DEVELOPMENT OF DENTAL PLAQUE ON EPOXY RESIN CROWNS IN M A N

cles in many but not all cases and the predominance of coccal forms over filamentous forms. Theilade and Theilade conducted cultural as well as electron micro­ scopic studies of 1 to 3-day old plaque collected on celluloid strips. Although cultivable organisms included predominantly gram-positive cocci (64-90%) and gram-negative cocci (3-16%), some gram-positive and gram-negative rods were also recovered. Fusobacteria and filaments were noted on smears in low numbers. N o spirillar forms were observed. Schroeder and DeBoever described the ultrastructure of 7 to 14 day old dental plaque collected on enamel surfaces. They noted the presence of acquired pellicles on dental cuticle remnants at the plaque-tooth interface. The bulk of the plaque was described as consisting of "colony-like clusters of mor­ phologically different, but taxonomically unidentified microorganisms'' which were rather haphazardly ar­ ranged. Cocci tended to be common near the tooth interface. Filamentous forms were preferentially oriented perpendicular to the tooth surface. Differences noted in the predominant morphology of plaque organisms (fila­ mentous versus coccal forms) were attributed to possible variations due to the anatomic location of the plaque (approximal versus facial or lingual). Despite the great variation noted in cell morphology, the authors pointed out the lack of available information to permit relating these cells to taxonomically defined organisms. N o systematic electron microscopic study is yet available on the gradual building up of plaque on a clean surface into a fully developed mature plaque. 6

A Light and Electron Microscopic Study

7

by MAX

A . LISTGARTEN, D.D.S.*

HARRY E. M A Y O , ROLLANDE

D.D.s.f

TREMBLAY‡

BACTERIAL PLAQUE HAS been studied with various methods. Björn and Carlsson followed development of bacterial plaque with a stereo microscope after initial cleansing of the surface of anterior teeth. Bacterial accumulations were made visible by means of a fuchsin containing disclosing solution. Weakly stained material was visible after 24 hours over most of the crown surface. Stronger staining was detected along cracks. Between 1 and 4 days, colony-like formations were noted which appeared to coalesce by 7 days. Some histological studies of plaque development have utilized celluloid strips as a supporting surface for the collection of bacterial p l a q u e . These studies have indicated that initially a cuticle, free of bacteria, is formed. It became colonized within 3 to 5 days by a predominantly coccal flora of gram-positive and gram-negative organisms as well as a few rods and filaments. Filamentous forms increased in number after the 7th day and by 12 days formed the main bulk of plaque bacteria. Using a smear technique, Löe et al. showed that the proportion of the various bacterial forms varied with plaque age. The early coccal flora was found to be replaced with rods and filamentous forms by 2 to 4 days, with vibrios and spirochetes appearing after 6-10 days. However, the technique used did not permit a study of these organisms in their undisturbed relationship to one another. Electron microscopic studies have been carried out on 1 to 7 day old plaque by Frank and Houver. They obtained surface enamel biopsies with plaque from different volunteers following an initial prophylaxis. They noted the presence of acquired pelli­ 1

Most plaque studies have dealt with supragingival plaque formation. Because of the anatomic relation of plaque to the periodontal tissues, it is likely that subgin­ gival plaque is of even greater importance than supragin­ gival plaque in the pathogenesis of advanced periodontal disease (periodontitis, periodontosis). Furthermore, the bacterial population of the outer layers of subgingival plaque may be in turn more important in the pathogen­ esis of periodontal disease than the deeper plaque population since it is in intimate contact with the host tissues. In this report, a method will be presented for the construction of epoxy resin crowns which were worn by human volunteers in order to study the sequential development of bacterial plaque on surfaces of reproduci­ ble shape and location. This technique provides a simple and convenient method to study the ultrastructure of dental plaque with minimal disturbance of its internal structure. The morphology of plaque collected with this technique will be described at varying time intervals of up to two months and the validity of the model discussed. The findings will be related to the probable growth pattern of human dental plaque. The significance of certain structural features of subgingival plaque will be discussed in relation to a possible preferential participa­ tion of certain microorganisms in the pathogenesis of periodontal disease.

2,3

4

5

Center for Oral Health Research and School of Dental Medicine, University of Pennsylvania, 4001 Spruce Street, Philadelphia, Pennsyl­ vania 19174. This project was supported by Grant No. DE-02623 from the United States Public Health Service to the Center for Oral Health Research. * Professor of Periodontics. †Post-Graduate student of Periodontal Prosthesis. t Electron microscopy and histology technician.

10

Dental Plaque

Volume 46 Number l

MATERIALS AND M E T H O D S

Construction of Epoxy Resin Crowns: Patients were selected on the basis of their need for a full crown restoration. The tooth was prepared and the full crown restoration fabricated in the usual manner. A n orangewood stick, approximately ½ to l inch long was attached to the occlusal surface by means of sticky wax (Fig. I). A n accurate silicone impression of the finished crown was made. We found " H i g h - P u t e § a silicone based impression material, suitable for this purpose. The impression material is available as a kit consisting of a heavy bodied silicone base and a lighter bodied wash impression material. A small cup was filled with the heavy bodied material and a depression created in the center with finger pressure to a size slightly larger than the diameter of the finished crown. A n adequate amount of silicone wash was then used to fill the depression in the heavy bodied impression material and to cover all the surfaces of the finished crown. While holding the silicone covered crown by the orangewood stick, it was gently pressed into the wash material occupying the central depression. After the impression material set, the sili­ cone was cut away, if necessary, to expose the occlusal surface only of the crown. The crown was then removed from the impression material by means of the orangewood stick. This left an accurate silicone impression of the finished crown (Fig. 2). The epoxy resin used to fabricate the crown was of the same type as that which is generally used in electron microscope laboratories. The resin was mixed in the usual manner and preheated to approximately 60°C. The silicone impression tray was preheated to the same temperature. Preheating of the impression and the resin facilitated the flow of the resin into the impression. The impression was filled with resin by means of a disposable syringe fitted with a blunted, 20 gauge needle. The needle was introduced into the impression to the level of the cervical margin and the impression filled by forcing the resin to flow from this location into the more occlusal portion of the impression (Fig. 3). The resin filling the impression was then heated in an oven at 65°C for 1-2 hours. A t that time the im­ pression was examined carefully under a dissecting microscope. A n y air bubbles appearing in the resin were brought to the surface by means of a shaved tooth pick. Polymerization was completed in the oven at 65°C for 3 days. The polymerized crown was withdrawn from the impression material. With the exception of the occlusal surface, which is adjusted to the existing occlusion at the time of insertion, the epoxy resin was a faithful replica of the previously fabricated gold crown (Fig. 4). The same impression can be used to construct several crowns. If time is a factor, several impressions can be made of the gold crown so that several epoxy resin crowns can be processed simultaneously. ,,

8

§ Accurate Set, Inc., Patterson, N.J. 07501.

11

Collection of Human Dental Plaque: This report is based on data collected from a group of 6 subjects selected among students and staff of the School of Dental Medicine at the University of Pennsylvania. The partici­ pants were chosen on the basis of their need for a full crown restoration on a posterior tooth and their willing­ ness to participate in the project to its completion. In compensation, they were fitted with the needed restora­ tion at no charge. Initially, the experiment was designed to collect plaque samples at 1 and 3 days, 1 and 3 weeks and 2 months following insertion of the epoxy resin crowns. Only four of the six subjects completed the original sampling protocol. In the others, some crowns had to be taken out prior to their scheduled removal date because of me­ chanical failure. If the timing was appropriate, such crowns were used to provide duplicate samples for a particular time interval. In other cases, the crowns had to be discarded. The clinical appearance of epoxy crowns in a heavy plaque former are shown at intervals of 1 and 3 days, 3 weeks and 2 months (Figs. 5-8). At least 5 crowns were available from each subject, each of whom contributed at least 1 plaque sample for each of at least 4 different time intervals. For each time interval, at least 5 crowns were studied. The order in which the short term samples (1-7 days) were obtained was purposefully varied so that the bacterial composition of these samples would not be biased by the sequence in which they were taken. The sequence in which the samples were obtained in each subject and the buccal G I and PI I scores at the time of sampling are recorded in Table 1. For example, in the case of subject J E , the 1-week samples included both the initial as well as the 5th or final sample of this series. The 2nd, 3rd and 4th samples corresponded to the 3 week, 1 day and 3 day crowns respectively. None of the subjects in this study had initial G I or PI I scores greater than 1 at the time of their selection. Following preparations of the teeth for a fully crown restoration, a temporary acrylic crown was constructed which was worn by the patient for 1-3 weeks prior to beginning the experiment. A t the beginning of the experiment, none of the PI I or G I scores exceeded a value of 1. At the first seating, the temporary crown was removed and the temporary cement cleaned off the prepared tooth surface. After seating the epoxy resin crown, the occlu­ sion was adjusted and the crown cemented to the tooth with Aquachem (a calcium hydroxide and water mixture) or Tenacin (a zinc oxyphosphate cement). The subjects were instructed to avoid chewing on that side and to avoid as much as possible cleaning the tooth during their usual oral hygiene practice. N o attempt was made in this study to alter the oral hygiene habits of the participants or to obtain dento-gingival units entirely free of plaque or inflammation. Processing of Plaque Specimens: A t appropriate inter9, 1 0

12

Listgarten, Mayo,

Tremblay

J. PeriodontoL January, 1975

Dental Plaque

Volume 46 Number I

13

TABLE 1

Sampling Sequence with Buccal GI and PI I Scores at Time of Sampling

vals, the crowns were removed without disturbing the marginal half of the crown surface and immediately transferred to an ice cold mixture of 5% glutaraldehyde and 4% paraformaldehyde fixative buffered to p H 7.2 with 0.1 M sodium cacodylate buffer. After 2-3 hours prefixation, the fixative was replaced with several changes of 0.185M sodium cacodylate buffer. The crown was then post-fixed in 2% s-collidine buffered osmic acid (pH 7.2, 350 mOsm) for 2 hours at 4 ° C , stained with 0.5% uranyl acetate in 50% ethanol, dehydrated and completely embedded in epon. Special care was taken during the transfer of solutions to not disturb the delicate microbial network on the crown surface. Solutions were added and removed by slow pipetting. Unnecessary contact with the crown was carefully avoided. Following polymerization of the epon at 65°C for 3 days, the embedded marginal half of the crown was cut out with a rotary saw and the periphery of the crown subdivided into vertical slices, approximately 1 mm thick, taken 11

from the buccal, lingual, mesial and distal surfaces. These slices were reembedded in epon prior to sectioning at 1 µm. These sections were collected on glass slides and stained by the following technique: Stock solutions were prepared as follows and left to age for at least 2 weeks: Stock solution A Stock solution B Stock solution C

1% methylene blue in 1% sodium borate 1% azure II in water 1% toluidine blue 0 in water

8

Solutions were filtered prior to use and mixed in the ratio of 1 part A : 3 parts B: 6 parts C . One micron thick epon sections mounted on glass slides were flooded with the stain on a hot plate for 3 minutes. The temperature of the hot plate was adjusted so that the stain just steamed when heated. After 3 minutes the stain was rinsed off with water. The slides were dried and coverslipped in the usual manner with balsam or epon. Selected blocks were

Plate I Note: Unless otherwise indicated, the bar in the lower right corner of the electron micrographs corresponds to a measurement of I µm. F I G U R E 1. Wooden stick attached to occlusal surface of final crown with sticky wax. F I G U R E 2. Silicone impression of crown with occlusal surface exposed. F I G U R E 3. Epoxy resin is injected with a blunted needle into cervical portion of the impression. F I G U R E 4. Polymerized crown prior to occlusal trimming fits exactly onto amalgam die of prepared molar. F I G U R E 5. Epoxy crown in situ after 24 hours. F I G U R E 6. Epoxy crown in situ after 3 days. F I G U R E 7. Epoxy crown in situ after 3 weeks. F I G U R E 8. Epoxy crown in situ after 2 months. Note the marked gingivitis and the accumulated plaque along the cervical part of the crown.

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Tremblay

J. Periodontol. January, 1975

Dental Plaque

Volume 46 Number 1

trimmed and sectioned at 0.075 fim for electron micro­ scopic study. Sections were collected on bare, 75 x 300 mesh grids, routinely stained with uranyl acetate and lead citrate and examined in a Philips E M 300 electron microscope.

RESULTS

One day Plaque samples obtained after 24 hours consisted primarily of coccal forms of varying size and in various arrangements. The quantity of plaque varied from practi­ cally no bacterial deposits to a well organized bacterial mat up to 80 thick. In the subject with minimal plaque deposition ( P R ) , the epon surfaces appeared either bare or covered with adherent squamous epithelial cells. Isolated coccal forms could be occasionally de­ tected at widely separated sites on the epoxy resin surface. In the most highly structured plaque sample (subject A G ) , the microbial population was organized in columnar microcolonies oriented with their long axis perpendicular to the crown surface (Fig. 9). Some sections of the plaque from this subject consisted of closely packed coccal forms without any detectable colonial organization. Other stages intermediate between these two extremes included the patchy distribution of rather uniform coccal layers, 2-10 cells thick, and bacterial clumping at discrete sites along the surface of the crown. Electron microscopy of 1 day plaque revealed the presence of a large variety of morphologic types of coccal cells and occasional, isolated, branching filaments (Fig. 11). In addition to differences in size, the cells demon­ strated cell walls with morphologic features commonly associated with both gram-positive as well as gram-nega­ tive cell walls. In some cells, delicate projections radiated from the cell wall (Fig. 10). Occasionally, a well defined intercellular matrix was evident in the portion of the plaque adjacent to the crown (Fig. 12). In some speci­ mens, a thin, dense pellicle, less than 1 µm thick, was interposed between the resin surface and the plaque (Figs. 11 and 12). A t high magnification, the pellicle in some samples demonstrated myelin figures (Fig. 12, insert), an observation which suggests the presence in the pellicle of phospholipids. 12

15

Three days Little change was noted between the 1 day and 3 day samples. Although cocci still predominated, a few rods and filaments were noted in many specimens. The patients who had provided crowns at the 1 day interval with little if any plaque deposits still failed to demon­ strate substantial plaque deposits after 3 days. On the other hand, the patient who had formed a well structured bacterial plaque after 1 day also exhibited a highly organized bacterial plaque with well defined columnar microcolonies consisting primarily of rather large cocci frequently aggregated in columnar clusters (Figs. 13 and 14). The loosely adapted cell wall of these cells possessed ultrastructural features which were neither typical of gram-positive nor gram-negative bacteria (Fig. 20). The rather densely packed layer of microcolonies adhering to the crown was covered by loosely arranged bacteria with occasional filamentous forms, some of which were partly covered by cocci in so-called "corn cob" formations. The ultrastructural features of the organisms associated with "corn cobs" revealed distinct morphological differences between the organisms forming the " c o b " and those forming the "kernels" (Fig. 15). Besides their obvious difference in shape, the organisms had distinctly different cytoplasmic and cell wall structures. These organisms have been described in detail in a previous publication. 13

One week The tendency which had been noted at earlier time intervals for certain subjects to form more plaque than others continued through the 1 week time interval. Significantly, the slow plaque formers at the one week time interval continued to form plaque with a predomi­ nance of cocci. Some subjects who had demonstrated distinct columnar microcolonies in plaque at the 3 day interval now had plaque with less distinct microcolonies. Others still exhibited well defined columnar arrangments of bacteria within plaque (Figs. 16-18). Although a few filaments were generally observable within plaque, the largest number appeared to initially colonize the surface of the predominantly coccal plaque (Fig. 16). Subsequently, the filamentous forms appeared to invade the underlying bacterial mass, gradually replac­ ing the predominantly coccal forms with a primarily filamentous flora (Fig. 17).

Plate 2 F I G U R E 9. Light micrograph of 1 day plaque in heavy plaque former. Note columnar shaped microcolonies which extend perpendicularly away from the crown surface(s). x 1,350. F I G U R E 10. One-day plaque. Coccal bacteria with fine, filamentous projections radiating from the cell wall periphery, x 23,600. F I G U R E 11. One-day plaque. The majority of bacteria are coccal shaped cells. Note the presence of isolated, elongated bacteria, including a branching filament (arrow) and of an electron dense pellicle (P) at the plaque-crown interface, x 3,800. F I G U R E 12. One-day plaque. Note the general orientation of bacterial cells in a plane perpendicular to the crown surface. The interbacterial matrix (M) after fixation has a fibrillar appearance. The diversity of structural features in the bacterial cells suggest that several bacterial types are able to colonize the crown surface. P, pellicle at the plaque-crown interface, x 23,600. (Insert: Pellicle at higher magnification exhibits myelin figures resembling fingerprints. This suggests the presence ofphospholipids, x 86,000.)

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J. Periodontol. January, 1975

Dental Plaque

Volume 46 Number I

Most subjects at this time interval exhibited a mixed plaque flora containing a substantial proportion of rods and filaments in addition to the coccal forms demon­ strated earlier. The thicker plaque near the gingival sulcus area, generally contained few if any columnar microcolonies and relatively more filamentous forms. In general, the more mature flora was noted primarily near the gingival crevice area. Coronal to this site, the flora tended to be more similar to that observed at earlier time intervals (Figs. 19 and 20). Three weeks The most distinct changes in plaque structure noted at 3 weeks consisted of a shift from a mixed coccofilamentous plaque near the gingival sulcus area to a predomi­ nantly filamentous plaque (Fig. 21). Interestingly, the predominantly filamentous plaques did not show any evidence of residual coccal microcolonies in the older portions of the plaque near the crown surface. The surface of these filamentous plaques was frequently overlaid by a loose covering of well developed "corn cob" formations. Electron micrographs of "corn cobs" at this and later time intervals revealed that the central filament and the surrounding cocci could differ morphologically from the cells seen at the 3 day interval (Fig. 22). In addition, the extracellular material connecting the fila­ ment and the cocci appeared much coarser than that noted at the 3 day period. Some of the peripheral coccal cells appeared to be undergoing division. Subjects which at earlier stages had shown a tendency toward little plaque formation continued to exhibit thin, predomi­ nantly coccal plaques. The heaviest plaque former (sub­ ject A G ) also demonstrated spirillar microorganisms associated with subgingival plaque formed at the apical border of the crown. In the light microscopic sections, these organisms appeared as a fuzzy covering on the surface of the dense, predominantly filamentous plaque (Fig. 23). Electron microscopy of this region indicated that spirochetes made up the bulk of the spirillar organisms (Fig. 24). It should be noted again that while

17

these changes in plaque structure were visible in the sulcus region, the more coronal plaque exhibited mor­ phological features similar to those observed at the earlier time intervals, with the most coronal plaque closely resembling the earliest plaque samples. Two months Samples collected at the 2 month interval generally provided the most voluminous amounts of plaque as estimated by the PI I or in microscopic sections. Both PI I and G I scores at this time interval were at least 2 or greater. The general features of plaque observed at 3 weeks were also evident at 2 months. The bulk of the plaque was composed of densely packed filamentous microorganisms oriented more or less perpendicularly to the crown surface. Where the crown margin was located subgingivally, the shape of the subgingival plaque was molded to the outline of the bacteria-filled sulcus (Fig. 25). The portion of the plaque extending subgingivally could be frequently identified by the presence of desqua­ mated epithelial cells and leucocytes on its external surface (Figs. 25 and 26). The most superficial microbial layer of the subgingival plaque, that is the portion of the plaque in close proximity to the sulcular and junctional epithelium, was composed of a fuzzy layer of thin bacterial cells, including numerous spirochetes (Figs. 26 and 27). This spirochete-rich layer frequently contained bacterial aggregates resembling the bristle brushes used to clean test tubes (Fig. 27). The spirochete-rich layer on the outer surface of subgingival plaque, which had been noted initially in only one of the 3 week samples, was now a feature of all but one of the 2 month old samples. O f the samples demonstrating spirochetes, the latter always occurred on the approximal surfaces, and in 2 subjects on the buccal surface of a maxillary and mandibular molar as well. The only crown without spirochetes was obtained from a volunteer ( P R ) who had been a poor plaque former from the beginning of the experiment and whose plaque at all time intervals tended to be predominantly coccal in

Plate 3 F I G U R E 13. Three-day plaque. The bulk of the plaque consists of densely packed bacteria arranged into well-defined, columnar microcolonies extending from the crown surface (S). Some filamentous forms extend beyond the densely packed bacterial mass. They may be associated with cocci in "corn cob" formations (arrows), x 1,050. F I G U R E 14. Three-day plaque. The cluster of large, coccal bacteria (between arrows) corresponds to those composing the clear microcolonies of Figure 13. x 4,200. F I G U R E 15. Three-day plaque. Cross-section of "corn cob" formation consisting of a large, central filament (F) surrounded by cocci (C) with thin, filamentous projections radiating from their cell wall. Note morphological difference in cell wall structure between filamentous and coccal cells, x 45,000. F I G U R E 16. One-week plaque. Columnar microcolonies are still well-defined. Note numerous filaments attached to the surface of this predominantly coccal plaque. S, epon surface, x 860. F I G U R E 17. One-week plaque. Area adjacent to that shown in Figure 16. Filamentous forms appear to invade and replace the coccal forms occupying the deeper layers. S, epon surface, x 860. F I G U R E 18. One-week plaque. Columnar microcolonies consisting of coccal microorganisms of three different types (labeled A, B, C) on the basis of cell morphology. Also note variation in relative volume of associated intermicrobial matrix, x 3,250.

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Listgarten, Mayo, Tremblay

J. PeriodontoL January, 1975

F I G U R E 19. One-week plaque. Microcolony of large coccal forms (A) appears to outgrow the adjacent microcolony of smaller, densely stained cocci (B). Note filaments and numerous "corn cob" formations attached to the plaque surface. S, epon surface, x 1,150. F I G U R E 2 0 . One-week plaque. Large coccal forms (A) adjacent to filamentous bacteria near surface of plaque shown in Figure 19. The ultrastructure of the cell wall of the coccal forms is not typical of that associated with either gram-positive or gram-negative cell walls, x 18,000. F I G U R E 2 1 . Three-week supragingival plaque, near gingival margin. The bulk of the microbial flora consists of filamentous microorganisms. Some "corn cob" formations are present at the surface (arrows), x 660. F I G U R E 2 2 . Cross-section of "corn cob" from 2 month old plaque. A coarse, fibrillar material attaches the cocci (C) to the central filament (F). Also note the morphological difference between the cells composing this "corn cob" and the "corn cob" in Figure 15. x 22,500.

Dental Plaque

Volume 46 Number l

structure. After 2 months, many coccal forms were still in evidence in the sulcus region of this subject, while the filamentous forms remained relatively low in numbers. Electron microscopy of the densely packed microorga­ nisms forming the bulk of 2 month old plaque indicated the presence of many filamentous forms with variable cell wall patterns, generally oriented perpendicularly to the crown surface. While the cell wall patterns could sometime be related to those which have been associated with the gram staining properties of certain bacteria (Fig. 28), this was not always the case (Fig. 29). Some sections revealed the presence of unidentified, relatively small, electron dense, rod-shaped microorganisms, measuring O . l 5 - 0 . 2 µ m in diameter, which were not detectable by light microscopy (Fig. 30). The loose plaque on the external surface of subgingival plaque consisted predominantly of spirochetes of the intermediate type, according to the classification of Listgarten and Socransky. Because these organisms were studied in sections only, it was not possible to determine the exact distribution and origins of the axial filaments. In close association with spirochetes, the external layer contained thin, filamentous bacteria, of approximately the same diameter as intermediate size spirochetes, i.e. about 0.3 µm and 5-6 µm long (Fig. 31). The ultrastructure of their cell wall could not be readily related to their gram staining properties (Figs. 32 and 33). In addition to the above, numerous micro­ organisms were present with cell wall features typical of those described for gram-negative bacteria, while other cell wall patterns were difficult to relate to the gram staining properties of the cells. Many bacteria were flagellated and appeared randomly distributed in this portion of the plaque. Most of these had not been observed in supragingival plaque or at earlier time in­ tervals. Some bacteria were present in highly struc­ tured formations consisting of a central axis with spokes radiating more or less perpendicularly away from the axis. In these structures, somewhat reminiscent of bristle brushes, the central axis was formed by one or more large filaments approximately 1 µm in diameter, closely attached to one another by an amorphous ma­ trix. The radiating bacteria forming the "bristles" of the "brush" appeared to be of two main types. A large flagellated filament, approximately 0.75-1 µm in diameter (Figs. 27 and 35), and a thin, gram-nega­ tive filament, approximately 0.3-0.4 µm in diameter (Figs. 23 and 34). These could be found alone or in combinations. Cross-sections of these formations varied somewhat in appearance depending on the relative proportion of the large and thin filaments making up the peripheral bacterial fringe. 14

In general, no mineralization of collected plaque was observed at any of the time intervals studied. The only exception was noted in plaque samples collected from the buccal surface of a maxillary molar in subject H S . In this subject some mineralization was noted in samples ob­

19

tained after one or more weeks. Mineralization occurred both in a diffuse pattern as well as by peripheral growth of a central nidus of mineralization. Where mineraliza­ tion was diffuse, it appeared to begin in close contact with or actually within bacterial cells with various cell types showing distinct differences in their mineralization patterns. Areas of focal mineralization appeared to grow by preferential mineralization of the intercellular matrix at the periphery. DISCUSSION

As indicated previously, plaque has been studied on a variety of artificial and natural substances. It is reasona­ ble to question whether an epoxy resin surface provides an adequate substance for the collection of human plaque. Is the chemical composition of the crown critical in determining the qualitative composition of acquired pellicle? Is the latter important in determining the qualitative composition of the overlying plaque? Finally, does such plaque resemble that which might be expected to form on a natural tooth surface? Only partial answers can be given to these questions. It is obvious from the results obtained by numerous investigators that hydroxyapatite is able to adsorb proteins with specific characteristics when it is exposed to complex protein mixtures. It is unlikely, however, that the initial specificity exhibited by a "clean" hydroxyapa­ tite surface persists once the various binding sites are saturated. Subsequent accumulation of proteins and other organic molecules probably becomes rapidly inde­ pendent of the nature of the original adsorbing surface. H a y points out that the composition of the specifically adsorbed proteins differs markedly from that of micro­ scopically visible pellicles and discusses some of the mechanisms which could account for pellicle growth following the initial adsorption of specific proteins. Although no data is available to this writer on the formation of "dental pellicles" on epoxy resin surfaces, it is likely that such pellicles are quite different in composi­ tion from those formed on apatite surfaces. The observa­ tion in our material of myelin figures in pellicles formed on epon surfaces suggests that phospholipids may be an important component of such pellicles. Whether the chemical composition of acquired pelli­ cles is critical in determining the pattern of colonization of the tooth or other artificial substrates is a question which cannot be answered satisfactorily at this time be­ cause of insufficient experimental data. Rölla and Mathiesen indicated that the affinity of six com­ mercially available dextrans was greater for hydroxy­ apatite "coated" with salivary proteins than for un­ treated hydroxyapatite. If dextrans or other glucans are important in the early colonization of the tooth sur­ face such differential affinity for "coated" and "uncoated" surfaces might be important. However, there is presently no indication that this affinity would be different for the type of pellicle which forms on a 15-20

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21

22,

2 3

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J. Periodontol. January, 1975

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Dental Plaque

Volume 46 Number l

hydroxyapatite surface as contrasted to that which forms on an epoxy resin crown. If salivary proteins contribute to the agglutination of bacteria as sugfested by Gibbons and Spinell, it is conceivable that bacteria coated with salivary proteins would also exhibit differential affinities for substrates with various surface properties. Whether this is, in fact, the case is not known nor is it clear to what extent differences in the initial colonization of a tooth or epoxy resin surface would affect the bacterial composition of mature plaque. 24

25

Recent reports from Berthold et a l . and Berthold et a l . indicate that if comparisons are made using various substrates with respect to the composition of accumu­ lated plaque, certain differences and similarities are apparent. These investigators noted that by scanning and transmission electron microscopy, the morphology of 2and 5-day old plaque formed on enamel was similar to that formed on polished "Vestopal W , " a type of epoxy resin. However, different microbial patterns were noted on Mylar films, or on Vestopal surfaces which had been previously sandblasted. Although morphologic criteria may be crude parameters for comparing the similarity or difference of such plaque samples, it should be noted that cultural techniques also suffer certain drawbacks. For example, the presently inadequate taxonomic classifica­ tion of the oral flora, sampling problems, the differential ability of certain bacterial strains to grow in vitro and the poorly understood effect of various methods of bacterial dispersion on the comparative recovery of various bacte­ ria may all distort to varying degrees the true picture of the microbial composition of dental plaque. 26

The results reported here basically agree with those of other investigators who have studied the developmental stages of plaque on smooth s u r f a c e s and on celluloid s t r i p s . Briefly, the earliest flora is predomi­ nantly coccal in nature with gram-positive microorga­ nisms appearing more numerous in the early stages than gram-negative ones. With time, filamentous forms ap­ pear and eventually spirillar organisms including spiro­ chetes. In our experimental situation, substantial num­ 4,27-29

2,3

bers of spirochetes were only noted in the 3 week and 2 month samples, an observation which is consistent with those of Loe et al and Mackler and Crawford. Scanning electron micrographs of early plaque indi­ cate that following the attachment of bacteria to the tooth surface, the latter proliferate to form dome-like colonies. These in turn expand until adjacent colonies merge. Our light micrographs of early plaque support the concept that continued growth of microcolonies must proceed vertically, that is, away from the crown surface, since lateral growth is no longer possible. This would account for the columnar microcolonies in early plaque. The principle underlying plaque growth at this stage bears a striking similarity to that which has led to the proliferation of skyscrapers in crowded cities. The co­ lumnar microcolonies observed in the early stages of plaque formation and in the coronal portion of older plaques represent good evidence that plaque thickening occurs by bacterial growth within plaque. Surface appo­ sition, as suggested by Gibbons and VanHoute and VanHoute, may allow filamentous forms to colonize the surface of the predominantly coccal plaque. How­ ever, it is again the growth inward of these filamentous forms which may be responsible for the eventual appear­ ance of predominantly filamentous plaques. Such a scheme would be dependent on the possession by these filamentous bacteria of enzymes capable of breaking down the structural integrity of underlying coccal forms. Preliminary data (Socransky, personal communication) indicate that certain filamentous microorganisms do, indeed, possess such enzymatic capability. There is also evidence of substantial cell lysis in the deeper layers of well developed filamentous plaque, an observation which is compatible with the postulation that plaque remodel­ ing may occur through active replacement of coccal microcolonies by filamentous bacteria. 4

28

30

22

23

It is also conceivable that some filaments may initially colonize the tooth as coccal forms and that later these cells assume filamentous shapes more typical of those observed in mature plaque. It is known that certain

Plate 5 F I G U R E 23. Three-week subgingival plaque. Spiral forms predominate along plaque surface facing gingival tissue. Note bacterial aggregations consisting of large, centrally located filaments, surrounded by a halo of thin, filamentous bacteria (arrows), x 1,750. F I G U R E 24. Three-week plaque. Spirochetes of intermediate size near the surface of the subgingival plaque shown in Figure 23. x 14,000. F I G U R E 25. Two-month old plaque. Apical extension of plaque into sulcus. Note desquamated epithelial cells and leukocytes on plaque surface facing the gingival tissue (arrows), x 200. F I G U R E 26. Higher magnification of outer surface of plaque shown in Figure 25. A fuzzy, bacterial layer (between arrows) covers the outer surface of subgingival plaque, x 550. F I G U R E 27. The external surface of subgingival plaque consists of a flora with many spirochetes, bacterial formations (B) resembling "bristle brushes" (here cut in cross-sections) and occasional mammalian cells (C). x 1,400. F I G U R E 28. Two-month old plaque. It contains many filamentous bacteria, some of which may appear coccus-like in cross-section (A). The cell wall structure of some bacteria conforms with that described for gram-positive organisms (A) or gram-negative organisms (not shown). Membranous vesicles between the cells (V) are likely the results of cell lysis, x 30,000. F I G U R E 29. Two-month old plaque. Filamentous forms near surface. Certain cells (A and B) have distinct cell walls, not readily related to the gram staining property of cell, x 26,000.

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organisms, for example Actinomyces naeslundii and Actinomyces viscosus, can exist in both coccal as well as filamentous forms, the environmental conditions being largely responsible for the predominant morphology. Since Actinomyces are also able to colonize smooth tooth surfaces, it is possible that some of these bacteria may be responsible for the altered cellular morphology which seems to occur in the deep portion of mature plaque. It should be noted that the ultrastructural appearance of bacterial cells in contact with the crown surface is frequently consistent with that which has been reported for A. naeslundii and A. viscosus. 31

32

Despite our inability at this time to identify most plaque bacteria by their morphology alone, it is possible by microscopic techniques to localize the preferential distribution of certain microorganisms within plaque. The observation of a spirochete-rich layer on the outer surface of subgingival plaque, where it is in close contact with the gingival tissues of the host, constitutes poten­ tially valuable information which can be readily obtained with such means. It should be noted that we have observed a similar spirochete-rich layer on the outer surface of subgingival plaque formed on the root surface of teeth extracted from patients because of advanced periodontal disease (Listgarten, in preparation). These results are compatible with the observations of Schultz-Haudt et a l . who, on the basis of examining plaque smears from patients with and without clinically detectable gingivitis, concluded that with gingivitis, the percentage of spirochetes shifted from 0.6 to 17%. These observations also agree with those of Jones resulting from a scanning electron microscopic study of teeth affected with periodontal disease, and the earlier studies of Bass who reported a "preponderance of spirochetes" between the deeper portion of the plaque and the pocket wall. This investigator also reported that he could pick material from this zone "which consists of practically pure spirochetes." It is perhaps appropriate to recall that in acute necrotizing ulcerative gingivitis ( A N U G ) , the microbial layer in intimate contact with the ulcerated gingival lesion also consists largely of intermediate size spirochetes. Although neither the pathogenesis of chronic periodontitis nor that of A N U G is known, the association of spirochetes with certain forms of periodon­ tal disease may be significant. It is unfortunate that the difficulties inherent in isolating and cultivating oral 3 3

34

35

36,

3 7

23

spirochetes, and their reluctance to grow in gnotobiotic animal models have prevented thus far a more compre­ hensive enquiry into the potential role these organisms may have in the etiology of certain forms of periodontal disease. Spirochetes as a group have characteristic morphologi­ cal features which are unique to these microorga­ nisms. The variety of ultrastructural features ob­ servable in other plaque organisms suggests that certain morphological parameters, for example cell wall ultrastructure, may be potentially useful in the identification of other types of bacteria. In order to be able to relate the ultrastructural features of certain microorganisms to specific species, it will be necessary to compare the morphology of the wild type to that of cells grown in pure cultures. In order to identify the microorganisms in vivo, it will be necessary to utilize specific markers based on the serological properties of cultivable species. Such an approach should provide a clearer understanding of the spatial arrangement of specific microorganisms in plaque and help in the location of microorganisms which, because of numbers or critical location, appear to be important in the etiology of certain forms of periodontal disease. 38-40

SUMMARY

A method was presented to fabricate epoxy resin crowns to be worn by human subjects requiring full crown restorations. These crowns were utilized in six young adults to study the internal structure of plaque after plaque formation periods of 1 and 3 days, 1 and 3 weeks and 2 months. This study confirmed previous findings that early plaque contains primarily coccal forms, with a shift to predominantly filamentous forms by 3 weeks. Early plaque growth seems to occur by the formation of columnar microcolonies which coalesce and grow by cell division within the colony in a direction perpendicular to the crown surface. Filamentous micro­ organisms appear in large numbers by 1 week. They appear to colonize the surface of the predominantly coccal plaque, eventually growing into it and replacing the coccal forms. The subgingival, mature plaque con­ tains many motile forms including bacteria with unusual cell wall ultrastructures. Certain bacteria combine into distinctive bacterial aggregations resembling "corn cobs" and "test tube brushes," the latter occurring exclusively in subgingival plaque. Spirochetes appear to grow pref-

Plate 6 F I G U R E 30. microscopy, F I G U R E 31. spirochetes F I G U R E 32. F I G U R E 33.

Two-month plaque. Small, electron-dense microorganisms (arrows) can be seen which are not detectable by light x 12,000. Two-month old subgingival plaque. Predominant microorganisms on the gingival tissue side of the plaque include (S) and thin filamentous microorganisms (F). x 7,800. Cross-section through the spirochetes (S) and filaments (F) of the types illustrated in Figure 31. x 52,000. Longitudinal section through the filaments illustrated in Figures 31 and 32. x 44,000.

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erentially on the external surface of subgingival plaque in close contact to the gingival tissue of the deepened sulcus. Their high concentration in the external layer of subgin­ gival plaque suggests that because of their strategic location they may play an important role in the etiology of periodontal disease. Studies of well preserved plaque, possibly combined with the use of serological markers, can serve a useful role in identifying certain microorga­ nisms in dental plaque. Because of their numbers and/or location in relation to periodontal tissues, some of these bacteria may warrant further studies as potential etiologic agents of certain forms of periodontal disease.

16. Hay, D. I.: The Interaction of Human Parotid Salivary Proteins with Hydroxyapatite. Arch. Oral Biol., 18:1517, 1973. 17. McGaughey, C . and Stowell, E . C : The Adsorption of Human Salivary Proteins and Porcine Submaxillary Mucin by Hydroxyapatite. Arch. Oral Biol., 12:815, 1967. 18. Ericson, T . : Salivary Glycoproteins: Composition and Adsorption to Hydroxyl Apatite in Relation to the Formation of Dental Pellicles and Calculus. Acta Odontol. Scand., 26:3, 1968. 19. Mayhall, C . W.: Concerning the Composition and Source of the Acquired Pellicle of Human Teeth. Arch. Oral Biol., 15:1327, 1970. 20. S ö n j u , T . and R ö l l a , G . : Chemical Analysis of the Acquired Pellicle Formed in Two Hours on Cleaned Human Teeth in vivo. Rate of Formation and Amino Acid Analysis. Caries Res., 7:30, 1973.

REFERENCES

1. Björn, H . and Carlsson, J . : Observations on a Dental Plaque Morphogenesis. Odontol. Revy., 15:23, 1964. 2. Mandel, I. D . , Levy, B. M . and Wasserman, B. H . : Histochemistry of Calculus Formation. J . PeriodontoL, 28:132, 1957. 3. Hazen, S. P. and Zander, H . A . : Four-Week Old Calculus. J . Dent. Res., 38:710, 1959. Abstract. 4. Loe, H . , Theilade, E . and Jensen, S. B.: Experimental Gingivitis in M a n . J . PeriodontoL, 36:177, 1965. 5. Frank, R. M . and Houver, G . : A n Ultrastructural Study of Supragingival Dental Plaque Formation, in Dental Plaque, M c H u g h , W . D . , ed., E . S. Livingstone, Edinburgh, 1970, pp. 85-108. 6. Theilade, E . and Theilade, J . : Bacteriological and Ultrastructural Studies of Developing Dental Plaque, in Dental Plaque, M c H u g h , W . D . , ed., E . S. Livingstone, Edinburgh, 1970, pp. 27-40. 7. Schroeder, H . E . and DeBoever, J . : The Structure of Microbial Dental Plaque, in Dental Plaque, M c H u g h , W . D., ed., E . S. Livingstone, Edinburgh, 1970, pp. 49-74. 8. Luft, J . H . : Improvements in Epoxy-Resin Embedding Methods. J . Biophys. Biochem. Cytol., 9:409, 1961. 9. Loe, H . and Silness, J . : Periodontal Disease in Preg­ nancy. I. Prevalence and Severity. Acta Odontol. Scand., 21:533, 1963. 10. Silness, J . and L ö e , H . : Periodontal Disease in Preg­ nancy. II. Correlation between Oral Hygiene and Periodontal Condition. Acta Odontol. Scand., 22:121 1964. 11. Karnovsky, M . J . : A Formaldehyde-Glutaraldehyde Fixative of High Osmolarity for Use in Electron Microscopy. J . Cell Biol., 27:137A, 1965. 12. Stoeckenius, W.: A n Electron Microscopic Study of Myelin Figures. J . Biophys. Biochem. CytoL, 5:491, 1959. 13. Listgarten, M . A . , M a y o , H . and Amsterdam, M . : Ultrastructure of the Attachment Device between Coccal and Filamentous Microorganisms in "Corn C o b " Formations of Dental Plaque. Arch. Oral Biol., 18:651, 1973. 14. Listgarten, M . A . and Socransky, S. S.: Electron Microscopy as an A i d in the Taxonomic Differentiation of Oral Spirochaetes. Arch. Oral Biol., 10:127, 1965. 15. Hay, D . I.: The Adsorption of Salivary Proteins by Hydroxyapatite and Enamel. A r c h . Oral Biol., 12:937, 1967.

25

21. R ö l l a , G . and Mathiesen, P.: The Adsorption of Salivary Proteins and Dextrans to Hydroxylapatite, in Dental Plaque, M c H u g h , W . D . , ed., E . S. Livingstone, Edinburgh, 1970, pp. 129-140. 22. Gibbons, R. J . and vanHoute, J . : O n the Formation of Dental Plaques. J . PeriodontoL, 44:347, 1973. 23. vanHoute, J . : Characteristics of Dental Surfaces Re­ lated to Plaque Adherence, Growth and Pathogenicity, in Den­ tal Plaque: Interfaces, Rowe, N . H . ed., University of M i c h ­ igan, A n n Arbor, 1973, pp. 25-46. 24. Gibbons, R. J . and Spinell, D . M . : Salivary-Induced Aggregation of Plaque Bacteria, in Dental Plaque, M c H u g h , W. D . , ed., E . S. Livingstone, Edinburgh, 1970, pp. 207-215. 25. Berthold, P., Berthold, C . - H . and S ö d e r , P.-Ö.: The Growth of Dental Plaque on Different Materials: A n Ultrastructure Study. J . Dent. Res., 50:1221, 1971. Abstract. 26. Berthold, C . - H . , Berthold, P. and S ö d e r , P . - Ö : The Growth of Dental Plaque on Different Materials. Swed. Dent. J.: 64:863, 1971. 27. Eastcott, A . D . and Stallard, R. E . : Sequential Changes in Developing Human Dental Plaque as Visualized by Scanning Electron Microscopy. J . PeriodontoL, 44:218, 1973. 28. Mackler, S. B. and Crawford, J . J.: Plaque Development and Gingivitis in the primary Dentition. J . PeriodontoL, 44:18, 1973. 29. McDougall, W . A . : Studies on the Dental Plaque. II. The Histology of the Developing Interproximal Plaque. Aust. Dent. J . , 8:398, 1963. 30. Saxton, C . A . : Scanning Electron Microscope Study of the Formation of Dental Plaque. Caries Res., 7:102, 1973. 31. Socransky, S. S.: Relationship of Bacteria to the Etiology of Periodontal Disease. J . Dent. Res., 49: (suppl. N o . 2) 203, 1970. 32. Girard, A . E . and Jacius, B. H . : Ultrastructure of Actinomyces viscosus and Actinomyces naeslundii. Arch. Oral Biol., 19:71, 1974. 33. Schultz-Haudt, S., Bruce, M . A . and Bibby, M . G . : Bacterial Factors in Non-specific Gingivitis. J . Dent. Res., 33:454, 1954. 34. Jones, S. J . : The Tooth Surface in Periodontal Disease. Dent. Pract. Dent. R e c , 22:462, 1972. 35. Bass, C . C : The Relation of the Inner Border of Bacterial Film on the Tooth within the Gingival Crevice to the

Plate 7 F I G U R E 34. Two-month plaque. Cross-section through a "bristle brush" formation in which the central filament (F) is surrounded by "bristles" consisting of thin filaments (FF) and occasional spirochetes (S). x 18,500. F I G U R E 35. Two-month plaque. Oblique section through a "bristle brush" formation consisting of large flagellated filaments (FF) forming the "bristles" and a single central filament (F) forming the axis of the "brush."*, artifact, x 12,000.

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Zone of Disintegrating Epithelial Attachment Cuticle. Oral Surg., 2:1580, 1949. 36. Listgarten, M . A . : Electron Microscopic Observations on the Bacterial Flora of Acute Necrotizing Ulcerative Gingivi­ tis. J . Periodontol., 36:328, 1965. 37. Listgarten, M . A . and Lewis, D . W . : The Distribution of Spirochetes in the Lesion of Acute Necrotizing Ulcerative Gingivitis. A n Electron Microscopic and Statistical Survey. J . Periodontol., 38:379, 1967.

38. Listgarten, M . A . and Socransky, S. S.: Electron Microscopy of Axial Fibrils, Outer Envelope and Cell Division of Certain Oral Spirochetes. J . Bacteriol., 88:1087, 1964. 39. Ovcinnikov, N . M . and Delektorskij, V . V . : Morphology of Treponema pallidum. Bull. W . H . O . , 35:223, 1966. 40. Pillot, J . and Reiter, A . : Structure des Spirochetes. I. Etude des Genres Treponema, Borrelia et Leptospira au Microscope Electronique. A n n . Inst. Pasteur, 108:791, 1965.

Announcements UNIVERSITY OF PENNSYLVANIA SCHOOL OF DENTAL MEDICINE The University of Pennsylvania School of Dental Medicine an­ nounces the following continuing education course: Title: Endo-Perio Therapy Faculty: Frederic M . Chacker, D.D.S., Associate Professor of Periodontology and Seymour Oliet, D.D.S., Director of Endodontics. Date: March 5, 1975 Description: Diagnosis and treatment of these complicated lesions are explained in terms applicable to general practice. Salvaging condemned teeth. For further information, contact: Division of Continuing Education, University of Pennsylvania School of Dental Medicine, Philadelphia, PA. 19174. UNIVERSITY OF K E N T U C K Y C O L L E G E OF DENTISTRY The University of Kentucky College of Dentistry announces the following continuing education courses: Title: Preclinical and Technique Course in Osseous Surgery (Pre­ requisite—basic course in periodontics) Faculty: Periodontics staff, University of Kentucky College of Dentistry Dates: February 13, 14, 1975. This is a laboratory and technique course in the recognition, de­ scription and correction of the alveolar bone defects of periodontitis. The concepts and techniques of osseous surgery will be presented. Each participant will use models with simulated bone defects to correct. Basic periodontal flap procedures (used for access to the alveolar bone) will be reviewed and suturing techniques done on models. Title: Concepts and Techniques of Mucogingival Surgery: A semi­ nar-laboratory course. Faculty: Periodontics staff, University of Kentucky College of Dentistry Dates: March 20, 21, 1975. Mucogingival surgery—its indications, goals, objectives and tech­ niques will be presented. Soft tissue surgical procedures will be discussed for 1) gaining access to the underlying alveolar bone, 2) establishing attached gingiva at and around teeth prior to any restorative procedures or crown preparation and 3) correction of soft tissue depth. Full thickness, split thickness and laterally positioned flaps will be discussed, as well as types of incisions and the indications and use of free gingival grafts (soft tissue autografts). The laboratory component will be the practice of the procedures and techniques on hog jaws, including various suturing techniques. Title: Surgical Periodontics—Participation in Treatment (Prerequisite—a basic periodontics course plus the University of Kentucky laboratory course in osseous and/or mucogingival surgery) Faculty: Periodontics Staff, University of Kentucky College of Dentistry

Dates: April 10, 11, 1975 A seminar on the management of patients for periodontal surgery. Participants in the course will work with and assist periodontics graduate students and residents in doing periodontal surgery. The patients are under the continuing care of the graduate students. Cases will be extensively reviewed and documented prior to surgery. Two such experiences will occur during the course. For further information contact: Dr. Harry E. Moore, Director of Continuing Education, University of Kentucky College of Dentistry, Albert B. Chandler Medical Center, Lexington, Kentucky 40506. BROOKDALE DENTAL CENTER OF NEW YORK UNIVERSITY On Friday, May 16 and Saturday, May 17, 1975, the Department of Periodontics at New York University College of Dentistry will celebrate its 50th Anniversary at the Plaza Hotel in New York City. The meeting will consist of a two-day scientific session devoted to the theme: Periodontics, Retrospect and Prospect. Speakers for the sessions will include outstanding national authorities—among them: Drs. H . Slavkin, S. Socransky, J. Jacoway, A. Melcher, E. Tonna, H . Zander, L . Cohen, T. O'Leary, R. Stallard, and F. Beube. The scientific sessions will be followed by a gala dinner-dance on Saturday night. For further information, please contact the Department of Periodon­ tics, Brookdale Dental Center of New York University, 421 First Avenue, New York, N Y 10010. NORTHWESTERN UNIVERSITY D E N T A L SCHOOL Northwestern University Dental School announces the following continuing education course: Periodontics II (Periodontics I is a prerequisite) Faculty: Dr. Henry Crossetti Dates: March 12, 13, 1975. This course is designed to provide the general practitioner with a sound understanding of modern surgical concepts and the rationale for their clinical application. For further information contact: Dr. Clifford H . Miller, Associate Dean, Northwestern University Dental School, 311 East Chicago Avenue, Chicago, Illinois 60611. UNIVERSITY OF TORONTO F A C U L T Y OF DENTISTRY The University of Toronto Faculty of Dentistry announces the following continuing education course: Periodontics Faculty: Professor J. E. Speck Dates: March 5, 12, 19, 26 and April 2, 23, 1975 The course will include lectures, demonstrations and student partici­ pation. For further information contact: Dr. G . S. Beagrie, Assistant Director, Division of Postgraduate Dental Education, University of Toronto Faculty of Dentistry, 124 Edward Street, Toronto, Ontario, Canada.