3D polyurethane scaffolds for exploring osteogenic differentiation and mechanical stimulation of mesenchymal stromal cells

Bone marrow-derived mesenchymal stromal cells (MSC) support bone regeneration by differentiating into osteogenic lineages in response to biochemical and mechanical cues such as fluid-flow and compression, processes replicable in vitro using bioreactors and 3D scaffolds. Human MSC were cultured on medium-stiffness and hard 3D polyurethane (PU) scaffolds, characterized by atomic force microscopy, scanning electron microscopy (SEM), and stiffness measurements, and subjected to fluid-flow and compression. Two-dimensional PU and polystyrene (PS) substrates served as controls. Cultures were maintained in expansion or osteogenic medium. Cell attachment and morphology were analyzed by SEM and F-actin staining; osteogenic differentiation was evaluated by qPCR. Alkaline phosphatase activity was quantified, and extracellular matrix (ECM) formation and mineralization were assessed using Picrosirius Red, Alizarin Red, and inductively coupled plasma mass spectrometry (ICP-MS). Mechanosensitivity was analyzed via FOS and PTGS2 expression, YAP/TAZ nuclear localization, and Wnt, Notch, and cAMP signaling. Cells adhered and spread throughout the scaffolds, exhibiting pore-spanning morphologies. Gene expression showed material- and time-dependent upregulation of osteoblastic and osteocytic markers, with PU scaffolds eliciting stronger responses than PS, even without osteogenic supplements. The scaffolds also supported ECM formation and mineralization. Mechanical stimulation via fluid-flow and compression induced time- and stimulus-dependent regulation of mechanoresponsive genes and activation of key mechanotransduction pathways, with distinct expression patterns for each loading mode. In this study, we developed a standardized, cost-effective, and easy-to-handle 3D PU scaffold that effectively supports hMSC adhesion and promotes osteogenic differentiation. The cells' mechanoresponse further highlights the scaffold's suitability as a versatile platform for in vitro studies under physiologically relevant conditions.