Probiotic-loaded Calcium Alginate/fucoidan Hydrogels For Promoting Oral Ulcer Healing

Objective: To prepare a probiotic-loaded calcium alginate/fucoidan hydrogel with remarkable wet-tissue adhesion,suitable swelling and mechanical properties,sustained probiotic release,excellent storage durability and wound healing promotion for oral mucosal ulcer.Herein,the hydrogels were characterized,their mechanical properties and swelling properties were evaluated,and their antimicrobial properties,cell compatibility,and in vivo treatment of oral ulcers were evaluated.Methods: In this study,alginate was cross-linked with Ca2+ to form hydrogel scaffold.Probiotics and different concentrations of fucoidan(0 mg/m L,2.5 mg/m L,5 mg/m L,7.5 mg/m L)were added to hydrogel scaffold to prepare probiotics-loaded calcium alginate/fucoidan composite hydrogels.Scanning electron microscope(SEM)and Fourier transform infrared spectroscopy(FTIR)were used to analyze the composition and morphology of hydrogels.The water absorption and water content of hydrogels were measured respectively by swelling experiment and water content experiment,and the mechanical strength of hydrogels was measured by compression experiment.Colony forming unit assay(CFU assay)was used to observe the release of probiotics from the hydrogels and storage hydrogels for a month.The antimicrobial activity of the hydrogels against Staphylococcus aureus(S.aureus)and Candida.albicans(C.albicans)was determined by CFU assay as well.The biocompatibility of hydrogels was evaluated by CCK-8.The effect of hydrogels on cell migration was evaluated by cell scratch assay.Finally,the oral ulcer model of SD rats was established,and hydrogel was placed on the oral ulcer of SD rats to evaluate the therapeutic effect of hydrogel on oral ulcerations in vivo.Using Graph Pad PRISM 8.0 and Origin Pro 2021 software,the data were statistically analyzed using the statistical method of one-way analysis of variance(ANOVA).Results: The probiotics-loaded calcium alginate/fucoidan composite hydrogels were threedimensional networks of polymer chains capable of holding large amounts of water which can be made into different shapes on demand.Hydrogel stuck tightly to the deformed porcine skin and was not flushed out by running water.SEM showed that when the concentration of fucoidan was higher than 5 mg/m L,the pores became smaller and irregular,especially whenthe concentration of fucoidan increased to 7.5 mg/m L.FTIR showed characteristic peaks of alginate,fucoidan and probiotics.The swelling test and water content test showed that the hydrogel had a low swelling ratio and high water content.Compression experiments showed that when the concentration of fucoidan was lower than 5 mg/m L,the hydrogel had good mechanical strength,while when the concentration of fucoidan was 7.5 mg/m L,the mechanical strength of the hydrogel decreased.The probiotic release test showed that the probiotics could be released from the hydrogels,and the probiotics in the hydrogels could still survive after storage for a month.The hydrogels had a good antimicrobial activity against S.aureus and C.albicans.When the concentration of fucoidan was lower than 5 mg/m L,the antifungal activity of the hydrogel increased with the increase of fucoidan concentration,but the antifungal activity of the hydrogel decreased when the concentration of fucoidan reached 7.5 mg/m L.The CCK-8 experiment showed that the addition of probiotics and fucoidan had no significant effect on the biocompatibility of the L929 cells after 3 days of culture.There was no significant difference between the hydrogel group and the control group when the fucoidan content was lower than 2.5 mg/m L.However,hydrogels containing 5 and 7.5 mg/m L fucoidan inhibited cell migration.The results of animal experiments showed that the mucosa of the hydrogel group was intact,without congestion,with mild epithelial hyperplasia and regular collagen arrangement,and CD31 staining showed strong positive expression of CD31.Conclusion: The probiotic-loaded calcium alginate/fucoidan composite hydrogels for oral ulcer healing were successfully developed in this work.The composite hydrogels possessed proper swelling properties,sustained probiotic release,excellent storage durability,and effective antimicrobial ability.Importantly,the composite hydrogel expressed superior performance in promoting ulcer healing in vivo.Thus,the composite hydrogel has great potential as an oral mucosal patch for healing ulceration.