Effect of gels containing stannous fluoride on oral bacteria - an in vitro study C. C. Tseng, DDS, MS* L. F. Wolff, PhD, DDSt D. M. Aeppli, P h D t
Key words: Bacterial inhibition, oral bacteria,
(Received for publication May 1991. Revised September 1991. Accepted December 1991.)
Introduction It is well established that stannous fluoride (SnF,) is very effective in preventing caries,' In recent years, SnF, use in concentrations between 0.02-1.64 per cent has resulted in a reduction of human periodontal disease.'-' The mechanism, however, through which SnF, prevents dental caries and reduces periodontal disease has not been completely explained. On the other hand, most investigators have agreed that the reduction in solubility of tooth surfaces by formation of relatively insoluble stannous phosphate compounds provides one form of protection induced by SnF, t~eatment.~.' Another and possibly more important protective mechanism of SnF, is its antimicrobial effect on the bacteria found in dental plaque.* This antimicrobial effect on oral bacteria has been attributed in large measure to the divalent cation, tin (Sn+ +). It is believed the tin in SnF, may exert its protective effect by interacting with negatively charged plaque components to alter bacterial adhesion/cohesi~n.~~'~ In addition to the antibacterial effectiveness of the tin ion, fluoride itself has been shown to decrease acid production by bacteria probably through the mechanism of inhibiting the enzyme enolase." Although clinical studies have been performed evaluating the clinical effectiveness of SnF, on both caries and periodontal d i ~ e a s e ~ ~there ' ~ - ' is~ little information available demonstrating the in vitro growth-inhibiting effect of SnF, on the wide variety
*Periodontal Division, Dental Department, National Cheng Kung University Medical Center, Tainan, Taiwan, ROC.
?Department of Preventive Sciences, School of Dentistry, University of Minnesota, USA.
stannous fluoride.
Abstract The purposes of this investigation were to evaluate and compare the antimicrobial effect of (1) twelve 0.4 per cent stannous fluoride (SnF,) commercial products and (2)different concentrations of SnF, (range = 0.02 to 3.28 per cent). The antibacterial inhibitory effect of various SnF, gels was evaluated as to their effectiveness against oral plaque bacteria including strains of S. mutans, S. sanguis, S. sobrinus, A. viscosus, A. actinomycetemcomitans, and 8.intermedius. When twelve different commercial preparations of 0.4 per cent SnF, were compared for inhibitory effect on plaque bacteria, several of the SnF, preparations were significantly more effective in inhibiting oral bacteria (pc0.05). With increasing concentration of SnF2,there was a comparable increase in the inhibitory effect on the oral bacteria tested (r* ranged from 0.867to 0.996). SnF, at a concentration of 0.4 per cent had a similar antibacterial effect to 0.12 per cent chlorhexidine. This in vitro study demonstrated that certain SnF, products are highly effective in inhibiting the growth of bacteria often found in plaque, and this inhibitory effect is directly related to the concentration of the SnF2.
368
Australian Dental Journal 1992;37(5):368-73.
of bacteria commonly found in plaque associated with dental disease. Therefore, the purpose of this study was to evaluate the antimicrobial effect of various commercial 0.4 per cent SnF, preparations on oral plaque bacteria and also to compare the inhibitory effect of various concentrations of SnF, with 0.12 per cent chlorhexidine.
Materials and methods Bacterial cultures Bacterial strains which have been associated with human dental caries, gingivitis and periodontitis were selected for use in this study. The exception was Escherichia coli which was a non-oral microorganism. Microbial strains chosen were Streptococcus mutans strain RL19, Streptococcus sobrinus strain 671 5, Streptococcus sanguis strain S4124 and S81, Actinomyces viscosus strain AVR and T-JY-2BY Actinobacillus actinomycetemcomitans strain Y4, Bacteroides intermedius, a wild type strain, and E. coli. Growth and test media Log phase cultures of all strains S. mutans, S. sobrinus, S. sanguis, A. viscosus, A. actinomycetemcomitans, and E. coli were grown in Todd-Hewitt broth.$ Bacteroides intermedius was grown to log phase in a basal broth medium which consisted of 1.0 per cent m/v trypticase,§ 1.0 per cent mlv proteopeptone,$ 0.5 per cent m/v yeast extract,$O.5 per cent m/v sodium chloride, 1 10 pglmL vitamin K-hemin solution,! and horse serum** at a final concentration of 4 per cent. All bacterial cultures were incubated anaerobically at 37°C. The agar medium on which the bacterial inhibition assay was performed included trypticase soy agars 5 per cent defibrinated sheep blood, 0.0005 per cent hemin,! and 0.00005 per cent menadione.1 Agar bacterial inhibition assay The agar bacterial inhibition assay is a modification of the method described earlier by Wolff and Duncan16 and is similar to the method described by Ostela and Tenovuo (1990)? A log phase broth culture of the test strain of bacteria was adjusted with a spectrophotometertt to an optical density of 0.3 (E540) using sterile Todd Hewitt broth for S. mutans, S. sobrinus, S. sanguis, A viscosus,
$Difco Laboratories, Detroit, MI, USA. BBBL Microbiology Systems, Cockeysville, MD, USA. ]EM Science, Cherry Hill, NJ, USA. (Sigma Chemical Co., St Louis, MO, USA. **Gibco Laboratories, Grand Island, New York, MY, USA. TtSpectronic 20, Bausch & Lomb, Rochester, NY, USA. Australian Dental Journal 1992;37:5.
A. actinomycetemcomitans and E. coli or a basal broth for B. intermedius as the diluent standard. Then a 1:400 dilution of the adjusted bacterial broth culture was made using Todd Hewitt or, in the case of B. intermedius, basal broth. One millilitre of this dilution was pipetted onto the supplemented blood agar plate and the agar surface flooded. The excess fluid was aspirated off with a pipette and the agar surface allowed to dry for 15 minutes. Bacteriological procedures involving preparation of the lawn culture using S. mutans, S. sobrinus, S. sanguis, A. viscosus, A. actinomycetemcomitans and E. coli were performed on a laboratory bench top. Laboratory procedures for preparation of the lawn culture for B. intermedius, were performed in an anaerobic chamber$$ which had an atmosphere of 10 per cent H,, 10 per cent CO, and 80 per cent N,. After the surface of the agar plates was dry, holes were punched into the agar with a sterile cork puncher. The diameter of all wells punched into the agar was 65 mm. A sterile tuberculin syringe was used to add 75 pL of the antiplaque or control agent to the appropriate wells. Glycerinegg served as the negative control and 0.12 per cent chlorhexidinell 1 served as the positive control. Glycerine was the carrying solution for the SnF, gels. The SnF, gels were delivered into wells of the agar plates in the anaerobic chamber to prevent or decrease the risk of oxidation of the stannous ions to the stannic ions. Bottles containing the 0.4 per cent SnF, gels were not opened prior to use in the bacterial inhibition assay. The 0.4 per cent SnF, gels used in these experiments included Gel Kam, Activus, Omnii Gel, Basic Gel, Ultra Gel, Gel Tin, Iradicav, Gel Pro, Flo Gel, Perfect Choice, Easy Gel, and Quick Gel (Table 1). Duplicate plates were used for each experiment. After the antiplaque agent and controls were added to the wells, agar plates were incubated at 37 "Cfor seven days in the anaerobic chamber. The diameter of the bacterial zone of inhibition was then measured to the nearest 0.1 mm with a boley gauge. The experimental protocol for the bacterial inhibition assay is shown in Fig. 1 . Statistical analysis For multiple comparison of mean zone of bacterial inhibition by twelve 0.4 per cent SnF, commercial products, Student-Neuman-Keuls (SNK)
$$Coy Manufacturing Co., Ann Arbor, MI, USA. BBColumbia Chemical Industries Inc., Columbus, WN, USA I [Procter & Gamble Co., Cincinnati, Ohio, USA. 369
Table 1. Stannous fluoride gels (0.4 per cent) used in this investigation Gel
Manufacturerlsupplier
Gel-Kam Activus Omnii Gel Basic Gel Ultra-Gel Gel-Tin Iradicav Gel-Pro Flo-Gel Perfect Choice Easy Gel Quick-Gel
Scherer Laboratory Inc., Dallas, Texas, USA. Scherer Laboratory Inc., Dallas, Texas, USA. Dunhall Pharmaceuticals Inc., Stafford, Texas, USA. Basic Dental Products Inc., Miami, Florida, USA. Dental Resources Inc., Long Lake, Minnesota, USA. Young Dental, Maryland Hgts., Maryland, USA. Johnson & Johnson Dental Products Company, East Windsor, New Jersey, USA. Supro Inc., Noblesville, Indiana, USA. Continental Quest Corp., Indianapolis, Indiana, USA. Challenge Products Inc., Osage Beach, Missouri, USA. Du-More Inc., Rogers, Arkansas, USA. Ultra Care Products, Lansdale, Philadelphia, USA.
was utilized. SNK method is applicable for pairwise comparison, with the sample sizes from each group being equal.” T o evaluate the relationship between the zone of bacterial inhibition (Y) and the concentration of SnF, (X), simple linear regression analysis was performed. The zone of inhibition was treated as
the dependent variable and the concentrations of SnF, as the independent variable and three basic assumptions, normality, independence and straight line relationships, were made. The strength of the linear relationship between X and Y was represented in terms of r2 which may vary between 0 and 1. A large value of r2 indicates a strong linear relationship between X and Y.
Results Twelve 0.4 per cent SnF2 products were evaluated for their antimicrobial effect in the agar inhibition assay. Gel Kam, Omnii Gel, Flo Gel, Perfect Choice, and Quick Gel were most effective at inhibiting growth of all bacterial test strains (Table 2). Activus and Gel Pro also showed a similar effect on inhibiting the test strains with the exception of S. sanguis strain S-81.On the other hand, Easy Gel, Basic Gel, Ultra Gel, Gel Tin and Iradicav showed a statistically significant smaller zone of bacterial inhibition for specific bacterial test strains, p I0.05. In the agar inhibition assay, B. inrerrnedius was consistently more sensitive to the SnF, gels, as indicated by the greater zone of bacterial inhibition, while the non-oral bacterium, E. coli, was least sensitive to the SnF, gels. The experimental results described in the previous section demonstrated that several SnF2gels exhibited more antibacterial inhibition than other
r- -0 1:400
dilution
Broth culture of bacterial test strain adjusted to optical density of 0.3 ( E d
0.4% SnF2
Bacterial test strain flooded onto blood agar plate
Allowed to dry for 15 minutes
Zones of bacterial inhibition measured with a boley gaug
punched into agar with identical diameter and depth. Then antiplaque agent or control added to well
Chlorhexidine 0.12% (positive control) Lawn of the bacterial test strain Incubate at 370 C in anaerobic chamber for 5-7 days
Fig. 1 . -Protocol for the bacterial inhibition assay. 370
Australian Dental Journal 1992;37:5.
Table 2.
Mean zones" of bacterial inhibition by twelve 0.4% SnF, preparations 0.4% SnF, preparations
Bacterial test strain
Gel Omnii Kam Gel
Advus
Flo Gel
Perfect Choice
S. mutans
1.75 (0.07) 1.73 (0.00) 2.07 (0.02) 1.67 (0.02) 2.15 (0.07) 2.18 (0.04) 1.77 (0.02) 2.67 (0.07) 0.97 (0.03)
1.65 (0.10) 1.68 (0.04) 2.21 0.18
1.78 (0.18) 1.64 (0.06) 2.18 (0.26) 1.85 (0.07) 2.06 (0.01) 2.01 (0.08) 1.69 (0.05) 2.58 (0.03) 1.11 (0.15)
1.83 (0.22) 1.82 (0.10) 2.31 (0.06) 1.64 (0.01) 2.13 (0 04) 2:Ol (0 08) 1170 (0.07) 2.69 (0.05) 1.00 (0.07)
(RL19) S. sobrinus (6715) S. sanguis (S4124) S. sanguis (S81) A. viscosus (AW A. viscosus (T-JY-2B) A. actinomycetemcomitans (Y4) B. intermedius
E. coli
1.77 (0.05) 1.77 (0.02) 2.33 (0.18) 1.65 (0.07) 2.13 (0.04) 2.15 (0.00) 1.74 (0.02) 2.66 (0.06) 0.91 (0.01)
3 0.07 2.16 (0.08) 2.13 (0.04) 1.80 (0.07) 2.70 (0.00) 0.94 (0.02)
Easy Gel
Quick Gel
kl(y:?
Basic Gel
1 1 . 1 5 1.82 1 1.62 0.00 2.23
l-%%l
I I y.21: 2.25 0.14 0.86 (0.02)
(0.07) 2.07
0.01 1.70
(0.02) 0.07 1.73 1.65 (0.04) (0.07) 2.15 1.79 (0 14) 2:05 (0.07) I(0.03) 1.82 1.50 (0.04) y.02: 2.66 2.03 (0.02) 0.13 1.03 0.96 (0.02) (0.15)
Ultra Gel
Gel Tin
Iradicav
Gel Pro
1.62
2.00
1.96
0.07 1.85
0.11 1.99
0.04 2.00
1.98 (0.03) 1.65 (0.07) 2.03 (0.04) 1.53 (0.04) 2.08 (0.06) 2.12 (0.05) 1.65 (0.00) 2.59 (0.01) 0.88 (0.04)
fl , 0.07 1.63 (0.04) 2.04 (0.01) 2.05 (0.07) 1.60 y.06: 2.35
,, , I
,:y:Jli: :&Jl:, 0.06 1.96
I(0.03) 1.54 (0.06) 2.53
::::
(0.03)
I(0.05)
I
0.09 1.92 (0.19) 2.05
I 1
?% y.00: 2.43
I
:::
(0.01)
*Values represent mean of duplicate experiments. The value of parentheses below the mean is the SD. Mean measurements outlined in box showed a statisticallysignificant smaller zone of bacterial inhibition than other measurements for a specific bacterial test strain. All values are expressed in cm.
SnF, gels. One of these gels, Gel Kam, was chosen to determine how various concentrations of this SnF, product would affect test micro-organisms in the agar bacterial inhibition assay. The mean zones of bacterial inhibition with different concentration of SnF, may be seen in Table 3. These data demonstrated that as SnF, concentration decreased from 3.28 per cent to 0.02 per cent, there was a linear decrease in the mean zone of bacterial inhibition for all strains. There was a statistically significant, high correlation between the zone of bacterial inhibition and concentration of SnF,, with rz between 0.867 and 0.996,p I0.002.The 0.4 per cent SnF, solution gave a similar zone of bacterial inhibition as 0.12 per cent chlorhexidine on test strains ofS. mutans, S. sobrinus, A. viscosus, A. actinomycetemwmitans and B. intermedius. A 0.02 per cent SnF, solution had minimum or no effect on the test strains. Discussion The results of this investigation indicated that the majority of the SnF, products evaluated have considerable antimicrobial activity in the agar bacterial inhibition assay. These findings firther support the observation made by other investigators who reported that SnF, inhibited the growth of S. mutans and A. viscosus.L8*L9 However, it was observed that not all the commercial 0.4 per cent SnF, products evaluated in the agar bacterial inhibition assay were equally effective in inhibiting Australian Dental Journal 1992;37:5.
bacterial growth. Several commercial 0.4 per cent SnF, products were found to exhibit greater antimicrobial activity than many of the other SnF, products tested. Because of the variable findings regarding the antimicrobial effectiveness of different 0.4 per cent SnF, gels, it may be prudent to evaluate these SnF, products or other antimicrobial agents in an inexpensive in vitro assay prior to investigating their effectiveness in clinical studies. An agar bacterial inhibition assay has been widely used to evaluate bacterial resistance to antibioticYz0 and has also been used to test the antimicrobial activity of pulpcapping agents.,' The agar bacterial inhibition assay utilized in this investigation, however, was different or a modification from the test traditionally used to evaluate bacterial sensitivity to antibiotics and was similar to the method recently described by Ostela and Tenovuo.8 In this study, a known quantity of a suspected antimicrobial agent was placed into wells prepared in agar plates onto which a lawn culture of a specific bacterium was placed. In antibiotic susceptibility tests, a known quantity of an antibiotic is generally incorporated into a disc and then placed onto a lawn culture of the test bacterium. The technique used for antibiotic sensitivity testing with a disc was not used in this study because there was a concern the antimicrobial agent, and in this case SnF,, would bind to the disc and not be released or only partially be released into the agar to inhibit bacterial 371
Table 3. Mean zones* of bacterial inhibition with different concentrations of SnF, ~
Bacterial test strain
S.mutans (RL19) S. sobrinus (6715) S. sanguis (S4124) S. sanguis (S8 1) A. viscosus
(AVR)
A. viscosus (T-JY-2B) A. actinomycetemcomitans (Y4) B. intennedius E. coli
Stannous fluoride concentrations ("70) 3.28
1.64
0.8
0.4
0.2
0.1
0.02
4.15 (0.07) 3.45 (0.07) 4.05 (0.07) 2.95 (0.07) 4.20 (0.21) 3.68 (0.04) 3.15 (0.00) 4.30 (0.14) 1.98 (0.03)
3.20 (0.07) 2.77 (0.09) 3.60 (0.06) 2.75 (0.01) 3.24 (0.01) 2.93 (0.05) 2.52 (0.00) 3.65 (0.06) 1.52 (0.05)
2.42 (0.04) 2.16 (0.06) 2.75 (0.07) 2.46 (0.01) 2.83 (0.11) 2.56 (0.06) 2.23 (0.1 1) 3.21 (0.04) 1.18 (0.02)
2.00 (0.00) 1.81 (0.15) 2.20 (0.00) 2.07 (0.02) 2.28 (0.04) 2.23 (0.06) 1.95 (0.00) 2.83 (0.04) 1.00 (0.07)
1.78 (0.04) 1.60 (0.00) 1.83 (0.1 1) 1.79 (0.01) 1.91 (0.06) 1.86 (0.04) 1.69 (0.05) 2.31 (0.01) 0.80 (0.01)
1.51 (0.01) 1.43 (0.03) 1.41 (0.06) 1.59 (0.02) 1.30 (0.07) 1.58 (0.04) 1.46 (0.06) 1.95 (0.08) 0.73 (0.04)
0.80 (0.01)
1.15 (0.07) 0.91 (0.01) 0.93 (0.11) 0.91 (0.01) 0.83 (0.04) 0.85 (0.07) 1.18 (0.00) 0.70 (0.04)
Chlorhexidine 0.12% (positive control) 2.00 (0.00) 1.78 (0.04) 1.95 (0.07) 1.72 (0.09) 2.53 (0.1 1) 2.32 (0.03) 1.95 (0.00) 2.80 (0.07) 1.30 (0.07)
r*t 0.987 0.956 0.996 0.97 0.988 0.977 0.984 0.988 0.867
'MeankSD (cm). Each experiment performed in duplicate and the mean determined. The value in parentheses below the mean is the SD. tr' =Correlation coefficient between zone of bacterial inhibition and concentration of SnF, (for r2 t 0.867, p < 0.002).
growth. Therefore, it was decided to prepare wells in the agar where the problem of the antimicrobial agent binding to a disc would not be a factor. Also, many in vitro tests evaluate antimicrobial susceptibility of a chemotherapeutic agent using a broth dilution assay m e t h ~ d .Because ~ ~ . ~ ~it was desirable to compare the actual concentration of the commercial SnF, products without a dilution factor, the agar inhibition assay was used. Several limitations are still inherent in the agar inhibition assay. For example, diffusion of test agents through the agar medium could be a problem. Binding of an ionic charged antimicrobial agent to the negatively charged agar may reduce its bacterial inhibition effectiveness. In vitro results using this bacterial inhibition assay, therefore, may not be completely transferable to a clinical situation. It was a concern that 0.4 per cent SnF, may lose activity as it aged. The rationale for this was the stannous ion (Sn+ + ) may become oxidized to the stannic ion (Sn+ + + +). In effect, it is believed this would reduce its antibacterial activity and possibly reduce its perceived clinical effectiveness. Experiments in this study, therefore, included only SnF, gels which were not opened prior to their evaluation. It was demonstrated in an earlier investigation, however, that after SnF, bottles were opened and used in a clinical situation for 45 days,1s the residual gel had lost 25 per cent of its available stannous ion. However, in the agar bacterial inhibition assay, the same SnF, gel lost less than 12.5 per cent of its antimicrobial activity. 372
Data reported in this investigation indicated an approximately linear relationship between the concentrations of SnF2 and the antimicrobial activity on the test strains of bacterial. While 3.28 per cent SnF, showed a high antimicrobial activity, 0.02 per cent SnF, had minimal or no activity on the test strains. These observations suggest that 0.02 per cent SnF, may not be a useful antiplaque agent. On the other hand, 0.4 per cent SnF, was as effective as 0.12 per cent chlorhexidine in the agar bacterial inhibition assay. Although, 0.12 per cent chlorhexidine has been found to be very effective as an antiplaque and antigingivitis agent in clinical s t u d i e ~ ,0.4 ~ ~per .~~ cent SnF, was not observed to be effective in reducing plaque and improving gingival health. While both 0.4 per cent SnF, and 0.12 per cent chlorhexidine possess a similar in vitro antimicrobial activity, a potential reason for the lower clinical effectiveness of SnF, in studies by Wolff et d i 5 and Tolle et aP6 could be due to its lower substantivity when compared with chlorhexidine.,' On the other hand, because of the excellent in vitro antimicrobial activity of 0.4 per cent SnF,, it may be useful as a chemotherapeutic agent incorporated within an intraoral slow release device28so that its substantivity could be artlficially increased and, in this way, perhaps increase its clinical effectiveness. The antibacterial clinical effectiveness of SnF, could also be increased by combining it with other antimicrobial agents. In fact, Ostela and Tenovuo* recently demonstrated the combination amine Australian Dental Journal 1992;37:5.
fluoride and SnF, was very effective in inhibiting S. mutans growth although it was not as active against Lactobcilli casei. Another means to increase the clinical effectiveness of SnF, is to utilize a higher concentration of the SnF,. The 0.8 per cent SnF2, with 2000 ppm fluoride, has significantly more antibacterial activity than the 0.4 per cent SnF, in the in virro agar bacterial inhibition assay. However, the higher concentration of 0.8 per cent SnF, may result in more problems with staining and possibly t a ~ t e . ' ~ . These ~ ~ , ~ 'concerns along with safety would have to be addressed in future studies involving 0.8 per cent or higher concentrations of SnF,. Acknowledgements The authors would like to thank Dr William Liljemark and Ms Lana Iverson, School of Dentistry, University of Minnesota, for their kind help to complete this project. This study was supported by the Cargill Foundation. References 1. Radike AW, Gish CW, Peterson JK, King JD, Segreto VA. Clinical evaluation of stannous fluoride as an anticaries mouthrinse. J Am Dent Assoc 1973;86:404-8. 2. Boyd RL, Leggott PJ, Robertson PB. Effect of selfadministered daily irrigation with 0.02% SnF, in periodontal disease activity. J Clin Periodontol 1985;12:420-31. 3. Mazza JE, Newman MG, Sims TN. Clinical and antimicrobial effect of stannous fluoride on periodontitis. J Clin Periodontal 1981;8:203-12. 4. Tinanoff N, Manwell MA. Clinical and microbiological effects of daily brushing with either NaF or SnF, gels in subjects with fixed or removable dental prostheses. J Clin Periodontol 1989;16:284-90. 5. Wolff LF, Bakdash MB, Pihlstrom BL, Bandt CL, Aeppli DM. The effect of professional and home subgingival irrigation with antimicrobial agents on gingivitis and early periodontitis. J Dent Hyg 1989; 63:222-5. 6. Muhler JC, Huysen GV. Solubility of enamel protected by sodium fluoride and other compounds. J Dent Res 1947;26:119-27. 7. Wei SHY, Forbes WC. Electron microprobe investigations of stannous fluoride reactions with enamel surfaces. J Dent Res 1974;53:51-6. 8. Ostela I, Tenovuo J. Antibacterial activity of dental gels containing combinations of amine fluoride, stannous fluoride, and chlorhexidine against cariogenic bacteria. Scand J Dent Res 1990;98:1-7. 9. Skjorland G, Gjermo P, Rlooa G. Effect of some polyvalent cations on plaque formation in oioo. Scand J Dent Res 1978;86:103-7. 10. Tinanoff N, Brady JM, Gross A. The effect of NaF and SnFzmouthrinses on bacterial colonization of tooth enamel: TEM and SEM studies. Caries Res 1976;10:415-26. 11. Marsh P, Martin M. Oral microbiology. 2nd edn. England Van Nostrand Reinhold, 1984:66-70,96. 12. Boyd RL, Leggott PJ, Robertson PB. Effects on gingivitis of two different 0.4% SnF, gels. J Dent Res 1988;67:503-7. Australian Dental Journal 1992;37:5.
13. Swango PA. The use of topical fluorides to prevent dental caries in adults. J Am Dent Assoc 1983;107:447-50. 14. Tinanoff N. Stannous fluoride in clinical dentistry. In: Wei SHY, ed. Clinical uses of fluorides. Philadelphia: Lea & Febiger, 1984;25-34. 15. Wolff LF, Pihlstrom BL, Bakdash MB, Aeppli DM, Bandt CL. EfTect of toothbrushing with 0.4% stannous fluoride and 0.22% sodium fluoride gel on gingivitis over eighteen months. J Am Dent Assoc 1989;119:283-9. 16. Wolff LF, Duncan JL. Studies on a bacterial substance produces by Group A streptococci. J Gen Microbiol 197431:413-24. 17. Kleinbaum DG, Kupper LL. Applied regression analysis and other multivariable methods. North Scituate, MA, USA: Duxbury Press, 1978:71-6, 268-71. 18. Lilienthal B. Inhibition of acid formation from carbohydrates by stannous fluoride and stannous chlorofluoride. Aust Dent J 1956;1:165-73. 19. Tinanoff N, Camosci DA. Microbiological ultrastructural and spectroscopicanalyses of the anti-tooth-plaqueproperties of fluoride compounds - in oitro. Arch Oral Biol 1980;25:531-4. 20. Hedgecock LW. Medical technology. Philadelphia: Lea & Febiger, 1967;204-11. 21. Lardo EA, Pappas J, Tyler K, Stanley HR, Walder C. In virro antimicrobial activity of six pulp-capping agents. Oral Surg Oral Med Oral Pathol. 1986;61:197-200. 22. Newbrun E, Hoover C, Ryder MI. Bactericidal action of bicarbonate ion on selected periodontal pathogenic microorganisms. J Periodontol 1984;55:658-67. 23. Yoon NA, Newman MG. Antimicrobial effect of fluorides on Bucteroides meluninogenicus subspecies and Bucteroides asucchurolyticus. J Clin Periodontal 1980;7:489-94. 24. Grossman E, Rettr G, Sturzenberger OP, Rosa MD, Dickinson TD, Ferretti GAYLudlam GE, Meckel AH. The effects of a chlorhexidinemouthrinse on gingivitis in adults. J Periodont Res 1986;21:Suppl 16:33-43. 25. Lang NP, Brecx MC. Chlorhexidine digluconate - an agent for chemical plaque control and prevention of gingival inflammation. J Periodont Res 1986;21:Suppl 16:74-89. 26. Tolle SL, Bauman DB, Allen DS. Effects of fluoride gels on plaque and gingival health. Dent Hyg 1987;62:280-4. 27. Bonesvoll P, Rolla G. Retention of Sn after mouth rinses. Caries Res 1978;12:112. 28. Mirth DB, Adderly DD, Amsbaugh SM, Monell-Torrens E, Li SN, Bowen WH. Inhibition of experimental dental caries using an intraoral fluoride-releasingdevice. J Am Dent ASSW1983; 107:55-8. 29. Lerevett DH, McHugh WD, Jensen OE. Dental caries and staining after twenty-eight months of rinsing with stannous fluoride or sodium fluoride. J Dent Res 1986;65:424-7. 30. Muhler JC. Stain from fluoride toothpaste. Br Dent J 1963;15:3.
Address for correspondenceheprints: Dental Department, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 70428, Taiwan ROC. 373
Key words: Bacterial inhibition, oral bacteria,
(Received for publication May 1991. Revised September 1991. Accepted December 1991.)
Introduction It is well established that stannous fluoride (SnF,) is very effective in preventing caries,' In recent years, SnF, use in concentrations between 0.02-1.64 per cent has resulted in a reduction of human periodontal disease.'-' The mechanism, however, through which SnF, prevents dental caries and reduces periodontal disease has not been completely explained. On the other hand, most investigators have agreed that the reduction in solubility of tooth surfaces by formation of relatively insoluble stannous phosphate compounds provides one form of protection induced by SnF, t~eatment.~.' Another and possibly more important protective mechanism of SnF, is its antimicrobial effect on the bacteria found in dental plaque.* This antimicrobial effect on oral bacteria has been attributed in large measure to the divalent cation, tin (Sn+ +). It is believed the tin in SnF, may exert its protective effect by interacting with negatively charged plaque components to alter bacterial adhesion/cohesi~n.~~'~ In addition to the antibacterial effectiveness of the tin ion, fluoride itself has been shown to decrease acid production by bacteria probably through the mechanism of inhibiting the enzyme enolase." Although clinical studies have been performed evaluating the clinical effectiveness of SnF, on both caries and periodontal d i ~ e a s e ~ ~there ' ~ - ' is~ little information available demonstrating the in vitro growth-inhibiting effect of SnF, on the wide variety
*Periodontal Division, Dental Department, National Cheng Kung University Medical Center, Tainan, Taiwan, ROC.
?Department of Preventive Sciences, School of Dentistry, University of Minnesota, USA.
stannous fluoride.
Abstract The purposes of this investigation were to evaluate and compare the antimicrobial effect of (1) twelve 0.4 per cent stannous fluoride (SnF,) commercial products and (2)different concentrations of SnF, (range = 0.02 to 3.28 per cent). The antibacterial inhibitory effect of various SnF, gels was evaluated as to their effectiveness against oral plaque bacteria including strains of S. mutans, S. sanguis, S. sobrinus, A. viscosus, A. actinomycetemcomitans, and 8.intermedius. When twelve different commercial preparations of 0.4 per cent SnF, were compared for inhibitory effect on plaque bacteria, several of the SnF, preparations were significantly more effective in inhibiting oral bacteria (pc0.05). With increasing concentration of SnF2,there was a comparable increase in the inhibitory effect on the oral bacteria tested (r* ranged from 0.867to 0.996). SnF, at a concentration of 0.4 per cent had a similar antibacterial effect to 0.12 per cent chlorhexidine. This in vitro study demonstrated that certain SnF, products are highly effective in inhibiting the growth of bacteria often found in plaque, and this inhibitory effect is directly related to the concentration of the SnF2.
368
Australian Dental Journal 1992;37(5):368-73.
of bacteria commonly found in plaque associated with dental disease. Therefore, the purpose of this study was to evaluate the antimicrobial effect of various commercial 0.4 per cent SnF, preparations on oral plaque bacteria and also to compare the inhibitory effect of various concentrations of SnF, with 0.12 per cent chlorhexidine.
Materials and methods Bacterial cultures Bacterial strains which have been associated with human dental caries, gingivitis and periodontitis were selected for use in this study. The exception was Escherichia coli which was a non-oral microorganism. Microbial strains chosen were Streptococcus mutans strain RL19, Streptococcus sobrinus strain 671 5, Streptococcus sanguis strain S4124 and S81, Actinomyces viscosus strain AVR and T-JY-2BY Actinobacillus actinomycetemcomitans strain Y4, Bacteroides intermedius, a wild type strain, and E. coli. Growth and test media Log phase cultures of all strains S. mutans, S. sobrinus, S. sanguis, A. viscosus, A. actinomycetemcomitans, and E. coli were grown in Todd-Hewitt broth.$ Bacteroides intermedius was grown to log phase in a basal broth medium which consisted of 1.0 per cent m/v trypticase,§ 1.0 per cent mlv proteopeptone,$ 0.5 per cent m/v yeast extract,$O.5 per cent m/v sodium chloride, 1 10 pglmL vitamin K-hemin solution,! and horse serum** at a final concentration of 4 per cent. All bacterial cultures were incubated anaerobically at 37°C. The agar medium on which the bacterial inhibition assay was performed included trypticase soy agars 5 per cent defibrinated sheep blood, 0.0005 per cent hemin,! and 0.00005 per cent menadione.1 Agar bacterial inhibition assay The agar bacterial inhibition assay is a modification of the method described earlier by Wolff and Duncan16 and is similar to the method described by Ostela and Tenovuo (1990)? A log phase broth culture of the test strain of bacteria was adjusted with a spectrophotometertt to an optical density of 0.3 (E540) using sterile Todd Hewitt broth for S. mutans, S. sobrinus, S. sanguis, A viscosus,
$Difco Laboratories, Detroit, MI, USA. BBBL Microbiology Systems, Cockeysville, MD, USA. ]EM Science, Cherry Hill, NJ, USA. (Sigma Chemical Co., St Louis, MO, USA. **Gibco Laboratories, Grand Island, New York, MY, USA. TtSpectronic 20, Bausch & Lomb, Rochester, NY, USA. Australian Dental Journal 1992;37:5.
A. actinomycetemcomitans and E. coli or a basal broth for B. intermedius as the diluent standard. Then a 1:400 dilution of the adjusted bacterial broth culture was made using Todd Hewitt or, in the case of B. intermedius, basal broth. One millilitre of this dilution was pipetted onto the supplemented blood agar plate and the agar surface flooded. The excess fluid was aspirated off with a pipette and the agar surface allowed to dry for 15 minutes. Bacteriological procedures involving preparation of the lawn culture using S. mutans, S. sobrinus, S. sanguis, A. viscosus, A. actinomycetemcomitans and E. coli were performed on a laboratory bench top. Laboratory procedures for preparation of the lawn culture for B. intermedius, were performed in an anaerobic chamber$$ which had an atmosphere of 10 per cent H,, 10 per cent CO, and 80 per cent N,. After the surface of the agar plates was dry, holes were punched into the agar with a sterile cork puncher. The diameter of all wells punched into the agar was 65 mm. A sterile tuberculin syringe was used to add 75 pL of the antiplaque or control agent to the appropriate wells. Glycerinegg served as the negative control and 0.12 per cent chlorhexidinell 1 served as the positive control. Glycerine was the carrying solution for the SnF, gels. The SnF, gels were delivered into wells of the agar plates in the anaerobic chamber to prevent or decrease the risk of oxidation of the stannous ions to the stannic ions. Bottles containing the 0.4 per cent SnF, gels were not opened prior to use in the bacterial inhibition assay. The 0.4 per cent SnF, gels used in these experiments included Gel Kam, Activus, Omnii Gel, Basic Gel, Ultra Gel, Gel Tin, Iradicav, Gel Pro, Flo Gel, Perfect Choice, Easy Gel, and Quick Gel (Table 1). Duplicate plates were used for each experiment. After the antiplaque agent and controls were added to the wells, agar plates were incubated at 37 "Cfor seven days in the anaerobic chamber. The diameter of the bacterial zone of inhibition was then measured to the nearest 0.1 mm with a boley gauge. The experimental protocol for the bacterial inhibition assay is shown in Fig. 1 . Statistical analysis For multiple comparison of mean zone of bacterial inhibition by twelve 0.4 per cent SnF, commercial products, Student-Neuman-Keuls (SNK)
$$Coy Manufacturing Co., Ann Arbor, MI, USA. BBColumbia Chemical Industries Inc., Columbus, WN, USA I [Procter & Gamble Co., Cincinnati, Ohio, USA. 369
Table 1. Stannous fluoride gels (0.4 per cent) used in this investigation Gel
Manufacturerlsupplier
Gel-Kam Activus Omnii Gel Basic Gel Ultra-Gel Gel-Tin Iradicav Gel-Pro Flo-Gel Perfect Choice Easy Gel Quick-Gel
Scherer Laboratory Inc., Dallas, Texas, USA. Scherer Laboratory Inc., Dallas, Texas, USA. Dunhall Pharmaceuticals Inc., Stafford, Texas, USA. Basic Dental Products Inc., Miami, Florida, USA. Dental Resources Inc., Long Lake, Minnesota, USA. Young Dental, Maryland Hgts., Maryland, USA. Johnson & Johnson Dental Products Company, East Windsor, New Jersey, USA. Supro Inc., Noblesville, Indiana, USA. Continental Quest Corp., Indianapolis, Indiana, USA. Challenge Products Inc., Osage Beach, Missouri, USA. Du-More Inc., Rogers, Arkansas, USA. Ultra Care Products, Lansdale, Philadelphia, USA.
was utilized. SNK method is applicable for pairwise comparison, with the sample sizes from each group being equal.” T o evaluate the relationship between the zone of bacterial inhibition (Y) and the concentration of SnF, (X), simple linear regression analysis was performed. The zone of inhibition was treated as
the dependent variable and the concentrations of SnF, as the independent variable and three basic assumptions, normality, independence and straight line relationships, were made. The strength of the linear relationship between X and Y was represented in terms of r2 which may vary between 0 and 1. A large value of r2 indicates a strong linear relationship between X and Y.
Results Twelve 0.4 per cent SnF2 products were evaluated for their antimicrobial effect in the agar inhibition assay. Gel Kam, Omnii Gel, Flo Gel, Perfect Choice, and Quick Gel were most effective at inhibiting growth of all bacterial test strains (Table 2). Activus and Gel Pro also showed a similar effect on inhibiting the test strains with the exception of S. sanguis strain S-81.On the other hand, Easy Gel, Basic Gel, Ultra Gel, Gel Tin and Iradicav showed a statistically significant smaller zone of bacterial inhibition for specific bacterial test strains, p I0.05. In the agar inhibition assay, B. inrerrnedius was consistently more sensitive to the SnF, gels, as indicated by the greater zone of bacterial inhibition, while the non-oral bacterium, E. coli, was least sensitive to the SnF, gels. The experimental results described in the previous section demonstrated that several SnF2gels exhibited more antibacterial inhibition than other
r- -0 1:400
dilution
Broth culture of bacterial test strain adjusted to optical density of 0.3 ( E d
0.4% SnF2
Bacterial test strain flooded onto blood agar plate
Allowed to dry for 15 minutes
Zones of bacterial inhibition measured with a boley gaug
punched into agar with identical diameter and depth. Then antiplaque agent or control added to well
Chlorhexidine 0.12% (positive control) Lawn of the bacterial test strain Incubate at 370 C in anaerobic chamber for 5-7 days
Fig. 1 . -Protocol for the bacterial inhibition assay. 370
Australian Dental Journal 1992;37:5.
Table 2.
Mean zones" of bacterial inhibition by twelve 0.4% SnF, preparations 0.4% SnF, preparations
Bacterial test strain
Gel Omnii Kam Gel
Advus
Flo Gel
Perfect Choice
S. mutans
1.75 (0.07) 1.73 (0.00) 2.07 (0.02) 1.67 (0.02) 2.15 (0.07) 2.18 (0.04) 1.77 (0.02) 2.67 (0.07) 0.97 (0.03)
1.65 (0.10) 1.68 (0.04) 2.21 0.18
1.78 (0.18) 1.64 (0.06) 2.18 (0.26) 1.85 (0.07) 2.06 (0.01) 2.01 (0.08) 1.69 (0.05) 2.58 (0.03) 1.11 (0.15)
1.83 (0.22) 1.82 (0.10) 2.31 (0.06) 1.64 (0.01) 2.13 (0 04) 2:Ol (0 08) 1170 (0.07) 2.69 (0.05) 1.00 (0.07)
(RL19) S. sobrinus (6715) S. sanguis (S4124) S. sanguis (S81) A. viscosus (AW A. viscosus (T-JY-2B) A. actinomycetemcomitans (Y4) B. intermedius
E. coli
1.77 (0.05) 1.77 (0.02) 2.33 (0.18) 1.65 (0.07) 2.13 (0.04) 2.15 (0.00) 1.74 (0.02) 2.66 (0.06) 0.91 (0.01)
3 0.07 2.16 (0.08) 2.13 (0.04) 1.80 (0.07) 2.70 (0.00) 0.94 (0.02)
Easy Gel
Quick Gel
kl(y:?
Basic Gel
1 1 . 1 5 1.82 1 1.62 0.00 2.23
l-%%l
I I y.21: 2.25 0.14 0.86 (0.02)
(0.07) 2.07
0.01 1.70
(0.02) 0.07 1.73 1.65 (0.04) (0.07) 2.15 1.79 (0 14) 2:05 (0.07) I(0.03) 1.82 1.50 (0.04) y.02: 2.66 2.03 (0.02) 0.13 1.03 0.96 (0.02) (0.15)
Ultra Gel
Gel Tin
Iradicav
Gel Pro
1.62
2.00
1.96
0.07 1.85
0.11 1.99
0.04 2.00
1.98 (0.03) 1.65 (0.07) 2.03 (0.04) 1.53 (0.04) 2.08 (0.06) 2.12 (0.05) 1.65 (0.00) 2.59 (0.01) 0.88 (0.04)
fl , 0.07 1.63 (0.04) 2.04 (0.01) 2.05 (0.07) 1.60 y.06: 2.35
,, , I
,:y:Jli: :&Jl:, 0.06 1.96
I(0.03) 1.54 (0.06) 2.53
::::
(0.03)
I(0.05)
I
0.09 1.92 (0.19) 2.05
I 1
?% y.00: 2.43
I
:::
(0.01)
*Values represent mean of duplicate experiments. The value of parentheses below the mean is the SD. Mean measurements outlined in box showed a statisticallysignificant smaller zone of bacterial inhibition than other measurements for a specific bacterial test strain. All values are expressed in cm.
SnF, gels. One of these gels, Gel Kam, was chosen to determine how various concentrations of this SnF, product would affect test micro-organisms in the agar bacterial inhibition assay. The mean zones of bacterial inhibition with different concentration of SnF, may be seen in Table 3. These data demonstrated that as SnF, concentration decreased from 3.28 per cent to 0.02 per cent, there was a linear decrease in the mean zone of bacterial inhibition for all strains. There was a statistically significant, high correlation between the zone of bacterial inhibition and concentration of SnF,, with rz between 0.867 and 0.996,p I0.002.The 0.4 per cent SnF, solution gave a similar zone of bacterial inhibition as 0.12 per cent chlorhexidine on test strains ofS. mutans, S. sobrinus, A. viscosus, A. actinomycetemwmitans and B. intermedius. A 0.02 per cent SnF, solution had minimum or no effect on the test strains. Discussion The results of this investigation indicated that the majority of the SnF, products evaluated have considerable antimicrobial activity in the agar bacterial inhibition assay. These findings firther support the observation made by other investigators who reported that SnF, inhibited the growth of S. mutans and A. viscosus.L8*L9 However, it was observed that not all the commercial 0.4 per cent SnF, products evaluated in the agar bacterial inhibition assay were equally effective in inhibiting Australian Dental Journal 1992;37:5.
bacterial growth. Several commercial 0.4 per cent SnF, products were found to exhibit greater antimicrobial activity than many of the other SnF, products tested. Because of the variable findings regarding the antimicrobial effectiveness of different 0.4 per cent SnF, gels, it may be prudent to evaluate these SnF, products or other antimicrobial agents in an inexpensive in vitro assay prior to investigating their effectiveness in clinical studies. An agar bacterial inhibition assay has been widely used to evaluate bacterial resistance to antibioticYz0 and has also been used to test the antimicrobial activity of pulpcapping agents.,' The agar bacterial inhibition assay utilized in this investigation, however, was different or a modification from the test traditionally used to evaluate bacterial sensitivity to antibiotics and was similar to the method recently described by Ostela and Tenovuo.8 In this study, a known quantity of a suspected antimicrobial agent was placed into wells prepared in agar plates onto which a lawn culture of a specific bacterium was placed. In antibiotic susceptibility tests, a known quantity of an antibiotic is generally incorporated into a disc and then placed onto a lawn culture of the test bacterium. The technique used for antibiotic sensitivity testing with a disc was not used in this study because there was a concern the antimicrobial agent, and in this case SnF,, would bind to the disc and not be released or only partially be released into the agar to inhibit bacterial 371
Table 3. Mean zones* of bacterial inhibition with different concentrations of SnF, ~
Bacterial test strain
S.mutans (RL19) S. sobrinus (6715) S. sanguis (S4124) S. sanguis (S8 1) A. viscosus
(AVR)
A. viscosus (T-JY-2B) A. actinomycetemcomitans (Y4) B. intennedius E. coli
Stannous fluoride concentrations ("70) 3.28
1.64
0.8
0.4
0.2
0.1
0.02
4.15 (0.07) 3.45 (0.07) 4.05 (0.07) 2.95 (0.07) 4.20 (0.21) 3.68 (0.04) 3.15 (0.00) 4.30 (0.14) 1.98 (0.03)
3.20 (0.07) 2.77 (0.09) 3.60 (0.06) 2.75 (0.01) 3.24 (0.01) 2.93 (0.05) 2.52 (0.00) 3.65 (0.06) 1.52 (0.05)
2.42 (0.04) 2.16 (0.06) 2.75 (0.07) 2.46 (0.01) 2.83 (0.11) 2.56 (0.06) 2.23 (0.1 1) 3.21 (0.04) 1.18 (0.02)
2.00 (0.00) 1.81 (0.15) 2.20 (0.00) 2.07 (0.02) 2.28 (0.04) 2.23 (0.06) 1.95 (0.00) 2.83 (0.04) 1.00 (0.07)
1.78 (0.04) 1.60 (0.00) 1.83 (0.1 1) 1.79 (0.01) 1.91 (0.06) 1.86 (0.04) 1.69 (0.05) 2.31 (0.01) 0.80 (0.01)
1.51 (0.01) 1.43 (0.03) 1.41 (0.06) 1.59 (0.02) 1.30 (0.07) 1.58 (0.04) 1.46 (0.06) 1.95 (0.08) 0.73 (0.04)
0.80 (0.01)
1.15 (0.07) 0.91 (0.01) 0.93 (0.11) 0.91 (0.01) 0.83 (0.04) 0.85 (0.07) 1.18 (0.00) 0.70 (0.04)
Chlorhexidine 0.12% (positive control) 2.00 (0.00) 1.78 (0.04) 1.95 (0.07) 1.72 (0.09) 2.53 (0.1 1) 2.32 (0.03) 1.95 (0.00) 2.80 (0.07) 1.30 (0.07)
r*t 0.987 0.956 0.996 0.97 0.988 0.977 0.984 0.988 0.867
'MeankSD (cm). Each experiment performed in duplicate and the mean determined. The value in parentheses below the mean is the SD. tr' =Correlation coefficient between zone of bacterial inhibition and concentration of SnF, (for r2 t 0.867, p < 0.002).
growth. Therefore, it was decided to prepare wells in the agar where the problem of the antimicrobial agent binding to a disc would not be a factor. Also, many in vitro tests evaluate antimicrobial susceptibility of a chemotherapeutic agent using a broth dilution assay m e t h ~ d .Because ~ ~ . ~ ~it was desirable to compare the actual concentration of the commercial SnF, products without a dilution factor, the agar inhibition assay was used. Several limitations are still inherent in the agar inhibition assay. For example, diffusion of test agents through the agar medium could be a problem. Binding of an ionic charged antimicrobial agent to the negatively charged agar may reduce its bacterial inhibition effectiveness. In vitro results using this bacterial inhibition assay, therefore, may not be completely transferable to a clinical situation. It was a concern that 0.4 per cent SnF, may lose activity as it aged. The rationale for this was the stannous ion (Sn+ + ) may become oxidized to the stannic ion (Sn+ + + +). In effect, it is believed this would reduce its antibacterial activity and possibly reduce its perceived clinical effectiveness. Experiments in this study, therefore, included only SnF, gels which were not opened prior to their evaluation. It was demonstrated in an earlier investigation, however, that after SnF, bottles were opened and used in a clinical situation for 45 days,1s the residual gel had lost 25 per cent of its available stannous ion. However, in the agar bacterial inhibition assay, the same SnF, gel lost less than 12.5 per cent of its antimicrobial activity. 372
Data reported in this investigation indicated an approximately linear relationship between the concentrations of SnF2 and the antimicrobial activity on the test strains of bacterial. While 3.28 per cent SnF, showed a high antimicrobial activity, 0.02 per cent SnF, had minimal or no activity on the test strains. These observations suggest that 0.02 per cent SnF, may not be a useful antiplaque agent. On the other hand, 0.4 per cent SnF, was as effective as 0.12 per cent chlorhexidine in the agar bacterial inhibition assay. Although, 0.12 per cent chlorhexidine has been found to be very effective as an antiplaque and antigingivitis agent in clinical s t u d i e ~ ,0.4 ~ ~per .~~ cent SnF, was not observed to be effective in reducing plaque and improving gingival health. While both 0.4 per cent SnF, and 0.12 per cent chlorhexidine possess a similar in vitro antimicrobial activity, a potential reason for the lower clinical effectiveness of SnF, in studies by Wolff et d i 5 and Tolle et aP6 could be due to its lower substantivity when compared with chlorhexidine.,' On the other hand, because of the excellent in vitro antimicrobial activity of 0.4 per cent SnF,, it may be useful as a chemotherapeutic agent incorporated within an intraoral slow release device28so that its substantivity could be artlficially increased and, in this way, perhaps increase its clinical effectiveness. The antibacterial clinical effectiveness of SnF, could also be increased by combining it with other antimicrobial agents. In fact, Ostela and Tenovuo* recently demonstrated the combination amine Australian Dental Journal 1992;37:5.
fluoride and SnF, was very effective in inhibiting S. mutans growth although it was not as active against Lactobcilli casei. Another means to increase the clinical effectiveness of SnF, is to utilize a higher concentration of the SnF,. The 0.8 per cent SnF2, with 2000 ppm fluoride, has significantly more antibacterial activity than the 0.4 per cent SnF, in the in virro agar bacterial inhibition assay. However, the higher concentration of 0.8 per cent SnF, may result in more problems with staining and possibly t a ~ t e . ' ~ . These ~ ~ , ~ 'concerns along with safety would have to be addressed in future studies involving 0.8 per cent or higher concentrations of SnF,. Acknowledgements The authors would like to thank Dr William Liljemark and Ms Lana Iverson, School of Dentistry, University of Minnesota, for their kind help to complete this project. This study was supported by the Cargill Foundation. References 1. Radike AW, Gish CW, Peterson JK, King JD, Segreto VA. Clinical evaluation of stannous fluoride as an anticaries mouthrinse. J Am Dent Assoc 1973;86:404-8. 2. Boyd RL, Leggott PJ, Robertson PB. Effect of selfadministered daily irrigation with 0.02% SnF, in periodontal disease activity. J Clin Periodontol 1985;12:420-31. 3. Mazza JE, Newman MG, Sims TN. Clinical and antimicrobial effect of stannous fluoride on periodontitis. J Clin Periodontal 1981;8:203-12. 4. Tinanoff N, Manwell MA. Clinical and microbiological effects of daily brushing with either NaF or SnF, gels in subjects with fixed or removable dental prostheses. J Clin Periodontol 1989;16:284-90. 5. Wolff LF, Bakdash MB, Pihlstrom BL, Bandt CL, Aeppli DM. The effect of professional and home subgingival irrigation with antimicrobial agents on gingivitis and early periodontitis. J Dent Hyg 1989; 63:222-5. 6. Muhler JC, Huysen GV. Solubility of enamel protected by sodium fluoride and other compounds. J Dent Res 1947;26:119-27. 7. Wei SHY, Forbes WC. Electron microprobe investigations of stannous fluoride reactions with enamel surfaces. J Dent Res 1974;53:51-6. 8. Ostela I, Tenovuo J. Antibacterial activity of dental gels containing combinations of amine fluoride, stannous fluoride, and chlorhexidine against cariogenic bacteria. Scand J Dent Res 1990;98:1-7. 9. Skjorland G, Gjermo P, Rlooa G. Effect of some polyvalent cations on plaque formation in oioo. Scand J Dent Res 1978;86:103-7. 10. Tinanoff N, Brady JM, Gross A. The effect of NaF and SnFzmouthrinses on bacterial colonization of tooth enamel: TEM and SEM studies. Caries Res 1976;10:415-26. 11. Marsh P, Martin M. Oral microbiology. 2nd edn. England Van Nostrand Reinhold, 1984:66-70,96. 12. Boyd RL, Leggott PJ, Robertson PB. Effects on gingivitis of two different 0.4% SnF, gels. J Dent Res 1988;67:503-7. Australian Dental Journal 1992;37:5.
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Address for correspondenceheprints: Dental Department, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 70428, Taiwan ROC. 373
