on activated human FLS in RA. Sinomenine, an alkaloid isolated from the root of Sinomenium acutum, has been used to alleviate the symptoms of rheumatic diseases. Liang Miao San (LMS), composed of the herbs Rhizoma Atractylodis (Cangzhu) and Cotex Phellodendri (Huangbai), is another traditional Chinese medicine formula for RA treatment. Since the potential anti-inflammatory activities of sinomenine and LMS have been demonstrated, we investigated the in vitro anti-inflammatory effects of sinomenine and LMS on inflammatory cytokine TNF-alpha activation of human normal and RA-FLS and the underlying intracellular mechanisms. In the present study, sinomenine was found to significantly inhibit TNF-alpha induced cell surface expression of VCAM-1 and release of inflammatory cytokine and chemokine IL-6, CCL2 and CXCL8 from both normal and RA-FLS (all p < 0.05). Our results provide a new insight into the differential anti-inflammatory activities of sinomenine and LMS through the suppression of TNF-alpha activated FLS by modulating distinct intracellular signaling pathways in RA, and help to provide a biochemical basis for the development of a cost-effective human synoviocyte model for the future screening of traditional Chinese medicine (TCM) possessing potential anti-rheumatic activities. (Abstract shortened by UMI.) / IL-27, a novel member of the IL-12 family that is produced early by antigen-presenting cells (APCs), can promote T cell proliferation as well as the production of interferon-gamma by naive T lymphocytes. Recent studies have found that elevated expression of IL-27 has been detected in the synovial membranes and fluid of RA. Herein we investigated the in vitro effects of IL-27, alone or in combination with inflammatory cytokine TNF-alpha or IL-Ibeta on the pro-inflammatory activation of human primary FLS isolated from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also studied. We found that the plasma concentration of IL-27 in RA patients (n=112) was significantly higher than that in control subjects (n=46). Both normal and RA-FLS constitutively express functional IL-27 receptor heterodimer, gp130 and WSX-1, with more potent IL-27-mediated activation of signal transducers and activators of transcription (STAT)1 in RA-FLS. IL-27 was found to induce significantly higher cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 and release of inflammatory cytokine IL-6, chemokine CCL2, CXCL9, CXCL10 and matrix metalloproteinase (MMP)-1 of RA-FLS than that of normal FLS (all p < 0.05). The above findings therefore provide a new insight into the IL-27-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation of RA and may have important therapeutic implications. / In the present study, we have investigated the mechanisms of the activation of human fibroblast-like synoviocytes (FLS) induced by various stimuli including interleukin (IL)-27, tumor necrosis factor (TNF)-alpha and IL-beta. The activation of human FLS was studied in terms of the release of cytokines and chemokines and the expression of adhesion molecules. / We investigated the in vitro effects of uric acid crystals, alone or in combination with inflammatory cytokine TNF-alpha or IL-beta on the pro-inflammatory activation of human FLS from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also determined. In the present study, uric acid crystals were found to result in a significant increase of inflammatory cytokine IL-6, chemokine CXCL8 and MMP-1 from both normal and RA-FLS (all p < 0.05). Moreover, additive or synergistic effect was observed in the combined treatment of uric acid crystals and TNF-alpha or IL-1beta on the release of IL-6, CXCL8 and MMP-1 from both normal and RA-FLS. Further investigations showed that the release of inflammatory cytokine, chemokine and matrix metalloproteinase stimulated by uric acid crystals was differentially regulated by intracellular activation of extracellular signal-regulated kinase (ERK) and JNK pathways. Our results therefore provide a new insight into the endogenous danger signal uric acid crystals-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation, and also provide biochemical basis for the development of new modality for inflammatory rheumatic diseases. / Chen, Dapeng. / Adviser: Wong Chun Kwok. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 203-240). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.