Objective:Chronic non-healing diabetic wounds and ulcers are one of the most common complications of diabetes,which can be fatal,lead to amputations.Susceptibility to infection and impaired angiogenesis are the two central reasons for the clinical consequences associated with chronic non-healing diabetic wounds.Herein,we successfully developed calcium ion(Ca2+)cross-linked sodium alginate(SA)hydrogels with both pro-angiogenesis and antibacterial properties for diabetic wound healing.Methods:CCK8,transwell assay,and tube formation assay were performed to assess the effects of deferoxamine(DFO),copper nanoparticles(Cu-NPs)and combination of DFO and Cu-NPs on human umbilical venous endothelial cells(HUVECs).The antibacterial property of Cu-NPs was assessed using the colony forming assay.The Ca2+cross-linked SA hydrogels were synthesized.The hydrogels were characterized the microstructure.Then,the gelling time,water content and swelling ratio of the hydrogels were calculated.Furthermore,rheological tests,compression tests and degradation experiments were carried out to characterized the hydrogels.Calcein-AM/propidium iodide(Calcein-AM/PI)staining assay,CCK-8 assay and hemolysis test were conducted to assess the cytotoxicity of the hydrogel in vitro.Wound healing,transwell assay,and tube formation assay were conducted to assess the influence of hydrogels on migration and tube formation capabilities of HUVECs.The contents of reactive oxygen species(ROS)in HUVECs were analyzed using a ROS detection kit.The antibacterial properties of the hydrogels were assessed using colony forming assay,hoechst 33342/PI staining assay,and microtiter plate biofilm assay.Furthermore,the full-thickness skin wound models of diabetic C57BL/6were made to evaluate the effects of the hydrogels on wound healing.The wound areas at different time points were calculated.Histopathology analysis was conducted to assess the effects of the hydrogels on wound healing rate,collagen deposition,vascular system formation,ROS formation,and inflammatory responses of the diabetic wound.Furthermore,the levels of hypoxia-inducible factor 1 alpha(HIF-1α)and vascular endothelial growth factor(VEGF)were detected using immunohistochemical staining.Results:DFO and Cu-NPs worked synergistically to enhance the proliferation,migration,and angiogenesis of HUVECs in vitro.Results of colony formation assay indicated Cu-NPs were effective against E.coli and S.aureus in a dose-dependent manner in vitro.An SA hydrogel containing both DFO and Cu-NPs(SA-DFO/Cu)was prepared using a Ca2+cross-linking method.The hydrogels possessed relatively smooth surface and multilayered,interconnected porous structure in the scanning electron microscope images.All hydrogels contained~97%water,and the swelling ratio of hydrogels was approximately 3700%.The hydrogel reached a plateau of release of DFO at 12 hours and a plateau of release of Cu at 24 hours in vitro.The hydrogels could withstand a compressive strain up to 80%without any observable structural damage.For the rheological test,the storage modulus was greater than the loss modulus,indicating that the hydrogel had solid properties.The degradation experiments indicated degradability of the hydrogel.The results of biocompatibility evaluation revealed that the hydrogels exhibited minimal cytotoxic effects.Furthermore,the SA-DFO/Cu could significantly enhance the ability of migration and tube formation and decrease the level of ROS in HUVECs.The results of antibacterial property assays indicated that the hydrogel exhibited antibacterial properties in vitro.SA-DFO/Cu significantly accelerated diabetic wound healing and collagen deposition,improved angiogenesis,and reduced formation of ROS and long-lasting inflammation in a mouse model of diabetic wound.Mechanistically,DFO and Cu-NPs synergistically stimulated the levels of HIF-1αand VEGF in vivo.Conclusions:Given the pro-angiogenesis,antibacterial and healing properties,the Ca2+cross-linked SA hydrogel possesses high potential for clinical application in refractory wounds. |