| Tooth defect is the most common disease in oral clinic,and caries is the main cause of it.Salivary proteins adhere to the tooth surface and promote the adhesion of bacteria,forming dental plaque.Cariogenic bacteria,represented by Streptococcus mutans(S.mutans),accumulate and colonize on the tooth surface,produce acid,and eventually cause hard tissue defects.How to identify caries at the early stage,intervene and interrupt its further development,and restore the defective tooth tissue,will provide a new idea for the prevention and treatment of caries.In this study,inspired by natural dental plaque,a bacteria-responsive versatile drug delivery system was constructed to explore the effect and mechanism of preventing caries and promoting enamel restoration,so as to provide new ideas for the research and development of oral functional materials.Part Ⅰ Construction and characterization of the dental plaque-inspired versatile nanosystemObjective: The main cause of tooth defect is demineralization of hard tissue caused by acid production of cariogenic bacteria.Inspired by natural dental plaque,this study aimed to construct a bacteria-responsive intelligent drug delivery system,which could achieve antibacterial interface and tooth defect restoration at the same time.Methods: Firstly,the block copolymer of 3-maleimidopropionic acid-poly(ethylene glycol)-block-poly(L-lysine)/phyenylboronic acid(MAL-PEG-b-PLL/PBA)was synthesized and self-assembled into micellar nanoparticles with tannic acid and sodium fluoride(PMs@Na F).The catechol group of tannic acid formed a p Hsensitive cleavage borate bond with phenylboronic acid,and the electrostatic interaction between tannic acid and PLL stabilized the structure of the micelles.In addition,the key adhesion peptide of the natural salivary protein was modified with cysteine(SAP),and was then conjugated to the above nanoparticles to construct the bacteria-responsive versatile nanosystem(PMs@Na F-SAP).Transmission electron microscopy(TEM),fourier transform infrared spectroscopy(FTIR)and nuclear magnetic resonance(NMR)were used to confirm the successful construction of the nanosystem.Dynamic light scattering(DLS),lyophilization and gel permeation chromatography(GPC)were used to characterize the particle size,concentration,molecular weight,polydispersity and zeta potential of the nanosystem.High performance liquid chromatography(HPLC)and ion exchange chromatography(IEC)were used to explore the drugloading capacity of the nanosystem and the drug release at different p H values.The adhesion of the nanosystem to the tooth surface was observed by confocal laser scanning microscope(CLSM),and the effect of saliva buffering on the adhesion was also evaluated.The biocompatibility of PMs@Na F-SAP was evaluated by cell counting kit-8(CCK-8)assay and cytoskeleton staining assay.Results: After concentration and proportion optimization,the micellar nanoparticle was successfully constructed and conjugated with the peptide.The characterization results showed that PMs@Na F-SAP had a spherical core-shell structure,which could simultaneously load tannic acid and sodium fluoride.Rapid release at acidic p H enabled more accurate targeted drug delivery.At the same time,PMs@Na FSAP could effectively adhere to the enamel surface and resist the buffering effect of saliva.In addition,PMs@Na F-SAP had good biocompatibility.Conclusion: The bacteria-responsive versatile nanosystem was successfully constructed,which could specifically adhere to the tooth surface and withstand the buffering effect of saliva.It could specifically identify the cariogenic microenvironment in the early stage and release drugs on demand.Meanwhile,it had good biocompatibility,which laid a foundation for subsequent biological activities.Part Ⅱ Antibacterial properties of the versatile nanosystem in vitroObjective: Oral biofilm plays a key role in bacterial resistance and caries progression.Current antimicrobial strategies have many limitations when applied to oral biofilms.This study aimed to investigate the antibacterial ability of the nanosystem in vitro from two aspects: antibacterial adhesion property and anti-cariogenic biofilm ability.Methods: Firstly,OD600 was used to detect the cell density of S.mutans in order to evaluate the antibacterial activity and the ability of inhibiting biofilm at different p H values.Surface contact angle tests were performed on PMs@Na F-SAP pretreated or untreated hydroxyapatite tablets to assess the effect of the material on hydrophilic or hydrophobic changes and S.mutans adhesion.For further evaluation,the antibacterial adhesion and anti-cariogenic biofilm capacity of the nanosystem were investigated.In the experiment of antibacterial adhesion,enamel samples were prepared and randomly divided into 2 groups.One group was pretreated with artificial saliva,and the other group was not.The enamel surfaces were treated with dd H2 O,PMs@Na F,SAP or PMs@Na F-SAP,respectively,and S.mutans was then cultured on the samples.The biofilm was observed with live/dead bacterial staining and CLSM,and fluorescence intensity was analyzed.Colony forming units(CFU)counting was conducted by dilution plate method.Biofilm volumes were assessed using crystal violet staining.For the study of anti-cariogenic biofilm ability,S.mutans biofilm was precultured on enamel samples,and then treated with dd H2 O,PMs@Na F,SAP or PMs@Na F-SAP,respectively.Live/dead bacterial staining,CLSM,fluorescence intensity analysis,CFU counting and crystal violet staining were conducted for qualitative and quantitative analysis of biofilms.Finally,16 Sr RNA PCR was used to explore the antibacterial mechanism of PMs@Na F-SAP against S.mutans.Results: In vitro antibacterial results showed that PMs@Na F-SAP displayed higher antibacterial and antibiofilm activity against S.mutans at acidic p H.On the one hand,PMs@Na F-SAP could effectively inhibit the adhesion,aggregation and biofilm formation of cariogenic bacteria on the tooth surface,providing a strong bacterial defense barrier.On the other hand,it showed targeting specificity to biofilms,and could effectively penetrate and kill the bacteria in cariogenic biofilms.In addition,the expression of biofilm formation and quorum-sensing(QS)system-related genes of S.mutans were significantly down-regulated under PMs@Na F-SAP,which was consistent with the above epigenetic studies.Conclusion: The versatile nanosystem constructed in this study could release antibacterial agents on demand under acidic p H in the early stage of caries,and achieve effective biofilm control.On the one hand,it could provide efficient anti-bacterial adhesion to establish bacterial defense,and on the other hand,it could achieve efficient biofilm destruction.Part Ⅲ Performance of versatile nanosystem in restoring early enamel cariesObjective: In order to innovate the existing post-defect restoration strategies for tooth defects caused by caries,this study applied PMs@Na F-SAP as a pretreatment agent on the enamel surface to investigate its ability to inhibit enamel demineralization and promote remineralization,and to explore its restoration effect on the morphology,structure and mechanical properties of tooth defects.Methods: In this study,the effects of the nanosystem were investigated in two aspects: inhibiting enamel demineralization and promoting remineralization restoration.In the experiment of demineralization inhibition,the enamel samples were etched and rinsed,and randomly divided into 4 groups,which were pretreated with dd H2 O,PMs@Na F,SAP or PMs@Na F-SAP,respectively.After rinsing,the samples were immersed in demineralized solution and incubated at 37℃ for 5 h,rinsed by ultrasound and dried.Enamel demineralization was observed by field emission scanning electron microscope(FESEM).Inductively coupled plasmaatomic emission spectroscopy(ICP-AES)was used to detect the loss of Ca and P after demineralization.Diffraction of x-rays(XRD)and energy dispersive spectroscopy(EDS)were used to detect the crystal structure and basic composition of enamel surface.The loss of enamel surface hardness was measured by Vickers microhardness test.The loss of mechanical properties and surface roughness changes were accessed by atomic force microscope(AFM).In the experiment of promoting remineralization,enamel samples were also etched and rinsed,and randomly divided into 4 groups,which were pretreated with dd H2 O,PMs@Na F,SAP or PMs@Na F-SAP,respectively.After rinsing,the samples were immersed in remineralized solution at 37℃ and incubated for 24 h,rinsed by ultrasound and dried.FESEM,ICP-AES,XRD,EDS,Vickers microhardness test and AFM were used to evaluate the restoration of the morphology,structure and mechanical properties of the enamel after reminerization.Results: The results of defects restoration study showed that enamel pretreated with PMs@Na F-SAP had lower demineralization degree and higher structural integrity.The loss of Ca and P was significantly reduced,showing less enamel structural damage.At the same time,PMs@Na F-SAP promoted the enamel remineralization and the formation of oriented and uniform hydroxyapatite crystals,which significantly improved the microhardness of enamel and orderly regulated crystal growth.The mineral phase analysis confirmed that the composition of the mineral deposits after PMs@NaF-SAP treatment was mainly hydroxyapatite crystals.Conclusion: The versatile nanosystem constructed in this study could effectively inhibit enamel demineralization and promote the restoration of tooth defects,so as to realize the early identification,intervention and interruption of the further development of caries,and restore the demineralized tooth tissue at the same time.It had great potential to be applied to the clinical practice of tooth defect restoration.Part Ⅳ In vivo study of the nanosystem for caries prevention and defect restorationObjective: The aim of this study was to explore the effect of the nanosystem in preventing caries and restoring defects by constructing a caries model in rats,so as to provide theoretical basis for the clinical transformation of oral functional materials.Methods: In this study,a caries model was pre-established in rodents.All rats were orally inoculated with S.mutans at the beginning of the experiment.The rats were randomly divided into 5 groups(6 rats in each group),and the teeth of rats were topically treated dd H2 O,PMs@Na F,SAP,PMs@Na F-SAP or chlorhexidine at regular intervals every day for 4 weeks.The body condition and weight of the rats were recorded daily.At the end of the experiment,oral microbiota samples were collected with sterile swabs,and rats were sacrificed.The jaw samples were stained with murexide,and prepared for caries scoring according to Keyes’ system.The incidence and severity of smooth-surface and sulcal-surface lesions were observed by stereomicroscopy.The mineral density of enamel were evaluated by microcomputed tomography CT(micro-CT),and the demineralization sites were observed by sagittal section images.Oral microbiota samples were detected by 16 S r RNA gene amplicon sequencing.The gingival and palatal tissues were processed for histopathological analysis.Results: In vivo study showed that the application of PMs@Na F-SAP significantly reduced the incidence and severity of smooth-surface and sulcal-surface lesions.The enamel was more intact,the mineral density was higher,and fewer demineralization sites were displayed.Further in vivo biocompatibility studies showed that PMs@Na F-SAP had no obvious adverse effects on the surrounding soft tissues,the composition and diversity of oral microorganisms.Conclusion: The versatile nanosystem constructed in this study could kill the cariogenic bacteria during the onset of caries and promote the restoration of tooth defects.Application to rodent caries models facilitated the development of clinically effective mouthwash,spray,paint or related anti-caries products.The nanosystem is expected to constitute major progress for the clinical transformation of intelligent drug-controlled release system in diseases prevention and treatment. |
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