This Article Abstract Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Eliasson, L. Articles by Lingström, P. Search for Related Content PubMed PubMed Citation Articles by Eliasson, L. Articles by Lingström, P. Social Bookmarking                         What's this?

Dental Plaque pH and Micro-organisms during Hyposalivation

¹ Department of Cariology, Faculty of Odontology, The Sahlgrenska Academy at Göteborg University, Box 450, SE-405 30 Göteborg, Sweden;
² Department of Oral Microbiology, Faculty of Odontology, The Sahlgrenska Academy at Göteborg University, Box 450, SE-405 30 Göteborg, Sweden; and
³ Department of Health Sciences, Kristianstad University, SE-291 88 Kristianstad, Sweden

Correspondence: ^* corresponding author, lars.e{at}telia.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
We have previously reported that minor gland and whole saliva flow rates and salivary proteins showed differences in individuals with primary Sjögren’s syndrome or head and neck radiation therapy, compared with controls (Eliasson et al., 2005). We now hypothesize that pH and number of acidogenic micro-organisms in dental plaque as well as saliva buffering capacity also differ in these individuals. Plaque pH was measured by the microtouch method up to 60 min after a sucrose rinse. Plaque collected from the same sites was analyzed for counts of total and acidic micro-organisms. Compared with their controls, the irradiated group but not the Sjögren’s syndrome group displayed significantly lower plaque pH, increased numbers of lactobacilli and Candida species, as well as reduced buffering capacity. Stepwise regression tests suggested that the buccal minor-salivary-gland secretion rate in the test groups and counts of mutans streptococci in the controls were of significant importance for dental plaque pH.

Key Words: buffer pH • hyposalivation • micro-organisms • plaque pH • saliva

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Reduced salivation occurs in individuals suffering from primary Sjögren’s syndrome or those who have been treated by radiation therapy for cancer in the head and neck region (Baum et al., 1985; Almståhl and Wikström, 2003). In primary Sjögren’s syndrome, lymphatic infiltration of the salivary glands reduces the function of the organs (List et al., 1998). The reduced secretion rates in irradiated patients are due to irreversible fibrosis and atrophy of the gland parenchyma (Rode et al., 2001), as well as damage to extraglandular blood vessels or nerve structures. A better residual salivary function could therefore be expected among primary Sjögren’s syndrome patients compared with radiation therapy patients, due to the ability of primary Sjögren’s syndrome salivary glands to respond to stimulation (Rhodus, 1997; Nusair and Rubinow, 1999). Changes in whole saliva composition in parallel with a reduced saliva flow rate have been reported for irradiated and primary Sjögren’s syndrome patients (Almståhl et al., 2001a; Andrews and Griffiths, 2001). We have previously reported that the minor salivary gland flow rate and protein concentrations are affected among both groups of individuals, but to a greater extent in the radiation therapy group (Eliasson et al., 2005).

Decreased salivary flow leads to reduced pH and an increase in the number of acidogenic micro-organisms in saliva and dental plaque (Lingström and Birkhed, 1993; Almståhl et al., 2001b; Vuotila et al., 2002; Almståhl et al., 2003). This condition increases the risk of both coronal and root caries (Ravald and List, 1998), as well as oral mucosal lesions (Samaranayake et al., 1988).

Despite the known association of reduced saliva flow with increased numbers of acidogenic micro-organisms in individuals with hyposalivation (Keene and Fleming, 1987; Weerkamp et al., 1987; Almståhl et al., 2003), there are no reports on the plaque acidogenicity and microflora of these individuals.

The aim of the present study was to examine the pH response to a sugar challenge in dental plaque, as well as acidogenic micro-organisms in that plaque, for individuals with hyposalivation due to primary Sjögren’s syndrome and radiation therapy. Moreover, the importance of acidogenic micro-organisms, stimulated whole saliva buffering capacity, and whole and minor gland saliva secretion rates on plaque acidogenicity was analyzed.

	MATERIALS & METHODS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Patients
Ten primary Sjögren’s syndrome patients, diagnosed, according to the Copenhagen criteria, 10 yrs prior to the investigation (Manthorpe et al., 1986), and ten patients who ended their radiation treatment (64.6 Gy) 3–5 yrs before the study participated. The sample size was considered sufficient, based on our previous experience from studies on these groups of patients (Almståhl and Wikström, 2003). All the patients in the primary Sjögren’s syndrome group were women with a mean age of 63.2 yrs (range, 42–76 yrs). In the radiation therapy group, four were women, and the mean age was 54.9 yrs (range, 38–76 yrs). The average number of teeth was 22.7 (range, 12–26) and 23.8 teeth (range, 15–28) in the primary Sjögren’s syndrome and radiation therapy groups, respectively. In both groups, the mean secretion rate was 0.4 mL/min of stimulated and < 0.1 mL/min of resting whole saliva (Eliasson et al., 2005). Two control groups (n = 10 per group)—matched with regard to gender, age (± 5 yrs), and number of teeth (± 5)—were also studied. None of the individuals in the test and control groups showed any signs of caries by visual inspection. Eight persons in the primary Sjögren’s syndrome group and four in the radiation therapy group used prescribed medication on a daily basis. Four preparations in the primary Sjögren’s syndrome group and one in the radiation therapy group were drugs that may reduce salivation. Among the controls, one person in each group used medication (for asthma and minor heart conditions, respectively), none with any reported adverse effects on salivation. No individual had undergone antibiotic treatment during the preceding 3 mos. The study was approved by the Ethics Committee at Göteborg University, Göteborg, Sweden, and written informed consent was obtained from all participants.

Study Design
Each participant visited the laboratory at the Department of Cariology once between 8 a.m. and 1 p.m. Prior to the visit, they were instructed not to clean their teeth proximally for 3 days, not to brush their teeth the same day, and not to eat or drink for at least 2 hrs prior to the test.

Plaque-pH Measurements
Plaque pH was measured by the microtouch method (Lingström et al., 1993), where a microelectrode (Beetrode^®, MEHP-1, W.P. Instruments Inc., New Haven, CT, USA) was connected to an Orion SA 720 pH/ISE Meter (Orion Research Inc., Boston, MA, USA), equipped with a reference electrode (MERE 1, W.P. Instruments Inc.). A salt bridge was established in a 3 M KCl solution between the reference electrode and a finger of the test participant. The electrode was calibrated against standard buffers before each reading (Scheie et al., 1992). Measurements were performed at two proximal dental sites: in the anterior and in the premolar/molar regions. With two exceptions, both sites were in the upper jaw. No metal fillings were present at the sites of measurements. After registering the resting pH (baseline = 0 min), a mouthrinse was performed with 10 mL of 10% sucrose for 1 min. Plaque pH was then recorded 2, 5, 10, 15, 20, 30, 40, 50, and 60 min after the start of the experiment. During this period, the participants were told to remain still and to avoid talking.

Micro-organisms in Plaque
Directly after the pH measurements, plaque was collected from the same 2 sites by means of sterile toothpicks. The cultivation media were Brucella agar (BBL Microbiological Systems, Cockeysville, MD, USA) with 50 mL/L of defibrinated horse blood, 20 mL/L of hemolyzed human blood, and 0.5 mg/L of menadione, Mitis-Salivarius-Bacitracin (MSB) agar (Gold et al., 1973), Rogosa agar (Difco, Detroit, MI, USA), and Sabouraud-Dextrose agar (Difco) with 100 mg/L of tetrazolium chloride (Sabouraud T). After the samples were diluted, a 100-µL quantity was inoculated onto each of the agar plates. Brucella agar plates were incubated by the hydrogen combustion technique at 36°C for 5–7 days. Plates with MSB agar and Rogosa agar were incubated in 90% CO₂ and 10% N₂ at 36°C for 3–5 days. Plates with Sabouraud T agar were incubated aerobically at 36°C for 3–5 days. The total number of bacteria was calculated from the growth on Brucella agar plates. Mutans streptococci were determined from their typical morphology on MSB agar (Emilson, 1983). Lactobacilli were identified as Gram-positive rods growing on Rogosa agar plates. The number of Candida species was calculated from the growth of lusterless and creamy whitish-pink, or pink, colonies on Sabouraud T agar plates.

Buffering Capacity
Stimulated saliva samples were collected in ice-chilled tubes after participants chewed on a piece of paraffin. The buffering capacity was determined by routine methods (Ericsson, 1959) within 15 min after the collection.

Statistical Methods
From each individual pH curve, the minimum pH and maximum pH drop were calculated. The AUC (area under the curve, pH x min) was calculated for pH 5.7 (AUC_5.7) and for pH 6.2 (AUC_6.2). The microbial data were logarithmically transformed. Since no statistically significant differences in pH and microbial counts were found between the two dental sites, the mean of the two was used for all further calculations. Mean pH values were calculated for each time point for each test and control group, i.e., primary Sjögren’s syndrome group, radiation therapy group, and their matched controls. Unpaired, two-tailed t tests were used for comparisons of variables between the test groups and their respective control groups. We used a stepwise regression model to test the importance of minor gland, stimulated and resting whole saliva secretion rates, stimulated whole saliva buffer capacity, and the numbers of lactobacilli, Candida species, and mutans streptococci on plaque acidogenicity, measured as both AUC_6.2 and AUC_5.7. Separate models were applied to two groups, the hyposalivators and their controls, respectively. StatView^® computer software (Abacus, Berkeley, CA, USA) was used for the analyses. p < 0.05 was regarded as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
The plaque-pH response to a sugar challenge in primary Sjögren’s syndrome, radiation therapy, and their respective control groups during the 60-minute test period is shown in the Fig. Compared with the controls, the primary Sjögren’s syndrome group displayed no significant differences in the measured plaque-pH variables. The curves representing the response in the primary Sjögren’s syndrome group and their controls were almost identical. A significant difference in pH response was found between the radiation therapy group and their controls at all time points between 15 and 60 min (p < 0.05–0.01). In the radiation therapy group, significantly reduced minimum and final pH, as well as increased AUC_6.2 and AUC_5.7, were found (Table 1).

View larger version (11K): [in this window] [in a new window]   Figure. Mean values of plaque-pH response to sugar challenge among patients with primary Sjögren’s syndrome (pSS) and head- and neck-irradiated patients (RT) and their matched controls (n = 10 in each group). The estimations are based on the mean from two different sites in each individual. Error bars at 5, 30, and 50 min represent SD.

The stepwise regression test models, with the present findings and the previously obtained secretion data (Eliasson et al., 2005), were applied separately to two groups: hyposalivators and controls, respectively. The tests revealed only one statistically significant factor of importance for plaque pH in the primary Sjögren’s syndrome and radiation therapy group. A reduced buccal gland saliva flow rate was correlated to high AUC_6.2 and AUC_5.7. This effect was not seen among the controls. In this case, the number of mutans streptococci was the only factor that correlated significantly to plaque acidogenicity, measured as AUC_5.7.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
In the present study of individuals with hyposalivation due to primary Sjögren’s syndrome or head and neck radiation therapy and their respective controls matched with respect to gender, age, and number of teeth, it was found that the irradiated individuals, but not the primary Sjögren’s syndrome individuals, had a plaque with higher acidogenic capacity than their controls. This paralleled our findings of reduced whole saliva buffer pH and previously reported buccal gland secretion rate (Eliasson et al., 2005) only in the radiation therapy group.

Previous studies have shown that a low stimulated whole saliva secretion rate and buffering pH produce a pronounced drop in pH and a slow recovery to neutral pH in the dental plaque after a sugar rinse (Abelson and Mandel, 1981; Lingström and Birkhed, 1993). This results in conditions favoring acidogenic micro-organisms (Hase and Birkhed, 1988). These conditions were found in the present study in the case of the radiation therapy individuals, who, in addition to reduced stimulated and resting whole saliva flow rates, had increased acidogenic plaque and low saliva buffer capacity compared with their healthy controls. Despite reduced whole and labial saliva flow rates (Eliasson et al., 2005), neither the plaque acidogenicity nor the buffer capacity differed significantly between the primary Sjögren’s syndrome group and their controls. This may be due to large individual variations in saliva secretion rate and buffering pH in primary Sjögren’s syndrome individuals and in healthy women during or after menopause (Laine and Leimola-Virtanen, 1996; Almståhl et al., 2001b). Although not statistically significant, the two control groups differed in terms of buffering capacity and saliva secretion rates (Eliasson et al., 2005). These differences might similarly result from variations between and among women, but also from differences between men and women (Heintze et al., 1983), since the groups deviated with respect to both individual age and gender.

In addition to the known importance of whole saliva, our studies further suggest that minor gland saliva has an important effect on the acidogenicity of dental plaque during hyposalivation. In our parallel study, a reduced buccal gland saliva flow was seen only in the irradiated patients, and the regression tests revealed that the buccal minor gland saliva secretion rate was more important than the whole saliva flow rate and buffer capacity for plaque acidity among the hyposalivators. Our findings therefore suggest that the secretion from the buccal glands, delivered close to the teeth, could affect the dental plaque more than whole saliva in these patients during resting conditions. Our finding that mutans streptococci were important for the drop and recovery of plaque-pH (AUC_5.7) after a sugar challenge in the controls agrees with previous data for healthy individuals with a normal whole saliva flow (Aranibar Quiroz et al., 2003).

Our research group has previously reported that hyposalivation of different origins, i.e., Sjögren’s syndrome and head and neck radiation, could affect the oral microflora and saliva differently (Almståhl et al., 2001a, 2003). Contrary to our previous findings (Almståhl et al., 2001b), we did not find significantly increased numbers of lactobacilli in individuals with primary Sjögren’s syndrome compared with their controls. This discrepancy is probably due to the fact that plaque here was collected where the pH was measured, i.e., mostly between unrestored teeth with comparably few bacterial retention sites. The present findings of higher numbers of lactobacilli and Candida species in the dental plaque of radiation therapy patients compared with primary Sjögren’s syndrome patients was consistent with our previous data on the microbiota in these groups of patients (Almståhl et al., 2003). Our studies suggest that lactobacilli, which are thought to be favored by retention sites that are more common in primary Sjögren’s syndrome than radiation therapy patients (Almståhl et al., 2003), could be favored to an even greater extent by acidogenic conditions resulting from a low saliva flow rate and buffer capacity.

In conclusion, we found an acidogenic response to sugar challenge, concomitant with a shift toward a more acidogenic microflora, in proximal dental plaque only in the group of individuals with hyposalivation due to radiation therapy. Furthermore, the buccal gland secretion rate was important in relation to plaque acidogenicity during hyposalivation, whereas the number of mutans streptococci was important in individuals with normal saliva secretion rates.

	ACKNOWLEDGMENTS

 
This project was supported by Patentmedelsfonden för Odontologisk Profylaxforskning, the Göteborg Dental Society, and Svenska Tandläkarnes Inköpsförenings Stiftelse för Forskning och Studier. We would like to thank Ann-Charlott Börjesson for her excellent technical assistance.

Received for publication May 3, 2005. Revision received November 4, 2005. Accepted for publication December 12, 2005.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 

• Abelson DC, Mandel ID (1981). The effect of saliva on plaque pH in vivo. J Dent Res 60:1634–1638.
• Almståhl A, Wikström M (2003). Electrolytes in stimulated whole saliva in individuals with hyposalivation of different origins. Arch Oral Biol 48:337–344.[Medline] [Order article via Infotrieve]
• Almståhl A, Wikström M, Groenink J (2001a). Lactoferrin, amylase and mucin MUC5B and their relation to the oral microflora at hyposalivation of different origins. Oral Microbiol Immunol 16:345–352.[CrossRef][Medline] [Order article via Infotrieve]
• Almståhl A, Wikström M, Kroneld U (2001b). Microflora in oral ecosystems in primary Sjögren’s syndrome. J Rheumatol 28:1007–1013.[Medline] [Order article via Infotrieve]
• Almståhl A, Wikström M, Stenberg I, Jakobsson A, Fagerberg-Mohlin B (2003). Oral microbiota associated with hyposalivation of different origins. Oral Microbiol Immunol 18:1–8.[CrossRef][Medline] [Order article via Infotrieve]
• Andrews N, Griffiths C (2001). Dental complications of head and neck radiotherapy: Part 1. Aust Dent J 46:88–94.[Medline] [Order article via Infotrieve]
• Aranibar Quiroz EM, Lingström P, Birkhed D (2003). Influence of short-term sucrose exposure on plaque acidogenicity and cariogenic microflora in individuals with different levels of mutans streptococci. Caries Res 37:51–57.[Medline] [Order article via Infotrieve]
• Baum BJ, Bodner L, Fox PC, Izutsu KT, Pizzo PA, Wright WE (1985). Therapy-induced dysfunction of salivary glands: implications for oral health. Spec Care Dentist 5:274–277.[CrossRef][Medline] [Order article via Infotrieve]
• Eliasson L, Almståhl A, Lingström P, Wikström M, Carlén A (2005). Minor gland saliva flow rate and proteins in subjects with hyposalivation due to Sjögren’s syndrome and radiation therapy. Arch Oral Biol 50:293–299.[Medline] [Order article via Infotrieve]
• Emilson CG (1983). Prevalence of Streptococcus mutans with different colonial morphologies in human plaque and saliva. Scand J Dent Res 91:26–32.[Medline] [Order article via Infotrieve]
• Ericsson Y (1959). Clinical investigations of the salivary buffering action. Acta Odontol Scand 17:131–165.
• Gold OG, Jordan HV, van Houte J (1973). A selective medium for Streptococcus mutans. Arch Oral Biol 18:1357–1364.[CrossRef][Medline] [Order article via Infotrieve]
• Hase JC, Birkhed D (1988). Salivary glucose clearance, dry mouth and pH changes in dental plaque in man. Arch Oral Biol 33:875–880.[Medline] [Order article via Infotrieve]
• Heintze U, Birkhed D, Björn H (1983). Secretion rate and buffer effect of resting and stimulated whole saliva as a function of age and sex. Swed Dent J 7:227–238.[Medline] [Order article via Infotrieve]
• Keene HJ, Fleming TJ (1987). Prevalence of caries-associated microflora after radiotherapy in patients with cancer of the head and neck. Oral Surg Oral Med Oral Pathol 64:421–426.[Medline] [Order article via Infotrieve]
• Laine M, Leimola-Virtanen R (1996). Effect of hormone replacement therapy on salivary flow rate, buffer effect and pH in perimenopausal and postmenopausal women. Arch Oral Biol 41:91–96.[Medline] [Order article via Infotrieve]
• Lingström P, Birkhed D (1993). Plaque pH and oral retention after consumption of starchy snack products at normal and low salivary secretion rate. Acta Odontol Scand 51:379–388.[Medline] [Order article via Infotrieve]
• Lingström P, Imfeld T, Birkhed D (1993). Comparison of three different methods for measurement of plaque-pH in humans after consumption of soft bread and potato chips. J Dent Res 72:865–870.
• List T, Lundeberg T, Lundström I, Lindström F, Ravald N (1998). The effect of acupuncture in the treatment of patients with primary Sjögren’s syndrome. A controlled study. Acta Odontol Scand 56:95–99.[CrossRef][Medline] [Order article via Infotrieve]
• Manthorpe R, Oxholm P, Prause JU, Schiødt M (1986). The Copenhagen criteria for Sjögren’s syndrome. Scand J Rheumatol Suppl 61:19–21.[Medline] [Order article via Infotrieve]
• Nusair S, Rubinow A (1999). The use of oral pilocarpine in xerostomia and Sjögren’s syndrome. Semin Arthritis Rheum 28:360–367.[CrossRef][Medline] [Order article via Infotrieve]
• Ravald N, List T (1998). Caries and periodontal conditions in patients with Sjögren’s syndrome. Swed Dent J 22:97–103.[Medline] [Order article via Infotrieve]
• Rhodus NL (1997). Oral pilocarpine HCl stimulates labial (minor) salivary gland flow in patients with Sjögren’s syndrome. Oral Dis 3:93–98.[Medline] [Order article via Infotrieve]
• Rode M, Smid L, Budihna M, Gaspersic D, Rode M, Soba E (2001). The influence of pilocarpine and biperiden on pH value and calcium, phosphate, and bicarbonate concentrations in saliva during and after radiotherapy for head and neck cancer. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 92:509–514.[Medline] [Order article via Infotrieve]
• Samaranayake LP, Robertson AG, MacFarlane TW, Hunter IP, MacFarlane G, Soutar DS, et al. (1988). The effect of chlorhexidine and benzydamine mouthwashes on mucositis induced by therapeutic irradiation. Clin Radiol 39:291–294.[Medline] [Order article via Infotrieve]
• Scheie AA, Fejerskov O, Lingström P, Birkhed D, Manji F (1992). Use of palladium touch microelectrodes under field conditions for in vivo assessment of dental plaque pH in children. Caries Res 26:44–52.[Medline] [Order article via Infotrieve]
• Vuotila T, Ylikontiola L, Sorsa T, Luoto H, Hanemaaijer R, Salo T, et al. (2002). The relationship between MMPs and pH in whole saliva of radiated head and neck cancer patients. J Oral Pathol Med 31:329–338.[Medline] [Order article via Infotrieve]
• Weerkamp AH, Wagner K, Vissink A, ‘s-Gravenmade EJ (1987). Effect of the application of a mucin-based saliva substitute on the oral microflora of xerostomia patients. J Oral Pathol 16:474–478.[Medline] [Order article via Infotrieve]

Journal of Dental Research, Vol. 85, No. 4, 334-338 (2006)
DOI: 10.1177/154405910608500410


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This Article Abstract Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Eliasson, L. Articles by Lingström, P. Search for Related Content PubMed PubMed Citation Articles by Eliasson, L. Articles by Lingström, P. Social Bookmarking                         What's this?