Ex vivo recapitulation of bone mechanobiology in a bioengineered 3D lamellar bone organoid

Introduction: Bone is a mechanically adaptive dynamic tissue that undergoes repeated remodeling to maintain its mechanical structure in response to changing physical stresses. Osteocytes, comprising over 90% of bone cells, serve as primary mechanostats, detecting a broad range of mechanical forces and responding by adjusting secretion profiles to regulate bone metabolism. Individual osteocytes form rich dendritic networks within the lamellar structure of the mineralized collagen matrix. Their unique morphology and surrounding extracellular matrix milieu are known to play a critical role in bone mechanobiology. However, detailed investigation remains daunting due to the intrinsic challenge of accessing osteocytes through the opaque bone matrix and maintaining long-term viability and function outside of the body. Here, we report a bioengineered 3D bone organoid that replicates essential bone tissue complexity and processes, including osteocyte-driven mechanotransduction.

Materials and

Methods: An osteoid-inspired demineralized bone paper (DBP) was prepared by thin (20 µm) sectioning of demineralized bovine bone matrix in a vertical direction. Primary murine osteoblasts from DsRed mice femurs and tibias were seeded on the DBP. By layering multiple osteoblast-seeded DBPs, a 3D bone model including surface osteoblasts and subsurface osteocytes was established. To simulate mechanical stimulation, a bimodal mechanoculture device was created that can deliver vibrational and compressive mechanical forces to the 3D bone model in a standard multi-well plate with time control. Osteoblast-to-osteocyte differentiation was confirmed by morphological changes and expression of lineage-specific genes. Mechano-responsiveness was characterized by monitoring secretion profile changes, including OPG and RANKL. Mechanotransduction-induced changes in bone metabolism were functionally characterized by monitoring multinucleated osteoclast differentiation from bone marrow mononuclear cells.

Results, Conclusions, and Discussions: The DBP induces in vivo-relevant mineralized collagen formation by osteoblasts. When multiple DBPs are layered, osteoblasts robustly differentiate into osteocytes while depositing minerals on both sides of the DBPs. By exploiting this additive approach, we have established a 3D bone organoid that includes surface osteoblasts and subsurface osteocytes in a controlled and reproducible manner. The resulting free-standing 3D bone organoid enables the application of compressive and vibrational mechanical forces. We demonstrate the feasibility of recapitulating bone mechanobiology by applying defined bimodal mechanical forces in a programmable manner and monitoring subsequent changes in the secretion profiles of regulatory molecules. We envision that DBP-based additively manufactured 3D bone organoids will contribute significantly to various aspects of bone mechanobiology.

Acknowledgements (Optional):