The main goal of this diploma thesis is to determine the role of hyaluron acid and phospholipids on friction and lubrication of articular cartilage in regard to optimization of viscosupplements. This is carried out by measuring the coefficient of friction of the articular cartilage with several lubricants. Cartilage is lubricated particularly by a conventional viscosuplement, optimized viscosuplementation with phospholipids and model synovial fluid. In order to observe the function of those viscosuplements in the human body, both are mixed with the model synovial fluid, ubiquitous in human joints, in given ratio. Experiments revealed high friction when it comes to convectional viscosupplementation as opposed to low friction of the optimized viscosuplement with phospholipids. The same situation occurs when cartilage is lubricated with those viscosuplements mixed with model synovial fluid which might lead to development of a new, better, viscosupplementation based on hyaluron acid and phospholipids.

A polyconvex continuum-level proteoglycan Cauchy stress function was developed based on the continuum electromechanical Poisson-Boltzmann unit cell model for proteoglycan interactions. The resulting proteoglycan model was combined with a novel collagen fibril model and a ground substance matrix material to create a polyconvex constitutive finite element model of articular cartilage. The true collagen fibril modulus , and the ground substance matrix shear modulus , were varied to obtain the best fit to experimental tension, confined compression, and unconfined compression data for native explants and explants cultured in insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1). Results indicate that culture in IGF-1 results in a weakening of the COL fibers compared to native explants, and culture in TGF-β1 results in a strengthening of the COL fibers compared to native explants. These results elucidate the biomechanical changes in collagen fibril modulus, and ground matrix shear modulus following in vitro culture with IGF-1 and TGF-β1. Understanding the constitutive effects of growth factor stimulated culture may have applications in AC repair and tissue engineering.

A poroelastic finite element model of a heterogeneous articular cartilage disc was created to examine the tissue response to low amplitude (± 2% strain), low frequency (0.1 Hz) dynamic unconfined compression (UCC). A strong correlation has been made between the relative fluid velocity and stimulation of glycosaminoglycan synthesis. A contour plot of the model shows the relative fluid velocity during compression exceeds a trigger value of 0.25 μm/s at the radial periphery. Dynamic UCC biochemical results have also reported a higher glycosaminoglycan content in this region versus that of day 0 specimens. Fluid velocity was also found not to be the dominant physical mechanism that stimulates collagen synthesis; the heterogeneity of the fluid velocity contour plot conflicts with the homogeneous collagen content from the biochemical results. It was also found that a Tresca (shear) stress trigger of 0.07 MPa could provide minor stimulation of glycosaminoglycan synthesis. A feasibility study on modeling a heterogeneous disc was conducted and found convergence issues with the jump in properties from the superficial to middle layers of the disc. It is believed that the superficial layer contains material properties that allow the tissue to absorb much of the compressive strain, which in turn increases pressure and causes convergence issues in ABAQUS. The findings in this thesis may help guide the development of a growth and remodeling routine for articular cartilage.

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 is a degenerative joint disease which affects the entire joint; however, one of its hallmarks is the progressive degeneration of the articular cartilage layer. Patients suffering from osteoarthritis exhibit chronic pain, stiffness, and a decreased range of motion, greatly affecting their quality of life. No drugs have been approved to stop the progression of osteoarthritis and focus solely on the management of symptoms. This is partly due to the challenges in delivering drugs to afflicted joints, and specifically to cartilage due to its lack of vasculature. While intra-articular injection holds promise for the local administration of drugs, small molecules are rapidly cleared from the synovial fluid. As a result, there is a need to develop effective drug delivery strategies to improve residence times in the joint to elicit a sustained therapeutic effect. Previous studies identified polyphosphate as a pro-anabolic molecule, promoting glycosaminoglycan and collagen accumulation in cartilage constructs. Therefore, polyphosphate may be a therapeutic of interest to address the degeneration of articular cartilage in patients suffering from osteoarthritis. In this study, calcium-polyphosphate and strontium-polyphosphate particles were synthesized and characterized as a potential drug carrier into articular cartilage. Physicochemical characterization revealed that the particles exhibit a spherical morphology, have a negative zeta potential, and are nanoscale in size. Biological characterization in chondrocytes confirmed cellular uptake of the particles and demonstrated a size and concentration-dependent cytotoxicity at high concentrations. Furthermore, treatment of chondrocytes with these particles resulted in a reduction in metabolic activity and cell proliferation, confirming biological effects. Preliminary studies using cartilage explants suggest that the particles can penetrate and be retained in cartilage tissue. Therefore, from the results obtained within this study, the polyphosphate-based particles may be a potential drug delivery strategy for delivery into articular cartilage.

Disertační práce se zabývá experimentálním studiem viskosuplementů na bázi kyseliny hyaluronové, které se aplikují do synoviálních kloubů postižených osteoartrózou. Hlavní pozornost byla věnována objasnění vlivu koncentrace a molekulové hmotnosti kyseliny hyaluronové na tření v kontaktu kloubí chrupavky resp. změnám tření v kontaktu po smíchání osteoartritické synoviální kapaliny s exogenní kyselinou hyaluronovou. Důležitou součástí experimentů bylo rovněž studium reologických vlastností synoviální kapaliny a kyseliny hyaluronové. Výsledky ukázaly, že molekulová hmotnost kyseliny hyaluronové významně ovlivňuje viskozitu a viskoelastické vlastnosti roztoku. Výrazná závislost mezi reologickými vlastnostmi kyseliny hyaluronové a třením v kontaktu však nebyla pozorována. Přimíchání kyseliny hyaluronové do synoviální kapaliny způsobí výrazný pokles součinitele tření v kontaktu. Rozdíly mezi viskosuplementy obsahující kyselinu hyaluronovou s různou molekulovou hmotností ale nijak výrazné nejsou. Nicméně, výsledky poukazují na možné ovlivnění režimu mazání v důsledku vysoké molekulové hmotnosti kyseliny hyaluronové. Tyto původní výsledky rozšiřují pochopení mechanizmů, ke kterým dochází v kloubu bezprostředně do vstříknutí kyseliny hyaluronové a mohou být použity při dalším vývoji viskosuplementů či v klinické praxi.

Knee pain can alter lower-extremity neuromechanics and often results in functional disability. The relationship between lower-extremity neuromechanical alterations, due to anterior knee pain, and articular cartilage condition is unclear. The purpose of this study was to determine the independent effect of anterior knee pain during running on articular cartilage condition, as reflected by serum cartilage oligomeric matrix protein concentrations and muscle cocontraction duration. Seven men and five women completed a 30-min run in three different sessions: control (no infusion), sham (isotonic saline infusion), and pain (hypertonic saline infusion). Saline was infused into the right infrapatellar fat pad for the duration of the run. Subject-perceived pain was recorded every 3 min on a 100-mm visual analog scale. During the run, bilateral electromyography was recorded for five leg muscles, and heel and toe markers were used to track foot position. During the 30-min run of the pain session average subject-perceived pain was 27.8 (SD = 2.3 mm) and 19.7 (SD = 1.9) mm greater than during the control (0.0 mm) and sham (8.1 mm) session, respectively (p < 0.01). Knee pain while running did not result in changes in muscular cocontraction duration (p = 0.13). Blood samples were drawn prior to the run, immediately following the run, and 60 min following the run. Samples were analyzed using enzyme-linked immunosortbent assay to determine serum cartilage oligomeric matrix protein concentration. Average serum cartilage oligomeric matrix protein concentration was 14% greater at immediate post run (132.19 ± 158.61 ng/ml; Range = 22.61-290.81 ng/ml) relative to pre run (116.02 ± 118.87 ng/ml; Range = 19.81-234.89 ng/ml) (p < 0.01), and 18% less at 60 min post run (108.45 ± 171.78 ng/ml; Range = 20.84-280.23 ng/ml) relative to immediate post run (Figure 4; p < 0.01). Serum cartilage oligomeric matrix protein did not significantly differ between baseline and 60 min post-exercise (p = 0.29). There was not a difference in cartilage oligomeric matrix protein concentration between sessions. Knee pain while running does not cause an increase in serum cartilage oligomeric matrix protein concentration (p = 0.29). There are two important findings from this study. First, anterior knee pain during a 30 min running session does not appear to independently affect cartilage oligomeric matrix protein concentrations. This implies other factors, aside from anterior knee pain alone, influence articular cartilage degradation during movement that occurs while individuals are experiencing anterior knee pain. Second, the present experimental anterior knee pain model can be used to evaluate the independent effects of anterior knee pain over an extended duration while subjects perform a dynamic activity like running.

Osteoarthritis (OA) is a degenerative condition of cartilage and is the leading cost of disability in the United States. Motion analysis experiments in combination with knee-joint finite element (FE) analysis may be used to identify exercises that maintain knee-joint osteochondral (OC) loading at safe levels for patients at high-risk for knee OA, individuals with modest OC defects, or patients rehabilitating after surgical interventions. Therefore, a detailed total knee-joint FE model was developed by modifying open-source knee-joint geometries in order to predict OC tissue stress and strain during the stance phase of gait. The model was partially validated for predicting the timing and locations of maximum contact parameters (contact pressure, contact area, and principal Green-Lagrangian strain), but over-estimated contact parameters compared with both published in vivo studies and other FE analyses of the stance phase of gait. This suggests that the model geometry and kinematic boundary conditions utilized in this FE model are appropriate, but limitations in the material properties used, as well as potentially the loading boundary conditions represent primary areas for improvement.

Please note: we were unable to immediately open the spreadsheet below. We repaired the spreadsheet file with Excel, and have a copy of it in storage. If you have difficulty opening the spreadsheet, please write to us at open-help@bu.edu. / Osteoarthritis (OA) is a painful, chronic, non-inflammatory disease affecting 140 million people worldwide that alters synovial joint structure and function. OA progressively breaks down hyaline cartilage, the hydrated tissue that provides a smooth, nearly frictionless surface and distributes loads applied to articulating joint surfaces. The loss of glycosaminoglycans (GAGs) from the extracellular matrix of cartilage is an early marker of OA. Therefore, imaging methods that quantify the GAG content of cartilage are of interest. This work investigates the synthesis and development of three cationic contrast agents (CAs) for imaging articular cartilage (AC): CA4+, an iodinated small molecule, and tantalum oxide nanoparticles (Ta2O5 NPs) for x-ray Computed Tomography (CT) imaging; and Gadopentetate-dilysine (Gd(DTPA)Lys2), a gadolinium small molecule for Magnetic Resonance (MR) imaging. These cationic contrast agents are attracted to the strong negative fixed charge of extracellular GAG and, therefore, infiltrate cartilage. This work begins with an overview of CT and MR imaging basic principles, current clinical CAs and contrast enhanced imaging of AC. First, the large-scale (50 g) synthesis of CA4+ is described and the partitioning over time of CA4+ into ex vivo AC is correlated to GAG content and cartilage mechanical properties. Similar partitioning studies are applied to anionic, neutral and cationic Ta2O5 NPs, where the cationic NP exhibited substantially greater affinity for AC. Moreover, by maintaining the positive charge on the NP surface and introducing a polyethylene glycol coating, a NP formulation is described for successful in vivo cartilage imaging. Next described is the MRI CA, Gd(DTPA)Lys2, which affords an equivalent T1 signal in cartilage at 1/10th the effective dosage of anionic gadopentetate. Finally, the equilibrium partitioning of the small molecule CT and MRI CAs is directly compared to GAG content and mechanical properties in human finger AC. In summary, results show cationic CAs strongly correlate to both GAG and mechanical properties and distribute in direct proportion to GAG unlike anionic CAs. The use of cationic CAs to quantify the biochemical and mechanical changes of AC may aid drug discovery and improve clinical assessment and intervention of OA for future patients. / 2017-11-03T00:00:00Z

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24830号 / 医博第4998号 / 新制||医||1067(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 後藤, 慎平, 教授 河本, 宏, 教授 羽賀, 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM