Bioactive reinforcing bioink for hybrid bioprinting of implantable bone

Project Information

BioForceInk

Grant agreement ID: 101189449

DOI

10.3030/101189449

Project closed

EC signature date 20 August 2024

Start date 1 September 2024

End date 28 February 2026

Funded under

• European Research Council (ERC)

Total cost

No data

EU contribution

€ 150 000,00

Coordinated by

TECHNION - ISRAEL INSTITUTE OF TECHNOLOGY
Israel

Periodic Reporting for period 1 - BioForceInk (Bioactive reinforcing bioink for hybrid bioprinting of implantable bone)

Reporting period: 2024-09-01 to 2026-02-28

Summary of the context and overall objectives of the project

Despite the transformative potential of 3D bioprinting for tissue engineering, replicating the intrinsic mechanical robustness and biological complexity of native bone tissues remains a challenge. Current bioprinting strategies often struggle to balance the structural requirements of hard tissues, such as bone, with the delicate, cell-conductive environments necessary for viability and function. This project addresses these limitations through the use of BioForceInk, a novel bioactive reinforcing bioink engineered for hybrid bioprinting. Composed of thermosensitive microparticles, BioForceInk is optimized for room-temperature extrusion and undergoes phase transition at 37°C to form a stiff, porous scaffold. This dual-material approach allows for the simultaneous deposition of high-strength structural supports alongside soft, cell-laden hydrogels, creating a biomimetic environment that provides both immediate mechanical integrity and long-term osteo-inductive and osteo-conductive potential.
The primary objective of this project is to bridge the gap between mechanical support and biological functionality by developing a growth factor-loaded BioForceInk enriched with osteogenic and angiogenic factors. By utilizing this ink in tandem with cell-laden bioinks, we aim to create complex, vascularized bone implants via a single-step fabrication process. The project further seeks to evaluate the regenerative potential of these bioprinted constructs within a critical-size bone loss model. Ultimately, this innovation aims to advance bone bioprinting toward clinical application by providing a tunable, personalized platform for the development of mechanically robust and biologically functional bone implants, with the potential for broader adaptation across various tissue types.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project’s outcome presents a significant breakthrough in extrusion 3D bioprinting by successfully formulating a bioink suitable for creating robust mechanical and biologically functional 3D bioprinted bone constructs. This bioink has a biphasic release of growth factors which recapitulates the natural bone healing process. The developed technology allows for the maturation of both bone and vascularization, which are critical to the upscaling of 3D printed bone constructs.
We have shown the efficacy of enriched BioForceInk 3D printed implants, suggesting that this bioink could be developed as a convenient, off-the-shelf product for bone regeneration. However, to ensure the technology’s entrance to the market, we are further testing its efficacy.