Effects Of Strontium/Calcium Crosslinked Alginate Hydrogel On Osteogenic Differentiation Of Mouse Precursor Osteoblasts

Objective:Strontium chloride and calcium chloride were mixed at different ratios to prepare strontium-containing alginate hydrogels.Partial physical and chemical properties of the hydrogels were examined,and the osteogenic differentiation of mouse MC3T3-E1cells on hydrogel scaffolds was studied.Methods:According to different ratios of calcium ions and strontium ions in the cross-linking agent,5 groups?Sr0,Sr1,Sr2,Sr3,and Sr4 groups,respectively,Sr0 as the control group?of electrolytes with a final concentration of 4 g/L of cross-linking agent were prepared.The electrolyte and the 2%wt sodium alginate solution with a pH value of 7.4were mixed and cross-linked at the same ratio to prepare a circular hydrogel with a diameter of 15 mm and a thickness of 1 mm.The strontium ion precipitation concentration of each group of hydrogels immersed in phosphate-buffered saline?PBS?at 1,3,5,10,and 14 days was detected by inductively coupled plasma spectrometer?ICP?.Hydrogels were immersed in PBS containing lysozyme to calculate the degradation rate of each group of hydrogels.Mouse MC3T3-E1 cells were inoculated on the surface of the hydrogel and replaced with bone culture medium.The expression of alkaline phosphatase?ALP?in cells of each group was detected on days 3,7,and 14.MC3T3-E1 cells were inoculated with hydrogels in each group,and alizarin red staining was performed on day 21 of osteogenesis to observe the formation of calcium nodules.MC3T3-E1 cells were inoculated with hydrogels from each group,and on the 3rd,7th,14th,and 21st days of osteogenesis,the expression of bone-specific genes of each component was detected by real-time fluorescence quantitative PCR?RT-qPCR?.MC3T3-E1 cells were inoculated with hydrogels in each group.On the 21st day of osteogenesis,the expression of extracellular osteoblast-related proteins was detected by Western Blot method.Results:The ICP results showed that the Sr²⁺concentration of each group of solutions gradually decreased.From the 3rd day,the Sr²⁺concentration gradually decreased and stabilized at an approximately constant rate.The change trend of the Sr2 group was the most stable.The results of hydrogel degradation rate showed that as the concentration of Sr²⁺in the cross-linking agent increased,the rate of hydrogel degradation decreased.The Sr2 group had the highest ALP activity,and there was a statistical difference compared with other groups?P<0.05?.The results of calcium nodules showed that the number of calcium nodules formed in the Sr2 group was large and the area of a single calcium nodule was large,while the Sr0 and Sr4 groups had almost no calcium nodules.RT-qPCR results showed that the osteogenesis-related genes in the Sr2 group showed high expression compared with other groups.Western Blot results showed that the expression of osteogenesis-related proteins in the Sr2 group was also significantly higher.Conclusion:Compared with the control group,as the concentration of strontium ions in the cross-linking agent increased,the degradation rate of alginate hydrogel gradually decreased.The ion release trend gradually slowed down with the increase of strontium ion concentration,but the ion release gradually accelerated with the further increase of strontium ion concentration,and the ion release trend of the Sr2 group was the most stable.According to the results of osteogenic differentiation experiments,it can be seen that the hydrogel in the Sr2 group promotes osteogenic differentiation more significantly.Therefore,when calcium and strontium are used as the crosslinker of alginate hydrogel in the same ratio,the hydrogel performance is most obviously improved,which provides theoretical support for the development and application of new injectable bone tissue engineering materials.