3D Printing Of Cerium-based MOF Hydrogels With Dual Enzyme Activities For Diabetic Wound Healing

Objective:Diabetic wounds that are difficult to heal and do not heal.To this end,we designed a 3D-printed hydrogel dressing with both antibacterial and free radical scavenging effects,verified and evaluated its performance,and finally validated the healing effect of the hydrogel on diabetic wounds through animal experiments,providing a new strategy for diabetic wound treatment.Methods:（1）Material preparation and characterization:In the double network hydrogel system containing acrylamide hydrogel and sodium alginate hydrogel,the organic ligand 1,3,5’-benzenetricarboxylic acid（BTC）of metal-organic backbone（MOF）was added to form the precursor ink for 3D printing,and the primary network was formed by UV light irradiation during the printing process,and the prints were immersed in50 mg/m L of cerium nitrate solution after the completion of printing to complete the construction of the secondary network.And also complete the one-step synthesis of trivalent cerium-based MOF（Ce-BTC MOF）to finally form a double network hydrogel（CBH）containing Ce-BTC MOF,followed by the dropwise addition of 1 m L of H₂O₂solution on the surface of CBH to form a double network hydrogel（MMH）containing mixed valence cerium-based MOF（MVC-MOF）.Afterwards,double network hydrogels C3H and C4H without BTC were synthesized by the same method（where C3H is the double network hydrogel without H2O2 treatment and C4H is the double network hydrogel with H2O2 treatment）.Scanning electron microscopy（SEM）,Fourier transform infrared spectroscopy（FT-IR）,X-ray diffractometry（XRD）,and X-ray photoelectron spectroscopy（XPS）were used to characterize CBH,MMH,C3H,and C4H.The water content,swelling rate,and tensile properties of this hydrogels were tested to study the physical properties of the hydrogels,and the rheological properties of the hydrogels were tested to study the 3D printable properties of this hydrogels.（2）Evaluation of free radical scavenging and discoloration performance:3,3’,5,5’-tetramethylbenzidine（TMB）colorimetric method,Fenton’s experiment and o-triphenol autoxidation method were used to evaluate the free radical scavenging performance of this hydrogels,respectively.The standard curve of H₂O₂correlation with glucose content was produced by indigo carmine fading method,followed by the production of CBH standard colorimetric cards by dropwise addition of different concentrations of H₂O₂（500,100,50,10,5,2.5,1,0.5,0.1,0.05,0 M）,and the color correlation between CBH and MMH resulting from the interconversion between CBH and MMH was investigated by in vivo and ex vivo colorimetric experiments.The correlation between color change and glucose concentration was investigated by in vitro and in vivo colorimetric experiments.（3）Antibacterial and biocompatibility evaluation:The antibacterial properties of the hydrogels were tested using the plate count method.The survival rate of fibroblasts was evaluated by MTT method and AM/PI staining method.（4）Wound healing evaluation:A diabetic rat dorsal skin total defect model was established with control,CBH,MMH,C3H and C4H groups,respectively,to observe the effect of skin healing and collect tissue samples on days 7,14 and 21 of treatment,using histopathological analysis（H&E,PAS and TNF-α,IL-1,IL-6immunohistochemistry）of wound healing effect and its possible causes.Results:（1）The presence of Ce-BTC MOF and MVC-MOF was successfully observed in CBH and MMH by SEM,and the size of MVC-MOF was smaller than that of Ce-BTC MOF.Physicochemical properties and surface morphology analysis proved the successful preparation of Ce-BTC MOF and MVC-MOF and their presence in CBH and MMH.The water content of all our synthesized hydrogels is above 90%,and the swelling rate can be more than 300%.The stretchability of CBH is demonstrated in tensile experiments,and the results of rheological experiments show that the hydrogels meet the 3D printing conditions.（2）The colorimetric method of TMB confirmed that MVC-MOF and MMH have oxidase-like properties,and the Fenton assay and the autoxidation of o-phenylene triol confirmed that CBH can effectively scavenge·OH and MMH is more effective for·O^2-scavenging,respectively,while the interconversion between CBH and MMH showed a glucose concentration-dependent color change,which can realize the indirect monitoring of periwound glucose indirect monitoring of periwound glucose content.（3）The plate counting method confirmed that both CBH and MMH groups could produce strong inhibition of Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus.The results of cytotoxicity assay showed that the prepared hydrogels were non-toxic to cells（cell viability>80%）.（4）The results of animal experiments showed that the diabetic wounds in the CBH treatment group basically healed completely within 21 days;histological and PAS staining confirmed that CBH and MMH could significantly promote epithelial proliferation and glycogenolysis;immunohistochemical staining results of TNF-α,IL-1 and IL-6 showed that CBH could effectively reduce the common inflammatory factors in diabetic wounds.Conclusions:This 3D printed hydrogel has excellent antibacterial properties,free radical scavenging properties and good cytocompatibility,and can achieve complete healing of 1 cm²diabetic wounds within 21 days,so this hydrogel will overturn the traditional treatment of diabetic wounds and provide a new therapeutic idea for the clinical application of diabetic wound dressings.