No description available.

The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases.

The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases.

The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Ce mémoire porte sur l’étude de pyrophosphates de calcium hydratés (CPP : Ca2P2O7•nH2O), composés rencontrés dans des micro-calcifications pathologiques associées à l’arthrose et, dans certains cas, responsables d’arthropathie destructive. Ces cristaux, présents dans les articulations de patients arthritiques, possèdent un fort potentiel inflammatoire susceptible d’engendrer une dégradation aigüe du cartilage. Cependant, les mécanismes de formation des phases de CPP et d’activation de leur potentiel inflammatoire n’ont pas encore été entièrement décrits. Nous nous sommes intéressés à l’étude des conditions de formation des différentes phases de pyrophosphates de calcium hydratés in vitro ainsi qu’à la caractérisation fine de chacune des phases d’intérêt biologique avec des outils de laboratoire et de grands instruments afin de mieux comprendre leurs propriétés physico-chimiques et d’améliorer leur identification in vitro et in vivo. Dans un premier temps, un protocole de synthèse a été établi permettant la synthèse de quantités importantes de chacune des phases pures de CPP (CPP amorphe, CPP dihydratés monoclinique et triclinique et CPP tétrahydraté). Les conditions de synthèse associées à la formation de chacune de ces phases, pH et température notamment, ont été explorées. Des échantillons purs ont été utilisés comme références pour les différentes études physico-chimiques et structurales qui ont ensuite été menées. Les échantillons de référence ont été caractérisés finement, d’un point de vue structural avec notamment la résolution de plusieurs structures cristallines (diffraction des rayons X et des neutrons, sur poudre et monocristal) mais aussi au travers de différentes analyses spectroscopiques (spectroscopies FTIR et Raman, RMN du solide) et d’analyses de la morphologie des cristaux (microscopies électroniques à balayage et en transmission, diffraction électronique). Chacune de ces analyses complémentaires, couplées à des modélisations ab initio, a permis de préciser les hypothèses suggérant un rôle de la surface des cristaux dans le potentiel inflammatoire de ces phases. Une troisième partie est consacrée à l’exploration de différentes techniques de synthèse mettant en œuvre différents milieux (cristallisation en solution et en gel). Ces expériences ont permis d’établir des comparaisons avec les processus de formation observés in vivo et d’évolution in vitro à haute température des phases de CPP. Finalement des études ex vivo de cartilages calcifiés seront présentées, mettant en évidence les avantages de ces techniques de caractérisation de laboratoire comme outils de diagnostic. Ce travail permet ainsi de préciser les mécanismes physico-chimiques liés aux différentes phases de CPP in vitro et in vivo afin de mieux comprendre la formation de ces phases et leur potentiel inflammatoire associé, tout en améliorant les possibilités de diagnostic des arthropathies microcristallines. / The present work concerns the study of hydrated calcium pyrophosphates (CPP: Ca2P2O7•nH2O), a group of phases detected in pathological microcalcifications and associated with arthritis. These crystals are frequently observed in the synovial fluid of arthritic patients and they were described as having a high inflammatory potential which could induce a severe degradation of cartilage. However, the mechanisms involved in the formation of the CPP crystals and the activation of their inflammatory potential are not fully understood. This work is focused on the study of the synthesis conditions of CPP in vitro and on the fine characterization of CPP phases of biological interest using laboratory equipments and large-scale facilities. The aim of this work was to describe the physico-chemical properties of these materials, including inflammatory potential, and to improve their identification in vivo and in vitro. First, a synthesis protocol was designed for the production of significant amounts of pure samples for each of the CPP phases (amorphous CPP, monoclinic and triclinic dihydrated CPP and tetrahydrated CPP). Different conditions, including pH and temperature, were studied to achieve the synthesis of reference materials. These samples were precisely characterized using complementary techniques to determine their crystalline structures (powder and single crystal X-ray diffraction and neutron diffraction) as well as using spectroscopic (FTIR and Raman spectroscopies, MAS-NMR) and morphologic analyses (SEM, TEM and electron diffraction). These analyses, combined with ab initio modeling, clarified the hypotheses concerning the role of the crystal surface on the adsorption properties of CPP crystals and their inflammatory potential. The third part of this thesis is focused on the study of CPP synthesis conditions, by using different experimental setups to study crystallization in solution and in gel. A comparison with in vivo formation processes and in vitro high temperature evolution phenomena of these phases was established based on the results of these experiments. Finally, ex vivo analyses of pathological cartilage are presented, highlighting the advantages of different laboratory characterizations as medical diagnostic tools. This work contributes to clarify the physico-chemical characteristics of CPP phases in vitro and in vivo, to improve the knowledge on the formation and the evolution of these phases, their properties including inflammatory potential, and to facilitate their identification in vivo.

Το αντικείμενο της παρούσας μεταπτυχιακής διατριβής ειδίκευσης ήταν η μελέτη των φυσικοχημικών και μηχανικών ιδιοτήτων παθολογικά αλλοιωμένων ανθρώπινων μηνίσκων. Οι μηνίσκοι είναι ινοχόνδρινοι μηνοειδείς δίσκοι που παρεμβάλλονται μεταξύ των αρθρικών επιφανειών του μηριαίου κονδύλου και της κνημιαίας γλύνης. Αποτελούνται ως επί το πλείστον από ίνες κολλαγόνου και χονδροκύταρα. Περιέχουν ακόμα γλυκοσαμινογλυκάνες, ελλαστίνη και DNA. Συμβάλλουν καθοριστικά στη σωστή λειτουργία της άρθρωσης καθώς είναι επιφορτισμένοι με λειτουργίες, όπως η προστασία των αρθρικών επιφανειών από τη φθορά, η λίπανση και η σταθεροποίηση της άρθρωσης. Η σημαντικότερη ίσως μεταβολική νόσος των μηνίσκων είναι η χονδρασβεστίωση (chondrocalcinosis) κατά την οποία παρατηρείται ανάπτυξη ανόργανων αλάτων στην επιφάνεια ή/και στο σώμα του ιστού. Για τη μελέτη των ιδιοτήτων των παθολογικά αλλοιωμένων ανθρώπινων μηνίσκων χρησιμοποιήθηκαν τεχνικές μηχανικής και φυσικοχημικής ανάλυσης. Με Δυναμική Μηχανική Ανάλυση επιχειρήθηκε να αξιολογηθεί η επίδραση των διαφόρων τύπων παθολογικών αλλοιώσεων στις δυναμικές μηχανικές ιδιότητες των ιστών. Οι ασβεστοποιημένοι ιστοί και οι εναποθέσεις τους μελετήθηκαν με φασματοσκοπικές τεχνικές RAMAN, FT-RAMAN, FT-IR καθώς επίσης και με απεικονιστικές τεχνικές οπτικής μικροσκοπίας, σαρωτικής μικροσκοπίας ηλεκτρονίων (SEM) και μικροσκοπίας ατομικής δύναμης (AFM). Επιπλέον, για πρώτη φορά έγινε διασύγκριση της ικανότητας τριών διαφορετικών διατάξεων RAMAN (FT-RAMAN: 1064 nm, Dispersion RAMAN: 633 nm και Dispersion RAMAN: 325 nm) να εντοπίσουν την παρουσία εναποθέσεων στους ιστούς. Στην παρούσα μελέτη εξετάστηκαν 63 μηνισκικά μοσχεύματα από οστεοαρθριτικούς ασθενείς οι οποίοι υποβλήθηκαν σε ολική αρθροπλαστική γόνατος στο Περιφερειακό Πανεπιστημιακό Νοσοκομείο Πατρών. Τα μοσχεύματα αυτά εξετάστηκαν από ιστοπαθολόγο και διαγνώστηκαν οι παθολογικές αλλοιώσεις που έφερα καθένας από αυτούς. Με βάση τα ιστοπαθολογικά αποτελέσματα έγινε κατηγοριοποίηση των δοκιμίων και συσχέτιση των αποτελεσμάτων αυτών με τα αποτελέσματα των προαναφερθέντων τεχνικών. Από τα αποτελέσματα της δυναμικής μηχανικής ανάλυσης φάνηκαν μικρές αλλά όχι στατιστικά σημαντικές διαφοροποιήσεις μεταξύ ασβεστοποιημένων μηνίσκων και μηνίσκων που παρουσίαζαν μυξωματοειδή εκφυλισμό και κύστες (σύμφωνα με τα αποτελέσματα της ιστοπαθολογικής εξέτασης). Τα αποτελέσματα της περιθλασιμετρίας ακτίνων Χ των ασβεστοποιημένων ιστών έδειξαν παρουσία μείγματος τρικλινούς και μονοκλινούς διένυδρου πυροφωσφορικού ασβεστίου (CPPD) υψηλής κρυσταλλικότητας καθώς και μικροκρυσταλλικές εναποθέσεις απατιτικής σύστασης (HAP). Οι τεχνικές μικροσκοποίας (οπτική, SEM και AFM) επιβεβαίωσαν τον παραπάνω ισχυρισμό αποκαλύπτοντας το χαρακτηριστικό τρόπο ανάπτυξης κάθε τύπου εναπόθεσης. Τα αποτελέσματα της φασματοσκοπικής ανάλυσης με χρήση των τεχνικών RAMAN – FT-RAMAN – και FT-IR έδειξαν την παρουσία επίσης χαρακτηριστικών κορυφών του CPPD και του HAP. Οι εναποθέσεις από CPPD ανιχνεύθηκαν στο 82.6 % των ασβεστοποιημένων δειγμάτων (19 στα 23) ενώ από απατίτη στο 17.4% (4 στα 23) Aναφορικά με τα RAMAN, επισημαίνεται ότι ανεξάρτητα από το μήκος κύματος που χρησιμοποιήθηκε και παρά την ισχυρή μπάντα φθορισμού που επεδείκνυαν τα φάσματα, οι χαρακτηριστικές κορυφές των εναποθέσεων ήταν εμφανείς. / The aim of the present master thesis study was the physicochemical and biomechanical characterization of pathological human menisci. Menisci are semilunar shaped fibrocartilaginous tissues that lie between the femoral condyle and tibia plateau. They mainly consist of collagen fibers and chondrocytes. Additionally glycosaminoglycans, elastin and DNA are present in the tissue. Menisci play an important role in the good function of human knee joint by distributing the applied loads, contributing to the joint’s stability and lubricating the joint’s area. The most common metabolic meniscal lesion is the chondrocalcinosis where inorganic phases of calcium or sodium salts are deposited in/on the tissue. Pathologically altered human menisci were studied by mechanical and physicochemical analysis techniques. Dynamic Mechanical Analysis was used to investigate the effects of degeneration lesions on the dynamic mechanical properties of the tissue. Calcified tissues and the corresponding encrustations were studied by spectroscopic techniques such as RAMAN, FT-RAMAN, FT-IR as well as imaging techniques such as optical microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Moreover, for the first time a comparison of the ability of three different RAMAN setups (FT-RAMAN: 1064 nm, Dispersion RAMAN: 633 nm and Dispersion RAMAN:325 nm) to detect calcification on the tissues was performed. The clinical sample comprised of 63 menisci with histopathologicaly confirmed alterations that were surgically extracted from osteoarthritic patients due to total knee arthroplasty in the University Hospital of Patras. Samples were classified according to the histopathological evaluation and a correlation of histopathological with mechanical or spectroscopic findings was attempted. Dynamic mechanical analysis results showed small but no statistically significant difference between the group of calcified and the group of mucoid and cystic degenerated tissues (as determined after histopathological examination) X-ray diffraction analysis of the menisci encrustations revealed highly crystallized depositions of triclinic and monoclinic Calcium Pyrophosphate Dihydrate (CPPD) and some samples with poorly crystallized apatitic depositions (HAP). Microscopic techniques (optical, SEM and AFM) confirmed the presence of CPPD and HAP and in addition revealed information regarding their growth mechanism.The results of spectroscopical analysis using RAMAN – FT-RAMAN and FT-IR showed also the characteristic peaks of the inorganic phase (CPPD or HAP) presented in/on the tissue. CPPD encrustations were detected in 82.6 % (19 of 23) of the calcified samples while apatite in 17.4 % (4 of 23). Moreover, it is notable that all RAMAN setups showed the characteristic peaks of CPPD and HAP regardless of the excitation source and the strong fluorescence band.

L’arthrose affecte une dizaine de millions de français. La présence de microcristaux calciques notamment ceux de pyrophosphates de calcium (CPP) dihydratés (Ca2P2O72H2O ; mono- et/ou triclinique : respectivement m- et/ou t-CPPD) au sein de l’articulation est l’un des facteurs aggravant identifié de cette maladie. Cependant, les conditions et mécanismes de formation de ces cristaux ainsi que l’ordre d’apparition de ces deux phases de CPPD restent méconnus ou n’ont pas encore été entièrement décrits et les traitements existants sont seulement symptomatiques. Dans ce mémoire nous nous sommes intéressés à l’étude de l’effet d’additifs ioniques sur la formation et les cinétiques de croissance cristalline de cristaux de CPP qui constitue une étape clé pour la mise au point à moyen ou long terme d’une stratégie thérapeutique visant à empêcher la formation, transformer ou dissoudre ces cristaux. La synthèse in vitro par précipitation des phases pures de CPP hydratés d’intérêt biologique (m-CPPD, t-CPPD et CPP monoclinique tétrahydraté ) a été réalisée afin d’identifier les paramètres opératoires conduisant à leur apparition et d’analyser l’effet d’additifs ioniques (Mg2+, Cu2+, Zn2+, Fe3+ ou S2O32-) lors de leur précipitation en conditions maîtrisées. La méthode de synthèse établie par Gras et al. a été transposée en réacteur agité. Il s’agit d’une précipitation avec ajout simultané de deux solutions mères respectivement de nitrate de calcium et de pyrophosphate de potassium dans une solution tampon d’acétate d’ammonium. Les composés obtenus ont été caractérisés par diffraction des rayons X, résonance magnétique nucléaire du solide, spectroscopies FTIR et RAMAN, microscopie électronique à balayage (MEB) et spectrométrie à plasma à couplage inductif. Grâce à une analyse semi-quantitative par diffraction des RX, nous avons montré que la présence d’additifs ioniques (Mg2+, Zn2+, Fe3+ ou Cu2+) lors de la précipitation change significativement les proportions des phases de CPP du produit final. Ainsi nous montrons que les ions Mg2+, Cu2+ ou Zn2+ orientent la formation de cristaux de m-CPPD lors de la précipitation en conditions maîtrisées de t-CPPD, et selon les conditions l’ion Fe3+ peut totalement inhiber la cristallisation de t-CPPD. Des analyses fines par RMN du solide 31P ont permis de déterminer les modes d’action de ces additifs. Les ions Cu2+ et Mg2+ ont notamment agi par substitution au Ca2+ dans la maille de CPP. Nous avons aussi mis en œuvre la méthode de croissance cristalline à composition constante pour étudier la germination et la croissance cristalline à température, pH, sursaturation et force ionique constants de t-CPPD sur une semence de t-CPPD en présence ou non d’additif ionique. La semence est introduite dans une solution sursaturée métastable de CPP à pH 6,2 incluant l’additif ionique et placée dans un réacteur agité et thermostaté à 37°C. La sursaturation du milieu est calculée à l’aide du logiciel Phreeqc et les temps de germination et vitesses initiales de croissance cristalline ont été déterminés. Les cristaux ont été analysés par spectroscopie FTIR et MEB. Nous avons mis en évidence l’influence d’additifs ioniques (Mg2+, Zn2+ ou Cu2+) sur les paramètres cinétiques de germination et croissance cristalline de t-CPPD. Ainsi les ions Cu2+ et Mg2+ ont respectivement inhibé partiellement et totalement la croissance cristalline de t-CPPD. Les résultats obtenus dans cette étude pourraient aider à expliquer la formation in vivo de cristaux de m-CPPD au sein des articulations de patients arthritiques alors que cette phase pure est difficile à obtenir in vitro par précipitation sans ions autres que Ca2+ et P2O74-. Ces résultats cinétiques pourront donner une indication sur l’évolution du ratio des deux phases (m-CPPD et tCPPD) formées in vivo dans les articulations arthrosiques et des pistes de traitement pour éviter la formation de ces cristaux inflammatoires ou orienter la formation de phases non inflammatoires. / Osteoarthritis (OA) affects about ten million people in France. The presence of microcrystals including those of calcium pyrophosphate (CPP) dihydrates (Ca2P2O7•2H2O; mono- and/or triclinic: m-CPPD and/or t-CPPD respectively) in the joints has been identified as an aggravating factor of OA. However the conditions of formation of both CPPD phases and their order of appearance remain unknown or have not been totally described and the existing treatments are only symptomatic. In the present work we were interested in the effect of ionic additives on the formation and the kinetics of the crystallization of CPP which is a key step in the medium to long term development of a therapeutic strategy to prevent the formation of these crystals, their transformation or their dissolution. At first the in vitro synthesis by precipitation of hydrated CPP pure phases of biological interest (mCPPD, t-CPPD and monoclinic CPP tetrahydrated ) have been realized to identify the operating parameters (concentration, pH, temperature) leading to their formation and to analyze the effect of ionic additives (Mg2+, Cu2+, Zn2+, Fe3+ or S2O32-) on their precipitation under controlled conditions. To achieve this objective, the synthesis method developed by Gras et al. was transposed in stirred reactor. It consists in a precipitation by simultaneous addition of two reagent solutions of calcium nitrate and potassium pyrophosphate in an ammonium acetate buffer solution. Powders synthesized were analyzed by X-ray diffraction, solid state nuclear magnetic resonance, FTIR and RAMAN spectroscopies, scanning electron microscopy and inductively coupled plasma atomic emission spectroscopy. Thanks to a semiquantitative analysis by X-ray diffraction, we showed that the presence of ionic additives (Mg2+, Zn2+, Fe3+ or Cu2+) during the precipitation significantly modified the proportions of CPP phases constituting the final product. We showed that Mg2+, Cu2+ or Zn2+ promote the formation of m-CPPD crystals during the precipitation under controlled conditions for t-CPPD phase synthesis and depending on the conditions Fe3+ ion can completely inhibit t-CPPD phase crystallization. 31P solid state NMR fine analyzes have allowed determining the mode of action of these ionic additives. Thus, Cu2+ or Mg2+ ions have substituted for Ca2+ in the CPP lattice. Additionally, we have implemented the method of constant composition crystal growth to study the nucleation and crystal growth at constant temperature, pH, supersaturation and ionic strength of tCPPD on seeds of t-CPPD crystals in the presence or not of ionic additive. The seed was introduced into a supersaturated metastable solution of calcium pyrophosphate at pH 6.2 including the ionic additive and placed in a stirred reactor maintained at 37°C. Supersaturation of the medium was calculated using Phreeqc software and nucleation time and the initial rate of crystal growth were determined. The resulting crystals were analyzed by FTIR spectroscopy and scanning electron microscopy. We were able to highlight the influence of ionic additives (Mg2+, Zn2+ or Cu2+) on the kinetic parameters of nucleation and crystal growth of t-CPPD. Thereby, Cu2+ and Mg2+ ions have respectively inhibited partially and totally the growth of the tCPPD crystals. These results may help to explain why m-CPPD phase is identified in vivo in the joint of arthritic patients while this pure phase is difficult to obtain by in vitro precipitation (very narrow temperature-pH domain in a medium free of any ionic additives). These results could also provide indications on the order of appearance and potential evolutions of m-CPPD and tCPPD phases formed in vivo in osteoarthritic joints and provide some clues to avoid the formation of CPPD crystals with inflammatory potential or orient the formation of noninflammatory CPP phases