Porous hydrogel promoting spontaneous organoid formation for bone reconstruction: implementation in a perfusion bioreactor

The physiological environment of living tissues is inherently three-dimensional (3D), posing a significant challenge for in vitro tissue engineering to recapitulate the complex cell-cell and cell-matrix interactions that govern tissue development and regeneration. This thesis addresses the critical need for physiologically relevant bone tissue models by integrating advances in cell biology, biomaterials, microfluidics, and computational modeling to investigate the interplay of scaffold architecture, matrix properties, and fluid dynamics on osteoprogenitor cell behavior within a perfusion bioreactor. A multidisciplinary approach was employed, focusing on the development and optimization of co-culture systems using human mesenchymal stem cells (MSCs) and endothelial cells (HUVECs) spheroids embedded in porous pullulan/dextran scaffolds supplemented with hydroxyapatite (HAp) particles (PUDNA/HAp). The perfusion bioreactor platform was engineered to mimic the size and structure of critical-sized bone defects, providing a dynamic environment for continuous nutrient and oxygen exchange. Computational fluid dynamics (CFD) were leveraged to characterize local oxygen gradients, pH, and mechanical stimuli, ensuring the maintenance of physiologically ideal conditions throughout the culture period. The first set of experiments established and validated a robust protocol for dynamic 3D co-culture using immortalized lines, demonstrating that perfusion flow combined with optimized scaffold geometry supports high cell viability (>94%), enhanced proliferation, spontaneous spheroid formation, and superior osteogenic and angiogenic differentiation. Imaging and computational analyses revealed that cell spatial behavior and activity were strongly influenced by local oxygenation and mechanical cues. Spheroid size and distribution was found to be regulated by the interconnected and porous architecture of the scaffold. Subsequent studies compared the osteogenic and angiogenic potential of two human primary MSC subpopulations: bone marrow-derived MSCs (BMSCs) and stem cells from human exfoliated deciduous teeth (SHEDs) in co-culture with HUVECs. Dynamic perfusion culture significantly improved cell viability and vascular organization in both groups. However, only BMSC/HUVEC spheroids exhibited robust osteogenic differentiation and mineral deposition, while SHED/HUVEC spheroids displayed a pronounced pericytic phenotype, demonstrating the intrinsic differences among different MSCs when coupled with angiogenic sources. Finally, the in vitro engineered constructs (BMSC/HUVEC) were translated into an in vivo setting by implanting cell-laden and cell-free scaffolds into large femoral defects in nude rats. Longitudinal micro-computed tomography (µCT) and histological analyses demonstrated progressive mineralization and bone regeneration across all groups, emphasizing the osteoconductive, osteoinductive, and biocompatible properties of PUDNA/HAp scaffolds. While osteogenesis was comparable between groups, dynamic preconditioning in vitro markedly enhanced angiogenesis and trabecular connectivity in vivo, highlighting the importance of biophysical cues in functional tissue regeneration. These results contribute to the field of bone tissue engineering by establishing a comprehensive framework for the design and evaluation of prevascularized 3D co-culture systems within dynamic bioreactor environments. By integrating computational modeling, advanced imaging techniques, and translational animal models, this multidisciplinary approach enhances our understanding of the cellular and molecular mechanisms that drive both osteogenesis and vascular organization. Importantly, this work can provide insight for the development of robust, cGMP-compliant translational protocols aimed at repairing large bone defects, enabling more predictive, ethical, and clinically relevant preclinical evaluation of biomaterials and therapeutic strategies.