Construction Of Injectable Hydrogel Based On ROS And Fluoride-releasing For Caries Management

Objectives: This study aimed to construct a bifunctional injectable alginate hydrogel loaded with Cu@Mn nanozymes and sodium fluoride to control caries pathogenic bacteria and promote enamel remineralization,which will provide a new strategy for caries management.Methods:(1)Preparation of Cu@Mn nanozymes and bifunctional injectable hydrogels: Cu@Mn nanozymes were prepared by gas-assisted soft template solvothermal preparation and high-temperature calcination.The morphologies,structures,chemical composition,element valence states,element distribution,particle size distribution,specific surface area and so on were characterized.Cu@Mn nanozymes and sodium fluoride(F)were loaded to obtain the bifunctional injectable SA-Cu@Mn-F hydrogels,which was composed of two pre-gels A and B.Component A consisted of a homogeneous colloid mixture of calcium carbonate,SA,Cu@Mn nanozymes,and F.Component B consisted of a homogeneous colloid mixture of SA and D-(+)-glucose-δ-lactone.The micromorphologies,swelling properties and compressive strength of SA-Cu@Mn-F hydrogels were evaluated by FE-SEM,swelling analysis and compression test,respectively.(2)Assessment of catalytic activity and photothermal performance of the injectable SACu@Mn-F hydrogels:3,3 ’,5,5’-tetramethylbenzidine(TMB)test and methyl violet(MV)test were used to characterize the catalytic activity and the ability of catalytic production of ROS(n=3).Irradiating near-infrared light(NIR)could improve local temperature to promote catalytic activity.Under the irradiation of NIR(wavelength: 808 nm,power: 1.0 W/cm2),the temperature value of the detection area would be recorded every 30 s to evaluate the photothermal performance(n=3).The photothermal stability(n=3)of the injectable SA-Cu@Mn-F hydrogels was also investigated after five laser ON/OFF cycles.(3)In vitro assessment of antibacterial property,mineralization,and cytotoxicity: After 2h of co-incubation of SA-Cu@Mn-F hydrogels and planktonic S.mutans,the bacterial colony counting method and live/dead bacterial staining test were used to evaluate the inhibitory effect on bacteria,and the distribution of ROS was detected by DCFH-DA fluorescent probe.Subsequently,the bacterial colony counting method and live/dead bacterial staining were also adopted to estimate the disruptive and inhibitive effects of SA-Cu@Mn-F hydrogels on dental plaque biofilms.Covered with SA-Cu@Mn-F hydrogels,enamel slices were immersed in artificial saliva for 3 d.The morphologies and composition of the mineralized layer were characterized by FE-SEM and XRD.After being treated with 6 wt % citric acid solution for 1 min,the content of calcium and phosphorus ions in the treated solution and the morphologies were evaluated by ICP-OES and FE-SEM to assess the acid resistance of the mineralized layer.Then,SA-Cu@Mn-F hydrogels were co-cultured with human gingival fibroblasts(HGF)for 1,4,and 7 d.The cell viability and cell morphologies were detected by CCK-8 assays and fluorescent staining to evaluate the biocompatibility in vitro.Incubated with SD rat blood for 30 min,the hemolysis rate was calculated to evaluate the blood compatibility of SA-Cu@Mn-F hydrogels.(4)In vivo assessment of the effect of the injectable SA-Cu@Mn-F hydrogels on caries management: Caries models were constructed,and the maxillary molars of rats were treated at corresponding time points.The bacterial colony counting method was used to record the changes in bacteria,and Keyes’ scoring was used to assess the degree of dental caries.Finally,in vivo biocompatibility of SA-Cu@Mn-F hydrogels for pre-clinical application was evaluated via histological analysis of the major organs(heart,liver,spleen,lung,kidney)and oral mucosa(tongue,palate,buccal mucosa).Results: Cu@Mn Nanozymes and dual-function injectable SA-Cu@Mn-F hydrogels were successfully prepared.SA-Cu@Mn-F hydrogels could be available and the hydrogels can convert into a solid gel within 3 minutes at 37 ℃.SA-Cu@Mn-F hydrogels had OXD-,POD-like catalytic activity,and excellent photothermal performance.Planktonic S.mutans were inhibited and dental plaque biofilms were destroyed via high temperature generated by photothermal conversion and ROS generated by catalysis under NIR.In the demineralized enamel model,SA-Cu@Mn-F hydrogels induced enamel remineralization to form a dense and distributed mineralized layer with a thickness of 4.3μm.XRD results showed that the mineralized layer is mainly composed of hydroxyapatite and fluorapatite,and is resistant to acid erosion and demineralization.When co-cultured with HGF or the blood of SD rats,SA-Cu@Mn-F hydrogels did not show an obvious effect on cell viability and cause hemolysis.In the animal caries model,SA-Cu@Mn-F hydrogels combined with NIR can effectively inhibit the growth of bacteria,and decrease the incidence and severity of dental caries.Little damage to the main organs and oral mucosa showed good systemic and local biocompatibility.Conclusions: In this study,an injectable anti-caries hydrogel loaded with Cu@Mn nanozymes and sodium fluoride was developed.The hydrogel would provide a new idea for the prevention and treatment of dental caries,and has potential clinical application prospects.