Structure-Driven Bioactivity of Chitosan-Agarose-Gelatin Hydrogels Functionalized with Tannic Acid-Cu ²⁺ /Sr ²⁺ Complexes

Hydrogels represent a versatile platform for regenerative applications; nevertheless, their translation is often limited by insufficient biofunctionality and poorly defined structure-property relationships in multicomponent networks. In chitosan-based hydrogels, performance strongly depends on intrinsic polymer characteristics, requiring precise control of blending and functionalization. Herein, a chitosan-agarose-gelatin blend was functionalized via tannic acid (TA) complexation with Cu²⁺, Sr²⁺, or a dual Cu²⁺/Sr²⁺ system. Chitosan (CS) variants differing in deacetylation degree (DDA), molecular weight (MW), and biopolymer origin were systematically evaluated to establish design-performance relationships. Effective functionalization occurred only for shrimp-derived chitosan with DDA >90%, confirming the decisive role of polymer chemistry in network formation. Within this design window, MW (medium and high) controlled structural organization, degradation kinetics, mechanical performance, and antibacterial efficacy. TA-metal complexes enabled tunable antioxidant and antibacterial responses, with TA-Cu²⁺/Sr²⁺ combined with high-MW chitosan yielding the most balanced multifunctional profile. This formulation preserved a porous architecture, exhibited controlled biodegradation (∼4.5% per month), maintained structural stability over one month, and reached compressive strength and modulus of ∼25.5 kPa and ∼32.6 kPa, respectively. Strong antioxidant capacity and broad-spectrum antibacterial effects were observed as well. Importantly, despite the incorporation of Sr²⁺ for its reported osteoconductive potential, no osteogenic stimulation was observed under the tested conditions. This finding indicates that further optimization of ion ratios and/or TA concentration is required to achieve clinically relevant multibiofunctional hydrogels for bone-related applications.