Construction of biomimetic hydroxyapatite bilayer scaffolds via DLP 3D printing for bio-root regeneration.

Digital light processing (DLP) 3D printing of hydroxyapatite (HA) bioceramics offers a promising strategy for fabricating complex, patient-specific bio-root scaffolds with high resolution and bioactivity. However, achieving adequate mechanical integrity while guiding the formation of a periodontal ligament (PDL)-like enthesis through structural optimisation remains a major challenge. Inspired by interfacial micromorphological cues that regulate both intrinsic mechanical properties and cellular responses, this study proposed a novel bone-root integrated design for a DLP 3D-printed bio-root scaffold. The scaffold incorporated a diamond-shaped core optimised through finite element analysis and recapitulated the differential pore structure of the native tooth-bone interface, thereby exhibiting superior mechanical properties compared with conventional hollow bio-root scaffolds. Furthermore, it provided an optimised microenvironment that supported the proliferation and expansive growth of human dental follicle stem cells, promoted the oriented formation of enthesis-like fibres, and facilitated osteogenic differentiation through physical contact guidance and metabolic modulation in vitro. In vivo, subcutaneous implantation in nude mice and evaluation in a rat calvarial defect model demonstrated that the bilayer bone-root scaffold promoted the regeneration of PDL-like structures and differentiation into periodontium-like tissue. Transcriptomic profiling further revealed activation of key signalling pathways related to osteogenesis and angiogenesis, including the upregulation of extracellular matrix proteins, activation of the BMP-2/Runx-2 axis, and chemokine networks that support vascular ingrowth. Collectively, this bilayer bone-root structural design provides a transferable paradigm that balances mechanical durability with biological inductive capacity, offering a practical template for next-generation bio-root scaffolds adaptable to diverse biomaterials.