Effect Of Multi-ion Crosslinked Sodium Alginate Hydrogel On The Adhesion And Proliferation Of Osteogenic Precursor Cells

OBJECTIVE: Strontium chloride,magnesium chloride and zinc chloride were used as cross-linking agents to synthesize the composite alginate brine gel containing strontium,magnesium and zinc.The physical and chemical properties of the alginate brine gel containing strontium,magnesium and zinc were detected,and the adhesion and proliferation of mouse MC3T3-E1 cells inoculated on the cross-linked alginate brine gel containing strontium,magnesium and zinc were detected.METHODS: According to different ion types in the crosslinking agent,5 groups of calcium,strontium,magnesium and zinc were prepared with the mass ratio of respectively A(2:0:0:0),B(2:2:0:0),C(2:2:2:0),D(2:2:0:0.5),E(2:2:2:0.5)electrolyte(group A for reference group),and were mixed and cross-linked with sodium alginate solution with the final concentration of 2%wt in equal proportion,respectively,to prepare5 groups with a diameter of 1.5 cm round hydrogel.The toxicity of alginate saline gel extract to osteogenic precursor cells was detected using MTT cytotoxicity assay kit at day1,2,3,4,and 5.The swelling rate of hydrogels in each group in double steam water and phosphate buffer solution(PBS)was detected.Degradation rate of hydrogels soaked in lysozyme containing PBS was detected.The microstructures of hydrogels were observed by scanning electron microscopy(SEM).Strontium,zinc and magnesium ions in the gel were all released rapidly and in large quantities on the first day.With the extension of time,the release concentrations of strontium and zinc ions gradually stabilized,while magnesium ions were released rapidly,and there was almost no precipitation of magnesium ions on the fifth day.One day after inoculating mouse pre-osteogenesis precursor MC3T3-E1 cells on the hydrogel surface of each group,the cell adhesion morphology was observed by scanning electron microscopy.MC3T3-E1 cells were inoculated with hydrogel in each group,and cell proliferation was detected by CCK8 kit at day 1,3 and 5.MC3T3-E1 cells in each group were inoculated with hydrogel.At the12 h,24h and 48 h,the cells were stained with calcein-AM and propidium iodide(PI),respectively,to observe the adhesion and proliferation of the cells on the surface of the gel.MC3T3-E1 cells in each group were inoculated with hydrogel,and the expressions of adhesion and proliferation specific genes in each group were detected by real-time fluorescent quantitative PCR(RT-q PCR)RESULTS: The results of cytotoxicity test showed that the sodium alginate hydrogel extracts of each group had no cytotoxicity to osteogenic precursor cells.The introduction of multi-ion crosslinking agent could change the swelling rate of alginate brine gel in double steam water and PBS,and the differences were statistically significant(P<0.05).The results of hydrogel degradation showed that with the introduction of strontium,zinc and magnesium ions,the hydrogel degradation rate decreased gradually.With the introduction of multi-ions,the microstructure of the gel changed significantly,and the pore size of each group increased gradually,while the microstructure of group E was relatively uniform.ICP results show that each hydrogel leaching liquid of strontium ions in 1,3,5 days to release concentration decreases gradually,and released into the leach liquor of strontium ion content and the original is proportional to the introduction of the strontium ions in water gel content,C and E group of hydrogel leaching liquid of magnesium ions quick release in 1 day,3 days to release concentration fell sharply,to 5days,there was no precipitation of magnesium ions.The release of zinc ions in the hydrogel leaching solution of group D and E was faster on the first day,and the release concentration gradually decreased on the third and fifth day.The results of in vitro experiments showed that the introduction of multi-ion crosslinking agent could effectively improve the adhesion and proliferation of osteogenic precursor cells on the gel surface,and group E showed a more significant effect on promoting the adhesion and proliferation of cells.At the same time,compared with the other groups,adhesion and proliferation-related genes in group E were highly expressed with statistical significance(P<0.05).Conclusion: Multi-ion crosslinking can effectively improve the physical and chemical properties of alginate gel,and then improve the adhesion and proliferation of osteogenic precursor cells on the gel surface.The results showed that when the mass ratio of Ca,Sr,Mg and Zn was 2:2:2:0.5,the composite hydrogel had the most significant effect on promoting cell adhesion and proliferation.The results provide a theoretical basis for the application of alginate gel as a new injectable scaffold for bone tissue engineering.