Preparation Of Strontium-loaded Photocrosslinked Hyaluronic Acid Hydrogel And Its Effect On MC3T3-E1 Cells

Objectives:The repair of bone defects is an urgent clinical problem,and bone tissue engineering represented by hyaluronic acid hydrogel is an effective solution.Hyaluronic acid has excellent biocompatibility and basic properties,but its biological activity is not clear,while strontium ions have clear osteogenic induction activity,and hyaluronic acid hydrogel scaffolds loaded with strontium were prepared by photocrosslinked,and its basic properties and biological activity were analyzed,which provided a theoretical basis for the further application of hyaluronic acid in the field of bone defect repair.Methods:（1）Preparation and basic performance analysis of photocrosslinked hyaluronic acid hydrogel loaded with strontium:Hyaluronic acid was modified by methacrylic anhydride and methacryloydated hyaluronic acid was characterized by Fourier transform infrared spectroscopy.A mixed solution of 3%methacrylated hyaluronic acid and 0.5%mass fraction SrCl₂ was prepared,and a photocrosslinked hyaluronic acid hydrogel（Sr/HM）loaded with strontium was prepared by ultraviolet light irradiation crosslinking in the presence of photoinitiator LAP.The experiments were divided into Sr/HM group and HM group,and the microstructure of each group was observed by scanning electron microscopy,and the basic properties of each group were analyzed by swelling performance,mechanical properties and degradation performance tests.（2）Study of the effect of strontium-loaded photocrosslinked hyaluronic acid hydrogel on MC3T3-E1 cells:Sr/HM leach liquor was prepared with reference to standard GB/T 16886.12-2017/ISO 10993-12:2012"Biological evaluation of medical devices-Part 12:Sample preparation and reference materials",and the biocompatibility and biological effects of Sr/HM on MC3T3-E1 cells were studied by co-culture of leach liquor and cells.The experiments were divided into Sr/HM group,HM group and blank control group,and CCK-8 experiments analyzed the biocompatibility and effect on cell proliferation ability of each group.Scratch experiments analyzed the effect of each group on cell migration ability;Alkaline phosphatase activity assay,alizarin red staining,and real-time PCR assay analyzed the effects of each group on the osteogenic differentiation ability of cells.Results:（1）Preparation and basic performance analysis of photocrosslinked hyaluronic acid hydrogel loaded with strontium:Methacrylated hyaluronic acid was successfully prepared by methacrylic anhydride modified hyaluronic acid,Fourier transform infrared spectroscopy confirmed the above results,and Sr/HM was successfully prepared by ultraviolet light irradiation crosslinking.The microstructure showed that Sr/HM and HM were polymers with a porous three-dimensional network structure,with a pore diameter of about 300-400μm.The compressive strength of Sr/HM and HM is 45.87 and 48.24 KPa,respectively,and the elastic modulus of Young’s is 15.65 and 13.53 KPa.The swelling rates of Sr/HM and HM in PBS buffer were 3606±126 and 3667±107%,respectively.Sr/HM and HM were immersed in PBS buffer with a hyaluronidase concentration of 30 U/m L for 7d degradation rates of66.38±5.08 and 64.20±3.42%,respectively.（2）Study of the effect of strontium-loaded photocrosslinked hyaluronic acid hydrogel on MC3T3-E1 cells:We successfully prepared Sr/HM extract and co-cultured MC3T3-E1 cells,and the results of CCK-8experiments showed that the OD values of 50%HM group,50%Sr/HM group,100%HM group,100%Sr/HM group and blank control group were 0.28±0.01,0.29±0.01,0.27±0.02,0.29±0.01 and 0.30±0.00 after 1d,and 1.40±0.18,1.68±0.12,1.59±0.05,1.81±0.23 and 1.33±0.18 after 3d,Sr/HM had no significant effect on the activity of MC3T3-E1 cells,and promoted the proliferation capacity of MC3T3-E1 cells（p<0.05）,and since the concentration factor does not affect cell activity,a 100%concentration leachate is selected for subsequent experiments.The scratch experiment results showed that the mobility of HM group,Sr/HM group and blank control group was 30.85±2.54,28.30±3.86 and 30.62±1.88%,respectively,Sr/HM had no significant effect on the migration ability of MC3T3-E1 cells（p>0.05）.The results of alkaline phosphatase activity detection showed that the ALP activity of HM group,Sr/HM group and blank control group was 15.45±1.79,18.45±2.11 and 14.40±0.91U/L,respectively,and Sr/HM had a promoting effect on the alkaline phosphatase activity of MC3T3-E1 cells（p<0.05）,and the results of alizarin red staining showed that the Sr/HM group had a deeper staining degree and a larger area than that of HM group and blank control group.Semi-quantitative analysis showed that the staining area of each group was 14.17±1.67,69.37±3.17 and 15.38±0.86%,respectively,and Sr/HM promoted the osteogenic mineralization process of MC3T3-E1 cells（p<0.05）,and the real-time PCR results showed that the relative expression of Runx2 gene in HM group,Sr/HM group and blank control group was 1.20±0.33,2.11±0.35 and 1±0.13,the relative expression of OCN gene was 1.29±0.17,1.48±0.37 and 1±0.17,and Sr/HM promoted the upregulation of the expression of Runx2 gene（p<0.05）.Conclusions:（1）Sr/HM is a three-dimensional structure of biological scaffold material with excellent porosity,mechanical properties,swelling properties and degradation properties,which are Similar to the basic performance of HM;（2）Sr/HM has excellent biocompatibility and can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells.Sr/HM is expected to be further applied as a new type of biological scaffold material in the field of bone defect repair.