During the project, we developed and optimized a reinforcing bioink (BioForceInk) enriched with osteoinductive and osteoconductive (BMP2, hydroxyapatite) and angiogenic (VEGF) growth factors. The formulation of the BioForceInk was optimized for dual growth factor delivery, achieving a biphasic kinetic profile. This design facilitates an immediate release of VEGF to initiate angiogenesis, followed by the prolonged delivery of BMP2 to support osteogenesis, thereby emulating the endogenous cascade of fracture repair.
To characterize the functional properties of the enriched BioForceInk, we 3D printed acellular constructs and quantified their growth factor release profile using ELISA assays. Next, we fabricated hybrid constructs by co-printing the enriched BioForceInk in tandem with a soft bioink laden with endothelial and mesenchymal stem cell. Immunostaining of these constructs confirmed in-vitro their capacity to improve vascularization and osteogenic differentiation. Finally, to assess translational potential, we implanted these constructs in a rat critical-sized cranial defect model. Subsequent micro-CT imaging and histological immunostaining demonstrated the construct’s capacity for in vivo bone regeneration.
Main Achievements:
1. VEGF and BMP2 enriched BioForceInk formulation showing biphasic kinetic profile:
The BioForceInk is composed of microparticles of PLGA/PEG/hydroxyapatite blend that are created using a double emulsion approach. The particles are then combined with a carrier liquid composed of aqueous Pluronic f127 and glycerol blend to achieve an extrudable paste. Incubation of this extruded paste at 37°C induces the material sintering and PEG diffusion, resulting in the formation of porous PLGA/hydroxyapatite mass. To achieve the biphasic kinetic profile, BMP2 was incorporated into the PLGA during the fabrication process of the double emulsion, while VEGF was added to the carrier liquid phase combined with 1% collagen type I. The use of collagen into the structure did not alter the sintering temperature of the material, however, it increased the stiffness of the printed construct, likely due to the presence of crosslinked fibers within the construct.
2. In-vitro enhanced vascularization and osteogenic differentiation:
Hybrid 3D printed constructs composed of the enriched BioForceInk and endothelial and mesenchymal stem cell laden soft bioink promoted vascularization and osteogenic differentiation in-vitro.
3. In-vivo enhanced intra-membranous ossification, and calcification foci:
Implantation of 3D printed constructs promoted intra-membranous ossification, and calcification foci across the entire implant volume. These results were superior to growth-factor-free controls, confirming the essential role of the bioactive factors.
4. Efficacy of growth factor enriched BioForceInk implants suggests that this bioink could be developed as a convenient, off-the-shelf product for bone regeneration.
