D e n t a l Plaque:
N a t u r e , F o r m a t i o n and
Effects*
BY I R W I N D . M A N D E L , * * B.S., D.D.S., NEW YORK, NEW YORK
IN 1897, J. Leon Williams demonstrated in a series of ground sections the presence of a thick felt like mass of microorganisms covering the surface where decay had commenced. He described this mass as "so dense and adhesive as to make it highly improbable that the enamel is affected by any acid other than that which is being excreted by the bacteria at the very point where they are attached to the enamel. The thick glutinous like mass of fungi also prevents the excreted acid from being washed away." 1
During most of the seventy years dental plaque has been studied it has been considered almost exclusively in relation to dental caries. It is only during the last decade that plaque has been widely implicated in calculus formation and periodontal disease. We now consider the deleterious effects of plaque accumulation and metabolism as bidirectional-outwards toward the soft tissues as well as inwards toward the tooth. Three general aspects of plaque will be considered: (1) morphology and composition (chemical and bacteriological), (2) manner of formation, (3) pathological implications. MORPHOLOGY OF PLAQUE
Present knowledge of the structure of dental plaque is derived mainly from three types of investigations: (1) study of extracted teeth, (2) examination of pieces of enamel and teeth inserted into prosthetic appliances and worn in the mouth for varying periods of time, (3) examination of plastic strips attached to teeth (usually lingual of lower anteriors) for varying periods. In most instances only supragingival plaque was investigated. *Presented as part of a symposium: Periodontal Home Care—Recent Trends, American Academy of Pericdontology, Las Vegas, Nevada, November 4, 1965. **Division of Stomatology, School of Dental and Oral Surgery, Columbia University, New York, New York. The original data contained herein are from investigation supported by Public Health Service Grant DE-01554 and The Bristol-Myers Co.
Page
5/357
Page
6/358
MANDEL
It is difficult to offer a precise description of plaque because it is a variable and dynamic entity. A n "umbrella" type of description would consider plaque as an acquired gel-like mat, closely adherent to a tooth or restoration surface. The mat is composed of an organic film (cuticle or pell i c l e ) , microbial masses and their products, organic and inorganic components from oral secretions, shed epithelial cells and blood cells. The major components are the cuticle, microbial masses and intermicrobial matrix.
matic rather than metachromatic) and to be strongly P . A . S . positive.
Dental plaque should be differentiated from materia alba and food debris. Plaque has a definite architecture discernible with the light or electron microscope. In its mature form at least, it is mainly a product of microbial growth. Since it is closely adherent to the tooth or restoration surface it is not removed by rinsing or a moderate water spray. A s M u h l e m a n n and Schroeder pointed out, materia alba, although composed i n large part of bacteria, doesn't usually exhibit any particular structure. It is a product of accumulation, composed of m i crobial masses, desquamated epithelial cells and blood cells which adhere loosely to surfaces of dental plaques, teeth, restorations or gingiva. Material alba can be removed by water sprays or even forceful rinsing. F o o d debris, unless impacted interproximally, is readily dislodged by lip and tongue movements, rinsing or gentle streams of water.
A n electron microscopic study of deposits formed on plastic strips disclosed the presence of a cuticle i n all sections examined. The cuticle varied from .05-.4 miera i n thickness, which agrees well with the light microscope studies of M c D o u g a l l .
2
DETAILED STRUCTURE-ACQUIRED
A number of other investigators studying tooth surface deposits reported the nearly constant presence of an acquired c u t i c l e . ' Remnants of the primary cuticle (Nasmyth's membrane) are only rarely seen i n functional teeth. Since the cuticle is thin and transparent it is difficult to visualize. A disclosing solution such as basic fuchsin stains it a light purplish red. 4
5
6
3
T h e h i s t o c h e m i c a l c h a r a c t e r i s t i c s of the cuticle have been rather intensively studied. ' The most significant observations are on the comparability between the natural cuticles and films formed directly from saliva. M e c k e l compared: (1) cuticles floated off from freshly extracted teeth; (2) cuticles formed on enamel segments worn i n partial dentures; (3) cuticles formed in vitro on enamel incubated i n saliva, and (4) dried salivary films. These four organic films stained almost identically. 4
5
7
5
Bacteria does not seem to be of consequence i n the formation of cuticle since in the i n vitro studies of M e c k e l inhibition of bacterial growth by antibiotics did not effect the formation of the films. "The only essential factor leading to the formation of an artificial cuticle i n vitro was the presence of saliva." 5
CUTICLES
In an extensive study of interproximal plaques on extracted teeth M c D o u g a l l found a rather uneven distribution with a thickness of 1-3 miera (1 thousandth of a millimeter) at the marginal ridge to over 60 miera gingival to the contact point. H e noted that attachment to the enamel surface frequently involved growth within lamellae, wedge-shaped defects and other defect areas. A l l mature plaques on enamel i n cluded an acquired cuticle—a homogeneous, continuous structure .3-.8 miera wide. H e found the cuticle to be intensely blue when stained with toluide-blue (orthochro3
W h e n the four types of films were exposed to a number of enzymes, differences were noted. The films formed i n vitro were more readily affected by the enzymes than the cuticular material found on extracted teeth. M e c k e l suggests that denaturation and polymerization of the cuticular proteins with increasing age of the film could account for its greater resistance. 5
Page
DENTAL PLAQUE
The staining reactions of the cuticles suggest that they are composed of modified mucoproteins or glycoproteins from saliva together with lipid material. The specific composition and the actual mechanism of formation remains to be established. Some further data on composition and several theories of formation w i l l be discussed later i n this papen The incidence of acquired cuticles in the subgingival plaque and the source of such material has not as yet been subjected to any systematic study. The recent interest in gingival fluid should act as a stimulus for such studies. M I C R O B I A L MASSES
In the mature plaque the microorganisms are the dominant components. Tremendous numbers of bacteria cluster together. Total microscopic counts indicate the presence of about 250 million organisms per mg. of wet weight of p l a q u e . ' Since a pure culture of streptococcus exhibits the same number to weight relationship, it is reasonable to assume that most of the weight of the plaque is due to bacteria. 8
9
8
Microscopic examination of the fully formed plaque usually discloses a mass of filaments at right angles to the cuticle near the enamel surface. A t the periphery, the filaments are curving and irregular. In the outer portion of the plaque, that is, the younger areas, cocci and other small bacterial forms can be noted. W i t h gram stain about two thirds of the plaque is usually gram positive. 10
When plaques are examined bacteriologically a different picture emerges. A l t h o u g h the filaments occupy so much volume, they are never the dominant organism on a quantitative basis. In fact, i n young plaques they are barely present at all. Although the actual number varies from study to study there seems to be general agreement that i n supragingival plaque gram positive cocci are the dominant organisms (40-50% ), followed by gram positive rods ( 1 0 - 4 0 % ) , gram negative rods ( 1 0 - 1 5 % ) .
7/359
Filaments have been reported as 4 % or less (down to almost 0% ) A 1 1
A n interesting observation, noted by us in 1 9 5 7 , and corroborated i n a number of studies, was that the proportion of cocci decreased and the number of filaments i n creased with age of the plaque. This morphological observation was recently quantitated bacteriologically by H o w e l l , R i z z o , and P a u l . The most dramatic change occurred between 14 and 21 days when the proportion of filamentous forms jumped from 4-18%. In our studies this change seemed to take place somewhat earlier. This may have been due to patient variation or to the inherent difficulty i n relating histological to bacteriological techniques. 12
1 1
W h e n material from the gingival crevice area (not true plaque but non-adherent material) was examined by Socransky et a l . and Gibbons et a l . , two major differences from supragingival plaque were noted. In the crevice Bacteroides melaninogenicus occurred at a level of about 5% (2.6% i n the normal group, 7.8% i n the periodontal). It rarely i f ever occurred i n supragingival plaque. This organism is especially interesting because it is a producer of collagenase. The second important difference was the presence of 1% spirochetes i n the crevice and only 0 - . l % i n supragingival areas. The rest of the flora was comparable. T o date no very significant differences have been found in the nature of the crevicular flora when periodontally normal and involved patients have been compared. There was a tendency for an increase i n numbers of bacteria per gram of wet weight i n the periodontally i n volved group (except for total streptococci), especially the spirochetes and B . melaninogenicus. N o etiologic significance could be deduced. The only really significant observation was the overall proliferation of the flora. The authors pointed out that "it was often difficult to remove 10 mg. of bacterial debris from the gingival crevice area of a 'normal' individual: however, one could obtain with ease 100 mg. or more of 'bacterial debris' from a periodontally involved individual." 1 3
14
1 3
Page
8/360
MANDEL INCLUSIONS
TABLE 1
O n occasion non-bacterial cells can be seen. These are usually shed epithelial cells or leukocytes i n varying stages of disruption. Fibrous material and nondescript food debris are also seen occasionally. Where plaques are adjacent to soft tissue (with resultant inflammatory changes) large numbers of cells (especially leukocytes) can be seen on the surface of the plaque.
Cuticular Material from Dentures*
10
CHEMICAL COMPOSITION
It is difficult to determine the chemical composition of dental plaque i n any definitive way because it is such a variable and polyglot material. Several laboratories, however, have undertaken the task of systematic analysis and some meaningful data is available. M i l l i n and S m i t h analyzed the material adhering to denture surfaces after thorough brushing i n a stream of water. The material remaining was virtually sterile and was probably cuticle or pellicle. F r o m the analytical findings (Table 1) it would appear that i f the cuticular material was salivary i n origin (which seems very likely) much of the carbohydrate was split off by the bacteria, leaving mainly the protein core and the closely associated hexoses. The calcium and phosphorous content is similar (as we shall see) to concentrations usually found i n early plaques.
% Dry
Weight
70.0 11.6 1.9 4.0 1.0
N a t u r e , F o r m a t i o n and
Effects*
BY I R W I N D . M A N D E L , * * B.S., D.D.S., NEW YORK, NEW YORK
IN 1897, J. Leon Williams demonstrated in a series of ground sections the presence of a thick felt like mass of microorganisms covering the surface where decay had commenced. He described this mass as "so dense and adhesive as to make it highly improbable that the enamel is affected by any acid other than that which is being excreted by the bacteria at the very point where they are attached to the enamel. The thick glutinous like mass of fungi also prevents the excreted acid from being washed away." 1
During most of the seventy years dental plaque has been studied it has been considered almost exclusively in relation to dental caries. It is only during the last decade that plaque has been widely implicated in calculus formation and periodontal disease. We now consider the deleterious effects of plaque accumulation and metabolism as bidirectional-outwards toward the soft tissues as well as inwards toward the tooth. Three general aspects of plaque will be considered: (1) morphology and composition (chemical and bacteriological), (2) manner of formation, (3) pathological implications. MORPHOLOGY OF PLAQUE
Present knowledge of the structure of dental plaque is derived mainly from three types of investigations: (1) study of extracted teeth, (2) examination of pieces of enamel and teeth inserted into prosthetic appliances and worn in the mouth for varying periods of time, (3) examination of plastic strips attached to teeth (usually lingual of lower anteriors) for varying periods. In most instances only supragingival plaque was investigated. *Presented as part of a symposium: Periodontal Home Care—Recent Trends, American Academy of Pericdontology, Las Vegas, Nevada, November 4, 1965. **Division of Stomatology, School of Dental and Oral Surgery, Columbia University, New York, New York. The original data contained herein are from investigation supported by Public Health Service Grant DE-01554 and The Bristol-Myers Co.
Page
5/357
Page
6/358
MANDEL
It is difficult to offer a precise description of plaque because it is a variable and dynamic entity. A n "umbrella" type of description would consider plaque as an acquired gel-like mat, closely adherent to a tooth or restoration surface. The mat is composed of an organic film (cuticle or pell i c l e ) , microbial masses and their products, organic and inorganic components from oral secretions, shed epithelial cells and blood cells. The major components are the cuticle, microbial masses and intermicrobial matrix.
matic rather than metachromatic) and to be strongly P . A . S . positive.
Dental plaque should be differentiated from materia alba and food debris. Plaque has a definite architecture discernible with the light or electron microscope. In its mature form at least, it is mainly a product of microbial growth. Since it is closely adherent to the tooth or restoration surface it is not removed by rinsing or a moderate water spray. A s M u h l e m a n n and Schroeder pointed out, materia alba, although composed i n large part of bacteria, doesn't usually exhibit any particular structure. It is a product of accumulation, composed of m i crobial masses, desquamated epithelial cells and blood cells which adhere loosely to surfaces of dental plaques, teeth, restorations or gingiva. Material alba can be removed by water sprays or even forceful rinsing. F o o d debris, unless impacted interproximally, is readily dislodged by lip and tongue movements, rinsing or gentle streams of water.
A n electron microscopic study of deposits formed on plastic strips disclosed the presence of a cuticle i n all sections examined. The cuticle varied from .05-.4 miera i n thickness, which agrees well with the light microscope studies of M c D o u g a l l .
2
DETAILED STRUCTURE-ACQUIRED
A number of other investigators studying tooth surface deposits reported the nearly constant presence of an acquired c u t i c l e . ' Remnants of the primary cuticle (Nasmyth's membrane) are only rarely seen i n functional teeth. Since the cuticle is thin and transparent it is difficult to visualize. A disclosing solution such as basic fuchsin stains it a light purplish red. 4
5
6
3
T h e h i s t o c h e m i c a l c h a r a c t e r i s t i c s of the cuticle have been rather intensively studied. ' The most significant observations are on the comparability between the natural cuticles and films formed directly from saliva. M e c k e l compared: (1) cuticles floated off from freshly extracted teeth; (2) cuticles formed on enamel segments worn i n partial dentures; (3) cuticles formed in vitro on enamel incubated i n saliva, and (4) dried salivary films. These four organic films stained almost identically. 4
5
7
5
Bacteria does not seem to be of consequence i n the formation of cuticle since in the i n vitro studies of M e c k e l inhibition of bacterial growth by antibiotics did not effect the formation of the films. "The only essential factor leading to the formation of an artificial cuticle i n vitro was the presence of saliva." 5
CUTICLES
In an extensive study of interproximal plaques on extracted teeth M c D o u g a l l found a rather uneven distribution with a thickness of 1-3 miera (1 thousandth of a millimeter) at the marginal ridge to over 60 miera gingival to the contact point. H e noted that attachment to the enamel surface frequently involved growth within lamellae, wedge-shaped defects and other defect areas. A l l mature plaques on enamel i n cluded an acquired cuticle—a homogeneous, continuous structure .3-.8 miera wide. H e found the cuticle to be intensely blue when stained with toluide-blue (orthochro3
W h e n the four types of films were exposed to a number of enzymes, differences were noted. The films formed i n vitro were more readily affected by the enzymes than the cuticular material found on extracted teeth. M e c k e l suggests that denaturation and polymerization of the cuticular proteins with increasing age of the film could account for its greater resistance. 5
Page
DENTAL PLAQUE
The staining reactions of the cuticles suggest that they are composed of modified mucoproteins or glycoproteins from saliva together with lipid material. The specific composition and the actual mechanism of formation remains to be established. Some further data on composition and several theories of formation w i l l be discussed later i n this papen The incidence of acquired cuticles in the subgingival plaque and the source of such material has not as yet been subjected to any systematic study. The recent interest in gingival fluid should act as a stimulus for such studies. M I C R O B I A L MASSES
In the mature plaque the microorganisms are the dominant components. Tremendous numbers of bacteria cluster together. Total microscopic counts indicate the presence of about 250 million organisms per mg. of wet weight of p l a q u e . ' Since a pure culture of streptococcus exhibits the same number to weight relationship, it is reasonable to assume that most of the weight of the plaque is due to bacteria. 8
9
8
Microscopic examination of the fully formed plaque usually discloses a mass of filaments at right angles to the cuticle near the enamel surface. A t the periphery, the filaments are curving and irregular. In the outer portion of the plaque, that is, the younger areas, cocci and other small bacterial forms can be noted. W i t h gram stain about two thirds of the plaque is usually gram positive. 10
When plaques are examined bacteriologically a different picture emerges. A l t h o u g h the filaments occupy so much volume, they are never the dominant organism on a quantitative basis. In fact, i n young plaques they are barely present at all. Although the actual number varies from study to study there seems to be general agreement that i n supragingival plaque gram positive cocci are the dominant organisms (40-50% ), followed by gram positive rods ( 1 0 - 4 0 % ) , gram negative rods ( 1 0 - 1 5 % ) .
7/359
Filaments have been reported as 4 % or less (down to almost 0% ) A 1 1
A n interesting observation, noted by us in 1 9 5 7 , and corroborated i n a number of studies, was that the proportion of cocci decreased and the number of filaments i n creased with age of the plaque. This morphological observation was recently quantitated bacteriologically by H o w e l l , R i z z o , and P a u l . The most dramatic change occurred between 14 and 21 days when the proportion of filamentous forms jumped from 4-18%. In our studies this change seemed to take place somewhat earlier. This may have been due to patient variation or to the inherent difficulty i n relating histological to bacteriological techniques. 12
1 1
W h e n material from the gingival crevice area (not true plaque but non-adherent material) was examined by Socransky et a l . and Gibbons et a l . , two major differences from supragingival plaque were noted. In the crevice Bacteroides melaninogenicus occurred at a level of about 5% (2.6% i n the normal group, 7.8% i n the periodontal). It rarely i f ever occurred i n supragingival plaque. This organism is especially interesting because it is a producer of collagenase. The second important difference was the presence of 1% spirochetes i n the crevice and only 0 - . l % i n supragingival areas. The rest of the flora was comparable. T o date no very significant differences have been found in the nature of the crevicular flora when periodontally normal and involved patients have been compared. There was a tendency for an increase i n numbers of bacteria per gram of wet weight i n the periodontally i n volved group (except for total streptococci), especially the spirochetes and B . melaninogenicus. N o etiologic significance could be deduced. The only really significant observation was the overall proliferation of the flora. The authors pointed out that "it was often difficult to remove 10 mg. of bacterial debris from the gingival crevice area of a 'normal' individual: however, one could obtain with ease 100 mg. or more of 'bacterial debris' from a periodontally involved individual." 1 3
14
1 3
Page
8/360
MANDEL INCLUSIONS
TABLE 1
O n occasion non-bacterial cells can be seen. These are usually shed epithelial cells or leukocytes i n varying stages of disruption. Fibrous material and nondescript food debris are also seen occasionally. Where plaques are adjacent to soft tissue (with resultant inflammatory changes) large numbers of cells (especially leukocytes) can be seen on the surface of the plaque.
Cuticular Material from Dentures*
10
CHEMICAL COMPOSITION
It is difficult to determine the chemical composition of dental plaque i n any definitive way because it is such a variable and polyglot material. Several laboratories, however, have undertaken the task of systematic analysis and some meaningful data is available. M i l l i n and S m i t h analyzed the material adhering to denture surfaces after thorough brushing i n a stream of water. The material remaining was virtually sterile and was probably cuticle or pellicle. F r o m the analytical findings (Table 1) it would appear that i f the cuticular material was salivary i n origin (which seems very likely) much of the carbohydrate was split off by the bacteria, leaving mainly the protein core and the closely associated hexoses. The calcium and phosphorous content is similar (as we shall see) to concentrations usually found i n early plaques.
% Dry
Weight
70.0 11.6 1.9 4.0 1.0
