Osteoarthritis (OA), a degenerative disease of articular joints, is the leading cause of chronic disability in the US and affects more than a third of adults over 65 years old. Due to the obesity epidemic and an aging population, the prevalence of OA is expected to rise in both young and old adults. There are no disease modifying OA drugs. Therefore, providing any treatment options that delay the onset or progression of OA is highly desirable. The scope of this dissertation examines two different strategies to promote translational therapies for OA. The first approach investigated whether Smad ubiquitin regulatory factor 2 (Smurf2), an E3 ubiquitin ligase, could be a potential therapeutic target for OA. The second approach examined the incorporation of small chemical residues to enhance the physical and bioactivity of a bioinert scaffold for cartilage tissue repair. Overexpression of Smurf2 in chondrocytes was shown to accelerate spontaneous OA development in mice. We hypothesized that reduced Smurf2 expression could slow the progression of OA and enhance the performance of cells for cartilage repair. By performing surgical destabilization of the medial meniscus (DMM) on Smurf2-deficient mice, loss of Smurf2 was shown to mitigate OA changes in young mice but this protection diminished in older mice. Assessment of Smurf2-deficient chondrocytes in vitro revealed an upregulation of chondrogenic genes compared to wild-type; however, these differences were not seen at the protein level, deterring its potential use for cell-based therapies. During the course of this study, new insights about how age and sex affects different joint compartments in response to DMM surgery were also uncovered. These results broadened existing understanding of DMM-induced OA in mice but also questioned the validity of such a model to identify disease modifying targets that are translatable to OA in humans with advanced age. Due to a lack of innate repair mechanisms in cartilage, damage to cartilage increases the risk of developing OA early. Tissue engineering provides a unique strategy for repairing damaged cartilage by delivering cells in a well-controlled environment that can promote the formation of neotissue. We hypothesized that synthetic chemical residues could enhance the mechanical properties of a bioinert scaffold and promote matrix production of encapsulated chondrocytes. Covalent incorporation of small anionic or zwitterionic chemical residues in a polyethylene glycol-based hydrogel improved its stiffness and resistance to fluid flow, however, the resulting physical environment can also exert a dominant negative effect on matrix production of encapsulated chondrocytes. These results suggest that modulating the biosynthesis of chondrocytes with biochemical signals requires a concurrent reduction in any conflicting mechanotransduction signaling, emphasizing the importance of a degradable system to promote new cartilage formation. In summary, this dissertation establishes Smurf2 as a modulator of OA progression but implies that other factors such as age or protein(s) with redundant Smurf2 functions may play a role in limiting its effect as a therapeutic target. This work also reveals fundamental biology about how chondrocytes behave in response to physical and chemical cues in their microenvironment, which will aid in the design of better scaffolds for cartilage tissue engineering.

Magnetic resonance imaging (MRI) has become the most important modality for assessment of pathological changes in the knee cartilage. The assessment of the cartilage is usually made by a set of anatomical MRI images with different sequences. Newer techniques, that map various in MRI parameters, have been developed and allows changes in an earlier stage of the disease. One of these techniques is T2 mapping. The goal of this thesis was to compare this newer technique, T2 mapping, with the standard MRI assessment for assessment of articular cartilage on distal femur in the knee. The purpose was to assess the cartilage with these two different methods and analyze its outcomes. Eight subjects were included in this study and scanned with a 3.0 T or 1.5 T MRI machine. A specific MRI knee protocol was used for the standard MRI assessment, and a multi-echo sequence was used for the T2 mapping. The T2 map was created and analyzed in the program IntelliSpace Portal. Both the standard MRI assessment and the T2 map showed changes in the knee cartilage. The result showed either indication for damage cartilage or healthy cartilage. The standard assessment showed cartilage lesion in three subjects and no lesion in five subjects. The same outcomes were with the T2 mapping. However, not all results were equal. The T2 mapping also showed higher values in the trochlea area where no indications for changes were found in the standard assessment. This study showed similar results for both the standard assessment and the T2 map. Both methods could identify damage and is, therefore, useful for assessment of the knee cartilage. The outcomes of the different methods differ, and the assessment is therefore made in different ways. The T2 mapping can be analyzed both visual and quantitative. The outcomes were both a color map of the knee but also results in graphs and values. The standard assessment is only assessed from grayscale images. The best outcomes from the T2 mapping was when it only was changes within the cartilage and not when the cartilage lesion was adjacent to an underlying bone lesion. Based on what was examined in this work, the best result was when T2 mapping was used together with the anatomical images used in the standard assessment. The conclusion is that the standard assessment is necessary when it comes to make a damage assessment and perform damage marking as for Episurf. The T2 mapping is, however, an interesting method and will be more useful with more applications in the future. It is therefore exciting to keep an eye on the technology and its development. / Magnetisk resonanstomografi (MR) har blivit den viktigaste modaliteten vid bedömning av patologiska förändingar i knäbrosket. Bedömningen av brosket görs vanligtvis med hjälp av anatomiska MR bilder som är skannade med olika sekvenser för att få olika viktningar på bilderna. En nyare teknik, T2 mappning, som kartlägger olika MR prameterar, har utvecklats för att med hjälp av andra parametrar analysera knäbrosket. Den här tekniken har resulterat i att förändringar i brosket kan upptäckas vid ett tidigare stadie i sjukdomsförloppet. Målet med det här examensarbetet var att jämföra de olika teknikerna, T2 mappning och MR-standardbedömningen, för att bedöma ledbrosket på distala lårbenet i knäet. Syftet var att bedöma brosket utifrån dessa olika metoder samt att analysera och jämföra dess resultat. Åtta subjekt ingick i studien och skannades med en 3,0 T eller 1,5 T MR-maskin. Ett specifikt MR-knäprotokoll användes för att skanna sekvenserna som ingick i standard bedömningen och en multi-ekosekvens användes för T2 mappningen. T2-mappningen skapades och analyserades sedan i programmet IntelliSpace Portal. Både standard MR-bedömningen och T2-mappningen visade tydliga förändringar i brosket. Resultatet visade antingen indikationer på skadat eller friskt brosk. Standardbedömningen visade broskskador hos tre subjekt och inga broskskador hos fem subjekt. Samma resultat visades med T2-mappningen. Däremot skilde sig vissa resultat mellan T2 mappningen och standardbedömningen. Då denna studie visade liknande resultat för både standardbedömningen och T2-mappningen, är båda metoderna användbara för bedömning av knäbrosket. De olika metoderna har olika utfall vilket gör att bedömningen sker på olika sätt. I T2 mapping får man ut både en färgkarta över knät men också grafter och värden som kan användas. I standardbedömningen görs bedömningen bara utifrån olika gråskalebilder. T2 mappningen var mest användbar när det var tydliga förändingar i bara brosket och inte när skadan mest var i benet. Det bästa resultatet var däremot när T2 mappning användes tillsammans med standardbedömningen. Slutsatsen är att standardbedömningen är nödvändig när det kommer till att bedömma skador och göra en skademarkering så som för Episurf. T2 mapping är däremot en väldigt intressant teknik men är idag inte en vanlig teknik inom diagnostiken och saknar just nu något tydligt användningsområde. Däremot, finns det stor potential och kommer troligtvis bli vanligare och få fler användingsområden i framtiden.

Articular cartilage is a specialized connective soft tissue that resides onthe ends of long-bones, transfers the load smoothly between the bones in diarthrodialjoints by providing almost frictionless, wear resistant sliding surfacesduring joint articulation. Focal chondral or osteochondral defects in articularcartilage are common and show limited capacity for biological repair. Furthermore,changes in the bio-mechanical forces at the defect site may makethe tissue more susceptible to continued degeneration. Alternatively, the contouredfocal resurfacing metal implant can be used to treat such full thicknesscartilage defects. Physiological and biomechanical studies on animal modelswith metal implant have shown good clinical outcomes. However, the mechanicalbehavior of cartilage surrounding the implant is not clearly known withrespect to the joint function after treating such defects with metal implantsand also to improve the implant design. We developed a simple 3-dimensionalfinite element model by approximating one of the condyles of the sheep kneejoint. Parametric study was conducted in the simulations to verify differentprofiles for the implant, positioning of the implant with respect to cartilagesurface, defect size and to show the mechanical sealing effect due to the wedgeshape of the implant. We found the maximal deformations, contact pressuresand stresses which constitute the mechanical behavior of cartilages. We alsoconfirmed that using a metal implant to fill the full thickness chondral defectsis more beneficial than to leave the defect untreated from mechanical point ofview. The implant should be positioned slightly sunk into the cartilage basedon the defect size, in order to avoid damage to the opposing surface. The largerthe defect size, the closer the implant should be to the flush. We also simulatedthe time dependent behavior of the cartilages. In all the simulations, a staticaxial loading was considered. The wedge shape of the implant provided themechanical sealing of the cartilage surrounding the implant. The determineddeformations in the cartilages immediately surrounding the implant are instrumentalin predicting the sticking-up of the implant into the joint cavity whichmay damage opposing soft tissues. / <p>QC 20101125</p>

Гидроцефалия – это синдром или симптом нарушения ликвородинамики, вызванный самыми различными заболеваниями. Врожденная гидроцефалия ассоциируется с нарушениями двигательной активности у детей раннего возраста. Целью работы стала оценка влияния разработанной программы физической реабилитации на степень выраженности двигательных нарушений у детей от 6 месяцев до 2 лет с гидроцефалией. В исследовании приняли участие 38 детей с гидроцефалией после оперативной коррекции ликвородинамики. В качестве методов исследования степени выраженности двигательных нарушений использовали шкалу оценки моторных функций GMFCS, шкалу оценки мышечной силы, модифицированную шкалу Ашфорта, ортостатическую устойчивость гемодинамических параметров оценивали при вертикализации. Согласно полученным результатам, предложенная программа физической реабилитации позволила снизить степень выраженности двигательных нарушений, улучшить мышечный тонус, повысить двигательную активность у детей с гидроцефалией. Также результаты апробации программы позволили подтвердить значимость восстановления подвижности мелких суставов и мышцы, а также вертикализации для профилактики осложнений, обусловленных ограничениями двигательной активности у детей с гидроцефалией. / Hydrocephaly is a syndrome or symptom of impaired liquorodynamics caused by a variety of diseases. Congenital hydrocephalus is associated with movement disorders in young children. The aim of the work was to assess the impact of the developed program of physical rehabilitation on the severity of motor disorders in children from 6 months to 2 years old with hydrocephaly. The study involved 38 children with hydrocephaly after surgical correction. As methods for studying the severity of motor disorders, the GMFCS scale for assessing motor functions, the scale for assessing muscle strength, the modified Ashworth scale, and the orthostatic stability of hemodynamic parameters were assessed during verticalization. According to the results obtained, the proposed program of physical rehabilitation allowed to reduce the severity of movement disorders, improve muscle tone, and increase motor activity in children with hydrocephalus. Either, the results of approbation of the program made it possible to confirm the importance of restoring the mobility of small joints and muscles, as well as verticalization for the prevention of complications caused by restrictions on motor activity.

Post-traumatic osteoarthritis (PTOA) of the knee is a complication resulting from direct injury to the joint, such as anterior cruciate ligament and meniscus tears, and accounts for approximately 12% of all OA cases. The economic and clinical impact of PTOA is also greater than idiopathic OA, as patients are younger and often more active, requiring treatments for symptomatic OA over a greater fraction of their lifetime. A common strategy to manage pain and inflammation associated with PTOA is the intraarticular administration of corticosteroids. However, these injections are limited due to the requirement of high-doses imposed by synovial joint clearance rates and their resulting systemic side effects. In addition, currently used broad-spectrum corticosteroids are palliative and not curative, stemming from incomplete knowledge of specific mechanisms that drive cartilage degeneration and other joint pathologies. Thus, most patients with PTOA eventually undergo surgical procedures such as osteochondral graft transplantation for focal defects and in more severe cases, total knee arthroplasty. As such, the studies presented in this dissertation (i) offer specific insights into mechanisms by which traumatic injury can drive joint degeneration and (ii) present novel strategies to modulate joint responses to pathological factors by leveraging sustained drug-delivery platforms. In Part I, mechanistic assessments of human cartilage and synovium responses to insults are conducted to identify novel pathways that may lead to impaired joint homeostasis. First, a direct consequence of traumatic injury, hemarthrosis, is explored as a potential contributor to the development of PTOA specifically through contributions by red blood cells. We demonstrate for the first time the differential roles of erythrocytes in their intact and lysed states through measures of oxidative stress and changes to metabolomic profiles in the context of ferroptosis. Furthermore, we demonstrate the therapeutic potential of Ferrostatin-1, a lipophilic radical scavenger in inhibiting pathological changes to cartilage and its crosstalk with the neighboring synovium in an in vitro model of hemophilic arthropathy. Second, a strategy to prevent an indirect consequence of traumatic injury, arthrofibrosis, is presented in an in vitro model of joint contraction. Fibrosis and the presence of hyperplastic synovium are implicated in the progression of OA through pathological shifts in tissue composition as well as secreted factors that promote cartilage degeneration and the maintenance of a pro-inflammatory joint environment. A type I transforming growth factor beta-1 receptor inhibitor, SB-431542, is encapsulated in polymeric microspheres for the prophylactic treatment of arthrofibrosis through sustained low-dose drug delivery to circumvent the challenges associated with resident joint clearance rates. Utilizing human-based in vitro models of cartilage and synovium pathology, we present novel mechanisms and therapeutic strategies to prevent pathological changes following traumatic joint injury that may contribute to the development of PTOA. In Part II, the sustained delivery platform introduced in Part I is extended to the treatment of PTOA. Osteochondral graft transplantation is currently the clinical gold standard for large focal cartilage lesions. However, allograft procedures are limited due to the lack of available donor tissues and autografts are associated with complications due to donor-site morbidity. In both cases, grafts are subject to failure, potentially in part due to the continual presence of pro-inflammatory factors following surgical procedure. In this section, we present cellular agarose hydrogels embedded with dexamethasone-releasing microspheres that are integrated with a titanium base as a functional tissue-engineered alternative to native osteochondral allografts. These allogenic tissue-engineered grafts were assessed in an in vivo preclinical canine model in their ability to maintain clinical function and to modulate the inflammatory response over the course of 12 months. We successfully demonstrated the feasibility of using engineered grafts by comparing clinical measures of range of motion, function, lameness, and pain, as well as modified cartilage graft scores, against native osteochondral allograft controls. In addition, improvements in the histopathological scoring of neighboring synovial and meniscal tissues indicate the therapeutic capacity of dexamethasone released from within the joint to modulate the inflammatory response up to one-year post-implantation. Taken together, the studies presented in this dissertation identify novel mechanisms behind pathological changes to the cartilage and synovium that may contribute to the development of PTOA following injury. Potential therapeutic targets, inhibitory compounds, and delivery strategies are also assessed using human-based in vitro models of disease and further validated in an in vivo canine model through a clinically relevant timeframe. Ultimately, we demonstrate for the first time, the use of dual-function tissue-engineered grafts in a weight-bearing region of the knee joint to circumvent limitations associated with the clinical gold standard for the treatment of large focal cartilage defects.

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Articular cartilage functions to reduce the mechanical stresses associated with diarthrodial joint movement, protecting these joints over a lifetime of use. Tissue function is maintained through the balance between synthesis and resorption (i.e., metabolism) of extracellular matrix (ECM) by articular chondrocytes (ACs). Two important hormonal regulators of cartilage metabolism are interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I). These factors have antagonistic effects on chondrocyte activity, and during the progression of osteoarthritis, IL-1 is thought to promote chondrocyte hyporesponsiveness to IGF-I. To better understand how the anabolic (IGF-I) and catabolic (IL-1) stimuli are linked within articular cartilage, we examined the mechanisms by which IL-1 regulates the IGF-I signaling system of ACs. Equine chondrocytes from non-arthritic stifle joints were multiplied over serial passages, re-differentiated in alginate beads, and stimulated with recombinant equine IL-1β. Chondrocytes were assayed for type I IGF receptor (IGF-IR), IGF binding proteins (IGFBPs), and endogenously-secreted IGF-I. Our experimental findings solidify the significance of IL-1 as a key regulator of IGF-I signaling within articular cartilage, demonstrating that regulation of the IGF-I system occurs through both direct (transcription) and indirect (proteolysis) mechanisms. These results have implications for molecular therapies (e.g., gene transfer) directed at reversing osteoarthritic cartilage deterioration. The presented research concerns not only cartilage biology but also tissue engineering strategies for cartilage repair. Alginate hydrogel culture has been reported to re-establish chondrocytic phenotype following monolayer expansion, but studies have not addressed effects on the signaling systems responsible for chondrocyte metabolism. We investigated whether chondrocyte culture history influences the IGF-I system and its regulation by IL-1. ACs expanded by serial passaging were either encapsulated in alginate beads or maintained on tissue culture plastic (TCP). Bead and TCP cells were plated at high-density, stimulated with IL-1β, and assayed for expression of IGF-I signaling mediators. Intermediate alginate culture yielded disparate basal levels of IGF-IR and IGFBP-2, which were attributed to differential transcription. The distinct mediator profiles coincided with varied effects of exogenous IL-1β and IGF-I on collagen Ia1 expression and cell growth rate. This study demonstrates that culture strategy impacts the IGF-I system of ACs, likely impacting their capability to mediate cartilage repair. / Ph. D.

In der Diagnostik und Reparatur von hyalinem Gelenkknorpel sind neue Methoden zur Quantifizierung von Struktur und mechanischer Belastbarkeit gefragt, um die Behandlung von Knorpelschäden an Millionen von Patienten weltweit zu verbessern. Mittels hochfrequentem, fokussierten Ultraschall werden Oberflächenparameter für Reflektivität und Rauheit an Gelenkknorpel bestimmt. Es wird gezeigt wie die Oberflächenneigung kontrolliert werden kann. Die Ergebnisse vermitteln ein besseres Verständnis über die Zusammensetzung der Ultraschallsignale aus reflektierten und gestreuten Komponenten. 3D Ultraschallscans von Knorpelregeneraten erlauben die Defektstellen volumetrisch zu Quantifizieren. Die Proben wurden zusätzlich nach etablierten Bewertungssystemen benotet, welche auf makroskopischer Beurteilungen, MRT-Scans und Histologie basieren. Die ultraschallbasierten Volumendaten zeigten dabei gute Korrelationen mit den Punktwertungen. Die im Labor verwendeten Messaufbauten zur biomechanischen Charakterisierung von Gelenkknorpel können am Patienten nicht angewandt werden. Daher können Ärzte die Festigkeit von Knorpel bisher nur mittels manueller Palpation abschätzen. Diese Arbeit entwickelt eine Methode der Ultraschall-Palpation (USP), die es erlaubt, die während der manuellen Palpation erzeugte Kraft und Deformation, basierend auf Ultraschallechos, aufzunehmen. Es wurde einen Prototyp entwickelt womit gezeigt werden konnte, dass USP eine ausreichende Genauigkeit und Reproduzierbarkeit aufweist. Wiederholte Messungen können zusätzlich zeitabhängige biomechanische Parameter von Knorpel ableiten. Zusammenfassend zeigt diese Arbeit verbesserte und neue Möglichkeiten zur strukturellen und biomechanischen Charakterisierung von hyalinem Gelenkknorpel bzw. den Ergebnissen von Knorpelreparatur basierend auf Ultraschalldaten. Diese Methoden haben das Potenzial die Diagnostik von Gelenkknorpel und die Quantifizierung von Knorpelreparatur zu verbessern. / In the diagnostics and repair of hyaline articular cartilage, new methods to quantify structure and mechanical capacity are required to improve the treatment of cartilage defects for millions of patients worldwide. This thesis uses high frequency focused ultrasound to derive surface parameters for reflectivity and roughness from articular cartilage. It is shown how to control the inclination dependency to gain more reliable results. Furthermore, the results provided a better understanding of the composition of ultrasonic signals from reflected and scattered components. 3D ultrasound scans of cartilage repair tissue were performed to quantify defect sites after cartilage repair volumetrically. The samples were also graded according to established scoring systems based on macroscopic evaluation, MRI scans and histology. The ultrasound-based volumetric parameters showed good correlation with these scores. Complex biomechanical measurement setups used in laboratories cannot be applied to the patient. Therefore, currently physicians have to estimate the stiffness of cartilage by means of manual palpation. In the last part of this thesis, a method denoted as ultrasound palpation is developed, which allows for measuring the applied force and strain during manual palpation in real time, solely based on the evaluation of the time of flight of ultrasound pulses. A prototype was developed and its measurement accuracy and reproducibility were characterized. It could be shown that ultrasound palpation has sufficient accuracy and reproducibility. Additionally, by repeated measurements it was possible to derive time-dependent biomechanical parameters of cartilage. In summary, this work shows improved and new possibilities for structural and biomechanical characterization of hyaline articular cartilage and the outcomes of cartilage repair based on ultrasound data. The methods have the potential to improve the diagnostics of articular cartilage and quantification of its repair.