Effect of Salivary Pellicle Formation Time on In Vitro Attachment and Demineralization by Streptococcus mutans R. T. ZAHRADNIK, D. PROPAS, and E. C. MORENO Forsyth Dental Center, 140 Fenway, Boston, Massachusetts 02115
Enamel subsurface demineralization induced by Streptococcus mutans was significantly reduced by seven-day saliva pre-treatments conducive to the formation of enamel pellicles. A two-hour saliva pre-treatment was ineffective. Results suggest that the protection provided by long-term pellicles may relate to changes in ionic transport rates rather than cell attachment.
J Dent Res 57(11-12):1036-1042, Nov.-Dec. 1978
Introduction. The tooth surfaces in situ are covered by a proteinaceous film generally referred to as the acquired pellicle.l,2 Similar films can be formed on clean, pumiced enamel surfaces in relatively short times by exposure to oral secretions, 3,4 and a selective adsorption of salivary macromolecules has been advanced3-5 as the mechanism involved in the initial stages of the development of this integument. The acquired pellicle is the surface to which oral bacterial cells become attached to teeth,6 thus initiating the formation of dental plaque. Therefore, the possibility exists of the pellicle affecting the cariogenic process by reducing bacterial attachment or by introducing a marked degree of specificity for the adsorption of oral microorganisms. This possibility has been studied through the use of adsorption models involving saliva-treated synthetic apatite surfaces or dental enamel and cultures of isolated oral micro-organisms.7-10 The results of such investigations indicate that the saliva treatment enhances the attachment of
This investigation was supported, in part, by U.S.P.H.S. Grant DE-03187 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland. Received for publication January 9, 1978. Accepted for publication April 5, 1978. 1036
some organisms, whereas for others the opposite is true. There are inconsistencies among the published results which seem to reflect the specific experimental conditions of each model. Nevertheless, taken in toto, the data indicate that the acquired pellicle may alter the relative distribution of microorganisms initially colonizing the human tooth surfaces. The acquired pellicle may not only affect the nature of the dental plaque but also the transport of matter from and to the tooth enamel. Recently, it has been reportedl 1 that salivary pellicles, developed in vitro on a pressed disk of hydroxyapatite (HA), display ionic permselectivity (i.e., ionic transport is slower than that of neutral molecules). The degree of permselectivity was found to increase with longer exposure times of the disk to saliva, and this dependence on formation time seems to parallel observations on the level of protection provided by acquired pellicles (developed on extracted teeth) against enamel subsurface demineralization by acid buffers. 1 These studies emphasize the importance of considering more than bacterial cell attachment when one attempts to understand the role of the acquired pellicle in the natural caries process. The physical properties of this membrane may act to inhibit caries development; furthermore, the time of pellicle formation appears to be an important consideration in any in vitro research which attempts to mimic conditions existing at the saliva-tooth interface. The protection observed for salivatreated teeth against acid buffers may be lost under oral conditions where any salivary film on the teeth may be susceptible to degradation by bacteria in plaque.12-15 A model system has been developed16 to test whether an acquired pellicle can affect the rate of subsurface demineralization when
Vol. 57No. 11^12
SHORT- AND LONG-TERM SALIVAR YPELLICLES
the acid challenge results from the direct colonization (on a tooth surface) by known strains of cariogenic micro-organisms. Preliminary results indicate that reduced mineral loss can indeed occur when an acquired pellicle is present on the challenged tooth surface. This is significant since it demonstrates that the pellicle does possess the ability to alter a "caries-like" subsurface demineralization process under conditions approaching those expected in the mouth. It therefore becomes extremely important to determine if reduced plaque accumulation can explain this observation or whether a second mechanism (e.g., permselectivity) must be invoked to account for the protection. Such information could assist in the search for practical ways to control caries by favorably modifying the tooth-saliva interface. The present investigation was initiated to study the effect of acquired salivary pellicles (and their formation time) on (1) enamel subsurface demineralization, (2) total plaque accumulation, and (3) initial bacterial cell attachment.
Experimental. Enamel demineralization. The technique developed to produce subsurface demineralization by Streptococcus mutans has been described previously.16 In the present case a total of 12 clinically sound premolar teeth were used (extracted for orthodontic purposes). The teeth were scaled, pumiced and split in half along the buccal-lingual plane so that one half could serve as a control for the other half of the buccal surface tested. The split teeth were covered with wax except for an 8 mm2 window. Short-term pellicles were developed by exposing the test halves of six teeth to stimulated parotid saliva (5 ml/tooth) for two hours with continuous stirring. Long-term pellicles were built up by exposing the test halves from the remaining six teeth to the parotid saliva for seven days, changing the saliva daily. Sodium azide was added to the parotid saliva (0.02%) to control bacterial growth in these experiments. It has previously been reported11 ,17 that pellicles developed using parotid, submandibular and whole clarified saliva are all capable of providing equivalent protection against acid buffer demineralization.
1037
Each group of saliva-exposed or control teeth was suspended in separate culture tubes containing trypticase soy broth with 2% sucrose, 200 ,ug/ml of streptomycinresistant Streptoccus mutans strain Ingbritt (IB 1600) of human origin.18 The teeth were incubated anaerobically at 370C for 5 days. Transfers to fresh culture media were made daily. The final culture medium was periodically checked for contamination using Gram staining and dark field microscopy. At the end of 5 days an adherent, granular plaque was present on the teeth. The enamel windows were cleaned of microbial deposits, the wax was removed, and the teeth were embedded in an epoxy resin. Enamel sections of 400 gm thickness were cut perpendicular to the window surface with a diamond saw. These sections were ground to a parallel thickness of 100 ,um, mounted in a specially made frame against a high resolution photographic film, and exposed to an x-ray beam (nickel-filtered, Cu radiation, 40 Kv, 200 ma) for 20 minutes. The resultant microradiographs were scanned (through the demineralized region) with a high resolution microdensitometer using an effective slit of 5 ,um x 100 gm, and the optical densities were continuously recorded. In this fashion, quantitative microradiography was used to determine the effect of salivary pellicles on the subsurface demineralization induced by colonization of IB1 600. The extent of demineralization was assessed by measuring the following three parameters16: maximum depth at which mineral loss could be detected, maximum mineral loss in the body of the lesion, and thickness of the intact enamel surface layer.
Assessment of total plaque. The effect of acquired pellicles on accumulation of microbial deposits was tested using a fluorometric determination of total DNA in the plaque samples scaled from control and pellicle-coated tooth surfaces. Ethidium bromide was used as the fluorescent probe, following the procedure outlined by Donkersloot and coworkers.19 A total of 18 teeth were used in this experiment (6 each for the control, short-term and long-term pellicle groups). The waxing step had to be modified to
1038
ZAHRA DNIK E T AL.
allow for a window of defined surface area so that the total plaque could be normalized to a unit of tooth surface area. An 18 mm2 window was produced by waxing around a parafilm circle of that surface area. The parafilm was removed after waxing. The microbial deposits were carefully scaled from each window at the completion of the 5-day incubation period with IB1600. The plaque samples were washed over a filter and then incubated for one hour at 37 C with 0.3 N KOH to eliminate RNA interference with the fluorometric determination of DNA. The samples were neutralized and then mixed with a tris buffer containing the ethidium bromide. Standards containing calf thymus DNA were treated in the same fashion. The DNA per individual plaque sample was determined spectrofluorometrically; these values were then converted to number of cells per plaque sample. The conversion factor for this procedure was determined by measuring the DNA/ml in an aliquot from a 24-hour T soy broth culture of IB1600 and then obtaining a value for the cell concentration in this broth by direct microscopic count in a Petroff Hauser counting chamber. Since the surface area of the window (from which the microbial deposit was scaled) is known, the total plaque accumulation on each tooth can be reported as the total number of cells per unit surface area of enamel.
Initial cell attachment. Initial colonization of saliva-treated and control enamel surfaces was evaluated by the direct observation of enamel surfaces using incident light microscopy. In this procedure it was necessary to have enamel specimens with flat polished surfaces (for focusing purposes). Therefore, an approach similar to that reported by ¢rstavik et al. 8 was used. Cores were taken from the buccal surfaces of 18 premolar teeth using a hollow bore diamond drill (I.D. 3 mm). The cores were ground to produce a flat polished enamel face. A group of 3 cores was treated for 2 hours with parotid saliva (as described above); a second group received the 7-day saliva treatment, and a third group served as a control. The three groups of cores were incubated with IB 1600 for 5 hours. They were then removed, washed, dried, fixed in 95% ethanol, and stained with crystal violet
JDent Res November-December 1978
(1 min). Cell attachment was assessed by observing the stained micro-organisms on the enamel surfaces with an incident light microscope (x256). A duplicate run was performed using the remaining nine enamel cores.
Results. Results concerning the effects of shortterm and long-term salivary pellicles on demineralization of enamel by IB1600 are shown in Table 1. The mean and standard deviation (6 tooth samples in each group) are given for each of the three parameters used to characterize the extent of demineralization. It is apparent that a 7-day pellicle, developed in vitro on extracted teeth, significantly decreases the extent of mineral loss, which is in agreement with an initial study previously reported.16 However, no statistically significant difference in the demineralization pattern was found between the controls and the enamel having 2-hour pellicles. It is interesting that a thicker intact surface layer was observed for the 7-day pellicle treatments than for their corresponding controls. The possibility that the results shown in Table 1 could be related to a decreased number of bacteria colonizing on the salivatreated surface was examined by determining quantitatively the effect of the pellicle on total microbial accumulation. In Table 2, total plaque is expressed in terms of number of bacterial cells per unit surface area of enamel. Two runs are reported, with the mean and standard deviation given for each group of three teeth. Results of the two runs are comparable, i.e., the trends are the same although the absolute magnitude of cells found between runs, for a given test group, may vary. The in vitro bacterial accumulation on the two salivatreated groups is similar and not statistically different from that found on the controls. However, a trend toward greater plaque accumulation on the enamel surfaces with acquired pellicles is evident. Thus, the effect of pellicles on plaque development in our model system would tend to be opposite to that expected if the observed pellicle protection against demineralization (Table 1) were related to a reduction in the number of acid-producing S. mutans cells on salivatreated vs. control tooth surfaces.
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1039
TABLE 1 ENAMEL DEMINERALIZATION AFTER 5-DAY INCUBATION WITH IB1600 2-hr Pellicle
Control Depth of lesion (um) Maximum mineral loss (%) Intact layer thickness (Am)
+ 6.9* 22.3 + 2.9 23.7 ± 3.8
118
Pellicle 116 + 11.7 23.0 ± 2.8 24.7 ± 4.3
7-day Pellicle Control Pellicle 115 + 13.8 23.3 ± 4.3 21.7 ± 5.9
106 + 4.3 15.7 ± 2.1 31.7 ± 3.1
*Standard deviation
Since the decreased demineralization in the presence of pellicle does not appear to be related to a decreased microbial deposit after 5 days of incubation with IB1600, we examined the possibility that the observed protection could have resulted from a delay in the initial microbial colonization, which was not apparent after 5 days of incubation. Typical photographs of initial cell attachment are included in Figures 1 and 2. In Figure 1 the left half shows the level of cell attachment found on control enamel surfaces after 5 hours' incubation with lB 1600. The right half shows the degree of initial colonization when a 2-hr pellicle is allowed to develop on the polished enamel core prior to the bacterial exposure. There was an enhancement in the rate of attachment when a short-term pellicle was present, and this feature was evident in all six cores in this group. Figure 2 contains a typical example comparing control and 7-day pellicle coated enamel after 5 hours of incubation with IB 1600. These results and those found for the other cores within the groups show a consistent and marked increase in the bacterial attachment rate to the enamel surfaces having a 7-day pellicle.
TABLE 2 TOTAL PLAQUE AFTER 5 DAY INCUBATION
WITH IB1600
Control 2 hr pellicle 7 day pellicle
Run I
Run II
109 cells/cm2
109 cells/cm2
2.01 ± 0.47* 3.04 ± 0.93 2.99 ± 0.91
0.69 ± 0.47 1.52 ± 0.76 2.01 ± 0.95
*Standard deviation
The initial kinetics of cell attachment observed in these last studies should reflect conditions occurring early in the demineralization experiments because the incubation steps were carried out in identical fashion. Since the IB 1600 cells were grown in sucrosecontaining medium, there was some clumping of the attached cells; no attempt was made to quantitatively evaluate the total numbers of adsorbed cells on the various enamel surfaces, since the trends were evident by visual inspection. Results for incubation times shorter than 5 hours displayed the same type of response; however, the total number of attached microorganisms was proportionally less.
Discussion. The results of the present investigation show that development of salivary pellicles on tooth enamel over a period of seven days protects this tissue against demineralization induced by cariogenic micro-organisms (see Table 1). This protection is not apparent for short-term pellicles (i.e., pellicles formed by exposing enamel to saliva for two hours prior to colonization of IB1600). Furthermore, the observed protection and its dependence upon the pellicle age seem to be unrelated to the amount of microbial accumulation on the tooth surface. Thus, the number of cells recovered per unit surface area of exposed enamel having short-term pellicles was not significantly different from the number of cells recovered from the enamel having 7-day pellicles (see Table 2). The mechanism by which the long-term pellicle protects enamel against demineralization is not fully understood at present, but it seems to be associated with some properties of the pellicle that depend on its age
1040
ZAHRADNIK ETA L.
J De}nt Res No ve i n b er-Dece in ber 19 78
Bcterial cell attachmiient to enamel sLurfaces after 5 hoLrs' incubation wxith an inoculun 1I 1. a--of S. mutans strain IB1600. Left, untreated sLrface; right, surface treated with parotid saliva for 2 lhours. or formation time. It is not known whether this time dependence reflects alterations in composition or in the ultrastructure of the integument, or both. There are several reports concerning the composition of short-term pellicles developed in situ and in vitro,3,4720-22 but only limited work has been conducted on possible compositional changes as the pellicle ages.3,23 Although the composition of short-term pellicles developed in situ and in vitro is quite similar, it appears to be significantly different from that of old natural pellicles of undetermined age.3 The few reports24'25 related to ultrastructural changes upon pellicle aging have not been done in a systematic fashion although a significant first effort in this direction has been made recently by Lie.26 This latter investigator followed the formation of pellicle on apatitic surfaces in the oral environment using scanning and transmission electron microscopy. His results, covering the first 48 hrs of pellicle formation, showed an increase in thickness (up to about 400 nm) accompanied by distinct
changes in the pellicle morphology; the older the pellicle, the more compact it appears, with a granular structure replacing the more open globular or fibrillar structures found in early pellicles. In some cases, Lie26 reported the formation of laminated structures in granular pellicles after 24 hrs. Thus, the scant information gathered so far indicates that most probably compositional and ultrastructural changes do occur as the pellicle ages, which undoubtedly change its physical properties. In the process of enamel deminerahization that results in mineral loss from the subsurface region, an important factor is the rate of transport of matter from the enamel surface to the demineralizing medium.27 It has been reported11,17 that this rate is greatly reduced by the presence of 7-day pellicles (i.e., the mineral loss is substantially reduced), whereas no protection was observed with short-term pellicles. Furthermore, it was observed11,17 that the ionic permselectivity of salivary pellicles adsorbed on apatite disks increased with increasing development times. Therefore, the perm-
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SHORT- AND LONG-TERM SALIVARY PELLICLES
1 041
Fig. 2 -Effect of a 7-day pellicle on the initial rate of cell attachment. Both enamel surfaces were incubated for 5 hours with S. mutans strain IB1600. Left, untreated surface; right, surface having a 7-day pellicle.
selectivity appears to be related to the reported protective action of the salivary pellicle. The results of the present investigation are consistent, then, with the idea that the permselectivity of the pellicle is probably related to the observed protection against demineralization induced by lB1600 the enhancement in the as reported here pellicle permselectivity being associated with the types of compositional and ultrastructural changes that occur upon aging and that have been reported by other investigators.3,26 Furthermore, it has been observed that the properties of natural acquired pellicles also change with time, displaying an enhanced acid resistance after several days of development.28 There are two acknowledged mechanisms by which an acquired pellicle may interfere with the natural caries process: (1) by altering the specificity or magnitude of microbial colonization, and (2) by affecting the transport rates related to enamel mineral loss. The present results show that the presence of a salivary pellicle increases both
the initial rate of cell attaclhment to the tooth surface (Figures 1 and 2) and the total microbial accumulation (Table 2). These results, in fact, made it possible to ascertain unequivocally the protective action of the pellicle against demineralization. However, no strain different from IB1600 (grown in a sucrose-containing medium) was used in this investigation. The results, therefore, do not eliminate the possibility that both mechanisms may be operative under in vivo conditions. Several reports7 -10 indicate that the attachment of some strains of Streptococci to apatitic surfaces is enhanced by a salivary pre-treatment of the adsorbent surfaces, while it is reduced for other strains. It appears then that, in the oral environment, the selective attachment induced by the acquired pellicle may determine the kind of dental plaque developed and, thereby, its cariogenic potential. This interesting possibility could be studied with the model system used in this investigation in conjunction with pure and mixed cultures of oral micro-organisms.
1042
J Dent Res November-December 1 978
ZAHRADNIK ETAL. REFERENCES
1. DAWES, C.; JENKINS, G. N.; and TONGE, C. H.: The nomenclature of the integuments of the enamel surface of teeth. Br. Dent. J. 115:65-68, 1963. 2. MECKEL, A.H.: The formation and properties of organic films of teeth. Arch. Oral Biol. 10:585-597, 1965. 3. MAYHALL, C. W.: Concerning the composition and source of the acquired enamel pellicle of human teeth. Arch. Oral Biol. 15:1327-1341, 1970. 4. SO NJU, T.; and ROLLA, G.: Chemical analysis of the acquired pellicle formed in two hours on cleaned human teeth in vivo. Caries Res. 7: 30-38, 1973. 5. HAY, D. I.: The adsorption of salivary proteins by hydroxyapatite and enamel. Arch. Oral Biol. 12:937-946, 1967. 6. SAXTON, C. A.: Scanning electron microscope study of the formation of dental plaque. Caries Res. 7:102-119, 1973. 7. MCGAUGHEY, C.; FIELD, B. D.; and STOWELL, E. C.: Effects of salivary proteins on the adsorption of cariogenic Streptococci by hydroxyapatite. J. Dent. Res. 50:917-922, 1971. 8. 0RSTAVIK, D.; KRAUS, F. W.; and HENSHAW, L. C.: In vitro attachment of Streptococci to the tooth surface. Infect. Immun. 9:794-800, 1974. 9. GIBBONS, R. J.; MORENO, E. C.; and SPINELL, D. M.: Model delineating the effects of a salivary pellicle on the adsorption of Streptococcus miteor onto hydroxyapatite. Infect. Immun. 14:11091112, 1976. 10. LILJEMARK, W. F.; and SCHAUER, S. V.: Competitive binding among oral Streptococci to hydroxyapatite. J Dent. Res. 56:157-165, 1977. 11. ZAHRADNIK, R. T.; MORENO, E. C.; and BURKE, E. J.: Effect of saliva pellicle on enamel subsurface demineralization in vitro. J. Dent. Res. 55:664-670, 1976. 12. LEACH, S. A.; and SAXTON, C. A.: An electron microscope study of the acquired pellicle and plaque formed on the enamel of human incisors. Arch. Oral Biol. 11:10811094, 1966. 13. FRANK, R. M.; and HOUVER, G.: An ultrastructural study of human supragingival dental plaque formation; in McHugh, Dental Plaque (Thomas, Dundee) 1970. 14. NEWMAN, H. N.: The organic films on enamel surfaces. 1. The vestigial enamel organ. Brit. Dent. J. 135:64-67, 1973.
15. NEWMAN, H. N.: The organic films on enamel surfaces. 2. The dental plaque. Brit. Dent. J. 135: 101-106, 1973. 16. ZAHRADNIK, R. T.; PROPAS, D.; and MORENO, E. C.: In vitro enamel demineralization by Streptococcus mutans in the presence of salivary pellicles. J. Dent. Res. 56:1107-1110, 1977. 17. MORENO, E. C.: The effect of acquired pellicle on enamel demineralization. Proc. Int. Symp. Acid Etch Tech., Silverstone, L. N. and Dogon, I. L. (eds.): St. Paul, North Central Publ. Co., pp. 1-12, 1975. 18. KRASSE, B.: Human Streptococci and experimental caries in hamsters. Arch. Oral Biol. 11:429-436, 1966. 19. DONKERSLOOT, J. A.; ROBRISH, S. A.; and KRICHEVSKY, M. I.: Fluorometric determination of deoxyribonucleic acid in bacteria with ethidium bromide. Applied
20.
21.
22.
23.
24.
Microbiology 24:179-183, 1972. S6NJU, T.; CHRISTENSEN, T. B.; KORNSTAD, L.; and ROLLA, G.: Electron microscopy, carbohydrate analyses and biological activities of the proteins adsorbed in two hours to tooth surfaces in vivo. Caries Res. 8:113-122, 1974. ORSTAVIK, D.; and KRAUS, F. W.: The acquired pellicle: Immunofluorescent demonstration of specific proteins. J. Oral Path. 2:68-76, 1973. KRAUS, F. W.; ORSTAVIK, D.; HURST, D. C.; and COOK, C. H.: The acquired pellicle: variability and subject-dependence of specific proteins. J. Oral Pathol. 2:165173, 1973. BELCOURT, A.; FRANK, R. M.; and HOUVER, G.: Analyse des acids amines de la pellicule exogene acquise et des proteins de l'6mail superficiel chez l'homme. J. Biol. Buccale 2:161-179, 1974. LIE, T.: Pellicle formation on hydroxyapatite splints attached to the human dentition: Morphologic confirmation of the concept of adsorption. Arch. Oral Biol. 20:739-742, 1975.
25. TINANOFF, N.; GLICK, P. L.; and WEBER, D. F.: Ultrastructure of organic films on the enamel surface. Caries Res. 10:19-32, 1976. 26. LIE, T.: Scanning and transmission electron microscope study of pellicle morphogenesis. Scand. J. Dent. Res. 85:217-231, 1977. 27. MORENO, E. C.; and ZAHRADNIK, R. T.: Chemistry of enamel subsurface demineralization in vitro. J. Dent. Res. 53:226-235, 1974. 28. TURNER, E. P.: The integument of the enamel surface of the human tooth: II. The acquired enamel cuticle. Dent. Practitioner 8:373-382, 1958.
Enamel subsurface demineralization induced by Streptococcus mutans was significantly reduced by seven-day saliva pre-treatments conducive to the formation of enamel pellicles. A two-hour saliva pre-treatment was ineffective. Results suggest that the protection provided by long-term pellicles may relate to changes in ionic transport rates rather than cell attachment.
J Dent Res 57(11-12):1036-1042, Nov.-Dec. 1978
Introduction. The tooth surfaces in situ are covered by a proteinaceous film generally referred to as the acquired pellicle.l,2 Similar films can be formed on clean, pumiced enamel surfaces in relatively short times by exposure to oral secretions, 3,4 and a selective adsorption of salivary macromolecules has been advanced3-5 as the mechanism involved in the initial stages of the development of this integument. The acquired pellicle is the surface to which oral bacterial cells become attached to teeth,6 thus initiating the formation of dental plaque. Therefore, the possibility exists of the pellicle affecting the cariogenic process by reducing bacterial attachment or by introducing a marked degree of specificity for the adsorption of oral microorganisms. This possibility has been studied through the use of adsorption models involving saliva-treated synthetic apatite surfaces or dental enamel and cultures of isolated oral micro-organisms.7-10 The results of such investigations indicate that the saliva treatment enhances the attachment of
This investigation was supported, in part, by U.S.P.H.S. Grant DE-03187 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland. Received for publication January 9, 1978. Accepted for publication April 5, 1978. 1036
some organisms, whereas for others the opposite is true. There are inconsistencies among the published results which seem to reflect the specific experimental conditions of each model. Nevertheless, taken in toto, the data indicate that the acquired pellicle may alter the relative distribution of microorganisms initially colonizing the human tooth surfaces. The acquired pellicle may not only affect the nature of the dental plaque but also the transport of matter from and to the tooth enamel. Recently, it has been reportedl 1 that salivary pellicles, developed in vitro on a pressed disk of hydroxyapatite (HA), display ionic permselectivity (i.e., ionic transport is slower than that of neutral molecules). The degree of permselectivity was found to increase with longer exposure times of the disk to saliva, and this dependence on formation time seems to parallel observations on the level of protection provided by acquired pellicles (developed on extracted teeth) against enamel subsurface demineralization by acid buffers. 1 These studies emphasize the importance of considering more than bacterial cell attachment when one attempts to understand the role of the acquired pellicle in the natural caries process. The physical properties of this membrane may act to inhibit caries development; furthermore, the time of pellicle formation appears to be an important consideration in any in vitro research which attempts to mimic conditions existing at the saliva-tooth interface. The protection observed for salivatreated teeth against acid buffers may be lost under oral conditions where any salivary film on the teeth may be susceptible to degradation by bacteria in plaque.12-15 A model system has been developed16 to test whether an acquired pellicle can affect the rate of subsurface demineralization when
Vol. 57No. 11^12
SHORT- AND LONG-TERM SALIVAR YPELLICLES
the acid challenge results from the direct colonization (on a tooth surface) by known strains of cariogenic micro-organisms. Preliminary results indicate that reduced mineral loss can indeed occur when an acquired pellicle is present on the challenged tooth surface. This is significant since it demonstrates that the pellicle does possess the ability to alter a "caries-like" subsurface demineralization process under conditions approaching those expected in the mouth. It therefore becomes extremely important to determine if reduced plaque accumulation can explain this observation or whether a second mechanism (e.g., permselectivity) must be invoked to account for the protection. Such information could assist in the search for practical ways to control caries by favorably modifying the tooth-saliva interface. The present investigation was initiated to study the effect of acquired salivary pellicles (and their formation time) on (1) enamel subsurface demineralization, (2) total plaque accumulation, and (3) initial bacterial cell attachment.
Experimental. Enamel demineralization. The technique developed to produce subsurface demineralization by Streptococcus mutans has been described previously.16 In the present case a total of 12 clinically sound premolar teeth were used (extracted for orthodontic purposes). The teeth were scaled, pumiced and split in half along the buccal-lingual plane so that one half could serve as a control for the other half of the buccal surface tested. The split teeth were covered with wax except for an 8 mm2 window. Short-term pellicles were developed by exposing the test halves of six teeth to stimulated parotid saliva (5 ml/tooth) for two hours with continuous stirring. Long-term pellicles were built up by exposing the test halves from the remaining six teeth to the parotid saliva for seven days, changing the saliva daily. Sodium azide was added to the parotid saliva (0.02%) to control bacterial growth in these experiments. It has previously been reported11 ,17 that pellicles developed using parotid, submandibular and whole clarified saliva are all capable of providing equivalent protection against acid buffer demineralization.
1037
Each group of saliva-exposed or control teeth was suspended in separate culture tubes containing trypticase soy broth with 2% sucrose, 200 ,ug/ml of streptomycinresistant Streptoccus mutans strain Ingbritt (IB 1600) of human origin.18 The teeth were incubated anaerobically at 370C for 5 days. Transfers to fresh culture media were made daily. The final culture medium was periodically checked for contamination using Gram staining and dark field microscopy. At the end of 5 days an adherent, granular plaque was present on the teeth. The enamel windows were cleaned of microbial deposits, the wax was removed, and the teeth were embedded in an epoxy resin. Enamel sections of 400 gm thickness were cut perpendicular to the window surface with a diamond saw. These sections were ground to a parallel thickness of 100 ,um, mounted in a specially made frame against a high resolution photographic film, and exposed to an x-ray beam (nickel-filtered, Cu radiation, 40 Kv, 200 ma) for 20 minutes. The resultant microradiographs were scanned (through the demineralized region) with a high resolution microdensitometer using an effective slit of 5 ,um x 100 gm, and the optical densities were continuously recorded. In this fashion, quantitative microradiography was used to determine the effect of salivary pellicles on the subsurface demineralization induced by colonization of IB1 600. The extent of demineralization was assessed by measuring the following three parameters16: maximum depth at which mineral loss could be detected, maximum mineral loss in the body of the lesion, and thickness of the intact enamel surface layer.
Assessment of total plaque. The effect of acquired pellicles on accumulation of microbial deposits was tested using a fluorometric determination of total DNA in the plaque samples scaled from control and pellicle-coated tooth surfaces. Ethidium bromide was used as the fluorescent probe, following the procedure outlined by Donkersloot and coworkers.19 A total of 18 teeth were used in this experiment (6 each for the control, short-term and long-term pellicle groups). The waxing step had to be modified to
1038
ZAHRA DNIK E T AL.
allow for a window of defined surface area so that the total plaque could be normalized to a unit of tooth surface area. An 18 mm2 window was produced by waxing around a parafilm circle of that surface area. The parafilm was removed after waxing. The microbial deposits were carefully scaled from each window at the completion of the 5-day incubation period with IB1600. The plaque samples were washed over a filter and then incubated for one hour at 37 C with 0.3 N KOH to eliminate RNA interference with the fluorometric determination of DNA. The samples were neutralized and then mixed with a tris buffer containing the ethidium bromide. Standards containing calf thymus DNA were treated in the same fashion. The DNA per individual plaque sample was determined spectrofluorometrically; these values were then converted to number of cells per plaque sample. The conversion factor for this procedure was determined by measuring the DNA/ml in an aliquot from a 24-hour T soy broth culture of IB1600 and then obtaining a value for the cell concentration in this broth by direct microscopic count in a Petroff Hauser counting chamber. Since the surface area of the window (from which the microbial deposit was scaled) is known, the total plaque accumulation on each tooth can be reported as the total number of cells per unit surface area of enamel.
Initial cell attachment. Initial colonization of saliva-treated and control enamel surfaces was evaluated by the direct observation of enamel surfaces using incident light microscopy. In this procedure it was necessary to have enamel specimens with flat polished surfaces (for focusing purposes). Therefore, an approach similar to that reported by ¢rstavik et al. 8 was used. Cores were taken from the buccal surfaces of 18 premolar teeth using a hollow bore diamond drill (I.D. 3 mm). The cores were ground to produce a flat polished enamel face. A group of 3 cores was treated for 2 hours with parotid saliva (as described above); a second group received the 7-day saliva treatment, and a third group served as a control. The three groups of cores were incubated with IB 1600 for 5 hours. They were then removed, washed, dried, fixed in 95% ethanol, and stained with crystal violet
JDent Res November-December 1978
(1 min). Cell attachment was assessed by observing the stained micro-organisms on the enamel surfaces with an incident light microscope (x256). A duplicate run was performed using the remaining nine enamel cores.
Results. Results concerning the effects of shortterm and long-term salivary pellicles on demineralization of enamel by IB1600 are shown in Table 1. The mean and standard deviation (6 tooth samples in each group) are given for each of the three parameters used to characterize the extent of demineralization. It is apparent that a 7-day pellicle, developed in vitro on extracted teeth, significantly decreases the extent of mineral loss, which is in agreement with an initial study previously reported.16 However, no statistically significant difference in the demineralization pattern was found between the controls and the enamel having 2-hour pellicles. It is interesting that a thicker intact surface layer was observed for the 7-day pellicle treatments than for their corresponding controls. The possibility that the results shown in Table 1 could be related to a decreased number of bacteria colonizing on the salivatreated surface was examined by determining quantitatively the effect of the pellicle on total microbial accumulation. In Table 2, total plaque is expressed in terms of number of bacterial cells per unit surface area of enamel. Two runs are reported, with the mean and standard deviation given for each group of three teeth. Results of the two runs are comparable, i.e., the trends are the same although the absolute magnitude of cells found between runs, for a given test group, may vary. The in vitro bacterial accumulation on the two salivatreated groups is similar and not statistically different from that found on the controls. However, a trend toward greater plaque accumulation on the enamel surfaces with acquired pellicles is evident. Thus, the effect of pellicles on plaque development in our model system would tend to be opposite to that expected if the observed pellicle protection against demineralization (Table 1) were related to a reduction in the number of acid-producing S. mutans cells on salivatreated vs. control tooth surfaces.
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TABLE 1 ENAMEL DEMINERALIZATION AFTER 5-DAY INCUBATION WITH IB1600 2-hr Pellicle
Control Depth of lesion (um) Maximum mineral loss (%) Intact layer thickness (Am)
+ 6.9* 22.3 + 2.9 23.7 ± 3.8
118
Pellicle 116 + 11.7 23.0 ± 2.8 24.7 ± 4.3
7-day Pellicle Control Pellicle 115 + 13.8 23.3 ± 4.3 21.7 ± 5.9
106 + 4.3 15.7 ± 2.1 31.7 ± 3.1
*Standard deviation
Since the decreased demineralization in the presence of pellicle does not appear to be related to a decreased microbial deposit after 5 days of incubation with IB1600, we examined the possibility that the observed protection could have resulted from a delay in the initial microbial colonization, which was not apparent after 5 days of incubation. Typical photographs of initial cell attachment are included in Figures 1 and 2. In Figure 1 the left half shows the level of cell attachment found on control enamel surfaces after 5 hours' incubation with lB 1600. The right half shows the degree of initial colonization when a 2-hr pellicle is allowed to develop on the polished enamel core prior to the bacterial exposure. There was an enhancement in the rate of attachment when a short-term pellicle was present, and this feature was evident in all six cores in this group. Figure 2 contains a typical example comparing control and 7-day pellicle coated enamel after 5 hours of incubation with IB 1600. These results and those found for the other cores within the groups show a consistent and marked increase in the bacterial attachment rate to the enamel surfaces having a 7-day pellicle.
TABLE 2 TOTAL PLAQUE AFTER 5 DAY INCUBATION
WITH IB1600
Control 2 hr pellicle 7 day pellicle
Run I
Run II
109 cells/cm2
109 cells/cm2
2.01 ± 0.47* 3.04 ± 0.93 2.99 ± 0.91
0.69 ± 0.47 1.52 ± 0.76 2.01 ± 0.95
*Standard deviation
The initial kinetics of cell attachment observed in these last studies should reflect conditions occurring early in the demineralization experiments because the incubation steps were carried out in identical fashion. Since the IB 1600 cells were grown in sucrosecontaining medium, there was some clumping of the attached cells; no attempt was made to quantitatively evaluate the total numbers of adsorbed cells on the various enamel surfaces, since the trends were evident by visual inspection. Results for incubation times shorter than 5 hours displayed the same type of response; however, the total number of attached microorganisms was proportionally less.
Discussion. The results of the present investigation show that development of salivary pellicles on tooth enamel over a period of seven days protects this tissue against demineralization induced by cariogenic micro-organisms (see Table 1). This protection is not apparent for short-term pellicles (i.e., pellicles formed by exposing enamel to saliva for two hours prior to colonization of IB1600). Furthermore, the observed protection and its dependence upon the pellicle age seem to be unrelated to the amount of microbial accumulation on the tooth surface. Thus, the number of cells recovered per unit surface area of exposed enamel having short-term pellicles was not significantly different from the number of cells recovered from the enamel having 7-day pellicles (see Table 2). The mechanism by which the long-term pellicle protects enamel against demineralization is not fully understood at present, but it seems to be associated with some properties of the pellicle that depend on its age
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ZAHRADNIK ETA L.
J De}nt Res No ve i n b er-Dece in ber 19 78
Bcterial cell attachmiient to enamel sLurfaces after 5 hoLrs' incubation wxith an inoculun 1I 1. a--of S. mutans strain IB1600. Left, untreated sLrface; right, surface treated with parotid saliva for 2 lhours. or formation time. It is not known whether this time dependence reflects alterations in composition or in the ultrastructure of the integument, or both. There are several reports concerning the composition of short-term pellicles developed in situ and in vitro,3,4720-22 but only limited work has been conducted on possible compositional changes as the pellicle ages.3,23 Although the composition of short-term pellicles developed in situ and in vitro is quite similar, it appears to be significantly different from that of old natural pellicles of undetermined age.3 The few reports24'25 related to ultrastructural changes upon pellicle aging have not been done in a systematic fashion although a significant first effort in this direction has been made recently by Lie.26 This latter investigator followed the formation of pellicle on apatitic surfaces in the oral environment using scanning and transmission electron microscopy. His results, covering the first 48 hrs of pellicle formation, showed an increase in thickness (up to about 400 nm) accompanied by distinct
changes in the pellicle morphology; the older the pellicle, the more compact it appears, with a granular structure replacing the more open globular or fibrillar structures found in early pellicles. In some cases, Lie26 reported the formation of laminated structures in granular pellicles after 24 hrs. Thus, the scant information gathered so far indicates that most probably compositional and ultrastructural changes do occur as the pellicle ages, which undoubtedly change its physical properties. In the process of enamel deminerahization that results in mineral loss from the subsurface region, an important factor is the rate of transport of matter from the enamel surface to the demineralizing medium.27 It has been reported11,17 that this rate is greatly reduced by the presence of 7-day pellicles (i.e., the mineral loss is substantially reduced), whereas no protection was observed with short-term pellicles. Furthermore, it was observed11,17 that the ionic permselectivity of salivary pellicles adsorbed on apatite disks increased with increasing development times. Therefore, the perm-
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1 041
Fig. 2 -Effect of a 7-day pellicle on the initial rate of cell attachment. Both enamel surfaces were incubated for 5 hours with S. mutans strain IB1600. Left, untreated surface; right, surface having a 7-day pellicle.
selectivity appears to be related to the reported protective action of the salivary pellicle. The results of the present investigation are consistent, then, with the idea that the permselectivity of the pellicle is probably related to the observed protection against demineralization induced by lB1600 the enhancement in the as reported here pellicle permselectivity being associated with the types of compositional and ultrastructural changes that occur upon aging and that have been reported by other investigators.3,26 Furthermore, it has been observed that the properties of natural acquired pellicles also change with time, displaying an enhanced acid resistance after several days of development.28 There are two acknowledged mechanisms by which an acquired pellicle may interfere with the natural caries process: (1) by altering the specificity or magnitude of microbial colonization, and (2) by affecting the transport rates related to enamel mineral loss. The present results show that the presence of a salivary pellicle increases both
the initial rate of cell attaclhment to the tooth surface (Figures 1 and 2) and the total microbial accumulation (Table 2). These results, in fact, made it possible to ascertain unequivocally the protective action of the pellicle against demineralization. However, no strain different from IB1600 (grown in a sucrose-containing medium) was used in this investigation. The results, therefore, do not eliminate the possibility that both mechanisms may be operative under in vivo conditions. Several reports7 -10 indicate that the attachment of some strains of Streptococci to apatitic surfaces is enhanced by a salivary pre-treatment of the adsorbent surfaces, while it is reduced for other strains. It appears then that, in the oral environment, the selective attachment induced by the acquired pellicle may determine the kind of dental plaque developed and, thereby, its cariogenic potential. This interesting possibility could be studied with the model system used in this investigation in conjunction with pure and mixed cultures of oral micro-organisms.
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J Dent Res November-December 1 978
ZAHRADNIK ETAL. REFERENCES
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