3D-BMSC Spheroids Enhance Bone Repair Associated with H-Type Vessels and Immunomodulation

The therapeutic potential of bone marrow mesenchymal stem cells (BMSCs) in bone tissue engineering (BTE) is compromised by functional decline during conventional two-dimensional (2D) expansion. We hypothesized that chitosan film-based three-dimensional (3D) culture rejuvenates BMSC potency, synergistically promoting angiogenesis and immunomodulation for vascularized bone regeneration. Mouse BMSCs were cultured into spheroids on chitosan films. Their stemness, proliferation, migration, senescence, osteogenic, and proangiogenic potential were compared with 2D cultures. Paracrine effects were evaluated by treating human umbilical vein endothelial cells (HUVECs) and RAW264.7 macrophages with BMSC-conditioned medium (CM). In vivo, gelatin methacryloyl (GelMA) hydrogel-encapsulated 3D-BMSC spheroids were implanted into mouse critical-size cranial defects. Compared to 2D counterparts, 3D-BMSCs exhibited increased stemness, proliferation, migration, delayed senescence, osteogenic differentiation, and enhanced proangiogenic potential. We observed that conditioned medium from 3D-BMSCs (3D-CM) was associated with in vitro angiogenesis and orchestrated a proreparative microenvironment by promoting M2 macrophage polarization and suppressing M1 inflammation. In vivo, GelMA + 3D-BMSC spheroids achieved greater bone regeneration, which was accompanied by a proreparative immune microenvironment and enhanced CD31/EMCN-positive H-type-like vessel formation. This chitosan film-based 3D culture system effectively augments BMSC therapeutic potency, simultaneously enhancing intrinsic cell properties and orchestrating a proregenerative microenvironment, thereby offering a promising experimental platform for critical-sized bone defect repair.