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Antibacterial surface properties of fluoride-containing resin-based sealants
journal of dentistry 38 (2010) 387–391
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Antibacterial surface properties of fluoride-containing resin-based sealants Supawadee Naorungroj a, Hong-Hong Wei b, Roland R. Arnold b,c, Edward J. Swift Jr.a, Ricardo Walter a,* a
The University of North Carolina at Chapel Hill, School of Dentistry, Department of Operative Dentistry, Chapel Hill, NC 27599-7450, USA The University of North Carolina at Chapel Hill, School of Dentistry, North Carolina Oral Health Institute, Chapel Hill, NC 27599-7450, USA c The University of North Carolina at Chapel Hill, School of Dentistry, Department of Diagnostic Sciences & General Dentistry, Chapel Hill, NC 27599-7450, USA b
article info
abstract
Article history:
Objectives: The aim of the present study was to determine the antibacterial properties of
Received 17 September 2009
three resin-based pit and fissure sealant products: Clinpro (3M ESPE), Embrace (Pulpdent),
Received in revised form
and UltraSeal XT plus (Ultradent).
11 January 2010
Methods: The antibacterial effects of the sealants were tested in both an agar diffusion assay
Accepted 12 January 2010
and a planktonic growth inhibition assay using Streptococcus mutans and Lactobacillus acidophilus. The materials were applied to paper and enamel disks in the former and on the side walls of 96-well microtiter plates on the latter.
Keywords:
Results: All materials showed either diffusible or contact antibacterial effects in the agar
Sealants
diffusion assays. The effect was diminished when enamel disks were used as substrate. In
Fluoride
the planktonic growth inhibition assay, Clinpro had its effect reduced, but retained activity
Antibacterial
against both bacteria over time. L. acidophilus was more sensitive than S. mutans to UltraSeal. Embrace retained antibacterial activity against both bacteria over time. Conclusions: Within the limitations of this in vitro study it can be concluded that all materials are capable of contact inhibition of L. acidophilus and S. mutans growth. Embrace has the longer lasting antibacterial activity when in solution, especially against S. mutans. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Sealants have been used for decades as a preventive measure against caries development in susceptible pits and fissures. Their beneficial effect, which has been recently corroborated in a meta-analysis,1 depends largely on retention.2 This mechanical retention is achieved by etching enamel with phosphoric acid,3–5 which also reduces the bacterial count that may be present on the treated surfaces.6 Once the area is sealed, the isolation of the bacteria underneath the sealant
from oral nutrients helps minimize the development of caries.7 It has been suggested that this positive effect could possibly be enhanced by adding fluoride to the sealant material.8 Fluoride is able to incorporate into enamel, making it more resistant to acid degradation; it also has antibacterial effects against cariogenic oral bacteria.9 However, although it seems obvious that fluoride would improve the protective effect of sealants, equivocal results have been reported. Clinical studies where sealants were applied to caries-free surfaces have
* Corresponding author at: The University of North Carolina at Chapel Hill, School of Dentistry, Department of Operative Dentistry, 433 Brauer Hall CB #7450, Chapel Hill, NC 27599-7450, USA. Tel.: +1 919 966 2770; fax: +1 919 966 5660. E-mail address: [email protected] (R. Walter). 0300-5712/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2010.01.005
388
journal of dentistry 38 (2010) 387–391
shown no significant difference in caries inhibition or retention rate between conventional and fluoride-containing sealants.10–12 On the other hand, in vitro studies have demonstrated the potential inhibitory effect of some resinbased fluoride-containing sealants on cariogenic oral bacteria.8,13,14 This potential antibacterial effect may be advantageous, for instance, when caries is inadvertently left in the surface to be sealed.7 In vitro studies testing antibacterial potential of materials are often performed with mutans streptococci and lactobacilli species, which are believed to be the two major groups of bacteria involved in the caries process.9 In those groups are included Streptococcus mutans and Lactobacillus acidophilus, respectively. These bacteria produce acids, primarily lactic acid, that diffuse into the enamel and dissolve the mineral crystals to initiate the caries process.15 This study does not evaluate the demineralization effects of those bacteria in enamel but uses enamel as a substrate in one of the experiments. To the best knowledge of the authors, no other study has used enamel as a substrate to study the antibacterial effects of sealants. The purpose of this study is to evaluate the antibacterial surface properties of three commercially available resinbased fluoride-containing sealants using an agar diffusion test to determine the inhibition of surface growth and a planktonic growth inhibition test to determine the inhibition in broth culture. The hypothesis is that the resin-based fluoride-containing sealants have antibacterial activities under both test conditions against both S. mutans and L. acidophilus.
2.
Materials and methods
Materials used in the study are listed in Table 1. They were tested against S. mutans strain ATCC 10449 (serotype c) and L. acidophilus strain ATCC 4356 using assays that measure the diffusible inhibition of bacterial growth on an agar surface and that estimate the antimicrobial activity by determining the inhibition of bacterial growth in broth culture. While the first experiment studied the ability of the materials to inhibit bacterial growth on a surface (simulating plaque formation), the second determined the materials’ action against the bacteria in liquid suspension (such as saliva). All procedures were performed under aseptic conditions. The same operator applied the materials in all of the assays.
2.1.
Agar diffusion assay—paper disk
Each material was applied to 6-mm sterile paper disks (Becton Dickinson and Company, Sparks, MD, USA) using the sealants’ applicator. Approximately 12 mg of the materials were placed onto the paper disk and light-activated for 20 s using a Demetron A.2 LED curing device (Kerr Corporation, Orange, CA, USA) with an output of 1000 W/cm2. Blank disks (no material applied) were used as controls. Inocula from frozen stock cultures were cultivated in Wilkins-Chalgren (W-C) broth (Oxoid Ltd., Basingstoke, Hampshire, England, UK) at 37 8C in ambient atmosphere, after being screened by Gram-staining to confirm purity. Loopful inoculations of S. mutans and L. acidophilus were transferred to 9 mL of appropriate broth and incubated at 37 8C under anaerobic conditions. Bacterial suspensions were prepared to 0.5 MacFarland standard16 and diluted to a 1:10 concentration with W-C broth. Two hundred microliters of the 1:10 dilution were then taken and spread-plated using a ‘‘hockey stick’’ on a turntable to ensure confluent bacterial distribution on the plates. Test specimens were immediately placed on the freshly inoculated agar plates and aerobically incubated for 48 h at 37 8C. Each plate contained four disks; one of each sealant group and a blank disk. This assay was performed four times. The diameters of the zone of inhibition of bacterial growth around the disks were measured using a caliper. If a zone of inhibition was not evident, the disk was removed to determine if there was growth under it. If inhibition was evident under the disk this outcome was treated as a qualitative observation indicating retention of antibacterial activity on the contacted surface. Diffusible activity was defined as a zone of inhibition greater than the 6-mm diameter of the disk. Both bacterial strains tested yielded confluent growth under the blank disks.
2.2.
Agar diffusion assay—enamel disk
Thirty-two enamel disks (6 mm in diameter and 2 mm thick) were cut from the labial surface of 32 lower anterior bovine teeth and were sterilized using ethylene oxide gas at the beginning of the experiment. The enamel surfaces of the disks were acid-etched with 35% phosphoric acid (Scotchbond Phosphoric Etchant, 3M ESPE, St. Paul, MN, USA) for 15 s, thoroughly rinsed with distilled water, and dried with compressed air. Materials were applied and the tests
Table 1 – Materials composition. Material
Type
Manufacturer
Composition
Clinpro sealant
Pit and fissure sealant
3M ESPE, St. Paul, MN, USA
Triethylene glycol dimethacrylate, bisphenol A diglycidyl ether dimethacrylate, tetrabutylammonium tetrafluoroborate, silane treated silica
Embrace WetBond pit and fissure sealant
Pit and fissure sealant
Pulpdent Corporation, Watertown, MA, USA
Di–tri multifunctional monomers in an acid-integrating network, fluoride
UltraSeal XT plus
Pit and fissure sealant
Ultradent Products, Inc., South Jordan, UT, USA
Diurethane dimethacrylate, bisphenol A diglycidyl ether dimethacrylate, fluoride
389
journal of dentistry 38 (2010) 387–391
conducted as described for the paper disks in the agar diffusion assay as described above.
2.3.
Planktonic growth inhibition
For this test, the materials were applied to the walls of individual wells of a 96-well, flat-bottomed microtiter plate (Greiner Bio-One GmbH, Germany) and the bacterial outgrowth measured with a spectrophotometer. Approximately 5 mg of each material was brushed to the sidewalls of the wells and light-activated for 20 s using the Demetron A.2 unit with an output of 1000 W/cm2. Those were tested after 30 min, 24 h, and 48 h of washing in distilled water, or after no washing. Controls were non-treated wells incubated with the bacteria. This experiment was performed in triplicate wells in triplicate microtiter plates. A volume of 200 ml of a bacterial suspension (3 103 CFU/ well of S. mutans and 3 105 CFU/well of L. acidophilus) was added into each well. This volume was sufficient to cover the entire painted surface of the treated sidewall. The antibacterial activities of the tested materials were measured as inhibition ofbacterialgrowthcomparedtothatinuntreatedcontrolwellsas determined by measuring change in optical density [OD (biomass)]atl650 nmusingaVMaxMicroplateReader(Molecular Devices, Sunnyvale, CA) after incubation for 16 h at 37 8C. Optical density value <0.1 was considered total inhibition of bacterial growth with no recoverable CFU from the well.
3.
Results
Results are shown in Tables 2–5. All materials had demonstrable inhibition of bacterial surface growth when tested on paper disks. Based on diffusible zones of inhibition, L. acidophilus was more sensitive to Clinpro than was S. mutans. Clinpro was more effective against L. acidophilus than were the
Table 2 – Zones of surface growth inhibition (diameter in mm, mean W SD) of pit and fissure sealants against S. mutans and L. acidophilus using the agar diffusion test— paper disk. n = 4.
L. acidophilus S. mutans
Control
Clinpro
Embrace
UltraSeal
0.0 0.0 0.0 0.0
17.6 2.8 6.8 0.5
6.0 0.0a 7.9 1.3
6.0 0.0a 6.0 0.0a
a Inhibition zones = 6 mm should be interpreted as non-diffusible contact inhibition of the bacterial lawn under the disk.
Table 3 – Zones of surface growth inhibition (diameter in mm, mean W SD) of pit and fissure sealants against S. mutans and L. acidophilus using agar diffusion method— enamel disk. n = 4. Control L. acidophilus S. mutans
0.0 0.0 0.0 0.0
Clinpro
Embrace
9.8 0.3 6.0 0.0b
a
<6.0 6.5 0.0
UltraSeal 6.0 0.0b <6.0a
a
Inhibition zones indicated as <6 mm are attributable to the irregular contact surface of the manually prepared disks. b Inhibition zones = 6 mm should be interpreted as non-diffusible contact inhibition of the bacterial lawn under the disk.
other two products. In contrast, Embrace had better activity against S. mutans than against L. acidophilus. UltraSeal had no discernible diffusible activity against either strain, but was effective at contact inhibition of the surface growth of both strains. In other words, no bacterial growth was noticed on the bacterial lawn where in contact with the test disk. The diffusible activities of the products applied to enamel disks were uniformly less than those with the paper disks (Table 3). The zones indicated as <6 mm observed with the dentin disks are attributable to the irregular contact surface of the manually prepared disks. Zones 6 mm should be interpreted
Table 4 – Bacterial growth as optical densities (mean W SD) of L. acidophilus suspensions exposed to pit and fissure sealants. n = 9.
Control Clinpro Embrace UltraSeal a
No wash
30-min wash
24-h wash
48-h wash
0.455 0.019 0.075 0.010 0.140 0.029a 0.056 0.002
0.431 0.014 0.075 0.005 0.086 0.005 0.086 0.003
0.429 0.017 0.077 0.003 0.098 0.029 0.067 0.005
0.405 0.012 0.103 0.026 0.098 0.065 0.106 0.026
Increased optical density in the no wash wells of Embrace is due to the product (artifact) and not due to bacterial growth.
Table 5 – Bacterial growth as optical densities (mean W SD) of S. mutans suspensions exposed to pit and fissure sealants. n = 9.
Control Clinpro Embrace UltraSeal a
No wash
30-min wash
24-h wash
48-h wash
0.441 0.024 0.068 0.007 0.117 0.018a 0.051 0.002
0.557 0.060 0.073 0.005 0.088 0.008 0.066 0.001
0.423 0.019 0.053 0.003 0.054 0.003 0.341 0.044
0.398 0.021 0.113 0.028 0.054 0.004 0.433 0.026
Increased optical density in the no wash wells of Embrace is due to the product (artifact) and not due to bacterial growth.
390
journal of dentistry 38 (2010) 387–391
as direct, non-diffusible (contact) inhibition of the bacterial lawn under the disk. In the planktonic growth inhibition assay, both bacterial strains achieved reproducible OD in the untreated control wells (Tables 4 and 5). Clinpro had reduced activity against both bacteria after two 24-h washes (48-h wash column), but it was still active compared to the controls. UltraSeal quickly lost activity against S. mutans with washing, but activity was retained after the 30-min wash. L. acidophilus was more sensitive than S. mutans to UltraSeal. Embrace retained its antibacterial activity against both bacteria over time.
4.
Discussion
Results of the agar diffusion and planktonic growth inhibition assays showed that all of the tested materials have antibacterial effects. Therefore, the hypothesis that resin-based fluoride-containing sealants have antibacterial effect against S. mutans and L. acidophilus was accepted. The diffusibility of the sealants when in contact with the bacteria and their potential to inhibit the bacteria growth was evaluated using agar diffusion assays. Each of the materials was able to inhibit bacterial growth when applied to paper and enamel disks. However, smaller inhibition zones were observed when the materials were applied to enamel than to paper disks. The reason for that is unclear. Nevertheless, enamel is the substrate involved clinically and should be the carrier option in in vitro studies. Most studies testing the antibacterial performance of sealants are performed using punched wells.13,14 To the best of our knowledge, no study has been published using enamel disks as the carrier. Inhibiting bacterial metabolism by fluoride has been reported as a result of diffusion of hydrogen fluoride into the bacterial culture.17 Even though all of the tested sealants contain fluoride, Clinpro and Embrace were the only materials to show discernible inhibition zones when tested on enamel disks against L. acidophilus and S. mutans. Another study has shown that Teethmate-F (Kuraray Medical Inc., Tokyo, Japan) was the only active fluoride-containing sealant with shortterm antibacterial effects against S. mutans.8 The lack of antibacterial effects of some fluoride-containing sealants might relate to the method of incorporation of that ion into the material. Methods used to incorporate fluoride into sealants might impact on the amount of fluoride released and its characteristics. The two main approaches of fluoride incorporation are: (1) addition of soluble fluoride salt to unpolymerized resin; and (2) incorporation of an organic fluoride compound to unpolymerized resin. It has been suggested that mechanical properties of sealants produced by the first method are decreased because fluoride releases from the dissolution of soluble fluoride salt. Meanwhile, incorporation of fluoride via the second method has shown long-term and low-level fluoride release.18 The results for Clinpro somewhat agree with that. Clinpro has an organic fluoride compound, tetrabutylammonium, tetrafluoroborate, which could be responsible for the relatively stable antibacterial effects over time. On the other hand, Embrace and UltraSeal are manufactured by the first method (communication with manufacturers). While
the former retained its antibacterial effects longer, the latter poorly performed against S. mutans in the planktonic growth inhibition assay. Those results are somewhat contradictory considering that both materials have fluoride added in the same manner. Different amounts of available fluoride in those materials may be the cause for such distinct results. Also, notice that the increased OD in the no wash wells of Embrace is due to the product and not due to bacterial growth. Immediately after incubation, the solution in the wells with Embrace disks became milky. That was likely to interfere with the OD readings. Washing gets rid of this artifact, but does not seem to reduce antibacterial activity. The release of fluoride is recognized as a ‘‘burst effect’’ that declines after the first few days. In a study with four different fluoride-containing sealants, none exhibited antibacterial activity after one month of aging.13 In the planktonic growth inhibition assay in this study, Embrace retained its antibacterial effect longer than the other materials. The clinical relevance of this finding is uncertain at this time as sealants potentially might be recharged with fluoride.19 A limitation of the study is the set-up used for the planktonic growth inhibition assay. That was performed in 96-well microtiter plates, which are made of plastic. The results of this assay may be overestimated considering the substrate, i.e., plastic, used. Nonetheless, all materials tested may present antibacterial effects in vivo, which may help in prevention of caries development.
5.
Conclusions
All of the tested sealant materials were capable of inhibiting L. acidophilus and S. mutans growth when in contact. Embrace had the more persistent antibacterial activity when in solution, especially against S. mutans. Such in vitro effects reflect in vivo antibacterial activities of fluoride-containing sealants placed on enamel.
references
1. Griffin SO, Oong E, Kohn W, Vidakovic B, Gooch BF, et al. CDC Dental Sealant Systematic Review Work Group. The effectiveness of sealants in managing caries lesions. Journal of Dental Research 2008;87:169–74. 2. Simonsen RJ. Cost effectiveness of pit and fissure sealant at 10 years. Quintessence International 1989;20:75–82. 3. Wendt LK, Koch G, Birkhed D. On the retention and effectiveness of fissure sealant in permanent molars after 15–20 years: a cohort study. Community Dentistry and Oral Epidemiology 2001;29:302–7. 4. Kanellis MJ, Warren JJ, Levy SM. Comparison of air abrasion versus acid etch sealant techniques: six-month retention. Pediatric Dentistry 1997;19:258–61. 5. Baratieri LN, Monteiro S. Influence of acid type (phosphoric or maleic) on the retention of pit and fissure sealant: an in vivo study. Quintessence International 1994;25:749–55. 6. Emilson CG, Bergenholtz G. Antibacterial activity of dentinal bonding agents. Quintessence International 1993;24:511–5. 7. Mertz-Fairhurst EJ, Schuster GS, Fairhurst CW. Arresting caries by sealants: results of a clinical study. Journal of the American Dental Association 1986;112:194–7.
journal of dentistry 38 (2010) 387–391
8. Loyola-Rodriguez JP, Garcia-Godoy F. Antibacterial activity of fluoride release sealants on mutans streptococci. The Journal of Clinical Pediatric Dentistry 1996;20:109–11. 9. Featherstone JD. The science and practice of caries prevention. Journal of American Dental Association 2000;131:887–99. 10. Morphis TL, Toumba KJ. Retention of two fluoride pit-andfissure sealants in comparison to a conventional sealant. International Journal of Paediatric Dentistry 1998;8:203–8. 11. Koch MJ, Garcı´a-Godoy F, Mayer T, Staehle HJ. Clinical evaluation of Helioseal F fissure sealant. Clinical Oral Investigations 1997;1:199–202. 12. Jensen OE, Billings RJ, Featherstone JD. Clinical evaluation of Fluoroshield pit and fissure sealant. Clinical Preventive Dentistry 1990;12:24–7. 13. Matalon S, Slutzky H, Mazor Y, Weiss EI. Surface antibacterial properties of fissure sealants. Pediatric Dentistry 2003;25:43–8. 14. Menon Preetha V, Shashikiran ND, Reddy VV. Comparison of antibacterial properties of two fluoride-releasing and a
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nonfluoride-releasing pit and fissure sealants. Journal of the Indian Society of Pedodontics and Preventive Dentistry 2007;25:133–6. Featherstone JD. The continuum of dental caries–evidence for a dynamic disease process. Journal of Dental Research 2004;83(Spec Iss C):C39–42. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology 1966;45: 493–6. Whitford GM, Schuster GS, Pashley DH, Venkateswarlu P. Fluoride uptake by Streptococcus mutans 6715. Infection and Immunity 1977;18:680–7. National Institute of Dental Research. Fluoride-releasing sealants. Journal of the American Dental Association 1985;110:90. Kavaloglu Cildir S, Sandalli N. Compressive strength, surface roughness, fluoride release and recharge of four new fluoride-releasing fissure sealants. Dental Materials Journal 2007;26:335–41.
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Antibacterial surface properties of fluoride-containing resin-based sealants Supawadee Naorungroj a, Hong-Hong Wei b, Roland R. Arnold b,c, Edward J. Swift Jr.a, Ricardo Walter a,* a
The University of North Carolina at Chapel Hill, School of Dentistry, Department of Operative Dentistry, Chapel Hill, NC 27599-7450, USA The University of North Carolina at Chapel Hill, School of Dentistry, North Carolina Oral Health Institute, Chapel Hill, NC 27599-7450, USA c The University of North Carolina at Chapel Hill, School of Dentistry, Department of Diagnostic Sciences & General Dentistry, Chapel Hill, NC 27599-7450, USA b
article info
abstract
Article history:
Objectives: The aim of the present study was to determine the antibacterial properties of
Received 17 September 2009
three resin-based pit and fissure sealant products: Clinpro (3M ESPE), Embrace (Pulpdent),
Received in revised form
and UltraSeal XT plus (Ultradent).
11 January 2010
Methods: The antibacterial effects of the sealants were tested in both an agar diffusion assay
Accepted 12 January 2010
and a planktonic growth inhibition assay using Streptococcus mutans and Lactobacillus acidophilus. The materials were applied to paper and enamel disks in the former and on the side walls of 96-well microtiter plates on the latter.
Keywords:
Results: All materials showed either diffusible or contact antibacterial effects in the agar
Sealants
diffusion assays. The effect was diminished when enamel disks were used as substrate. In
Fluoride
the planktonic growth inhibition assay, Clinpro had its effect reduced, but retained activity
Antibacterial
against both bacteria over time. L. acidophilus was more sensitive than S. mutans to UltraSeal. Embrace retained antibacterial activity against both bacteria over time. Conclusions: Within the limitations of this in vitro study it can be concluded that all materials are capable of contact inhibition of L. acidophilus and S. mutans growth. Embrace has the longer lasting antibacterial activity when in solution, especially against S. mutans. # 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Sealants have been used for decades as a preventive measure against caries development in susceptible pits and fissures. Their beneficial effect, which has been recently corroborated in a meta-analysis,1 depends largely on retention.2 This mechanical retention is achieved by etching enamel with phosphoric acid,3–5 which also reduces the bacterial count that may be present on the treated surfaces.6 Once the area is sealed, the isolation of the bacteria underneath the sealant
from oral nutrients helps minimize the development of caries.7 It has been suggested that this positive effect could possibly be enhanced by adding fluoride to the sealant material.8 Fluoride is able to incorporate into enamel, making it more resistant to acid degradation; it also has antibacterial effects against cariogenic oral bacteria.9 However, although it seems obvious that fluoride would improve the protective effect of sealants, equivocal results have been reported. Clinical studies where sealants were applied to caries-free surfaces have
* Corresponding author at: The University of North Carolina at Chapel Hill, School of Dentistry, Department of Operative Dentistry, 433 Brauer Hall CB #7450, Chapel Hill, NC 27599-7450, USA. Tel.: +1 919 966 2770; fax: +1 919 966 5660. E-mail address: [email protected] (R. Walter). 0300-5712/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2010.01.005
388
journal of dentistry 38 (2010) 387–391
shown no significant difference in caries inhibition or retention rate between conventional and fluoride-containing sealants.10–12 On the other hand, in vitro studies have demonstrated the potential inhibitory effect of some resinbased fluoride-containing sealants on cariogenic oral bacteria.8,13,14 This potential antibacterial effect may be advantageous, for instance, when caries is inadvertently left in the surface to be sealed.7 In vitro studies testing antibacterial potential of materials are often performed with mutans streptococci and lactobacilli species, which are believed to be the two major groups of bacteria involved in the caries process.9 In those groups are included Streptococcus mutans and Lactobacillus acidophilus, respectively. These bacteria produce acids, primarily lactic acid, that diffuse into the enamel and dissolve the mineral crystals to initiate the caries process.15 This study does not evaluate the demineralization effects of those bacteria in enamel but uses enamel as a substrate in one of the experiments. To the best knowledge of the authors, no other study has used enamel as a substrate to study the antibacterial effects of sealants. The purpose of this study is to evaluate the antibacterial surface properties of three commercially available resinbased fluoride-containing sealants using an agar diffusion test to determine the inhibition of surface growth and a planktonic growth inhibition test to determine the inhibition in broth culture. The hypothesis is that the resin-based fluoride-containing sealants have antibacterial activities under both test conditions against both S. mutans and L. acidophilus.
2.
Materials and methods
Materials used in the study are listed in Table 1. They were tested against S. mutans strain ATCC 10449 (serotype c) and L. acidophilus strain ATCC 4356 using assays that measure the diffusible inhibition of bacterial growth on an agar surface and that estimate the antimicrobial activity by determining the inhibition of bacterial growth in broth culture. While the first experiment studied the ability of the materials to inhibit bacterial growth on a surface (simulating plaque formation), the second determined the materials’ action against the bacteria in liquid suspension (such as saliva). All procedures were performed under aseptic conditions. The same operator applied the materials in all of the assays.
2.1.
Agar diffusion assay—paper disk
Each material was applied to 6-mm sterile paper disks (Becton Dickinson and Company, Sparks, MD, USA) using the sealants’ applicator. Approximately 12 mg of the materials were placed onto the paper disk and light-activated for 20 s using a Demetron A.2 LED curing device (Kerr Corporation, Orange, CA, USA) with an output of 1000 W/cm2. Blank disks (no material applied) were used as controls. Inocula from frozen stock cultures were cultivated in Wilkins-Chalgren (W-C) broth (Oxoid Ltd., Basingstoke, Hampshire, England, UK) at 37 8C in ambient atmosphere, after being screened by Gram-staining to confirm purity. Loopful inoculations of S. mutans and L. acidophilus were transferred to 9 mL of appropriate broth and incubated at 37 8C under anaerobic conditions. Bacterial suspensions were prepared to 0.5 MacFarland standard16 and diluted to a 1:10 concentration with W-C broth. Two hundred microliters of the 1:10 dilution were then taken and spread-plated using a ‘‘hockey stick’’ on a turntable to ensure confluent bacterial distribution on the plates. Test specimens were immediately placed on the freshly inoculated agar plates and aerobically incubated for 48 h at 37 8C. Each plate contained four disks; one of each sealant group and a blank disk. This assay was performed four times. The diameters of the zone of inhibition of bacterial growth around the disks were measured using a caliper. If a zone of inhibition was not evident, the disk was removed to determine if there was growth under it. If inhibition was evident under the disk this outcome was treated as a qualitative observation indicating retention of antibacterial activity on the contacted surface. Diffusible activity was defined as a zone of inhibition greater than the 6-mm diameter of the disk. Both bacterial strains tested yielded confluent growth under the blank disks.
2.2.
Agar diffusion assay—enamel disk
Thirty-two enamel disks (6 mm in diameter and 2 mm thick) were cut from the labial surface of 32 lower anterior bovine teeth and were sterilized using ethylene oxide gas at the beginning of the experiment. The enamel surfaces of the disks were acid-etched with 35% phosphoric acid (Scotchbond Phosphoric Etchant, 3M ESPE, St. Paul, MN, USA) for 15 s, thoroughly rinsed with distilled water, and dried with compressed air. Materials were applied and the tests
Table 1 – Materials composition. Material
Type
Manufacturer
Composition
Clinpro sealant
Pit and fissure sealant
3M ESPE, St. Paul, MN, USA
Triethylene glycol dimethacrylate, bisphenol A diglycidyl ether dimethacrylate, tetrabutylammonium tetrafluoroborate, silane treated silica
Embrace WetBond pit and fissure sealant
Pit and fissure sealant
Pulpdent Corporation, Watertown, MA, USA
Di–tri multifunctional monomers in an acid-integrating network, fluoride
UltraSeal XT plus
Pit and fissure sealant
Ultradent Products, Inc., South Jordan, UT, USA
Diurethane dimethacrylate, bisphenol A diglycidyl ether dimethacrylate, fluoride
389
journal of dentistry 38 (2010) 387–391
conducted as described for the paper disks in the agar diffusion assay as described above.
2.3.
Planktonic growth inhibition
For this test, the materials were applied to the walls of individual wells of a 96-well, flat-bottomed microtiter plate (Greiner Bio-One GmbH, Germany) and the bacterial outgrowth measured with a spectrophotometer. Approximately 5 mg of each material was brushed to the sidewalls of the wells and light-activated for 20 s using the Demetron A.2 unit with an output of 1000 W/cm2. Those were tested after 30 min, 24 h, and 48 h of washing in distilled water, or after no washing. Controls were non-treated wells incubated with the bacteria. This experiment was performed in triplicate wells in triplicate microtiter plates. A volume of 200 ml of a bacterial suspension (3 103 CFU/ well of S. mutans and 3 105 CFU/well of L. acidophilus) was added into each well. This volume was sufficient to cover the entire painted surface of the treated sidewall. The antibacterial activities of the tested materials were measured as inhibition ofbacterialgrowthcomparedtothatinuntreatedcontrolwellsas determined by measuring change in optical density [OD (biomass)]atl650 nmusingaVMaxMicroplateReader(Molecular Devices, Sunnyvale, CA) after incubation for 16 h at 37 8C. Optical density value <0.1 was considered total inhibition of bacterial growth with no recoverable CFU from the well.
3.
Results
Results are shown in Tables 2–5. All materials had demonstrable inhibition of bacterial surface growth when tested on paper disks. Based on diffusible zones of inhibition, L. acidophilus was more sensitive to Clinpro than was S. mutans. Clinpro was more effective against L. acidophilus than were the
Table 2 – Zones of surface growth inhibition (diameter in mm, mean W SD) of pit and fissure sealants against S. mutans and L. acidophilus using the agar diffusion test— paper disk. n = 4.
L. acidophilus S. mutans
Control
Clinpro
Embrace
UltraSeal
0.0 0.0 0.0 0.0
17.6 2.8 6.8 0.5
6.0 0.0a 7.9 1.3
6.0 0.0a 6.0 0.0a
a Inhibition zones = 6 mm should be interpreted as non-diffusible contact inhibition of the bacterial lawn under the disk.
Table 3 – Zones of surface growth inhibition (diameter in mm, mean W SD) of pit and fissure sealants against S. mutans and L. acidophilus using agar diffusion method— enamel disk. n = 4. Control L. acidophilus S. mutans
0.0 0.0 0.0 0.0
Clinpro
Embrace
9.8 0.3 6.0 0.0b
a
<6.0 6.5 0.0
UltraSeal 6.0 0.0b <6.0a
a
Inhibition zones indicated as <6 mm are attributable to the irregular contact surface of the manually prepared disks. b Inhibition zones = 6 mm should be interpreted as non-diffusible contact inhibition of the bacterial lawn under the disk.
other two products. In contrast, Embrace had better activity against S. mutans than against L. acidophilus. UltraSeal had no discernible diffusible activity against either strain, but was effective at contact inhibition of the surface growth of both strains. In other words, no bacterial growth was noticed on the bacterial lawn where in contact with the test disk. The diffusible activities of the products applied to enamel disks were uniformly less than those with the paper disks (Table 3). The zones indicated as <6 mm observed with the dentin disks are attributable to the irregular contact surface of the manually prepared disks. Zones 6 mm should be interpreted
Table 4 – Bacterial growth as optical densities (mean W SD) of L. acidophilus suspensions exposed to pit and fissure sealants. n = 9.
Control Clinpro Embrace UltraSeal a
No wash
30-min wash
24-h wash
48-h wash
0.455 0.019 0.075 0.010 0.140 0.029a 0.056 0.002
0.431 0.014 0.075 0.005 0.086 0.005 0.086 0.003
0.429 0.017 0.077 0.003 0.098 0.029 0.067 0.005
0.405 0.012 0.103 0.026 0.098 0.065 0.106 0.026
Increased optical density in the no wash wells of Embrace is due to the product (artifact) and not due to bacterial growth.
Table 5 – Bacterial growth as optical densities (mean W SD) of S. mutans suspensions exposed to pit and fissure sealants. n = 9.
Control Clinpro Embrace UltraSeal a
No wash
30-min wash
24-h wash
48-h wash
0.441 0.024 0.068 0.007 0.117 0.018a 0.051 0.002
0.557 0.060 0.073 0.005 0.088 0.008 0.066 0.001
0.423 0.019 0.053 0.003 0.054 0.003 0.341 0.044
0.398 0.021 0.113 0.028 0.054 0.004 0.433 0.026
Increased optical density in the no wash wells of Embrace is due to the product (artifact) and not due to bacterial growth.
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journal of dentistry 38 (2010) 387–391
as direct, non-diffusible (contact) inhibition of the bacterial lawn under the disk. In the planktonic growth inhibition assay, both bacterial strains achieved reproducible OD in the untreated control wells (Tables 4 and 5). Clinpro had reduced activity against both bacteria after two 24-h washes (48-h wash column), but it was still active compared to the controls. UltraSeal quickly lost activity against S. mutans with washing, but activity was retained after the 30-min wash. L. acidophilus was more sensitive than S. mutans to UltraSeal. Embrace retained its antibacterial activity against both bacteria over time.
4.
Discussion
Results of the agar diffusion and planktonic growth inhibition assays showed that all of the tested materials have antibacterial effects. Therefore, the hypothesis that resin-based fluoride-containing sealants have antibacterial effect against S. mutans and L. acidophilus was accepted. The diffusibility of the sealants when in contact with the bacteria and their potential to inhibit the bacteria growth was evaluated using agar diffusion assays. Each of the materials was able to inhibit bacterial growth when applied to paper and enamel disks. However, smaller inhibition zones were observed when the materials were applied to enamel than to paper disks. The reason for that is unclear. Nevertheless, enamel is the substrate involved clinically and should be the carrier option in in vitro studies. Most studies testing the antibacterial performance of sealants are performed using punched wells.13,14 To the best of our knowledge, no study has been published using enamel disks as the carrier. Inhibiting bacterial metabolism by fluoride has been reported as a result of diffusion of hydrogen fluoride into the bacterial culture.17 Even though all of the tested sealants contain fluoride, Clinpro and Embrace were the only materials to show discernible inhibition zones when tested on enamel disks against L. acidophilus and S. mutans. Another study has shown that Teethmate-F (Kuraray Medical Inc., Tokyo, Japan) was the only active fluoride-containing sealant with shortterm antibacterial effects against S. mutans.8 The lack of antibacterial effects of some fluoride-containing sealants might relate to the method of incorporation of that ion into the material. Methods used to incorporate fluoride into sealants might impact on the amount of fluoride released and its characteristics. The two main approaches of fluoride incorporation are: (1) addition of soluble fluoride salt to unpolymerized resin; and (2) incorporation of an organic fluoride compound to unpolymerized resin. It has been suggested that mechanical properties of sealants produced by the first method are decreased because fluoride releases from the dissolution of soluble fluoride salt. Meanwhile, incorporation of fluoride via the second method has shown long-term and low-level fluoride release.18 The results for Clinpro somewhat agree with that. Clinpro has an organic fluoride compound, tetrabutylammonium, tetrafluoroborate, which could be responsible for the relatively stable antibacterial effects over time. On the other hand, Embrace and UltraSeal are manufactured by the first method (communication with manufacturers). While
the former retained its antibacterial effects longer, the latter poorly performed against S. mutans in the planktonic growth inhibition assay. Those results are somewhat contradictory considering that both materials have fluoride added in the same manner. Different amounts of available fluoride in those materials may be the cause for such distinct results. Also, notice that the increased OD in the no wash wells of Embrace is due to the product and not due to bacterial growth. Immediately after incubation, the solution in the wells with Embrace disks became milky. That was likely to interfere with the OD readings. Washing gets rid of this artifact, but does not seem to reduce antibacterial activity. The release of fluoride is recognized as a ‘‘burst effect’’ that declines after the first few days. In a study with four different fluoride-containing sealants, none exhibited antibacterial activity after one month of aging.13 In the planktonic growth inhibition assay in this study, Embrace retained its antibacterial effect longer than the other materials. The clinical relevance of this finding is uncertain at this time as sealants potentially might be recharged with fluoride.19 A limitation of the study is the set-up used for the planktonic growth inhibition assay. That was performed in 96-well microtiter plates, which are made of plastic. The results of this assay may be overestimated considering the substrate, i.e., plastic, used. Nonetheless, all materials tested may present antibacterial effects in vivo, which may help in prevention of caries development.
5.
Conclusions
All of the tested sealant materials were capable of inhibiting L. acidophilus and S. mutans growth when in contact. Embrace had the more persistent antibacterial activity when in solution, especially against S. mutans. Such in vitro effects reflect in vivo antibacterial activities of fluoride-containing sealants placed on enamel.
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