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International audience; Osteogenesis imperfecta (OI), also known as "brittle bone disease", is a rare genetic disorder of the skeleton, whose most benign form I corresponds to autosomal dominant mutations in the genes encoding type I collagen (COLA1, COLA2). Several associated skeletal manifestations are often observed but, surprisingly, while dentin defects often reflect genetic bone disorders, about half of OI patients have no obvious oral manifestations. Here, we investigated the collagen, mineral and mechanical properties of dentin from deciduous teeth collected from patients with mild form of OI and displaying no obvious clinical signs of dentinogenesis imperfecta. For the first time, an increase in the hardness of OI dentin associated with an increase in mineral content compared to healthy patients was reported. In addition, OI altered the tissue characteristics of the dentin-enamel junction but the interfacial gradient was preserved. The impact of changes in molecular structure due to mutations in OI was assessed by Raman microspectroscopy. Our results highlighted a change in the hydroxyproline-proline ratio in direct association with collagen mineralization. Our findings suggest that the evaluation of teeth could be an important aid for mild types of OI that are often difficult to diagnose clinically and provide experimental evidence that hydroxyproline content should be considered in future studies on collagen-based biomaterials.

International audience; A cell-mechanobiological model is used for the prediction of bone density variation in rat tibiae under medium and high mechanical loads. The proposed theoretical-numerical model has only four parameters that need to be identified experimentally. It was used on three groups of male Wistar rats under sedentary, moderate intermittent and continuous running scenarios over an eight week period. The theoretical numerical model was able to predict an increase in bone density under intermittent running (medium intensity mechanical load) and a decrease of bone density under continuous running (higher intensity mechanical load). The numerical predictions were well correlated with the experimental observations of cortical bone thickness variations, and the experimental results of cell activity enabled us to validate the numerical results predictions. The proposed model shows a good capacity to predict bone density variation through medium and high mechanical loads. The mechanobiological balance between osteoblast and osteoclast activity seems to be validated and a foreseen prediction of bone density is made available.

International audience; While human bone morphogenetic protein-2 (BMP-2) is a promising growth factor for bone regeneration, a major challenge in biomedical applications is finding an optimal carrier for its delivery at the site of injury. Because of their natural affinities for growth factors (including BMP-2) as well as their role in instructing cell function, cultured cell-derived extracellular matrices (ECM) are of special interest. We hereby hypothesized that a “bony matrix” containing mineralized, osteogenic ECM is a potential efficacious carrier of BMP-2 for promoting bone formation and, therefore, compared the efficacy of the decellularized ECM derived from osteogenic-differentiated human mesenchymal stem cells (hMSCs) to the one obtained from ECM from undifferentiated hMSCs. Our results provided evidence that both ECMs can bind BMP-2 and promote bone formation when implanted ectopically in mice. The osteoinductive potential of BMP-2, however, was greater when loaded within an osteogenic MSC-derived ECM; this outcome was correlated with higher sequestration capacity of BMP-2 over time in vivo. Interestingly, although the BMP-2 mainly bound onto the mineral crystals contained within the osteogenic MSC derived-ECM, these mineral components were not involved in the observed higher osteoinductivity, suggesting that the organic components were the critical components for the matrix efficacy as BMP-2 carrier.

International audience

International audience; Most of the last century, bone remodeling models have been proposed based on the observation that bone density is dependent on the intensity of the applied mechanical loads. Most of these cortical or trabecular bone remodeling models are related to the osteocyte mechanosensitivity, and they all have a direct correlation between the bone mineral density and the mechanical strain energy. However, experiments on human athletes show that high-intensity sport activity tends not to increase bone mineral density but rather has a negative impact. Therefore, it appears that the optimum bone mineral density would develop for “medium”-intensity activity (or medium mechanical loads) and not for the highest-intensity one.In this work, we propose a new continuum approach based on bone cell activ-ity being either positive or negative as a function of the intensity of the applied mechanical load. At standard earth gravity without exercise, bone homeostasis is observed with cell activity being at equilibrium. When “medium loads” such as “low-intensity” or “optimized” sport activity are applied, cells are activated and an increase of bone density occurs. On the other hand, “high-intensity loads” such as over-training lead to bone density decrease or bone degradation. Our results are in agreement with the literature and enable us to foresee applications such as optimal sport training for best physical conditions.

International audience; The fixation of hydrogels to biological tissues is a major challenge conditioning the development of implants and surgical techniques. Here, coatings of procoagulant nanoparticles are devised which use the presence of blood to create adhesion between hydrogels and soft internal organs. Those nanostructured coatings are simply adsorbed at the hydrogel surfaces and can rapidly activate the formation of an interfacial blood clot acting as an adhesive joint. This concept is demonstrated on pig liver capsules with model poly(ethylene-glycol) membranes that are intrinsically poorly adhesive. In the absence of blood, ex vivo peeling tests show that coatings with aggregates of bare silica nanoparticles induce a 2- to 4-fold increase in adhesion energy as compared to the uncoated membrane (3 ± 2 J m–2). This effect is found to scale with the specific surface area of the coating. The highest adhesion energies produced by these nanoparticle-coated membranes (10 ± 5 J m–2) approach the value obtained with cyanoacrylate glue (33 ± 11 J m–2) for which tearing of the tissue is observed. Ex vivo pull-off tests show an adhesion strength of coated membranes around 5 ± 1 kPa, which is significantly reduced when operating in vivo (1.0 ± 0.5 kPa). Nevertheless, when blood is introduced at the interface, the in vivo adhesion strength can be improved remarkably with silica coatings, reaching 4 ± 2 kPa after 40 min contact. In addition, these silica-coated membranes can seal and stop the bleeding produced by liver biopsies very rapidly (

Study Design In vitro disk explant culture. Objective Notochordal cells (NCs) have been shown to upregulate matrix production by nucleus pulposus (NP) cells in coculture. To examine the translation of these in vitro results to a nativelike setting, the regenerative potential of NCs injected into NP tissue was assessed in this study. Methods NP explants were cultured after injection with NCs in phosphate-buffered saline (PBS) or with PBS alone (sham). At days 0 and 42, cell viability and morphology, water, DNA, sulfated glycosaminoglycan and hydroxyproline content, and gene expression of anabolic markers were analyzed. Results NCs remained viable during culture, but their morphology changed. The biochemical content remained unchanged, except for the DNA content in the NC group. Overall ACAN expression remained unchanged, whereas COL2A1 decreased during culture. Conclusions No overall anabolic response was observed when NCs were injected into NP explants. NCs were found to survive but did not display the typical NC morphology by the end of the culture period.

International audience; In tissue engineering and regenerative medicine, stem cell-specifically, mesenchymal stromal/stem cells (MSCs)-therapies have fallen short of their initial promise and hype. The observed marginal, to no benefit, success in several applications has been attributed primarily to poor cell survival and engraftment at transplantation sites. MSCs have a metabolism that is flexible enough to enable them to fulfill their various cellular functions and remarkably sensitive to different cellular and environmental cues. At the transplantation sites, MSCs experience hostile environments devoid or, at the very least, severely depleted of oxygen and nutrients. The impact of this particular setting on MSC metabolism ultimately affects their survival and function. In order to develop the next generation of cell-delivery materials and methods, scientists must have a better understanding of the metabolic switches MSCs experience upon transplantation. By designing treatment strategies with cell metabolism in mind, scientists may improve survival and the overall therapeutic potential of MSCs. Here, we provide a comprehensive review of plausible metabolic switches in response to implantation and of the various strategies currently used to leverage MSC metabolism to improve stem cell-based therapeutics. Significance statement: Lack of success of stem cell-based therapies has been largely attributed to the massive cell death observed post-transplantation, which is caused by the metabolic shock these cells experience as they transition from in vitro to a hostile, injured site in vivo. The metabolism in mesenchymal stem cells (MSCs), specifically, is highly sensitive to cellular and environmental cues. In order to improve cell survival rate posttransplantation, it is important that scientists understand, and take into account, the needs and demands of MSC metabolism as they design the next generation of MSC-based therapies.

International audience; Nucleic acid-based therapy has shown great promise in accelerating bone regeneration as well as other diseases. Nucleic acids used in gene therapy mainly are either plasmid DNA (pDNA) or RNAs. Although pDNA therapy has been extensively studied for decades with encouraging preclinical and clinical results, side effects, and low efficiency associated with nuclear trafficking are hard to bypass. Unlike pDNA, RNAs (mRNA, siRNA, miRNA) exert their function in the cytoplasm, thereby being more efficient in hard-to-transfect cells such as primary osteoblasts. RNA interference-based gene silencing represents a negative regulation which knockdown the expression of antagonists that impair osteogenesis process. In contrary, mRNA therapy for osteogenesis represents a positive regulation which delivers mRNA encoding growth factors to accelerate bone regeneration. This review presents a comprehensive summary of the mRNA and siRNA-based therapies and the targets for bone re-generation in case of bone defect and osteoporosis.

Bone marrow-derived multipotent stromal cells (BMMSCs) represent an attractive therapeutic modality for cell therapy in type 2 diabetes mellitus (T2DM)-associated complications. T2DM changes the bone marrow environment, however, its effects on BMMSC properties remain unclear. The present study aimed at investigating select functions and differentiation of BMMSCs harvested from the T2DM microenvironment as potential candidates for regenerative medicine. BMMSCs were obtained from Zucker diabetic fatty (ZDF, an obese-T2DM model) rats and their lean littermates (ZL, controls), and cultured under normoglycemic conditions. The BMMSCs derived from ZDF animals were fewer in number, with limited clonogenicity (by 2-fold), adhesion (by 2.9-fold), proliferation (by 50%), migration capability (by 25%), and increased apoptosis rate (by 2.5-fold) compared to their ZL counterparts. Compared to the cultured ZL-BMMSCs, the ZDF-BMMSCs exhibited (i) enhanced adipogenic differentiation (increased number of lipid droplets by 2-fold, upregulation of the Pparg, AdipoQ, and Fabp genes), possibly due to having been primed to undergo such differentiation in vivo prior to cell isolation, and (ii) different angiogenesis-related gene expression in vitro and decreased proangiogenic potential after transplantation in nude mice. These results provided evidence that the T2DM environment impairs BMMSC expansion and select functions pertinent to their efficacy when used in autologous cell therapies.

Study Design Preclinical in vitro culture of human degenerated nucleus pulposus (NP) tissue. Objective Cyclooxygenase 2 inhibitors (e.g., celecoxib) inhibit prostaglandin E2 (PGE2) production, and they have been shown to upregulate regeneration of articular cartilage. In this study, we developed an explant culture system for use with human tissue and tested the potential of celecoxib. Methods NP explants were cultured with or without 1 μM of celecoxib and were analyzed at days 0 and 7 for biochemical content (water, sulfated glycosaminoglycans, hydroxyproline, and DNA), gene expression (for disk matrix anabolic and catabolic markers), and PGE2 content. Results Water and biochemical contents as well as gene expression remained close to native values after 1 week of culture. PGE2 levels were not increased in freshly harvested human NP tissue and thus were not reduced in treated tissues. Although no anabolic effects were observed at the dosage and culture duration used, no detrimental effects were observed and some specimens did respond by lowering PGE2. Conclusions Human degenerated NP explants were successfully cultured in a close to in vivo environment for 1 week. Further research, especially dosage-response studies, is needed to understand the role of PGE2 in low back pain and the potential of celecoxib to treat painful disks.

International audience; Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately.

International audience; Study design: In vitro analysis.Objective: The aim of this study was to assess the effect of three-dimensional (3D) printing of porous titanium on human mesenchymal stem cell (hMSC) adhesion, proliferation, and osteogenic differentiation.Summary of background data: A proprietary implant using three-dimensional porous titanium (3D-pTi) that mimics trabecu-lar bone structure, roughness, porosity, and modulus of elasticity was created (Ti-LIFE technology™, Spineart SA Switzerland). Such implants may possess osteoinductive properties augmenting fusion in addition to their structural advantages. However, the ability of 3D-pTi to affect in vitro cellular proliferation and osteogenic differentiation remains undefined.Methods: Disks of 3D-pTi with a porosity of 70% to 75% and pore size of 0.9 mm were produced using additive manufacturing technology. 2D Ti6Al4V (2D-Ti) and 2D polyetheretherketone (2D-PEEK) disks were prepared using standard manufacturing process. Tissue culture plastic (TCP) served as the control surface. All discs were characterized using 2D-micros-copy, scanning electron microscopy (SEM), and x-ray micro-computed tomography. Forty thousand hMSCs were seeded on the disks and TCP and cultured for 42 days. hMSC morphology was assessed using environmental SEM and confocal imaging following phalloidin staining. hMSC proliferation was evaluated using DNA fluorescent assay. hMSC differentiation was assessed using RT-qPCR for genes involved in hMSC osteogenic differentiation and biochemical assays were performed for alkaline phosphatase activity (ALP) and calcium content.Results: 3D-pTi lead to a higher cell number as compared to 2D-Ti and 2D-PEEK at D21, D28 and D42. ALP activity of hMSCs seeded into 3D-pTi scaffolds was as high as or higher than that of hMSCs seeded onto TCP controls over all time points and consistently higher than that of hMSCs seeded onto 2D-Ti scaffolds. However, when ALP activity was normalized to protein content, no statistical differences were found between all scaffolds tested and TCP controls.Conclusion: 3D-pTi provides a scaffold for bone formation that structurally mimics cancellous bone and improves hMSC adhesion and proliferation compared to 2D-Ti and PEEK.

Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00820-8.

The deterioration of the musculoskeletal system is a serious health concern for long-term space missions. The accumulated information over the past decades of space flights showed that microgravity impacts significantly the musculoskeletal system with muscle atrophy and bone loss. Until now, it has been difficult to make reasonable predictions of the bone loss for prolonged space missions due to the lack of in-space experimental data and weak understanding of the mechanobiological bone mechanisms. On earth, the healthy musculoskeletal degradation is mainly age related with osteoporosis and delayed fracture healing. A better understanding of the bone mechanobiological functions could help us improve our model predictions of the musculoskeletal health system during long-term space missions. We develop a numerical model able to predict the bone loss at the mesoscopic scale (bone trabecula) in microgravity. The model is able to correlate the calculated bone degradation mechanism with data available in the literature showing the effective bone density loss measured experimentally. An optimization algorithm is used for an average bone microstructure distribution and long-term prediction. Extrapolation is made to link the local bone loss at the structural scale with the corresponding effective bone strength. The first part of the paper details the extraction of the bone microstructure using micro-CT images and numerical model development. Next, the degradation and optimization schemes are detailed. Finally, some results are presented for long-term degradation.

International audience; Repair and reconstruction of large bone defects remain a significant challenge. Cell construct, containing mesenchymal stem cells (MSCs) and scaffold, is a promising strategy for addressing and treating major orthopedic clinical conditions. However, the design of an ideal cell construct for engineering bone faces two critical challenges (i) matching the scaffold degradation rate to that of new bone formation and (ii) preventing the massive cell death post-implantation (caused by disruption of oxygen and nutrient supply). We will hereby primarily focus on the challenge of survival of MSCs post-implantation. Increasing evidence indicates that metabolic regulation plays a critical role in cell fate and functions. In cell metabolism, glucose is considered the major metabolic substrate to produce ATP via glycolysis when the availability of oxygen is limited. In this paper, we delineate the essential roles of glucose on MSC survival. We aim to provide a different perspective which highlights the importance of considering glucose in the development of tissue engineering strategies in order to improve the efficiency of MSC-based cell constructs in the repair of large bone defects.

International audience; Reports on the association of periodontal disease (PD) with systemic lupus erythematosus (SLE) have regularly been published. PD is a set of chronic inflammatory conditions linked to a dysbiotic microbial biofilm, which affects the periodontal tissues, resulting eventually in their destruction and contributing to systemic inflammation. SLE is a multi-system chronic inflammatory autoimmune disease that has a wide range of clinical presentations, touching multiple organ systems. Many epidemiological studies have investigated the two-way relationship between PD and SLE, though their results are heterogeneous. SLE and PD are multifactorial conditions and many biological-based hypotheses suggest common physiopathological pathways between the two diseases, including genetics, microbiology, immunity, and environmental common risk factors. By focusing on recent clinical and translational research, this review aimed to discuss and give an overview of the relationship of SLE with PD, as well as looking at the similarities in the immune-pathological aspects and the possible mechanisms connecting the development and progression of both diseases.

Currently, medical imaging modalities involving X-rays play an essential role in diagnosing this pathology thanks to the good visualization of bone structure. The fluoroscopy on the other hand is used in the operating room to assist and guide the surgical gesture, now available with mobile systems which can adapt to restricted areas of operating rooms. These systems can generate two-dimensional (2D) images such as of radiography or three-dimensional (3D) images such as the scanner, based on the principle of tomography which is used in the diagnosis of pathologies. The generation of these images in 2D/3D dimensions is possible thanks to the mobile C-arm or O-arm already used for surgery. However, these devices have one major disadvantage: the amount of radiation dose they create. When exposed to high radiation doses, they become dangerous threat to both patients and operators. In this thesis, for we will try to respond to two problematics: For which tomographic configurations, for a strategy dose reduction for applications in orthopaedical surgery? what reconstruction methods are best suited to the use of morphometric criteria? How to improve the diagnosis of osteo-articular pathologies based on the analysis of 3D scanner images? I will address these objectives, on the one hand, in the field of orthopaedical surgery. To respond to the first two objectives, our approach has been experimental. The knee specimen was firstly imaged by a prototype C-arm coupling a cone-beam X-ray source to a plane sensor. This prototype was developed by our partners in the FUI-3D4Carm project with the aim of being integrated on a C-arm to get an intraoperative 3D image. We tested several dose reduction scenarios with 2 types of reconstruction algorithms, 1) classical Felkamp (FDK) and 2) Iterative (SART). We tested the reduction of the intensity (mAs), the number of projections and the angular range on the quality of the images. Various evaluation approaches were performed by expert qualitative assessment, digital image quality indicators based on grey levels, structure and finally segmentation methods. On the other hand, in the context of rheumatology, the objective is to quantify the variations in joint width as a biomarker using 3D images for the diagnosis of osteoarthritis in order to answer our third objective. We studied variations in joint spacing at the initial time and at 36 months in a longitudinal study of patients suffering from osteoarthritis. To carry out this quantification, a hybrid segmentation method was developed. It is based on two segmentation methods: a classification segmentation to select the bone part, followed by an active contour method, called "Snake", to extract the joint space. We have been able to demonstrate which criteria for which images are potentially usable for orthopaedic surgery: i) an intensity greater than 5mAs, ii) a minimum threshold angular range of 170°, iii) a minimum number of projections of 200. Then we were also able to demonstrate that the choice of the reconstruction algorithm depends on its use. Indeed, the FDK algorithm gives a better visualization of the bone structure and observers tend to favor it. Whereas images reconstructed with the SART algorithm offer a better similarity on geometric criteria and allow a more stable segmentation. Finally, we were able to obtain preliminary results on the quantification of joint spacing using a semi-automated segmentation method on patients suffering from osteoarthritis. We have identified good segmentation results in 60% of the cases, which suggests that this method is potentially interesting but still needs to be improved., De nos jours, les modalités utilisant des rayons X joue un rôle essentiel dans le diagnostic des pathologies rhumatismales en particulier l’arthrose. Cette modalité permet une bonne visualisation de la structure osseuse essentielle dans ce domaine. En chirurgie orthopédique, la radiographie garde un rôle essentiel avant, pendant et après la chirurgie, la fluoroscopie X basée sur des systèmes mobiles étant adaptée à cet environnement pour aider le geste opératoire. Ces modalités peuvent générer soit des images bidimensionnelles (2D) comme en radiographie soit des images tridimensionnelles (3D) avec le scanner basé sur le principe de la tomographie. L’approche 3D est néanmoins indispensable pour rendre compte de la complexité des structures osseuses. Des méthodes dérivées de la radiographie et plus rarement du scanner permettent de suivre quantitativement l’évolution de l’interligne articulaire. Des arceaux mobiles en forme de C (C-arm) ou de O (O-arm) sont utilisés pour la chirurgie et génèrent des images 2D rarement 3D. Néanmoins, ces appareils présentent comme inconvénient majeur le caractère irradiant qui doit être réduit pour les patients et les manipulateurs. Dans cette thèse, nous tenterons de répondre à trois problématiques : 1) quelles sont les conditions limites pour l’obtention d’images exploitables pour la chirurgie orthopédique dans l’optique de réductions de dose, 2) quelles méthodes de reconstruction sont les plus adaptées pour l’utilisation de critères morphométriques, 3) Comment améliorer le diagnostic de pathologie ostéo-articulaire tel que la gonarthrose à partir de l’analyse d’images 3D de scanners. Pour répondre aux deux premiers objectifs, notre démarche a été expérimentale. Une pièce anatomique de genou a été imagée tout abord par un prototype de C-arm couplant une source X à faisceau conique à un capteur plan. Ce prototype a été développé par nos partenaires du projet FUI-3D4Carm avec comme but d’être intégré sur un arceau C-Arm pour obtenir d’une image 3D en peropératoire. Nous avons testé plusieurs scénarios de diminution de dose avec 2 types d’algorithmes de reconstruction, 1) classique de type Felkamp (FDK) et 2) Itératif (SART). Nous avons testé la diminution de l’ampérage (mAs), du nombre de projections et de la plage angulaire sur la qualité des images. Différentes approches d’évaluation ont été abordées par une appréciation qualitative d’experts, des indicateurs numériques de qualité des images basés sur les niveaux de gris, la structure et pour finir des méthodes de segmentation. Pour répondre à notre troisième objectif, nous avons étudié les variations de l’interligne articulaire au temps initial et à 36 mois dans d’une étude longitudinale de patientes souffrant de gonarthrose. Pour procéder à cette quantification, une méthode de segmentation hybride a été développée. Elle repose sur deux méthodes de segmentation : une segmentation par classification pour sélectionner la partie osseuse, suivie d’une méthode du contour actif, nommé « Snake », pour extraire l’interligne articulaire. Nous avons pu démontrer qu’il existe des critères pour lesquels des images sont potentiellement exploitables pour la chirurgie orthopédique : i) avoir une intensité supérieure à 5mAs, ii) un seuil minimum de plage angulaire de 170°, iii) un nombre de projections minimum de 200. Puis nous avons pu également démontrer que le choix de l’algorithme de reconstruction dépend de son utilisation. En effet, l’algorithme FDK donne une meilleure visualisation de la structure osseuse et les observateurs ont tendance à le privilégier. Tandis que les images reconstruites avec l’algorithme SART offrent une meilleure similarité sur des critères géométriques et permettent une segmentation plus stable. Enfin, nous avons pu obtenir des résultats préliminaires sur la quantification de l’interligne articulaire à l’aide d’une méthode de segmentation semi-automatisée sur des patientes souffrant d’arthrose.

Spectral computed tomography acquires energy-resolved data that allows recovery of densities of constituents of an object. This can be achieved by decomposing the measured spectral projection into material projections, and passing these decomposed projections through a tomographic reconstruction algorithm, to get the volumetric mass density of each material. Material decomposition is a nonlinear inverse problem that has been traditionally solved using model-based material decomposition algorithms. However, the forward model is difficult to estimate in real prototypes. Moreover, the traditional regularizers used to stabilized inversions are not fully relevant in the projection domain.In this study, we propose a deep-learning method for material decomposition in the projection domain. We validate our methodology with numerical phantoms of human knees that are created from synchrotron CT scans. We consider four different scans for training, and one for validation. The measurements are corrupted by Poisson noise, assuming that at most 105 photons hit the detector. Compared to a regularized Gauss-Newton algorithm, the proposed deep-learning approach provides a compromise between noise and resolution, which reduces the computation time by a factor of 100.

It is now well established that physical exercise has unquestionable beneficial effects on bone tissue and metabolism. However, no consensus regarding the osteogenic effects of exercise on healthy or healing bone has been reached yet. The objective of this work is (i) to evaluate the effects of two running protocols (continuous vs. intermittent) on the parameters of bone quality and bone metabolism, and (ii) to evaluate the preventive effects of these running protocols on bone repair in Wistar rats. We demonstrated here that high-intensity interval-running has positive effects on cortical bone quality, while moderate continuous running has beneficial effects on trabecular bone but is detrimental to cortical bone. These effects also depend on the bone (tibia vs. femur). In addition, our data suggest that both running protocols accelerate the repair of a non-critical bone defect in the tibia but not in the femur, possibly by modulating bone resorption. Associating different types of running is more beneficial to bone health than just one. As part of the refinement of animal models, the use of the tibia would be more suitable to study bone healing than the femur, Il est désormais bien établi que la pratique d’un exercice physique a des effets bénéfiques sur le tissu osseux et son métabolisme. Cependant, aucun consensus n’a encore été établi en ce qui concerne les effets ostéogéniques de l’exercice sur l’os sain ou en cours de réparation. L’objectif de ce travail est d’une part d’évaluer les effets de deux protocoles de course (continue vs. Intermittente) sur les paramètres de la qualité osseuse et le métabolisme osseux, d’autre part d’évaluer les effets de la pratique préventive de ces protocoles de course sur la réparation osseuse chez le rat Wistar. Ces travaux mettent en évidence un effet bénéfique de la course intermittente avec intervalles de haute intensité sur la qualité de l’os cortical tandis que la course continue modérée a des effets bénéfiques sur la qualité de l’os trabéculaire mais néfastes sur l’os cortical. Ces effets dépendent également de l’os étudié (tibia vs. fémur). De plus, nos données suggèrent que les deux protocoles de course accélèrent la réparation d’un défaut osseux de taille non critique dans le tibia mais pas dans le fémur, probablement par modulation de la résorption osseuse. La pratique de différents types de course serait plus bénéfique à la santé osseuse qu’un seul. Dans le cadre du raffinement des modèles animaux, l’utilisation du tibia serait plus adaptée à l’étude de la guérison osseuse que le fémur

Introduction Les modalites d’imagerie qui aident a mieux comprendre le processus arthrosique sont la radiographie, l’IRM, la tomographie X, les ultrasons, la DXA et le TEP. A ce jour, seuls l’IRM et la tomographie sont des modalites tri-dimensionnelles (3D). L’analyse du cartilage sous forme de cartographie 3D ont ete largement developpe en IRM avec des applications in vivo reservees a la recherche clinique. L’arthroscanner permet de visualiser la surface du cartilage et la presence de calcifications mais est tres peu utilise en pratique clinique. La tomographie spectrale a comptage de photons est une nouvelle technologie qui est l’extension en multi-energie de la tomographie a double energie qui a montre son utilite pour la detection des cristaux d’urate de sodium. L’acces a des images mono-energetiques permet d’ameliorer le contraste des tissus mous habituellement non visibles en scanner classique en particulier le cartilage et le menisque. Nous proposons dans ce travail le developpement de biomarqueurs a type d’analyse morphologique quantitative du cartilage. Materiels et methodes Nous avons utilise un prototype (Philips Healthcare, Haifa) situe au CERMEP, Lyon, il s’agit appareil clinique dote d’un tube a rayons X classique et d’un detecteur a conversion directe de photons resolutif en energie qui permet l’obtention de voxels isotropes de 250 × 250 × 250 microns. L’acquisition dure quelques minutes. Cet appareil permet de recuperer une serie d’images mono-energetiques de 30 keV a 120 keV, nous avons selectionne les images a 60 keV qui offraient le meilleur contraste pour la visualisation du cartilage. Pour ce faire, nous avons etudie 24 specimens de genoux (15 normaux et 9 arthrosiques avec presence d’osteophytes), 5 hommes (âge moyen : 75,6 ± 14,3 ans), 17 femmes (87,7 ± 9,7 ans). Nous avons analyse le tibia et le femur separement des compartiments femoro-tibial medial et lateral. Nous avons utilise le logiciel itk-SNAP 3,8.0 selon un processus semi-automatique avec segmentation manuelle et interpolation. Le parametre mesure est l’epaisseur moyenne du cartilage en mm par la methode 3D des spheres. Resultats Les epaisseurs moyennes (ep_moys) du tibia lateral etaient de 2,82 ± 0,44 pour les normaux (Nx) versus 1,96 ± 0,94 mm pour les arthrosiques (OA), au tibia medial, les ep_moys etaient de 2,14 ± 0,36 Nx versus 1,95 ± 0,5 mm OA. Au femur lateral, les ep_moys etaient de 1,74 ± 0,34 Nx versus 1,32 ± 0,5 mm OA et au femur medial les ep_moys etaient de 1,35 ± 0,3 Nx versus 1,28 ± 0,7 mm OA. La difference a ete evalue a l’aide du test t et les differences etaient significatives au compartiment lateral pour le tibia (p = 0,002), femoral (p = 0,01), la difference n’etait pas significative au tibia medial et limite au niveau du femur medial (p = 0,04). Conclusion Le scanner spectral a comptage de photons est un nouvel outil qui permettra d’analyser sur la meme image des parametres quantitatifs 3D du cartilage et de l’os sous chondral pour ameliorer la comprehension du phenomene arthrosique et mieux suivre l’evolution naturelle ou sous l’effet des traitements.

Oro-facial bone loss is a major problem in oral and maxillofacial rehabilitation. They have various and varied causes: cancer, cysts, cleft palates, infections or trauma. The management of such bone defects requires the use of bone grafts. Autogenous bone or autograft is the standard gold but has disadvantages: i) the need for a second surgical site, ii) morbidity, iii) increased operating time, iv) limited amount of bone to be removed. To overcome these pitfalls, alternative therapeutics to bone autograft are used, in particular bone substitutes derived from human (allografts), animal (xenografts) or synthetic (alloplastic) bone tissue. Allografts are a credible alternative to autografts and are nowaday widely used in reconstructive and regenerative surgery. These allografts are either mineralized (FDBA: Freeze-Dried Bone Allograft) or demineralized (DFDBA: Demineralized Freeze-Dried Bone Allograft) and come in powder or gel form depending on the support used in the case of DBM (Demineralized Bone Matrix). This PhD project aims to show the osteoformative potential of a new bone allograft formulation, partially demineralized as an injectable paste, in two different models for rat calvaria; it is a PDBM (Partially Demineralized Bone Matrix). The first model is the rat calvarial defect model. Critical size defects (8 mm diameter) and sub-critical size defects (6 mm and 4 mm diameter) are performed on rat calvaria and then left empty or filled with the PDBM. Micro sacnner (µCT) and histological results showed the positive effect of PDBM in the bone repair of 6 and 8 mm diameter defects. Similarly, molecular biology (RT-PCR) showed the positive effect of PDBM in the early expression of genes involved in osteoformation. The 2nd model is a vertical bone augmentation model. A bone chamber (custom-made cylinder of Polyetheretherketone) of 6 mm high and 5 mm in diameter is attached to the rat calvarias after perforating it with 3 small holes 1 mm in diameter. The bone chambers are filled with either PDBM or Bio-Oss®. The µCT and histological results revealed an osteoformative potential for PDBM in the bone chambers. These results support the idea that allografts are a viable option for the treatment of periodontal and maxillofacial defects.; Les pertes osseuses de la sphère oro-faciale constituent un problème majeur dans la réhabilitation orale et maxillo-faciale. Elles ont des causes diverses et variées : cancers, kystes, fentes palatines, infections ou traumatismes. La prise en charge de tels défauts osseux nécessite l’utilisation de greffes osseuses. L’os autogène ou autogreffe constitue le gold standard mais présente des inconvénients: i) la nécessité de recourir à un second site opératoire, ii) morbidité subséquente, iii) une augmentation du temps opératoire, iv) une quantité limitée d’os à prélever. Pour pallier à ces écueils, des alternatives thérapeutiques à l’autogreffe osseuse sont utilisés notamment des substituts osseux dérivés des tissus osseux humains (allogreffes), animaux (xénogreffes) ou synthétiques (alloplastiques). Les allogreffes constituent une bonne alternative aux autogreffes et sont, aujourd’hui, largement utilisées en chirurgie reconstructive et régénérative. Ces allogreffes sont soit minéralisées (FDBA : Freeze-Dried Bone Allograft) ou déminéralisées (DFDBA : Demineralized Freeze-Dried Bone Allograft) et se présentent sous forme de poudre ou de gel selon le support utilisé dans le cas des DBM (Demineralized Bone Matrix). Ce projet de Doctorat avait pour but de montrer le potentiel ostéoformateur d’une nouvelle formulation d’allogreffe osseuse, partiellement déminéralisée sous forme de pâte injectable, dans deux modèles différents au niveau de la calvaria de rat : il s’agit d’une Matrice Osseuse Partiellement Déminéralisée (MOPD). Le 1er modèle est celui de défauts de calvaria de rat. Des défauts de taille critique (8 mm de diamètre) et sub-critique (6 mm et 4 mm de diamètre) sont réalisés sur la calvaria des rats puis laissés vides ou comblés avec la MOPD. Les résultats du micro scanner (µCT) et ceux histologiques ont montré l’effet positif de la MOPD dans la réparation osseuse des défauts de 6 et 8 mm de diamètre. De même, la biologie moléculaire (RT-PCR) a montré l’effet positif de la MOPD dans l’expression précoce des gènes impliqués dans l’ostéoformation. Le 2e modèle est celui d’augmentation osseuse verticale. Une chambre à os en Polyétherétherkétone (PEEK) de 6 mm de haut et 5 mm de diamètre est fixée sur la calvaria des rats après avoir perforé cette dernière de 3 petits trous de 1 mm de diamètre. Les chambres à os sont remplies soit de MOPD soit d’os bovin déprotéinisé (Bio-Oss®). Les résultats du µCT et ceux histologiques ont révélé un potentiel ostéoformateur de la MOPD dans les chambres à os. Ces résultats confortent l’idée que les allogreffes sont une option viable pour le traitement des défauts parodontaux et maxillo-faciaux.

Summary Most organs and tissues in the body, including bone, can repair after an injury due to the activation of endogenous adult stem/progenitor cells to replace the damaged tissue. Inherent dysfunctions of the endogenous stem/progenitor cells in skeletal repair disorders are still poorly understood. Here, we report that Fgfr3Y637C/+ over-activating mutation in Prx1-derived skeletal stem/progenitor cells leads to failure of fracture consolidation. We show that periosteal cells (PCs) carrying the Fgfr3Y637C/+ mutation can engage in osteogenic and chondrogenic lineages, but following transplantation do not undergo terminal chondrocyte hypertrophy and transformation into bone causing pseudarthrosis. Instead, Prx1Cre;Fgfr3Y637C/+ PCs give rise to fibrocartilage and fibrosis. Conversely, wild-type PCs transplanted at the fracture site of Prx1Cre;Fgfr3Y637C/+ mice allow hypertrophic cartilage transition to bone and permit fracture consolidation. The results thus highlight cartilage-to-bone transformation as a necessary step for bone repair and FGFR3 signaling within PCs as a key regulator of this transformation., Graphical Abstract, Highlights • Fgfr3Y367C activating mutation in skeletal stem/progenitor cells prevents bone healing • Intrinsic deficiencies in transplanted Prx1Cre;Fgfr3Y637C/+ PCs cause pseudarthrosis • Prx1Cre;Fgfr3Y637C/+ PCs cannot support cartilage-to-bone transformation • Wild-type PCs can rescue the Prx1Cre;Fgfr3Y637C/+ pseudarthrosis phenotype, Julien et al. report a severe bone repair phenotype associated with pseudarthrosis and functional impairment of periosteal cells (PCs) in Prx1Cre;Fgfr3Y637C/+ mice. PCs carrying the Fgfr3Y367C activating mutation cannot undergo cartilage-to-bone transformation thus uncovering an essential role of this transformation step in bone healing.

International audience; Allogenic demineralized bone matrix has been developed as a reliable alternative to the autologous bone graft. In the present study, we assessed the osteoformation potential of a partially demineralized bone matrix (PDBM) in a paste form obtained without an added carrier. This formulation included the preparation of cancelous bone from femoral heads after decellularision, delipidation, demineralization in HCl and autoclaving at 121 • C. Structural and biochemical characteristics of PDBM were determined using FTIR (Fourier transform infrared spectroscopy), hydroxyproline, DNA content assays, and optical ellipsometry. The osteoformation potential was evaluated in 8-, 6-, and 4-mm-diameter rat-calvarial bone defects by in vivo micro-CT analysis, performed immediately after surgery on days 0, 15, 30, 45, and 60. Moreover, histological and histomorphometric analyses were done on day 60. PDBM was compared to cancelous bone powder (BP) before its partial demineralization. The expression levels of selected inflammation-, angiogenesis-, and bone-related genes were also investigated by RT-PCR, 3, 7, and 14 days after surgery. Compared to the control group, the PDBM group exhibited a significant increase (p < 0.05) in radiopacity in 8-mm-and 6-mm-diameter defects at all time points tested. On day 60, the amount of newly-formed bone was greater (16 and 1.6 folds; p < 0.001; respectively) compared to that in control defects. No bone formation was observed in defects filled with BP regardeless of the size. In 8-mm-diameter defect, PDBM was effective enough to induce the upregulation of genes pertinent to inflammation (i.e., TNFα, IL-6, and IL-8), angiogenesis (i.e., VEGF, VWF), and osteogenesis (ALP, RUNX2, BGLAP, SP7) by day 3 after surgery. This study showed that the tested PDBM deeply influences the early critical events involved in bone regeneration and exhibits efficient osteoformation capacity, making it an attractive graft option for treating defects in periodontal and maxillofacial areas.

International audience; Oxidative stress (OS) plays a pivotal role in diabetes mellitus (DM) onset, progression, and chronic complications. Hyperglycemia-induced reactive oxygen species (ROS) have been shown to reduce insulin secretion from pancreatic β-cells, to impair insulin sensitivity and signaling in insulin-responsive tissues, and to alter endothelial cells function in both type 1 and type 2 DM. As a powerful antioxidant without side effects, astaxanthin (ASX), a xanthophyll carotenoid, has been suggested to contribute to the prevention and treatment of DM-associated pathologies. ASX reduces inflammation, OS, and apoptosis by regulating different OS pathways though the exact mechanism remains elusive. Based on several studies conducted on type 1 and type 2 DM animal models, orally or parenterally administrated ASX improves insulin resistance and insulin secretion; reduces hyperglycemia; and exerts protective effects against retinopathy, nephropathy, and neuropathy. However, more experimental support is needed to define conditions for its use. Moreover, its efficacy in diabetic patients is poorly explored. In the present review, we aimed to identify the up-to-date biological effects and underlying mechanisms of ASX on the ROS-induced DM-associated metabolic disorders and subsequent complications. The development of an in-depth research to better understand the biological mechanisms involved and to identify the most effective ASX dosage and route of administration is deemed necessary.

The architectural features of synthetic bone grafts are key parameters for regulating cell functions and tissue formation for the successful repair of bone defects. In this regard, macroporous structures based on triply-periodic minimal surfaces (TPMS) are considered to have untapped potential. In the present study, custom-made implants based on a gyroid structure, with (GPRC) and without (GP) a cortical-like reinforcement, were specifically designed to fit an intended bone defect in rat femurs. Sintered hydroxyapatite implants were produced using a dedicated additive manufacturing technology and their morphological, physico-chemical and mechanical features were characterized. The implants' integrity and ability to support bone ingrowth were assessed after 4, 6 and 8 weeks of implantation in a 3-mm-long, femoral defect in Lewis rats. GP and GPRC implants were manufactured with comparable macro- to nano-architectures. Cortical-like reinforcement significantly improved implant effective stiffness and resistance to fracture after implantation. This cortical-like reinforcement also concentrated new bone formation in the core of the GPRC implants, without affecting newly formed bone quantity or maturity. This study showed, for the first time, that custom-made TPMS-based bioceramic implants could be produced and successfully implanted in load-bearing sites. Adding a cortical-like reinforcement (GPRC implants) was a relevant solution to improve implant mechanical resistance, and changed osteogenic mechanism compared to the GP implants. STATEMENT OF SIGNIFICANCE: Architectural features are known to be key parameters for successful bone repair using synthetic bioceramic bone graft. So far, conventional manufacturing techniques, lacking reproducibility and complete control of the implant macro-architecture, impeded the exploration of complex architectures, such as triply periodic minimal surfaces (TPMS), which are foreseen to have an unrivaled potential for bone repair. Using a new additive manufacturing process, macroporous TPMS-based bioceramics implants were produced in calcium phosphate, characterized and implanted in a femoral defect in rats. The results showed, for the first time, that such macroporous implants can be successfully implanted in anatomical load-bearing sites when a cortical-like outer shell is added. This outer shell also concentrated new bone formation in the implant center, without affecting new bone quantity or maturity.

Background Calcium pyrophosphate dihydrate deposition disease (CPPD) has been reported to involve the spine, but few systematic studies have been published. Objective To further characterize the spinal involvement with CPPD by a review of CPPD patients hospitalized in a rheumatology department. Methods We retrospectively reviewed data for patients consecutively admitted with a diagnosis of CPPD in the rheumatology department of Lariboisiere hospital in Paris, France over 5 years by using a standardized protocol and electronic case report forms. Imaging studies were also reviewed. Results Spinal CPPD was diagnosed in 37/152 (24.3%) CPPD patients. Patient with spinal involvement had more widespread peripheral CC. The cervical (n = 21) and lumbar (n = 19) segments were most involved. CT-scan was more sensitive than plain radiographs for detecting spinal calcifications. Crown dens syndrome was a prominent feature of cervical involvement. Inflammatory sterile spondylo-discitis was observed in 6 patients. Lesions were frequently multiple and were classified into 4 types. Ruling out septic discitis required image-guided biopsies in 3 patients. Sacroiliac involvement included calcification in 5 patients and severe sterile destructive arthropathy and joint fusion in one patient each. Degenerative changes were common, and CPPD could not be implicated because of the patients ‘age and lack of a control population. In 12 patients, severe clinical features requiring hospitalization were related to such degenerative changes. Conclusion Symptomatic involvement of the spine was observed in 24% of this series of hospitalized CPPD patients. Specific entities were the cause of hospitalisation in 25 of the 37 patients with spinal calcification and included inflammatory pain related to crystal deposits and destructive arthropathy of the spine and sacroiliac joints. Discitis exhibited a wide range of MRI features and biopsies were needed to rule out infection in 3 of the 6 discitis.

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3 · 10 6 cells/mL) and NPCs + BMSCs (6 · 10 6 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10ng/mL transforming growth factor- b 1 (TGF- b 1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9 , A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.

Introduction Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs. Methods Canine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture. Results NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa. Conclusions No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.

Bone marrow stromal cells (BMSCs) have shown promising potential to stop intervertebral disc degeneration in several animal models. In order to restore a healthy state, though, this potential should be further stimulated. Notochordal cells (NCs), influential in disc development, have been shown to stimulate BMSC differentiation, but it is unclear how this effect will translate in an environment where resident disc cells (nucleus pulposus cells [NPCs]) could also influence BMSCs. The goal of this study was, therefore, to evaluate the effects of NCs on BMSCs when cocultured with NPCs, in a simplified 3D in vitro system. Bovine BMSCs and NPCs were mixed (Mix) and seeded into alginate beads. Using culture inserts, the Mix was then cocultured with porcine NCs (alginate beads) and compared to coculture with empty beads or porcine skin fibroblasts (SFs, alginate beads). NPCs alone were also cocultured with NCs, and BMSCs alone cultured under chondrogenic conditions. The effects of coculture conditions on cell viability, matrix production (proteoglycan and collagen), and gene expression of disc markers (aggrecan, type II collagen, and SOX9) were assessed after 4 weeks of culture. The NC phenotype and gene expression profile were also analyzed. Coculture with NCs did not significantly influence cell viability, proteoglycan production, or disc marker gene expression of the Mix. When compared to NPCs, the Mix produced the same amount of proteoglycan and displayed a higher expression of disc marker, indicating a stimulation of the BMSCs (and/or NPCs) in the Mix. Additionally, during the 4 weeks of culture, the NC phenotype changed drastically (morphology, gene expression profile). These results show that NCs might not be as stimulatory for BMSCs in an NPC-rich environment, as believed from individual cultures. This absence of effects could be explained by a mild stimulation provided by (de)differentiating NCs and the costimulation of BMSCs and NPCs by each other.

Tooth development is regulated by a series of reciprocal inductive signaling between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial–mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissue formation. We report a delay in enamel deposition and formation that is clearly distinguishable in the growing incisor and associated with a significant reduction of MMP-3 and MMP-20 expression in tooth germs of KO mice. Insufficient basement membrane degradation is evidenced by a persistent laminin immunostaining, resulting in a delay of both odontoblast and ameloblast differentiation. Consequently, enamel volume and thickness are decreased in adult mutant teeth but enamel maturation and tooth morphology are normal, as shown by micro-computed tomographic (micro-CT), nanoindentation, and scanning electron microscope analyses. In addition, the dentino-enamel junction appears as a rough calcified layer of approximately 10 ± 5 μm thick (mean ± SD) in both molars and growing incisors of KO adult mice. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. The mild tooth phenotype observed in EMMPRIN KO mice suggests that the direct effect of EMMPRIN may be limited to a short time window, comprised between basement membrane degradation allowing direct cell contact and calcified matrix deposition.

The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human, bone marrow-derived stromal cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, i.e., 0.5 unit more alkaline than that in two other cell-containing material (hydroxyapatite/tricalcium phosphate (HA/TCP) and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics which consistently promoted formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, that displayed a pH of around 7.8, promoted 20-30 fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared to the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, ALP activity and gene expression of RUNX2, ALP and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤ 7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. Formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (> 7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells. ispartof: Tissue Engineering Part A vol:20 issue:13 pages:1827-1840 ispartof: location:United States status: published

Purpose: Bone marrow stromal cells (BMSCs) have been proposed to complement the declining population of nucleus pulposus cells (NPCs) found in a degenerative intervertebral disc. Although able to stop degeneration, they could not produce enough matrix to restore a healthy state. Looking at development, when a large amount of matrix is produced, the disc also contains notochordal cells (NCs), potential progenitors or regulators of NPCs. The aim of the study was, therefore, to combine NCs to a BMSC/NPC mix and evaluate their effects on cell phenotype and matrix production, in long-term culture. Methods: In a 3D hydrogel, NCs were co-cultured in different ratios with BMSCs and/or NPCs. Matrix production, cell morphology, and gene expression of disc markers were assessed after 4 weeks of culture. Results: At day 28, BMSCs/NPCs highly expressed disc matrix markers (type II collagen and aggrecan) and produced disc matrix up to 30 % of values obtained for the positive control (BMSCs under TGFß stimulation). The addition of NCs only slightly up-regulated marker expression (6-12x increase); an up-regulation not reflected at the matrix level. During the 4 weeks of culture, however, the NC phenotype changed drastically (morphology, disc marker expression). Conclusion: In contrast to previously reported short-term studies, long-term co-cultures with NCs had no substantial effects on BMSCs and NPCs, most likely due to the loss of the NC native phenotype during culture. It, therefore, appears critical to maintain this specific phenotype for a long-term effect of the NCs. © 2013 Springer-Verlag.

After assessing cell viability (CV), tissue-engineered constructs are often discarded, as current CV assays commonly require specific (fluorescent) dyes to stain cells and may need scaffold/tissue digestion before quantifying the live and dead cells. Here, we demonstrate and evaluate how cellular auto-fluorescence can be exploited to facilitate a noninvasive CV estimation in three-dimensional scaffolds using two advanced microscopy methods. Mixtures of live and dead C2C12 myoblasts (0%, 25%, 50%, 75%, and 100% live cells) were prepared, and CV was determined before seeding cells into collagen carriers using the trypan blue (TB) assay. Cell-seeded collagen gels ([CSCGs], n = 5/cell mixture) were produced by mixing collagen solution with the live/dead cell mixtures (7 x 10(6) cells/mL). After polymerization, two-photon microscopy (TPM) and confocal microscopy images of the CSCG were acquired (n = 30 images/CSCG). It was found that live and dead cells systematically emit auto-fluorescent light with different spectral characteristics. Viable cells showed predominantly blue fluorescence with a peak emission around 470 nm, whereas dead cells appeared to mainly emit green fluorescent light with a peak intensity around 560 nm. For TPM, live and dead cells were distinguished spectrally. For confocal images, the intensity ratio of images taken with band-pass filters was used to distinguish live from dead cells. CV values obtained with both TPM and confocal imaging did not significantly differ from those acquired with the established TB method. In comparison to TPM, confocal microscopy was found to be less accurate in assessing the exact CV in constructs containing mostly live or dead cells. In summary, monitoring cellular auto-fluorescence using advanced microscopy techniques allows CV assessment requiring no additional dyes and/or scaffold digestion and, thus, may be especially suitable for tissue-engineering studies where CV is measured at multiple time points.

Because bone marrow-derived stromal cells (BMSCs) are able to generate many cell types, they are envisioned as source of regenerative cells to repair numerous tissues, including bone, cartilage, and ligaments. Success of BMSC-based therapies, however, relies on a number of methodological improvements, among which better understanding and control of the BMSC differentiation pathways. Since many years, the biochemical environment is known to govern BMSC differentiation, but more recent evidences show that the biomechanical environment is also directing cell functions. Using in vitro systems that aim to reproduce selected components of the in vivo mechanical environment, it was demonstrated that mechanical loadings can affect BMSC proliferation and improve the osteogenic, chondrogenic, or myogenic phenotype of BMSCs. These effects, however, seem to be modulated by parameters other than mechanics, such as substrate nature or soluble biochemical environment. This paper reviews and discusses recent experimental data showing that despite some knowledge limitation, mechanical stimulation already constitutes an additional and efficient tool to drive BMSC differentiation.

Purpose: Intimal smooth muscle cell (SMC) hyperplasia is a main component of the arterial wall response to injury. We have investigated the capacity of a water-soluble nonanticoagulant functionalized dextran (E9) in inhibition of SMC growth in vitro and in vivo. Methods: E9 was obtained with chemical substitutions with anionic and hydrophobic groups on the dextran backbone. SMC proliferation (cell counting, thymidine uptake, cell cycle analysis) was followed in culture in the presence of E9. Western blot analysis against phosphorylated mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinase 1/2, and assessment of MAPK activity on serum-stimulated SMCs also were investigated. Binding/displacement experiments, electron microscopy, and cell fractionations were used to follow the binding and internalization of radiolabeled and fluorescentlabeled E9. New Zealand white rabbit iliac arteries were injured with balloon dilatation and stent deployment. Animals were treated for 14 days with saline solution or E9 (5 mg/kg injected subcutaneously, twice daily). Morphometric analyses were carried out in each group (n = 6 arteries, 18 sections). Results: Nonanticoagulant E9 inhibited SMC proliferation in vitro. Tyrosine phosphorylation of MAPK 1/2 and MAPK activity were inhibited with E9 within 5 minutes of incubation. The binding and rapid cytoplasmic internalization of the synthetic compound was evidenced, but, in contrast to heparin, we did not detect any nuclear localization of the antiproliferative E9. In the in vivo model, qualitative modifications of neointimal structure with a thinner fibrocellular neointima were noticed after E9 treatment. Morphometric analyses of stented arteries in E9-treated animals indicated an important reduction (P

It is now well established that physical exercise has unquestionable beneficial effects on bone tissue and metabolism. However, no consensus regarding the osteogenic effects of exercise on healthy or healing bone has been reached yet. The objective of this work is (i) to evaluate the effects of two running protocols (continuous vs. intermittent) on the parameters of bone quality and bone metabolism, and (ii) to evaluate the preventive effects of these running protocols on bone repair in Wistar rats.We demonstrated here that high-intensity interval-running has positive effects on cortical bone quality, while moderate continuous running has beneficial effects on trabecular bone but is detrimental to cortical bone. These effects also depend on the bone (tibia vs. femur). In addition, our data suggest that both running protocols accelerate the repair of a non-critical bone defect in the tibia but not in the femur, possibly by modulating bone resorption. Associating different types of running is more beneficial to bone health than just one. As part of the refinement of animal models, the use of the tibia would be more suitable to study bone healing than the femur.; Il est désormais bien établi que la pratique d’un exercice physique a des effets bénéfiques sur le tissu osseux et son métabolisme. Cependant, aucun consensus n’a encore été établi en ce qui concerne les effets ostéogéniques de l’exercice sur l’os sain ou en cours de réparation. L’objectif de ce travail est d’une part d’évaluer les effets de deux protocoles de course (continue vs. intermittente) sur les paramètres de la qualité osseuse et le métabolisme osseux, d’autre part d’évaluer les effets de la pratique préventive de ces protocoles de course sur la réparation osseuse chez le rat Wistar. Ces travaux mettent en évidence un effet bénéfique de la course intermittente avec intervalles de haute intensité sur la qualité de l’os cortical tandis que la course continue modérée a des effets bénéfiques sur la qualité de l’os trabéculaire mais néfastes sur l’os cortical. Ces effets dépendent également de l’os étudié (tibia vs. fémur). De plus, nos données suggèrent que les deux protocoles de course accélèrent la réparation d’un défaut osseux de taille non critique dans le tibia mais pas dans le fémur, probablement par modulation de la résorption osseuse. La pratique de différents types de course serait plus bénéfique à la santé osseuse qu’un seul. Dans le cadre du raffinement des modèles animaux, l’utilisation du tibia serait plus adaptée à l’étude de la guérison osseuse que le fémur.