Microfluidique des milieux poreux : application au génie tissulaire osseux

En orthopédie, l’utilisation d’un implant hybride constitué d’un substrat poreux dans lequel sont cultivées des cellules osseuses provenant du patient est une technique de greffe actuellement développée. La culture des cellules est effectuée au sein d’un bioréacteur, où l’implant est perfusé pendant plusieurs semaines. La perfusion permet l’apport d’oxygène nécessaire aux cellules pour leur développement. La maîtrise des phénomènes de transport au sein de l’implant est donc essentielle à la réussite d’une telle culture cellulaire. L’étude ici présentée, a abouti à la mise en place d’un modèle simplifié permettant de résoudre par simulation numérique un problème de transport d’oxygène stationnaire en milieu poreux. Ce modèle prend en compte l’influence de la géométrie complexe de l’implant sur les vitesses locales du fluide nutritif perfusé et un terme « puits » non linéaire imposé par la spécificité de la consommation cellulaire. La résolution numérique faiblement couplée de l’équation de convection-diffusion et celles de Navier Stokes est alors nécessaire. Une longueur de pénétration de l’oxygène dans le milieu poreux est calculée, l’hétérogénéité locale chimique et mécanique de l’environnement de culture mise en évidence, et l’influence de différents paramètres tels que la vitesse de perfusion ou la concentration initiale en oxygène du fluide nutritif est analysée de manière théorique. Bien qu’il soit nécessaire de compléter ce modèle, nous pouvons ainsi envisager de choisir une taille d’implant adaptée et de définir des conditions de culture optimales préalables à la réalisation d’un processus de culture performant

Bone Regeneration in Sheep Using Acropora Coral, a Natural Resorbable Scaffold, and Autologous Mesenchymal Stem Cells

International audienc

Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression

International audienceMesenchymal stromal cells (MSCs) seeded onto biocompatible scaffolds have been proposed for repairing bone defects. When transplanted in vivo, MSCs (expanded in vitro in 21% O 2) undergo temporary oxygen deprivation due to the lack of pre-existing blood vessels within these scaffolds. In the present study, the effects of temporary (48-hour) exposure to hypoxia (<1% O 2) on primary human MSC survival and osteogenic potential were investigated. Temporary exposure of MSCs to hypoxia had no effect on MSC survival, but resulted in (i) persistent (up to 14 days post exposure) down-regulation of cbfa-1/Runx2, osteocalcin and type I collagen and (ii) permanent (up to 28 days post exposure) up-regulation of osteopontin mRNA expressions. Since angiogenesis is known to contribute crucially to alleviating hypoxia, the effects of temporary hypoxia on angiogenic factor expression by MSCs were also assessed. Temporary hypoxia led to a 2-fold increase in VEGF expression at both the mRNA and protein levels. Other growth factors and cytokines secreted by MSCs under control conditions (namely bFGF, TGFb1 and IL-8) were not affected by temporary exposure to hypoxia. All in all, these results indicate that temporary exposure of MSCs to hypoxia leads to limited stimulation of angiogenic factor secretion but to persistent down-regulation of several osteoblastic markers, which suggests that exposure of MSCs transplanted in vivo to hypoxia may affect their bone forming potential. These findings prompt for the development of appropriate cell culture or in vivo transplantation conditions preserving the full osteogenic potential of MSCs

Proangiogenic and Prosurvival Functions of Glucose in Human Mesenchymal Stem Cells upon Transplantation

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The paracrine effects of human induced pluripotent stem cells promote bone-like structures via the upregulation of BMP expression in a mouse ectopic model

Abstract Use of human induced pluripotent stem cells (h-iPSCs) for bone tissue engineering is most appealing, because h-iPSCs are an inexhaustible source of osteocompetent cells. The present study investigated the contribution of undifferentiated h-iPSCs and elucidated aspects of the underlying mechanism(s) of the involvement of these cells to new bone formation. Implantation of undifferentiated h-iPSCs seeded on coral particles in ectopic sites of mice resulted in expression of osteocalcin and DMP-1, and in mineral content similar to that of the murine bone. The number of the implanted h-iPSCs decreased with time and disappeared by 30 days post-implantation. In contrast, expression of the murine osteogenic genes at day 15 and 30 post-implantation provided, for the first time, evidence that the implanted h-iPSCs affected the observed outcomes via paracrine mechanisms. Supporting evidence was provided because supernatant conditioned media from h-iPSCs (h-iPSC CM), promoted the osteogenic differentiation of human mesenchymal stem cells (h-MSCs) in vitro. Specifically, h-iPSC CM induced upregulation of the BMP-2, BMP-4 and BMP-6 genes, and promoted mineralization of the extracellular matrix. Given the current interest in the use of h-iPSCs for regenerative medicine applications, our study contributes new insights into aspects of the mechanism underlying the bone promoting capability of h-iPSCs

Human Mesenchymal Stem Cell Failure to Adapt to Glucose Shortage and Rapidly Use Intracellular Energy Reserves Through Glycolysis Explains Poor Cell Survival After Implantation

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A Perfusion Bioreactor for Engineering Bone Constructs: An In Vitro and In Vivo Study

International audienceA perfusion bioreactor, which was designed based on fluidized bed concepts, was validated for the culture of bone constructs of clinically relevant size. For this study, natural coral has been used as three-dimensional scaffolds. This biomaterial is a microporous, biocompatible, osteoconductive, and absorbable scaffold. This perfusion bioreactor provided a stable environment in terms of osmolarity, pH, and, most importantly, oxidative stress. Bone constructs engineered in this system resulted in significantly higher cell proliferation and homogenous cell distribution than those cultured under static conditions. Particularly relevant to the production of bioengineered bone in a clinical setting, custom-made bone constructs (each one with volume up to 30 cm 3) could be produced using a such perfusion bioreactor. Last, but not least, the bone constructs of clinically relevant volume thus produced were shown to be osteogenic when transplanted subcutaneously in sheep

NADPH oxidase 4 deficiency attenuates experimental osteoarthritis in mice

Objective: Low-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In this study, we evaluated the role of NOX4 on joint homoeostasis after destabilisation of the medial meniscus (DMM) in mice. Methods: Experimental OA was simulated on cartilage explants using interleukin-1β (IL-1β) and induced by DMM in wild-type (WT) and NOX4 knockout (NOX4-/-) mice. We evaluated NOX4 expression, inflammation, cartilage metabolism and oxidative stress by immunohistochemistry. Bone phenotype was also determined by micro-CT and histomorphometry. Results: Whole body NOX4 deletion attenuated experimental OA in mice, with a significant reduction of the OARSI score at 8 weeks. DMM increased total subchondral bone plate (SB.Th), epiphysial trabecular thicknesses (Tb.Th) and bone volume fraction (BV/TV) in both NOX4-/-and wild-type (WT) mice. Interestingly, DDM decreased total connectivity density (Conn.Dens) and increased medial BV/TV and Tb.Th only in WT mice. Ex vivo, NOX4 deficiency increased aggrecan (AGG) expression and decreased matrix metalloproteinase 13 (MMP13) and collagen type I (COL1) expression. IL-1β increased NOX4 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in WT cartilage explants but not in NOX4-/-. In vivo, absence of NOX4 increased anabolism and decreased catabolism after DMM. Finally, NOX4 deletion decreased synovitis score, 8-OHdG and F4/80 staining following DMM. Conclusion: NOX4 deficiency restores cartilage homoeostasis, inhibits oxidative stress, inflammation and delays OA progression after DMM in mice. These findings suggest that NOX4 represent a potential target to counteract for OA treatment.</p