Abstract
Printing technology is a leading strategy for creating customized 3D matrices for tissue engineering. Our study developed an injectable nanocomposite hydrogel (bHAGel) for high-fidelity 3D extrusion printing composed of gelatin (Gel) and magnesium-doped biomimetic hydroxyapatite (bHA) particles that mimics a bone extracellular matrix. bHA particles, synthesized through a bioinspired mineralization process, acted as multifunctional additives, modulating rheology for printability, ensuring homogeneous phase distribution, enabling excellent model fidelity, and providing osteoinductive cues. The optimized hydrogel formulation enables the fabrication of porous scaffolds with interconnected macro- and microporosity via extrusion-based printing and freeze-drying. This key feature promoted cell infiltration and nutrient diffusion during tissue engineering procedures. Biological validation involves tailoring 3D scaffolds to fit a perfusion bioreactor chamber supporting seamless handling, seeding, and long-term culturing without scaffold removal or repositioning. Dynamic in vitro experiments with donor-derived human bone marrow stromal cells assessed the constructs' stability, ability to maintain geometry and perfusability, cytocompatibility and osteoconductivity, as well as robust osteogenic differentiation over 28 days. A more complex dynamic coculture model further demonstrated that the scaffold supports osteoclastogenesis under physiological, osteoblast-mediated conditions. Altogether, bHAGel scaffolds provided a customizable, bioactive platform suitable for engineering bone-mimetic organoids under dynamic conditions. Their modularity and biological relevance could be exploited in bone regeneration, disease modeling, and drug testing.