Architecture-Driven Preclinical Bone Regeneration in 3D-Printed Hydroxyapatite Scaffolds with Local Nanomechanical Insights

Critical-sized bone defects remain a major clinical challenge due to their limited self-healing capacity and the inability of current grafting materials to achieve both structural and mechanical integration. In this work, three-dimensional (3D) printed hydroxyapatite (HA) scaffolds with grid and honeycomb architectures and tunable infill densities (30-70%) were fabricated to investigate how architecture-controlled micromechanics influences bone regeneration. The scaffolds were evaluated in rat cranial (non-load-bearing) and tibial (load-bearing) defect models representing distinct mechanical environments. Micro-computed tomography (micro-CT), histological analyses, and high-speed nanoindentation (HSN) were used to quantify bone ingrowth, tissue distribution, and local stiffness. Grid scaffolds with 30% infill supported significantly greater bone volume fraction and more homogeneous regeneration, while HSN confirmed that the regenerated bone approached the modulus and hardness of native tissue. Comparable scaffold degradation and bone maturation were observed across both anatomical sites. These results show that scaffold architecture influences local nanomechanical properties and bone formation patterns, providing design principles for engineering reproducible, site-specific 3D-printed HA scaffolds. The findings further highlight that bioactive-free HA scaffolds can effectively support high-quality bone regeneration, offering a clinically translatable strategy for reconstructing critical-sized bone defects in orthopedic and craniofacial applications.