(227e) Bioengineered 3D Bone Marrow Assembloids

In vitro reconstruction of trabecular bone marrow holds great potential for advancing both basic and applied research. However, the sharply distinct yet intermingled microenvironments of rigid bone and semi-solid marrow present significant challenges, as single biomaterials are insufficient to replicate the intrinsic biophysical complexity of bone marrow. Here, we report a modular tissue-engineering strategy to create a 3D bone marrow assembloid by integrating separately prepared 3D bone and marrow organoids. For the bone component, we fabricated an osteoid-inspired demineralized bone paper to generate constructs that recapitulate surface and subsurface bone architecture while supporting dynamic remodeling. For the marrow component, we employed sinusoid-mimicking porous hydrogel scaffolds that support bone marrow stromal cells and facilitate intimate co-culture with hematopoietic cells within spherical cavities. Assembly of these engineered bone and marrow organoids successfully recapitulated key features of bone marrow tissue complexity. We demonstrate that the bone surface attracts mononuclear cells and supports continuous remodeling under biochemical stimulation. Hematopoietic cells exhibit significantly prolonged mitogenic activity in the assembloid compared to marrow-only cultures. Finally, in a proof-of-concept experiment, we simulated bone marrow transplantation by irradiating the assembloid and introducing GFP-labeled bone marrow cells. Together, these findings establish the 3D bone marrow assembloid as a versatile platform for replicating native tissue complexity and modeling clinical scenarios, offering new opportunities for fundamental and translational bone marrow research.