Articular cartilage injuries occur frequently in the knee joint. Several methods have been implemented clinically, to treat osteochondral defects but none have been able to produce a long term, durable solution. Photopolymerizable cartilage tissue engineering approaches appear promising; however, fundamentally, forming a stable interface between the tissue engineered cartilage and native tissue, mainly subchondral bone and native cartilage, remains a major challenge. The overall objective of this research is to find a solution for the current problem of dislodgment of tissue engineered cartilage at the defect site for the treatment of degraded cartilage that has been caused due to knee injuries or because of mild to moderate level of osteoarthritis. For this, an in-vitro model was created to analyze the integration of tissue engineered cartilage with the bone, healthy and diseased cartilage over time. We investigated the utility of hydroxyapatite (HA) nanoparticles to promote controlled bone-growth across the bone-cartilage interface in an in vitro engineered tissue model system using bone marrow derived stem cells. We also investigated the application of HA nanoparticles to promote enhance integration between tissue engineered cartilage and native cartilage both in healthy and diseased states. Samples incorporated with HA demonstrated significantly higher interfacial shear strength (at the junction between engineered cartilage and engineered bone and also with diseased cartilage) compared to the constructs without HA (p < 0.05), after 28 days of culture. These findings indicate that the incorporation of HA nanoparticles permits more stable anchorage of the injectable hydrogel-based engineered cartilage construct via augmented integration between bone and cartilage.

O ligamento cruzado anterior é o ligamento mais lesado dentre os ligamentos do joelho. Apesar de um aprimoramento das técnicas de reconstrução, existe ainda um porcentual de pacientes que apresenta resultado funcional insatisfatório. Possivelmente a causa dessa instabilidade rotatória estaria nas estruturas localizadas na porção anterolateral do joelho, não reconstruídas nas reconstruções intra-articulares isoladas. Uma dessas estruturas seria o ligamento anterolateral, estrutura estudada com detalhes nos últimos anos, ainda com controvérsias em relação a seus parâmetros anatômicos, histológicos e de visualização por ressonância magnética. Neste estudo, foram realizadas dissecções de 112 cadáveres para avaliação dos parâmetros anatômicos e histológicos do ligamento anterolateral, sendo que 13 deles foram submetidos ao exame de ressonância magnética previamente às dissecções. O estudo do ligamento anterolateral por ressonância magnética foi realizado também em 42 pacientes. Como resultados, o ligamento anterolateral foi encontrado com constância na região anterolateral do joelho, com origem próxima ao epicôndilo lateral, trajeto anterodistal em direção à tíbia e inserção na periferia do menisco lateral e na região anterolateral da tíbia, entre o tubérculo de Gerdy e a cabeça da fíbula. Sua análise histológica mostrou a presença de tecido conectivo denso e bem organizado, semelhante a tecido ligamentar. A avaliação por ressonância magnética mostrou parâmetros de origem, inserção e trajeto semelhantes às dissecções anatômicas, embora não sendo possível a visualização completa dessa estrutura em todos os exames. Existiu correlação entre as medidas encontradas nos exames de ressonância magnética e nas dissecções, exceto em relação à espessura do ligamento anterolateral / The anterior cruciate ligament is the most injured ligament of the knee. Despite an improvement in reconstruction techniques, there is still a percentage of patients with poor functional outcome after its reconstruction. Possibly, the cause of this rotational instability would be the structures located in the anterolateral portion of the knee, not addressed in the isolated intra-articular reconstructions. One of such structures would be the anterolateral ligament, a structure studied in detail in recent years, with controversies regarding its anatomical, histological and magnetic resonance imaging parameters. In this study, dissections of 112 cadavers were performed to evaluate anatomical and histological parameters of anterolateral ligament, and 13 of them underwent magnetic resonance imaging examination prior to dissection. The anterolateral ligament study by magnetic resonance imaging was also performed in 42 patients. As a result, the anterolateral ligament was found with constancy in the anterolateral region of the knee, with origin near the lateral epicondyle, antero-distal path towards the tibia and insertion in the periphery of the lateral meniscus and in the anterolateral region of the proximal tibia, between Gerdy\'s tubercle and the fibular head. Its histological analysis showed the presence of dense and well-organized connective tissue, similar to a ligamentous tissue. The magnetic resonance imaging evaluation showed origin, insertion and path parameters similar to the anatomical dissections, although it is not possible to fully visualize this structure in all the magnetic resonance imaging exams. There was a correlation between the measurements found in magnetic resonance imaging scans and dissections, except in relation to the anterolateral ligament thickness

Zielstellung: Die Ruptur des vorderen Kreuzbandes (VkB) ist die häufigste Ursache einer Lahmheit beim Hund. Im Gegensatz zu der Humanmedizin, wo die anatomische intraartikuläre Rekonstruktion des vorderen Kreuzbandes als Therapie der Wahl gilt, wird die intraartikuläre Rekonstruktion beim Hund nur selten durchgeführt und hat bis jetzt nicht dauerhaften Erfolg. Die anatomische Platzierung der Bohrkanäle ist bei Menschen für den Erfolg der Operation bei Menschen entscheidend. Erstes Ziel der Studie war die Bestimmung der radiologischen Lage des Zentrums des femoralen vorderen Kreuzbandursprungs beim Hund. Zweites Ziel war die Entwicklung und Erprobung eines Zielgerätes für die arthroskopisch-assistierte, anatomische vordere Kreuzbandrekonstruktion beim Hund. Material und Methode: A. Radiologische Studie: Die kraniale Begrenzung des femoralen Ursprungs des vorderen Kreuzbandes (VK) wurde mit einem röntgendichten Draht bei 49 Femora orthopädisch gesunder Hunde (KM > 20 kg) markiert. Anschließend wurde eine Computertomographie und 3D- Rekonstruktion jedes Femurs angerfertigt, anhand derer der Ursprung manuell segmentiert und das Zentrum berechnet wurde. Schließlich wurden, basierend auf den 3D-Modellen, virtuelle Röntgenbilder in zwei Ebenen berechnet. An diesen wurde die Position des berechneten Zentrums mit drei unterschiedlichen Methoden bestimmt (4x4-Gitterbox-Methode und prozentuale Position für die medio-laterale Projektion; Ziffernblattmethode für die disto-proximale Projektion). B. Zielgerät: Hintergliedmaßen (n = 12) von 6 Hundekadavern (KM ≥20 kg) wurden verwendet. Eine Gliedmaße jedes Kadavers wurde zufällig ausgewählt und die kaudo-kraniale Lage des Zentrums des vorderen Kreuzbandansatzes (vKBA) in medio-lateralen Röntgenbildern berechnet und anschließend auf ein justierbares Zielgerät übertragen. Unter arthroskopischer Kontrolle wurde das Zielgerät hinter der lateralen Kondyle eingehakt und ein Steinmann Pin von extra nach intraartikulär platziert. Die Position der resultierenden Bohrkanäle wurde sowohl röntgenologisch bestimmt als auch dreidimensional mit dem anatomischen Zentrum des vKBA der kontralateralen Hintergliedmaßen verglichen. Ergebnisse: A. Radiologische Studie: In der medio-lateralen Projektion befand sich das Zentrum des femoralen Kreuzbandursprungs im zweiten Rechteck von proximal in der kaudalen Spalte. Die mittlere prozentuale kaudo-kraniale und proximo-distale Position war 20,2 % (± 2,2), beziehungsweise 33,8% (± 3,7). Im disto-proximalen Röntgenbild lag in 97,6 % der Femora das Zentrum des femoralen Kreuzbandursprungs zwischen 14:00 und 15:00 Uhr. B. Zielgerät: In allen postoperativen Röntgenaufnahmen lagen die sechs Bohrkanäle im bzw. nahe dem Zentrum des vKBA. Die 3D- Messungen ergaben eine mediane Abweichung der Bohrkanalposition im Vergleich zum anatomischen Zentrum der kontralateralen Seite von 0,6 mm (Bereich:0,2– 0,9 mm). Schlussfolgerung: Die erarbeiteten Referenzwerte können für die Planung sowie die intra- und postoperative Kontrolle der femoralen Bohrung verwendet werden. Die Verwendung eines justierbaren Zielgerätes ermöglicht die präzise anatomische Platzierung des femoralen Bohrkanals für die intraartikuläre Rekonstruktion des vorderen Kreuzbandes. Die beschriebene Methode wird helfen, eine Fehlplatzierung des femoralen Bohrkanals im Zuge der intraartikulären vorderen Kreuzbandplastik zu reduzieren. In Kombination mit dem bereits beschriebenen tibialen Zielgerät sind nun die technischen Voraussetzungen für die arthroskopisch-assistierte anatomische vordere Kreuzbandplastik in der Tiermedizin gegeben. / Objective: Cranial cruciate ligament (CrCL) pathology is the most frequent cause of lameness in dogs. In contrast to human medicine, where anatomic reconstruction of the ACL is considered the treatment of choice, intra-articular repair in dogs is not commonly performed and until now has not met with enduring success. Accurate tunnel placement has been shown to be crucial in obtaining a successful outcome after anterior cruciate ligament reconstruction in humans. The first aim of our study was to define the radiographic location of the center of the femoral attachment of the CrCL in dogs, for the pre- operative planning as well as post-operative control of anatomical placement of the femoral tunnel. Second aim of the study was to develop and validate an aiming device for arthroscopic femoral tunnel placement. Materials and Methods: A. Radiographic study: Using femora from 49 adult, orthopedically sound dogs (BW ≥ 20 kg), a radiopaque marker was placed on the cranial border of the femoral footprint of the CrCL. Computed tomography and 3D reconstruction of each femur was performed subsequently, followed by manual segmentation of the footprint on the 3D models and calculation of its center. Finally, virtual digital radiographs in two planes were produced and the location of the calculated center of the CrCL was expressed using three different methods (4x4 box grid method and percentage position for the medio-lateral projection; o’clock position for the disto-proximal projection). B. Aiming device: Hindlimbs (n=12) of 6 cadaveric dogs weighing ≥20 kg were used. One hindlimb from each cadaver was randomly chosen and the caudo- cranial position of the CrCL center was calculated, on standard medio-lateral stifle radiographs, and transferred onto to an adjustable aiming device. During stifle arthroscopy the aiming device was inserted and guide pin placed from extra-to-intra-articular. The position of the resulting bone tunnel was evaluated on stifle radiographs and also compared with the anatomic center of each contralateral hindlimb, in the three dimensional (3D) space. Results: A. Radiographic study: In the medio-lateral radiographs the center of the femoral footprint was consistently located in the second rectangle from the top of the most caudal column of the 4x4 grid. The mean percentage caudo- cranial and proximo-distal location was 20.2% (± 2.2) and 33.8% (± 3.7), respectively. In the disto-proximal radiograph, the o’clock position of the CrCL center was between 2 and 3 o’clock in 97.6% of the femora. B. Aiming device: According to the postoperative radiographs, the location of all 6 intra-articular tunnel openings was consistent with the results of the radiographic study. In 3D space, arthroscopic femoral drilling resulted in a median deviation of the drill tunnels of 0.6 mm around the CrCL center. All tunnel openings were located within the CrCL insertion. Conclusions: The reported data can be used to plan and verify the placement of the femoral tunnel opening during intra-articular anatomic CrCL repair. The use of the aiming device suggests that arthroscopic femoral tunnel placement can be achieved with high precision. The measurement for the device can be derived from a standard medio-lateral radiograph of the stifle, which is part of the diagnostic work up of every dog with lameness localized in the stifle. The proposed technique may reduce femoral tunnel misplacement when performing intra-articular CrCL repair in dogs. In combination with the described technique for arthroscopic tibial tunnel drilling, arthroscopic assisted anatomic reconstruction of the CrCL in dogs can be achieved.

1 Abstract Introduction Autologous transplants of the cartilage tissue from the pinna is commonly used in reconstructive surgery of the nasal skeleton. The present study used animal models to elucidate responses of the auricular cartilage to its damage or transplantation to ectopic sites. Histomorphological analysis of changes observed in auricular cartilage including immunohistochemical study of different isoforms of actin and S-100 proteins was performed. Human articular cartilage prepared by in vitro cultivation using artificial scaffolds was also studied after its transplantation. Aims of the study The aim was to study histological changes and expression of chondrocytic markers (α- SMA and S-100 proteins) in intact, artificially traumatised, or in a human auricular cartilage cultivated in culture medium. An attempt to grow human auricular cartilage chondrocytes implanted in vitro into various types of three dimensional scaffolds aimed at testing chondrocyte survival and phenotype both in the culture and after transplantation to immunodeficient mice. A human auricular cartilage transplanted into the nasal skeleton of patients during a reconstruction surgery should be submitted to a histomorphological examination. Research assumed also comparison of the auricular cartilage responses to a damage,...

Indiana University-Purdue University Indianapolis (IUPUI) / Articular cartilage is subjected to dynamic mechanical loading during normal daily activities. This complex mechanical loading, including cell deformation and interstitial fluid flow, affects chondrocyte mechano-chemical signaling and subsequent cartilage homeostasis and remodeling. Focal adhesion kinase (FAK) and Src are known to be main mechanotransduction proteins, but little is known about the effect of mechanical loading on FAK and Src under its varying magnitudes and types. In this study, we addressed two questions using C28/I2 chondrocytes subjected to the different types and magnitudes of mechanical loading: Does a magnitude of the mechanical loading affect activities of FAK and Src? Does a type of the mechanical loading also affect their activities? Using fluorescence resonance energy transfer (FRET)-based FAK and Src biosensor in live C28/I2 chondrocytes, we monitored the effects of interstitial fluid flow and combined effects of cell deformation/interstitial fluid flow on FAK and Src activities. The results revealed that both FAK and Src activities in C28/I2 chondrocytes were dependent on the different magnitudes of the applied fluid flow. On the other hand, the type of mechanical loading differently affected FAK and Src activities. Although FAK and Src displayed similar activities in response to interstitial fluid flow only, simultaneous application of cell deformation and interstitial fluid flow induced differential FAK and Src activities possibly due to the additive effects of cell deformation and interstitial fluid flow on Src, but not on FAK. Collectively, the data suggest that the intensities and types of mechanical loading are critical in regulating FAK and Src activities in chondrocytes.

Univerzita Karlova v Praze 1. lékařská fakulta Postgraduální doktorský program v biomedicíně Studijní obor: Experimentální chirurgie Disertační práce MUDr. Jan Klouda: Preoperative planning of the reconstructive surgery of the forefoot Abstract The presented thesis describes a study aimed at the change of articular surface position in the region of first metatarsophalangeal joint after operative reconstruction due to hallux valgus deformity, done by performing Lapidus arthrodesis. The study evaluates a cohort of patients, who underwent surgery in Department of Orthopaedics, First Faculty of Medicine, Charles University in Prague at Motol University Hospital from 2010 to 2015. The results and clinical implications of the aforementioned retrospective study were published in 2018. The observers evaluated several defined angles on preoperative and postoperative radiographs of the forefoot. The angles were thoroughly selected with regard to the exact representation of first metatarsophalangeal joint articular surface position in relation to the axes of first metatarsal and the whole forefoot. The executed measurements allowed us to assess the overall effectivity of the performed surgery in deformity correction, as well as to describe possible pitfalls and disadvantages resulting from the change of structural...

Einleitung: Die Osteoarthritis ist für einen Großteil der Lahmheiten beim Pferd verantwortlich. Durch die starken Belastungen der equinen Gelenke schreitet die Osteoarthritis unweigerlich fort und setzt diese Tiere einem hohen Leidensdruck aus. Bisherige Therapien reichen nicht aus, um in osteoarthritischen Gelenken die physiologische Integrität des Knorpels wiederherzustellen. Humane und caprine nasale Knorpelzellen zeigten in präklinischen und klinischen Studien ein hohes Regenerationspotential und eine hohe Integrität nach autologer Implantation in Defekte des Gelenkknorpels. Dies wurde auf ihr „Hox-Gen-negatives“ Expressionsprofil zurückgeführt, das sich nach der Implantation dem des artikulären Knorpels anpasste. Ziel der Studie: Das Hox-Genexpressionsprofil nasaler Chondrozyten sollte mit denen artikulärer Chondrozyten in der Zellkultur verglichen werden, um den nasalen Knorpel auch beim Pferd als mögliche autologe Knorpelquelle zu identifizieren. Tiere, Material und Methoden: Knorpelgewebe wurde von 7 verstorbenen Pferden aus dem Nasenseptum und einem vorderen sowie hinterem Fesselgelenk entnommen, Chondrozyten isoliert und bis zur vierten Passage zweidimensional kultiviert. Während der Kultivierung wurden die Chondrozyten alle 3 bis 4 Tage mittelseines eigens erstellten Beurteilungsbogens evaluiert. Zellen aus der ersten (T1) und der dritten (T2) Subkultivierung wurden lysiert, die RNA extrahiert und in cDNA umgeschrieben. Es folgte eine qPCR, um die Expressionslevel von drei Hox-Genen (A3, D1, D8) und zwei knorpeltypischen Genen (SOX9, Kollagen II) an den drei verschiedenen Lokalisationen während T1 und T2 zu bestimmen. Die Quantifizierung der relativen Genexpression erfolgte anschließend mit der ΔΔCT-Methode unter Verwendung von RPL32 und GAPDH als Housekeeping-Gene. Zur statistischen Auswertung wurden die Multiple Lineare Regression, eine einfaktorielle Varianzanalyse (ANOVA) und ein zweiseitiger t-Test herangezogen. Die Signifikanzniveaus aller statistischen Tests wurden auf α= 0,05 festgesetzt. Ergebnisse: Die Hox-Genexpressionen unterschieden sich in Bezug auf die drei Lokalisation und die Messzeitpunkte nicht signifikant voneinander. Die nasalen Chondrozyten wiesen während der ersten Subkultivierung gegenüber den artikulären Chondrozyten signifikant höhere Kollagen-II-Expressionen auf. Eine „Hox-Gen-negative“ Expression konnte für die Tierart Pferd nicht bestätigt werden. Die vorliegende Arbeit zeigt, dass Pferde wahrscheinlich ein speziesspezifisches Hox- Gen-Expressionsmuster aufweisen und dass die equinen Hox-Genexpressionsprofile statistisch signifikanten individuellen Einflüssen unterliegen. Schlussfolgerung: Das equine Hox-Genexpressionsprofil unterliegt statistisch signifikanten individuellen Einflüssen, die bei einer potenziellen Zelltherapie zu beachten sind. Nasale Chondrozyten eignen sich beim Pferd aufgrund ihrer genetischen Ähnlichkeit, bezogen auf die Expressionen der untersuchten Hox-Gene, zu artikulären Chondrozyten und ihrer hohen Bereitschaft zur Kollagen-II-Bildung wahrscheinlich als potenzielle Quelle für die autologe chondrozytäre Implantation (ACI).:1. Einleitung ...................................................................................................... 1 2. Literaturübersicht .......................................................................................... 2 2.1 Knorpel ..................................................................................................... 2 2.1.1 Allgemeiner Überblick ....................................................................... 2 2.1.2 Chondrogenese und Regeneration .................................................. 3 2.1.3 Intraartikulärer Hyaliner Knorpel ....................................................... 3 2.1.4 Extraartikulärer Hyaliner Knorpel....................................................... 6 2.2 Osteoarthritis bei Mensch und Pferd ........................................................ 8 2.2.1 Bedeutung und Ätiologie ................................................................... 8 2.2.2 Pathogenese ..................................................................................... 9 2.2.3 Diagnostik ........................................................................................ 10 2.2.4 Bisherige Therapieansätze .............................................................. 12 2.3 Knorpelgewebe in der Forschung .............................................................13 2.3.1 Kultivierung von Chondrozyten ......................................................... 13 2.3.2 Tiermodelle in der Osteoarthritisforschung ....................................... 15 2.3.3 Neue Therapieansätze ..................................................................... 17 2.3.3.1 Stammzellbasierte Verfahren ...................................................... 17 2.3.3.2 Artikuläre autologe Chondrozyten .............................................. 19 2.3.3.3 Nasale Chondrozyten ................................................................. 21 2.4 Hox-Gene ................................................................................................ 22 2.4.1 Allgemeiner Überblick ..................................................................... ..22 2.4.2 Regulation der Hox-Gene ................................................................. 23 2.4.3 Erkrankungen im Zusammenhang mit Hox-Genen ........................... 25 3. Ziel der Studie ........................................................................................... 27 4. Publikation ................................................................................................ 28 5. Diskussion................................................................................................. 45 5.1 Primer ....................................................................................................46 5.2 Auswahl der Messzeitpunkte und Proben ............................................. 47 5.3 Hox-Genprofile kultivierter equiner Chondrozyten ................................ 49 5.4 Individuelle Hox-Genprofile ................................................................... 50 5.5 SOX9 und Kollagen II .............................................................................51 5.6 Hox-Genprofile im Vergleich verschiedener Spezies ............................. 53 5.7 Ausblick ................................................................................................ 53 6. Schlussfolgerung ...................................................................................... 55 7. Zusammenfassung ................................................................................... 56 8. Summary .................................................................................................. 58 9. Referenzen .............................................................................................. 60 9.1 Literaturverzeichnis .............................................................................. 60 9.2 Abbildungsverzeichnis.......................................................................... 71 10. Anhang .................................................................................................. 72 10.1 Evaluierungsbogen Chondrozytenkulturen ........................................ 72 10.2 Auszug aus der Korrelationsmatrix ..................................................... 73 10.3 Publikationsverzeichnis Christiane Storch .......................................... 74 11. Danksagung .......................................................................................... 75 / Introduction: Osteoarthritis is responsible for most of the lameness in horses. Due to severe stress on equine joints, osteoarthritis inevitably progresses and results in a high degree of suffering. Current therapeutic options are not sufficient to restore the physiological integrity of the cartilage in osteoarthritic joints. Human and caprine nasal chondrocytes demonstrated high regenerative potential and integrity after autologous implantation into articular cartilage defects in preclinical and clinical studies. This was attributed to their “Hox gene negative” expression profile, matching the profile of articular cartilage after implantation. Aim of the study: The Hox gene expression profile of nasal chondrocytes was compared with those of articular chondrocytes in a cell culture to address nasal cartilage as a possible autologous source also in horses. Animals, Material and Methods: Cartilage was harvested from the nasal septum, one anterior and one posterior fetlock joint of deceased 7 horses, chondrocytes were isolated and cultured two-dimensionally until the fourth passage. During cultivation, chondrocytes were evaluated every 3 to 4 days using a specially designed assessment sheet. Cells were harvested during the first (T1) and third (T2) subcultivation. RNA was extracted and transcribed into cDNA. Subsequently, qPCR was performed to determine the expression levels of three Hox genes (A3, D1, D8) and two tissue-identifying genes (SOX9, collagen II) of the three locations after T1 and T2. Quantification of relative gene expression was performed with the ΔΔCT method using RPL32 and GAPDH as housekeeping genes. Multiple linear regression, one-way analysis of variance (ANOVA), and two-tailed t-test were used for statistical analysis. The significance levels of all statistical tests were set at α= 0.05. Results: Hox gene expressions were not significantly different in terms of localization and measurement time points. Nasal chondrocytes exhibited significantly higher collagen II expression than articular chondrocytes during the first subcultivation. 'Hox gene negative' expression could not be confirmed in horses. This study demonstrates that equine Hox gene expression pattern is likely species-specific and that equine Hox gene expression profiles are subject to statistically significant individual influences. Conclusion: The equine Hox gene expression profile is subject to statistically significant individual influences that should be considered in potential cell therapy. Nasal chondrocytes are probably suitable as a potential source for autologous chondrocyte implantation (ACI) in horses due to their genetic similarity, in terms of the expressions of the examined Hox genes, to articular chondrocytes and their high propensity for collagen II formation.:1. Einleitung ...................................................................................................... 1 2. Literaturübersicht .......................................................................................... 2 2.1 Knorpel ..................................................................................................... 2 2.1.1 Allgemeiner Überblick ....................................................................... 2 2.1.2 Chondrogenese und Regeneration .................................................. 3 2.1.3 Intraartikulärer Hyaliner Knorpel ....................................................... 3 2.1.4 Extraartikulärer Hyaliner Knorpel....................................................... 6 2.2 Osteoarthritis bei Mensch und Pferd ........................................................ 8 2.2.1 Bedeutung und Ätiologie ................................................................... 8 2.2.2 Pathogenese ..................................................................................... 9 2.2.3 Diagnostik ........................................................................................ 10 2.2.4 Bisherige Therapieansätze .............................................................. 12 2.3 Knorpelgewebe in der Forschung .............................................................13 2.3.1 Kultivierung von Chondrozyten ......................................................... 13 2.3.2 Tiermodelle in der Osteoarthritisforschung ....................................... 15 2.3.3 Neue Therapieansätze ..................................................................... 17 2.3.3.1 Stammzellbasierte Verfahren ...................................................... 17 2.3.3.2 Artikuläre autologe Chondrozyten .............................................. 19 2.3.3.3 Nasale Chondrozyten ................................................................. 21 2.4 Hox-Gene ................................................................................................ 22 2.4.1 Allgemeiner Überblick ..................................................................... ..22 2.4.2 Regulation der Hox-Gene ................................................................. 23 2.4.3 Erkrankungen im Zusammenhang mit Hox-Genen ........................... 25 3. Ziel der Studie ........................................................................................... 27 4. Publikation ................................................................................................ 28 5. Diskussion................................................................................................. 45 5.1 Primer ....................................................................................................46 5.2 Auswahl der Messzeitpunkte und Proben ............................................. 47 5.3 Hox-Genprofile kultivierter equiner Chondrozyten ................................ 49 5.4 Individuelle Hox-Genprofile ................................................................... 50 5.5 SOX9 und Kollagen II .............................................................................51 5.6 Hox-Genprofile im Vergleich verschiedener Spezies ............................. 53 5.7 Ausblick ................................................................................................ 53 6. Schlussfolgerung ...................................................................................... 55 7. Zusammenfassung ................................................................................... 56 8. Summary .................................................................................................. 58 9. Referenzen .............................................................................................. 60 9.1 Literaturverzeichnis .............................................................................. 60 9.2 Abbildungsverzeichnis.......................................................................... 71 10. Anhang .................................................................................................. 72 10.1 Evaluierungsbogen Chondrozytenkulturen ........................................ 72 10.2 Auszug aus der Korrelationsmatrix ..................................................... 73 10.3 Publikationsverzeichnis Christiane Storch .......................................... 74 11. Danksagung .......................................................................................... 75

Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. One of the common forms of articular cartilage disease which has a huge impact on patient¿s life is arthritis. Research on cartilage cell/tissue engineering will help patients to improve their physical activity by replacing or treating the diseased/damaged cartilage tissue. Cartilage cell, called chondrocyte is embedded in the matrix (Lacunae) and has round shape in vivo. The in vitro monolayer culture of primary chondrocyte causes morphological change characterized as dedifferentiation. Transforming growth factor-beta (TGF-¿), a cytokine superfamily, regulates cell function, including differentiation and proliferation. The effect of TGF-¿1, 2, 3, and their manipulated forms in biological regulation of primary chondrocyte was investigated in this work. A novel method was developed to isolate and purify the primary chondrocytes from knee joint of neonate Sprague-Dawley rat, and the effect of some supplementations such as hyaluronic acid and antibiotics were also investigated to provide the most appropriate condition for in vitro culture of chondrocyte cells. Addition of 0.1mg/ml hyaluronic acid in chondrocyte culture media resulted an increase in primary chondrocyte proliferation and helped the cells to maintain chondrocytic morphology. TGF-¿1, 2 and 3 caused chondrocytes to obtain fibroblastic phenotype, alongside an increase in apoptosis. The healing process of the wound closure assay of chondrocyte monolayers were slowed down by all three isoforms of TGF-¿. All three types of TGF-¿ negatively affected the strength of chondrocyte adhesion. TGF-¿1, 2 and 3 up regulated the expression of collagen type-II, but decreased synthesis of collagen type-I, Chondroitin sulfate glycoprotein, and laminin. They did not show any significant change in production of S-100 protein and fibronectin. TGF-¿2, and 3 did not change expression of integrin-¿1 (CD29), but TGF-¿1 decreased the secretion of this adhesion protein. Manipulated TGF-¿ showed huge impact on formation of fibroblast like morphology of chondrocytes with chondrocytic phenotype. These isoforms also decreased the expression of laminin, chondroitin sulfate glycoprotein, and collagen type-I, but they increased production of collagen type-II and did not induce synthesis of fibronectin and S-100 protein. In addition, the strength of cell adhesion on solid surface was reduced by manipulated TGF-¿. Only manipulated form of TGF-¿1 and 2 could increase the proliferation rate. Manipulation of TGF-¿ did not up regulate the expression of integrin-¿1in planar culture system. The implications of this R&D work are that the manipulation of TGF-¿ by combination of TGF-¿1, 2, and 3 can be utilized in production of superficial zone of cartilage and perichondrium. The collagen, fibronectin and hyaluronic acid could be recruited for the fabrication of a biodegradable scaffold that promotes chondrocyte growth for autologous chondrocyte implantation or for formation of cartilage.

Contexte Un objectif important de la prise en charge de l'arthrite juvénile oligoarticulaire serait d'altérer le cours de la maladie à l'aide d'une thérapie hâtive. Nous avons étudié l'effet des injections intra-articulaires de corticostéroïdes hâtives sur les chances d'atteindre un décompte d'articulation active de zéro et une maladie inactive. Méthode Les données démographiques, cliniques et thérapeutiques des patients avec oligoarthrite juvénile enrôlés dans une étude prospective longitudinale pancanadienne ont été collectées pendant 2 ans. Une injection hâtive était définie comme étant reçue dans les 3 premiers mois suivant le diagnostic. Les équations d'estimation généralisées ont été utilisées pour l'analyse statistique. Résultats Trois cent dix patients ont été inclus. Cent onze (35.8%) ont reçu une injection hâtive. Ces derniers avaient une maladie plus active lors de l'entrée dans l'étude. Les patients exposés à une injection hâtive avaient une chance similaire d'obtenir un décompte d'articulation active de zéro, OR 1.52 (IC95% 0.68-3.37), p=0.306 mais étaient significativement moins à risque d'avoir une maladie inactive, OR 0.35 (IC95% 0.14-0.88), p=0.026. Interprétation Dans cette cohorte de 310 patients avec oligoarthrite juvénile, les injections hâtives de corticostéroïdes n'ont pas mené à une probabilité plus élevée d'atteindre un décompte d'articulation active de zéro ou une maladie inactive. Des problématiques méthodologiques intrinsèques à l'utilisation de données observationnelles pour fins d'estimation d'effets thérapeutiques auraient pu biaiser les résultats. Nous ne pouvons affirmer avec certitude que les injections hâtives n'améliorent pas le décours de la maladie. Des études prospectives adressant les limitations soulevées seront requises pour clarifier la question. / Background One of the goals in oligoarticular juvenile idiopathic arthritis would be to alter the disease course with early therapy. We examined the association between early intra-articular corticosteroid injections and the achievement of an active joint count of zero and inactive disease during the first two years after study enrollment. Methods We included oligoarticular juvenile idiopathic arthritis patients enrolled into a prospective longitudinal cohort across Canada. Demographic, clinical and treatment-related information were collected. Early intra-articular corticosteroid injections was defined as having received the first injection within 3 months of diagnosis. Generalized estimating equations were used for data analysis. Results A total of 310 patients were included, of whom 111 (35.8%) received an early injection. Participants who received an early injection had more severe disease at baseline. Patients exposed to early injections had a similar chance to achieve an active joint count of zero, OR 1.52 (95%CI 0.68-3.37), p=0.306 but were significantly less likely to achieve inactive disease, OR 0.35 (95%CI 0.14-0.88), p=0.026. Interpretation In this cohort of 310 oligoarticular juvenile idiopathic arthritis patients, early intra-articular corticosteroid injections did not result in an increased risk of achieving an active joint count of zero or inactive disease. Methodological issues encountered when estimating treatment effect using observational data might have biased the estimates obtained. Firm conclusion on the inefficacy of early injections in improving outcomes in this population cannot be drawn from this study. Prospective studies addressing the limitations raised will be needed to clarify if early injections can alter the disease course.

O presente estudo destinou-se a avaliar se os resultados clínicos iniciais da viscossuplementação poderiam ser melhorados com a adição de corticosteróide. As injeções intra-articulares são usadas há muitos anos no tratamento da osteoartrite dos joelhos, principalmente com suspensões cristalinas de corticosteróides. A viscossuplementação é uma intervenção relativamente nova, atualmente recomendada no tratamento da osteoartrite. Trata-se da injeção de ácido hialurônico exógeno em articulações diartrodiais, visando, além de restaurar as propriedades reológicas do líquido sinovial, efeitos modificadores da doença osteoartrite. Revisões sistemáticas mostram que a melhora clínica ocorre em duas a cinco semanas após a viscossuplementação. Comparando-se a viscossuplementação com a injeção intraarticular com corticosteróides, dados recentes sugerem maior eficiência no alívio da dor nas quatro primeiras semanas após a infiltração com corticosteróides, similaridade dos procedimentos ao redor da quarta semana e melhores resultados com a viscossuplementação após a oitava semana. Este inicio de ação mais tardio, associado a relatos de sinovite reacional após a viscossuplementação podem desencorajar médicos e pacientes ao uso desta modalidade de tratamento. No presente estudo foram avaliados 104 pacientes em tratamento para osteoartrite do joelho no grupo de doenças osteometabólicas do Instituto de Ortopedia do Hospital das Clínicas da FMUSP. Os pacientes foram randomizados em dois grupos. Um dos grupos foi denominado VS e recebeu uma única injeção intra-articular de 6ml de Hylan GF-20 (Synvisc One®-Genzyme) no joelho estudado. O segundo grupo foi denominado VS+T e recebeu uma injeção intra-articular de 6ml de Hylan GF-20 (Synvisc One®-Genzyme) mais 1ml (20mg) de Hexacetonido de Triancinolona (Triancil®-Apsen). Foram aplicados a escala visual analógica de dor (EVA) e os questionários de WOMAC e Lequesne uma semana antes da injeção e após uma, quatro, 12 e 24 semanas. Os dois grupos com 52 pacientes cada eram homogêneos. Na primeira semana, o WOMAC e a EVA apresentaram melhores resultados no Grupo VS+T (p < 0,01) em relação ao Grupo VS. Na quarta semana não houve diferença entre os grupos. Ambos apresentaram resultados similares nas semanas 12 e 24. Concluiu-se que a adição de hexacetonido de triancinolona melhorou os resultados clínicos da viscossuplementação no curto prazo, sem interferir nos resultados a longo prazo ou na incidência de efeitos adversos / The present study aims to assess if the initial results of viscosupplementation can be improved by the addition of corticosteroid. Intraarticular injections have been used for many years to treat arthritis and other painful articular disorders, mainly using long-lasting crystalline corticosteroid suspensions. Viscosupplementation is a relatively new intervention that is now widely used and recommended for the treatment of knee osteoarthritis. It is comprised of the injection of exogenous hyaluronic acid in diarthrodial joints, in order to restore the rheological properties of synovial fluid and also to promote osteoarthritis disease-modifying effects. Several placebo-controlled studies reported that clinical improvement began only within two to five weeks after viscosupplementation. When comparing viscosupplementation versus intraarticular injection of corticosteroid, recent data suggest that from baseline to week four, intraarticular steroid were more effective for pain relief. By the fourth week, however, both provided similar relief, but beyond the eighth week, hyaluronic acid provided greater pain reduction. The mechanism of action of hyaluronic acid, with delayed onset of pain/functional improvement, combined with reports of reactional sinovitis may discourage physicians and patients regarding this treatment modality. The present study evaluated 104 patients receiving usual care for knee osteoarthritis at the University of São Paulo Medical Center. Patients were randomized to receive either a single intra-articular injection of 6ml of Hylan GF-20 (Synvisc One®-Genzyme) (Group VS) or a single intra-articular injection of 6ml of Hylan GF-20 (Synvisc One®-Genzyme) plus 1ml (20mg) of Triamcinolone Hexacetonide (Triancil®-Apsen) (Group VS+T). VAS, WOMAC and Lequesne questionnaires were applied one week prior the injection, and after one, four, 12 and 24 weeks. The two groups with 52 patients each were homogeneous. At week one, WOMAC and VAS showed better results for Group VS+T compared to Group VS (p < 0,05). At week four the scores did not show statistically significant differences. The groups showed similar results at weeks 12 and 24. In conclusion, the addition of triamcinolone improved first-week symptom and functional scores of viscosupplementation, but not beyond. It did not seem to alter the likelihood of adverse effects