Development of New Bone Substitutes Supporting Vascularization and Mineralization Using 3D Bioprinting

Abstract

Critical-size bone defects (CBD) remain a major clinical challenge, and their rising incidence has intensified the need for effective and customizable bone substitutes. Successful repair of CBD requires the coordinated establishment of a functional vascular network along with mineralized matrix formation. By enabling precise spatial organization of materials, cells, and growth factors, 3D bioprinting provides a powerful platform for bone regeneration through the fabrication of customized, biomimetic scaffolds. Accordingly, the overall objective of this thesis was to develop new bone substitutes capable of supporting both vascularization and mineralization using 3D bioprinting. The specific objectives were to: 1. develop and characterize a new fibrinogen/gelatin methacryloyl (GelMA)-based bioink with vasculogenic and osteogenic potential; and 2. fabricate scaffolds using the newly developed bioink, either as a stand-alone system or by injecting the bioink into 3D-printed cylindrical polycaprolactone (PCL) grids serving as a reinforcement phase, and compare their physicochemical, mechanical, and biological properties. Three fibrinogen/gelatin methacryloyl (GelMA) ink formulations (with different fibrinogen concentrations) were evaluated for their rheological properties and printability. Following this characterization, the inks were supplemented with vascular endothelial growth factor (VEGF165) and bone morphogenetic protein 2 (BMP 2), and assessed for their ability to enable sustained growth factor release from 3D-printed disk-shaped scaffolds. All inks exhibited suitable rheological properties, including shear-thinning behavior, demonstrated good printability, and enabled sustained growth factor release from scaffolds. To evaluate cell viability, vasculogenic and osteogenic responses, disk-shaped scaffolds were printed with the newly developed bioink containing a mid-range concentration of fibrinogen, supplemented with growth factors, and laden with human bone marrow-derived mesenchymal stem cells (hBM-MSC) and human umbilical vein endothelial cells (HUVEC). Cell viability remained high throughout 21 days of in vitro culture. Immunohistochemical analysis revealed progressive alignment and sprouting of CD31 positive cells, indicative of early vasculogenesis, alongside early osteogenic differentiation, as confirmed by histochemical analysis of alkaline phosphatase (ALP) activity. In parallel, scaffolds were fabricated by printing cylindrical PCL grids that served as a reinforcement phase, into which the bioink was injected. The compressive modulus of these scaffolds under unconfined compression was comparable to values reported for trabecular bone. Finally, an alginate-heparinized alginate/collagen-based ink containing hydroxyapatite nanopowder (HAnp) and supplemented with BMP-2 was developed to fabricate acellular scaffolds. Sustained BMP-2 release from 3D-printed disk-shaped scaffolds was observed over 21 days, likely due in part to the conjugation of alginate to heparin. The released BMP-2 remained biologically active, as evidenced by early osteogenic differentiation of hBM-MSC cultured in 2D with supernatants from BMP-2-supplemented scaffolds. In conclusion, this work demonstrated the potential of a fibrinogen/GelMA-based bioink, supplemented with VEGF165 and BMP-2, to enable vascularization and mineralization in 3D-printed scaffolds for bone regeneration in non-load-bearing applications. When the bioink was injected into a cylindrical PCL grid, the resulting scaffold was mechanically reinforced, supporting its use in load-bearing applications. Additionally, the alginate/collagen-based acellular ink containing heparinized alginate enabled sustained release of bioactive BMP 2 from scaffolds, which could facilitate the recruitment of endogenous cells and promote their osteogenic differentiation in vivo. Together, these findings highlight the potential of the newly developed inks for the fabrication of scaffolds supporting bone regeneration in a broad range of applications.