- Email: [email protected]
Effect of nanomaterial incorporation on the mechanical and microbiological properties of dental porcelain
RESEARCH AND EDUCATION
Effect of nanomaterial incorporation on the mechanical and microbiological properties of dental porcelain Izabela Ferreira, DDS,a Carla Larissa Vidal, MS,b André Luís Botelho, PhD,c Paulo Sérgio Ferreira, MLT,d Mariana Lima da Costa Valente, PhD,e Marco Antonio Schiavon, PhD,f and Andréa Cândido dos Reis, PhDg Nanotechnology has enabled ABSTRACT the development of silver Statement of problem. Dental porcelain restorations are subject to biological failures related to nanoparticles (AgNPs)1-3 that secondary caries and periodontal disease leading to prosthesis replacement. have a broad-spectrum antiPurpose. The purpose of this in vitro study was to explore the microbiological and mechanical microbial effect, including properties of dental porcelain incorporated with different percentages of silver vanadate against Streptococcus mutans, (b-AgVO3) through microbiological analysis, roughness tests, and the Vickers microhardness test. the most cariogenic microor4-6 7 Material and methods. IPS InLine porcelain specimens were made by using a cylindrical Teflon matrix ganism. Holtz et al rein the dimensions of 8×2 mm. For the control group, the porcelain was manipulated according to the ported on the use of AgNPs as manufacturer’s instructions. The groups incorporating the nanomaterial were prepared with 2.5%, 5%, an antibacterial additive to and 10% of b-AgVO3, which was added proportionally by mass to the porcelain powder. In vitro water-based paint and varnish microbiologic analysis, roughness tests, and the Vickers microhardness test were performed. formulations. This nanoResults. Against Streptococcus mutans, the control group showed no inhibition halo (0 mm). All material was shown to have an groups with AgVO3 showed a zone of inhibition, the highest for the group with 10% (30 mm) and antibacterial activity against then the groups with 2.5% (9 mm) and 5% (17 mm). For Vickers microhardness, no statistically several types of multidrugsignificant difference (P<.05) was observed between the evaluated groups. The group with 10% of resistant bacteria strains, AgVO3 had the highest mean roughness and was statistically different (P<.001) from the other groups. including methicillin-resistant Conclusions. Adding b-AgVO3 to dental porcelain demonstrated antimicrobial effectiveness at all Staphylococcus aureus (MRSA). concentrations (2.5%, 5%, and 10%), with no effect on Vickers microhardness. The 10% group AgNPs can be applied in huhad higher roughness than the other groups. (J Prosthet Dent 2019;-:---) mid domestic environments provide an efficient antimicrobial activity against gram(such as kitchens, toilets, and sports locker rooms), and positive bacteria, including Staphylococcus aureus,7 its use in hospital environments may improve sanitary 7 and has attracted interest in dentistry.11-17 The bactericonditions for health-care workers. 8-10 cidal effect occurs by the release of silver and vanadium The use of AgNPs in dental materials has been ions that bind to the cell membrane and the thiol groups facilitated because of the combination of vanadate present in the enzymes of bacterial metabolisms.7,15 nanowires to form a nanostructured silver vanadate b-AgVO3 has been used to improve the biological decorated with AgNPs (b-AgVO3) that prevented its properties of dental materials. The addition of b-AgVO3 agglomeration. This material has been reported to
a
Student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. Graduate student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. c Postdoctoral student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. d Laboratory Technician, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. e Postdoctoral student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. f Professor, Department of Natural Sciences, Federal University of São João del-Rei (UFSJ), São João del-Rei, Brazil. g Professor, Department of Dental Materials and Prosthodontics, Ribeiraeo Preto Dental School, University of Saeo Paulo (USP), Ribeiraeo Preto, Brazil. b
THE JOURNAL OF PROSTHETIC DENTISTRY
1.e1
1.e2
Clinical Implications The addition of nanostructured silver vanadate to dental porcelain may reduce biofilm formation without compromising mechanical properties. This antibacterial action may improve the longevity of an oral rehabilitation.
to acrylic resins showed bactericidal effects against Candida albicans, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa.12,13 The addition of b-AgVO3 to endodontic cements has been reported to have a bactericidal effect against E. faecalis, P. aeruginosa, and Escherichia coli.14 The incorporation of b-AgVO3 into a soft denture liner has a bactericidal effect against P. aeruginosa, E. faecalis, and C. albicans and improves the adhesion properties between the liner and the denture base material.16 Also, b-AgVO3 can be incorporated into an irreversible hydrocolloid as an antimicrobial agent without promoting adverse effects on physicalmechanical properties.17 The combination of the antimicrobial properties of b-AgVO3 with the excellent properties18-30 of dental porcelain can produce an improved dental material. The purpose of the present study was to evaluate the microbiological and mechanical properties of dental porcelain incorporated with different percentages of bAgVO3 through in vitro microbiological analysis, roughness tests, and the Vickers microhardness test. The null hypotheses were that the addition of b-AgVO3 would not provide antibacterial activity or change the mechanical properties of the dental porcelain. MATERIAL AND METHODS A nanostructured silver vanadate was synthesized by a precipitation reaction between silver nitrate (99.8% AgNO3; Merck) and ammonium vanadate (99% NH4VO3; Merck). Initially, 0.9736 g of NH4VO3 and 1.3569 g of AgNO3 were dissolved in 200 mL of distilled water. Then, the AgNO3 solution was added, drop by drop, to the NH4VO3 solution under constant agitation at 65 C. A precipitate was obtained and washed with distilled water and absolute alcohol, filtered, and dried in a vacuum line for 10 hours to obtain the silver vanadate powder. A scanning transmission electron microscope (STEM) (Magellan 400L; FEI Co) was used to observe the morphology and presence of silver nanoparticles on the surface of the crystals.7,31 A commercial porcelain (dentin A3, IPS InLine; Ivoclar Vivadent AG) was used. The specimens (8×2 mm) were made by using a cylindrical Teflon mold. For the control group, the porcelain was manipulated according THE JOURNAL OF PROSTHETIC DENTISTRY
Volume
-
Issue
-
to the manufacturer’s instructions. The groups incorporating the nanomaterial were prepared with 2.5%, 5%, and 10% of b-AgVO3, which was added proportionally by mass to the porcelain powder. After the homogenization of the powders, the corresponding liquid was mixed following the manufacturer’s instructions. The mixtures were then manipulated, and the specimens were prepared by using the vibration-condensation method and sintered in a dental porcelain furnace according to the firing schedules recommended by the manufacturers. The porcelain disks were fired over refractory wool (initial temperature of 403 C; heating rates of 60 C/min; firing temperatures of 910 C). Once the firing schedule ended, the furnace door was opened only 10% until the temperature inside the oven reached 300 C, whereupon it was opened completely. The specimens were then submitted to a second firing (900 C), following a fast cooling protocol (45 C/s). The specimens of the different groups were sterilized by using ethylene oxide. An assay was performed by using the agar diffusion method.16 The inhibition halo test was performed to determine the inhibitory effect of the porcelain against Streptococcus mutans after incubation for 48 hours and 7 days17 For this, the specimens were prepared according to the percentage of AgVO3 (0%, 2.5%, 5%, and 10%), with dimensions of 3×2 mm. The test specimens (n=3) were positioned on Petri dishes on a solidified base layer of culture medium (SB 20, modified), and then 8 mL of the microorganism solution incorporated into the sterilized culture medium was distributed on the plates, involving the specimens. The plates were incubated at 37 C in a microbiological greenhouse for 48 hours. The inhibition zones around the specimens were measured by using a dry tip compass and a millimeter ruler. The 7-day specimens were incubated under the same conditions. The inhibition zone was determined by measuring the diameter (mm) in 2 perpendicular directions, repeated 3 times, and the arithmetic mean was determined. Two mechanical assays were performed to evaluate the performance of the material: roughness and Vickers microhardness. For roughness testing, specimen preparation, technical standard, and the analysis following International Organization for Standardization (ISO) 9693-1.32 This analysis was performed by using a roughness meter (SJ201P; Mitutoyo Corp). The analyzer tip of the roughness meter touched the workpiece and traveled 4 mm, making 3 measurements in each test body in the direction of its largest diameter. The Vickers microhardness reading (n=10) was performed in a microhardness tester (HMV-2; Shimadzu), with a load of 2 N, for 20 seconds. Three readings were performed in each test body in 3 regions. The mean of the 3 values was calculated. Ferreira et al
-
2019
1.e3
Figure 1. Transmission electron microscopy of nanostructured silver vanadate (original magnification ×400 000).
Figure 2. Inhibition halo in tested groups. A, Control group. B, Group 2.5%. C, Group 5%. D, Group 10%.
Table 1. Average of dimensions of inhibition halos formed around specimens (mm)
Table 2. Mean ±standard deviation of Vickers microhardness and superficial roughness of IPS InLine porcelain in different concentrations of AgVO3
Groups
Streptococcus mutans
Vickers Microhardness
Roughness
Control 0%
0
Group 2.5%
9
Group
Mean (SD)
Mean (SD)
Group 5%
17
Control
542.46 ±23.96A
1.76 ±0.72A
Group 10%
30
2.5%
503.10 ±48.60A
3.37 ±1.35A
5%
535.83 ±36.93A
3.51 ±2.09A
A
7.71 ±1.77B
10%
The normal distribution of the data was verified by using the Shapiro-Wilk test, and the ANOVA and Tukey multiple comparison test (a=.05) were applied. RESULTS The SEM micrographs revealed that the silver vanadate nanowires presented dimensions at nanometric and micrometric scales for diameter and length, respectively, and were coated with semispherical metallic silver nanoparticles capable of maintaining a high contact surface with microorganisms (Fig. 1). For the microbiological test of agar diffusion, the mean values of the halos formed around the specimens are reported in Table 1. Against S. Mutans, the control group, without AgVO3, did not present an inhibition halo (0 mm). All groups with AgVO3 presented an inhibition zone, with the highest values observed for the group with 10% (30 mm). The group with 2.5% (9 mm) and 5% (17 mm) presented intermediate values. Time did not influence the dimensions of the halos (Fig. 2). For the Vickers microhardness, no statistically significant difference was observed (P<.05) between the IPS InLine porcelain groups evaluated (Table 2). In the analysis of the superficial roughness, the concentration of 10% was statistically different (P<.001) from the other groups and presented the highest mean value (Table 2). Ferreira et al
550.70 ±85.35
Rows with same uppercase letters statistically similar (P>.05).
DISCUSSION The results of this study support the rejection of the null hypothesis that the addition of b-AgVO3 would not provide antibacterial activity or change the mechanical properties of the dental porcelain. Antimicrobial agents have been incorporated into mouthwashes, dental creams, dental restorations, and endodontic cements.30 Examples of these agents are chlorhexidine,33 benzalkonium chloride,34 and antibiotics.35 Turkun et al36 and Derafshi et al37 attempted to incorporate chlorhexidine digluconate into porcelain, but without success, and the antimicrobial capacity was not evaluated. The authors are unaware of previous studies evaluating the antimicrobial capacity and the mechanical properties of b-AgVO3 in dental porcelain. The expansion of nanotechnology has allowed the synthesis of different types of nanomaterials, including zinc, titanium, copper, and silver.1,2 Although some bacteria have become antibiotic resistant, they are less prone to develop resistance against metallic nanoparticles,1,2 especially silver nanoparticles (AgNPs).3 The present study explored the incorporation of bAgVO3 by analyzing the formation of inhibition halos against S. mutans bacteria, as it is commonly found in the oral cavity and when adhered to the biofilm is the main organism responsible for caries.5,6 The addition of THE JOURNAL OF PROSTHETIC DENTISTRY
1.e4
nanomaterial inhibited bacterial growth in all groups. These results are consistent with those obtained by Castro et al,13 who incorporated AgVO3 in acrylic resins and reported an effect against Candida albicans, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa. Baharav et al30 analyzed microhardness in aluminamodified porcelain and found no statistically significant difference between the ceramics. In the present study, the incorporation of b-AgVO3 did not lead to a statistically significant difference in hardness among the control groups and those modified with the nanomaterial. In the present study, roughness was analyzed by comparing the groups incorporated with nanomaterial and the control group. Only the group incorporated with the nanomaterial in the concentration of 10% presented statistically significant difference. de Kok et al19 analyzed porcelain modified with lithium disilicate and compared the roughness of the specimens before and after polishing, concluding that polished restorations are less likely to fail. Alao et al20 analyzed the roughness of porcelain modified with lithium metasilicate after different porcelain processing, concluding that roughness is influenced by the type of processing. High roughness can lead to a radical fracture when a force is applied.19 Recent literature has demonstrated the promising future of b-AgVO3 in dentistry.8,9,16,17 Silver ions have low toxicity to human cells and a long useful life against bacteria.10 However, the toxicity of b-AgVO3 in human cells is not yet fully clarified,15 suggesting the need for further studies of the nanomaterial. The nanoparticles have a yellowish color,31 so their application may be limited because porcelain incorporated with b-AgVO3 can lead to color change and esthetic concerns; the resulting grayish color11 may limit its use to posterior teeth. CONCLUSIONS Based on the findings of this in vitro study, the following conclusions were drawn: 1. Addition of b-AgVO3 to dental porcelain demonstrated antimicrobial effectiveness at all concentrations (2.5%, 5%, and 10%), with no effect on Vickers microhardness. 2. The 10% group had higher roughness than the other groups. REFERENCES 1. Allaker RP. The use of nanoparticles to control oral biofilm formation. J Dent Res 2010;89:1175-86. 2. Borzabadi-Farahani A, Borzabadi E, Lynch E. Nanoparticles in orthodontics, a review of antimicrobial and anti-caries applications. Acta Odontol Scand 2014;72:413-7. 3. Cheng H, Li Y, Huo K, Gao B, Xiong W. Long-lasting in vivo and in vitro antibacterial ability of nanostructured titania coating incorporated with silver nanoparticles. J Biomed Mater Res A 2014;102:3488-99.
THE JOURNAL OF PROSTHETIC DENTISTRY
Volume
-
Issue
-
4. Cheng L, Weir MD, Zhang K, Wu EJ, Xu SM, Zhou X, et al. Dental plaque microcosm biofilm behavior on calcium phosphate nanocomposite with quaternary ammonium. Dent Mater 2012;28:853-62. 5. Meizhen OU, Juni L. Norspermidine changes the basic structure of S. mutans biofilm. Mol Med Rep 2017;15:210-20. 6. Tu Y, Ling X, Chen Y, Wang Y, Zhou N, Chen H. Effect of S. mutans and S. sanguinis on growth and adhesion of P. Gingivalis and their ability to adhere to different dental materials. Med Sci Monit 2017;15:5439-45. 7. Holtz RD, Lima BA, Souza Filho AG, Brocchi M, Alves OL. Nanostructured silver vanadate as a promising antibacterial additive to water-based paints. Nanomedicine 2012;8:935-40. 8. de Castro DT, Valente MLDC, Aires CP, Alves OL, Dos Reis AC. Elemental ion release and cytotoxicity of antimicrobial acrylic resins incorporated with nanomaterial. Gerodontology 2017;34:320-5. 9. de Castro DT, do Nascimento C, Alves OL, de Souza Santos E, Agnelli JAM, Dos Reis AC. Analysis of the oral microbiome on the surface of modified dental polymers. Arch Oral Biol 2018;93:107-14. 10. Corrêa JM, Mori M, Sanches HL, da Cruz AD, Poiate E Jr, Poiate IA. Silver nanoparticles in dental biomaterials. Int J Biomater 2015;2015:1-9. 11. de Castro DT, Valente ML, Agnelli JA, Lovato da Silva CH, Watanabe E, Siqueira RL, et al. In vitro study of the antibacterial properties and impact strength of dental acrylic resins modified with a nanomaterial. J Prosthet Dent 2016;115:238-46. 12. Castro DT, Holtz RD, Alves OL, Watanabe E, Valente ML, Silva CH, et al. Development of a novel resin with antimicrobial properties for dental application. J Appl Oral Sci 2014;22:442-9. 13. de Castro DT, Valente ML, da Silva CH, Watanabe E, Siqueira RL, Schiavon MA, et al. Evaluation of antibiofilm and mechanical properties of new nanocomposites based on acrylic resins and silver vanadate nanoparticles. Arch Oral Biol 2016;67:46-53. 14. Vilela Teixeira AB, Vidal CL, de Castro DT, da Costa Valente ML, OliveiraSantos C, Alves OL, et al. Effect of incorporation of a new antimicrobial nanomaterial on the physical-chemical properties of endodontic sealers. J Conserv Dent 2017;20:392-7. 15. Vilela Teixeira AB, Silva CCH, Alves OL, Dos Reis AC. Endodontic sealers modified with silver vanadate: antibacterial, compositional, and setting time evaluation. Biomed Res Int 2019;2019:1-9. 16. Kreve S, Oliveira VC, Bachmann L, Alves OL, Reis ACD. Influence of AgVO3 incorporation on antimicrobial properties, hardness, roughness and adhesion of a soft denture liner. Sci Rep 2019;9:1-9. 17. de Castro DT, Kreve S, Oliveira VC, Alves OL, Dos Reis AC. Development of an impression material with antimicrobial properties for dental application. J Prosthodont 2019;2019:1-7. 18. Kruzic JJ, Arsecularatne JA, Tanaka CB, Hoffman MJ, Cesar PF. Recent advances in understanding the fatigue and wear behavior of dental composites and ceramics. J Mech Behav Biomed Mater 2018;88:504-33. 19. de Kok P, Pereira GKR, Fraga S, de Jager N, Venturini AB, Kleverlaan CJ. The effect of internal roughness and bonding on the fracture resistance and structural reliability of lithium disilicate ceramic. Dent Mater 2017;33: 1416-25. 20. Alao AR, Stoll R, Song XF, Abbott JR, Zhang Y, Abduo J, et al. Fracture, roughness and phase transformation in CAD/CAM milling and subsequent surface treatments of lithium metasilicate/disilicate glass-ceramics. J Mech Behav Biomed Mater 2017;74:251-60. 21. Rashid H, Sheikh Z, Misbahuddin S, Kazmi MR, Qureshi S, Uddin MZ. Advancements in all-ceramics for dental restorations and their effect on the wear of opposing dentition. Eur J Dent 2016;10:583-8. 22. Silva TSO, Freitas AR, Pinheiro MLL, do Nascimento C, Watanabe E, Albuquerque RF. Oral biofilm formation on different materials for dental implants. J Vis Exp 2018;24:136. 23. Willard A, Gabriel Chu TM. The science and application of IPS e.Max dental ceramic. Kaohsiung J Med Sci 2018;34:238-42. 24. Silva NR, Bonfante EA, Rafferty BT, Zavanelli RA, Rekow ED, Thompson VP, et al. Modified Y-TZP core design improves all-ceramic crown reliability. J Dent Res 2011;90:104-8. 25. Conrad HJ, Seong WJ, Pesun IJ. Current ceramic materials and systems with clinical recommendations: a systematic review. J Prosthet Dent 2007;98: 389-404. 26. Pjetursson BE, Thoma D, Jung R, Zwahlen M, Zembic A. A systematic review of the survival and complication rates of implant-supported fixed dental prostheses (FDPs) after a mean observation period of at least 5 years. Clin Oral Implants Res 2012;23:22-38. 27. Jung RE, Zembic A, Pjetursson BE, Zwahlen M, Thoma DS. Systematic review of the survival rate and the incidence of biological, technical, and aesthetic complications of single crowns on implants reported in longitudinal studies with a mean follow-up of 5 years. Clin Oral Implants Res 2012;23: 2-21. 28. Pjetursson BE, Sailer I, Makarov NA, Zwahlen M, Thoma DS. Corrigendum to "All-ceramic or metal-ceramic tooth-supported fixed dental prostheses (FDPs)? A systematic review of the survival and complication rates. Part II: Multiple-unit FDPs" [Dental Materials 2015;31:624e639]. Dent Mater 2017;33:48-51.
Ferreira et al
-
2019
29. Pihlgren K, Forsberg H, Sjödin L, Lundgren P, Wänman A. Changes in tooth mortality between 1990 and 2002 among adults in Västerbotten County, Sweden: influence of socioeconomic factors, general health, smoking, and dental care habits on tooth mortality. Swed Dent J 2011;35:77-88. 30. Baharav H, Laufer BZ, Mizrachi A, Cardash HS. Effect of different cooling rates on fracture toughness and microhardness of a glazed alumina reinforced porcelain. J Prosthet Dent 1996;76:19-22. 31. Holtz RD, Souza Filho AG, Brocchi M, Martins D, Durán N, Alves OL. Development of nanostructured silver vanadates decorated with silver nanoparticles as a novel antibacterial agent. Nanotechnology 2010;21: 185102. 32. International Organization for Standardization. ISO 9693e1. Dentistry compability testing. Part 1: Metal-ceramic systems. Geneva: International Organization for Standardization; 2012. ISO Store Order: OP-184149 (Date: 2017-06-09). Available at: http://www.iso.org/iso/home.html. 33. Ruiz-Linares M, Bailón-Sánchez ME, Baca P, Valderrama M, FerrerLuque CM. Physical properties of AH Plus with chlorhexidine and cetrimide. J Endod 2013;39:1611-4. 34. Gjorgievska E, Apostolska S, Dimkov A, Nicholson JW, Kaftandzieva A. Incorporation of antimicrobial agents can be used to enhance the antibacterial effect of endodontic sealers. Dent Mater 2013;29:29-34.
Ferreira et al
1.e5
35. Hoelscher AA, Bahcall JK, Maki JS. In vitro evaluation of the antimicrobial effects of a root canal sealer-antibiotic combination against Enterococcus faecalis. J Endod 2006;32:145-7. 36. Turkun M, Cal E, Toman M, Toksavul S. Effects of dentin disinfectants on the shear bond strength of all-ceramics to dentin. Oper Dent 2005;30:453-60. 37. Derafshi R, Khorshidi H, Kalantari M, Ghaffarlou I. Effect of mouthrinses on color stability of monolithic zirconia and feldspathic ceramic: an in vitro study. BMC Oral Health 2017;17:129. Corresponding author: Dr Andréa Cândido dos Reis Department of Dental Materials and Prosthodontics Ribeirão Preto Dental School FORP-USP. Av. Do Café, s/n 14040-904 Ribeirão Preto, SP BRAZIL Email: [email protected] Copyright © 2019 by the Editorial Council for The Journal of Prosthetic Dentistry. https://doi.org/10.1016/j.prosdent.2019.10.012
THE JOURNAL OF PROSTHETIC DENTISTRY
Effect of nanomaterial incorporation on the mechanical and microbiological properties of dental porcelain Izabela Ferreira, DDS,a Carla Larissa Vidal, MS,b André Luís Botelho, PhD,c Paulo Sérgio Ferreira, MLT,d Mariana Lima da Costa Valente, PhD,e Marco Antonio Schiavon, PhD,f and Andréa Cândido dos Reis, PhDg Nanotechnology has enabled ABSTRACT the development of silver Statement of problem. Dental porcelain restorations are subject to biological failures related to nanoparticles (AgNPs)1-3 that secondary caries and periodontal disease leading to prosthesis replacement. have a broad-spectrum antiPurpose. The purpose of this in vitro study was to explore the microbiological and mechanical microbial effect, including properties of dental porcelain incorporated with different percentages of silver vanadate against Streptococcus mutans, (b-AgVO3) through microbiological analysis, roughness tests, and the Vickers microhardness test. the most cariogenic microor4-6 7 Material and methods. IPS InLine porcelain specimens were made by using a cylindrical Teflon matrix ganism. Holtz et al rein the dimensions of 8×2 mm. For the control group, the porcelain was manipulated according to the ported on the use of AgNPs as manufacturer’s instructions. The groups incorporating the nanomaterial were prepared with 2.5%, 5%, an antibacterial additive to and 10% of b-AgVO3, which was added proportionally by mass to the porcelain powder. In vitro water-based paint and varnish microbiologic analysis, roughness tests, and the Vickers microhardness test were performed. formulations. This nanoResults. Against Streptococcus mutans, the control group showed no inhibition halo (0 mm). All material was shown to have an groups with AgVO3 showed a zone of inhibition, the highest for the group with 10% (30 mm) and antibacterial activity against then the groups with 2.5% (9 mm) and 5% (17 mm). For Vickers microhardness, no statistically several types of multidrugsignificant difference (P<.05) was observed between the evaluated groups. The group with 10% of resistant bacteria strains, AgVO3 had the highest mean roughness and was statistically different (P<.001) from the other groups. including methicillin-resistant Conclusions. Adding b-AgVO3 to dental porcelain demonstrated antimicrobial effectiveness at all Staphylococcus aureus (MRSA). concentrations (2.5%, 5%, and 10%), with no effect on Vickers microhardness. The 10% group AgNPs can be applied in huhad higher roughness than the other groups. (J Prosthet Dent 2019;-:---) mid domestic environments provide an efficient antimicrobial activity against gram(such as kitchens, toilets, and sports locker rooms), and positive bacteria, including Staphylococcus aureus,7 its use in hospital environments may improve sanitary 7 and has attracted interest in dentistry.11-17 The bactericonditions for health-care workers. 8-10 cidal effect occurs by the release of silver and vanadium The use of AgNPs in dental materials has been ions that bind to the cell membrane and the thiol groups facilitated because of the combination of vanadate present in the enzymes of bacterial metabolisms.7,15 nanowires to form a nanostructured silver vanadate b-AgVO3 has been used to improve the biological decorated with AgNPs (b-AgVO3) that prevented its properties of dental materials. The addition of b-AgVO3 agglomeration. This material has been reported to
a
Student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. Graduate student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. c Postdoctoral student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. d Laboratory Technician, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. e Postdoctoral student, Department of Dental Materials and Prosthodontics, Ribeirão Preto Dental School, University of São Paulo (USP), Ribeirão Preto, Brazil. f Professor, Department of Natural Sciences, Federal University of São João del-Rei (UFSJ), São João del-Rei, Brazil. g Professor, Department of Dental Materials and Prosthodontics, Ribeiraeo Preto Dental School, University of Saeo Paulo (USP), Ribeiraeo Preto, Brazil. b
THE JOURNAL OF PROSTHETIC DENTISTRY
1.e1
1.e2
Clinical Implications The addition of nanostructured silver vanadate to dental porcelain may reduce biofilm formation without compromising mechanical properties. This antibacterial action may improve the longevity of an oral rehabilitation.
to acrylic resins showed bactericidal effects against Candida albicans, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa.12,13 The addition of b-AgVO3 to endodontic cements has been reported to have a bactericidal effect against E. faecalis, P. aeruginosa, and Escherichia coli.14 The incorporation of b-AgVO3 into a soft denture liner has a bactericidal effect against P. aeruginosa, E. faecalis, and C. albicans and improves the adhesion properties between the liner and the denture base material.16 Also, b-AgVO3 can be incorporated into an irreversible hydrocolloid as an antimicrobial agent without promoting adverse effects on physicalmechanical properties.17 The combination of the antimicrobial properties of b-AgVO3 with the excellent properties18-30 of dental porcelain can produce an improved dental material. The purpose of the present study was to evaluate the microbiological and mechanical properties of dental porcelain incorporated with different percentages of bAgVO3 through in vitro microbiological analysis, roughness tests, and the Vickers microhardness test. The null hypotheses were that the addition of b-AgVO3 would not provide antibacterial activity or change the mechanical properties of the dental porcelain. MATERIAL AND METHODS A nanostructured silver vanadate was synthesized by a precipitation reaction between silver nitrate (99.8% AgNO3; Merck) and ammonium vanadate (99% NH4VO3; Merck). Initially, 0.9736 g of NH4VO3 and 1.3569 g of AgNO3 were dissolved in 200 mL of distilled water. Then, the AgNO3 solution was added, drop by drop, to the NH4VO3 solution under constant agitation at 65 C. A precipitate was obtained and washed with distilled water and absolute alcohol, filtered, and dried in a vacuum line for 10 hours to obtain the silver vanadate powder. A scanning transmission electron microscope (STEM) (Magellan 400L; FEI Co) was used to observe the morphology and presence of silver nanoparticles on the surface of the crystals.7,31 A commercial porcelain (dentin A3, IPS InLine; Ivoclar Vivadent AG) was used. The specimens (8×2 mm) were made by using a cylindrical Teflon mold. For the control group, the porcelain was manipulated according THE JOURNAL OF PROSTHETIC DENTISTRY
Volume
-
Issue
-
to the manufacturer’s instructions. The groups incorporating the nanomaterial were prepared with 2.5%, 5%, and 10% of b-AgVO3, which was added proportionally by mass to the porcelain powder. After the homogenization of the powders, the corresponding liquid was mixed following the manufacturer’s instructions. The mixtures were then manipulated, and the specimens were prepared by using the vibration-condensation method and sintered in a dental porcelain furnace according to the firing schedules recommended by the manufacturers. The porcelain disks were fired over refractory wool (initial temperature of 403 C; heating rates of 60 C/min; firing temperatures of 910 C). Once the firing schedule ended, the furnace door was opened only 10% until the temperature inside the oven reached 300 C, whereupon it was opened completely. The specimens were then submitted to a second firing (900 C), following a fast cooling protocol (45 C/s). The specimens of the different groups were sterilized by using ethylene oxide. An assay was performed by using the agar diffusion method.16 The inhibition halo test was performed to determine the inhibitory effect of the porcelain against Streptococcus mutans after incubation for 48 hours and 7 days17 For this, the specimens were prepared according to the percentage of AgVO3 (0%, 2.5%, 5%, and 10%), with dimensions of 3×2 mm. The test specimens (n=3) were positioned on Petri dishes on a solidified base layer of culture medium (SB 20, modified), and then 8 mL of the microorganism solution incorporated into the sterilized culture medium was distributed on the plates, involving the specimens. The plates were incubated at 37 C in a microbiological greenhouse for 48 hours. The inhibition zones around the specimens were measured by using a dry tip compass and a millimeter ruler. The 7-day specimens were incubated under the same conditions. The inhibition zone was determined by measuring the diameter (mm) in 2 perpendicular directions, repeated 3 times, and the arithmetic mean was determined. Two mechanical assays were performed to evaluate the performance of the material: roughness and Vickers microhardness. For roughness testing, specimen preparation, technical standard, and the analysis following International Organization for Standardization (ISO) 9693-1.32 This analysis was performed by using a roughness meter (SJ201P; Mitutoyo Corp). The analyzer tip of the roughness meter touched the workpiece and traveled 4 mm, making 3 measurements in each test body in the direction of its largest diameter. The Vickers microhardness reading (n=10) was performed in a microhardness tester (HMV-2; Shimadzu), with a load of 2 N, for 20 seconds. Three readings were performed in each test body in 3 regions. The mean of the 3 values was calculated. Ferreira et al
-
2019
1.e3
Figure 1. Transmission electron microscopy of nanostructured silver vanadate (original magnification ×400 000).
Figure 2. Inhibition halo in tested groups. A, Control group. B, Group 2.5%. C, Group 5%. D, Group 10%.
Table 1. Average of dimensions of inhibition halos formed around specimens (mm)
Table 2. Mean ±standard deviation of Vickers microhardness and superficial roughness of IPS InLine porcelain in different concentrations of AgVO3
Groups
Streptococcus mutans
Vickers Microhardness
Roughness
Control 0%
0
Group 2.5%
9
Group
Mean (SD)
Mean (SD)
Group 5%
17
Control
542.46 ±23.96A
1.76 ±0.72A
Group 10%
30
2.5%
503.10 ±48.60A
3.37 ±1.35A
5%
535.83 ±36.93A
3.51 ±2.09A
A
7.71 ±1.77B
10%
The normal distribution of the data was verified by using the Shapiro-Wilk test, and the ANOVA and Tukey multiple comparison test (a=.05) were applied. RESULTS The SEM micrographs revealed that the silver vanadate nanowires presented dimensions at nanometric and micrometric scales for diameter and length, respectively, and were coated with semispherical metallic silver nanoparticles capable of maintaining a high contact surface with microorganisms (Fig. 1). For the microbiological test of agar diffusion, the mean values of the halos formed around the specimens are reported in Table 1. Against S. Mutans, the control group, without AgVO3, did not present an inhibition halo (0 mm). All groups with AgVO3 presented an inhibition zone, with the highest values observed for the group with 10% (30 mm). The group with 2.5% (9 mm) and 5% (17 mm) presented intermediate values. Time did not influence the dimensions of the halos (Fig. 2). For the Vickers microhardness, no statistically significant difference was observed (P<.05) between the IPS InLine porcelain groups evaluated (Table 2). In the analysis of the superficial roughness, the concentration of 10% was statistically different (P<.001) from the other groups and presented the highest mean value (Table 2). Ferreira et al
550.70 ±85.35
Rows with same uppercase letters statistically similar (P>.05).
DISCUSSION The results of this study support the rejection of the null hypothesis that the addition of b-AgVO3 would not provide antibacterial activity or change the mechanical properties of the dental porcelain. Antimicrobial agents have been incorporated into mouthwashes, dental creams, dental restorations, and endodontic cements.30 Examples of these agents are chlorhexidine,33 benzalkonium chloride,34 and antibiotics.35 Turkun et al36 and Derafshi et al37 attempted to incorporate chlorhexidine digluconate into porcelain, but without success, and the antimicrobial capacity was not evaluated. The authors are unaware of previous studies evaluating the antimicrobial capacity and the mechanical properties of b-AgVO3 in dental porcelain. The expansion of nanotechnology has allowed the synthesis of different types of nanomaterials, including zinc, titanium, copper, and silver.1,2 Although some bacteria have become antibiotic resistant, they are less prone to develop resistance against metallic nanoparticles,1,2 especially silver nanoparticles (AgNPs).3 The present study explored the incorporation of bAgVO3 by analyzing the formation of inhibition halos against S. mutans bacteria, as it is commonly found in the oral cavity and when adhered to the biofilm is the main organism responsible for caries.5,6 The addition of THE JOURNAL OF PROSTHETIC DENTISTRY
1.e4
nanomaterial inhibited bacterial growth in all groups. These results are consistent with those obtained by Castro et al,13 who incorporated AgVO3 in acrylic resins and reported an effect against Candida albicans, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa. Baharav et al30 analyzed microhardness in aluminamodified porcelain and found no statistically significant difference between the ceramics. In the present study, the incorporation of b-AgVO3 did not lead to a statistically significant difference in hardness among the control groups and those modified with the nanomaterial. In the present study, roughness was analyzed by comparing the groups incorporated with nanomaterial and the control group. Only the group incorporated with the nanomaterial in the concentration of 10% presented statistically significant difference. de Kok et al19 analyzed porcelain modified with lithium disilicate and compared the roughness of the specimens before and after polishing, concluding that polished restorations are less likely to fail. Alao et al20 analyzed the roughness of porcelain modified with lithium metasilicate after different porcelain processing, concluding that roughness is influenced by the type of processing. High roughness can lead to a radical fracture when a force is applied.19 Recent literature has demonstrated the promising future of b-AgVO3 in dentistry.8,9,16,17 Silver ions have low toxicity to human cells and a long useful life against bacteria.10 However, the toxicity of b-AgVO3 in human cells is not yet fully clarified,15 suggesting the need for further studies of the nanomaterial. The nanoparticles have a yellowish color,31 so their application may be limited because porcelain incorporated with b-AgVO3 can lead to color change and esthetic concerns; the resulting grayish color11 may limit its use to posterior teeth. CONCLUSIONS Based on the findings of this in vitro study, the following conclusions were drawn: 1. Addition of b-AgVO3 to dental porcelain demonstrated antimicrobial effectiveness at all concentrations (2.5%, 5%, and 10%), with no effect on Vickers microhardness. 2. The 10% group had higher roughness than the other groups. REFERENCES 1. Allaker RP. The use of nanoparticles to control oral biofilm formation. J Dent Res 2010;89:1175-86. 2. Borzabadi-Farahani A, Borzabadi E, Lynch E. Nanoparticles in orthodontics, a review of antimicrobial and anti-caries applications. Acta Odontol Scand 2014;72:413-7. 3. Cheng H, Li Y, Huo K, Gao B, Xiong W. Long-lasting in vivo and in vitro antibacterial ability of nanostructured titania coating incorporated with silver nanoparticles. J Biomed Mater Res A 2014;102:3488-99.
THE JOURNAL OF PROSTHETIC DENTISTRY
Volume
-
Issue
-
4. Cheng L, Weir MD, Zhang K, Wu EJ, Xu SM, Zhou X, et al. Dental plaque microcosm biofilm behavior on calcium phosphate nanocomposite with quaternary ammonium. Dent Mater 2012;28:853-62. 5. Meizhen OU, Juni L. Norspermidine changes the basic structure of S. mutans biofilm. Mol Med Rep 2017;15:210-20. 6. Tu Y, Ling X, Chen Y, Wang Y, Zhou N, Chen H. Effect of S. mutans and S. sanguinis on growth and adhesion of P. Gingivalis and their ability to adhere to different dental materials. Med Sci Monit 2017;15:5439-45. 7. Holtz RD, Lima BA, Souza Filho AG, Brocchi M, Alves OL. Nanostructured silver vanadate as a promising antibacterial additive to water-based paints. Nanomedicine 2012;8:935-40. 8. de Castro DT, Valente MLDC, Aires CP, Alves OL, Dos Reis AC. Elemental ion release and cytotoxicity of antimicrobial acrylic resins incorporated with nanomaterial. Gerodontology 2017;34:320-5. 9. de Castro DT, do Nascimento C, Alves OL, de Souza Santos E, Agnelli JAM, Dos Reis AC. Analysis of the oral microbiome on the surface of modified dental polymers. Arch Oral Biol 2018;93:107-14. 10. Corrêa JM, Mori M, Sanches HL, da Cruz AD, Poiate E Jr, Poiate IA. Silver nanoparticles in dental biomaterials. Int J Biomater 2015;2015:1-9. 11. de Castro DT, Valente ML, Agnelli JA, Lovato da Silva CH, Watanabe E, Siqueira RL, et al. In vitro study of the antibacterial properties and impact strength of dental acrylic resins modified with a nanomaterial. J Prosthet Dent 2016;115:238-46. 12. Castro DT, Holtz RD, Alves OL, Watanabe E, Valente ML, Silva CH, et al. Development of a novel resin with antimicrobial properties for dental application. J Appl Oral Sci 2014;22:442-9. 13. de Castro DT, Valente ML, da Silva CH, Watanabe E, Siqueira RL, Schiavon MA, et al. Evaluation of antibiofilm and mechanical properties of new nanocomposites based on acrylic resins and silver vanadate nanoparticles. Arch Oral Biol 2016;67:46-53. 14. Vilela Teixeira AB, Vidal CL, de Castro DT, da Costa Valente ML, OliveiraSantos C, Alves OL, et al. Effect of incorporation of a new antimicrobial nanomaterial on the physical-chemical properties of endodontic sealers. J Conserv Dent 2017;20:392-7. 15. Vilela Teixeira AB, Silva CCH, Alves OL, Dos Reis AC. Endodontic sealers modified with silver vanadate: antibacterial, compositional, and setting time evaluation. Biomed Res Int 2019;2019:1-9. 16. Kreve S, Oliveira VC, Bachmann L, Alves OL, Reis ACD. Influence of AgVO3 incorporation on antimicrobial properties, hardness, roughness and adhesion of a soft denture liner. Sci Rep 2019;9:1-9. 17. de Castro DT, Kreve S, Oliveira VC, Alves OL, Dos Reis AC. Development of an impression material with antimicrobial properties for dental application. J Prosthodont 2019;2019:1-7. 18. Kruzic JJ, Arsecularatne JA, Tanaka CB, Hoffman MJ, Cesar PF. Recent advances in understanding the fatigue and wear behavior of dental composites and ceramics. J Mech Behav Biomed Mater 2018;88:504-33. 19. de Kok P, Pereira GKR, Fraga S, de Jager N, Venturini AB, Kleverlaan CJ. The effect of internal roughness and bonding on the fracture resistance and structural reliability of lithium disilicate ceramic. Dent Mater 2017;33: 1416-25. 20. Alao AR, Stoll R, Song XF, Abbott JR, Zhang Y, Abduo J, et al. Fracture, roughness and phase transformation in CAD/CAM milling and subsequent surface treatments of lithium metasilicate/disilicate glass-ceramics. J Mech Behav Biomed Mater 2017;74:251-60. 21. Rashid H, Sheikh Z, Misbahuddin S, Kazmi MR, Qureshi S, Uddin MZ. Advancements in all-ceramics for dental restorations and their effect on the wear of opposing dentition. Eur J Dent 2016;10:583-8. 22. Silva TSO, Freitas AR, Pinheiro MLL, do Nascimento C, Watanabe E, Albuquerque RF. Oral biofilm formation on different materials for dental implants. J Vis Exp 2018;24:136. 23. Willard A, Gabriel Chu TM. The science and application of IPS e.Max dental ceramic. Kaohsiung J Med Sci 2018;34:238-42. 24. Silva NR, Bonfante EA, Rafferty BT, Zavanelli RA, Rekow ED, Thompson VP, et al. Modified Y-TZP core design improves all-ceramic crown reliability. J Dent Res 2011;90:104-8. 25. Conrad HJ, Seong WJ, Pesun IJ. Current ceramic materials and systems with clinical recommendations: a systematic review. J Prosthet Dent 2007;98: 389-404. 26. Pjetursson BE, Thoma D, Jung R, Zwahlen M, Zembic A. A systematic review of the survival and complication rates of implant-supported fixed dental prostheses (FDPs) after a mean observation period of at least 5 years. Clin Oral Implants Res 2012;23:22-38. 27. Jung RE, Zembic A, Pjetursson BE, Zwahlen M, Thoma DS. Systematic review of the survival rate and the incidence of biological, technical, and aesthetic complications of single crowns on implants reported in longitudinal studies with a mean follow-up of 5 years. Clin Oral Implants Res 2012;23: 2-21. 28. Pjetursson BE, Sailer I, Makarov NA, Zwahlen M, Thoma DS. Corrigendum to "All-ceramic or metal-ceramic tooth-supported fixed dental prostheses (FDPs)? A systematic review of the survival and complication rates. Part II: Multiple-unit FDPs" [Dental Materials 2015;31:624e639]. Dent Mater 2017;33:48-51.
Ferreira et al
-
2019
29. Pihlgren K, Forsberg H, Sjödin L, Lundgren P, Wänman A. Changes in tooth mortality between 1990 and 2002 among adults in Västerbotten County, Sweden: influence of socioeconomic factors, general health, smoking, and dental care habits on tooth mortality. Swed Dent J 2011;35:77-88. 30. Baharav H, Laufer BZ, Mizrachi A, Cardash HS. Effect of different cooling rates on fracture toughness and microhardness of a glazed alumina reinforced porcelain. J Prosthet Dent 1996;76:19-22. 31. Holtz RD, Souza Filho AG, Brocchi M, Martins D, Durán N, Alves OL. Development of nanostructured silver vanadates decorated with silver nanoparticles as a novel antibacterial agent. Nanotechnology 2010;21: 185102. 32. International Organization for Standardization. ISO 9693e1. Dentistry compability testing. Part 1: Metal-ceramic systems. Geneva: International Organization for Standardization; 2012. ISO Store Order: OP-184149 (Date: 2017-06-09). Available at: http://www.iso.org/iso/home.html. 33. Ruiz-Linares M, Bailón-Sánchez ME, Baca P, Valderrama M, FerrerLuque CM. Physical properties of AH Plus with chlorhexidine and cetrimide. J Endod 2013;39:1611-4. 34. Gjorgievska E, Apostolska S, Dimkov A, Nicholson JW, Kaftandzieva A. Incorporation of antimicrobial agents can be used to enhance the antibacterial effect of endodontic sealers. Dent Mater 2013;29:29-34.
Ferreira et al
1.e5
35. Hoelscher AA, Bahcall JK, Maki JS. In vitro evaluation of the antimicrobial effects of a root canal sealer-antibiotic combination against Enterococcus faecalis. J Endod 2006;32:145-7. 36. Turkun M, Cal E, Toman M, Toksavul S. Effects of dentin disinfectants on the shear bond strength of all-ceramics to dentin. Oper Dent 2005;30:453-60. 37. Derafshi R, Khorshidi H, Kalantari M, Ghaffarlou I. Effect of mouthrinses on color stability of monolithic zirconia and feldspathic ceramic: an in vitro study. BMC Oral Health 2017;17:129. Corresponding author: Dr Andréa Cândido dos Reis Department of Dental Materials and Prosthodontics Ribeirão Preto Dental School FORP-USP. Av. Do Café, s/n 14040-904 Ribeirão Preto, SP BRAZIL Email: [email protected] Copyright © 2019 by the Editorial Council for The Journal of Prosthetic Dentistry. https://doi.org/10.1016/j.prosdent.2019.10.012
THE JOURNAL OF PROSTHETIC DENTISTRY