Archs oral Bid. Vol. 35, No. 3, pp. 229-234, 1990 Printed in Great Britain. All rights reserved
0003-9969/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc
EFFECTS OF LOW AND HIGH FLUORIDE LEVELS ON RAT DENTAL CARIES AND SIMULTANEOUS DENTINE APPOSITION S. KORTELA~NEN Institute
of Dentistry, (Received
University
and M. LARMAS
of Oulu,
11 October
Aapistie
1988; accepted
3, SF-90220 13 September
Oulu 22, Finland 1989)
Summary-The effects of various fluoride levels in drinking water on the development of enamel and dentinal caries and on dentine apposition were determined in Osborne-Mendel rats fed ad libitum either on a 43% sucrose diet and distilled water supplemented with 0, 1, 7 or 19 parts/lo6 fluoride, or on a non-cariogenic diet. The jaws were sectioned sagittally and fissure caries was assessed. The areas of the dentinal lesions and simultaneously formed dentine were quantified after tetracycline staining. There were fewer and smaller caries lesions in all fluoride groups than in the non-fluoride groups. The anti-caries effects of fluoride at 1 and 7 parts/IO6 were similar, but both had significantly less effect than 19 parts/lo6 fluoride. Much smaller dentinal lesions were seen with 19 parts/lo6 fluoride, which appears to have been accompanied by an even greater reduction in dentine apposition. Dentine formation in the third molars was significantly reduced in the rats fed a cariogenic diet. These observations suggest that a high-sucrose diet interferes with the formation of dentine in the developing tooth. Fluoride has a multiple effect; besides reducing the formation and progression of caries, it may also affect the dentinal response normally seen in the development of a caries lesion. Key words:
fluoride,
caries,
dentine
apposition.
INTRODUCTION
fluoride in the drinking water far in excess of current practice; only a few such experiments have been conducted at fluoride concentrations less than 10 parts/lo6 (Larson et al., 1976; Spuller et al., 1986; Beiraghi et al., 1989). Accordingly, we have now examined the effect of fluoride concentrations between 0 and 19 parts/lo6 in drinking water on (i) the initiation of enamel fissure caries and its progression into the dentine, and (ii) the host response in the form of dentine apposition, using a new tetracycline staining method for the measurement of areas of dentine (Larmas and Kortelainen, 1989). This method also enables measurement of the size of the dentinal lesion.
The anti-caries potential of fluoride arises from (i) its ability to prevent or reduce the loss of minerals from the teeth through various demineralizationremineralization mechanisms, and (ii) its inhibitory effect on the glycolytic metabolism of the microorganisms in dental plaque and its ability to produce an acidic pH (reviewed by Keyes and Englander, 1975). A longitudinal !study on human subjects in the fluoridated area of Tie1 in the Netherlands showed them to have similar numbers of caries lesions to those of their non-fluoridated controls when enamel and dentinal lesions were both tabulated, but when enamel lesions were omitted fluoride was seen to produce a significant teduction (Groeneveld, 1985). It was concluded that fluoride may prevent many precavitation lesions from progressing to a more severe dentinal condition, or at least reduce the rate of progression. This may involve some mode(s) of action of fluoride not hitherto considered, namely cessation or reduction in the rate of lesion progression within the dentine. Fluoride is known ‘to increase the rate of proliferation of bone cells at 2.5-25 p M concentrations, both in monolayer cultures and in organ cultures from the calvaria of chick embryos, and to enhance the growth and mineralization of embryonic bone (Farley, Wergedal and Baylink, 1983). This observation led us to consider the possibility that various concentrations of fluoride might also alter odontoblastic activity and/or the mineralization of dentine, and thereby stop the progress of dentinal lesions. Earlier experiments with rodents have employed concentrations of oa
MATERIALS AND METHODS
Osborne-Mendel rats were weaned at 22 days of age, weighed, marked and distributed into 5 groups of 10. Four groups were fed ad libitum on a modified Stephan-Harris diet containing 43% sucrose (plus 22% wheat flour, 32% milk powder, 2% whole liver powder, 1% corn oil) and distilled water supplemented with 0 (sucrose control), 1, 7 or 19 parts/lo6 fluoride in the form of its sodium salt (Merck 6449). The mean daily water consumption per animal was 26 + 9, 22 + 5, 27 f 5, 25 f 4 ml for those in the 0, 1, 7 and 19 parts/lo6 fluoride groups, respectively. A non-sucrose control group of 10 rats was fed a pulverized pellet diet and distilled water ad Zibitum. The fluoride content of the rat chow was measured by a modified microdiffusion method after perchloric acid digestion (Dabeka, McKenzie and Conacher, 1979). An Orion fluoride-sensitive electrode 94-09
35,hD
229
230
S. KORTELAINEN and M. KARMAS Table 1. Incidence of fissure caries in the mandibular molars of the animals fed on a cariogenic diet (means + SD) Fluoride (parts/106) 0
Total Initial Advanced Very advanced
(A+T+B+C) (T+B+C) (B + C) (C)
1
12.0 f 0.0 11.6kO.S 9.7k2.1 2.1 + 2.0
was used with a pH-mV metre. The free fluoride content of the{ commercial rat chow was 0.05 parts/lo6 and that of the sucrose-supplemented diet 0.00 parts/lo6 fluoride; the total fluoride concentration was 50 and 5 parts/106, respectively. The animals were housed 2 per cage under normal atmospheric conditions at 21°C. Each animal was subjected to the same regimen of lighting (12 h of light and 12 h of dark) and the same times of feeding, human handling and noise. All the animals were given an intraperitoneal injection of oxytetracycline hydrochloride (30 mg/kg; Vendarcin”) at the onset of the experiment and 2 days before death to mark the areas of the dentinal lesions and of dentine apposition during the test period. They were all inoculated with a fresh suspension of Streptococcus mutans (ATCC 27351 K 1 Fitzgerald) on days 22 and 23 (i.e. days 1 and 2 of the experiment). On day 70 (day 49 of the experiment), all the animals were weighed and decapitated under ether anaesthesia. The lower jaws were defleshed and preserved in absolute ethanol for later sagittal sectioning by the technique of Keyes (1958). The mandibular molars were stained with Schiff’s reagent and fissure caries was assessed by the scoring method of Konig, Marthaler and Miihlemann (1958). In order to measure the size of the dentinal lesions and the amount of dentine formed, the molars were each examined under an Orthoplan Ploemopak microscope equipped with incident light fluorescent (wavelength 460 nm), upon which the tetracyclinestained lesion and the stripes in newly formed dentine could be readily seen (Larmas and Kortelainen, 1989). The main central transverse fissure of each
10.2 * 6.8 + 1.9 f 0.2 f
7 1.1 1.7 1.4 0.4
19
11.2* 1.2 9.2k2.1 4.6k2.1 0.0 & 0.0
9.8 f 5.4 k 0.1 * 0.0 +
1.3 2.8 0.3 0.0
lower mandibular molar was photographed with Kodak Ektachrome daylight film (400 ASA). The dentinal lesions and tetracycline-marked zones of dentine apposition were measured from the video images (Larmas and Kortelainen, 1989) by circumscribing their respective areas on a monitor (Salora 445 A RGB, Camera Hitachi VKM 98 E) using a serial “mouse” connected to a PCVision Frame Grabber (Imaging Technology, Inc., Woburn, Mass., U.S.A.) and an associated program (Image Measure program by Microscience, Federal Way, Wash., U.S.A.). The inter-examiner and intra-examiner errors in the determination of areas were insignificant, as reported earlier (Larmas and Kortelainen, 1989). An analysis of variance and Student’s f-test were used to test the significances of any differences in dentinal lesions and areas of simultaneously formed dentine between the experimental and control animals. An adaptation of a test for proportional frequencies (Hald, 1952) was used for the percentage differences in fissures. RESULTS There were no significant differences in bodyweight gain between the experimental and control groups. The experimental animals remained the same as the controls in physical appearance. Caries scoring
The animals fed a cariogenic diet and treated with fluoride at all of the concentrations tested had less caries than those which did not receive fluoride in
Table 2. Percentage reductions in the prevalence of total caries and dentinal caries in rats receiving 1, 7 or 19 parts/IO6 F I molar
[F-l I 7 19
2 molar
3 molar
Total
Dentinal
Total
Dentinal
Total
W)
(%I
WI
WI
@I
WI
18.3+ 15.0* 11.1
42.5*” 19.2* 40.2***
20.0* 15.0* 22.2’S
27.5” 27.5*+ 74.3*+*
5.0 16.7 22.2
60.0*** 44.4** 38.8**
Dentinal
Significances of the differences between the experimental animals and the sucrose controls: ***p < 0.001, **p < 0.01, *p < 0.05.
Plate 1 Fig. 1. (a) The main central transverse fissure of a second molar with an advanced dentinal lesion. (b) Schematic diagram of Fig. l(a). E = enamel, C = dentinal caries lesion, T = tetracycline stripe, PD = primary dentine, SD = secondary dentine.
Fluoride,
caries and dentine
(a)
(b)
Plate
1
apposition
231
232
S. KORTELAINEN and M. LARMAS
their drinking water (Table 1). The non-sucrose group did not develop any caries. The effect of fluoride was most pronounced in its reduction of the prevalence of dentinal lesions, the percentage reduction in which was much greater than that of total caries (Table 2). The qualitative score (Kiinig et al., 1958) corresponded well with the quantitative score (Larmas and Kortelainen, 1989) for the areas of tetracyclinemarked dentinal lesions (Plate Fig. 1). Effect
?f‘Jiuoride
on caries
The effect of fluoride varied in magnitude from one molar tooth or set of teeth to another (Text Fig. 2 top). Fluoride at the lower concentrations (1 or 7 parts/106) did not markedly reduce the size of the caries lesions in the second or third molars, whereas 1 part/l O6F reduced the area of dentinal lesions in the first molars, although 7 parts/IO6 did not. In all molars the highest concentration of fluoride (19 parts/106) significantly reduced the area of dentinal caries lesions (p < 0.001; Text Fig. 2 top) and the incidence of all types of caries (Table 1). Dentinal
response
The areas of dentine apposition were larger in the first molars than in the second or third molars (Fig. 2 bottom). Fluoride (1 and 19 parts/106) reduced the area of dentine in the first (p < 0.001) and second molars (p < 0.001 and p < 0.05), but 7 parts/lo6 fluoride had no effect. There was a significant difference between the 1 part/lo6 fluoride group and the sucrose controls in the formation of dentine in the third molars (p < 0.05). The most pronounced effect on the dentinal response to carious attack was achieved with 19 parts/lo6 fluoride, and was seen in all the molar teeth (Table 3). The formation of dentine in the non-sucrose control group did not differ from that of the sucrose control group in the case of the first and second molar
Dentin01
20 c
m
lstmolor
n
wr~es
I
2ndmolar
Table 3. Ratio of area of dentinal lesions to area of simultaneous dentine apposition in the animals receiving a cariogenic diet
F-l
1 molar
2 molar
3 molar
0 1 7 19
1:3.0 1:2.0 1:2.7 1:39.0
1:1.9 1: 0.6 1:1.6 1:33.0
1:8.6 1:3.7 I : 7.0 1:15.5
teeth. In the third molars, however, the dentine apposition was significantly larger in the non-sucrose group than in the groups fed a cariogenic diet (p
0003-9969/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc
EFFECTS OF LOW AND HIGH FLUORIDE LEVELS ON RAT DENTAL CARIES AND SIMULTANEOUS DENTINE APPOSITION S. KORTELA~NEN Institute
of Dentistry, (Received
University
and M. LARMAS
of Oulu,
11 October
Aapistie
1988; accepted
3, SF-90220 13 September
Oulu 22, Finland 1989)
Summary-The effects of various fluoride levels in drinking water on the development of enamel and dentinal caries and on dentine apposition were determined in Osborne-Mendel rats fed ad libitum either on a 43% sucrose diet and distilled water supplemented with 0, 1, 7 or 19 parts/lo6 fluoride, or on a non-cariogenic diet. The jaws were sectioned sagittally and fissure caries was assessed. The areas of the dentinal lesions and simultaneously formed dentine were quantified after tetracycline staining. There were fewer and smaller caries lesions in all fluoride groups than in the non-fluoride groups. The anti-caries effects of fluoride at 1 and 7 parts/IO6 were similar, but both had significantly less effect than 19 parts/lo6 fluoride. Much smaller dentinal lesions were seen with 19 parts/lo6 fluoride, which appears to have been accompanied by an even greater reduction in dentine apposition. Dentine formation in the third molars was significantly reduced in the rats fed a cariogenic diet. These observations suggest that a high-sucrose diet interferes with the formation of dentine in the developing tooth. Fluoride has a multiple effect; besides reducing the formation and progression of caries, it may also affect the dentinal response normally seen in the development of a caries lesion. Key words:
fluoride,
caries,
dentine
apposition.
INTRODUCTION
fluoride in the drinking water far in excess of current practice; only a few such experiments have been conducted at fluoride concentrations less than 10 parts/lo6 (Larson et al., 1976; Spuller et al., 1986; Beiraghi et al., 1989). Accordingly, we have now examined the effect of fluoride concentrations between 0 and 19 parts/lo6 in drinking water on (i) the initiation of enamel fissure caries and its progression into the dentine, and (ii) the host response in the form of dentine apposition, using a new tetracycline staining method for the measurement of areas of dentine (Larmas and Kortelainen, 1989). This method also enables measurement of the size of the dentinal lesion.
The anti-caries potential of fluoride arises from (i) its ability to prevent or reduce the loss of minerals from the teeth through various demineralizationremineralization mechanisms, and (ii) its inhibitory effect on the glycolytic metabolism of the microorganisms in dental plaque and its ability to produce an acidic pH (reviewed by Keyes and Englander, 1975). A longitudinal !study on human subjects in the fluoridated area of Tie1 in the Netherlands showed them to have similar numbers of caries lesions to those of their non-fluoridated controls when enamel and dentinal lesions were both tabulated, but when enamel lesions were omitted fluoride was seen to produce a significant teduction (Groeneveld, 1985). It was concluded that fluoride may prevent many precavitation lesions from progressing to a more severe dentinal condition, or at least reduce the rate of progression. This may involve some mode(s) of action of fluoride not hitherto considered, namely cessation or reduction in the rate of lesion progression within the dentine. Fluoride is known ‘to increase the rate of proliferation of bone cells at 2.5-25 p M concentrations, both in monolayer cultures and in organ cultures from the calvaria of chick embryos, and to enhance the growth and mineralization of embryonic bone (Farley, Wergedal and Baylink, 1983). This observation led us to consider the possibility that various concentrations of fluoride might also alter odontoblastic activity and/or the mineralization of dentine, and thereby stop the progress of dentinal lesions. Earlier experiments with rodents have employed concentrations of oa
MATERIALS AND METHODS
Osborne-Mendel rats were weaned at 22 days of age, weighed, marked and distributed into 5 groups of 10. Four groups were fed ad libitum on a modified Stephan-Harris diet containing 43% sucrose (plus 22% wheat flour, 32% milk powder, 2% whole liver powder, 1% corn oil) and distilled water supplemented with 0 (sucrose control), 1, 7 or 19 parts/lo6 fluoride in the form of its sodium salt (Merck 6449). The mean daily water consumption per animal was 26 + 9, 22 + 5, 27 f 5, 25 f 4 ml for those in the 0, 1, 7 and 19 parts/lo6 fluoride groups, respectively. A non-sucrose control group of 10 rats was fed a pulverized pellet diet and distilled water ad Zibitum. The fluoride content of the rat chow was measured by a modified microdiffusion method after perchloric acid digestion (Dabeka, McKenzie and Conacher, 1979). An Orion fluoride-sensitive electrode 94-09
35,hD
229
230
S. KORTELAINEN and M. KARMAS Table 1. Incidence of fissure caries in the mandibular molars of the animals fed on a cariogenic diet (means + SD) Fluoride (parts/106) 0
Total Initial Advanced Very advanced
(A+T+B+C) (T+B+C) (B + C) (C)
1
12.0 f 0.0 11.6kO.S 9.7k2.1 2.1 + 2.0
was used with a pH-mV metre. The free fluoride content of the{ commercial rat chow was 0.05 parts/lo6 and that of the sucrose-supplemented diet 0.00 parts/lo6 fluoride; the total fluoride concentration was 50 and 5 parts/106, respectively. The animals were housed 2 per cage under normal atmospheric conditions at 21°C. Each animal was subjected to the same regimen of lighting (12 h of light and 12 h of dark) and the same times of feeding, human handling and noise. All the animals were given an intraperitoneal injection of oxytetracycline hydrochloride (30 mg/kg; Vendarcin”) at the onset of the experiment and 2 days before death to mark the areas of the dentinal lesions and of dentine apposition during the test period. They were all inoculated with a fresh suspension of Streptococcus mutans (ATCC 27351 K 1 Fitzgerald) on days 22 and 23 (i.e. days 1 and 2 of the experiment). On day 70 (day 49 of the experiment), all the animals were weighed and decapitated under ether anaesthesia. The lower jaws were defleshed and preserved in absolute ethanol for later sagittal sectioning by the technique of Keyes (1958). The mandibular molars were stained with Schiff’s reagent and fissure caries was assessed by the scoring method of Konig, Marthaler and Miihlemann (1958). In order to measure the size of the dentinal lesions and the amount of dentine formed, the molars were each examined under an Orthoplan Ploemopak microscope equipped with incident light fluorescent (wavelength 460 nm), upon which the tetracyclinestained lesion and the stripes in newly formed dentine could be readily seen (Larmas and Kortelainen, 1989). The main central transverse fissure of each
10.2 * 6.8 + 1.9 f 0.2 f
7 1.1 1.7 1.4 0.4
19
11.2* 1.2 9.2k2.1 4.6k2.1 0.0 & 0.0
9.8 f 5.4 k 0.1 * 0.0 +
1.3 2.8 0.3 0.0
lower mandibular molar was photographed with Kodak Ektachrome daylight film (400 ASA). The dentinal lesions and tetracycline-marked zones of dentine apposition were measured from the video images (Larmas and Kortelainen, 1989) by circumscribing their respective areas on a monitor (Salora 445 A RGB, Camera Hitachi VKM 98 E) using a serial “mouse” connected to a PCVision Frame Grabber (Imaging Technology, Inc., Woburn, Mass., U.S.A.) and an associated program (Image Measure program by Microscience, Federal Way, Wash., U.S.A.). The inter-examiner and intra-examiner errors in the determination of areas were insignificant, as reported earlier (Larmas and Kortelainen, 1989). An analysis of variance and Student’s f-test were used to test the significances of any differences in dentinal lesions and areas of simultaneously formed dentine between the experimental and control animals. An adaptation of a test for proportional frequencies (Hald, 1952) was used for the percentage differences in fissures. RESULTS There were no significant differences in bodyweight gain between the experimental and control groups. The experimental animals remained the same as the controls in physical appearance. Caries scoring
The animals fed a cariogenic diet and treated with fluoride at all of the concentrations tested had less caries than those which did not receive fluoride in
Table 2. Percentage reductions in the prevalence of total caries and dentinal caries in rats receiving 1, 7 or 19 parts/IO6 F I molar
[F-l I 7 19
2 molar
3 molar
Total
Dentinal
Total
Dentinal
Total
W)
(%I
WI
WI
@I
WI
18.3+ 15.0* 11.1
42.5*” 19.2* 40.2***
20.0* 15.0* 22.2’S
27.5” 27.5*+ 74.3*+*
5.0 16.7 22.2
60.0*** 44.4** 38.8**
Dentinal
Significances of the differences between the experimental animals and the sucrose controls: ***p < 0.001, **p < 0.01, *p < 0.05.
Plate 1 Fig. 1. (a) The main central transverse fissure of a second molar with an advanced dentinal lesion. (b) Schematic diagram of Fig. l(a). E = enamel, C = dentinal caries lesion, T = tetracycline stripe, PD = primary dentine, SD = secondary dentine.
Fluoride,
caries and dentine
(a)
(b)
Plate
1
apposition
231
232
S. KORTELAINEN and M. LARMAS
their drinking water (Table 1). The non-sucrose group did not develop any caries. The effect of fluoride was most pronounced in its reduction of the prevalence of dentinal lesions, the percentage reduction in which was much greater than that of total caries (Table 2). The qualitative score (Kiinig et al., 1958) corresponded well with the quantitative score (Larmas and Kortelainen, 1989) for the areas of tetracyclinemarked dentinal lesions (Plate Fig. 1). Effect
?f‘Jiuoride
on caries
The effect of fluoride varied in magnitude from one molar tooth or set of teeth to another (Text Fig. 2 top). Fluoride at the lower concentrations (1 or 7 parts/106) did not markedly reduce the size of the caries lesions in the second or third molars, whereas 1 part/l O6F reduced the area of dentinal lesions in the first molars, although 7 parts/IO6 did not. In all molars the highest concentration of fluoride (19 parts/106) significantly reduced the area of dentinal caries lesions (p < 0.001; Text Fig. 2 top) and the incidence of all types of caries (Table 1). Dentinal
response
The areas of dentine apposition were larger in the first molars than in the second or third molars (Fig. 2 bottom). Fluoride (1 and 19 parts/106) reduced the area of dentine in the first (p < 0.001) and second molars (p < 0.001 and p < 0.05), but 7 parts/lo6 fluoride had no effect. There was a significant difference between the 1 part/lo6 fluoride group and the sucrose controls in the formation of dentine in the third molars (p < 0.05). The most pronounced effect on the dentinal response to carious attack was achieved with 19 parts/lo6 fluoride, and was seen in all the molar teeth (Table 3). The formation of dentine in the non-sucrose control group did not differ from that of the sucrose control group in the case of the first and second molar
Dentin01
20 c
m
lstmolor
n
wr~es
I
2ndmolar
Table 3. Ratio of area of dentinal lesions to area of simultaneous dentine apposition in the animals receiving a cariogenic diet
F-l
1 molar
2 molar
3 molar
0 1 7 19
1:3.0 1:2.0 1:2.7 1:39.0
1:1.9 1: 0.6 1:1.6 1:33.0
1:8.6 1:3.7 I : 7.0 1:15.5
teeth. In the third molars, however, the dentine apposition was significantly larger in the non-sucrose group than in the groups fed a cariogenic diet (p
