(108f) Additive Biomanufacturing of 3D Mature Lamellar Bone Structure Using Layered Demineralized Bone Paper

A major goal in bone organoid development is to establish a biomanufacturing strategy that faithfully replicates mature lamellar bone in a scalable and standardized manner. In vivo, repeated cycles of bone remodeling transform immature woven bone into mature lamellar bone, which is characterized by a highly organized, mineralized collagen matrix spanning multiple length scales. Although 3D bioprinting technologies have advanced, most rely on mechanical or enzymatic degradation of natural ECM to enable nozzle-based extrusion. This process compromises the structural integrity of the bone ECM despite preserving its biochemical composition. As a result, current bone organoids resemble non-structural woven bone and fail to capture the hierarchical architecture and physiological function of lamellar bone, underscoring the need for new biomanufacturing approaches.

To address this limitation, we developed an osteoid-mimicking demineralized bone paper (DBP) by thin-sectioning demineralized bovine compact bone. DBP preserves the native collagen alignment and microarchitecture at the centimeter scale while maintaining mechanical durability and optical transparency. It supports in vivo-like mineralization by osteoblasts and enables osteoblast–osteoclast-mediated bone remodeling under biochemical stimulation. In this study, we demonstrate additive biomanufacturing of 3D lamellar bone organoids by sequentially stacking osteoblast-seeded DBP in a layer-by-layer configuration, forming a biomimetic scaffold that preserves key structural features of native lamellar bone.

This approach avoids ECM-destructive processing and enables emergent properties not observed in single-layer cultures, including matrix-embedded osteocyte differentiation and layer-dependent regulation of bone remodeling. The resulting constructs exhibit tunable thickness and hierarchical organization of both surface and embedded cellular components, closely mimicking the structure and function of native lamellar bone in a controlled and reproducible manner. Compatible with robotic and automated workflows, this platform supports high-throughput, standardized production of bone organoids in multi-well formats. We envision that DBP-based, additively manufactured bone organoids will significantly advance preclinical research in bone development, disease modeling, and drug screening.