Porous hydrogel under perfusion: modeling and optimization of an in vitro model of bone defect reconstruction – HydrOs

Bone is a living tissue that is subjected to various stimuli. Cells receive mechanical, chemical or biophysical signals through their environment. The information translates into a capacity to adapt to any environmental modification. In the context of bone tissue engineering (BTE), this property can be exploited to produce osteogenic substitutes from Mesenchymal Stem Cells (MSCs) seeded within porous scaffolds cultured under dynamic conditions. Porous scaffold is required to accommodate cells and guide their growth and tissue regeneration in a 3D environment. Design variables for producing optimum scaffold architecture include the provision of adequate space for growth and the development of sufficient transport pathways within the porous material. To overcome nutrients transport limitations and to subject cells to optimal mechanical stresses, bioreactors are used. The production of osteogenic substitutes in a bioreactor lie on a rudimentary approach compared to those developed in process engineering, which rely on modeling and integrate the multi-scales and multi-physics aspects. It appears necessary to rationalize the running of bioreactors for bone tissue engineering. The originality of our approach lies in the development of methods to control and quantify the mass and momentum transfers within a perfusion bioreactor. Numerical simulations providing the stress and the nutrients concentration field experienced by the cells will systematically extend experiments.

At the interface of physics, mechanics and biology, HydrOs aims to produce vascularized osteogenic bone substitutes from a coculture of MSCs and endothelial cells (ECs) seeded in hydrogel scaffolds of polysaccharides supplemented, or not, with hydroxyapatite particles (HAp). They will be cultured in our experimental and numerical characterized perfusion bioreactor. Current knowledge regarding the fabrication of osteogenic porous scaffolds, the influence of its internal structuring on the formation of physiologically relevant biological structures, understanding of the influence of species transport and momentum mechanisms do not currently allow to envisage a realistic translation of the process to the clinic. Our ambition is to bring knowledge and methods in the field by carrying out the various tasks proposed in this project.

A key parameter is the understanding and control of the structuration of the scaffold porosity during hydrogel processing. In WP1, the morphological, mechanical and diffusional properties of hydrogel will be characterized. The mechanical environment and nutrients gradients will influence MSCs fate. In WP2, we will develop a digital twin of the bioreactor that will provide the nutrients concentration field and the stress field within the bioreactor in order to know the local conditions seen by the cells. For that purpose, we need to model the mechanical behavior of the swollen scaffolds, describe the interactions between the deformable scaffolds and the hydrodynamics within the bioreactor and model the transport of nutrients within the bioreactor. In WP3, a bioreactor will produce bone constructs from hydrogel of controlled porosity and mechanical properties (develop in WP1) seeded with a co-culture of MSCs and ECs then cultivated under optimized operating conditions given by the digital twin developed in WP2. Osteoblastic differentiation markers will be quantified and thanks to numerical simulation, correlate with hydrodynamics, stress field and mass transfers within the bioreactor. The potential of the bioreactor to improve a vascular network formation within spheroids will be assessed. In WP4, the potential of osteogenic cellularized bone grafts produced in the bioreactor and the evaluation of the added value of the supplementation of hydrogels with HAp for promoting osteoinduction will be assessed in vivo.