Fabrication of cell-laden hydrogel microcapsules of alginate and chitin fibrils using divalent and trivalent metal ions

Nanofiber-embedded 3D hydrogel constructs have garnered significant attention due to their versatile applications in drug delivery, cell therapy, tissue engineering, and regenerative medicine. These constructs are especially prized for their capacity to mimic the composition of the extracellular matrix (ECM) found in living tissues and organs. The unique chemical and mechanical properties of hydrogel microcapsules have made them particularly notable among various biomaterial constructs for their effectiveness in cell encapsulation, which aims to improve cell growth and proliferation. In this study, we developed alginate hydrogel microcapsules embedded with chitin nanofibrils, using divalent calcium ions and trivalent iron ions as crosslinking agents. An electrostatic encapsulation technique was utilized to create microcapsules with diameters ranging from 200–500 μm, and their physicochemical properties, rheological properties, size, and mechanical stability were evaluated. The rheological analysis demonstrated that the Fe³⁺ crosslinked hydrogel (AF0) and Fe³⁺/Ca²⁺ cross-linked hydrogel (AFC) have higher storage modulus than the Ca²⁺ crosslinked hydrogel (AC0). Additionally, FTIR analyses of AF0 and AFC demonstrated a broader –O–H stretching peak compared to that of AC0, suggesting that more hydroxyl groups of alginate chains are involved in crosslinking with ferric ions exhibiting extended mechanical stability compared to those crosslinked with calcium ions under in vitro physiological conditions. We also investigated the cellular responses to the composite hydrogels crosslinked with these divalent and trivalent metal ions through in vitro studies involving the seeding and encapsulation of NIH/3T3 fibroblast cells. Remarkably, both types of crosslinked microcapsules maintained excellent cell viability for up to 5 days. Our in vitro scratch assay demonstrated that media extracted from AF0 microcapsules facilitated faster wound closure compared to that extracted from AC0, suggesting that hydrogels crosslinked with Fe³⁺ ions promote enhanced cellular proliferation. These results suggest that calcium and ferric ion crosslinked alginate–chitin composite microcapsules provide a promising platform for developing 3D hydrogel constructs suitable for various biomedical applications, including wound healing models, tissue engineering, and drug toxicity testing.