3D/3.5D Extrusion-Printed Electroconductive Carbon-Nanofiber-Embedded Alginate-Gelatin Hydrogel-Based Grafts: Comprehensive Understanding of Process Science and Biocompatibility

The reconstruction of complex soft tissues, such as urethral and nerve tissues, requires constructs that integrate vascularization, lumen integrity, and innervation with clinically relevant mechanical and biophysical properties. Current tissue-engineered tubular constructs often fail due to limited strength, instability under physiological conditions, and insufficient electroactivity. This study demonstrates the unique role of carbon nanofibers (CNFs) in improving the structural fidelity of alginate-gelatin hydrogels for additive manufacturing (3D extrusion printing) and clinical applicability. CNF incorporation improved gel strength, viscoelasticity, printability, and buildability while tailoring stretchability, compressibility, swelling, degradation, antimicrobial activity, vascularization, and inflammatory response in 3D-printed scaffolds. In the 3.5D printing approach, the rapid transformation of flat sheets of the CNF-reinforced hydrogel inks to customized tubular constructs with lumen patency was accomplished. At 0.75% CNF addition, hydrogel inks showed a 1.57- and 2.5-fold improvement in viscoelastic range with respect to shear stress and shear strain and a 1.2-fold increase in elastic recoverability, alongside a 3.6-fold enhancement in fracture stress and a 2-fold increase in elastic modulus under uniaxial tension, and the highest electrical conductivity of 0.5 S/m. Micro-CT confirmed interconnected porous structures with pore volume fraction and pore tortuosity of 0.74 and 1.09, respectively. At the same time, the as-printed tubular grafts (3.8 cm in length, 3 mm internal diameter) exhibited smooth luminal surfaces (∼44-100 nm roughness). NIH-3T3 fibroblasts maintained >80% viability, while antimicrobial analysis revealed strong activity against <i>E. coli</i> and <i>S. aureus</i>. In vivo study in Wistar rats revealed normal regulation of different immune cell markers such as CD8, CD68, TNF-α, COX-2, and IL-6, shifting from acute to chronic inflammation and an ehnacement in vascularization by 30 days as evident from H&E, MTS, and vWF straining. No systemic toxicity in the vital organs was recorded. Collectively, these findings highlight CNF-reinforced alginate-gelatin hydrogels can serve as electroconductive, mechanically robust, and biologically responsive scaffolds with a translational potential for complex soft tissue regeneration.