Dual-Stage Crosslinking of Gelatin-Alginate Bioink Supplemented with Wharton’s Jelly to Generate 3D Bioprinted Scaffolds for Wound Healing Application

Incorporation of extracellular matrix (ECM) components into bioinks can enhance biological functionality but often compromises print fidelity and structural stability. This study developed a dual-stage calcium chloride (CaCl2) crosslinking strategy to incorporate Wharton’s jelly-derived ECM (WJ-ECM) into a gelatin-alginate bioink for bioprinted scaffold fabrication. A baseline formulation (BGA) and a WJ-ECM-supplemented formulation (BGAE, 1 mg/mL) were pre-crosslinked with 14 mM CaCl2 prior to extrusion, followed by secondary crosslinking in 0.5 M CaCl2 post-printing. Both formulations exhibited comparable viscosity (20–180 kcP) and high print fidelity (Pr = 0.99 ± 0.01 for BGA; 0.95 ± 0.01 for BGAE), with scaffolds displaying well-defined architecture and over 84% of pores within the target range (160–270 µm). FTIR analysis confirmed WJ-ECM incorporation without detectable alteration of the primary matrix structure. Both scaffolds were non-cytotoxic and supported fibroblast viability; BGAE constructs showed greater cell coverage over 14 days when surface-seeded and more stable fluorescence intensity through 28 days when encapsulated. In a murine thermal burn model, BGAE-treated wounds demonstrated more advanced re-epithelialization and more continuous epidermal coverage at day 14 compared to controls. These findings indicate that dual-stage crosslinking enables WJ-ECM integration while preserving printability, offering a practical platform for bioactive skin tissue engineering applications.