Rôle de la signalisation Wnt non-canonique dans l’étiologie de l’ostéoarthrose chez l’humain

Les études cliniques et in vitro suggèrent que la sclérose de l’os sous-chondral due aux ostéoblastes (Ob) anormaux est impliquée dans la progression de l’ostéoarthrose (OA). Les Ob OA humains isolés à partir d’os sous-chondral sclérosé montrent un phénotype altéré, un niveau réduit de signalisation Wnt/β-caténine canonique et une minéralisation in vitro réduite. Il existe également deux voies non-canoniques, Wnt/PKC et Wnt/PCP qui ont étés décrites dans la littérature. Cependant, il n’existe aucune étude qui traite de ces deux voies dans les Ob OA. Ces voies sont activées après qu’un ligand Wnt non-canonique tel que Wnt-5a se lie à un récepteur Wnt couplé à des corécepteurs de la voie non-canonique. Ceci enclenche, respectivement pour la voie Wnt/PKC-Ca2+ et Wnt/PCP, la phosphorylation de PKC (p-PKC) et la phosphorylation de JNK (p-JNK) et agit sur les cibles en aval. Nous avons voulu déterminer s’il était possible de constater des altérations dans les voies Wnt non-canoniques dans les Ob OA. Nous avons préparé des cultures primaires d’ostéoblastes sous-chondral humains à partir de plateaux tibiaux de patients OA subissant une arthroplastie totale du genou, ainsi qu’à partir de plateaux tibiaux recueillis à l’autopsie de patients « normaux ». L’expression des gènes impliqués dans les voies Wnt/PKC et Wnt/PCP a été évaluée par RT-qPCR et la production par Western Blot des protéines, ainsi que celle de p-PKC et p-JNK et que l’activité des facteurs NFAT et AP-1 utilisés par ces deux voies. L’activité phosphatase alcaline (ALPase) et la quantité d’ostéocalcine (OC) ont étés évaluées respectivement à l’aide d’hydrolyse de substrat et d’ELISA. Le niveau de minéralisation a été évalué par la coloration au rouge Alizarine. Nos résultats montrent que l’expression et la production de Wnt-5a étaient augmentées dans les Ob OA comparées aux Ob N et LGR5 était significativement plus élevée. De plus, l’expression de LGR5 est directement régulée via la stimulation ou la diminution de Wnt-5a, à la fois au niveau de l’ARNm et des protéines. Par ailleurs, Wnt-5a a stimulé la phosphorylation de JNK et de PKC ainsi que l’activité NFAT et AP-1. Les niveaux de minéralisation ainsi que d’activité ALPase et de sécrétion d’OC ont aussi été affectés par les changements du niveau de Wnt-5a. Ces résultats suggèrent que Wnt-5a, qui est augmentée dans les OA Ob, peut stimuler les voies Wnt non-canoniques et affecter le phénotype et la minéralisation des OA Ob humains.

EVIDENCE FOR MECHANICAL STRAINS INFLUENCE IN OSTEOPHYTE DEVELOPMENT

Purpose: Osteophytes are osteo-cartilaginous metaplastic tissue outgrowths of bone capped by cartilage usually found in degenerative and inflammatory joint disease. The presence and degree of maturity of osteophytes, along with joint space narrowing, are the main radiographic criteria for diagnosis and grading osteoarthritis (OA). Although osteophytes are known for being anatomic signs of advanced OA, they can occur in non-symptomatic joints, in joints with no other observable alterations, and in early stage OA. It remains unclear if they develop from molecular, physiological and/or mechanical stimuli. We hypothesized that mechanical strains play a role in osteophyte development. The overall objective of this thesis was to find evidence that osteophytes are influenced by mechanical strains. Methods: The first project was to develop a mechanically-induced osteophyte animal model. One single impact load that was reported to induce moderate joint damage was applied to the periosteum of the rat knee. Animals were sacrificed at four time points to characterize the evolution of damaged tissue and the joint by histology. A second study using human mature hip osteophytes was conducted to evaluate if mature osteophyte presented histological signs of proliferating and developmental processes. The histological characterization of mature osteophyte was used to compare findings of the mechanically-induced osteophyte in the animal model to validate the use of this rodent model in studying some aspect of osteophyte development of human. Lastly, a detailed three-dimensional (3D) radiological morphometric analysis was performed on microscopic computed tomography (µCT) scanned femoral heads collected from total hip arthroplasty patients presenting mature hip osteophytes. Quantitative morphometric measures of osteophytes internal structure was compared to three regions of the femoral head of known quality of organisation and mechanical constraint. Results and Conclusion: Osteophyte can be mechanically induced by a single load impact to the joint periosteum, indicating that a moderate trauma to the periosteal layer of the joint may play a role in osteophyte development. Mature osteophytes have proliferation, developing and remodelling zones and have trabecular structures. Mechanically-induced osteophytes and mature osteophytes presented similar histological composition. Mature osteophytes have organized internal structure. These results provide evidence that mechanical strain can influence osteophyte development.

Design et synthèse d’inhibiteurs d’une ectonucléotide pyrophosphatase/phosphodiestérase de type 1 (ENPP1) et leur activité anticancéreuse

La calcification de la valve aortique (CVA) est une maladie cardiovasculaire de plus en plus répandue, particulièrement en Amérique du Nord. Elle cause le rétrécissement de la valve aortique et le seul traitement actuellement disponible est le remplacement chirurgical. Des études menées par le Dr Patrick Mathieu (Institut de Cardiologie et de Pneumologie de Québec) ont montré qu’une surexpression d’une ectonucléotide pyrophosphatase/phosphodiestérase de type 1 (ENPP1) est à l’origine de cette sténose. Une solution à cette maladie serait donc de trouver un inhibiteur d’ENPP1. Inspirées des travaux du groupe de Pfizer visant ENPP1 pour le traitement de la chondrocalcinose articulaire et l’ostéoarthrite, quelques familles d’inhibiteurs de type quinazoline-4-pipéridine sulfamides (QPS) ont été synthétisés et testées in vitro. Une étude en modélisation moléculaire sur le site potentiel de liaison des inhibiteurs sur ENPP1 est en cours, en collaboration avec le Pr Patrick Lagüe (Université Laval, Département de biochimie, microbiologie et bio-informatique) et son équipe pour optimiser le design de la structure des composés. Les composés d’une des familles, les QPS-pyrimidine, ont été testés in vitro sur quelques lignées cellulaires cancéreuses (HT-1080, HT-29, M21 et MCF-7) pour mesurer leur activité antiproliférative. Ces composés ont une inhibition de croissance médiane (IC50) de l’ordre du micromolaire et représentent donc un point de départ intéressant pour la mise au point de nouveaux traitements anticancéreux.

Evaluación de desenlace: trasplante meniscal versus segunda meniscectomía

Introducción: El incremento de pacientes sintomáticos de rodilla y la osteoartrosis en jóvenes con limitadas posibilidades terapéuticas después de una meniscectomía, genera la búsqueda de alternativas terapéuticas. A pesar que es poco utilizado en Colombia, el trasplante meniscal es una propuesta para el manejo sintomático. Según cifras norteamericanas, se practican entre 700.000 a 1.500.000 artroscopias de rodilla anualmente, el 50% termina en meniscectomía y de este un 40% persisten sintomáticos. Métodos: Estudio de cohorte retrospectivo, con el objetivo de evaluar dolor (Escala Visual Análoga-EVA) y funcionalidad (Escala de Tegner y Lysholm) en los pacientes a quienes se les realizó trasplante meniscal o meniscectomía por segunda vez, entre los años 2007 a 2015. Resultados: A partir de los 6 meses la EVA mostró una tendencia a la mejoría en el grupo de trasplante meniscal, pasando de Moderado a Leve (p: <0.000). La Escala de Tegner y Lysholm cambió de Pobre a Bueno en el grupo de segunda meniscectomía (p= 0.008) y de Bueno a Excelente en el grupo trasplantado (p=0.225). La calificación promedio de la EVA en el grupo de trasplante presentó mejoría (p=<0.000), a diferencia del grupo de segunda meniscectomía (p=0.591). La escala de Tegner y Lysholm, mostró significancia estadística con tendencia a la mejoría en el grupo de segunda meniscectomía. Discusión: Los resultados muestran que con trasplante meniscal hay mejoría del dolor y la funcionalidad versus un segunda meniscectomía. Para fortalecer la evidencia de este tratamiento son necesarios estudios prospectivos complementarios.

Primary Joint Arthroplasty Surgery: Is the Risk of Major Bleeding Higher in Elderly Patients? A Retrospective Cohort Study

Increased risk of bleeding after major orthopedic surgery (MOS) has been widely documented in general population. However, this complication has not been studied in elderly patients. The purpose of this study is to determine whether the risk of major bleeding after MOS is higher in elderly patients, compared with those operated at a younger age. Methods: This retrospective cohort study included total hip and total knee arthroplasty patients operated during 5 consecutive years. The main outcome was the occurrence of major bleeding. Patients with other causes of bleeding were excluded. Relative risks (RRs) and confidence intervals (CIs) were calculated, anda multivariate analysis was performed. Results: A total of 1048 patients were included, 56% of patients were hip arthroplasties. At the time of surgery, 553 (53%) patients were older than 70 years. Patients aged >70 years showed an increased risk of major bleeding (RR: 2.42 [95% CI: 1.54-3.81]). For hip arthroplasty, the RR of bleeding was 2.61 (95%CI: 1.50-4.53) and 2.25 (95% CI: 1.03-4.94) for knee arthroplasty. After multivariate analysis, age was found to be independently associated with higher risk of major bleeding. Conclusion: According to European Medicines Agency criteria, patients aged 70 years are at a higher risk of major bleeding after MOS, result of a higher frequency of blood transfusions in this group of patients. Standardized protocols for blood transfusion in these patients are still required.

Relationship between biomarkers of cartilage in serum and degenerative joint disease in lusitanian horses

Cartilage degradation biomarkers are a potential tool for early diagnosis of degen- erative joint disease (DJD). In young horses, Coll2-1 and Coll2-1NO2 have been studied in serum and reported to be useful in the assessment of joint disease. Fib3-2 has been described to be higher in serum of humans with osteoarthritis but has not been assessed in horses. The aim of the current study was to evaluate biomarkers’ changes with age, sex, and exercise and correlate them with DJD. Blood collection and radiographic examination were performed in 51 Lusitanian horses. Moreover, inertial sensor-based detection of lameness was used to assess pain together with sub- jective examination. Females presented significantly higher concentrations of Coll2- 1 (P5.015) and Coll2-1NO2 (P5.014) compared to males. We found significant influence of high level of work in lower concentration of Coll2-1 (P5.001) and sig- nificant influence of sex in concentration of Coll2-1NO2 (P5.030). There was no influence of sex, age and work on Fib3-2. All biomarkers were increased in the DJD group (n535) compared to healthy controls (n516). This difference was significant for Coll2-1 (P5.015). When sorted by sex and age groups, significant difference in Coll2-1 between disease and healthy controls disappeared in old horses and females. Coll2-1 is a good marker of cartilage degradation in horses with DJD, being more specific in young horses and males. Fib3-2 may be further explored to help identify disease in particular cases.

Relationship between biomarkers of cartilage in serum and degenerative joint disease in Lusitano horses

Introduction: Cartilage degradation biomarkers are a potential tool for early diagnosis of degenerative joint disease (DJD). In young horses, Coll2-1 and Coll2-1NO2 have been studied in serum and reported to be useful in the assessment of joint disease. Fib3-2 has been described to be higher in serum of humans with osteoarthritis but was never assessed in horses. The aim of the current study was to evaluate biomarkers’ changes with age, sex and exercise and correlate them with DJD. Material and Methods: Blood collection and radiographic examination were performed in 51 Lusitanian horses. Moreover, inertial sensor-based detection of lameness was used to assess pain together with subjective examination. Results: Females presented significantly higher concentrations of Coll2-1 (p = 0.015) and Coll2-1NO2 (p = 0.014) compared to males. We have found significant influence of high level of work in lower concentration of Coll2-1 (p = 0.001) and significant influence of sex in concentration of Coll2-1NO2 (p = 0.030). There was no influence of sex, age and work on Fib3-2. All biomarkers were increased in the DJD group (n= 35) compared to healthy controls (n = 16). This difference was significant for Coll2-1 (p = 0.015). When sorted by sex and age groups, significant difference in Coll2-1 between disease and healthy controls disappeared in old horses and females. Discussion/ Conclusion: Coll2-1 is a good marker of cartilage degradation in horses with DJD, being more specific in young horses and males. Fib3-2 may be further explored to help identify disease in particular cases.

A single-sided NMR approach to study structural differences of bovine articular tissue

This thesis work has been developed in collaboration between the Department of Physics and Astronomy of the University of Bologna and the IRCCS Rizzoli Orthopedic Institute during an internship period. The study aims to investigate the sensitivity of single-sided NMR in detecting structural differences of the articular cartilage tissue and their correlation with mechanical behavior. Suitable cartilage indicators for osteoarthritis (OA) severity (e.g., water and proteoglycans content, collagen structure) were explored through four NMR parameters: T2, T1, D, and Slp. Structural variations of the cartilage among its three layers (i.e., superficial, middle, and deep) were investigated performing several NMR pulses sequences on bovine knee joint samples using the NMR-MOUSE device. Previously, cartilage degradation studies were carried out, performing tests in three different experimental setups. The monitoring of the parameters and the best experimental setup were determined. An NMR automatized procedure based on the acquisition of these quantitative parameters was implemented, tested, and used for the investigation of the layers of twenty bovine cartilage samples. Statistical and pattern recognition analyses on these parameters have been performed. The results obtained from the analyses are very promising: the discrimination of the three cartilage layers shows very good results in terms of significance, paving the way for extensive use of NMR single-sided devices for biomedical applications. These results will be also integrated with analyses of tissue mechanical properties for a complete evaluation of cartilage changes throughout OA disease. The use of low-priced and mobile devices towards clinical applications could concern the screening of diseases related to cartilage tissue. This could have a positive impact both economically (including for underdeveloped countries) and socially, providing screening possibilities to a large part of the population.

Efficacia del trattamento in acqua (Idrokinesiterapia) nei soggetti con diagnosi di osteoartrosi di ginocchio: revisione sistematica della letteratura

Background. One of the most diffused types of arthrities is Knee Osteoarthritis. Among symptoms found correlated to this disorder are: pain, joint stiffness, decresed muscle strenght. All these symptoms lead to a decreased function, difficulty in partcipation in physical activity, and decreased quality of life. Is Hydrotherapy increases strenght and mobility of patients for a better quality of life and society partecipation? Outcome. The aim of this Systematic Review is to verify efficacy of Hydrotherapy on upmentioned factors compared to conservative physical rehabilitation and self physical treatment. Study design. Systematc Review following The preffered Reporting Itemes For Systematic Reviews and Meta-analyses (PRISMA) statement checklist [3] Eligibility criteria. Chioice of RCTs which investigate changes in patients’ outcome who suffer from knee osteoarthritis applying Hydrotherapy treatment compared to conservative physical therapy treatment and self physical treatment. Method. Database research: PubMed, PEDro, Cochrane Central Register of Controlled Trial. Results. These reviewed studies show that Hydrotherapy induces improvements comparable with only gym treatment on muscle strenght, and step kinematic parameters, and it’s an ally of reduction of pain. Inaddition, Hydrotherapy is considered more effective physical treatment for knee orsteoarthrtis than the only conservative physical treatment and self physical treatment regarding parameters above mentioned. Conclusion. Hydrotherapy may be a valid alternative compared to only gym treatment, better than self physical treatment in pain relief and increases patients’ general physical health. Key words. Rehabilitation, Hydrotherapy, knee osteoarthritis, aquatic exercise, pain, self care, gait, balance.

L'efficacia della fisioterapia rispetto all'utilizzo di iniezioni intra articolari nella riabilitazione di pazienti con gonartrosi: una Revisione Sistematica della Letteratura

Background:La gonartrosi è una patologia degenerativa che colpisce l’articolazione del ginocchio. Rappresenta un grave problema di salute pubblica mondiale e comporta dolore e riduzione della funzionalità. Sono presenti studi che confrontano l’uso di iniezioni intra articolari con la fisioterapia per ridurre il dolore, ma ad oggi non vi è ancora un gold standard per la gestione della malattia. Scopo della ricerca:Valutare l’efficacia della fisioterapia rispetto all’uso di iniezioni intra articolari in pazienti affetti da gonartrosi, sul dolore e sulla qualità di vita. Disegno dello studio:Revisione Sistematica della Letteratura effettuata seguendo il PRISMA Statement. Metodi:La ricerca è stata condotta nelle banche dati PubMed, PEDro, Cochrane Library. Sono stati inclusi trial randomizzati controllati (RCT) in lingua inglese e italiana, che confrontavano l’efficacia del trattamento fisioterapico rispetto all’utilizzo di iniezioni intra articolari, con outcome relativi a riduzione del dolore e al miglioramento della qualità di vita. La qualità metodologica e il rischio di bias sono stati valutati utilizzando la PEDro Scale. Risultati:Cinque studi sono stati inclusi in questa Revisione Sistematica. Ci sono stati due studi che hanno dimostrato la maggiore efficacia di altri trattamenti rispetto alla fisioterapia. Altri due trials hanno confrontato il trattamento fisioterapico con l’uso di iniezioni di acido ialuronico e si sono osservati risultati pressoché uguali tra i gruppi. Solo uno studio ha dimostrato la superiorità del trattamento combinato di iniezioni e programma riabilitativo nella gestione della gonartrosi, rispetto al solo utilizzo di iniezioni intra articolari. Conclusioni:I risultati contrastanti che si sono osservati non permettono di affermare con certezza la superiorità della fisioterapia rispetto all’utilizzo di iniezioni intra articolari, sebbene rappresenti una valida scelta terapeutica grazie al suo basso costo e alla sua ridotta invasività.