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<p>Knee osteoarthritis (OA) is a common chronic disease that often occurs in older adults, affecting their quality of life. The purposes of this study were to examine 1) the relationship between physical activity and medial tibial cartilage volume and thickness in participants with clinical knee OA; and 2) the test-retest reliability of daily step counts produced by participants with knee OA.</p> <p>The study included 34 participants (age 60.6 ± 6.5 years; body mass index 28.5 ± 5.4 kg/m²). Dependent variables were medial tibial cartilage volume and thickness, measured from magnetic resonance images. Independent variables were average step counts and average time spent in light intensity activity, measured with an accelerometer. These data were then was analyzed using linear and second degree polynomial regression analyses to determine the relationship between dependent and independent variables along with age, sex and BMI as covariates. The test-retest reliability of step counts collected over 5 days was evaluated with an intraclass correlation coefficient (ICC).</p> <p>Average time engaged in light intensity activity, average daily step counts, sex and age accounted for 56.3% of the variance in medial tibial cartilage volume. Age and average time in light intensity activity explained 33.2% of the variance in medial tibial cartilage thickness. Results from the second degree polynomial analyses were not significant. The ICC for the daily step counts over first 5 days of 10 hours wear time was 0.929 (95% [CI] =0.883, 0.961).</p> <p>A weak linear relationship existed between physical activity and cartilage volume and thickness within the knee joint. The greatest medial tibial cartilage volume was found in men who were engaged in longer durations of low intensity physical activity. The test-retest of step counts data by participants with knee OA was very reliable over the 5 days. Findings from this study augment current knowledge of knee OA.</p> / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25186号 / 医博第5072号 / 新制||医||1072(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 安達 泰治, 教授 森本 尚樹, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

[Truncated abstract] Articular cartilage (AC) covers the surface of synovial joints providing a nearly frictionless bearing surface and distributing mechanical load. Joint trauma can damage the articular surface causing pain, loss of mobility and deformation. Currently there is no uniform treatment protocol for managing focal cartilage defects, with most treatment options targeted towards symptomatic relief but not limiting the progression into osteoarthritis (OA). Autologous chondrocyte implantation (ACI) and more recently matrix-induced autologous chondrocyte implantation (MACI), have emerged as promising methods for producing hyaline or hyaline-like repair tissue, however there remains some controversy regarding the exact histological nature of the tissue formed. Histological characterisation of AC repairs requires destructive tissue biopsy potentially inducing further joint pathology thereby negating the treatment effect. OA is recognised as a major cause of pain, loss of function and disability in Western populations, however the exact aetiology is yet to be elucidated. The assessment of both OA and cartilage repair has been limited to macroscopic observation, radiography, magnetic resonance imaging (MRI) or destructive biopsy. The development of non-destructive AC assessment modalities will facilitate further development of AC repair techniques and enable early monitoring of OA changes in both experimental animal models and clinical subjects. Confocal laser scanning microscopy (CLSM) is a type of fluorescence microscopy that generates high-resolution three-dimensional images from relatively thick sections of tissue. ... Biomechanical analysis suggested that the mechanical properties of MACI tissue remain inferior for at least three months. This study showed the potential of a multi-site sheep model of articular cartilage defect repair and validated its assessment via LSCA. Finally, the LSCA was used to arthroscopically image the cartilage of an intact fresh frozen cadaveric knee from a patient with clinically diagnosed OA. Images were correlated to ICRS (Outerbridge) Grades I-IV and histology. The LSCA gave excellent visualization of cell morphology and cell density to a depth of up to 200'm. Classical OA changes including clustering chondrocytes, surface fibrillation and fissure formation were imaged. Fair to moderate agreement was demonstrated with statistically significant correlations between all modalities. This study confirmed the viability of the LSCA for non-destructive imaging of the microstructure of the OA cartilage. In conclusion, the LSCA identified histological features of orthopaedic tissues, accurately quantified chondrocyte morphology and demonstrated classical OA changes. While the development and investigation of an ovine model of cartilage repair showed the treatment benefit of MACI, some biomechanical issues remain. Ultimately, the LSCA has been demonstrated as a reliable nondestructive imaging modality capable of providing optical histology without the need for mechanical biopsy. Medical Subject Headings (MESH): articular cartilage; autologous chondrocyte implantation; matrix-induced autologous chondrocyte implantation; biomechanics; cartilage; confocal microscopy; diagnosis; histology; image analysis; immunohistochemistry; magnetic resonance imaging; microscopy; osteoarthritis

<strong>Osteoarthritis</strong>, the most prevalent joint disease in the United Kingdom, is a progressive condition that results in end-stage full-thickness cartilage loss and has important social and economic impacts on society. Since cartilage lacks regenerative capabilities, it is essential to develop approaches to initiate and enhance cartilage regeneration. In this context, tissue engineering is emerging as an attractive approach for the regeneration of cartilage tissue damaged due to disease or trauma. A scaffold-free cartilage construct, analogous to those found during embryonic precartilage condensation, has received much attention as an alternative novel modality for cartilage <strong>tissue engineering</strong>. Cartilage repair with <strong>scaffold-free</strong> tissue more closely resembles the natural situation and mimics the features of the original tissue. Moreover, scaffold-free cartilage implants can overcome the complications caused by the use of suboptimal scaffolds by avoiding the need for a foreign scaffold at all. Culturing cells into tissue patches without the requirement for a scaffold can be achieved through <strong>cell sheet engineering</strong>, which uses thermo-responsive culture dishes. However, the high costs of the tissue culture consumables, and the relatively low cellular yield, makes this process less attractive. This thesis presents a novel method for generating shape-, size- and thickness-adjustable 3-dimensional scaffold-free cell pellet sheets for use as implantable biological cell patches for cartilage tissue engineering. This new technique of bioengineering scaffold-free cell pellet sheets proves to be reproducible, easily applicable, sizable and thickness adjustable. <strong>Stem cells</strong> have added a new thrust to tissue engineering. Their distinctive self-renewal and plasticity have not only optimized many tissue engineering developments, but also rendered feasible some applications which would otherwise be unattainable with somatic cells. Human mesenchymal stem cells (HMSCs) were used to examine the optimal condition for generating cell pellet sheets with this new method. Furthermore, the resultant differentiated pellet sheets were compared directly with HMSCs, human chondrocytes and human fibroblasts alone to evaluate the feasibility of using this cell pellet sheet for clinical applications in terms of their biological and mechanical properties. The results of this thesis suggest that the engineered scaffold-free, chondrogenic, differentiated MSC pellet sheet not only exhibits desirable biologic features similar to chondrocytes, but also demonstrates good integrative and viscoelastic potential that might offer exciting possibilities for the development of novel biologically-based clinical therapies. In summary the data presented herein indicate the following points: <table><ul style="list-style-type:square"><li>The differentiation of human MSCs into chondrogenic cells was achieved.</li> <li>A novel approach of centrifugal seeding on a PDMS surface was shown to effectively generate chondrogenic-differentiated cell pellet sheets without impairing the biological functions of chondrocytes.</li> <li>Various cell types such as human MSCs, human chondrocytes and human fibroblasts were found to respond well to the novel methodology and generated viable, cohesive, less shrinkable, and readily-detachable cell pellet sheets, the size and thickness of which could be adapted as required. The results obtained were superior to those obtained using the conventional thermo-responsive culture dish method.</li></table> This new methodology developed in this thesis provides an approach to in vitro cell pellet sheet generation which is closer to the physiological process of cartilage development and which proved valuable for the study of in vitro generation of scaffold-free cell patches as an important adjunct to many traditional cartilage restorative procedures. Future research on in vivo assessment of the cell sheet and the functional role of these sheets in repairing damaged cartilage is recommended.

A reproducible in vitro model of mechanically injured (scalpel cut) articular cartilage was developed in this work utilising bovine and human osteochondral tissue. Using fluorescence-mode confocal laser scanning microscopy (CLSM), the model allowed (1) spatial and temporal quantification of in situ (within the matrix) chondrocyte viability following a full thickness cartilage injury and (2) serial evaluation of three chondroprotective strategies in injured bovine and human articular cartilage: (a) medium osmolarity (b) medium calcium concentration and, (c) subchondral bone attachment to articular cartilage. Medium osmolarity significantly influenced superficial zone chondrocyte death in injured (scalpel cut) bovine and human articular cartilage. Greatest percentage cell death occurred at 0 mOsm (distilled water). Conversely, a raised medium osmolarity (600 mOsm) was chondroprotective. The majority of in situ cell death occurred within 2.5 hours of the experimental injury, with no further increase over 7 days. Exposure of articular cartilage to calcium-free media significantly decreased superficial zone chondrocyte death in injured (scalpel cut) articular cartilage compared with exposure to calcium-rich media (2-20 mM). In calcium-rich media, the extent of percentage cell death increased with increasing medium calcium concentration but remained localised to the superficial zone of injured articular cartilage over 7 days. However, in calcium-free media, there was an increase in percentage cell death within deeper zones of injured articular cartilage over 7 days. Excision of subchondral bone from injured (scalpel cut) articular cartilage resulted in an increase in chondrocyte death at 7 days that occurred in the superficial zone of injured as well as the adjacent uninjured regions of articular cartilage. However, the presence of subchondral bone in the culture medium prevented this increase in chondrocyte death within the superficial zone. Subchondral bone may have interacted with articular cartilage via soluble mediator(s) that influenced chondrocyte survival. In human articular cartilage, healthy subchondral bone also interacted with articular cartilage in explant culture and promoted in situ chondrocyte survival, while sclerotic subchondral bone was detrimental to chondrocyte viability. These findings are of translational relevance to fluid management systems used during open and arthroscopic articular surgery, clinical and experimental research into cartilage injury, repair and degeneration as well as current techniques of tissue engineering.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy