Bioactive Ion-Doped Monetite/Chitosan Thermosensitive Hydrogels with Dual Osteogenic and Pro-Angiogenic Stimuli for Bone Regeneration

Abstract

The development of multifunctional scaffolds capable of orchestrating the complex cascade of bone regeneration, cell recruitment, vascularization, and extracellular matrix deposition remains a significant challenge in regenerative medicine. This study introduces a dual-strategy biomaterial system. On one hand, pure monetite (CaP), cobalt-doped monetite (CoCaP), and magnesium-doped monetite (MgCaP) powders were synthesized via coprecipitation to deliver specific ionic cues. On the other hand, these bioactive powders were incorporated into a thermosensitive chitosan/β-glycerophosphate (β-GP) hydrogel to create an injectable, conformable composite. Then, comprehensive physicochemical characterization (X-ray diffraction, Raman, Fourier transform infrared (FTIR), and scanning electron microscopy/energy dispersive spectroscopy) confirmed phase-pure monetite with successful ionic incorporation. FTIR analysis of the hydrogels revealed characteristic bands for chitosan, with evidence of the establishment of electrostatic interactions between protonated amino groups and phosphate species. The composite hydrogels exhibited shear-thinning behavior, storage modulus (G′) exceeding loss modulus (G″), and tunable mechanical properties, with MgCaP composites displaying the highest stiffness. Indirect contact assays on CoCaP powders and hydrogels confirmed enhanced cell adhesion, migration, and expression of pro-angiogenic genes and matrix metalloproteinases. MgCaP formulations robustly upregulated osteogenic markers. Additionally, direct cell contact with composite hydrogels dramatically amplified transcriptional responses on adhesion, cell cycle, and osteogenic genes, particularly on MgCaP hydrogels. Altogether, this work demonstrates that ionic doping confers distinct and complementary bioactivities to monetite, pro-angiogenic/migratory Co2+, and osteogenic/matrix-synthetic Mg2+, and that chitosan hydrogels represent an injectable vehicle that amplifies bioactive signaling upon direct cell contact, thus offering a versatile and innovative platform for bone tissue engineering.