Bone is a dynamic tissue that undergoes repeated remodeling through the coordinated actions of bone-forming osteoblasts and bone-resorbing osteoclasts under spatiotemporal molecular regulation. Mechanosensory osteocytes, embedded within the bone matrix, play a major role in regulating bone metabolism via paracrine signaling. Developing bone organoids is essential for understanding bone biology, modeling bone-related diseases, and advancing bone-targeting drug development. Motivated by this, we have developed an osteoid-mimicking demineralized bone paper that preserves the multi-scale collagen structure of the bone matrix while retaining semi-transparency for imaging and mechanical durability for experimental handling. This demineralized bone paper directs osteoblasts to form a mineralized collagen matrix and supports osteoblast-osteoclast-mediated bone remodeling. By layering multiple osteoblast-seeded demineralized bone papers, we have created a 3D bone organoid in which osteoblasts robustly differentiate into osteocytes. This 3D organoid captures the complexity of bone surface and subsurface, demonstrating potential for replicating bone mechanobiology. Demineralized bone paper-based 3D bone organoids are expected to become an enabling preclinical model for various aspects of basic and applied research in bone health and disease.
