Antibacterial Application Of Phase-transited Lysozyme In Root Canal Therapy:A Preliminary Study

Objective:Phase-transited lysozyme(PTL)is a kind of amyloid-like protein obtained through the amyloid transformation of lysozyme by reducing agent.This new material possesses antibacterial,adhesion ability,chemical stability,good biocompatibility and nano-size dimension,with a rich variety of chemical groups on the surface,which create the conditions for surface chemical modification such as free radical active grafting and drug carrying.PTL has good antibacterial effect against planktonic bacteria such as Escherichia coli,Staphylococcus aureus and Candida albicans.At present,it had been applied to implant surface modification and dentin tubule closure.However,there has been no research about the application of PTL in root canal antibacterial therapy.Previous studies mainly focused on PTL nano-films formed on gas-liquid surface.There are few studies on the adhesion ability and antibacterial property of PTL products particle.Thus,in this study,we will synthesize amyloid-like protein PTL and conduct a preliminary study of the adhesion effect on dentin surface and antibacterial effect of PTL.Thus,it may provides a basis for preparation of PTL-based root canal antibacterial agents.Methods:Target solution was prepared by equal volume lysozyme and reductant tris(2-carbonyl ethyl)phosphate(tris(2-carboxyethyl)phosphine,TCEP).A preliminary determination of the particle size in the target solution was accomplished by the Tyndall effect experiment.The coverslips treated with the target solution were stained with Congo red and thioflavin T(Th T)respectively and then were observed under Optical Microscope(OM)and Confocal Laser Scanning Microscope(CLSM)to characterize whether lysozyme underwent amyloid transformation.The particle size and surface morphology of PTL product was further observed and identified by Transmission Electron Microscope(TEM)and Atomic Force Microscopy(AFM).26 Intact third molars collected from the Department of Maxillofacial Surgery,Dental Hospital of Tianjin Medical University were prepared into 4mm×3mm×2mm dentin slices and then polished by silicon carbide sandpaper.After smear layerremoval,sterilization and autoclaving,dentin slices were randomly divided into control group and experimental group.The dental slices in 2 groups were treated with HEPES and PTL solution respectively for 2 hours and rinsed with 3ml aseptic PBS solution for 3 times.The morphology and adhesion ability of PTL on dentin surface was evaluated by Field Emission Scanning Electron Microscope(FESEM).E.facealis(ATCC 29212)freeze-dried strain was cultured in broth medium(Brain Heart Infusion,BHI)to obtain E.facealis bacteria solution.The planktonic E.faecalis solution was treated with equal volume HEPES and PTL solution respectively for 2hours and the effect of PTL solution on planktonic E.faecalis was evaluated by crystal violet staining.The dentin slice biofilm samples which were obtained by E.faecalis cultivation on the dentin surface for 21 days were treated with HEPES and PTL solution for 2 hours.The effect of PTL solution on the biofilm of E.faecalis was observed by FESEM.E.faecalis were cultured in cell culture plates for 21 days to obtain biofilm.Control group and experimental group were treated with equal volume HEPES and PTL solution for 6 hours respectively and stained by Sytox Green to evaluate the effect of PTL solution on the E.faecalis biofilm.Results:The Tyndall Effect experiment preliminarily proved that the particles in supernate of the mixed solution of lysozyme and TCEP were nanoscale.The Congo red staining results of experimental group showed that light brick red coloring film and large particles scattered or agglomerated into clusters on the surface of the coverslips with deep brick red coloring.The results of Th T staining showed that experimental group appeared as film with light green fluorescence and scattered clusters with strong green fluorescence.The results of TEM and AFM proved that the spherical-like nanoscle particles of PTL aggregated each other into chains and networks,and particles could stably adhere to the surface of the silicon wafer.The FESEM results of dentin surface PTL coating showed that PTL particles adhered to both hydroxyapatite and fibrous network stably and part of particles entered into dentine tubules.The obvious agglutination effect of PTL solution on planktonic standard strains of E.faecalis was determined by crystal violet staining.FESEM figures of the E.faecalis biofilm on the dentin surface treated with PTLsolution showed large area of grape bead-like bacteria agglutination in the E.faecalis biofilm and a large number of bacteria adhered to the surface of PTL particles.Nucleic acid staining demonstrated that PTL has a more obvious destructive effect on bacterial membrane of E.faecalis biofilm than that of the control group.Statistical analysis of the dead bacteria number per unit area showed that the number of bacterial plasma membrane destruction per unit area in the PTL group was significantly more than that in the control group and showed significant difference(P<0.05).Conclusion:PTL with identification amyloid structure were successfully synthesized in this study.PTL particles were nanoscale and aggregated each other into chains and networks.It could stably adhere to the surface of silicon wafer and dentin.PTL had obvious bacterial agglutination effect on planktonic E.faecalis and biofilm.Moreover,it exerted antimicrobial effects by destroying bacterial membrane.PTL had the advantages of low cost,environmental protection,good biocompatibility,superior adhesion and antimicrobial properties.The new long-acting root canal irrigation based on PTL was expected to be prepared by further study.