The Stiffness of Bone Extracellular Matrix Derived Scaffolds Regulates Macrophage Function and Osteogenesis of Mesenchymal Stromal Cells

Bone extracellular matrix (ECM) derived biomaterials possess intrinsic osteoinductive properties, motivating their use as scaffolds for bone and osteochondral defect repair. However, their translation is limited by the need for scalable and reproducible ECM extraction protocols and by their poor mechanical properties when processed into porous scaffolds. Furthermore, it is unclear how the stiffness of ECM-derived scaffolds, a known regulator of mesenchymal stromal/stem cell (MSC) differentiation, impacts their ability to support osteogenesis. To address these challenges, this study first sought to develop a robust ECM extraction process from porcine bone, and secondly to fabricate solubilized bone ECM scaffolds with tuneable mechanical properties to assess how their stiffness influences osteogenesis of MSCs and macrophage phenotype. Using an optimised bone ECM extraction protocol, we produced scaffolds with a unidirectional pore architecture using ECM concentrations of 2%, 4%, and 6% w/v to examine the effect of matrix content on scaffold mechanical properties and osteogenic potential. Scaffold stiffness increased with bone ECM concentration, from 2 kPa for 2% ECM to 14 kPa for 6% ECM. All scaffolds displayed low levels of permanent deformation (<5%) following large strains, demonstrating their highly elastic nature. Increased scaffold stiffness also influenced MSC behaviour, with DNA content almost doubling over culture, and the stiffest scaffold supporting approximately 33% more mineral deposition and almost 100% more collagen deposition compared to the softer scaffolds. Furthermore, bone ECM scaffolds modulated the function of primary human macrophages, leading to a downregulation of the proinflammatory factors and an upregulation in angiogenic growth factors supportive of bone regeneration. In conclusion, we established a reliable protocol for bone ECM extraction from porcine tissue and demonstrated that scaffold stiffness can be finely tuned to regulate both MSC fate and macrophage polarization.