Alginate/Gelatin Hydrogel Scaffold Containing NCeO₂ As A Potential Osteogenic Nanomaterial For Bone Tissue Engineering

Objective:The aim of this search is to synthesize a gelatin-sodium alginate hydrogel loaded with nanoparticles of cerium dioxide（GA-n Ce O₂）,and to investigate its physicochemical properties,mechanical performance,enzyme-mimicking activity,in vitro biocompatibility,ability to promote osteogenic differentiation,and in vivo bone regeneration potential.The study further aims to explore the bone repairing ability of the GA-n Ce O₂ scaffold composite material and its suitability for bone regeneration.Methods:Nano cerium oxide（n Ce O₂）was synthesized by hydrothermal method with cerium nitrate hexahydrate（Ce（NO₃）₃·6H₂O）as raw material,and the morphology,particle size and potential of n Ce O₂ were analyzed;GA-n Ce O₂ freeze-dried scaffold was prepared by freeze-drying method with sodium Alginate（Alg）,Gelatin（Gel）and n Ce O₂in the ratio of 1:2:1.The surface morphology of the scaffold was observed by Scanning Electron Microscope（SEM）,and the chemical composition of the functional surface was analyzed by X-ray photoelectron spectroscopy（XPS）,Fourier Transform Infrared Spectroscopy（FTIR）and X-ray diffraction（XRD）Mechanical properties,in vitro degradation and swelling properties;Superoxide dismutase（SOD）assay kit and Catalase（CAT）assay kit were used to evaluate the mimic activity of n Ce O₂.The adhesion of MC3T3-E1 cells on freeze-dried scaffold was observed by SEM.CCK-8 method and living/dead cell staining method were used to detect the cell compatibility of scaffolds Alkaline Phosphatase（ALP）test kit and ALP staining were used to evaluate the early differentiation status of cells within 7 days and 14 days,and alizarin red staining（ARS）was used to detect the late mineralization ability of cells within 14 days and 21days.Real-time PCR was used to detect the expression levels of osteogenesis-related genes,including transcription factor 2（Runx-2）,ALP,Collagen-1（Col-1）and Osteocalcin（OCN）.The Sprague dawley（SD）rat femoral defect model was established.The experimental group and the blank group were implanted into the bone defect site respectively.The rats were killed at the 4th,6th and 8th weeks respectively,and the femoral specimens were collected.The healing of bone defect was evaluated by Micro-CT scanning and the bone regeneration was evaluated by Masson’s trichrome stain（Masson stain）and Hematoxylin and eosin stain（H&E stain）.Results:The results of SEM,EDS,FTIR and XRD showed that the GA-n Ce O₂ hydrogel scaffold was successfully synthesized.The scaffold had a typical three-dimensional porous structure with an average porosity of 70.61±1.94%;After adding n Ce O₂,the degradation rate of GA-n Ce O₂ hydrogel is higher than that of Alg and lower than that of Gel.The swelling rate of GA-n Ce O₂ hydrogel is similar to that of Gel/Alg（GA）,the mechanical properties are improved compared with that of GA,and the SOD and CAT activities of GA-n Ce O₂ hydrogel are similar to those of n Ce O₂ hydrogel.The extension and adhesion of MC3T3-E1 cells on the surface of GA-n Ce O₂ hydrogel scaffold were more obvious than those in other groups.The cell state showed that MC3T3-E1 cells developed more evenly,and thick and long processes extended around the cells.CCK-8results showed that the cell proliferation ability of GA-n Ce O₂ hydrogel group was higher than that of other groups;Live/dead cell staining showed that there was no obvious cytotoxicity in each group.The results of ALP test showed that the activity of ALP in GA and GA-n Ce O₂ scaffolds increased significantly on the 14th day,and the activity of ALP in GA-n Ce O₂ scaffolds was higher than that in GA and other scaffolds;The results of ARS showed that the cells in GA-n Ce O₂scaffold group showed the highest level of mineralization;The results of PCR showed that the expression levels of Runx-2,Col-1and OCN increased with time.Micro-CT images showed that the healing degree of femoral defect in GA-n Ce O₂ scaffold group was higher than that in blank group.H-E staining and Masson staining showed that new bone was formed and a large number of collagen fibers were deposited around the bone defect in GA-n Ce O₂ scaffold group.Conclusion:1.GA-n Ce O₂ composite hydrogel scaffold was successfully prepared by freeze-drying method.Physical property analysis and surface characterization showed that n Ce O₂ was successfully loaded into GA hydrogel scaffold.The porous scaffold had ideal porosity,excellent mechanical properties,suitable swelling degradation performance and enzyme simulation activity similar to n Ce O_2.2.GA-n Ce O₂ hydrogel scaffold has excellent biocompatibility and can promote the proliferation and osteogenic differentiation of MC3T3-E 1 cells in vitro.3.The results showed that GA-n Ce O₂ hydrogel scaffold has good degradation and bone regeneration ability in vivo,which is expected to provide a new idea for bone defect repair and treatment.