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

Area of Science:

• Biomaterials Science
• Stem Cell Biology
• Regenerative Medicine

Background:

• Two-dimensional culture of bone marrow mesenchymal stem cells (BMSCs) leads to functional decline, limiting their therapeutic efficacy in bone tissue engineering (BTE).
• Three-dimensional (3D) culture systems offer a potential strategy to overcome these limitations and enhance BMSC potency.

Purpose of the Study:

• To investigate the rejuvenating effect of chitosan film-based 3D culture on BMSC potency.
• To evaluate the synergistic promotion of angiogenesis and immunomodulation by 3D-cultured BMSCs for vascularized bone regeneration.

Main Methods:

• Mouse BMSCs were cultured in 3D spheroids on chitosan films and compared to conventional 2D cultures.
• Stemness, proliferation, migration, senescence, osteogenic, and proangiogenic potentials were assessed.
• Paracrine effects on endothelial cells and macrophages were evaluated using conditioned media. *In vivo* studies utilized GelMA hydrogel-encapsulated 3D-BMSC spheroids in mouse cranial defects.

Main Results:

• 3D-cultured BMSCs showed enhanced stemness, proliferation, migration, delayed senescence, osteogenic differentiation, and proangiogenic potential compared to 2D cultures.
• Conditioned medium from 3D BMSCs (3D-CM) promoted *in vitro* angiogenesis and M2 macrophage polarization, suppressing M1 inflammation.
• *In vivo*, GelMA + 3D-BMSC spheroids significantly improved bone regeneration with enhanced vascularization and a proreparative immune microenvironment.

Conclusions:

• Chitosan film-based 3D culture effectively rejuvenates BMSC therapeutic potency.
• This 3D culture system enhances intrinsic cell properties and orchestrates a proregenerative microenvironment.
• It presents a promising platform for repairing critical-sized bone defects through enhanced vascularized bone regeneration.