Parallelized multi-stimuli bioreactor for combined, physiologically relevant fluid shear stress and intermittent hydrostatic pressure in 3D bone tissue models

Regeneration of bone tissue is tightly regulated by its complex mechanical microenvironment, in which intraosseous hydrostatic pressure (HP) and interstitial fluid flow-induced shear stress (FSS) represent key mechanical cues. While the individual effects of these stimuli have been extensively investigated, their combined influence within physiologically relevant three-dimensional (3D) bone tissue models remains largely unexplored due to the lack of suitable experimental platforms. In this study, we present an automated, parallelized multi-stimuli bioreactor designed to deliver tunable combinations of FSS and intermittent HP to 3D bone tissue models. The platform integrates a direct perfusion bioreactor with a custom-designed, modular intermittent HP unit based on a solenoid pinch valve, enabling independent and combined control of flow rate and pressure waveforms. Bioreactor performance was characterized using two biomimetic polylactic acid (PLA) scaffold types, demonstrating the ability of the system to reproduce physiologically relevant ranges of HP (1.81-30.59 kPa) and FSS (0.48-683.26 mPa). Biological experiments using bone marrow-derived mesenchymal stem cells (BM-MSCs) suspended in a collagen matrix and seeded into PLA scaffolds, cultured for 7 days, showed that dynamic culture conditions significantly enhanced cell viability compared with static culture. Moreover, the combination of FSS and intermittent HP stimulation within daily activity-related ranges accelerated early osteogenic differentiation compared to direct perfusion alone. Overall, the proposed platform provides a versatile and robust investigation tool for advancing bone mechanobiology and tissue engineering research.