Objective: Physical exercise decreases disability and pain associated with chronic articular cartilage degradation. However, understanding of the pathology is lacking. In this study, the levels of 17 biomarkers of inflammation and cartilage degradation were measured in synovial fluid (SF) before and after a 30-minute run in able-bodied and previously-injured individuals. Materials & Methods: Four able-bodied recreational runners (3 men and 1 woman: 24 ± 2 years, 68 ± 7 kg, and 173 ± 9 cm) and 4 recreational runners who had undergone a unilateral anterior cruciate ligament reconstruction (ACLr) (2 men and 2 women: 23 ± 1 years, 71 ± 6 kg, and 175 ± 4 cm) were recruited to participate in this study. Using a saline-assisted method, SF was aspirated before and after both a 30-minute unloading and 30-minute exercise session. Samples were corrected for blood contamination and analyzed for 15 cytokines and 2 matrix metalloproteinases (MMPs). Mixed model analyses were used to determine the main effects of session, case/control status, pre/post aspirations, and the interactions between case/control status and pre/post aspirations. Results: Blood protein contamination was calculated and accounted for in 15 of 32 synovial fluid samples. Granulocyte colony stimulating factor (GCSF) was the only detectable cytokine of the 15 analyzed. No statistical differences were found in GCSF concentrations between pretreatment and posttreatment aspirations (p = 0.45), ACLr and able-bodied control groups (p = 0.60), or unloading and exercise sessions (p = 0.96). MMP-13 was undetectable. No statistical differences were found in MMP-3 between pretreatment and posttreatment aspirations (p = 0.15), ACLr and able-bodied control groups (p = 0.85), or unloading and exercise sessions (p = 0.14).Conclusions: Two (GCSF and MMP-3) of the 17 measured biomarkers were detectable. There were no significant differences in either GCSF or MMP-3 due to a 30-minute run or 30-minute unloading period in either the able-bodied or ACLr participants. Further, there were no significant differences between biomarker concentrations and case-control status. A novel method of controlling for blood contamination in synovial fluid samples was implemented.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21019号 / 医博第4365号 / 新制||医||1028(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 別所 和久, 教授 安達 泰治, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Articular cartilage damage and degeneration is a siginficant clinical problem which no technique has been able to adequately and reliably repair or regenerate. Recent research has investigated the use of cell-based therapies to treat focal cartilage lesions. In clinical practice proliferated autologous chondrocytes are used and clinical trials are investigating the use of mesenchymal stem cells. The aim of this thesis was to assess aspects of current cell-based therapy and to investigate the potential of perivascular stem cells for articular cartilage repair. The phenotype of expanded matrix-applied autologous chondrocytes utilised in current cell therapies was confirmed using immunocytochemistry and polymerase chain reaction (PCR) expression of hyaluronan and proteoglycan link protein 1 (HAPLN1), transcription factor sox-9 (SOX9) and aggrecan (ACAN). Quantitative real-time PCR demonstrated that they were down-regulated for expression of COL2A1, SOX9 and ACAN but up-regulated for COL1A1 compared to unproliferated chondrocytes. Confocal laser-scanning microscopy (CLSM) demonstrated a significant decrease in cell viability and density when the membranes were subjected to levels of trauma similar to those that could be experienced during surgery. Hyperosmolar solutions did not confer a chondroprotective effect. Pericytes and adventitial cells, collectively termed perivascular stem cells (PSCs), from the infra-patellar fat pad were identified using immunohistochemistry and isolated using enzymatic digestion and fluorescence-activated cell sorting (FACS). Cell identity was ascertained using PCR, FACS and mesenchymal differentiation (osteogenesis, adipogenesis and chondrogenesis). Quantitative real-time PCR analysis of micromass cultures indicated that PSCs displayed increased chondrogenic potential compared to mesenchymal stem cells. An ovine model of perivascular stem cells was developed and a pilot study using three sheep was undertaken to confirm the viability of the model. Autologous ovine PSCs were isolated and re-implanted into articular cartilage defects. Green fluorescent protein transfected cells were identified in the cartilage defect four weeks following re-implantation using CLSM. This thesis has examined aspects of matrix-applied autologous chondrocyte implantation for cell based cartilage repair and has identified a new source of prospectively identified and purified stem cells that have demonstrated increased chondrogenic potential compared to mesenchymal stem cells, which are commonly used in clinical research. The methods to identify and purify ovine perivascular stem cells were developed to investigate the use of autologous PSCs and to track the cells following implantation.

Abstract Primary hallmark of osteoarthritis (OA) is the progressive degeneration of articular cartilage. An accurate estimation of cartilage mechanics is important when analyzing the subject-specific function of the knee joint and risks for the onset and development of OA due to cartilage damage. Finite element (FE) modeling can help to estimate peak joint stresses and strains and explain how they could lead to OA. FE models of the knee joint during simplified gait were developed to define the level of material complexity required for 3D FE modeling of the knee joint in estimation of reliable tissue stresses and strains within articular cartilage of the knee joint; and to investigate the predictive value of FE modeling of the knee joint on the development and progression of radiographic OA within obese and normal weight subjects. It was found that maximum principal stresses and strains within articular cartilage in the knee joint during walking are highly sensitive to the material parameters of cartilage. It was not possible to match simultaneously stresses, strains and contact pressures between simplified (non-fibrillar) and advanced (fibrillar) models. Though, it was possible to find parameters for transversely isotropic models that enable the estimation of stresses and strains throughout the depth of cartilage similarly to more advanced fibril reinforced models. Locations of peak cumulative stresses in obese subjects at the baseline without radiographic OA showed a good agreement with the locations of cartilage loss and magnetic resonance imaging (MRI) based scoring in four year follow-up when they had developed OA. Simulated weight loss in obese subjects significantly reduced the highest cumulative stresses in cartilage to the level of normal weight subjects. The cartilage degeneration algorithm was able to predict subject-specific progression of OA similarly with MRI follow-up data and separate subjects with radiographic OA from healthy subjects. The computational FE models developed in this thesis represent useful tools to identify possible risk locations within the knee joint and how they relate to OA onset and progression. The presented methods have clinical potential in the diagnostics of knee joint OA in a subject-specific manner and in simulating the effect of interventions on the progression of OA thus helping with an effective treatment planning. / Tiivistelmä Nivelrikon tunnusomaisin piirre on nivelrustokudoksen rappeutuminen ja kuluminen. Nivelruston tehtävänä on tasata niveliin kohdistuvaa kuormitusta. Rustokudoksen mekaanisten ominaisuuksien määrittäminen on tärkeässä roolissa, kun halutaan arvioida tarkemmin polvinivelen toimintakykyä sekä rustokudoksen rappeutumista. Magneettikuvantamisen pohjalta tehtävä polvinivelen biomekaaninen tietokonemallinnus mahdollistaa rustokudoksen jännitys- ja puristusjakauman arvioinnin simuloidun kuormituksen aikana, mikä edelleen voi antaa vastauksia siihen, kehittyykö niveleen tulevaisuudessa nivelrikko, tai miten tietyn nivelrikkopotilaan sairaus etenee. Tämän tutkimuksen päätavoitteena oli kehittää kolmiulotteisia polvinivelen biomekaanisia tietokonemalleja, joiden perusteella simuloitiin normaalia kävelyä. Polvinivelen kolmiulotteinen geometria luotiin terveiden koehenkilöiden sekä nivelrikkopotilaiden magneettikuvista. Malleilla selvitettiin aluksi, miten monimutkaisena materiaalina nivelrusto tulee mallintaa, jotta mallin ennustama jännitys- ja puristusjakauma on silti realistinen. Tämän jälkeen tutkittiin, miten hyvin tietokonemallinnus ennustaa polvinivelrikon kehittymistä ja etenemistä sekä nivelruston rappeutumista ylipainoisilla potilailla. Tutkimuksessa havaittiin, että tietokonemallin ennustamat jännitys- ja puristusjakaumat nivelrustossa kävelyn aikana riippuvat merkittävästi nivelrustolle valitusta materiaalimallista ja sen parametreista. Tietokonemallien ennustamat nivelruston jännityskeskittymien sekä ruston rappeutumisen sijainnit vastasivat erittäin hyvin nivelrustokudoksen todellisen kulumisen sijainteja magneettikuvasta arvioituna neljän vuoden seuranta-ajan jälkeen. Tietokonemalleilla oli myös mahdollista simuloida painon pudotuksen vaikutusta, jolloin nivelrustokudoksen jännitys- ja puristusjakaumat palautuivat normaalien koehenkilöiden tasolle. Tässä tutkimuksessa kehitetyt polvinivelen tietokonemallit tarjoavat tutkijoille uuden työkalun paikallistaa sellaiset kohdat nivelpinnalta, joissa kuormituksen aiheuttama mekaaninen jännitys on suurta; nämä kohdat ovat kaikista riskialtteimpia nivelrikon kehittymiselle. Kehitettyjä malleja voidaan perustutkimuksen lisäksi jatkokehittää edelleen kohti kliinistä sovellusta, jolloin niitä voitaisiin hyödyntää esimerkiksi simuloitaessa erilaisten hoitojen vaikutusta kuormitusjakaumiin ja rustokudoksen rappeutumiseen.