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Hydrogel poreux pour la reconstruction osseuse : élaboration, caractérisation et mise en œuvre dans un bioréacteur à perfusion

Abstract : The reconstruction of large bone defects requires the implantation of scaffolds that are biocompatible, biodegradable and able to promote bone healing. This thesis focused on a porous biomaterial that had already demonstrated its osteo-inductive properties after implantation in rats and goats. This biomaterial is produced by freeze-drying of a chemically crosslinked polysaccharide-based (pullulan and dextran) hydrogel.First, we studied the influence of the process parameters on the properties of the biomaterial porous structure. The scaffolds were characterized at each step of the fabrication process: by dynamic rheometry during crosslinking, by electron cryo-microscopy just after freezing, by X-ray microtomography in the dry state and finally by confocal microscopy in the swollen state. It appears that the porous structure obtained at the end of freeze-drying strongly depends on the microstructure of the ice formed during the freezing stage: each pore results from the growth of one to a few crystals. Ice grains are mostly generated by secondary nucleation, this phenomenon is enhanced by the presence of the polymer network. Two parameters controlling the porous structure were particularly examined: the amount of crosslinker that reacts with the polysaccharides (which affects the correlation length of the polymer network), and the nucleation temperature at the onset of freezing. After sublimation of ice, the biomaterial becomes highly porous (92-94%).The seeding efficiency of the dried scaffolds was quantified using suspensions of narrow-sized calibrated microspheres and suspensions of cells: the seeding threshold is in the order of the average diameter of the dry pores. After swelling (occurring simultaneously with seeding), porosity is significantly lower (~ 30%) and the average diameter of the swollen pores is 2 to 4 times lower than in the dry state (depending on the crosslink density).Secondly, we investigated in vitro the interactions between the porous hydrogel scaffolds and osteo-competent cells derived from a mouse cell line. The experimental device was designed in order to mimic the physiological conditions. A perfused bioreactor was chosen because of its ability to generate a 3D environment with controlled shear stress and controlled solute concentration. Such a system should help to optimize the biomaterial while reducing the use of animal experiments. A multiscale characterization of the bioreactor tests was implemented: use of biomarkers and confocal microscopy at the spheroid and scaffold scales, magnetic resonance imaging at the bioreactor scale. We also investigated the hydrodynamics and the transport of oxygen within the bioreactor using computational fluid dynamics: fluid, hydrogel and spheroids were described at the MRI spatial resolution (i.e. 55 µm), NavierStokes equations and advection-diffusion equation were simulated using lattice-Boltzmann methods. These methods are indeed particularly suitable for complex geometries. The influence of organoid size and density on the oxygen concentration field was studied to optimize cell viability.This thesis provides key elements to control the microstructure of the porous hydrogel scaffolds and proposes a workflow to optimize the bone healing properties of the biomaterial by coupling tests in perfused bioreactors, experimental characterizations and numerical modelling.
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  • HAL Id : tel-02638200, version 1

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Jérôme Grenier. Hydrogel poreux pour la reconstruction osseuse : élaboration, caractérisation et mise en œuvre dans un bioréacteur à perfusion. Génie des procédés. Université Paris-Saclay, 2019. Français. ⟨NNT : 2019SACLC091⟩. ⟨tel-02638200⟩

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