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5-Mesenchymal stem cells stained with fluorescent dyes 2. Nucleus are in blue, microtubules in green, and actin filaments in red. 

5-Mesenchymal stem cells stained with fluorescent dyes 2. Nucleus are in blue, microtubules in green, and actin filaments in red. 

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Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold material, resolves mass transport limitations and provides physical stimuli, increasing the overall proliferation and differentiation of cells. Despi...

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... In order to determine the extent of biomass growth in the bioreactor, we estimate the surface area occupied by the cells and the tissue for each histological slice. Image treatment and segmentation are carried with ImageJ software [35] following the protocol reported in Chabanon [36] . Briefly, we define the total area occupied by the cells (Σcells) as the total area of blue pixels in each slice. ...
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Background Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. Methods In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. Results Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. Conclusion We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.
... Following the above remarks, we propose to study momentum transport in a general heterogeneous porous structure composed of two porous regions and an impermeable one. Although this system is directly inspired from biofilms in soil bioremediation processes, it should be noted that it is relevant to a plethora of other heterogeneous porous media of high industrial relevance, including fuel cells [70], catalytic reactors [50], bioreactor for tissue engineering [16,36,49], and biological environments [21,34,42]. Another feature often shared by these complex systems, is their hierarchical nature, where several structural length scales are present in cascade within each porous regions [1,17,26,41,[54][55][56]. ...
... Importantly, the physics describing transport phenomena at these boundaries is still an active research field [30,47,[65][66][67]. Finally, from a practical point of view, these structures are commonly encountered in a large range of applications relevant to chemical and biological engineering [16,23,27,48,70]. ...
... This analysis could be easily extended to more complex hierarchical porous media such as bioreactors for tissue engineering [34]. ...
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The perfusion of flow during cell culture induces cell proliferation and enhances cellular activity. Perfusion bioreactors offer a controlled dynamic environment for reliable in vitro applications in the tissue engineering field. In this work, to evaluate the effects of the operating parameters of a custom-made bioreactor, numerical simulations were performed to solve the fluid velocity profile inside the bioreactor containing multi-grid support that allows allocating of multiple seeded scaffolds at the same time. The perfusion system exhibited a uniform distribution of liquid velocities within the regions, suitable for cell growth on seeded scaffolds. The effects of the porous microstructure of scaffolds on the extracellular matrix deposition also play a crucial role during perfusion cultures. In the present study, a numerical simulation was implemented at the pore level of the scaffold for fluid flow through porous media during perfused culture. Micro-computed tomography was used to obtain the digital 3D image of the complex geometry of a PLLA scaffold, offering a detailed analysis from a volume-based methodology without simplifications of the results as for pore or Darcy's law-models. Predictions about the uniformity of the flow field through the scaffolds-bioreactor system have been assessed by quantifying the cell viability of a perfusion culture while using pre-osteoblastic cells seeded on 24 PLLA scaffolds for up to 6 days.
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The Tissue Engineering (TE) strategy is widely focused on the development of perfusion bioreactors to promote the production of three-dimensional (3D) functional tissues. To optimize tissue production, it is worth investigating the engineering parameters of a bioreactor system for identifying a beneficial range of operation variables. Mathematical and numerical modeling of a perfusion bioreactor is capable to provide relevant insights into the fluid flow and nutrients transport while predicting experimental data and exploring the impact of changing operating parameters, such as fluid velocities. In this work, the hydrodynamic parameters and oxygen transport were investigated using mathematical equations and Computational Fluid Dynamics (CFD) analysis modeling on a novel perfusion bioreactor working as an airlift external loop. Mathematical models and numerical simulation were associated with experimental results at different configurations to evaluate the effect of the reactor parameters on its hydrodynamics. All predictions of gas holdup and liquid circulation velocity from the modeling were in good agreement with the experimental data. A Poly-L-lactic acid (PLLA) scaffold was produced by Thermally Induced Phase Separation (TIPS) and used as 3D support for biological tests in static and dynamic conditions to assess cell viability and proliferation inside the bioreactor. The whole set of fluid-dynamic and biological results showed that our bioreactor environment offers the benefits of an in vitro potential system to produce functional engineered tissues.
Thesis
La reconstruction de larges défauts osseux nécessite l’implantation de matrices jouant le rôle d’échafaudage, biocompatibles, biodégradables et capables de promouvoir la régénération osseuse. Cette thèse porte sur un biomatériau poreux dont certaines formulations ont déjà démontré leur potentiel de régénération osseuse chez le rat et la chèvre. Il est obtenu par lyophilisation d’un hydrogel de polysaccharides (pullulane et dextrane) réticulé chimiquement.Dans un premier temps, on s’intéresse à l’influence des paramètres du procédé d’élaboration sur la structure poreuse du biomatériau. Les matrices sont caractérisées à chaque étape du procédé : par rhéométrie en mode dynamique lors de la réticulation, par cryomicroscopie électronique à l’issue de la congélation, par microtomographie à rayon X à l’état déshydraté et enfin par microscopie confocale à l’état solvaté. Il apparaît que la structure poreuse obtenue à l’issue de la lyophilisation dépend fortement de la microstructure de la glace formée lors de l’étape de congélation : chaque pore résulte de la croissance d’un à quelques cristaux. Le nombre et la taille des grains de glace après solidification complète sont étroitement liés à la germination secondaire, un phénomène qui est exacerbé par la présence du réseau polymère.Deux paramètres d’élaboration contrôlant la structure poreuse sont particulièrement examinés : d’une part la quantité de réticulant introduit lors de la synthèse de l’hydrogel (qui modifie la longueur de corrélation du réseau polymère), d’autre part la température de germination lors de la congélation. Après sublimation de la glace, le biomatériau obtenu est extrêmement poreux (92 − 94%).L’efficacité d’ensemencement des matrices déshydratées est quantifiée à l’aide de suspensions de microsphères de différents diamètres et de suspensions de cellules : le seuil de coupure est de l’ordre du diamètre moyen des pores secs. Après hydratation (concomitante à l’ensemencement), la porosité est nettement plus faible (~ 30%) et le diamètre moyen des pores hydratés diminue d’un facteur 2 à 4 en fonction de la densité de réticulation.Dans un second temps, cette thèse met en place une démarche d’étude in vitro portant sur les interactions des cellules osseuses (ostéoblastes de souris) avec le biomatériau. L’objectif est de disposer d’un système d’étude mimant les conditions physiologiques afin d’optimiser les propriétés de régénération osseuse du biomatériau. Le choix d’un dispositif in vitro permet de faire l’économie d’expérimentations animales. Un bioréacteur à perfusion est choisi comme modèle d’étude car il permet un environnement 3D où les transferts de matière peuvent être contrôlés. Une caractérisation multi-échelle est mise en place : marquage biologique et microscopie confocale à l’échelle des amas cellulaires et des matrices, imagerie par résonnance magnétique à l’échelle du bioréacteur. Celle-ci est complétée par la simulation numérique de l’hydrodynamique et du transport d’oxygène dissous dans le bioréacteur où chaque phase (fluide, hydrogel, amas cellulaires) est décrite avec une résolution spatiale de 55 µm correspondant à celle de l’IRM. Les équations de Navier-Stokes et l’équation de convection-diffusion sont simulées à l’aide de méthodes de Boltzmann sur réseau, particulièrement adaptées aux géométries complexes. On étudie l’influence de la taille des amas cellulaires et de la densité d’amas sur le champ de concentration d’oxygène en vue d’optimiser leur viabilité.Cette thèse donne des éléments clés pour contrôler la microstructure d’un hydrogel poreux destiné à l’ingénierie tissulaire et fournit un protocole expérimental d’étude en bioréacteur à perfusion couplé à une modélisation numérique pour optimiser les propriétés d’usage du biomatériau.