Regulatory mechanism of stem cell scaffold porosity and stiffness on the processes of intramembranous ossification and bone regeneration.

Bone defects remain a significant clinical challenge due to the limited regenerative capacity. Biomaterials can offer effective therapeutic approaches to address this challenge, where their physical properties play a critical role in determining the success of regeneration. Here, we fabricated cryogelated 3D porous scaffolds with independently controlled pore sizes and stiffness. These scaffolds were then seeded separately with bone marrow stromal cells (BMSCs) and MLO-A5 cells to create osteogenic cell-scaffold composites. To determine the optimal physical properties for promoting bone regeneration, we evaluated the scaffolds using both in vitro osteogenic cultures and a rat critical-sized calvarial defect model. Transcriptomic analysis was performed concurrently to explore the molecular link between physical scaffold cues and osteogenic differentiation. The results indicated that the small-pore high-stiffness scaffold exhibited the optimal osteogenic performance in vitro. In rats, the small pore scaffold groups also showed superior bone regeneration. Furthermore, transcriptomic analysis revealed that scaffold pore size significantly changed the expression of Apolipoprotein E (ApoE) and Osteoglycin (OGN). Our findings suggest a feasible strategy to enhance the efficiency of bone regeneration by leveraging the properties of tailored scaffolds, enabling personalized repair approaches.