Synergistic tri-modal reinforcement of chitosan–gelatin hydrogels with hydroxyapatite, functionalized carbon nanotubes, and zinc oxide for superior mechanical and biological performance

The clinical translation of hydrogel-based scaffolds for tissue engineering is often limited by the inherent trade-off between biocompatibility, mechanical integrity, and resistance to post-implantation infections. While natural polymer hydrogels closely mimic the extracellular matrix (ECM), they typically exhibit insufficient load-bearing capacity and inadequate antibacterial performance, restricting their application in mechanically demanding and infection-prone environments. The objective of this study was to develop and systematically evaluate a multifunctional chitosan–gelatin (CS–Gel) nanocomposite hydrogel capable of simultaneously addressing these limitations through a tri-modal reinforcement strategy. The hydrogels were fabricated via glutaraldehyde-mediated chemical cross-linking and reinforced with hydroxyapatite (HA, 1 wt.%), carboxyl-functionalized multi-walled carbon nanotubes (f-MWCNTs, 0.3 wt.%), and zinc oxide nanoparticles (ZnO, 1–3 wt.%). Comprehensive structural characterization using FTIR, XRD, and SEM confirmed homogeneous nanofiller dispersion and the formation of an interconnected porous microarchitecture. Progressive incorporation of ZnO resulted in microstructural densification, reduced pore size, enhanced compressive strength and Young’s modulus, and suppressed swelling and hydrolytic degradation compared with the ZnO-free control. In vitro cytocompatibility assays using MC3T3-E1 pre-osteoblastic cells demonstrated sustained cell viability exceeding 70% for all formulations. Furthermore, antibacterial evaluations revealed a strong, concentration-dependent inhibitory effect, with more than 80% bacterial inhibition against Staphylococcus aureus (S. aureus, Gram-positive) and Escherichia coli (E. coli, Gram-negative) at 3 wt.% ZnO. This systematic study demonstrates that tri-modal reinforcement effectively overcomes the mechanical, biological, and antibacterial limitations of conventional CS–Gel hydrogels, establishing a robust and rational design framework for next-generation multifunctional scaffolds suitable for load-bearing and infection-sensitive tissue engineering applications. • Tri-modal reinforced chitosan–gelatin hydrogels with HA (1 wt.%), f-MWCNTs (0.3 wt.%), and ZnO (1–3 wt.%). • FTIR, XRD, and SEM confirmed uniform nanofiller dispersion and well-integrated porous networks. • ZnO incorporation led to synergistic enhancement of compressive strength and Young’s modulus. • Swelling and hydrolytic degradation decreased with increasing ZnO, improving scaffold stability. • All formulations exhibited sustained cell viability (>70%) and concentration-dependent antibacterial activity, highlighting multifunctional bioactivity.