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Protein content and amino-acid content of consolidated carious lesions in human enamel and of experimental lesions in bovine enamel exposed to the human mouth
0003-9969/86$3.00+ 0.00 Pergamon Journals Ltd
Archs oral Biol. Vol. 31, No. 6, pp. 405410, 1986 Printed in Great Britain
PROTEIN CONTENT AND AMINO-ACID CONTENT OF CONSOLIDATED CARIOUS LESIONS IN HUMAN ENAMEL AND OF EXPERIMENTAL LESIONS IN BOVINE ENAMEL EXPOSED TO THE HUMAN MOUTH T. TERANAKA,’ T. KOULOURIDE~’ and W. T. BUTLER’ ‘Department of Restorative Dentistry, Kanagawa Dental College, Yokosuka, Japan and *Institute of Dental Research, University of Alabama, School of Dentistry, Birmingham, AL 35294, U.S.A. Summary-Consolidation is a natural defence reaction that results in arrest of enamel caries. Experimental consolidated lesions (ECL) were compared with naturally-consolidated lesions (NCL): ECL were obtained by exposing pre-softened, bovine-enamel slabs to the oral environment in 3 subjects for 2 h, 24 h or 7 days, and NCL were sampled from extracted human teeth with white or yellow spots of arrested caries. Protein content of ECL from 2 subjects were similar to each other and to that of NCL throughout the experimental period. The ECL of the other subject showed a gradual increase in protein content with significantly higher values at day 7. The predominant amino acids in ECL were glutamic acid, proline and alanine, and in NCL, glutamic acid, glycine and alanine. The amino-acid composition of the 7-day ECL was closer to that of NCL than were that of the 2 and 24 h ECL. I&a-oral ageing caused significant reductions in proline and glycine and pronounced increases in aspartic acid, threonine, alanine and leucine. Thus the adsorbed or incorporated organic material in the ECL changed from having components dissimilar to NCL to ones similar to NCL. This intra-oral model might be useful for studies of the organic material incorporated into enamel during the process of consolidation.
MATERIALS AND
INTRODUCTION
The term consolidated enamel lesions was given by Bibby (1932) to carious enamel lesions that were arrested. In some lesions this arrest can be attributed to a change in the conditions of microbial accumulatlon on the vulnerable surface, such as occurs on an approximal surface after the extraction of a neighbouring tooth. Conceivably, improvement in oral hygiene and fluoride treatment could also contribute to arrest by decreasing the cariogenic challenge or by increasing the remineralizing activity of saliva (Koulourides and Cameron, 1980). Consolidated lesions are more resistant to acid exposure than adjacent normal enamel (Beust, 1930; Bibby, 1932). They also have a higher proportion of organic material (Stack, 1954; Bhussry and Bibby, 1957) and fluoride (Little, Posen and Singer, 1962; Weatherell et al., 1977). Hay and Moreno (1979) and Moreno, Varughese and Hay (1979) demonstrated that salivary acidic proline-rich proteins and statherin bind to hydroxyapatite and inhibit the nucleation of calcium-phosphate salts; hence, in remineralization phenomena, certain salivary proteins or peptides could play an important role. Experimental cariogenic challenge to pre-softened enamel, with the influence of fluoride, increases fluoride incorporation and increases resistance of these experimentallyconsolidated lesions to in-vitro acid challenge (Koulourides and Housch, 1983). 0ur purpose was to investigate the incorporation of organic material into in-vitro enamel lesions exposed to the mouth, to compare the amino-acid composition of experimental enamel lesions (ECL), and to compare the amino-acid composition of ECL with that of naturally-consolidated lesions (NCL). 405
METHODS
Preparation and sampling of ECL
Rectangular enamel slabs (3 x 5 x 2 mm) were cut from the buccal surfaces of bovine permanent incisors. The slabs were mounted with sticky-wax on plexiglass blocks with the enamel surfaces as parallel with the base of the block as possible. The outer enamel layer was removed to an approximate depth of 100 pm by grinding against emery paper (Norton, Tufbak Durite, waterproof, 600-A grit) using a specially designed grinder-polisher (Koulourides et al., 1976). Artificial enamel lesions were produced by immersing the enamel slabs in 0.01 M sodium-lactate buffer, pH 4.0 (20ml/slab), containing 3.0 mM calcium, 1.8 mM phosphate and 1 per cent carboxyat 37°C for 4 days. Micromethylcellulose, radiography indicated a lesion depth of approx. 60 pm. The pre-softened slabs were fixed with stickywax into recesses drilled in the flanges of acrylic dentures; the enamel surface was exposed to the oral environment to produce ECL. In three groups of experiments, eight pre-softened slabs per group were exposed in the mouths of three subjects for either 2 h, 24 h or 7 days. For the 2-h experiment, the subjects were instructed not to drink or eat; for the other time periods, their diets were not restricted. The dentures with the slabs were worn continuously throughout each experimental period; they were cleaned by brushing with soft toothbrushes and a nonfluoridated toothpaste. After removal from the mouth, the slab surface was wiped with a cotton pellet under running deionized water to remove plaque and debris. The ECL
406
T.
~UANAKA
samples were collected by scraping the lesions with scalpel blades down to hard, normal enamel on plastic weighing trays. Control samples were prepared from lesions not exposed to the mouth.
et al.
Statistics The data were analysed statistically analysis of variance.
Preparation of NCL
by one-way
RESULTS
Freshly-extracted human teeth showing white or yellow enamel spots were used. Dental calculus and deposits were removed with periodontal scalers and the enamel surfaces cleaned with pumice. Because the NCL usually invades normal enamel in a conical pattern, the sampling was done with a no. 6 round bur rotated at low speed to prevent overheating. Care was taken to stop the grinding when sound enamel was reached. The NCL samples were collected on plastic weighing trays. Amino -acid analysis The samples were left at room temperature for 1.5 h, then weighed and hydrolysed with 2.0 ml of 6.0 M HCl for 24 h at 108°C. The hydrolysates were substantially soluble in distilled water. After evaporation of HCl in uacuo, the hydrolysates were dissolved in 1.Oml water and analysed for 19 amino acids using a Beckman 121-M amino-acid analyser (Butler, Finch and Miller, 1977). The presence of calcium and phosphate salts did not interfere with this analysis. The protein content of the various lesions was calculated from the amount of amino acids (nmol/ml) in hydrolysates of each sample and expressed as pg protein per mg (wet weight).
Protein content in the control, NCL and ECL The protein content of the carious lesions is shown in Fig. 1. The ECL samples from two subjects (A and B) were similar to each other and to the samples of NCL. However, in subject C, there was a gradual increase in the protein content of ECL with intra-oral ageing; the 7-day ECL had a significantly higher value than NCL (p < 0.01). The control slabs, not exposed to the mouth, contained less than 5 per cent of the protein in ECL. The coefficient of variation for NCL was much greater in controls and ECL, 103 and 41 per cent, respectively. Amino-acid composition of the controls The predominant amino acids were glycine, glutamic acid and aspartic acid. These three accounted for 43 per cent of the total amino acids. Hydroxyproline, cysteine and hydroxylysine were not detected. These samples contained approx. 23 per cent acidic, 12 per cent basic and 5 per cent aromatic amino acids. Amino-acid composition of the NCL (Table 1) The predominant amino acids were glutamic acid,
Subjects
*
g:
UIIIC
Cant
2h
ECL
24h
ECL
?day
ECL
NCL
Fig. 1. Protein content of control, 2-h, 24-h and ‘I-day ECL and of NCL. ECL data are shown in the individual subjects (A, B and C). Mean values (columns) and standard errors (bars) are given (n = 8). *p c 0.01, significantly from NCL.
Composition Table 1. Amino-acid composition*
of control and NCL
Controlt Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Vaiine Methionine Isoleucine Leucine Tyrosine Phenylalanine L,ysine Histidine Aqinine
of experimental consolidated lesions
NCL
Mean
SE
Mean
SE
87.18 39.90 80.55 139.52 35.69 207.22 47.39 23.81 51.82 33.36 80.68 16.38 32.01 62.87 31.57 30.06
8.02 4.62 6.83 9.16 1.57 2.68 3.27 2.16 2.97 1.16 15.88 3.81 2.15 5.09 6.32 2.06
97.06 39.54 58.32 144.50 63.57 123.88 134.11 50.45 31.701 30.71 61.17 22.25 28.50 52.48 17.02 45.08
4.67 1.99 2.60 5.25 5.40 9.06 11.64 1.61 6.84 4.41 2.78 1.55 1.28 7.09 2.24 2.35
*Residues per 1000 residues, n = 8. Hydroxyproline, cysteine and hydroxylysine were not detected. tPre-softened, but unexposed to the oral environment. In = 7. alanine and glycine; their contribution was approx. 40 per cent of the total in NCL. Hydroxyproline and
hydroxylysine were not detected. Compared to controls, the amino-acid composition of NCL was characterized by smaller amounts of glycine and larger amounts of alanine. Amino-acid compositions of 2-h, 24-h and 7-day ECL (Tables 2, 3 and 4)
The three major amino acids were proline, glycine and glutamic acid in the 2-h ECL; glutamic acid, glycine and proline in the 24-h ECL; and glutamic aad, glycine and aspartic acid in the 7-day ECL. These predominant amino acids accounted for 35-50 per cent of the total in each ECL. The amino-acid composition of the 2-h ECL had larger amounts of proline, glycine and histidine, and smaller amounts of aspartic acid, threonine, alanine, valine and leucine than the NCL (p < 0.01). The
407
proportions of amino acids among the three subjects were similar, except for differences in proline, tyrosine and histidine which were pronounced in the 2-h ECL, less so in the 24-h ECL and not apparent in the 7-day ECL compared with NCL samples. In the 24-h ECL (Table 3) basic amino-acid values were significantly higher (p < 0.01) and aromatic acid values were slightly higher than in NCL, but the acidic amino-acid values were almost the same as in NCL. Compared with the 2-h ECL, the proline value of the 24-h ECL was significantly reduced (p < O.OS), but it was still higher than for NCL (p < 0.01; Fig, 2a). Also, the glycine value decreased to become similar to that of NCL in the 24-h ECL (Fig. 2b). The values of proline and glycine in 7-day ECL were significantly reduced (Table 4) but there were pronounced increases in aspartic acid, threonine, alanine and leucine compared with the 2-h ECL (Tables 2 and 4; Fig. 2). These differences were statistically significant (p < 0.01). With ageing from 2-h to 7 days, the proportion of amino-acid compositions in ECL became similar to those of NCL. DISCUSSION
Earlier studies have shown an increase in the organic content of carious lesions compared with normal enamel (Stack, 1954; Bhussry and Bibby, 1957; Weatherell et al., 1977). In most of these, the nitrogen content of the lesions was taken as representative of the organic content. There have been few studies (Robinson, Weatherell and Hallsworth, 1983) of the amino-acid composition of material incorporated during the process of natural caries. We have directed our efforts toward the development of an intra-oral model of lesion consolidation. The selection of bovine enamel as a substrate was based on our earlier observation that its ground and polished surface, at a depth of lOO-200pm from the natural one, reacts predictably to demineralization and remineralization. Thus, with acid gels the developing subsurface lesion is almost parallel to the test surface (Koulourides and Housch, 1983). This facilitates
Table 2. Amino-acid composition* of 2-h ECL Subject A Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
B
C
Mean
SE
Mean
SE
Mean
SE
65.93 16.33 57.58 139.35 186.79 152.25 37.40 11.43 16.15 29.72 51.23 40.51 77.29 61.32 59.48
1.14 4.88 1.54 1.22 9.50 8.40 1.65 I .49 3.31 3.31 6.26 6.18 4.20 2.97 1.46
86.57 19.39 62.02 130.19 113.70 131.02 45.56 20.66 30.50 40.06 53.49 36.83 79.81 77.60 72.59
2.48 1.69 1.98 7.08 9.24 5.48 1.06 2.10 4.16 3.45 6.45 5.44 2.88 6.75 1.79
86.00 27.33 71.35 156.77 164.15 164.96 53.03 33.24 33.38 43.30 22.25 21.23 49.05 25.81 48.21
1.88
1.45 1.03 4.77 6.31 4.51 2.18 5.87 7.11 2.97 1.36 0.70 1.87 1.72 2.14
*Residues per 1000 residues, n = 8. Hydroxyproline, cysteine, methionine and hydroxylysine were not detected.
408
T.
TERANAKA
et al.
Table 3. Amino-acid composition* of 24-h ECL Subject A Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
B
C
Mean
SE
Mean
SE
Mean
SE
79.62 19.72 63.73 141.54 104.69 129.36 39.99 29.72 31.48 43.23 57.66 43.12 75.13 72.24 68.62
3.24 1.10 2.01 2.79 10.49 5.68 1.44 1.59 4.21 3.86 8.46 6.70 4.21 3.16 1.20
88.95 14.81 64.71 148.79 93.59 127.91 40.55 13.58 27.82 39.27 64.42 36.40 71.33 94.10 73.81
1.74 0.96 2.89 6.19 3.12 4.34 2.55 1.19 2.69 2.19 4.62 2.04 3.02 6.04 3.29
92.62 24.47 67.01 146.83 113.85 119.70 51.71 30.91 33.89 51.02 37.36 30.34 74.34 59.54 63.34
2.84 1.49 2.41 2.76 10.91 4.29 2.27 3.94 5.74 5.12 2.14 1.18 2.89 2.71 1.46
*Residues per 1000 residues, n = 8. Hydroxyproline, onine and hydroxylysine were not detected.
sampling of the layer that is altered by interaction between pre-softened enamel and the oral environment. The difference in mineral density between the
ECL and the underlying normal enamel is so great that the lesion can easily be sampled by scraping with a scalpel blade, and there is no appreciable contamination of the sample with unaffected tissue. The demineralization used in preparation of the lesions provided 2-4 mg wet weight of material for aminoacid analysis. Sampling is more difficult with NCL due to their conical shape and to the irregular boundaries between them and normal enamel; a slowly-rotating round bur is more appropriate than scraping with a blade. Although some of the NCL could have been left in the tooth, we feel that the material analysed was representative of NCL without appreciable contamination by normal enamel. We consider the two ways of sampling to be equivalent;
cysteine, methi-
both are mechanical, and both are stopped when the instrument reaches the much harder sound enamel. Sijnju and Rolla (1973) reported that the amount of new salivary pellicle on cleaned teeth increases for about 1.5 h and then levels off; the total amount of amino acids in pellicles from the buccal surfaces of approx. 24 teeth after 1.5 h was almost 100 pg. To assess the extent to which salivary pellicle might contribute to our samples, we exposed slabs of sound enamel in the mouth for 24 h and then collected pellicle samples with 2 per cent HCl using the method of Mayhall (1970). The mean protein content per slab of these pellicles was 1.1 pg, but the specimens of the 24-h ECL contained 34 pg per slab. Thus the pellicle content was approx. 3 per cent of the total ECL protein. It might be argued that surface pellicles formed on pre-softened enamel may have a different composition from those on normal enamel. We could
Table 4. Amino-acid composition* of 7-day ECL Subject A Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
B
C
Mean
SE
Mean
SE
Mean
SE
92.46 38.14 67.09 161.15 65.18 102.12 97.69 38.28 37.58 60.19 30.52 32.86 84.14 36.05 56.63
2.45 3.49 4.57 3.20 7.09 4.62 14.24 3.42 2.54 1.85 4.19 1.59 6.55 5.17 2.56
99.67 34.18 68.03 148.42 74.77 107.80 79.99 30.70 39.83 59.39 32.14 34.38 81.37 45.71 60.37
1.92 4.17 2.68 3.25 7.77 5.67 9.17 5.67 3.38 2.07 2.74 0.91 7.14 6.00 3.31
104.22 43.70 64.99 155.53 63.07 99.69 113.75 37.47 28.01 64.23 30.68 31.58 87.25 31.32 44.53
1.14 1.71 2.21 3.26 2.77 6.89 7.60 2.07 3.06 2.62 1.22 0.81 1.78 3.21 1.21
*Residues per 1000 residues, n = 8. Hydroxyproline, cysteine and hydroxylysine were not detected. Methionine was trace in each subject.
Composition
409
of experimental consolidated lesions
ECL B
2h
0
24h
cl
7dcJy
m
NCL
Subjects
I,_
:
(0)
: A,E,C
A
B
C
Thr
(b)
A GUY
E AlO
C
A
B
C
LOU
Fig. 2. Changes in enamel amino-acid composition with intra-oral ageing which were significantly different. Mean values (columns) and standard errors (bars) are given (n = 8). *p < 0.05. **p < 0.01, significantly different from NCL.
not examine this because pellicle sampling with HCl causes flotation of surface-adsorbed organic material by diisolving the underlying sound enamel. A similar procedure in this investigation would cause flotation of the whole ECL, making it impossible to separate
the pellicle from the organic material within the lesion. The protein content of control samples not exposed to the mouth was less than 5 per cent of the amount recovered from the ECL (Fig. 1). Therefore, more than 90 per cent of the ECL and NCL samples represented organic material incorporated into the lesion from the mouth. The environment for 2-h ECL was mainly saliva but the diet and microbial activity could have influenced the composition of the 24-h and 7-day ECL, as well as all the NCL samples. The larger standard error for the mean organic content of the NCL lesions indicates that conditions during their formation, or the state of their development, were more varied than for the ECL, in which they were controlled by the experimental design. This would be expected, as the teeth with NCL had been extracted from different individuals of unknown age and dental history. Although 2- and 24-h ECL had similar organic content in all three subjects, the 7-day ECL in one subject (C) was considerably higher than that of the NCL (p < O.Ol), while with the other two subjects the
difference was not appreciable. This greater incorporation of organic material seems to be related to the high caries activity of that subject. Our earlier studies with the intra-oral cariogenicity test (ICT) indicate that individuals have characteristic and different propensities to develop caries within 1 week in bovine enamel slabs exposed in their mouths (Ostrom et al., 1977). Our subjects A and B were classified as low in ICT caries, whereas subject C exhibited high cariogenic activity. This higher activity may have contributed to more extensive demineralization of the pre-softened enamel and less mineral deposition, thus leaving a larger proportion of microspaces to be filled with organic material. The amino-acid composition of the 2-h ECL was more like that of saliva than were the 24-h and 7-day ECL. With intra-oral ageing, ECL showed compositional changes, possibly due to the influence of bacteria and diet. Through such changes the proportions of amino acids of the 7-day ECL became close to that of the NCL. Similarity in amino-acid composition between 7-day ECL and NCL indicates that underlying reactions in the formation of the organic materials in the experimental and the natural lesions are also similar. Bennick et al. (1983) found that the percentage of proline-rich proteins, adsorbed on sound enamel in
T. TERANAKAet al.
410
vivo, increases during the first hour to about 37 per cent of the total extracted protein; any differences between the 1 and 24-h period are minor, but there is a gradual degradation of the proteins. They found that extracts of old acquired pellicle contain less than 0.1 per cent proline-rich proteins, and that pellicles more than 24-h old show degradation of the adsorbed proline-rich proteins. It is possible that the gradual increase in alanine was also a result of bacterial involvement, as bacterial cell walls usually have a high alanine content (Cummins, 1962). Zahradnik (1979) suggested that the presence of an adsorbed salivary pellicle on the surface of a tooth plays an important part in remineralizing phenomena when the tooth is placed in an inorganic mineralizing solution. Likewise, the organic material in consolidated lesions could affect the remineralization of both NCL and ECL. As pointed out by Zahradnik (1979), the incorporated organic material could actually retard mineralization in the surface layers of lesions and enhance ionic penetration into the subsurface.
The contribution of the organic material to remineralization and to the increased acid resistance of consolidated lesions requires further investigation. Acknowledgements-We gratefully acknowledge the contribution by MS A. Vitek of technical help in the amino-acid analysis. This work was supported by USPHS-NIDR Grant DE 02670 and a grant from the Warner-Lambert Company. REFERENCES
Bennick A., Chau G., Goodlin R., Abrams S., Tustian D. and Madapallimattan G. (1983) The role of human salivary acidic proline-rich proteins in the formation of acquired dental pellicle in vivo and their fate after adsorption to the human enamel surface. Archs oral Biol. 28, 19-27.
Beust T. B. (1930) Micro-organisms and caries. J. Am. dent. Ass. 17, 15361544. Bhussry B. R. and Bibby B. G. (1957) Surface changes in enamel. J. dent. Res. 36, 409416. Bibby B. G. (1932) The organic structure of dental enamel as a passive defense against caries. J. dent. Res. 12, 99-116. Butler W. T., Finch J. E. Jr and Miller E. J. (1977) The covalent structure of cartilage collagen. J. biol. Chem. 252, 639643.
Cummins C. S. (1962) Chemical composition and antigenic
structure of cell wall of Corynebacterium, Mycobacterium, Nocardia, Actinomyces and Arthrobacter. J. gen. Microbiol. 28, 35-50. Hay D. I. and Moreno E. C. (1979) Macromolecular inhibitors of calcium phosphate precipitation in human saliva. Their roles in providing a protective environment for the teeth. In: Proceedings ofa Workshop on Saliva and Dental Caries (Edited by Kleinberg I., Ellison S. A. and Mandel I. D.) Special Supplement on Microbiology Abstracts, pp. 45-58. IRL Press, Oxford. Koulourides T. and Cameron B. (1980) Enamel remineralisation as a factor in the pathogenesis of dental caries. J. oral Path. 9, 255-269. Koulourides T. and Housch T. (1983) Hardness testing and microradiography of enamel in relation to intraoral deand remineralisation. In: Demineralisation and Remineralisation of the Teeth (Edited by Leach S. A. and Edger W. M.) pp. 255272. IRL Press, Oxford. Koulourides T., Bodden R., Keller S., Manson-Hing L., Lastsra J. and Housch T. (1976) Cariogenicity of nine sugars tested with an intraoral device in man. Caries Res. 10, 42741. Little M. F., Posen J. and Singer L. (1962) Chemical and physical properties of altered and sound enamel. 3. Fluoride and sodium content. J. dent. Res. 41, 784-789. Mayhall C. W. (1970) Concerning the composition and source of the acquired enamel pellicle of human teeth. Archs oral Biol. 15, 1327-1341. Moreno E. C., Varughese K. and Hay D. I. (1979) Effect of human salivary proteins on the precipitation kinetics of calcium phosphate. Calc. Tissue-Znt. k, 7-16. Ostrom C. A.. Koulourides T.. Hickman F. and Phantumvanit P. (1977) Combined effects of sucrose and fluoride on experimental caries and on the associated microbial plaque. J. dent. Res. 56, 212-221. Robinson C., Weatherell J. A. and Hallsworth A. S. (1983) Alterations in the composition of permananent human enamel during caries attack. In: bemineralisation and Remineralisation of the Teeth (Edited bv Leach S. A. and Edger W. M.) pp: 209-223. I‘RL Press: Oxford. Sisnju T. and Rolla G. (1973) Chemical analysis of the acquired pellicle formed in two hours on cleaned human teeth in vitro. Caries Res. 7, 3&38. Stack M. V. (1954) The organic content of chalky enamel. Br. dent. J. %, 73-76.
Weatherell J. A., Deutsch D., Robinson C. and Hallsworth A. S. (1977) Assimulation of fluoride bv enamel throuehout the life of the tooth. Caries Res. il, 85115. ” Zahradnik R. T. (1979) Modification by salivary pellicles of in vitro enamel remineralisation. J. dent. Res. 58, 20662073.
Archs oral Biol. Vol. 31, No. 6, pp. 405410, 1986 Printed in Great Britain
PROTEIN CONTENT AND AMINO-ACID CONTENT OF CONSOLIDATED CARIOUS LESIONS IN HUMAN ENAMEL AND OF EXPERIMENTAL LESIONS IN BOVINE ENAMEL EXPOSED TO THE HUMAN MOUTH T. TERANAKA,’ T. KOULOURIDE~’ and W. T. BUTLER’ ‘Department of Restorative Dentistry, Kanagawa Dental College, Yokosuka, Japan and *Institute of Dental Research, University of Alabama, School of Dentistry, Birmingham, AL 35294, U.S.A. Summary-Consolidation is a natural defence reaction that results in arrest of enamel caries. Experimental consolidated lesions (ECL) were compared with naturally-consolidated lesions (NCL): ECL were obtained by exposing pre-softened, bovine-enamel slabs to the oral environment in 3 subjects for 2 h, 24 h or 7 days, and NCL were sampled from extracted human teeth with white or yellow spots of arrested caries. Protein content of ECL from 2 subjects were similar to each other and to that of NCL throughout the experimental period. The ECL of the other subject showed a gradual increase in protein content with significantly higher values at day 7. The predominant amino acids in ECL were glutamic acid, proline and alanine, and in NCL, glutamic acid, glycine and alanine. The amino-acid composition of the 7-day ECL was closer to that of NCL than were that of the 2 and 24 h ECL. I&a-oral ageing caused significant reductions in proline and glycine and pronounced increases in aspartic acid, threonine, alanine and leucine. Thus the adsorbed or incorporated organic material in the ECL changed from having components dissimilar to NCL to ones similar to NCL. This intra-oral model might be useful for studies of the organic material incorporated into enamel during the process of consolidation.
MATERIALS AND
INTRODUCTION
The term consolidated enamel lesions was given by Bibby (1932) to carious enamel lesions that were arrested. In some lesions this arrest can be attributed to a change in the conditions of microbial accumulatlon on the vulnerable surface, such as occurs on an approximal surface after the extraction of a neighbouring tooth. Conceivably, improvement in oral hygiene and fluoride treatment could also contribute to arrest by decreasing the cariogenic challenge or by increasing the remineralizing activity of saliva (Koulourides and Cameron, 1980). Consolidated lesions are more resistant to acid exposure than adjacent normal enamel (Beust, 1930; Bibby, 1932). They also have a higher proportion of organic material (Stack, 1954; Bhussry and Bibby, 1957) and fluoride (Little, Posen and Singer, 1962; Weatherell et al., 1977). Hay and Moreno (1979) and Moreno, Varughese and Hay (1979) demonstrated that salivary acidic proline-rich proteins and statherin bind to hydroxyapatite and inhibit the nucleation of calcium-phosphate salts; hence, in remineralization phenomena, certain salivary proteins or peptides could play an important role. Experimental cariogenic challenge to pre-softened enamel, with the influence of fluoride, increases fluoride incorporation and increases resistance of these experimentallyconsolidated lesions to in-vitro acid challenge (Koulourides and Housch, 1983). 0ur purpose was to investigate the incorporation of organic material into in-vitro enamel lesions exposed to the mouth, to compare the amino-acid composition of experimental enamel lesions (ECL), and to compare the amino-acid composition of ECL with that of naturally-consolidated lesions (NCL). 405
METHODS
Preparation and sampling of ECL
Rectangular enamel slabs (3 x 5 x 2 mm) were cut from the buccal surfaces of bovine permanent incisors. The slabs were mounted with sticky-wax on plexiglass blocks with the enamel surfaces as parallel with the base of the block as possible. The outer enamel layer was removed to an approximate depth of 100 pm by grinding against emery paper (Norton, Tufbak Durite, waterproof, 600-A grit) using a specially designed grinder-polisher (Koulourides et al., 1976). Artificial enamel lesions were produced by immersing the enamel slabs in 0.01 M sodium-lactate buffer, pH 4.0 (20ml/slab), containing 3.0 mM calcium, 1.8 mM phosphate and 1 per cent carboxyat 37°C for 4 days. Micromethylcellulose, radiography indicated a lesion depth of approx. 60 pm. The pre-softened slabs were fixed with stickywax into recesses drilled in the flanges of acrylic dentures; the enamel surface was exposed to the oral environment to produce ECL. In three groups of experiments, eight pre-softened slabs per group were exposed in the mouths of three subjects for either 2 h, 24 h or 7 days. For the 2-h experiment, the subjects were instructed not to drink or eat; for the other time periods, their diets were not restricted. The dentures with the slabs were worn continuously throughout each experimental period; they were cleaned by brushing with soft toothbrushes and a nonfluoridated toothpaste. After removal from the mouth, the slab surface was wiped with a cotton pellet under running deionized water to remove plaque and debris. The ECL
406
T.
~UANAKA
samples were collected by scraping the lesions with scalpel blades down to hard, normal enamel on plastic weighing trays. Control samples were prepared from lesions not exposed to the mouth.
et al.
Statistics The data were analysed statistically analysis of variance.
Preparation of NCL
by one-way
RESULTS
Freshly-extracted human teeth showing white or yellow enamel spots were used. Dental calculus and deposits were removed with periodontal scalers and the enamel surfaces cleaned with pumice. Because the NCL usually invades normal enamel in a conical pattern, the sampling was done with a no. 6 round bur rotated at low speed to prevent overheating. Care was taken to stop the grinding when sound enamel was reached. The NCL samples were collected on plastic weighing trays. Amino -acid analysis The samples were left at room temperature for 1.5 h, then weighed and hydrolysed with 2.0 ml of 6.0 M HCl for 24 h at 108°C. The hydrolysates were substantially soluble in distilled water. After evaporation of HCl in uacuo, the hydrolysates were dissolved in 1.Oml water and analysed for 19 amino acids using a Beckman 121-M amino-acid analyser (Butler, Finch and Miller, 1977). The presence of calcium and phosphate salts did not interfere with this analysis. The protein content of the various lesions was calculated from the amount of amino acids (nmol/ml) in hydrolysates of each sample and expressed as pg protein per mg (wet weight).
Protein content in the control, NCL and ECL The protein content of the carious lesions is shown in Fig. 1. The ECL samples from two subjects (A and B) were similar to each other and to the samples of NCL. However, in subject C, there was a gradual increase in the protein content of ECL with intra-oral ageing; the 7-day ECL had a significantly higher value than NCL (p < 0.01). The control slabs, not exposed to the mouth, contained less than 5 per cent of the protein in ECL. The coefficient of variation for NCL was much greater in controls and ECL, 103 and 41 per cent, respectively. Amino-acid composition of the controls The predominant amino acids were glycine, glutamic acid and aspartic acid. These three accounted for 43 per cent of the total amino acids. Hydroxyproline, cysteine and hydroxylysine were not detected. These samples contained approx. 23 per cent acidic, 12 per cent basic and 5 per cent aromatic amino acids. Amino-acid composition of the NCL (Table 1) The predominant amino acids were glutamic acid,
Subjects
*
g:
UIIIC
Cant
2h
ECL
24h
ECL
?day
ECL
NCL
Fig. 1. Protein content of control, 2-h, 24-h and ‘I-day ECL and of NCL. ECL data are shown in the individual subjects (A, B and C). Mean values (columns) and standard errors (bars) are given (n = 8). *p c 0.01, significantly from NCL.
Composition Table 1. Amino-acid composition*
of control and NCL
Controlt Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Vaiine Methionine Isoleucine Leucine Tyrosine Phenylalanine L,ysine Histidine Aqinine
of experimental consolidated lesions
NCL
Mean
SE
Mean
SE
87.18 39.90 80.55 139.52 35.69 207.22 47.39 23.81 51.82 33.36 80.68 16.38 32.01 62.87 31.57 30.06
8.02 4.62 6.83 9.16 1.57 2.68 3.27 2.16 2.97 1.16 15.88 3.81 2.15 5.09 6.32 2.06
97.06 39.54 58.32 144.50 63.57 123.88 134.11 50.45 31.701 30.71 61.17 22.25 28.50 52.48 17.02 45.08
4.67 1.99 2.60 5.25 5.40 9.06 11.64 1.61 6.84 4.41 2.78 1.55 1.28 7.09 2.24 2.35
*Residues per 1000 residues, n = 8. Hydroxyproline, cysteine and hydroxylysine were not detected. tPre-softened, but unexposed to the oral environment. In = 7. alanine and glycine; their contribution was approx. 40 per cent of the total in NCL. Hydroxyproline and
hydroxylysine were not detected. Compared to controls, the amino-acid composition of NCL was characterized by smaller amounts of glycine and larger amounts of alanine. Amino-acid compositions of 2-h, 24-h and 7-day ECL (Tables 2, 3 and 4)
The three major amino acids were proline, glycine and glutamic acid in the 2-h ECL; glutamic acid, glycine and proline in the 24-h ECL; and glutamic aad, glycine and aspartic acid in the 7-day ECL. These predominant amino acids accounted for 35-50 per cent of the total in each ECL. The amino-acid composition of the 2-h ECL had larger amounts of proline, glycine and histidine, and smaller amounts of aspartic acid, threonine, alanine, valine and leucine than the NCL (p < 0.01). The
407
proportions of amino acids among the three subjects were similar, except for differences in proline, tyrosine and histidine which were pronounced in the 2-h ECL, less so in the 24-h ECL and not apparent in the 7-day ECL compared with NCL samples. In the 24-h ECL (Table 3) basic amino-acid values were significantly higher (p < 0.01) and aromatic acid values were slightly higher than in NCL, but the acidic amino-acid values were almost the same as in NCL. Compared with the 2-h ECL, the proline value of the 24-h ECL was significantly reduced (p < O.OS), but it was still higher than for NCL (p < 0.01; Fig, 2a). Also, the glycine value decreased to become similar to that of NCL in the 24-h ECL (Fig. 2b). The values of proline and glycine in 7-day ECL were significantly reduced (Table 4) but there were pronounced increases in aspartic acid, threonine, alanine and leucine compared with the 2-h ECL (Tables 2 and 4; Fig. 2). These differences were statistically significant (p < 0.01). With ageing from 2-h to 7 days, the proportion of amino-acid compositions in ECL became similar to those of NCL. DISCUSSION
Earlier studies have shown an increase in the organic content of carious lesions compared with normal enamel (Stack, 1954; Bhussry and Bibby, 1957; Weatherell et al., 1977). In most of these, the nitrogen content of the lesions was taken as representative of the organic content. There have been few studies (Robinson, Weatherell and Hallsworth, 1983) of the amino-acid composition of material incorporated during the process of natural caries. We have directed our efforts toward the development of an intra-oral model of lesion consolidation. The selection of bovine enamel as a substrate was based on our earlier observation that its ground and polished surface, at a depth of lOO-200pm from the natural one, reacts predictably to demineralization and remineralization. Thus, with acid gels the developing subsurface lesion is almost parallel to the test surface (Koulourides and Housch, 1983). This facilitates
Table 2. Amino-acid composition* of 2-h ECL Subject A Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
B
C
Mean
SE
Mean
SE
Mean
SE
65.93 16.33 57.58 139.35 186.79 152.25 37.40 11.43 16.15 29.72 51.23 40.51 77.29 61.32 59.48
1.14 4.88 1.54 1.22 9.50 8.40 1.65 I .49 3.31 3.31 6.26 6.18 4.20 2.97 1.46
86.57 19.39 62.02 130.19 113.70 131.02 45.56 20.66 30.50 40.06 53.49 36.83 79.81 77.60 72.59
2.48 1.69 1.98 7.08 9.24 5.48 1.06 2.10 4.16 3.45 6.45 5.44 2.88 6.75 1.79
86.00 27.33 71.35 156.77 164.15 164.96 53.03 33.24 33.38 43.30 22.25 21.23 49.05 25.81 48.21
1.88
1.45 1.03 4.77 6.31 4.51 2.18 5.87 7.11 2.97 1.36 0.70 1.87 1.72 2.14
*Residues per 1000 residues, n = 8. Hydroxyproline, cysteine, methionine and hydroxylysine were not detected.
408
T.
TERANAKA
et al.
Table 3. Amino-acid composition* of 24-h ECL Subject A Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
B
C
Mean
SE
Mean
SE
Mean
SE
79.62 19.72 63.73 141.54 104.69 129.36 39.99 29.72 31.48 43.23 57.66 43.12 75.13 72.24 68.62
3.24 1.10 2.01 2.79 10.49 5.68 1.44 1.59 4.21 3.86 8.46 6.70 4.21 3.16 1.20
88.95 14.81 64.71 148.79 93.59 127.91 40.55 13.58 27.82 39.27 64.42 36.40 71.33 94.10 73.81
1.74 0.96 2.89 6.19 3.12 4.34 2.55 1.19 2.69 2.19 4.62 2.04 3.02 6.04 3.29
92.62 24.47 67.01 146.83 113.85 119.70 51.71 30.91 33.89 51.02 37.36 30.34 74.34 59.54 63.34
2.84 1.49 2.41 2.76 10.91 4.29 2.27 3.94 5.74 5.12 2.14 1.18 2.89 2.71 1.46
*Residues per 1000 residues, n = 8. Hydroxyproline, onine and hydroxylysine were not detected.
sampling of the layer that is altered by interaction between pre-softened enamel and the oral environment. The difference in mineral density between the
ECL and the underlying normal enamel is so great that the lesion can easily be sampled by scraping with a scalpel blade, and there is no appreciable contamination of the sample with unaffected tissue. The demineralization used in preparation of the lesions provided 2-4 mg wet weight of material for aminoacid analysis. Sampling is more difficult with NCL due to their conical shape and to the irregular boundaries between them and normal enamel; a slowly-rotating round bur is more appropriate than scraping with a blade. Although some of the NCL could have been left in the tooth, we feel that the material analysed was representative of NCL without appreciable contamination by normal enamel. We consider the two ways of sampling to be equivalent;
cysteine, methi-
both are mechanical, and both are stopped when the instrument reaches the much harder sound enamel. Sijnju and Rolla (1973) reported that the amount of new salivary pellicle on cleaned teeth increases for about 1.5 h and then levels off; the total amount of amino acids in pellicles from the buccal surfaces of approx. 24 teeth after 1.5 h was almost 100 pg. To assess the extent to which salivary pellicle might contribute to our samples, we exposed slabs of sound enamel in the mouth for 24 h and then collected pellicle samples with 2 per cent HCl using the method of Mayhall (1970). The mean protein content per slab of these pellicles was 1.1 pg, but the specimens of the 24-h ECL contained 34 pg per slab. Thus the pellicle content was approx. 3 per cent of the total ECL protein. It might be argued that surface pellicles formed on pre-softened enamel may have a different composition from those on normal enamel. We could
Table 4. Amino-acid composition* of 7-day ECL Subject A Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
B
C
Mean
SE
Mean
SE
Mean
SE
92.46 38.14 67.09 161.15 65.18 102.12 97.69 38.28 37.58 60.19 30.52 32.86 84.14 36.05 56.63
2.45 3.49 4.57 3.20 7.09 4.62 14.24 3.42 2.54 1.85 4.19 1.59 6.55 5.17 2.56
99.67 34.18 68.03 148.42 74.77 107.80 79.99 30.70 39.83 59.39 32.14 34.38 81.37 45.71 60.37
1.92 4.17 2.68 3.25 7.77 5.67 9.17 5.67 3.38 2.07 2.74 0.91 7.14 6.00 3.31
104.22 43.70 64.99 155.53 63.07 99.69 113.75 37.47 28.01 64.23 30.68 31.58 87.25 31.32 44.53
1.14 1.71 2.21 3.26 2.77 6.89 7.60 2.07 3.06 2.62 1.22 0.81 1.78 3.21 1.21
*Residues per 1000 residues, n = 8. Hydroxyproline, cysteine and hydroxylysine were not detected. Methionine was trace in each subject.
Composition
409
of experimental consolidated lesions
ECL B
2h
0
24h
cl
7dcJy
m
NCL
Subjects
I,_
:
(0)
: A,E,C
A
B
C
Thr
(b)
A GUY
E AlO
C
A
B
C
LOU
Fig. 2. Changes in enamel amino-acid composition with intra-oral ageing which were significantly different. Mean values (columns) and standard errors (bars) are given (n = 8). *p < 0.05. **p < 0.01, significantly different from NCL.
not examine this because pellicle sampling with HCl causes flotation of surface-adsorbed organic material by diisolving the underlying sound enamel. A similar procedure in this investigation would cause flotation of the whole ECL, making it impossible to separate
the pellicle from the organic material within the lesion. The protein content of control samples not exposed to the mouth was less than 5 per cent of the amount recovered from the ECL (Fig. 1). Therefore, more than 90 per cent of the ECL and NCL samples represented organic material incorporated into the lesion from the mouth. The environment for 2-h ECL was mainly saliva but the diet and microbial activity could have influenced the composition of the 24-h and 7-day ECL, as well as all the NCL samples. The larger standard error for the mean organic content of the NCL lesions indicates that conditions during their formation, or the state of their development, were more varied than for the ECL, in which they were controlled by the experimental design. This would be expected, as the teeth with NCL had been extracted from different individuals of unknown age and dental history. Although 2- and 24-h ECL had similar organic content in all three subjects, the 7-day ECL in one subject (C) was considerably higher than that of the NCL (p < O.Ol), while with the other two subjects the
difference was not appreciable. This greater incorporation of organic material seems to be related to the high caries activity of that subject. Our earlier studies with the intra-oral cariogenicity test (ICT) indicate that individuals have characteristic and different propensities to develop caries within 1 week in bovine enamel slabs exposed in their mouths (Ostrom et al., 1977). Our subjects A and B were classified as low in ICT caries, whereas subject C exhibited high cariogenic activity. This higher activity may have contributed to more extensive demineralization of the pre-softened enamel and less mineral deposition, thus leaving a larger proportion of microspaces to be filled with organic material. The amino-acid composition of the 2-h ECL was more like that of saliva than were the 24-h and 7-day ECL. With intra-oral ageing, ECL showed compositional changes, possibly due to the influence of bacteria and diet. Through such changes the proportions of amino acids of the 7-day ECL became close to that of the NCL. Similarity in amino-acid composition between 7-day ECL and NCL indicates that underlying reactions in the formation of the organic materials in the experimental and the natural lesions are also similar. Bennick et al. (1983) found that the percentage of proline-rich proteins, adsorbed on sound enamel in
T. TERANAKAet al.
410
vivo, increases during the first hour to about 37 per cent of the total extracted protein; any differences between the 1 and 24-h period are minor, but there is a gradual degradation of the proteins. They found that extracts of old acquired pellicle contain less than 0.1 per cent proline-rich proteins, and that pellicles more than 24-h old show degradation of the adsorbed proline-rich proteins. It is possible that the gradual increase in alanine was also a result of bacterial involvement, as bacterial cell walls usually have a high alanine content (Cummins, 1962). Zahradnik (1979) suggested that the presence of an adsorbed salivary pellicle on the surface of a tooth plays an important part in remineralizing phenomena when the tooth is placed in an inorganic mineralizing solution. Likewise, the organic material in consolidated lesions could affect the remineralization of both NCL and ECL. As pointed out by Zahradnik (1979), the incorporated organic material could actually retard mineralization in the surface layers of lesions and enhance ionic penetration into the subsurface.
The contribution of the organic material to remineralization and to the increased acid resistance of consolidated lesions requires further investigation. Acknowledgements-We gratefully acknowledge the contribution by MS A. Vitek of technical help in the amino-acid analysis. This work was supported by USPHS-NIDR Grant DE 02670 and a grant from the Warner-Lambert Company. REFERENCES
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Weatherell J. A., Deutsch D., Robinson C. and Hallsworth A. S. (1977) Assimulation of fluoride bv enamel throuehout the life of the tooth. Caries Res. il, 85115. ” Zahradnik R. T. (1979) Modification by salivary pellicles of in vitro enamel remineralisation. J. dent. Res. 58, 20662073.