Original Paper Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Received: September 3, 2013 Accepted after revision: February 20, 2014 Published online: October 9, 2014
Plaque pH in Caries-Free and Caries-Active Young Individuals before and after Frequent Rinses with Sucrose and Urea Solution Haidar Hassan a Peter Lingström b Anette Carlén a Departments of a Oral Microbiology and Immunology and b Cariology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Abstract Objective: To examine pH in the approximal dental biofilm after acid and alkali formation from sucrose and urea, after an adaptation period to these substances, in caries-free (CF) and caries-active (CA) individuals. Saliva flow and buffer capacity, and aciduric bacteria in saliva and plaque were also examined. Material and Methods: Twenty adolescents and young adults (15–21 years) with no caries (n = 10, Dm + iMFS = 0) or ≥1 new manifest lesions/year (n = 10, DmMFS = 3.4 ± 1.8) participated. After plaque sampling, interproximal plaque pH was measured using the strip method before (baseline) and up to 30 min (final pH) after random distribution of a 1-min rinse with 10 ml of 10% sucrose or 0.25% urea. This procedure was repeated after a 1-week adaptation period of rinsing 5 times/day with 10 ml of the selected solution. After a 2-week washout period the second solution was similarly tested. Mutans streptococci, lactobacilli and pH 5.2-tolerant bacteria were analyzed by culturing. Results: In the CF group, acid adaptation resulted in lowering of baseline and final plaque pH values after a sugar chal-
© 2014 S. Karger AG, Basel 0008–6568/14/0491–0018$39.50/0 E-Mail [email protected] www.karger.com/cre
lenge, and in increased numbers of bacteria growing at pH 5.2, which was increased also after alkali adaptation. In the CA group, the final pH was decreased after acid adaptation. No clear effects of alkali adaptation were seen in this group. Conclusion: One-week daily rinses with sucrose and urea had the most pronounced effect on the CF group, resulting in increased plaque acidogenicity from the sugar rinses and increased number of acid-tolerant plaque bacteria from both rinses. © 2014 S. Karger AG, Basel
Even if dental caries has decreased worldwide since the middle of the last century, it still constitutes a large problem for many individuals [Marthaler, 2004; Hugoson et al., 2008]. It is a multifactorial disease, which occurs according to the ecological plaque hypothesis [Marsh, 1994], when the balance in the microflora of the dental biofilm is disturbed by modifications in the oral environment. Dental biofilm activity is controlled by a number of environmental factors including fermentable carbohydrates and alkali. Stephan [1940] showed that the bacterial metabolism of glucose, resulting in a pH decrease of the dental biofilm, plays a major role in caries development. Frequent Assoc. Prof. Anette Carlén Department of Oral Microbiology and Immunology Institute of Odontology, University of Gothenburg Box 450, SE–405 30 Gothenburg (Sweden) E-Mail anette.carlen @ odontologi.gu.se
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Key Words Acid formation · Alkali formation · Biofilm adaptation · Plaque pH
Material and Methods Study Groups Healthy CF and CA adolescents and young adults (15–21 years) were randomly selected from the patient’s dental records among four Public Dental Clinics in and around Gothenburg City, Sweden. A total of 150 individuals were invited by letter and telephone call to participate in the study. The letter was sent to the parents or legal guardian in case of adolescents and to the young adult volunteers. Eleven individuals with and 12 without any caries, who met the inclusion and exclusion criteria, accepted to join the study. Ac-
In vivo Plaque pH after Sugar and Urea Rinses
cording to our pilot tests, 10 individuals/group were sufficient to reveal statistically significant lowered or increased plaque pH after 1-week rinsing with 10% sucrose or 0.25% urea solution, respectively. CF was defined as Dm + iMFS (decayed, missed and filled surfaces, both manifest and initial caries) = 0, CA as DmMFS ≥ 1/year new, primary, manifest caries lesions (occlusal and/or approximal) in the last 3 years. The inclusion criteria were: (1) all permanent teeth erupted, (2) regular attendance to a Public Dental Clinic, (3) normal stimulated salivary secretion rate, ≥1.0 ml/min, (4) no smoking and no snuffing. Exclusion criteria were: (1) had disease and medication, (2) not within the age range 15–21 years, (3) had deciduous teeth, (4) irregular attendance to Public Dental Clinic, (5) smoker and/or snuffer, (6) had received antibiotics within the last 3 months prior to the study. The study was approved by the Ethics Committee at the University of Gothenburg (Dnr 282-10) and followed the ethical considerations of the Helsinki Declaration. Written informed consent was obtained from the parents or legal guardian in case of adolescents or from the young adult volunteers, prior to the study. Study Design The study was designed as a single-blind (for the participants), randomized, controlled, two-leg cross-sectional clinical trial. It consisted of two test periods and two washout periods. Two test solutions, 10% sucrose and 0.25% urea, were distributed in a random order. All participants came to the laboratory 5 times (fig. 1). At the first visit, where medical and dental anamnesis was obtained, detailed information about the study was given, and the consent form was signed. At the end of this first visit, professional tooth cleaning using rubber cup and prophylactic paste (Cleaning RDA 170; CCS Clean Chemical Sweden, Borlänge, Sweden) as well as dental flossing without fluoride (Reach®, waxed floss, Johnson & Johnson, Canada) was performed. The second and fourth visits (before the 1-week adaptation periods) followed after a washout period of 2 weeks. The third and fifth visits followed after the 1-week adaptation periods where the participants rinsed with one of the two randomly selected test solutions. During these washout and test periods, the participants were asked to keep their regular dietary and oral hygiene habits. On the last day prior to all four visits, the participants were asked to refrain from oral hygiene and from eating and drinking during the last 2 h prior to the visit. An SMS message was sent daily as a reminder to all the participants during the adaptation periods and before every visit. At the four visits II–V (including start and end of the two adaptation periods), supragingival plaque samples were collected, plaque pH was measured before and up to 30 min after a 1-min rinse with the randomly selected test solution, and a stimulated saliva sample was collected. In addition, professional tooth cleaning as described above was performed at the end of the adaptation visits (III, V). All sampling and measurements were performed by one and the same operator (H.H.). Rinsing Regimen The two rinse solutions were coded A (10% sucrose) and B (0.25% urea) and were distributed in 10-ml portions in plastic tubes. Before each adaptation period, the participants were given
Caries Res 2015;49:18–25 DOI: 10.1159/000360798
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decreases of the dental biofilm pH activate acidogenic and aciduric microorganisms to produce acids and to adapt to low pH values, which can lead to significant tooth demineralization [Belli and Marquis, 1991; Takahashi and Yamada, 1999; Welin-Neilands and Svensäter, 2007]. A few in vivo studies have been conducted to investigate the ability of the dental biofilm to produce acid in relation to caries [Abelson and Mandel, 1981; Scheie et al., 1992; Dong et al., 1999; Lingström et al., 2000]. In one study, the in vivo adaptation of plaque bacteria to an acidic environment was demonstrated [Aranibar Quiroz et al., 2003]. The acidifying metabolism in plaque is followed by an alkalization phase, generating increased pH from ammonia after bacterial metabolism of e.g. urea and arginine and by the effects of saliva [Burne and Marquis, 2000; Kleinberg, 2002]. This phase neutralizes the acids and protects the bacteria from acid damage and leads to raised pH in the plaque [Kleinberg, 2002]. Recent studies have also demonstrated that a low ability to form alkaline substances from urea and arginine is associated with a higher caries activity [Shu et al., 2007; Nascimento et al., 2009; Gordan et al., 2010; Toro et al., 2010; Morou-Bermudez et al., 2011]. However, no study that examined the dental biofilm adaptation to alkali formation has been found. Our hypothesis was that frequent supply of sucrose and urea, respectively, affects the acid and alkali formation in the dental biofilm differently in caries-free (CF) and in caries-active (CA) individuals. The main aim of the present study was therefore to investigate, by in vivo pH measurements, the ability of the dental biofilm to form acid and alkali after short-term adaptation periods with daily exposure of sucrose and urea in relation to caries. Stimulated saliva secretion rate and buffer capacity as well as acid-tolerant bacteria in the saliva and plaque were also examined. To our knowledge, this study is the first attempt to investigate the dental biofilm adaptation to alkali formation.
I. Start
- Written consent - Medical and dental history - Professional tooth cleaning (PTC)
II. BAA/BBA
Washout (2 weeks)
- Plaque samples - Plaque pH - Saliva sample
III. AAA/ABA
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V. AAA/ABA
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Day 7 (test day): - Plaque samples - Plaque pH - Saliva sample - PTC
- Plaque samples - Plaque pH - Saliva sample
Washout (2 weeks)
Mouthrinse (1 week)
Day 7 (test day): - Plaque samples - Plaque pH - Saliva sample - PTC
Fig. 1. Study design. Sampling and pH measurements performed: visit II, before start of the first 1-week acid (BAA) or base adaptation
(BBA) period using a randomly selected 10% sucrose or 0.25% urea rinse; visit III, after the first acid (AAA) or base adaptation (ABA) period; visit IV, before the second 1-week acid (BAA) or base adaptation (BBA) period; visit V, after the second acid (AAA) or base adaptation (ABA) period.
Microbial Analysis The plaque and saliva samples were incubated at 37 ° C for 30 min before further dilution and plated onto agar medium essentially as previously described [Almståhl, 2001]. In short, after dilution to 10–4, 100 μl of the concentrated and diluted plaque sample solutions were plated on mitis salivarius-bacitracin agar [Gold et al., 1973], Rogosa SL agar and on pH 5.2 agar [Svensäter et al., 2003]. The number of colony-forming units (CFU) of mutans streptococci and lactobacilli was calculated after incubation of the mitis salivarius-bacitracin agar and Rogosa agar plates in 90% N2 and 10% CO2 at 37 ° C for 3–5 days, and identification of mutans streptococci and lactobacilli from their typical colony morphology. The total number of bacteria growing at pH 5.2 was enumerated after anaerobic incubation in 95% H2 and 5% CO2 for 7 days at 37 ° C. The saliva samples were diluted to 10–4 before 2 × 25 μl of the dilutions were plated on mitis salivarius-bacitracin agar and Rogosa SL agar plates. After incubation in 90% N2 and 10% CO2 at 37 ° C for 3–5 days, mutans streptococci and lactobacilli were identified and enumerated as above.
Plaque Samples Two supragingival dental plaque samples were collected from the interproximal sites between the upper right first and second molars, and the upper left first and second molars, respectively, using sterile toothpicks (TePe Birch, TePe, Malmö, Sweden). The samples were pooled in 3.5 ml VMGA III [Möller, 1966] transport medium and the samples were incubated onto agar plates within 24 h for microbial analyses. pH Measurements Supragingival plaque pH was measured using the pH strip method as previously described [Carlén et al., 2010]. Measurements were performed between the upper right and left second premolar and first molar, respectively; pH was measured before (baseline, 0 min) and at 2, 5, 10, 15, 20 and 30 min after a 1-min rinse with 10 ml of the test solution. A strip (Spezialindikator, Merck, Darmstadt, Germany) measuring pH 4.0–7.0 and pH 6.5– 10.0 was inserted into the interproximal area in order to test acid and alkali formation, respectively. Stimulated Saliva Stimulated saliva was collected by chewing on a piece of paraffin during 5 min and continuously spitting the obtained saliva into an ice-chilled tube. The secretion rate was determined after which 1 ml of the saliva was transferred to VMGA IIs [Möller, 1966] transport medium for microbial analysis within 24 h. Another milliliter of the saliva was used for determination of the buffer capacity according to Ericsson [1959].
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Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Statistical Analyses Statistical descriptive analyses were performed using IBM® SPSS® Statistics (version 19.0.0.1, IBM software, 2011). The mean approximal plaque pH (±SD) for all participants at the different time points was calculated for the two interproximal sites. The maximum pH fall and minimum pH after a 10% sucrose rinse and the maximum pH increase and maximum pH after a 0.25% urea rinse were also calculated. Changes in plaque pH during 30 min were calculated from the area of the curve below the critical pH of enamel (pH 5.7; AUC5.7) and of dentine (pH 6.2; AUC6.2) for acid formation and the area of the curve above pH 7.0 (AOC7.0) for alkali formation. AUC and AOC values were calculated using the computer program according to Larsen and Pearce [1997]. The bacterial numbers were transformed logarithmically and the mean ± SD was calculated. Student’s twosample, paired t test was used to analyze the significance of differences within the same group and nonpaired t test between the CF and CA groups; p < 0.05 was considered statistically significant.
Hassan /Lingström /Carlén
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sufficient numbers of tubes for five rinses/day during 1 week. They were instructed to keep the tubes in the refrigerator and only take out the necessary number of tubes when leaving home. They were further instructed to rinse with the 10-ml solution for 1 min early in the morning 2 h after tooth brushing, before lunch, after lunch, later in the afternoon and in the evening 2 h before tooth brushing. They were also asked to avoid eating or drinking 1 h after rinsing and at least 2 h between the rinses. To evaluate compliance, the participants were asked if they had followed the instructions given.
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Study Groups Out of the 23 volunteers included, 1 never attended, 1 got pulpitis and 1 moved to another city during the study. Thus, 20 of the volunteers completed the study: 10 CF individuals (Dm + iMFS = 0, 6 female and 4 male, 15–21 years, mean age 17.0 ± 1.8 years) and 10 CA individuals (DmMFS = 3.4 ± 1.8, 5 female and 5 male, 15–20 years, mean age 17.0 ± 1.6).
Dental Anamnesis The participants were asked about their oral hygiene habits and all reported that they brushed their teeth twice a day and used fluoridated toothpaste containing 1,450 ppm fluoride. 80% the CF individuals reported an infrequent use of additional fluoride-containing mouthrinses (Flux 900 ppm F), whereas 40% in the CA group used it regularly (once a day) as recommended by their dentist. Dental floss was reported to be used by 30 and 40% in the CF and CA groups, respectively. There were no differ-
In vivo Plaque pH after Sugar and Urea Rinses
Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Results
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Fig. 2. The approximal plaque pH prior to (0 min), and up to 30 min after a 1-min rinse with 10% sucrose (a, b) or 0.25% urea (c, d) in 10 CF (a, c) and 10 CA (b, d) individuals; pH measurements were performed before (BAA) and after (AAA) adaptation to acid and before (BBA) and after (ABA) adaptation to alkali by 1-week daily rinses with the test solutions 5 times/day.
Table 1. pH values in approximal plaque up to 30 min after a 10% sucrose rinse in CF and CA individuals before and after acid adaptation
CF
Baseline pH Max pH drop Min pH Final pH AUC5.7 AUC6.2
CA
acid adaptation before
acid adaptation after
p value
acid adaptation before
acid adaptation after
p value
6.6 ± 0.4 1.1 ± 0.4 5.5 ± 0.6 6.13 ± 0.5 4.9 ± 7.1 12.4 ± 12.6
6.3 ± 0.5 1.1 ± 0.3 5.2 ± 0.6 5.8 ± 0.6 9.3 ± 11.4 18.5 ± 16.6
0.03 0.59 0.07 0.04 0.11 0.06
6.3 ± 0.3 1.1 ± 0.6 5.2 ± 0.6 6.2 ± 0.5 7.2 ± 9.8 16.12 ± 13.7
6.2 ± 0.4 1.1 ± 0.4 5.1 ± 0.5 5.9 ± 0.4 8.4 ± 8.3 20.6 ± 10.9
0.32 0.93 0.30 0.02 0.39 0.06
AUC5.7, AUC6.2 = AUC at pH 5.7 and pH 6.2, respectively, up to 30 min after the sucrose rinse. p values for comparisons before and after adaptation within the respective groups.
Supragingival Plaque pH before and after Acid Adaptation The plaque pH curves obtained after a sugar challenge, before and after the adaptation period of daily rinses with 10% sucrose, are seen in figure 2a, b. After the adaptation period, the sugar challenge resulted in statistically significant lower pH values at time points 0 (baseline pH), 10, 15 and 30 min (final pH) in the CF group, and at 15 and 30 min in the CA group. The curves were at a lower level especially for the CF group, but had the same shape after adaptation as before. A clear tendency to an increased mean AUC6.2 value was seen within both groups after the rinsing period. In the CF group, the minimum pH also tended to be decreased (table 1). Baseline pH was higher in the CF group compared to the CA group (p = 0.05) before but not after the acid adaptation period. Supragingival Plaque pH before and after Alkali Adaptation The curves of plaque pH obtained after an alkali challenge, before and after the adaptation period of daily rinses with 0.25% urea, are seen in figure 2c, d. In the CA group only, somewhat higher pH values after the adaptation period were noticed at baseline and up to 20 min after the urea challenge (fig. 2d). The area of the curve above pH 7 (AOC7.0) was numerically but not statistically significantly increased from 12.3 ± 8.3 to 13.8 ± 6.5 after the 22
Caries Res 2015;49:18–25 DOI: 10.1159/000360798
adaptation in this group. There were also no statistically significant differences between or within the groups for any of the other pH variables, neither before nor after the adaptation period (data not shown). Saliva Secretion Rate and Buffer Capacity There were no statistically significant differences in saliva secretion rate and buffer capacity before and after the two rinsing periods, neither within nor between the two groups, although somewhat lower numerical values were seen for the CA group at all four measurements (mean secretion rate: 2.0 ± 0.8 ml/min for CF and 1.7 ± 0.7 ml/ min for CA; mean buffer capacity: 6.2 ± 1.6 for CF and 5.7 ± 1.4 for CA). Microbiological Analysis At the start of the study (visit II), the mean numbers of mutans streptococci were higher in the CA group both in plaque (1.2 ± 0.6 for CF; 2.1 ± 1.3 for CA; p = 0.05) and in saliva (2.3 ± 1.9 for CF; 5.2 ± 1.1 for CA; p = 0.0004). No mutans streptococci were detected in the plaque samples from 8 of the CF and 4 of the CA individuals. The corresponding numbers of saliva samples with no detected mutans streptococci were 6 in the CF and none in the CA group. The mean log values of microbial data for saliva and plaque before and after the acid and alkali adaptation periods are presented in table 2. Significant differences were seen in the CF group only where the level of total plaque bacteria growing at pH 5.2 was increased after both periods and that of salivary lactobacilli after the acid adaptation period. Hassan /Lingström /Carlén
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ences in the mean of daily intake of main meals (2.8 ± 0.8 for CF/2.8 ± 0.6 for CA) and in between snacks (1.7 ± 0.8 for CF/1.7 ± 0.7 for CA).
Table 2. Mean number of aciduric bacteria (±SD) in plaque and saliva samples in CF and CA individuals before and after acid adaptation as well as before and after alkali adaptation
Acid adaptation
p value
before
after
1.2 ± 0.6 1.1 ± 0.3 5.4 ± 0.6 2.8 ± 1.5 2.4 ± 0.8
1.1 ± 0.4 1.2 ± 0.4 5.8 ± 0.7 2.7 ± 1.5 3.2 ± 1.3
1.9 ± 1.1 1.9 ± 1.7 5.6 ± 1.1 4.7 ± 1.5 3.7 ± 1.6
1.7 ± 0.8 2.1 ± 1.6 5.8 ± 0.8 5.5 ± 0.7 3.8 ± 1.4
Alkali adaptation
p value
before
after
0.34 0.13 0.02 0.27 0.05
1.1 ± 0.4 1.1 ± 0.2 5.1 ± 0.9 2.7 ± 1.5 3.0 ± 1.2
1.3 ± 0.7 1.2 ± 0.4 5.7 ± 0.5 2.8 ± 1.5 3.3 ± 1.0
0.20 0.51 0.04 0.71 0.21
0.42 0.31 0.61 0.16 0.74
2.0 ± 1.3 2.2 ± 1.8 5.8 ± 0.8 4.7 ± 1.4 3.6 ± 1.3
2.4 ± 1.4 2.2 ± 1.8 5.3 ± 1.5 4.8 ± 1.5 4.0 ± 1.4
0.22 0.95 0.21 0.77 0.07
CF Plaque MS1 Plaque LB1 Plaque pH 5.22 Saliva MS3 Saliva LB3 CA Plaque MS1 Plaque LB1 Plaque pH 5.22 Saliva MS3 Saliva LB3
In this short cross-sectional clinical trial with 1-week periods of daily rinses with sugar and urea solutions, respectively, the largest effects on approximal supragingival plaque pH and aciduric bacteria in plaque and saliva were seen after daily sugar rinses in CF compared to CA individuals. Daily rinses with urea did not significantly affect plaque pH in either of the groups but resulted in increased aciduric plaque bacteria in the CF group. Despite overall lower levels of mutans streptococci, the most pronounced effect on pH after daily sugar rinses in the CF individuals harboring less mutans streptococci than the CA individuals is in concordance with a previous study where plaque pH was compared in groups of individuals harboring 106 mutans streptococci/ml saliva, respectively [Aranibar Quiroz et al., 2003]. In a recent study a larger number of adolescents grouped after their ability to lower plaque pH after a sugar rinse were compared. In that study there were no differences in the number of mutans streptococci, neither in saliva nor in plaque, between groups with more or less caries lesions [Aranibar Quiroz et al., 2014]. Different from the study by Aranibar Quiroz et al. [2003], where the number of salivary mutans streptococci in the high mutans group was increased after 1 week of sugar rinsing 5 times/day, no significant effect on these
bacteria was seen in any of the groups in the present study. Also different from the previous study, baseline pH was higher in the CF group than in the CA group before, but not after, the acid adaptation period. This finding before acid adaptation is in concordance with a recent study [Aranibar Quiroz et al., 2014], where a group with the lowest minimum pH had the lowest baseline pH and the highest number of caries lesions. One-week rinsing with urea did not have any major effect on plaque pH, although somewhat increased pH values were seen in the CA group during the first 5–10 min after a urea challenge. The baseline pH was also not different between the groups, neither before nor after the rinsing period. However, the pH 6.5–10 strips used here did not discriminate values around pH 7 as well as did the pH 4–7 strips used for the acid period. Therefore, the pH 4–7 strips may be preferable for baseline measurements in alkali as well as acid formation studies. Previous studies showed a correlation between low caries activity and high urease activity in the dental plaque [Shu et al., 2007; Nascimento et al., 2009; Gordan et al., 2010; Toro et al., 2010; Morou-Bermudez et al., 2011]. Although an increased plaque pH after urea rinsing should reflect the urease activity, a similar correlation was not revealed in the present study. This may be due to several reasons. One could be different caries activity of the subjects in the present and former studies. In the previous studies urease activ-
In vivo Plaque pH after Sugar and Urea Rinses
Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Discussion
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MS = Mutans streptococci; LB = lactobacilli; p values for comparisons before and after the respective adaptation periods within the two groups. 1 Number of bacteria (log CFU). 2 Total number of plaque bacteria growing on an agar plate at pH 5.2 (log CFU). 3 Number of bacteria (log CFU/ml).
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Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Takahashi and Nyvad, 2011; Liu et al., 2012]. A more pronounced effect on the bacteria in CF compared to CA individuals could result from an initially higher number of bacteria being adapted to an acidic milieu in the CA group and therefore, these individuals had more bacteria prone to produce acid and protective alkali already from the start of the study. A higher number of bacteria being prone to alkali formation could be one reason why a slight although nonsignificant effect of daily rinses with urea at a low concentration was seen in the CA group only. More lactobacilli in saliva after the sucrose-rinsing period in the CF group may reflect increased acidic oral conditions in these individuals. It could be summarized that a group of CF adolescents and young adults had a higher plaque pH before but not after an acid adaptation period compared with a CA group. The adaptation period increased acid formation after a sugar challenge more in the CF individuals. An alkali adaptation period did not have any significant effect on plaque pH in any of the groups, although a urea challenge resulted in slightly increased pH values in the CA group. Significant effects on the bacterial plaque flora were only found for the total numbers of bacteria growing at pH 5.2 in the CF group, where the number was increased both after the acid and the alkali adaptation periods. The type of bacteria present remains to be analyzed.
Acknowledgments We thank Mrs. Gunilla Hjort for excellent technical assistance. The study was founded by the grant TUAGBG-89603 (VG Region, Sweden) and the Swedish Patent Revenue Fund for Preventive Odontology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions Haidar Hassan (PhD resident) participated in designing and reporting of the study, performed all the tests and data analyses and wrote a first draft of the manuscript. Anette Carlén (study PI) was responsible for planning, performance and writing of the manuscript. Peter Lingström participated in the design and reporting of the study.
Disclosure Statement There are no potential conflicts of interest for any of the authors. The authors alone are responsible for the content and writing of the paper.
Hassan /Lingström /Carlén
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ity was further examined by the determination of ammonia produced in pooled plaque samples ex vivo. In the present study the net effect of urea on plaque pH was examined at specific interproximal sites in vivo with less plaque present and where the dynamic effects of several agents and activities, e.g. saliva flow, gingival crevicular fluid and buffering, could affect the urea penetration into the plaque and overall outcome. The urea concentration in the rinsing solution was also low. Preliminary studies using rinses with higher concentrations did, however, not result in notably higher plaque pH responses. Many individuals experienced these rinses as having a bitter taste, which could affect compliance and particularly when young individuals are involved. With the attempt to further assure compliance regarding rinsing, the participants were reminded by SMS messages. Our pilot tests involved adults only (21–31 years) and the caries activity was not considered. No significant differences after alkali adaptation in the present study may therefore be due to younger individuals with less caries than in our pilot tests. Furthermore, many of the individuals in the present study used products with fluoride in addition to toothpaste and it has been shown that fluoride could inhibit the urease activity [Barboza-Silva et al., 2005]. The participants were told to follow their regular oral hygiene habits since it was considered interesting to see how plaque pH could vary in CF and CA individuals under normal hygiene conditions. More individuals and/or individuals with a higher caries activity may be necessary to better evaluate differences in plaque pH between CF and CA individuals after challenges with sugar and urea. The saliva secretion rate and buffering capacity were reported to affect pH in plaque and be related to caries [Kleinberg, 2002; Varma et al., 2008]. In the present study the numerical values for these saliva variables were lower for the CA individuals, but the differences were not statistically different. An interesting finding was, however, found in the present study where the total number of plaque bacteria growing at pH 5.2 increased after both the acid and alkali adaptation periods in the CF group. Our findings of no effect of the rinses on mutans streptococci, together with previous reports of acidogenic and aciduric bacteria in plaque, suggest that other bacteria like non-mutans streptococci, bifidobacteria and Actinomyces spp. may be present and contribute to the pH fall [Aas et al., 2008; Mantzourani et al., 2009]. In order to protect themselves from the acidic milieu, the acidogenic bacteria produce alkali, which also other bacteria such as Campylobacter ureolyticus and Haemophilus parainfluenzae may do [Kilian and Schiott, 1975; Burne and Marquis, 2000;
References
In vivo Plaque pH after Sugar and Urea Rinses
Gordan VV, Garvan CW, Ottenga ME, Schulte R, Harris PA, McEdward D, Magnusson I: Could alkali production be considered an approach for caries control? Caries Res 2010;44:547–554. Hugoson A, Koch G, Helkimo AN, Lundin SA: Caries prevalence and distribution in individuals aged 3–20 years in Jonkoping, Sweden, over a 30-year period (1973–2003). Int J Paediatr Dent 2008;18:18–26. Kilian M, Schiott CR: Haemophili and related bacteria in the human oral cavity. Arch Oral Biol 1975;20:791–796. Kleinberg I: A mixed-bacteria ecological approach to understanding the role of the oral bacteria in dental caries causation: an alternative to Streptococcus mutans and the specific-plaque hypothesis. Crit Rev Oral Biol Med 2002;13:108– 125. Larsen MJ, Pearce EIF: A computer program for correlating dental plaque pH values, cH+, plaque titration, critical pH, resting pH and the solubility of enamel apatite. Arch Oral Biol 1997;42:475–480. Lingström P, van Ruyven FO, van Houte J, Kent R: The pH of dental plaque in its relation to early enamel caries and dental plaque flora in humans. J Dent Res 2000;79:770–777. Liu YL, Nascimento M, Burne RA: Progress toward understanding the contribution of alkali generation in dental biofilms to inhibition of dental caries. Int J Oral Sci 2012;4:135–140. Mantzourani M, Gilbert SC, Sulong HN, Sheehy EC, Tank S, Fenlon M, Beighton D: The isolation of bifidobacteria from occlusal carious lesions in children and adults. Caries Res 2009; 43:308–313. Marsh PD: Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263–271. Marthaler TM: Changes in dental caries 1953– 2003. Caries Res 2004;38:173–181. Möller ÅJR: Microbiological examination of root canals and periapical tissues of human teeth. Odontol Tidskr 1966;74:1–380.
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Morou-Bermudez E, Elias-Boneta A, Billings RJ, Burne RA, Garcia-Rivas V, Brignoni-Nazario V, Suárez-Pérez E: Urease activity as a risk factor for caries development in children during a three-year study period: a survival analysis approach. Arch Oral Biol 2011;56:1560–1568. Nascimento MM, Gordan VV, Garvan CW, Browngardt CM, Burne RA: Correlations of oral bacterial arginine and urea catabolism with caries experience. Oral Microbiol Immunol 2009;24:89–95. Scheie AA, Fejerskov O, Lingström P, Birkhed D, Manji F: Use of palladium touch microelectrodes under field conditions for in vivo assessment of dental plaque pH in children. Caries Res 1992;26:44–51. Shu M, Morou-Bermudez E, Suárez-Pérez E, Rivera-Miranda C, Browngardt CM, Chen YY, Magnusson I, Burne RA: The relationship between dental caries status and dental plaque urease activity. Oral Microbiol Immunol 2007; 22:61–66. Stephan RM: Changes in hydrogen-ion concentration on tooth surfaces and in carious lesions. J Am Dent Assoc 1940;27:718–723. Svensäter G, Borgström M, Bowden GH, Edwardsson S: The acid-tolerant microbiota associated with plaque from initial caries and healthy tooth surfaces. Caries Res 2003;37:395–403. Takahashi N, Nyvad B: The role of bacteria in the caries process: ecological perspectives. J Dent Res 2011;90:294–303. Takahashi N, Yamada T: Acid-induced acid tolerance and acidogenicity of non-mutans streptococci. Oral Microbiol Immunol 1999;14:43–48. Toro E, Nascimento MM, Suarez-Perez E, Burne RA, Elias-Boneta A, Morou-Bermudez E: The effect of sucrose on plaque and saliva urease levels in vivo. Arch Oral Biol 2010;55:249–254. Varma S, Banerjee A, Bartlett D: An in vivo investigation of associations between saliva properties, caries prevalence and potential lesion activity in an adult UK population. J Dent 2008; 36:294–299. Welin-Neilands J, Svensäter G: Acid tolerance of biofilm cells of Streptococcus mutans. Appl Environ Microbiol 2007;73:5633–5638.
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Aas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, Leys EJ, Paster BJ: Bacteria of dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol 2008;46:1407–1417. Abelson DC, Mandel ID: The effect of saliva on plaque pH in vivo. J Dent Res 1981;60:1634– 1638. Almståhl A, Wikström M, Kroneld U: Microflora in oral ecosystems in primary Sjögren’s syndrome. J Rheumatol 2001;28:1007–1013. Aranibar Quiroz EM, Alstad T, Campus G, Birkhed D, Lingström P: Relationship between plaque pH and different caries-associated variables in a group of adolescents with varying caries prevalence. Caries Res 2014;48:147–153. Aranibar Quiroz EM, Lingström P, Birkhed D: Influence of short-term sucrose exposure on plaque acidogenicity and cariogenic microflora in individuals with different levels of mutans streptococci. Caries Res 2003;37:51–57. Barboza-Silva E, Castro AC, Marquis RE: Mechanisms of inhibition by fluoride of urease activities of cell suspensions and biofilms of Staphylococcus epidermidis, Streptococcus salivarius, Actinomyces naeslundii and of dental plaque. Oral Microbiol Immunol 2005;20:323–332. Belli WA, Marquis RE: Adaptation of Streptococcus mutans and Enterococcus hirae to acid stress in continuous culture. Appl Environ Microbiol 1991;57:1134–1138. Burne RA, Marquis RE: Alkali production by oral bacteria and protection against dental caries. FEMS Microbiol Lett 2000;193:1–6. Carlén A, Hassan H, Lingström P: The ‘strip method’: a simple method for plaque pH assessment. Caries Res 2010;44:341–344. Dong YM, Pearce EI, Yue L, Larsen MJ, Gao XJ, Wang JD: Plaque pH and associated parameters in relation to caries. Caries Res 1999; 33: 428–436. Ericsson Y: Clinical investigations of the salivary buffering action. Acta Odontol Scand 1959;17: 131–165. Gold OG, Jordan HV, Van Houte J: A selective medium for Streptococcus mutans. Arch Oral Biol 1973;18:1357–1364.
Received: September 3, 2013 Accepted after revision: February 20, 2014 Published online: October 9, 2014
Plaque pH in Caries-Free and Caries-Active Young Individuals before and after Frequent Rinses with Sucrose and Urea Solution Haidar Hassan a Peter Lingström b Anette Carlén a Departments of a Oral Microbiology and Immunology and b Cariology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Abstract Objective: To examine pH in the approximal dental biofilm after acid and alkali formation from sucrose and urea, after an adaptation period to these substances, in caries-free (CF) and caries-active (CA) individuals. Saliva flow and buffer capacity, and aciduric bacteria in saliva and plaque were also examined. Material and Methods: Twenty adolescents and young adults (15–21 years) with no caries (n = 10, Dm + iMFS = 0) or ≥1 new manifest lesions/year (n = 10, DmMFS = 3.4 ± 1.8) participated. After plaque sampling, interproximal plaque pH was measured using the strip method before (baseline) and up to 30 min (final pH) after random distribution of a 1-min rinse with 10 ml of 10% sucrose or 0.25% urea. This procedure was repeated after a 1-week adaptation period of rinsing 5 times/day with 10 ml of the selected solution. After a 2-week washout period the second solution was similarly tested. Mutans streptococci, lactobacilli and pH 5.2-tolerant bacteria were analyzed by culturing. Results: In the CF group, acid adaptation resulted in lowering of baseline and final plaque pH values after a sugar chal-
© 2014 S. Karger AG, Basel 0008–6568/14/0491–0018$39.50/0 E-Mail [email protected] www.karger.com/cre
lenge, and in increased numbers of bacteria growing at pH 5.2, which was increased also after alkali adaptation. In the CA group, the final pH was decreased after acid adaptation. No clear effects of alkali adaptation were seen in this group. Conclusion: One-week daily rinses with sucrose and urea had the most pronounced effect on the CF group, resulting in increased plaque acidogenicity from the sugar rinses and increased number of acid-tolerant plaque bacteria from both rinses. © 2014 S. Karger AG, Basel
Even if dental caries has decreased worldwide since the middle of the last century, it still constitutes a large problem for many individuals [Marthaler, 2004; Hugoson et al., 2008]. It is a multifactorial disease, which occurs according to the ecological plaque hypothesis [Marsh, 1994], when the balance in the microflora of the dental biofilm is disturbed by modifications in the oral environment. Dental biofilm activity is controlled by a number of environmental factors including fermentable carbohydrates and alkali. Stephan [1940] showed that the bacterial metabolism of glucose, resulting in a pH decrease of the dental biofilm, plays a major role in caries development. Frequent Assoc. Prof. Anette Carlén Department of Oral Microbiology and Immunology Institute of Odontology, University of Gothenburg Box 450, SE–405 30 Gothenburg (Sweden) E-Mail anette.carlen @ odontologi.gu.se
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Key Words Acid formation · Alkali formation · Biofilm adaptation · Plaque pH
Material and Methods Study Groups Healthy CF and CA adolescents and young adults (15–21 years) were randomly selected from the patient’s dental records among four Public Dental Clinics in and around Gothenburg City, Sweden. A total of 150 individuals were invited by letter and telephone call to participate in the study. The letter was sent to the parents or legal guardian in case of adolescents and to the young adult volunteers. Eleven individuals with and 12 without any caries, who met the inclusion and exclusion criteria, accepted to join the study. Ac-
In vivo Plaque pH after Sugar and Urea Rinses
cording to our pilot tests, 10 individuals/group were sufficient to reveal statistically significant lowered or increased plaque pH after 1-week rinsing with 10% sucrose or 0.25% urea solution, respectively. CF was defined as Dm + iMFS (decayed, missed and filled surfaces, both manifest and initial caries) = 0, CA as DmMFS ≥ 1/year new, primary, manifest caries lesions (occlusal and/or approximal) in the last 3 years. The inclusion criteria were: (1) all permanent teeth erupted, (2) regular attendance to a Public Dental Clinic, (3) normal stimulated salivary secretion rate, ≥1.0 ml/min, (4) no smoking and no snuffing. Exclusion criteria were: (1) had disease and medication, (2) not within the age range 15–21 years, (3) had deciduous teeth, (4) irregular attendance to Public Dental Clinic, (5) smoker and/or snuffer, (6) had received antibiotics within the last 3 months prior to the study. The study was approved by the Ethics Committee at the University of Gothenburg (Dnr 282-10) and followed the ethical considerations of the Helsinki Declaration. Written informed consent was obtained from the parents or legal guardian in case of adolescents or from the young adult volunteers, prior to the study. Study Design The study was designed as a single-blind (for the participants), randomized, controlled, two-leg cross-sectional clinical trial. It consisted of two test periods and two washout periods. Two test solutions, 10% sucrose and 0.25% urea, were distributed in a random order. All participants came to the laboratory 5 times (fig. 1). At the first visit, where medical and dental anamnesis was obtained, detailed information about the study was given, and the consent form was signed. At the end of this first visit, professional tooth cleaning using rubber cup and prophylactic paste (Cleaning RDA 170; CCS Clean Chemical Sweden, Borlänge, Sweden) as well as dental flossing without fluoride (Reach®, waxed floss, Johnson & Johnson, Canada) was performed. The second and fourth visits (before the 1-week adaptation periods) followed after a washout period of 2 weeks. The third and fifth visits followed after the 1-week adaptation periods where the participants rinsed with one of the two randomly selected test solutions. During these washout and test periods, the participants were asked to keep their regular dietary and oral hygiene habits. On the last day prior to all four visits, the participants were asked to refrain from oral hygiene and from eating and drinking during the last 2 h prior to the visit. An SMS message was sent daily as a reminder to all the participants during the adaptation periods and before every visit. At the four visits II–V (including start and end of the two adaptation periods), supragingival plaque samples were collected, plaque pH was measured before and up to 30 min after a 1-min rinse with the randomly selected test solution, and a stimulated saliva sample was collected. In addition, professional tooth cleaning as described above was performed at the end of the adaptation visits (III, V). All sampling and measurements were performed by one and the same operator (H.H.). Rinsing Regimen The two rinse solutions were coded A (10% sucrose) and B (0.25% urea) and were distributed in 10-ml portions in plastic tubes. Before each adaptation period, the participants were given
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decreases of the dental biofilm pH activate acidogenic and aciduric microorganisms to produce acids and to adapt to low pH values, which can lead to significant tooth demineralization [Belli and Marquis, 1991; Takahashi and Yamada, 1999; Welin-Neilands and Svensäter, 2007]. A few in vivo studies have been conducted to investigate the ability of the dental biofilm to produce acid in relation to caries [Abelson and Mandel, 1981; Scheie et al., 1992; Dong et al., 1999; Lingström et al., 2000]. In one study, the in vivo adaptation of plaque bacteria to an acidic environment was demonstrated [Aranibar Quiroz et al., 2003]. The acidifying metabolism in plaque is followed by an alkalization phase, generating increased pH from ammonia after bacterial metabolism of e.g. urea and arginine and by the effects of saliva [Burne and Marquis, 2000; Kleinberg, 2002]. This phase neutralizes the acids and protects the bacteria from acid damage and leads to raised pH in the plaque [Kleinberg, 2002]. Recent studies have also demonstrated that a low ability to form alkaline substances from urea and arginine is associated with a higher caries activity [Shu et al., 2007; Nascimento et al., 2009; Gordan et al., 2010; Toro et al., 2010; Morou-Bermudez et al., 2011]. However, no study that examined the dental biofilm adaptation to alkali formation has been found. Our hypothesis was that frequent supply of sucrose and urea, respectively, affects the acid and alkali formation in the dental biofilm differently in caries-free (CF) and in caries-active (CA) individuals. The main aim of the present study was therefore to investigate, by in vivo pH measurements, the ability of the dental biofilm to form acid and alkali after short-term adaptation periods with daily exposure of sucrose and urea in relation to caries. Stimulated saliva secretion rate and buffer capacity as well as acid-tolerant bacteria in the saliva and plaque were also examined. To our knowledge, this study is the first attempt to investigate the dental biofilm adaptation to alkali formation.
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Fig. 1. Study design. Sampling and pH measurements performed: visit II, before start of the first 1-week acid (BAA) or base adaptation
(BBA) period using a randomly selected 10% sucrose or 0.25% urea rinse; visit III, after the first acid (AAA) or base adaptation (ABA) period; visit IV, before the second 1-week acid (BAA) or base adaptation (BBA) period; visit V, after the second acid (AAA) or base adaptation (ABA) period.
Microbial Analysis The plaque and saliva samples were incubated at 37 ° C for 30 min before further dilution and plated onto agar medium essentially as previously described [Almståhl, 2001]. In short, after dilution to 10–4, 100 μl of the concentrated and diluted plaque sample solutions were plated on mitis salivarius-bacitracin agar [Gold et al., 1973], Rogosa SL agar and on pH 5.2 agar [Svensäter et al., 2003]. The number of colony-forming units (CFU) of mutans streptococci and lactobacilli was calculated after incubation of the mitis salivarius-bacitracin agar and Rogosa agar plates in 90% N2 and 10% CO2 at 37 ° C for 3–5 days, and identification of mutans streptococci and lactobacilli from their typical colony morphology. The total number of bacteria growing at pH 5.2 was enumerated after anaerobic incubation in 95% H2 and 5% CO2 for 7 days at 37 ° C. The saliva samples were diluted to 10–4 before 2 × 25 μl of the dilutions were plated on mitis salivarius-bacitracin agar and Rogosa SL agar plates. After incubation in 90% N2 and 10% CO2 at 37 ° C for 3–5 days, mutans streptococci and lactobacilli were identified and enumerated as above.
Plaque Samples Two supragingival dental plaque samples were collected from the interproximal sites between the upper right first and second molars, and the upper left first and second molars, respectively, using sterile toothpicks (TePe Birch, TePe, Malmö, Sweden). The samples were pooled in 3.5 ml VMGA III [Möller, 1966] transport medium and the samples were incubated onto agar plates within 24 h for microbial analyses. pH Measurements Supragingival plaque pH was measured using the pH strip method as previously described [Carlén et al., 2010]. Measurements were performed between the upper right and left second premolar and first molar, respectively; pH was measured before (baseline, 0 min) and at 2, 5, 10, 15, 20 and 30 min after a 1-min rinse with 10 ml of the test solution. A strip (Spezialindikator, Merck, Darmstadt, Germany) measuring pH 4.0–7.0 and pH 6.5– 10.0 was inserted into the interproximal area in order to test acid and alkali formation, respectively. Stimulated Saliva Stimulated saliva was collected by chewing on a piece of paraffin during 5 min and continuously spitting the obtained saliva into an ice-chilled tube. The secretion rate was determined after which 1 ml of the saliva was transferred to VMGA IIs [Möller, 1966] transport medium for microbial analysis within 24 h. Another milliliter of the saliva was used for determination of the buffer capacity according to Ericsson [1959].
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Statistical Analyses Statistical descriptive analyses were performed using IBM® SPSS® Statistics (version 19.0.0.1, IBM software, 2011). The mean approximal plaque pH (±SD) for all participants at the different time points was calculated for the two interproximal sites. The maximum pH fall and minimum pH after a 10% sucrose rinse and the maximum pH increase and maximum pH after a 0.25% urea rinse were also calculated. Changes in plaque pH during 30 min were calculated from the area of the curve below the critical pH of enamel (pH 5.7; AUC5.7) and of dentine (pH 6.2; AUC6.2) for acid formation and the area of the curve above pH 7.0 (AOC7.0) for alkali formation. AUC and AOC values were calculated using the computer program according to Larsen and Pearce [1997]. The bacterial numbers were transformed logarithmically and the mean ± SD was calculated. Student’s twosample, paired t test was used to analyze the significance of differences within the same group and nonpaired t test between the CF and CA groups; p < 0.05 was considered statistically significant.
Hassan /Lingström /Carlén
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sufficient numbers of tubes for five rinses/day during 1 week. They were instructed to keep the tubes in the refrigerator and only take out the necessary number of tubes when leaving home. They were further instructed to rinse with the 10-ml solution for 1 min early in the morning 2 h after tooth brushing, before lunch, after lunch, later in the afternoon and in the evening 2 h before tooth brushing. They were also asked to avoid eating or drinking 1 h after rinsing and at least 2 h between the rinses. To evaluate compliance, the participants were asked if they had followed the instructions given.
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Study Groups Out of the 23 volunteers included, 1 never attended, 1 got pulpitis and 1 moved to another city during the study. Thus, 20 of the volunteers completed the study: 10 CF individuals (Dm + iMFS = 0, 6 female and 4 male, 15–21 years, mean age 17.0 ± 1.8 years) and 10 CA individuals (DmMFS = 3.4 ± 1.8, 5 female and 5 male, 15–20 years, mean age 17.0 ± 1.6).
Dental Anamnesis The participants were asked about their oral hygiene habits and all reported that they brushed their teeth twice a day and used fluoridated toothpaste containing 1,450 ppm fluoride. 80% the CF individuals reported an infrequent use of additional fluoride-containing mouthrinses (Flux 900 ppm F), whereas 40% in the CA group used it regularly (once a day) as recommended by their dentist. Dental floss was reported to be used by 30 and 40% in the CF and CA groups, respectively. There were no differ-
In vivo Plaque pH after Sugar and Urea Rinses
Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Results
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Fig. 2. The approximal plaque pH prior to (0 min), and up to 30 min after a 1-min rinse with 10% sucrose (a, b) or 0.25% urea (c, d) in 10 CF (a, c) and 10 CA (b, d) individuals; pH measurements were performed before (BAA) and after (AAA) adaptation to acid and before (BBA) and after (ABA) adaptation to alkali by 1-week daily rinses with the test solutions 5 times/day.
Table 1. pH values in approximal plaque up to 30 min after a 10% sucrose rinse in CF and CA individuals before and after acid adaptation
CF
Baseline pH Max pH drop Min pH Final pH AUC5.7 AUC6.2
CA
acid adaptation before
acid adaptation after
p value
acid adaptation before
acid adaptation after
p value
6.6 ± 0.4 1.1 ± 0.4 5.5 ± 0.6 6.13 ± 0.5 4.9 ± 7.1 12.4 ± 12.6
6.3 ± 0.5 1.1 ± 0.3 5.2 ± 0.6 5.8 ± 0.6 9.3 ± 11.4 18.5 ± 16.6
0.03 0.59 0.07 0.04 0.11 0.06
6.3 ± 0.3 1.1 ± 0.6 5.2 ± 0.6 6.2 ± 0.5 7.2 ± 9.8 16.12 ± 13.7
6.2 ± 0.4 1.1 ± 0.4 5.1 ± 0.5 5.9 ± 0.4 8.4 ± 8.3 20.6 ± 10.9
0.32 0.93 0.30 0.02 0.39 0.06
AUC5.7, AUC6.2 = AUC at pH 5.7 and pH 6.2, respectively, up to 30 min after the sucrose rinse. p values for comparisons before and after adaptation within the respective groups.
Supragingival Plaque pH before and after Acid Adaptation The plaque pH curves obtained after a sugar challenge, before and after the adaptation period of daily rinses with 10% sucrose, are seen in figure 2a, b. After the adaptation period, the sugar challenge resulted in statistically significant lower pH values at time points 0 (baseline pH), 10, 15 and 30 min (final pH) in the CF group, and at 15 and 30 min in the CA group. The curves were at a lower level especially for the CF group, but had the same shape after adaptation as before. A clear tendency to an increased mean AUC6.2 value was seen within both groups after the rinsing period. In the CF group, the minimum pH also tended to be decreased (table 1). Baseline pH was higher in the CF group compared to the CA group (p = 0.05) before but not after the acid adaptation period. Supragingival Plaque pH before and after Alkali Adaptation The curves of plaque pH obtained after an alkali challenge, before and after the adaptation period of daily rinses with 0.25% urea, are seen in figure 2c, d. In the CA group only, somewhat higher pH values after the adaptation period were noticed at baseline and up to 20 min after the urea challenge (fig. 2d). The area of the curve above pH 7 (AOC7.0) was numerically but not statistically significantly increased from 12.3 ± 8.3 to 13.8 ± 6.5 after the 22
Caries Res 2015;49:18–25 DOI: 10.1159/000360798
adaptation in this group. There were also no statistically significant differences between or within the groups for any of the other pH variables, neither before nor after the adaptation period (data not shown). Saliva Secretion Rate and Buffer Capacity There were no statistically significant differences in saliva secretion rate and buffer capacity before and after the two rinsing periods, neither within nor between the two groups, although somewhat lower numerical values were seen for the CA group at all four measurements (mean secretion rate: 2.0 ± 0.8 ml/min for CF and 1.7 ± 0.7 ml/ min for CA; mean buffer capacity: 6.2 ± 1.6 for CF and 5.7 ± 1.4 for CA). Microbiological Analysis At the start of the study (visit II), the mean numbers of mutans streptococci were higher in the CA group both in plaque (1.2 ± 0.6 for CF; 2.1 ± 1.3 for CA; p = 0.05) and in saliva (2.3 ± 1.9 for CF; 5.2 ± 1.1 for CA; p = 0.0004). No mutans streptococci were detected in the plaque samples from 8 of the CF and 4 of the CA individuals. The corresponding numbers of saliva samples with no detected mutans streptococci were 6 in the CF and none in the CA group. The mean log values of microbial data for saliva and plaque before and after the acid and alkali adaptation periods are presented in table 2. Significant differences were seen in the CF group only where the level of total plaque bacteria growing at pH 5.2 was increased after both periods and that of salivary lactobacilli after the acid adaptation period. Hassan /Lingström /Carlén
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ences in the mean of daily intake of main meals (2.8 ± 0.8 for CF/2.8 ± 0.6 for CA) and in between snacks (1.7 ± 0.8 for CF/1.7 ± 0.7 for CA).
Table 2. Mean number of aciduric bacteria (±SD) in plaque and saliva samples in CF and CA individuals before and after acid adaptation as well as before and after alkali adaptation
Acid adaptation
p value
before
after
1.2 ± 0.6 1.1 ± 0.3 5.4 ± 0.6 2.8 ± 1.5 2.4 ± 0.8
1.1 ± 0.4 1.2 ± 0.4 5.8 ± 0.7 2.7 ± 1.5 3.2 ± 1.3
1.9 ± 1.1 1.9 ± 1.7 5.6 ± 1.1 4.7 ± 1.5 3.7 ± 1.6
1.7 ± 0.8 2.1 ± 1.6 5.8 ± 0.8 5.5 ± 0.7 3.8 ± 1.4
Alkali adaptation
p value
before
after
0.34 0.13 0.02 0.27 0.05
1.1 ± 0.4 1.1 ± 0.2 5.1 ± 0.9 2.7 ± 1.5 3.0 ± 1.2
1.3 ± 0.7 1.2 ± 0.4 5.7 ± 0.5 2.8 ± 1.5 3.3 ± 1.0
0.20 0.51 0.04 0.71 0.21
0.42 0.31 0.61 0.16 0.74
2.0 ± 1.3 2.2 ± 1.8 5.8 ± 0.8 4.7 ± 1.4 3.6 ± 1.3
2.4 ± 1.4 2.2 ± 1.8 5.3 ± 1.5 4.8 ± 1.5 4.0 ± 1.4
0.22 0.95 0.21 0.77 0.07
CF Plaque MS1 Plaque LB1 Plaque pH 5.22 Saliva MS3 Saliva LB3 CA Plaque MS1 Plaque LB1 Plaque pH 5.22 Saliva MS3 Saliva LB3
In this short cross-sectional clinical trial with 1-week periods of daily rinses with sugar and urea solutions, respectively, the largest effects on approximal supragingival plaque pH and aciduric bacteria in plaque and saliva were seen after daily sugar rinses in CF compared to CA individuals. Daily rinses with urea did not significantly affect plaque pH in either of the groups but resulted in increased aciduric plaque bacteria in the CF group. Despite overall lower levels of mutans streptococci, the most pronounced effect on pH after daily sugar rinses in the CF individuals harboring less mutans streptococci than the CA individuals is in concordance with a previous study where plaque pH was compared in groups of individuals harboring 106 mutans streptococci/ml saliva, respectively [Aranibar Quiroz et al., 2003]. In a recent study a larger number of adolescents grouped after their ability to lower plaque pH after a sugar rinse were compared. In that study there were no differences in the number of mutans streptococci, neither in saliva nor in plaque, between groups with more or less caries lesions [Aranibar Quiroz et al., 2014]. Different from the study by Aranibar Quiroz et al. [2003], where the number of salivary mutans streptococci in the high mutans group was increased after 1 week of sugar rinsing 5 times/day, no significant effect on these
bacteria was seen in any of the groups in the present study. Also different from the previous study, baseline pH was higher in the CF group than in the CA group before, but not after, the acid adaptation period. This finding before acid adaptation is in concordance with a recent study [Aranibar Quiroz et al., 2014], where a group with the lowest minimum pH had the lowest baseline pH and the highest number of caries lesions. One-week rinsing with urea did not have any major effect on plaque pH, although somewhat increased pH values were seen in the CA group during the first 5–10 min after a urea challenge. The baseline pH was also not different between the groups, neither before nor after the rinsing period. However, the pH 6.5–10 strips used here did not discriminate values around pH 7 as well as did the pH 4–7 strips used for the acid period. Therefore, the pH 4–7 strips may be preferable for baseline measurements in alkali as well as acid formation studies. Previous studies showed a correlation between low caries activity and high urease activity in the dental plaque [Shu et al., 2007; Nascimento et al., 2009; Gordan et al., 2010; Toro et al., 2010; Morou-Bermudez et al., 2011]. Although an increased plaque pH after urea rinsing should reflect the urease activity, a similar correlation was not revealed in the present study. This may be due to several reasons. One could be different caries activity of the subjects in the present and former studies. In the previous studies urease activ-
In vivo Plaque pH after Sugar and Urea Rinses
Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Discussion
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MS = Mutans streptococci; LB = lactobacilli; p values for comparisons before and after the respective adaptation periods within the two groups. 1 Number of bacteria (log CFU). 2 Total number of plaque bacteria growing on an agar plate at pH 5.2 (log CFU). 3 Number of bacteria (log CFU/ml).
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Caries Res 2015;49:18–25 DOI: 10.1159/000360798
Takahashi and Nyvad, 2011; Liu et al., 2012]. A more pronounced effect on the bacteria in CF compared to CA individuals could result from an initially higher number of bacteria being adapted to an acidic milieu in the CA group and therefore, these individuals had more bacteria prone to produce acid and protective alkali already from the start of the study. A higher number of bacteria being prone to alkali formation could be one reason why a slight although nonsignificant effect of daily rinses with urea at a low concentration was seen in the CA group only. More lactobacilli in saliva after the sucrose-rinsing period in the CF group may reflect increased acidic oral conditions in these individuals. It could be summarized that a group of CF adolescents and young adults had a higher plaque pH before but not after an acid adaptation period compared with a CA group. The adaptation period increased acid formation after a sugar challenge more in the CF individuals. An alkali adaptation period did not have any significant effect on plaque pH in any of the groups, although a urea challenge resulted in slightly increased pH values in the CA group. Significant effects on the bacterial plaque flora were only found for the total numbers of bacteria growing at pH 5.2 in the CF group, where the number was increased both after the acid and the alkali adaptation periods. The type of bacteria present remains to be analyzed.
Acknowledgments We thank Mrs. Gunilla Hjort for excellent technical assistance. The study was founded by the grant TUAGBG-89603 (VG Region, Sweden) and the Swedish Patent Revenue Fund for Preventive Odontology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author Contributions Haidar Hassan (PhD resident) participated in designing and reporting of the study, performed all the tests and data analyses and wrote a first draft of the manuscript. Anette Carlén (study PI) was responsible for planning, performance and writing of the manuscript. Peter Lingström participated in the design and reporting of the study.
Disclosure Statement There are no potential conflicts of interest for any of the authors. The authors alone are responsible for the content and writing of the paper.
Hassan /Lingström /Carlén
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ity was further examined by the determination of ammonia produced in pooled plaque samples ex vivo. In the present study the net effect of urea on plaque pH was examined at specific interproximal sites in vivo with less plaque present and where the dynamic effects of several agents and activities, e.g. saliva flow, gingival crevicular fluid and buffering, could affect the urea penetration into the plaque and overall outcome. The urea concentration in the rinsing solution was also low. Preliminary studies using rinses with higher concentrations did, however, not result in notably higher plaque pH responses. Many individuals experienced these rinses as having a bitter taste, which could affect compliance and particularly when young individuals are involved. With the attempt to further assure compliance regarding rinsing, the participants were reminded by SMS messages. Our pilot tests involved adults only (21–31 years) and the caries activity was not considered. No significant differences after alkali adaptation in the present study may therefore be due to younger individuals with less caries than in our pilot tests. Furthermore, many of the individuals in the present study used products with fluoride in addition to toothpaste and it has been shown that fluoride could inhibit the urease activity [Barboza-Silva et al., 2005]. The participants were told to follow their regular oral hygiene habits since it was considered interesting to see how plaque pH could vary in CF and CA individuals under normal hygiene conditions. More individuals and/or individuals with a higher caries activity may be necessary to better evaluate differences in plaque pH between CF and CA individuals after challenges with sugar and urea. The saliva secretion rate and buffering capacity were reported to affect pH in plaque and be related to caries [Kleinberg, 2002; Varma et al., 2008]. In the present study the numerical values for these saliva variables were lower for the CA individuals, but the differences were not statistically different. An interesting finding was, however, found in the present study where the total number of plaque bacteria growing at pH 5.2 increased after both the acid and alkali adaptation periods in the CF group. Our findings of no effect of the rinses on mutans streptococci, together with previous reports of acidogenic and aciduric bacteria in plaque, suggest that other bacteria like non-mutans streptococci, bifidobacteria and Actinomyces spp. may be present and contribute to the pH fall [Aas et al., 2008; Mantzourani et al., 2009]. In order to protect themselves from the acidic milieu, the acidogenic bacteria produce alkali, which also other bacteria such as Campylobacter ureolyticus and Haemophilus parainfluenzae may do [Kilian and Schiott, 1975; Burne and Marquis, 2000;
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