Biodegradable and osteoconductive sodium alginate-gelatin/amorphous magnesium phosphate 3D-printed scaffolds for craniofacial bone regeneration

Abstract

This study aimed (1) to develop and characterize 3D-printed hydrogel-based scaffolds composed of sodium alginate and gelatin containing amorphous magnesium phosphate (AMP), and (2) to evaluate the scaffolds' biological response with alveolar bone-derived mesenchymal stem cells (aBMSCs). Hydrogel inks were prepared with sodium alginate, gelatin, calcium chloride, and varying AMP contents (0 %, 5 %, and 10 %). The scaffolds were fabricated using an extrusion-based 3D bioprinter. First, the formulated hydrogel-based inks were characterized for rheological behavior and printability. After printing, the scaffolds were assessed for morphology, chemical composition, mechanical properties, and swelling/degradation profiles. For in vitro cell-scaffold interaction, scaffolds were seeded with aBMSCs and analyzed for cell viability, matrix mineralization, and osteogenic gene expression via RT-qPCR. Statistical analysis was performed with ANOVA/Sidak or Tukey tests, with confidence intervals (α = 5 %). Rheological analysis showed that all inks exhibited shear-thinning behavior, more pronounced in AMP-containing formulations. Filament drop tests and printability assessments demonstrated filament uniformity and structural fidelity in AMP-containing inks. Morphological analysis revealed well-defined scaffold architecture with regular edges, and SEM confirmed smooth surface morphology with uniform AMP distribution. FTIR spectra displayed characteristic phosphate and polymer bands, while EDS confirmed the presence of magnesium and phosphorus in AMP-containing scaffolds. The swelling behavior increased over 24 h, and all 3D-printed scaffolds fully degraded within 35 days. All formulations supported increased cell viability over time (p ≤ 0.0092). AMP-containing scaffolds enhanced mineralized matrix deposition under osteogenic stimulation (p < 0.0001), particularly in the 10 % AMP group, and promoted upregulation of osteogenic genes (COL1A1, ALPL, and RUNX2). Clinical significance: This study demonstrated that incorporating AMP into alginate-based hydrogels combines printability, biodegradability, and osteoconductive properties. Previous AMP-containing biomaterials lacked optimization for material extrusion-based 3D printing or the synergistic combination with a gelatin-alginate network. This strategy represents an advance in the field, offering a potential biomaterial ink for the fabrication of personalized scaffolds for craniofacial bone regeneration, enabling synergistic modulation of rheology and early osteogenic stimulation.