Nanocomplexes Of Carboxymethyl Chitosan/Amorphous Calcium Phosphate Reduce Oral Bacteria Adherence And Biofilm Formation On Human Enamel Surface

ObjectivesNanocomplexes of carboxymethyl chitosan/amorphous calcium phosphate(CMC/ACP)have a biomimetic remineralisation function.However,its effect on dental cariogenic plaque formation remains unclear.In this study,an in vitro model of oral cariogenic bacterial biofilm on human enamel surface was established under laboratory conditions to further investigate the effect of CMC/ACP on oral bacteria growth,adherence,co-aggregation and biofilm formation on the enamel surface as well as the underlying mechanism.To provide experimental basis and reference for develop oral health care products with dual functions of promoting remineralization and inhibiting cariogenic plaque formation.Methods1.Preparation of enamel blocks and hydroxyapatite discsCollecting freshly extracted human third molars according to the inclusion criteria,enamel blocks(~4×3×2 mm3 in size)removed by sawing from the buccal or lingual surfaces of the third molars and polished with 400-to 2000-grit SiC paper.Hydroxyapatite discs(5.0 mm in diameter and 2.0 mm in thickness)were used as a substratum for confocal scanning laser microscopy analysis.All enamel blocks and hydroxyapatite discs were autoclaved and stored in sterile tubes.The study was approved by the ethics committee(TMUSHhMEC2017090).2.Preparation of salivary pellicleNon-stimulated whole saliva samples were collected from 10 healthy adult volunteers as previously described,and mixed and immediately centrifuged at 12,000×g for 20 min at 4°C.After sterile filtration,enamel blocks were treated with saliva for 30 min at 37°C to obtain salivary pellicle.3.Preparation and characterisation of CMC/ACP nanocomplexesA 1%(m/v)CMC/ACP mineral solution with biomimetic remineralisation function was prepared by Polyelectrolyte-mediated synthesis.The size and morphology of nanocomplexes formed by CMC/ACP nanoparticles were characterised by transmission electron microscopy(TEM)and selected area electron diffraction(SAED)at 110 Kv.4.Antibacterial test of CMC/ACP nanocomplexes1%CMC/ACP,CMC or deionized water were mixed with S.mutans,S.gordonii,and F.nucleatum and incubated for 24 h at 37°C under anaerobic conditions.The antibacterial activity of CMC/ACP was evaluated with the plate count method.5.Effect of CMC/ACP nanocomplexes on bacterial adhesionA 200-?l volume of streptococcal suspension was combined with an equal volume of CMC/ACP,CMC,or deionised water,and the mixture was added to the enamel blocks in a 48-well plate,followed by incubation at 37°C for 1 or 5 h.To quantify the bacteria adhering to the enamel blocks,samples were examined by scanning electron microscopy(SEM).Adherent cells in nine randomly selected3000-fold magnification fields were counted,and the number of bacteria in 1 mm~2was calculated.6.Effect of CMC/ACP nanocomplexes on biofilm formationA 200-?l volume of suspension was added to a 48-well plate with saliva-coated enamel blocks along with an equal volume of CMC/ACP,CMC,or deionised water,followed by incubation at 37°C for 24 h under anaerobic conditions.crystal violet was used to determine the amount of the formed biofilms.7.Effect of CMC/ACP nanocomplexes on bacteria co-aggregationA 24-hour biofilm of S.mutans or S.gordonii labeled with hexidium iodide(HI)was first formed on the surface of hydroxyapatite discs.Then,CFSE-labelled F.nucleatum suspension(OD600=1)was added to the saliva-coated hydroxyapatite disc along with an equal volume of CMC/ACP,CMC,or deionised water.The samples were then incubated for 24 h at 37°C under anaerobic conditions in the dark.The attachment of F.nucleatum to streptococcal biofilms was visualised by confocal laser scanning microscopy.For each sample,images were obtained from five randomly selected areas at 400-fold magnification and were analysed with image analysis software to determine the ratio of green to red fluorescence(GR ratio).8.Bacteria cell surface charge assayS.mutans or S.gordonii suspension was mixed with an equal volume of CMC/ACP,CMC,or deionised water and cultured for 1 h at 37°C under anaerobic conditions.The cytochrome c binding assay was used to estimate bacterial surface charge.The average zeta potential of CMC,CMC/ACP,untreated S.mutans and the mixture that mixed S.mutans suspension with CMC or CMC/ACP for 1 hour was measured using a Zetasizer Nano ZS 90 device at room temperature.9.Statistical analysisStatistical analysis was performed with SPSS v.22.0 software(IBM,Armonk,NY,USA).Differences between groups were evaluated by one-way analysis of variance with the Tukey-Kramer multiple comparisons post-hoc test and with the two-tailed unpaired t test.P<0.05 was considered statistically significant.Results1.CMC/ACP nanoparticles mainly constituted an amorphous phaseTEM showed the particles of CMC/ACP had a rough surface and had a diameter<100 nm.SAED analysis did not reveal an obvious dot or ring pattern characteristic of crystal structures,indicating that the CMC/ACP nanoparticles mainly constituted an amorphous phase.2.CMC/ACP has no bacteriostatic activityAfter culturing for 24 h,there were no significant differences among groups in terms of the number of live colonies on the agar plates.These results suggest that CMC and CMC/ACP do not interfere with the growth and viability of S.mutans,S.gordonii,and F.nucleatum at a concentration of 1%.3.CMC/ACP nanocomplexes significantly inhibited bacterial initial adhesionThe adherence of S.mutans and S.gordonii to saliva-coated enamel blocks was inhibited in the presence of 1%CMC/ACP by 89.7%and 86.1%(P<0.01),respectively,at 1 h and by 80.8%and 82.1%(P<0.01),respectively,at 5 h.There was no significant difference between CMC/ACP and CMC groups.4.CMC/ACP nanocomplexes significantly inhibited Biofilm formationBiofilm formation was decreased by 45.3%(S.mutans)and 44.0%(S.gordonii)after incubation for 24 h with 1%CMC/ACP(P<0.01).There was no significant difference between CMC/ACP and CMC groups.5.CMC/ACP nanocomplexes significantly inhibited bacteria co-aggregation1%CMC/ACP decreased the number of F.nucleatum cells bound to the pre-formed S.mutans or S.gordonii biofilm by 74.6%and 74.9%,respectively,(P<0.01).There was no significant difference between CMC/ACP and CMC groups in the above studies.6.CMC/ACP nanocomplexes significantly alters the surface charge of bacteria1%CMC/ACP reduced cytochrome c binding to S.mutans and S.gordonii cells by 12.6%and 11.9%,respectively(P<0.01),indicating that their surface charge was altered.The average zeta potential of CMC,CMC/ACP,and untreated S.mutans was-41.6,-28.1,and-38.2 mV,respectively;however,1 h after adding CMC or CMC/ACP,the zeta potential of S.mutans decreased to-31.3 and-22.1 mV,respectively,which was significantly lower than the value of untreated S.mutans suspension(P<0.05).Conclusions1.CMC can effectively stabilize ACP to form stable amorphous CMC/ACP nanocomposites.2.CMC/ACP do not interfere with the growth of S.mutans,S.gordonii,and F.nucleatum.3.CMC/ACP significantly inhibits the early adhesion of S.mutans and S.gordonii on enamel surface.4.CMC/ACP significantly inhibits the 24h biofilm formation of S.mutans and S.gordonii on enamel surface.5.CMC/ACP significantly inhibits the co-aggregation of F.nucleatum to S.mutans or S.gordonii.6.CMC/ACP significantly alters the surface charge of S.mutans.