Archa oral Bid. Vol. 24, pp. 15 lo 81. Pergamon Press Ltd. 1979.Printed m Great Britain.
PENETRATION
OF IONS
IN HUMAN
DENTAL
PLAQUE
B. MELSEN, 0. KAAE, G. RBLLA,*0. FEJERSKOV and T. KARRING Department of Ortodontics, Royal Dental College, Aarhus DK 8000, Denmark Summary--Columns of freshly-collected plaque were prepared in plastic tubes and the ion or potential substrate to be tested was applied. The columns were frozen at time intervals and sectioned. The amount of sample penetrated into the columns was detected by scintillation counting. The depth and level of penetration of calcium and phosphate was much greater than that of glucose and glucosamine. Within the two groups, the amount of calcium penetrated exceeded that of phosphate, and glucosamine penetrated slightly deeper than glucose, indicating that plaque may be permselective.
INTRODUffION Mandel (1974) suggested that diffusion processes in dental plaque might be related to cariogenicity and Stralfors (1950) stressed the importance of thickness of plaque. Other aspects of diffusion in plaque have been studied by Winkler and Dirks (195Q Huh, Blackwell and Fosdick (1959), Macdonald (1962) and Singer and Kleinberg (1969). Kleinberg (1970a) maintained that plaque is usually a floe, a porous aggregate of bacterial cells, where the diffusion rate is determined by the nature and amount of extracellular matrix present and that plaque is converted to a gel on exposure to dietary sucrose (Kleinberg, 1970b). Workers have suggested that fixed electric charges in the plaque matrix may influence the flow of ions to and from the enamel surface (Ellwood and Ellwood, 1966; Melvaer, Helgeland and Rolla, 1974; Ciardi et al., 1977). Our aim was to develop a model to study the penetration of substances in plaque especially calcium and phosphate ions. Glucose and glucosamine were added for comparison. MATERIALAND
METHODS
Plaque
During clinical examination of schoolchildren aged 7--13 yr, samples of plaque were collected with a flat plastic instrument from the smooth surfaces of teeth. Immediately after collection, the plaque was forced into sterile plastic tubes (internal diameter 2 mm) with the blunt instrument. When 5 mm of the tube were filled, the compressed plaque was moved a further 67mm into the tube by vacuum suction. About 2 cm of the tube containing the plaque samples was then cut off and stored in a plastic container at - 18°C humidified by a piece of wet cotton. Each tube contained about 15 mg of plaque (wet wt). The history of the plaque being unknown, the samples were pooled so that their origin would not affect the development of a method for the study of penetration of ions plaque. * Present address: Dental Faculty, University of Oslo, Oslo, Norway.
Penetration of different compounds in plaque The isotopes were obtained from Norsk Atominstitutt, Kjeller, Norway. At the top of the plaque column, 10~1 of a I-mM solution of [45Ca]-C1, (specific activity: 13 &i/mg), NazH32P-04 (specific activity: 10 $i/mg), C3H]respectively, was glucose or [3H]-glucosamine, applied. After 1, 3 or 9 min, the diffusion of the test isotope was stopped by freezing the tube in liquid nitrogen. A specially designed knife with two parallel razor blades was used to cut the sample into l-mm sections. The frozen water phase on top of the plaque column (which contained the major part of the label) could easily be removed because of the differences in physical properties. The sections were dried for 24 h at 105°C weighed and suspended in soluene 350 (Packard) at 60°C for scintillation counting in dimilume 30 (Packard). The amount of isotope in the different sections of the plaque column indicated the distribution of the test ions through the plaque. Sixteen plaque samples for each of the four compounds tested were performed with a diffusion time of 9 min. Additionally, the diffusion of phosphate and calcium were tested after 1 and 3 min. The procedure was performed at low temperatures (immediately after melting of the stored frozen cylinders).
Electron microscopy (EM) The structural composition of the plaque columns was examined by EM. Ten plaque columns were fixed for 24 h in a combined paraformaldehyde and glutaraldehyde fixative (Karnovsky, 1965) immediately after collection. In half of the specimens 3000parts/106 of Ruthenium Red was added to the fixative. After the tubes were washed in O.lOmM cacodylate buffer, pH 7.4, they were post-fixed for 2 h in 2 per cent osmium tetroxide. Again Ruthenium Red was added to the buffer used for washing and post-fixation in half of the material. Ultrathin sections were obtained from both ends of the tubes perpendicular to the long axis of the tubes. Only the first 334 sections of the material stained with Ruthenium Red were examined, in order to ensure that none of the negative staining reaction was due to a lack of diffusion of the dye into the plaque (Luft, 1971ab). After the specimens had been sectioned, they were examined in a Philips EM 301
76
B. Melsen
electron microscope without UMA and lead-citrate.
prior
staining
with
et al.
column by the scintillation counting procedure was expressed as a percentage of the total count/min applied to the column; (2) the nM of the ions per mg of plaque (dry weight) in each mm was calculated. For each substance and distance, a statistical description of the distribution was then carried out for the three different diffusion times studied (Tables 1 and 2). Departures from normality were tested by means of the Kolmogorov-Smirnov test. In calculation of the percentage, a large part of the variance could be accounted for by differences in the amount of plaque contained in each millimetre. As penetration follows a hyperbolic function (Sokal and Rohlf, 1969), the diffusions were transformed to the reciprocal values to obtain a linear regression. The slope and level of the 9-min diffusion lines were then compared.
Autoradiography
Additional plaque tubes were used for an autoradiographic analysis of the localization of the ions in cross-sections of the plaque. Each of the l-mm sections dere embedded in 4 per cent ice-cold buffered formaldehyde (pH 6.8-7) and after an initial cooling in carbon dioxide specimens were covered by gum arabic sucrose for freeze-sectioning. Cross-sections (7 pm thick) were produced 0.5, 1.5 and 2.5 mm from the top of the columns where the ions were applied. Autoradiographs of the dry sections were prepared by the dipping technique using Ks nuclear research emulsion from Ilford. At the time of dipping, half of the sections were stained with the Feulgen reagent and the other left unstained. The autoradiographs were exposed for 46 days at 4°C and then developed in amidol (Rogers, 1973). After fixation, the unstained sections were stained with haematoxylin-eosin, dehydrated, cleared and mounted.
RESULTS The penetrations of the four substances differed considerably. The 1- and 3-min penetrations were pilot trials and showed a large variability, although the data are presented to illustrate the time dependency of the penetration (Table 1). Statistical comparison of the penetration of the different substances was thus carried out only for the 9-min results (Table 2), from which it appears that the slope as
Statistics
The diffusion was evaluated in two different ways: (1) the count/min found in each mm of the plaque
Table 1. Percentage of calcium and phosphate ions penetrating plaque in 1 and 3 min Time Dist. (mm) 1
2 3 4 5
Phosphate n
1 min x
8 8 8 6 7
5.03 0.77 0.088 0.043 0.0086
Calcium
SE
n
3 min x
1.32 0.50 0.053 0.037 0.0040
8 8 7 8 7
1.99 0.44 0.056 0.0075 0.0214
1 min
3 min
SE
n
X
SE
n
1
SE
0.35 0.22 0.030 0.0025 0.0118
8 8 8 8 8
4.88 0.62 0.14 0.005 0.000
1.29 0.23 0.052 0.0033 0.0000
8 8 8 8 8
5.36 0.034 0.028 0.123 0.041
0.99 0.017 0.028 0.034 0.038
Table 2a. Percentage of substances penetrating plaque in 9 min (n = 16) Distance mm 1
2 3 4 5
CA*+
HPO;-
Glucose
X
SE
X
SE
x
SE
6.01 1.31 0.18 0.076 0.038
0.81 0.30 0.037 0.014 0.0055
10.41 2.86 1.40 1.59 0.78
1.76 0.51 0.25 0.37 0.16
1.24 0.36 0.068 0.009 0.005
0.11 0.097 0.017 0.002 osMI2
Glucosamine x SE 4.17 1.16 0.024 0.004 0.000
0.54 0.41 0.0108 0.0020 0.0000
Table 2b. Amounts (nM/mg) of substances penetrating plaque in 9 min (n = 16) Dist. mm
x
Phosphate SE
X
Calcium SE
x
Glucose SE
1 2 3 4 5
1.62 0.39 0.041 0.030 0.009
0.27 0.10 0.0075 0.0051 0.0013
2.62 0.66 0.38 0.42 0.21
0.43 0.12 0.07 0.11 0.04
0.57 0.15 0.019 0.0022 0.0014
0.09 0.06 0.006 0.0005 0.0005
Glucosamine X SE 1.47 0.32 0.0046 0.0013 0
0.31 0.11 0.0022 0.0007 0
Penetration of ions in human dental plaque
/mg
n
phosphate
‘mg
‘/nM/mg. calcium
3
2
100
glucosamine , ;gfucose
1
phosphate
LtTlzL
II_-
2
12345mm
r
‘mg glucose
3
4
5
mm
‘w
3 50
2
T 1
IL 1
2
3
4
Fig. 1. The distribution
5
mm
L 1
2
3
4
of the ions and metabolites plaque columns (9 min).
I t I i ii
!I 5
in the
well as the intercept of the regression lines fell into two groups (Text Fig. 2 and Table 3). The depth and level of penetration of calcium and phosphate was much greater than that of glucose and glucosamine. Within the two groups there was also a difference as the amount of calcium that penetrated into the plaque exceeded that of phosphate. The difference in the lines in Text Fig. 2 between calcium and phosphatz is ascribed to the difference in depth of penetration, i.e. 3, 4 or 5mm. Autoradiography
The radioactive labelling from the diffusing substances was distributed all over the plaque sections but was more concentrated in the spaces between larger aggregations of microorganisms indicating that the substances do not pass through the cells and only pass with difficulty through dense colonies of bacteria (Plate Fig. 3a). Elmron
ii 1/! I ,..........-* I I
mm
microscopy
Ultrastructural examination of the plaque columns shaMed large masses of microorganisms arranged in a haphazard way (Plate Figs. 4a and b), consisting of aggregations of microorganisms, mainly cocci and small rods often forming microcolonies (Plate Fig. 4b). Between the individual large masses, larger gaps containing long filaments occurred (Plate Fig. 4a). The amount and structure of the intermicrobial matrix varied greatly but appeared most often fibrillar in substructure. Most of the matrix appeared highly
1
2
3
. . . . . . . . ..*calcium I I 4
5
e mm.
Fig. 2. The amounts of the different penetrated represented as straight lines by the reciprocal transformation of the real values on the y axis (9 min).
electron dense in the specimens stained with Ruthenium Red (Plate Fig. 4c), particularly near Grampositive microorganisms. DISCUSSION
The model represents a convenient method for measuring the flow of ions and molecules through the plaque, provided that sensitive methods of analysis are available for the compounds tested. However, the model represents an artificial situation which cannot be directly compared to the inviuo situation. Thus, established plaque in situ has a typical structural arrangement which may account for different diffusion properties in the outer and inner parts. This layering is disturbed as shown on the electron micrographs. As the structural arrangement of the plaque columns differs markedly, this may explain
Table 3. The equations
Phosphate Calcium Glucose Glucosamine
for the regression in Fig. 2
y=
-
y=
-
y= y=
-
lines shown
41.12 + 25.18 x 0.55 + 0.96 x 315.91 + 187.29x 382.37 + 251.99 x
78
B. Melsen et al.
Fig. 5(a). The inhibition of penetration of anions in a narrow pore with fixed negative charges (Sollner, 1955). (b) Binding of anions in a wider pore by calcium bound equimolarly to negatively charged fixed negative charges (Weiss, 1958; Rolla and Bowen, 1977). (b) The situation in an ionic exchange column whereas (a) shows penetration of ions as it occurs in dental enamel (Waters, 1968). -
some of the variations in the results obtained. However, the overall consistency between the results of the large number of samples in each experimental group clearly indicates that the model is a useful tool for obtaining basic information about the diffusion properties of plaque. Some of the carbohydrate isotopes may have been metabolized during the penetration of plaque, but as the temperature was kept low and the time was short this was presumably not a major reaction. The plaque samples were randomly collected from Danish schoolchildren. A corresponding study employing plaque samples of known history (cariogenic and non-cariogenic) is in progress in our laboratory. It can be speculated that the selectivity of plaque may be partly based on the numerous fixed negatively-charged macromolecules known to be present. Sulphated glycoproteins have been demonstrated in the pellicle and in plaque (Rolla and Embery, 1976; Rolla, Melsen and SGnju, 1975) and lipoteichoic acid from the Gram-positive bacteria is also available in plaque (Ellwood and Ellwood, 1966; Melvier et al., 1974; Markham et al., 1975; Ciardi et al., 1977). The surfaces of Gram-positive bacteria have a net negative charge (Olsson, Glantz and Krasse, 1976). Fixed negative charges would repel anions like phosphate, whereas cations like calcium would be permitted to diffuse in the plaque (Text Fig. 5a). Another possible mechanism (Fig. 5b) is based on calcium being absorbed on acidic groups, therefore binding anions but not retaining cations. Such mechanisms are well established in soil chemistry (Weiss, 1958) and have been suggested as a possible mechanism for the binding of fluoride in plaque (Rolla and Bowen, 1977). It is not known which of the above mechanisms, if any, are involved in plaque, but it may depend on the actual pore size (Sollner, 1955). Tatevossian and Gould (1976) showed that the aqueous phase in human dental plaque contained high concentrations of sodium and potassium whereas that of inorganic phosphate was relatively low. Tatevossian, Edgar and Jenkins (1975) reported that ingestion of sugar with added phosphate gave only minor and inconsistent increases of phosphate in the plaque because of the slow penetration of phos-
phate in plaque. The concept of the significance of fixed negative charges in plaque was supported by the electron micrographs which showed that Ruthenium Red precipitated on the bacterial cell walls and in the intermicrobial matrix (Text Fig. 5~). Extracellular teichoic acid or salivary sulphated glycoproteins (mu&s) seem to be the most likely constituents of the plaque matrix to bind Ruthenium Red, as this reagent is known to have a high affinity for phosphate and sulphate groups. The penetrations of ions and molecules in plaque in viva at body temperature would involve additional problems like the metabolism and selective uptake by microorganism and is thus an extremely complex process. Experiments along the lines indicated here could, however, give information on the basic chemical properties of the plaque matrix. AcknowIedgemenr-This report was supported by the Danish State Research Foundation, grant No. 512-5159. REFERENCES
Ciardi J. E., Rolla G., Bowen W. H. and Reilly J. A. 1977. Adsorption of streptococcus mutant lipoteichoic acid hydroxyapatite. Sand. J. dent. Res. (in press). Ellwood J. C. and Ellwood D. C. 1966. Dental caries. Br. dent. J. 120.
Huh C., Blackwell R. Q. and Fosdick L. S. 1959. The diffusion of glucose through microbial plaques. J. dent. Rex 38, 569-576.
Karnovsky M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A-138A. Kleinberg I. 1970a. Biochemistry of the dental plaque. In: Advances in Oral Biology (Edited by Staple P. H.) Vol. 4, pp. 43-90. Academic Press, New York. Kleinberg I. 1970b. Formation and accumulation of acid on the tooth surface. J. dent. Res. 49, 13O(r1316. Luft J. H. 1971a. Ruthenium Red and violet I. Anat. Rec. 171, 347-368. Luft J. H. 1971b. Ruthenium Red and violet II. Amt. Rec. 171, 369416. Macdonald J. B. 1962. Microbiology of caries. In: Chemistry and Prevention of Dental Caries (Edited by Sognnaes R. F.) pp. 89-125. Charles C. Thomas, Springfield, Ill. Mandel I. D. 1974. Relation of saliva and plaque to caries. J. dent. Res. 53, 246266.
B. Melsen et al. Markham J. L., Know K. W., Wicken A. J. and Hewett M. J. 1975. Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun. 12, 378-386. Melvrer K. L., Helgeland D. and Rolla G. 1974. A charged component in purified polysaccharide preparations from Streptococcus mutans and Streptococcus sanguis. Archs oral Eiol. 19, 589-595. Olsson J., Glantz P. 0. and Krasse B. 1976. Electrophoretic mobility of oral streptococci. Archs oral Biol. il, 605609. Rogers A. W. 1973. Techniques of Autoradiography, 2nd Edn. Elsevier, Amsterdam. Rolla G., Melsen B. and Sonju T. 1975. Sulphated macromolecules in dental plaque in the monkey Macaca irus. 4rchs oral Biol. 20, 341-344.
19
Sokal R. R. and Rohlf F. J. 1969. Biometry. The Principles and Practice of Statistics in Biological Research. W. H. Freeman, San Francisco. Sollner K. 1955. The electrochemistry of porous membranes. In: Electrochemistry in Biology and Medicine (Edited by Schedlovski T.). Chap. 3, pp. 33-64. John Wiley, New York. Strftfors A. 1950. Investigations into the bacterial chemistry of dental plaques. Odont. Tidskr. 58, 153-341. Tatevossian A., Edgar W. M. and Jenkins G. N. 1975. Changes in the concentrations of phosphates in human plaque after ingestion of sugar with and without added phosphates. Archs oral Biol. 20, 617-662. Tatevossian A. and Gould C. T. 1976. The composition of the aqueous phase in human dental plaque. Archs oral Biol. 21, 319-323.
Rolla G. and Embery G. 1976. Sulphated glycoproteins in plaque and pellicle from Macaca irus. J. dent. Res. 55, (spec. issue B), Abstract 1056. Rolla G. and Bowen W. H. 1977. Concentration of fluoride in plaque-a possible mechanism. Stand. J. dent. Res. 85, 149-151.
Singer D. L. and Kleinberg I. 1969. NH3 formation in plaque in situ. Internat. Ass. for Dent. Res. Preprinted abstracts, 47th General Meeting, Abstract 637.
Waters N. E. 1968. Electrochemical properties of human dental enamel. Nature, Land. 219, 62-62. Weiss A. 1958. ijber lquimolaren Kationenaustausch bei niedrig geladenen Ionenaustauschern. Kolloid 158, 22228. Winkler K. C. and Dirks 0. B. 1958. The mechanism of the dental plaque. Int. dent. J. 8, 561-585.
Plate 1 overleaf
B. Melsen et al.
Plate 1. Fig. 3. Autoradiography are mainly concentrated
of the distribution of the radioactive ions. x 1600. (a) The penetrated ions in larger spaces between aggregation of microorganisms. (b) Some ions are also penetrating the aggregations of bacteria.
Fig. 4. Electron micrographs of the plaque columns. (a) An almost empty space is seen between two dense aggregations of plaque. x 5000. (b) A microcolony of densely-packed microorganisms surrounded by coccoid bacteria arranged in a loose matrix. x 5000. (c) Variations in Ruthenium Red positive material in the matrix and particularly along the cell periphery of some of the bacteria. x 28,ooO
Penetration
of ions in human dental plaque
Plate 1
81
PENETRATION
OF IONS
IN HUMAN
DENTAL
PLAQUE
B. MELSEN, 0. KAAE, G. RBLLA,*0. FEJERSKOV and T. KARRING Department of Ortodontics, Royal Dental College, Aarhus DK 8000, Denmark Summary--Columns of freshly-collected plaque were prepared in plastic tubes and the ion or potential substrate to be tested was applied. The columns were frozen at time intervals and sectioned. The amount of sample penetrated into the columns was detected by scintillation counting. The depth and level of penetration of calcium and phosphate was much greater than that of glucose and glucosamine. Within the two groups, the amount of calcium penetrated exceeded that of phosphate, and glucosamine penetrated slightly deeper than glucose, indicating that plaque may be permselective.
INTRODUffION Mandel (1974) suggested that diffusion processes in dental plaque might be related to cariogenicity and Stralfors (1950) stressed the importance of thickness of plaque. Other aspects of diffusion in plaque have been studied by Winkler and Dirks (195Q Huh, Blackwell and Fosdick (1959), Macdonald (1962) and Singer and Kleinberg (1969). Kleinberg (1970a) maintained that plaque is usually a floe, a porous aggregate of bacterial cells, where the diffusion rate is determined by the nature and amount of extracellular matrix present and that plaque is converted to a gel on exposure to dietary sucrose (Kleinberg, 1970b). Workers have suggested that fixed electric charges in the plaque matrix may influence the flow of ions to and from the enamel surface (Ellwood and Ellwood, 1966; Melvaer, Helgeland and Rolla, 1974; Ciardi et al., 1977). Our aim was to develop a model to study the penetration of substances in plaque especially calcium and phosphate ions. Glucose and glucosamine were added for comparison. MATERIALAND
METHODS
Plaque
During clinical examination of schoolchildren aged 7--13 yr, samples of plaque were collected with a flat plastic instrument from the smooth surfaces of teeth. Immediately after collection, the plaque was forced into sterile plastic tubes (internal diameter 2 mm) with the blunt instrument. When 5 mm of the tube were filled, the compressed plaque was moved a further 67mm into the tube by vacuum suction. About 2 cm of the tube containing the plaque samples was then cut off and stored in a plastic container at - 18°C humidified by a piece of wet cotton. Each tube contained about 15 mg of plaque (wet wt). The history of the plaque being unknown, the samples were pooled so that their origin would not affect the development of a method for the study of penetration of ions plaque. * Present address: Dental Faculty, University of Oslo, Oslo, Norway.
Penetration of different compounds in plaque The isotopes were obtained from Norsk Atominstitutt, Kjeller, Norway. At the top of the plaque column, 10~1 of a I-mM solution of [45Ca]-C1, (specific activity: 13 &i/mg), NazH32P-04 (specific activity: 10 $i/mg), C3H]respectively, was glucose or [3H]-glucosamine, applied. After 1, 3 or 9 min, the diffusion of the test isotope was stopped by freezing the tube in liquid nitrogen. A specially designed knife with two parallel razor blades was used to cut the sample into l-mm sections. The frozen water phase on top of the plaque column (which contained the major part of the label) could easily be removed because of the differences in physical properties. The sections were dried for 24 h at 105°C weighed and suspended in soluene 350 (Packard) at 60°C for scintillation counting in dimilume 30 (Packard). The amount of isotope in the different sections of the plaque column indicated the distribution of the test ions through the plaque. Sixteen plaque samples for each of the four compounds tested were performed with a diffusion time of 9 min. Additionally, the diffusion of phosphate and calcium were tested after 1 and 3 min. The procedure was performed at low temperatures (immediately after melting of the stored frozen cylinders).
Electron microscopy (EM) The structural composition of the plaque columns was examined by EM. Ten plaque columns were fixed for 24 h in a combined paraformaldehyde and glutaraldehyde fixative (Karnovsky, 1965) immediately after collection. In half of the specimens 3000parts/106 of Ruthenium Red was added to the fixative. After the tubes were washed in O.lOmM cacodylate buffer, pH 7.4, they were post-fixed for 2 h in 2 per cent osmium tetroxide. Again Ruthenium Red was added to the buffer used for washing and post-fixation in half of the material. Ultrathin sections were obtained from both ends of the tubes perpendicular to the long axis of the tubes. Only the first 334 sections of the material stained with Ruthenium Red were examined, in order to ensure that none of the negative staining reaction was due to a lack of diffusion of the dye into the plaque (Luft, 1971ab). After the specimens had been sectioned, they were examined in a Philips EM 301
76
B. Melsen
electron microscope without UMA and lead-citrate.
prior
staining
with
et al.
column by the scintillation counting procedure was expressed as a percentage of the total count/min applied to the column; (2) the nM of the ions per mg of plaque (dry weight) in each mm was calculated. For each substance and distance, a statistical description of the distribution was then carried out for the three different diffusion times studied (Tables 1 and 2). Departures from normality were tested by means of the Kolmogorov-Smirnov test. In calculation of the percentage, a large part of the variance could be accounted for by differences in the amount of plaque contained in each millimetre. As penetration follows a hyperbolic function (Sokal and Rohlf, 1969), the diffusions were transformed to the reciprocal values to obtain a linear regression. The slope and level of the 9-min diffusion lines were then compared.
Autoradiography
Additional plaque tubes were used for an autoradiographic analysis of the localization of the ions in cross-sections of the plaque. Each of the l-mm sections dere embedded in 4 per cent ice-cold buffered formaldehyde (pH 6.8-7) and after an initial cooling in carbon dioxide specimens were covered by gum arabic sucrose for freeze-sectioning. Cross-sections (7 pm thick) were produced 0.5, 1.5 and 2.5 mm from the top of the columns where the ions were applied. Autoradiographs of the dry sections were prepared by the dipping technique using Ks nuclear research emulsion from Ilford. At the time of dipping, half of the sections were stained with the Feulgen reagent and the other left unstained. The autoradiographs were exposed for 46 days at 4°C and then developed in amidol (Rogers, 1973). After fixation, the unstained sections were stained with haematoxylin-eosin, dehydrated, cleared and mounted.
RESULTS The penetrations of the four substances differed considerably. The 1- and 3-min penetrations were pilot trials and showed a large variability, although the data are presented to illustrate the time dependency of the penetration (Table 1). Statistical comparison of the penetration of the different substances was thus carried out only for the 9-min results (Table 2), from which it appears that the slope as
Statistics
The diffusion was evaluated in two different ways: (1) the count/min found in each mm of the plaque
Table 1. Percentage of calcium and phosphate ions penetrating plaque in 1 and 3 min Time Dist. (mm) 1
2 3 4 5
Phosphate n
1 min x
8 8 8 6 7
5.03 0.77 0.088 0.043 0.0086
Calcium
SE
n
3 min x
1.32 0.50 0.053 0.037 0.0040
8 8 7 8 7
1.99 0.44 0.056 0.0075 0.0214
1 min
3 min
SE
n
X
SE
n
1
SE
0.35 0.22 0.030 0.0025 0.0118
8 8 8 8 8
4.88 0.62 0.14 0.005 0.000
1.29 0.23 0.052 0.0033 0.0000
8 8 8 8 8
5.36 0.034 0.028 0.123 0.041
0.99 0.017 0.028 0.034 0.038
Table 2a. Percentage of substances penetrating plaque in 9 min (n = 16) Distance mm 1
2 3 4 5
CA*+
HPO;-
Glucose
X
SE
X
SE
x
SE
6.01 1.31 0.18 0.076 0.038
0.81 0.30 0.037 0.014 0.0055
10.41 2.86 1.40 1.59 0.78
1.76 0.51 0.25 0.37 0.16
1.24 0.36 0.068 0.009 0.005
0.11 0.097 0.017 0.002 osMI2
Glucosamine x SE 4.17 1.16 0.024 0.004 0.000
0.54 0.41 0.0108 0.0020 0.0000
Table 2b. Amounts (nM/mg) of substances penetrating plaque in 9 min (n = 16) Dist. mm
x
Phosphate SE
X
Calcium SE
x
Glucose SE
1 2 3 4 5
1.62 0.39 0.041 0.030 0.009
0.27 0.10 0.0075 0.0051 0.0013
2.62 0.66 0.38 0.42 0.21
0.43 0.12 0.07 0.11 0.04
0.57 0.15 0.019 0.0022 0.0014
0.09 0.06 0.006 0.0005 0.0005
Glucosamine X SE 1.47 0.32 0.0046 0.0013 0
0.31 0.11 0.0022 0.0007 0
Penetration of ions in human dental plaque
/mg
n
phosphate
‘mg
‘/nM/mg. calcium
3
2
100
glucosamine , ;gfucose
1
phosphate
LtTlzL
II_-
2
12345mm
r
‘mg glucose
3
4
5
mm
‘w
3 50
2
T 1
IL 1
2
3
4
Fig. 1. The distribution
5
mm
L 1
2
3
4
of the ions and metabolites plaque columns (9 min).
I t I i ii
!I 5
in the
well as the intercept of the regression lines fell into two groups (Text Fig. 2 and Table 3). The depth and level of penetration of calcium and phosphate was much greater than that of glucose and glucosamine. Within the two groups there was also a difference as the amount of calcium that penetrated into the plaque exceeded that of phosphate. The difference in the lines in Text Fig. 2 between calcium and phosphatz is ascribed to the difference in depth of penetration, i.e. 3, 4 or 5mm. Autoradiography
The radioactive labelling from the diffusing substances was distributed all over the plaque sections but was more concentrated in the spaces between larger aggregations of microorganisms indicating that the substances do not pass through the cells and only pass with difficulty through dense colonies of bacteria (Plate Fig. 3a). Elmron
ii 1/! I ,..........-* I I
mm
microscopy
Ultrastructural examination of the plaque columns shaMed large masses of microorganisms arranged in a haphazard way (Plate Figs. 4a and b), consisting of aggregations of microorganisms, mainly cocci and small rods often forming microcolonies (Plate Fig. 4b). Between the individual large masses, larger gaps containing long filaments occurred (Plate Fig. 4a). The amount and structure of the intermicrobial matrix varied greatly but appeared most often fibrillar in substructure. Most of the matrix appeared highly
1
2
3
. . . . . . . . ..*calcium I I 4
5
e mm.
Fig. 2. The amounts of the different penetrated represented as straight lines by the reciprocal transformation of the real values on the y axis (9 min).
electron dense in the specimens stained with Ruthenium Red (Plate Fig. 4c), particularly near Grampositive microorganisms. DISCUSSION
The model represents a convenient method for measuring the flow of ions and molecules through the plaque, provided that sensitive methods of analysis are available for the compounds tested. However, the model represents an artificial situation which cannot be directly compared to the inviuo situation. Thus, established plaque in situ has a typical structural arrangement which may account for different diffusion properties in the outer and inner parts. This layering is disturbed as shown on the electron micrographs. As the structural arrangement of the plaque columns differs markedly, this may explain
Table 3. The equations
Phosphate Calcium Glucose Glucosamine
for the regression in Fig. 2
y=
-
y=
-
y= y=
-
lines shown
41.12 + 25.18 x 0.55 + 0.96 x 315.91 + 187.29x 382.37 + 251.99 x
78
B. Melsen et al.
Fig. 5(a). The inhibition of penetration of anions in a narrow pore with fixed negative charges (Sollner, 1955). (b) Binding of anions in a wider pore by calcium bound equimolarly to negatively charged fixed negative charges (Weiss, 1958; Rolla and Bowen, 1977). (b) The situation in an ionic exchange column whereas (a) shows penetration of ions as it occurs in dental enamel (Waters, 1968). -
some of the variations in the results obtained. However, the overall consistency between the results of the large number of samples in each experimental group clearly indicates that the model is a useful tool for obtaining basic information about the diffusion properties of plaque. Some of the carbohydrate isotopes may have been metabolized during the penetration of plaque, but as the temperature was kept low and the time was short this was presumably not a major reaction. The plaque samples were randomly collected from Danish schoolchildren. A corresponding study employing plaque samples of known history (cariogenic and non-cariogenic) is in progress in our laboratory. It can be speculated that the selectivity of plaque may be partly based on the numerous fixed negatively-charged macromolecules known to be present. Sulphated glycoproteins have been demonstrated in the pellicle and in plaque (Rolla and Embery, 1976; Rolla, Melsen and SGnju, 1975) and lipoteichoic acid from the Gram-positive bacteria is also available in plaque (Ellwood and Ellwood, 1966; Melvier et al., 1974; Markham et al., 1975; Ciardi et al., 1977). The surfaces of Gram-positive bacteria have a net negative charge (Olsson, Glantz and Krasse, 1976). Fixed negative charges would repel anions like phosphate, whereas cations like calcium would be permitted to diffuse in the plaque (Text Fig. 5a). Another possible mechanism (Fig. 5b) is based on calcium being absorbed on acidic groups, therefore binding anions but not retaining cations. Such mechanisms are well established in soil chemistry (Weiss, 1958) and have been suggested as a possible mechanism for the binding of fluoride in plaque (Rolla and Bowen, 1977). It is not known which of the above mechanisms, if any, are involved in plaque, but it may depend on the actual pore size (Sollner, 1955). Tatevossian and Gould (1976) showed that the aqueous phase in human dental plaque contained high concentrations of sodium and potassium whereas that of inorganic phosphate was relatively low. Tatevossian, Edgar and Jenkins (1975) reported that ingestion of sugar with added phosphate gave only minor and inconsistent increases of phosphate in the plaque because of the slow penetration of phos-
phate in plaque. The concept of the significance of fixed negative charges in plaque was supported by the electron micrographs which showed that Ruthenium Red precipitated on the bacterial cell walls and in the intermicrobial matrix (Text Fig. 5~). Extracellular teichoic acid or salivary sulphated glycoproteins (mu&s) seem to be the most likely constituents of the plaque matrix to bind Ruthenium Red, as this reagent is known to have a high affinity for phosphate and sulphate groups. The penetrations of ions and molecules in plaque in viva at body temperature would involve additional problems like the metabolism and selective uptake by microorganism and is thus an extremely complex process. Experiments along the lines indicated here could, however, give information on the basic chemical properties of the plaque matrix. AcknowIedgemenr-This report was supported by the Danish State Research Foundation, grant No. 512-5159. REFERENCES
Ciardi J. E., Rolla G., Bowen W. H. and Reilly J. A. 1977. Adsorption of streptococcus mutant lipoteichoic acid hydroxyapatite. Sand. J. dent. Res. (in press). Ellwood J. C. and Ellwood D. C. 1966. Dental caries. Br. dent. J. 120.
Huh C., Blackwell R. Q. and Fosdick L. S. 1959. The diffusion of glucose through microbial plaques. J. dent. Rex 38, 569-576.
Karnovsky M. J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A-138A. Kleinberg I. 1970a. Biochemistry of the dental plaque. In: Advances in Oral Biology (Edited by Staple P. H.) Vol. 4, pp. 43-90. Academic Press, New York. Kleinberg I. 1970b. Formation and accumulation of acid on the tooth surface. J. dent. Res. 49, 13O(r1316. Luft J. H. 1971a. Ruthenium Red and violet I. Anat. Rec. 171, 347-368. Luft J. H. 1971b. Ruthenium Red and violet II. Amt. Rec. 171, 369416. Macdonald J. B. 1962. Microbiology of caries. In: Chemistry and Prevention of Dental Caries (Edited by Sognnaes R. F.) pp. 89-125. Charles C. Thomas, Springfield, Ill. Mandel I. D. 1974. Relation of saliva and plaque to caries. J. dent. Res. 53, 246266.
B. Melsen et al. Markham J. L., Know K. W., Wicken A. J. and Hewett M. J. 1975. Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun. 12, 378-386. Melvrer K. L., Helgeland D. and Rolla G. 1974. A charged component in purified polysaccharide preparations from Streptococcus mutans and Streptococcus sanguis. Archs oral Eiol. 19, 589-595. Olsson J., Glantz P. 0. and Krasse B. 1976. Electrophoretic mobility of oral streptococci. Archs oral Biol. il, 605609. Rogers A. W. 1973. Techniques of Autoradiography, 2nd Edn. Elsevier, Amsterdam. Rolla G., Melsen B. and Sonju T. 1975. Sulphated macromolecules in dental plaque in the monkey Macaca irus. 4rchs oral Biol. 20, 341-344.
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Sokal R. R. and Rohlf F. J. 1969. Biometry. The Principles and Practice of Statistics in Biological Research. W. H. Freeman, San Francisco. Sollner K. 1955. The electrochemistry of porous membranes. In: Electrochemistry in Biology and Medicine (Edited by Schedlovski T.). Chap. 3, pp. 33-64. John Wiley, New York. Strftfors A. 1950. Investigations into the bacterial chemistry of dental plaques. Odont. Tidskr. 58, 153-341. Tatevossian A., Edgar W. M. and Jenkins G. N. 1975. Changes in the concentrations of phosphates in human plaque after ingestion of sugar with and without added phosphates. Archs oral Biol. 20, 617-662. Tatevossian A. and Gould C. T. 1976. The composition of the aqueous phase in human dental plaque. Archs oral Biol. 21, 319-323.
Rolla G. and Embery G. 1976. Sulphated glycoproteins in plaque and pellicle from Macaca irus. J. dent. Res. 55, (spec. issue B), Abstract 1056. Rolla G. and Bowen W. H. 1977. Concentration of fluoride in plaque-a possible mechanism. Stand. J. dent. Res. 85, 149-151.
Singer D. L. and Kleinberg I. 1969. NH3 formation in plaque in situ. Internat. Ass. for Dent. Res. Preprinted abstracts, 47th General Meeting, Abstract 637.
Waters N. E. 1968. Electrochemical properties of human dental enamel. Nature, Land. 219, 62-62. Weiss A. 1958. ijber lquimolaren Kationenaustausch bei niedrig geladenen Ionenaustauschern. Kolloid 158, 22228. Winkler K. C. and Dirks 0. B. 1958. The mechanism of the dental plaque. Int. dent. J. 8, 561-585.
Plate 1 overleaf
B. Melsen et al.
Plate 1. Fig. 3. Autoradiography are mainly concentrated
of the distribution of the radioactive ions. x 1600. (a) The penetrated ions in larger spaces between aggregation of microorganisms. (b) Some ions are also penetrating the aggregations of bacteria.
Fig. 4. Electron micrographs of the plaque columns. (a) An almost empty space is seen between two dense aggregations of plaque. x 5000. (b) A microcolony of densely-packed microorganisms surrounded by coccoid bacteria arranged in a loose matrix. x 5000. (c) Variations in Ruthenium Red positive material in the matrix and particularly along the cell periphery of some of the bacteria. x 28,ooO
Penetration
of ions in human dental plaque
Plate 1
81
