Antibacterial Properties And Bond Strength Of Experimental Nano Silver-containing Orthodontic Cements

Enamel demineralization is a publicly recognized complication of orthodontic treatment with a fixed appliance. The placement of fixed orthodontic appliances creates a favorable environment for the proliferation of caries-associated microorganisms, with the higher risk of caries prevalence. Clinical observations have indicated that the common sites for demineralization are at the areas adjacent to the orthodontic bonding agents. For the purpose of decreasing the demineralization rate during orthodontic treatment, various antimicrobial agents have been incorporated into orthodontic adhesives. Fluoride and chlorhexidine are the most common preventive additives for orthodontic use. However, the effect of clinical application indicated that different degrees of caries white spot still could be seen in a lot of orthodontic patients. Despite some advances in orthodontic bonding materials and techniques in recent years, the enamel demineralization and dental caries around bracket remain unresolved. The silver ions exhibited high bactericidal activity against oral streptococci and silver bonding agent has a strong inhibitory effect on the bacterial biofilm formation and the growth of oral streptococcus mutans. To keep long time antibacterial activity without affecting the bond strength, silver nanoparticles were incorporated into resin-modified glass ionomer cement (RMGIC, GC Fuji ORTHO LC; GC Corporation, Tokyo, Japan) in this study to acquire experimental nano-silver containing orthodontic cements. A better antibacterial ability of nano silver-containing cement hoped to be obtained on the premise of meeting the clinical requirements by comparison of bonding strength, antibacterial ability, and the prevention of enamel demineralization by in vitro and animal caries disease model. We hope that this study will lay a foundation for further application research.Part one in vitro experimental studyExperiment one: composite nano-silver orthodontic adhesive sample preparationIn this experiment, nano inorganic silver antibacterial powder was incorporated into resin-modified glass ionomer cement through two kinds of physical mixing method of mechanical milling and anhydrous ethanol suspension, then nano silver-containing cements were obtained. The dispersion of nano silver in adhesive powder was observed by transmission electron microscope. The results showed that the powder particles of mechanical milling seemed to be smaller than the method of anhydrous ethanol suspension and about10-20nm sized nano-silver particles dispersed evenly in the powders. However, nano-silver particles present a reunion phenomenon in the samples obtained by anhydrous ethanol suspension method.Experiment two:agar diffusion test (ADT)The ADT was designed to test whether the adhesives containing an antimicrobial agent could permeate through agar to produce an inhibition zone and the internal antibacterial ingredients can free to the materials surface to play a continuous antimicrobial activity. After48h incubation, about13mm bacterial inhibition zones were observed around NSCs and ORTHO LC fresh specimens, with no significant differences between them. However, no inhibition zones were observed around any of the specimens aged for2days,1week, and2weeks, which should be interpreted as the fast consumption of fluoride and silver ion existing in the surface of the material. Another explanation for this result may be the silver ion non-releasing property from reinforced glass ionomer cement or the amount of silver ion from NSCs permeating into agar is not enough to produce bacterial inhibition zone.Experiment three:direct contact and aging testsThis experiment aimed at evaluating the antibacterial ability of fresh and aging sample material by measuring the turbidness change of bacterial growth after direct contact between test material and streptococcus mutans in96-well microtiter plates. The experimental results show that all fresh composite nano-silver materials show strong antimicrobial properties, however, with the extension of aging time, gradually lost all their antimicrobial properties until8weeks of aging. This in vitro evaluation method, to some extent, reflected that composite nano-silver antibacterial agent showed a better and stronger antibacterial ability, however could not reflect the real mouth situation and make relative real evaluation to its long-term antibacterial ability for the reason of a short observation time.Experiment four:the in vitro studies of bond strength both resin-modified glass ionomer cement and composite nano-silver antibacterial agentFreshly extracted upper premolar teeth were used for this study. This experiment aimed at exploring the best conditions for resin-modified glass ionomer cement clinical application and comparing the bonding strength between it and composite nano-silver antibacterial agent. The experimental results show that wet tooth surface does not favor the bonding of resin-modified glass ionomer cement, but the surface after dealing with the acid corrosion will significantly enhanced its bonding strength. The findings from this study indicated that the brackets bonded with all the tested material specimens had reached the ideal bond strength range of6to8Mpa, which is adequate for clinical use. However, a gradually decreasing trend of bond strength was seen with the increasing incorporation of nano silver base inorganic antibacterial powder into the glass ionomer cement.Part two animal experimentExperiment one:rat caries disease model establishmentThis experiment is to use the rat caries disease model to simulate the oral environment and then to use this environment to indirectly reflect antibacterial ability of the samples within the rat mouth. As a result, we successfully build the caries disease model in rats by implanting of streptococcus mutans to the rat mouths treated with antibiotics and feeding the caries feed Keyes2000to them. It will be a very meaningful try for the new research method.Experiment two:the implantation of material samplesIn this experiment, the preparation of sample teeth had been achieved by bonding braces base plates with bonding material on the surface of the buccal half crowns of the upper first premolar, and then fixed these samples under the rats’front teeth lingual root to ensure the material sample to save for a long time in the rats’mouth. Through long-term observation, we found that material samples’position did not affect rats’normal eating and there was almost no falls off phenomenon.Experiment three: the scanning electron microscope observation of bacteria growth on the surface of bonding materials in rats’mouth25samples would be dried and sprayed gold after taking out from rats’mouth under local anesthesia during one and three month of implantation respectively. Then, the scanning electron microscope observation would be conducted. As a result, in the group of Transbond XT, the growth of streptococcus mutans was very active, layer upon layer. Streptococcus mutans scattered or a small amount of them gathered into a cluster on the one month GC Fuji ORTHO LC surface and the number significantly increased and arranged in chains on the three month material surface. In groups of NSC2, NSC3and NSC4, the number of streptococcus mutans presented the downward trend and tended to disperse individually with the increase of nano silver content. The results showed that the incorporation of silver nanoparticles enhanced GC Fuji ORTHO LC the effect on adhesion restrain and kill to streptococcus mutans.Experiment four: the micro-CT observation of enamel demineralization around the bonding material in rats’mouth15samples was taken out from the rats whose molar decayed apparently under local anesthesia after three month of implantation, and removed the adhesives and the base plate from the teeth surface after cleaning, then micro-CT scanning was taken by10micrometer resolution. Enamel demineralization pits would be examined surrounding the areas of original adhesives after the3d imaging. As a result, in the3M Transbond XT group, there were many enamel demineralization pits on the dental crown sides, which form a clear boundary with buccal upper part. A few enamel demineralization pits were discovered on one dental crown side in the GC Fuji ORTHO LC group. No enamel demineralization pits were discovered on buccal neck and two sides in all NSC2, NSC3and NSC4groups. These results suggest that, in observation period, silver nanoparticles can effectively inhibit the bacterial adhesion around the material and the formation of plaque, and then prevent the occurrence or reduce the incidence of enamel demineralization.