Three-dimensional (3D) porous biocomposite hydrogel sponges consisting of chitosan (CS), silk fibroin (SF) and ZnO nanoparticles (ZnO NPs) were prepared by the freeze-drying method. The biocomposite was also loaded with tetracycline hydrochloride (TCH) and its wound healing efficacy was investigated in an attempt to enhance healing through the release of the drug. The chemical nature, composition, thermal properties and morphology of the sponges were revealed by FTIR, TGA, and SEM/EDX, analysis. The hydrogels presented the swelling degree in the range of 650-1180% at pH 7.4; 1300-3160% at pH 5.5 and 550-920% at pH 9.0, respectively. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay demonstrated the cytocompatibility of CS/SF/ZnO hydrogels against Wharton's jelly mesenchymal stem cells (WJ-MSCs). CS/SF/ZnO-0 and CS/SF/ZnO-0.5 hydrogels exhibited no cytotoxic effect. In addition, CS/SF/ZnO hydrogels exhibited antimicrobial activity against some tested gram-positive and gram-negative bacteria. Physical immobilization of TCH in hydrogel matrix resulted in sustained release of the drug for more than 2 days at pH 7.4. The results showed that CS/SF/ ZnO-0.5 hydrogel sponge is safe, convenient and could be effective for wound healing applications. WOS:000909694100001 Q2