Journal of Dentistry,
7, No. 2, 1979,
PP.
169-l
73. Printed
in Great Britain
Variations in the initial formation of pellicle and plaque on enamel in vivo D. Adams, H. D. K. Stipho and W. M. Murphy Department of Oral Biology, Dental School, Cardiff
ABSTRACT Four subjects wore acrylic baseplates with small pieces of enamel from unerupted teeth for periods of 24 and 48 hours. The rate of colonization of the pellicle with bacteria varied among the subjects in 2 of whom bacteria were not seen on the surface at 24 hours. Etching the enamel did not enhance the rate of colonization. Chlorhexidine treatment did not prevent bacterial adherence after 2 days, but etched enamel retained the antibacterial effect of chlorhexidine longer than normal enamel.
INTRODUCTION The initial stages of pellicle and plaque formation are of great interest to workers in the field of preventive dentistry, and in relation to surface phenomena in the mouth. Only a few reports deal with the initial appearance of pellicle (Eastcott and &Hard, 1973; Saxton, 1973). Differences among individuals in the rate of accumulation of pellicle and plaque have also only been mentioned rarely in the literature (Connor et al., 1976). The present report highlights the variation that was found in the growth of pellicle and plaque during a study of the effect of various chemical treatments on the accumulation of these coatings on enamel in viva. The chemical treatments included etching the enamel surface and applying chlorhexidine separately and together.
MATERIALS
AND METHODS
Small pieces of surface enamel were obtained by sectioning extracted, unerupted but sound human teeth. These pieces, after cleaning to remove remnants of developmental coatings, were worn in an acrylic baseplate for 24 or 48 hours. Triplicate samples were used in each baseplate and for every test period. Four subjects wore the plates and refrained from using oral hygiene procedures during this time. The surface of the enamel faced towards, but was not in contact with, the oral mucosa (Fig. I). The enamel pieces were either untreated, acidetched for 2 minutes with 50 per cent phosphoric acid, soaked in a 0.2 per cent solution of chlorhexidine gluconate or acidetched and treated with chlorhexidine solution. After 24 or 48 hours in the mouth the enamel pieces were removed from the appliance, rinsed and prepared for the scanning electron microscope (ISI Mini-SEM) by freeze-drying and goldcoating. Control specimens of untreated and treated enamel not worn in the mouth served as a baseline for the observations on the test pieces. In order to render the structureless pellicle visible in the SEM a scratch mark was made on the surface using a very fine pliable needle.
RESULTS The untreated control enamel did not have any residual developmental coverings when examined in the SEM and no evidence of the scratch mark was seen (Fig. 2). Bacteria were
170
Fig. 7. Baseplate with place with sticky wax. wards the mucosa.
Fig. 3. Example
Journal of Dentistry,
enamel segments held in The enamel surface is to-
of the scratch mark on enamel worn for 24 hours in the mouth. No bacteria were found in this specimen. SEM. (X52.1
Fig. 2. Control of developmental
Vol. ~/NO.
2
untreated enamel. No remnants coatings are visible. SEM. (X52.)
Fig. 4. Bacterial colonization subject after 24 hours. SEM.
on enamel (X 1800.)
from
one
not found on these control pieces but the prism markings and other features described by Boyde (1972) were well shown. There was some variation between the teeth in the number of perikymata and the pits described by Boyde, and in one case the tooth had an irregular granular appearance. After 24 hours in the mouth the enamel surface was covered by a thin film which was more obvious where the scratch by the fine needle traversed the surface (Fig. 3). This left a shallow depression with a heaping up of material on either side. No differences in the thickness of the pellicle on the enamel surfaces from the 4 subjects could be detected in this way. Two of the subjects did not appear to have any bacteria on the surface of the specimens, but from one of the other subjects the specimens were partially coated with bacteria. These bacteria formed in places a monolayer of coccal forms over the surface with spaces in between, which revealed the pellicle as a thin film obscuring the enamel prism markings (Fig. 4). In the remaining subject the bacteria formed chains of cocci and individual organisms were scattered over the surface, but on the whole the bacteria were more difficult to find than on the more heavily colonized specimens. There was little variation between the triplicate specimens from any one individual.
Adams et al.: Pellicle and plaque formation
in vivo
Fig. 5. Enamel from the same subject as in Fig. 4 after 48 hours in the mouth. This was the heaviest accumulation found in all the subjects. SEM. (X 1300.)
Fig. 6. Etched enamel after 24 hours. A patchy film covers the irregular surface. SEM. (X 1300.)
Fig. 7. Chlorhexidine-treated enamel after 48 hours. Rod forms make up a larger proportion of the bacteria than is the case with untreated enamel. SEM. (X 1500.)
After 48 hours in the mouth bacteria could be found on the enamel pieces from all the subjects, but the greatest accumulation was on the specimens from the subject with the heaviest colonization at 24 hours (Fig. 5). These specimens also showed a greater variety of forms, whereas from the other 3 subjects coccal forms predominated.
Acid-etched enamel The appearance of etched enamel in the SEM varied. In some places there was a loss of the prism boundaries whilst in others there was a loss of the core material In all cases a rough and irregular surface resulted from the acid treatment. After 24 hours in the mouth specimens had a patchy film which made the irregularities on the surface less distinct (Fig. 6). Bacteria could not be found on any of the specimens, though squamous cells were occasionally encountered. After 48 hours the enamel had a crusty layer on the surface, but this appearance varied from subject to subject. Clumps of bacteria were seen on the specimens from all the subjects, but the heaviest accumulation was found on those from the subject who had most bacteria on the untreated enamel.
172
Journal of Dentistry,
Vol. ~/NO.
2
Chlorhexidine-treated enamel These specimens did not show any bacterial colonization after 24 hours in the mouth but all had become covered with a thin pellicle similar to that seen on untreated enamel. After 48 hours bacterial clumps were found on the surface of all the specimens but there was a variation in the numbers of bacteria seen. Rod forms were more in evidence than on untreated enamel at the same stage (Fig. 7). The trend in quantity of bacteria was consistent with that found in the other tests.
Etched and chlorhexidine-treated enamel Specimens which were etched and treated with chlorhexidine before being worn in the mouth showed no bacterial contamination of the surface pellicle either at 24 or 48 hours. This was consistent in all the enamel pieces. Pellicle was found and in places epithelial cells adhered to it.
DISCUSSION The technique of wearing specimens on an appliance in the mouth has been used by several authors (Connor et al., 1976; Tinanoff et al., 1976; Ewers et al., 1977). The main advantage is that the pellicle and plaque can be studied in situ in a reproducible situation on the enamel surface. Drawing a fine probe across the surface was used by Saxton (1976) in an attempt to measure the thickness of the pellicle. The variation in the height of the side walls of the trough made this impossible and any estimate of the pellicle thickness is largely subjective. The work reported here differs from that of Tinanoff et al. (1976) in that all their specimens came from one individual and in the present study the enamel was treated chemically to alter its characteristics. In addition unerupted teeth have been used in an attempt to standardize the enamel surface by excluding the effects of previous trauma, oral hygiene measures and wear. Connor et al. (1976) and Ewers et al. (1977) used gold discs recessed into full upper dentures for 4 patients. It is interesting to note that they found considerable variation in the rate of plaque formation not only among patients but also from side to side in the same patient. Even in the small sample of 4 patients studied here there was wide individual variation, but the triplicate specimens showed no appreciable differences from each other. It is unlikely that bacteria were washed off some of the specimens and not others as all the specimens were treated alike. Acid-etching produces a comparatively rough surface which is considered to enhance the rate of plaque formation (Miner, 1973). We noted, however, a reduction in rate since none of the subjects accumulated bacteria until 4g hours when wearing the etched specimens. It is possible that the irregularities in the specimens masked the few bacteria that may have been present at 24 hours, but identification was not unduly difficult at 48 hours. The latter specimens did not show any greater accumulation when compared with untreated enamel. Chlorhexidine gluconate adsorbed to hydroxyapatite is released when the concentration in the environment is low (Rolla et al., 1970). It would appear that all the chlorhexidine had been leached from the enamel by 48 hours since plaque had begun to form at this stage. The chemical had not prevented pellicle formation, though the slight change in character of the bacteria towards rod forms suggests a residual influence of the chlorhexidine on the forming plaque. Chlorhexidine treatment of the etched enamel prevented bacterial accumulation even up to the 4%hour period. This probably reflects the increased area for adsorption created by
Adams
et al.:
Pellicle and plaque formation
in vivo
173
the etching process and hence an enhanced uptake of chlorhexidine. This was a similar effect to the incidental finding of Turesky et al. (1977) in a trial of other chemicals with chlorhexidine. Although the sample size of 4 subjects is small the amount of variation in the rate of plaque formation is surprising. Very little is known of the factors involved, though age of the individual, sex, oral hygiene experience and immunological activity of saliva probably are implicated. Whether the composition of the pellicle has an effect on the subsequent bacterial attraction is a subject which has attracted considerable attention from this Research Group, and the individual variation in its composition in relation to its surface properties would seem to merit further investigation. REFERENCES
Boyde A. (1972) Influence
of normal and abnormal
enamel structure
on cavity margins. Br.
Dent. J. 133,421-427.
C. M. and Taylor R. L. (1976) A study of in viuo plaque formation.
Connor J. N., Schoenfeld J. Dent Res. SS,481-488.
Eastcott A. D. and Stallard R. E. (1973) Sequential changes in developing human dental plaque as visualized by scanning electron microscopy. J. Periodontol. 44, 2 18-224. Ewers G. J., Taylor R. L. and Schoenfeld C. M. (1977) Scanning electron microscopy of human dental pellicle and initial plaque formation in viva. Amt. Dent J. 22, 462-467. Miner J. R. (1973) The nature of the denture base: a key factor in denture sore mouth (an SEM study). J. Prosthet. Dent. 29,250-255. Rolla G., Lee H. and Schiott C. R. (1970) The affinity of chlorhexidine for hydroxyapatite and salivary mucins. J. Periodont. Res. 5, 90-95. Saxton C. A. (1973) Scanning electron microscope study of the formation of dental plaque. Caries Res. 7, 102-l
19.
Saxton C. A. (1976) The effects of dentifrices on the appearance of the tooth surface observed by scanning electron microscopy. J. Periodont. Res. 11, 74-85. Tinanoff N., Gross A. and Brady J. M. (1976) The development of plaque on enamel. J. Periodont. Res. 11, 197-209. Turesky S., Warner V., Lin P. S. et al. (1977) Prolongation of antibacterial activity of chlorhexidine adsorbed to teeth. Effect of sulfates. J. Periodontal. 48, 646-649.
7, No. 2, 1979,
PP.
169-l
73. Printed
in Great Britain
Variations in the initial formation of pellicle and plaque on enamel in vivo D. Adams, H. D. K. Stipho and W. M. Murphy Department of Oral Biology, Dental School, Cardiff
ABSTRACT Four subjects wore acrylic baseplates with small pieces of enamel from unerupted teeth for periods of 24 and 48 hours. The rate of colonization of the pellicle with bacteria varied among the subjects in 2 of whom bacteria were not seen on the surface at 24 hours. Etching the enamel did not enhance the rate of colonization. Chlorhexidine treatment did not prevent bacterial adherence after 2 days, but etched enamel retained the antibacterial effect of chlorhexidine longer than normal enamel.
INTRODUCTION The initial stages of pellicle and plaque formation are of great interest to workers in the field of preventive dentistry, and in relation to surface phenomena in the mouth. Only a few reports deal with the initial appearance of pellicle (Eastcott and &Hard, 1973; Saxton, 1973). Differences among individuals in the rate of accumulation of pellicle and plaque have also only been mentioned rarely in the literature (Connor et al., 1976). The present report highlights the variation that was found in the growth of pellicle and plaque during a study of the effect of various chemical treatments on the accumulation of these coatings on enamel in viva. The chemical treatments included etching the enamel surface and applying chlorhexidine separately and together.
MATERIALS
AND METHODS
Small pieces of surface enamel were obtained by sectioning extracted, unerupted but sound human teeth. These pieces, after cleaning to remove remnants of developmental coatings, were worn in an acrylic baseplate for 24 or 48 hours. Triplicate samples were used in each baseplate and for every test period. Four subjects wore the plates and refrained from using oral hygiene procedures during this time. The surface of the enamel faced towards, but was not in contact with, the oral mucosa (Fig. I). The enamel pieces were either untreated, acidetched for 2 minutes with 50 per cent phosphoric acid, soaked in a 0.2 per cent solution of chlorhexidine gluconate or acidetched and treated with chlorhexidine solution. After 24 or 48 hours in the mouth the enamel pieces were removed from the appliance, rinsed and prepared for the scanning electron microscope (ISI Mini-SEM) by freeze-drying and goldcoating. Control specimens of untreated and treated enamel not worn in the mouth served as a baseline for the observations on the test pieces. In order to render the structureless pellicle visible in the SEM a scratch mark was made on the surface using a very fine pliable needle.
RESULTS The untreated control enamel did not have any residual developmental coverings when examined in the SEM and no evidence of the scratch mark was seen (Fig. 2). Bacteria were
170
Fig. 7. Baseplate with place with sticky wax. wards the mucosa.
Fig. 3. Example
Journal of Dentistry,
enamel segments held in The enamel surface is to-
of the scratch mark on enamel worn for 24 hours in the mouth. No bacteria were found in this specimen. SEM. (X52.1
Fig. 2. Control of developmental
Vol. ~/NO.
2
untreated enamel. No remnants coatings are visible. SEM. (X52.)
Fig. 4. Bacterial colonization subject after 24 hours. SEM.
on enamel (X 1800.)
from
one
not found on these control pieces but the prism markings and other features described by Boyde (1972) were well shown. There was some variation between the teeth in the number of perikymata and the pits described by Boyde, and in one case the tooth had an irregular granular appearance. After 24 hours in the mouth the enamel surface was covered by a thin film which was more obvious where the scratch by the fine needle traversed the surface (Fig. 3). This left a shallow depression with a heaping up of material on either side. No differences in the thickness of the pellicle on the enamel surfaces from the 4 subjects could be detected in this way. Two of the subjects did not appear to have any bacteria on the surface of the specimens, but from one of the other subjects the specimens were partially coated with bacteria. These bacteria formed in places a monolayer of coccal forms over the surface with spaces in between, which revealed the pellicle as a thin film obscuring the enamel prism markings (Fig. 4). In the remaining subject the bacteria formed chains of cocci and individual organisms were scattered over the surface, but on the whole the bacteria were more difficult to find than on the more heavily colonized specimens. There was little variation between the triplicate specimens from any one individual.
Adams et al.: Pellicle and plaque formation
in vivo
Fig. 5. Enamel from the same subject as in Fig. 4 after 48 hours in the mouth. This was the heaviest accumulation found in all the subjects. SEM. (X 1300.)
Fig. 6. Etched enamel after 24 hours. A patchy film covers the irregular surface. SEM. (X 1300.)
Fig. 7. Chlorhexidine-treated enamel after 48 hours. Rod forms make up a larger proportion of the bacteria than is the case with untreated enamel. SEM. (X 1500.)
After 48 hours in the mouth bacteria could be found on the enamel pieces from all the subjects, but the greatest accumulation was on the specimens from the subject with the heaviest colonization at 24 hours (Fig. 5). These specimens also showed a greater variety of forms, whereas from the other 3 subjects coccal forms predominated.
Acid-etched enamel The appearance of etched enamel in the SEM varied. In some places there was a loss of the prism boundaries whilst in others there was a loss of the core material In all cases a rough and irregular surface resulted from the acid treatment. After 24 hours in the mouth specimens had a patchy film which made the irregularities on the surface less distinct (Fig. 6). Bacteria could not be found on any of the specimens, though squamous cells were occasionally encountered. After 48 hours the enamel had a crusty layer on the surface, but this appearance varied from subject to subject. Clumps of bacteria were seen on the specimens from all the subjects, but the heaviest accumulation was found on those from the subject who had most bacteria on the untreated enamel.
172
Journal of Dentistry,
Vol. ~/NO.
2
Chlorhexidine-treated enamel These specimens did not show any bacterial colonization after 24 hours in the mouth but all had become covered with a thin pellicle similar to that seen on untreated enamel. After 48 hours bacterial clumps were found on the surface of all the specimens but there was a variation in the numbers of bacteria seen. Rod forms were more in evidence than on untreated enamel at the same stage (Fig. 7). The trend in quantity of bacteria was consistent with that found in the other tests.
Etched and chlorhexidine-treated enamel Specimens which were etched and treated with chlorhexidine before being worn in the mouth showed no bacterial contamination of the surface pellicle either at 24 or 48 hours. This was consistent in all the enamel pieces. Pellicle was found and in places epithelial cells adhered to it.
DISCUSSION The technique of wearing specimens on an appliance in the mouth has been used by several authors (Connor et al., 1976; Tinanoff et al., 1976; Ewers et al., 1977). The main advantage is that the pellicle and plaque can be studied in situ in a reproducible situation on the enamel surface. Drawing a fine probe across the surface was used by Saxton (1976) in an attempt to measure the thickness of the pellicle. The variation in the height of the side walls of the trough made this impossible and any estimate of the pellicle thickness is largely subjective. The work reported here differs from that of Tinanoff et al. (1976) in that all their specimens came from one individual and in the present study the enamel was treated chemically to alter its characteristics. In addition unerupted teeth have been used in an attempt to standardize the enamel surface by excluding the effects of previous trauma, oral hygiene measures and wear. Connor et al. (1976) and Ewers et al. (1977) used gold discs recessed into full upper dentures for 4 patients. It is interesting to note that they found considerable variation in the rate of plaque formation not only among patients but also from side to side in the same patient. Even in the small sample of 4 patients studied here there was wide individual variation, but the triplicate specimens showed no appreciable differences from each other. It is unlikely that bacteria were washed off some of the specimens and not others as all the specimens were treated alike. Acid-etching produces a comparatively rough surface which is considered to enhance the rate of plaque formation (Miner, 1973). We noted, however, a reduction in rate since none of the subjects accumulated bacteria until 4g hours when wearing the etched specimens. It is possible that the irregularities in the specimens masked the few bacteria that may have been present at 24 hours, but identification was not unduly difficult at 48 hours. The latter specimens did not show any greater accumulation when compared with untreated enamel. Chlorhexidine gluconate adsorbed to hydroxyapatite is released when the concentration in the environment is low (Rolla et al., 1970). It would appear that all the chlorhexidine had been leached from the enamel by 48 hours since plaque had begun to form at this stage. The chemical had not prevented pellicle formation, though the slight change in character of the bacteria towards rod forms suggests a residual influence of the chlorhexidine on the forming plaque. Chlorhexidine treatment of the etched enamel prevented bacterial accumulation even up to the 4%hour period. This probably reflects the increased area for adsorption created by
Adams
et al.:
Pellicle and plaque formation
in vivo
173
the etching process and hence an enhanced uptake of chlorhexidine. This was a similar effect to the incidental finding of Turesky et al. (1977) in a trial of other chemicals with chlorhexidine. Although the sample size of 4 subjects is small the amount of variation in the rate of plaque formation is surprising. Very little is known of the factors involved, though age of the individual, sex, oral hygiene experience and immunological activity of saliva probably are implicated. Whether the composition of the pellicle has an effect on the subsequent bacterial attraction is a subject which has attracted considerable attention from this Research Group, and the individual variation in its composition in relation to its surface properties would seem to merit further investigation. REFERENCES
Boyde A. (1972) Influence
of normal and abnormal
enamel structure
on cavity margins. Br.
Dent. J. 133,421-427.
C. M. and Taylor R. L. (1976) A study of in viuo plaque formation.
Connor J. N., Schoenfeld J. Dent Res. SS,481-488.
Eastcott A. D. and Stallard R. E. (1973) Sequential changes in developing human dental plaque as visualized by scanning electron microscopy. J. Periodontol. 44, 2 18-224. Ewers G. J., Taylor R. L. and Schoenfeld C. M. (1977) Scanning electron microscopy of human dental pellicle and initial plaque formation in viva. Amt. Dent J. 22, 462-467. Miner J. R. (1973) The nature of the denture base: a key factor in denture sore mouth (an SEM study). J. Prosthet. Dent. 29,250-255. Rolla G., Lee H. and Schiott C. R. (1970) The affinity of chlorhexidine for hydroxyapatite and salivary mucins. J. Periodont. Res. 5, 90-95. Saxton C. A. (1973) Scanning electron microscope study of the formation of dental plaque. Caries Res. 7, 102-l
19.
Saxton C. A. (1976) The effects of dentifrices on the appearance of the tooth surface observed by scanning electron microscopy. J. Periodont. Res. 11, 74-85. Tinanoff N., Gross A. and Brady J. M. (1976) The development of plaque on enamel. J. Periodont. Res. 11, 197-209. Turesky S., Warner V., Lin P. S. et al. (1977) Prolongation of antibacterial activity of chlorhexidine adsorbed to teeth. Effect of sulfates. J. Periodontal. 48, 646-649.
