Study Of Ag-Zn-Ca-Si Mesoporous Nanoparticles On The Control Of Enterococcus Faecalis Infection In Root Canals

Objectives:The purpose of this study was:(1)to synthesis the mesoporous calcium-silicon nanoparticles loaded with different silver-zinc atomic ratios(Ag-Zn-MCSNs)by template method,detect the physico-chemical properties of the nanoparticles and screen the nanoparticles that could stably release the optimal atomic ratio of silver to zinc;(2)to evaluate the infiltration ability in the dentinal tubules of mesoporous calcium-silica nanoparticles loaded with the optimal silver-zinc atomic ratio,the in vitro antibacterial effect against planktonic Enterococcus faecalis(E.faecalis),the biocompatibility,changes in the microhardness of the dentin and the ability to inhibit the biofilm on dentin slices;(3)to evaluate the preventive effect of mesoporous calcium-silica nanoparticles with different atomic ratio of silver to zinc in the root canal of Beagles against E.faecalis infection.Methods:Part 1:Mesoporous calcium-silicon nanoparticles loaded with different silver-zinc atomic ratio(Ag-Zn-MSCNs)were successfully synthesized by template method.The morphological properties of nanoparticles were characterized by using field emission-scanning electron microscopy(FE-SEM)and transmission electron microscopy(TEM).The Brunauer–Emmett–Teller and the Barrett–Joyner–Halenda(BET&BJH)analyses were used to examine the specific surface area,pore volume,and pore size distribution by nitrogen adsorption–desorption isotherms.The elemental composition of silver,zinc,calcium and silicon elements of nanoparticles was analyzed by energy dispersive spectrometry(EDS).The cumulative release amount of silver,zinc,calcium and silicon elements of nanoparticles was measured in Tris-HCl by inductively coupled plasma-atomic emission spectrometry(ICP-AES).The p H values of mesoporous calcium-silicon nanoparticles loaded with different silver-zinc atomic ratio were measured in double-distilled water.Part 2:Dentin slices was immersed in nanoparticles suspension and activated by ultrasonic vibration to evaluate the infiltration ability into dentinal tubules.A colony-forming units(CFUs)counting method was applied for the assessment of antibacterial ability of mesoporous calcium-silicon nanoparticles loaded with different silver-zinc atomic ratio against planktonic E.faecalis.To evaluate the biocompatibility of the nanoparticles,a CCK-8 kit was used.After co-cultivating the dentin slices with E.faecalis for 30 days,the dentin slices were taken out and incubated in Ag-Zn-MCSNs nanoparticles paste for 7 days.After 7 days,FE-SEM was used to observe the effect of nanoparticles on the mature biofilm of E.faecalis on dentin slices.Similarly,dentin slices were incubated with the nanoparticles paste for 14 days,and the effect of the material on the hardness of the dentin was evaluated by comparing the changes in the microhardness of the dentin before and after the treatment.Part 3:Beagles'bilateral mandibular second and third premolars were well instrumented,rinsed and dried.After shaping and cleaning,suspensions of mesoporous calcium-silicon nanoparticles with different atomic ratios of silver to zinc,calcium hydroxide paste,chlorhexidine solution and normal saline was randomly placed in the root canal of the Beagles.The pulp chambers were sealed for one week after ultrasonic vibration of the root canal.One week later,the E.faecalis suspension was added to the root canal,and then the pulp chambers were sealed with glass ionomer.The apical X-ray film was taken every two weeks to observe the lesions of the periapical tissues in each group.Eight weeks later,the experimental tooth and its surrounding alveolar bone were dissected in squares,and tissue sections were made for staining.Hematoxylin-eosin staining(HE)was used to observe the pathological changes of the periapical tissue.Brown&Brenn staining method was used to observe the distribution of bacteria in the dentinal tubules.Tartrate-resistant acid phosphatase(TRAP)staining measured the number of osteoclasts in a certain area of the periapical tissue.Results:Part 1:Mesoporous calcium-silicon nanoparticles loaded with different silver-zinc atomic ratio(MCSNs,Zn₁₀-MCSNs,Ag_0.5-MCSNs,Ag_0.5Zn₃-MCSNs,Ag_0.5Zn₁₀-MCSNs)were synthesized successfully.FE-SEM images showed that all nanoparticles were mostly in spherical shape with a diameter of approximate 100 nm.TEM showed that they all had well-ordered nanopores and channel structures.BET&BJH results showed these nanoparticles had high specific surface area and the pore diameter was mostly in the range of 2–4 nm.The EDS analysis spectrum and ICP-AES results confirmed the presence of Ag,Zn,Ca and Si elements in nanoparticles.Mesoporous calcium-silicon nanoparticles that stably release the optimal silver-zinc ratio(1:12)were successfully screened(Ag_0.5Zn₁₀-MCSNs).The p H of nanoparticles loaded with different silver-zinc atomic ratio were all alkaline.Part 2:Images acquired by FE-SEM revealed that the nanoparticles infiltrated into the dentinal tubules after passive ultrasonic treatment.All the silver-loaded nanoparticles showed strong antibacterial effects,among which Ag_0.5Zn₁₀-MCSNs was the strongest.Nanoparticles loaded with different silver-zinc atomic ratio were all had good biocompatibility.Observed by electron microscopy,compared with the MCSNs group,all the silver-zinc-loaded nanoparticles had a strong inhibitory effect against E.faecalis biofilm on the surface of the dentin slices.Besides,dentin slices medicated with the nanoparticles displayed no decrease in microhardness.Part 3:The radiographic examination of X-ray revealed that the mean radiolucent zone of the Ag_0.5Zn₁₀-MCSNs group and the CHX group was significant less than the blank control group.Evaluation by HE staining showed that Ag_0.5Zn₃-MCSNs and Ag_0.5Zn₁₀-MCSNs both had a tendency to reduce the inflammatory infiltration of the periapical tissue,and the ability to prevent E.faecalis infection in the root canal of Beagles was close to 2%CHX.Brown&Brenn staining and TRAP staining revealed that less bacteria could be identified in the dentinal tubules of the Ag_0.5Zn₁₀-MCSNs and the 2%CHX groups,but the number of periapical osteoclasts were not reduced.Conclusions:(1)In this study,mesoporous calcium-silicon nanoparticles loaded with the optimal silver-zinc atomic ratio were successfully synthesized by template method(Ag_0.5Zn₁₀-MCSNs).Those nanoparticles were mostly in spherical shape with a diameter of approximate 100 nm and had a high specific surface area.The nanoparticles could not only stably release silver and zinc elements,but also the released silver-zinc atom ratio could reach the optimal silver-zinc atom ratio(1:12)against E.faecalis.(2)Ag_0.5Zn₁₀-MCSNs showed excellent antibacterial activity against planktonic E.faecalis in vitro,good biocompatibility,and could penetrate into dentin tubules.It had a good inhibitory effect on the mature biofilm of E.faecalis on dentin slices.(3)The Ag-Zn-Ca-Si nanometer mesoporous particles had a good preventive effect on E.faecalis infection in the root canal of Beagles.Among them,Ag_0.5Zn₁₀-MCSNs showed better prevention effect than calcium hydroxide,and closed to 2%CHX,which could be used as a disinfectant in the root canal.