Chitosan/Bioactive Glass Microparticles Enriched with Therapeutic Metal Ions for Bone Tissue Engineering

The application of divalent bioactive metal ions, such as Cu²⁺, Zn²⁺, and Mn²⁺, emerges as a growth factor-free approach for bone defect regeneration. Delivery of those ions can be achieved by organic or inorganic phases through desirable rapid or sustainable release in order to stimulate specific cell responses. In this work, bioactive ions were incorporated into both phases, chitosan (Cht), via chelation reactions, and mesoporous bioactive glass nanoparticles (MBGNs), by doping. The BG/Cht composites with undoped and Cu-, Zn-, or Mn-doped MBGNs were produced as spherical microparticles with a narrow size distribution and an average size of 42-45 μm via electrohydrodynamic atomization. Swelling studies showed enhanced water uptake in the complete cell culture medium with values between 2.5 and 3.1 compared to phosphate-buffered saline (2.2-2.5). Ion release experiments in phosphate-buffered saline revealed a pronounced release of silicon and calcium up to 7 days for all samples. A sustained release of manganese ions from the MnBG/Cht sample was detected for up to 14 days. A precipitated layer of calcium phosphates on all composites, except on the MnBG/Cht samples, confirmed the materials' bioactivity after 21 days in simulated body fluid. Indirect cytotoxicity tests indicated that the materials were generally nontoxic to human osteosarcoma (MG-63) cells at concentrations below 1 mg/mL. However, direct contact assays with MG-63 and human dermal fibroblast (HDFa) cells revealed concentration-dependent cytotoxic effects, particularly for MnBG/Cht microparticles at a concentration of 0.5 mg/mL. Vascular endothelial growth factor (VEGF) expression analysis on MG-63 and HDFa cells demonstrated that only a higher concentration of MnBG/Cht significantly enhanced the angiogenic response in MG-63 cells, likely due to the decreased cell viability and oxidative stress generated by the redox activity of Mn²⁺ ions. Our results show that composite microparticles have good potential in the design of microparticulate systems with tailored properties through the combination of bioactive metal ions.