Abstract: BACKGROUND: Hydrogel materials have garnered significant attention in the field of tissue repair due to their excellent biocompatibility and biodegradability. However, the clinical application of hydrogel materials is hindered by their limited functionality, particularly the absence of antimicrobial and osteogenic properties. 
OBJECTIVE: To design hydrogels with both antibacterial and osteogenic properties for bone tissue repair.
METHODS: (1) Hydroxyapatite co-doped with copper and zinc ions was synthesized by chemical precipitation. Double-ion-doped hydroxyapatite, epigallocatechin gallate, and double-ion-doped hydroxyapatite + epigallocatechin gallate were added to a photoinitiator, respectively. Methacrylated gelatin was added to either the photoinitiator alone or the aforementioned photoinitiator, respectively. The hydrogels were cured under 405 nm ultraviolet light for 20 seconds to obtain methacrylated gelatin hydrogels (denoted as G), double-ion-doped hydroxyapatite/methacrylated gelatin hydrogels (denoted as G-Cu/Zn HA), epigallocatechin gallate-modified methacrylated gelatin hydrogels (denoted as G-E), and epigallocatechin gallate-modified double-ion-doped hydroxyapatite/methacrylated gelatin hydrogels (denoted as G-E-Cu/Zn HA). The micromorphology, compressive mechanical properties, swelling properties, degradation properties, and release kinetics of metal ions and epigallocatechin gallate of the four hydrogels were characterized. (2) Staphylococcus aureus (or Escherichia coli) cultures were co-cultured with each of the four hydrogel groups. The antibacterial properties of each hydrogel group were evaluated by agar plate coating, live/dead staining, and scanning electron microscopy. (3) The four hydrogel groups were co-cultured with MC3T3-E1 cells. The cytocompatibility of each hydrogel group was evaluated by live/dead staining and CCK-8 assay. After co-culture with MC3T3-E1 cells, osteogenic activity of each hydrogel group was evaluated by alkaline phosphatase staining, Alizarin Red S staining, and expression of osteogenesis-related genes after osteogenic induction.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy revealed that the cross-sections of all four hydrogel groups exhibited a loose and porous internal structure. The surfaces of the G and G-E hydrogels were relatively smooth, while the pore walls of the G-Cu/Zn HA and G-E-Cu/Zn HA hydrogels showed increased surface roughness. The compressive stresses of the G, G-E, G-Cu/Zn HA, and G-E-Cu/Zn HA hydrogels were 10.48, 12.91, 23.64, and 41.03 kPa, respectively. Compared with the G hydrogel, the G-E, G-Cu/Zn HA, and G-E-Cu/Zn HA hydrogels exhibited shortened overall swelling times and decreased swelling equilibrium rates. The degradation cycle of the G-E-Cu/Zn HA hydrogel was prolonged compared with the other three hydrogel groups. The G-Cu/Zn HA and G-E-Cu/Zn HA hydrogels continuously released Cu2+, Zn2+, and Ca2+ over 30 days, with the total amount of Cu2+ and Zn2+ released from the G-Cu/Zn HA hydrogel exceeding that from the G-E-Cu/Zn HA hydrogel. Compared with G-E hydrogel, G-E-Cu/Zn HA hydrogel significantly inhibited the burst release of epigallocatechin gallate. (2) Agar plate spread assay, live/dead staining, and scanning electron microscopy revealed that compared with G hydrogel, the other three hydrogel groups all exhibited antibacterial activity, with G-E-Cu/Zn HA hydrogel exhibiting the strongest inhibitory activity. (3) Live/dead staining and CCK-8 assay revealed that all four hydrogel groups exhibited good cytocompatibility. Alkaline phosphatase staining, Alizarin Red S staining, and osteogenesis-related gene expression assay revealed that G hydrogel exhibited the weakest osteogenic activity, while G-E-Cu/Zn HA hydrogel exhibited the strongest osteogenic activity. (4) These results demonstrate that the epigallocatechin gallate-modified dual-ion-doped hydroxyapatite/methacrylated gelatin composite hydrogel exhibits excellent antibacterial and osteogenic properties.

Key words: Cu2?, Zn2?, nanohydroxyapatite, epigallocatechin gallate, methacryloylated gelatin hydrogel, osteogenesis, antibacterial, biomaterial