J. Periodontal Res. 13: 17-23, 1978
Studies in plaque pathogenicity I. Plaque collection and limulus lysate screening of adherent and loosely adherent plaque D. H. EiNE, L. TABAK, H . OSHRAIN, A. SALKIND AND K. SIEGEL Division of Preventive Dentistry School of Dental and Oral Surgery Columbia University New York, New York, U.S.A. A study was undertaken to assay and compare the pathological potential of loosely adherent plaque (LAP) with that of adherent plaque (AP) using the limulus lysate assay (LLA). A technique was devised for collecting both AP and LAP from supra- as well as subgingival tooth areas under pyrogen-free conditions. Eive supragingival samples (AP & LAP) were collected from teeth (G.I. = < 1) after subjects had abstained from tooth cleaning for two weeks. Eive subgingival samples were collected from mylar strips one week after placement in periodontal pockets not less than 4 mm and not more than 7 mm in depth. All samples were lyophilized, reconstituted and analyzed for plaque mass by the quantitative ninhydrin reaction (Moore and Stein, 1954). Samples were analyzed for limulus lysate activity by a modification of the LLA of Levin and Bang (1964). Results from the five supragingival samples indicate that plaque mass, as determined by the ninhydrin reaction, was 4-5 fold greater in AP than in LAP. In the five subgingival samples, ninhydrin values for AP were two times higher than LAP. In spite of these differences in plaque mass, limulus activity in all LAP samples was consistently higher than in comparable AP samples when expressed as limulus activity per mole of amino acid. These preliminary findings suggest that subgingival LAP, situated in a position of strategic importance, is biologically active and deserves further study. (Accepted for publication March 4, 1977)
Introduction
Although it is generally accepted that adherent plaque plays an important role in the etiology of periodontal disease, little is known about the role of loosely adherent plaque in relation to this disease. Recently, several papers have suggested that various subgingival plaque bacteria, possessing characteristics of minimal adherence, could be involved in gingivitis Listgarten, Mayo & Tremblay 1975, Soames & Davies 1975) and periodontosis (Newman et al. 1973). On the basis of these
observations, a series of studies was undertaken to determine the pathological potential of loosely adherent plaque (LAP) and to compare this potential with that of adherent plaque (AP). To make this comparison it was necessary: 1) to develop a collection technique that would allow for the separation of AP from LAP; and 2) to develop an assay system to allow comparison of the pathological activity or potential of these materials. This paper will present the techniques
18
F I N E ,
T A B A K ,
O S H R A I N ,
Fig. 1. Photograph of the pyrogen-free maxillary mouthguard (M) with anterior window exposing four anterior teeth, Pyrogen-free funnel is used to collect wash of LAP.
used for collection of AP and LAP and the assay used to screen biological activity of collected samples. A technique for the specific determination of endotoxin in various plaque fractions and the chemotactic activity of plaque fractions will be presented in subsequent reports. Materials and Methods
All laboratory glassware was rendered pyrogen-free by heating to 180°C for three hours or by sterilization with ethylene oxide. All dilutions were made with sterile, pyrogen-free water, obtained from Abbott Laboratories (Chicago, 111.). 1) Sample collection from supragingival area Supragingival samples were taken primarily
S A L K I N D
Fig. 2. of the herent (LAP),
A N D S I E G E L
Diagram depicting the subgingivai placement mylar strip (S) and the accumulation of adplaque (AP) and loosely adherent plaque In vivo strip rests against root surface.
to determine the feasibility of the collection system and the sensitivity of the lysate assay to plaque samples. Five subjects with normal gingival health and having a gingival index of less than one (Loe & Silness 1963) were given a thorough prophylaxis to achieve a plaque index (Silness & Loe 1964) of zero. Custom mouthguards with a window exposing the labial surfaces of the maxillary incisors were constructed from study models (Fig. 1). Subjects abstained from all tooth cleaning procedures for two weeks. On the day of sampling the mouthguards were carefully inserted and the exposed four anterior teeth were washed with a gentle stream of pyrogen-free water delivered by syringe. Samples of loosely adherent plaque were collected in a test tube below a funnel held under the maxillary incisors (Fig. 1). Adherent plaque was
PLAOUE
a
•^9
PATHOGENICITY
19
0
Fig. 3. Diagram illustrating the strip removed with its adherent plaque (AP depicted as lines perpendicular to the strip) and loosely adherent plaque (LAP depicted as circles adjacent to the AP) intact (a); the strip being washed to remove the LAP (b); and, the strip being scraped to remove the AP (c).
collected with a curet from one of the maxillary incisors. 2) Sample collection from subgingival area Three subjects with periodontal pockets of less than 7 mm and greater than 4 mm were given a thorough prophylaxis and subgingival scaling and curettage to achieve a supragingival plaque index of zero. Five subgingival samples were collected from each of the three subjects by means of a mylar strip inserted subgingivally for one week (Oshrain, Salkind & Mandel 1968) (Fig. 2). The strip was carefully removed from the tooth and washed with pyrogenfree water for thirty seconds to collect nonadherent material (Fig. 3). 3) Sample processing a) Plaque mass measurement: All collected materials were frozen at — 20°C and then lyophilized. The lyophilized materials were then resuspended in 0.5 ml water, and boiled for no more than one minute. A
Fig. 4. Photograph of a positive (-F) and a negative
(-) limulus lysate test.
0.1 ml aliquot was hydrolyzed in 6 N HCl at 110°C for 18 hours in sealed tubes. The hydrolysate was dried in vacuo over solid NaOH pellets. The quantitative ninhydrin reaction measuring total amino acid content colorimetrically (Moore & Stein 1954) was used as an estimate of plaque mass. b) Limulus Lysate Activity Testing: A second 0.1 ml aliquot was tested with the limulus lysate assay, as described by Levin & Bang (1964) and modified by Wildfeuer et al. (1974). A 0.1 ml volume of limulus lysate (obtained from Associates of Cape Cod, Inc., Woods Hole, Mass.) was added to each 0.1 ml sample and allowed to incubate at 37°C in a water bath for one hour, and then for 15 minutes at 4°C. Tubes were inverted 180° and results were recorded as follows: + , firm adherent clot; ±, incomplete clot with a definite opacity, granularity and viscosity; —, no change in opacity, granularity and viscosity (Fig. 4). Appropriate controls of standard endotoxin (Klebsiella pneumoniae, obtained from the
20
F I N E ,
T A B A K ,
O S H R A I N ,
S A L K I N D
A N D S I E G E L
Table 1 Ninhydrin values and LLA from supragingival AP and LAP Limulus lysate activity' (LLA)
AA X 10-'
1:100
1:1000
ri8* Li9**
25,85 88.70
-1-
Li3**
146,00 33.75
M4* Ii6**
552,00 84,35
+
-1- -t-
r24*
635,00 51,70
-1-
4-
-t-
+
4-
MO*
1:100,000
4-
-
' - Standard - K. pneumoniae endotoxin active at 10.0 ng/ml * AP ** LAP 1 X 10* *«* 1 1 A\/
11 e
— niA
1.4 2.3 ,45 1,13
4-/-
-1- -1-
L22**
1 :10,000
++
-1-
++
Li5**
70,85 43,85
-1- +
ri2*
1:10
LLA value*** = LLA/moles AA X 10"
_
.6 2.9
++
Moles
Sample ^
4-
.18 11.8
4-
44-
1.6 19
V ini2
70,85 X 10-» [ = samples from the same strip
Food and Drug Administration), and water blanks were run with each assay. All assays were performed in triplicate and read double blind. Results
1) Plaque mass measurement All plaque mass-ninhydrin values that follow are expressed in moles of amino acid times 10-8. In the supragingival region, adherent plaque collected from the coronal surface after two weeks of abstaining from tooth cleaning had a mean relative plaque mass of 285.94 per tooth, as determined by the quantitative ninhydrin reaction. Supragingival loosely adherent plaque had a mean ninhydrin value of 60.47 for four anterior teeth, or 15.12 per tooth surface (Tables 1 and 3). In the subgingival region adherent plaque collected from a mylar strip placed subgingivally in a 7-mm pocket for one week had a mean relative cell mass of 140.99
per tooth, approximately half the value obtained after two weeks supragingivally. On the other hand, subgingival loosely adherent plaque collected after one week had a mean ninhydrin value of 95.31 per tooth, approximately six times the value obtained after two weeks from the supragingival region (Tables 2 and 3). 2) Limulus lysate activity testing The limulus lysate activity value (LLAV) was determined by dividing the highest dilution of the sample that had yielded a positive limulus test by the moles of amino acid of that sample. Sample number twelve (Table 1) provides an example of the calculation used. In this case, LLAV = LLA/ 1 X 104
mole A.A. =
- = .014 X 1012
70.85 X 10-8 or 1.4 X 1010 units of activity/mole. Values determined in this manner show that LAP activity levels were consistently higher thar those of AP and ranged from approximate
PLAQUE
21
PATHOGENICITY
Table 2 Ninhydrin values and LLA from subgingival AP and LAP
1:100
1:1,000
1:10,000
1:100,000
LLA value*** LLA/moles AA X 10"
4-
222
Moles Sample AA X 10-«
8**
238,50 45,00
44-
44-
44-
44-
r32* L30**
84,35 47,25
44-
44-
44-
+/
r36* L34**
123,65 217,00
44-
44-
44-
+/
r55*
44-
44-
44-
_
[56**
146,00 71.70
.07 .14
r52* [51**
112,45 95,60
4-
44-
4-/-
—
,009 ,11
.42
.12 .21
4-/
.08 .46
4-
4-
' Standard - K. pneumoniae endotoxin active at 10,0 ng/ml * AP ** LAP 1 X
*** LLAV = LLA/moles AA; e,g, sample* 7;
-^'
,0042 X
238,50 X
[ = samples from the same strip
ly two to sixty times higher in the five supragingival and five subgingival samples (Tables 1 and 2). Discussion
Dramatic differences appear to exist between the supragingival adherent and loosely adherent samples in terms of relative plaque mass/tooth surface. This result could be expected, in view of the constant ex-
posure of supragingival plaque to the mechanical cleansing afforded by the tongue, muscle action, and salivary flow. The higher levels of plaque mass found in the subgingival region for loosely adherent plaque as compared to that collected from the supragingival region (Table 3) is consistent with this view. In the subgingival area, as a result of the protected confines of the sulcular or pocket environment, many more motile non-adherent organisms can survive.
Table 3 Total and mean ninhydrin values for AP and LAP Supragingival
AP LAP
Subgingival
Total'
Mean/tooth surface^
Total'
Mean/tooth surface'
1429,70 302.35
285,94 15.12"
704,95 476,53
140,99 95,31
addition of all AP* and LAP** ninhydrin values from Table 1 addition of all AP* and LAP** ninhydrin values from Table 2 Total divided by the number of samples (5) (* = supragingival LAP divided by 20 (5 x 4) to achieve mean/tooth surface score)
22
F I N E ,
T A B / V K ,
O S H R A I N ,
sheltered from mechanical oral cleansing mechanisms. Loosely adherent plaque is most probably a collection of motile, anaerobic, amino acid utilizing organisms (Listgarten, Mayo & Tremblay 1975). The sulcus or pocket provides shelter, a reduced atmosphere with little or no oxygen and the amino acids necessary for growth of these fastidious microorganisms (Loesche 1968). In general, it appears that plaque mass does not correlate with the level of limulus lysate activity obtained (see Table 2, samples 55 and 56). Comparison of adherent with loosely adherent supragingival plaque reveals an equivalent limulus lysate activity despite a four times greater plaque mass in adherent plaque (Tables 1 and 3). Limulus lysate activity has been shown to have a statistically significant correlation with B-cell mitogenic and/or pyrogenic activity (Elin, Sandberg & Rosenstreich 1976; Johnson et al. 1976). Results of our study suggest that LAP may possess higher levels of mitogenic and pyrogenic activity than AP, as indicated by limulus activity per mole of amino acid of plaque. Previous claims that the limulus lysate assay was a specific assay for endotoxin presence (Levin et al. 1972) or that plaque stimulation of limulus lysate activity was the exclusive property of endotoxin (Shapiro et al. 1972) are inaccurate. In addition to endotoxin, substances such as ribonucleic acid (Elin & Wolff 1973) and peptidoglycans (Wildfeuer et al. 1975) have been shown to provoke a positive limulus test. Our results support the work of Cobb & Brown (1968), Powell (1969), Baboolal, Mlinek & Powell (1970), Simon et al. (1970) and Shapiro et al. (1972), and add the suggestion that material from loosely adherent as well as adherent plaque could play a role in the pathogenesis of periodontal inflammation. Loosely adherent material may have a
S A L K I N D
A N D
S I E G E L
significant pathological potential in the subgingival region. This is especially important when one takes into consideration the positional relationship of loosely adherent plaque to the sulcular epithelium. Whereas adherent plaque, by definition, adheres to the tooth and root surface, the bulk of loosely adherent plaque lies adjacent to the vulnerable sulcular epithelium.. A pathologically active material in this position could be responsible for tissue destruction. Therefore, one could speculate that removal of this less adherent plaque could have a clinical effect. A water irrigation device, capable of removing loosely adherent plaque, could conceivably have both beneficial and destructive effects. If the sulcular epithelium is ulcerated, the loosely adherent plaque could be forced subgingivally. If the sulcular epithelium is intact, removal of LAP could significantly decrease the susceptibility of the sulcular tissue to its inflammatory potential. A recent study has suggested that the use of a water irrigation device will reduce levels of gingival inflammation in an experimental gingivitis design (Wheatcroft & Sciantarelli 1974). Since adherent plaque is resistant to removal by water spray, it is possible that removal of this loosely adherent plaque could be responsible for the improvement reported.
Acknowledgements
This study was supported in part by Public Health Service Grant #DE 01554-15 from the National Institute of Dental Research and by a grant from the Colgate-Palmolive Company. The authors are indebted to Professor I. D. Mandel for his assistance throughout the study, to Jeanne Cole for her help editing the manuscript, and to Mrs. Zoila Noguerole for her typing help.
PLAOUE
PATHOGENICITY
References
Baboolal, R., Mlinek, A. & Powell, R. N. 1970. A study of the effects of gingival plaque extracts on cells cultured in vitro. J. Periodontal Res. 5: 248-254. Cobb, C. M. & Brown, L. R. 1967. The effects of exudate from the periodontal pocket on cell culture. Periodontics 5: 5-18. Elin, R. J., Sandberg, A. L. & Rosenstreich, D. L. 1976. Comparison of the pyrogenicity, limulus activity, mitogenicity and complement reactivity of several bacterial endotoxins and related compounds. J. Immunol. 117: 1238-1242. Elin. R. J. & Wolf, S. M. 1973. Nonspecificity of the limulus amebocyte lysate test: positive reactions with polynucleotides and proteins. /. Infect. Dis. 128: 349-352. Johnson, D. A., Chen, C. H., Dombrowski, J. C. & Nowotny, A. 1976. Role of bacterial products in periodontitis. I. Endotoxin content and immunogenicity of human plaque. J. Periodontal Res. 11: 349-359. Levin, J, & Bang, F. B. 1964. The role of endotoxin in the extracellular coagulation of limulus blood. Bull. Johns Hopkins Hosp. 115: 265-274. Levin, J., Boore, T. E., Young, N. S., Margolis, S., Zauber, N. P., Townes, A. S. & Bell, W. R. 1972. Gram-negative sepsis: detection of endotoxemia with the limulus test. Ann. Intern. Med. 76: 1-7. Loe, H. & Silness, J. 1963. Periodontal diease in pregnancy. I. Prevalence and severity. Acta odont. Scand. 21: 533-551. Listgarten, M. A., Mayo, H. E. & Tremblay, R. 1975. Development of dental plaque on epoxy resin crowns in man. /. Periodontol. 46: 10-26. Loesche, W. J. 1968. Importance of nutrition in gingival crevice microbial ecology. Periodontics 6: 245-249. Moore, S. & Stein, W. H. 1954. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. /. Biol. Chem. 111. 907-913. Newman, M., Williams, R., Crawford. A,, Manganiello, A. D. & Socransky, S. S. 1973.
23
Predominant cultivable microbiota of periodontitis and periodontosis. III. Periodontosis. J. Dent. Res., 52, I.A.D.R. Abstr., No. 290. Oshrain, H., Salkind, A. & Mandel, I. D. 1968. A method for collection of subgingival plaque and calculus. /. Periodontol. 39: 322-325. Powell, R. N. 1969. The effect of bacterial plaque on gingival epithelium in vitro. Dent. Practit. dent. Rec. 20: 139-142. Shapiro, L., Lodato, E. M., Courant, P. R. & Stallard, R. E. 1972. Endotoxin determinations in gingival inflammation. J. Periodontol. 42: 591-596. Silness, J. & Loe, H. 1964. Periodontal disease in pregnancy. II, Correlation between oral hygiene and periodontal condition. Acta odont. Scand. 11: 121-135. Simon, B., Goldman, H., Ruben, H. & Baker, E. 1970. The role of endotoxin in periodontal disease. II. Correlation of the amount of endotoxin in human gingival exudate with the clinical degree of inflammation. J. Periodontol. 41: 81-86. Soames, J. V. & Davies, R. M. 1975. The structure of subgingival plaque in a beagle dog. J. Periodontal Res. 9: 333-341. Wheatcroft, M, G. & Sciantarelli, E. 1974. The effect of oral water irrigation on the prevention of gingival inflammation. J. Am. Soc. Prev. Dent. 4: 38-39. Wildfeuer, A., Heymer, B., Spilker, D., Schleifer, K.-H., Vanek, E. & Haferkamp, O. 1975. Use of limulus assay to compare the biological activity of peptidoglycan and endotoxin. Z. lmmun.-forsch. Bd. 149: S258-264. Wildfeuer, A., Heymer, B., Schleifer, K. H. & Haferkamp, O. 1974. Investigations on the specificity of the limulus test for the detection of endotoxin. Appl. Microbiol. 28: 867871.
Address: Division of Preventive Dentistry School of Dental and Oral Surgery Columbia University New York, N.Y. 10032 U.S.A.
Studies in plaque pathogenicity I. Plaque collection and limulus lysate screening of adherent and loosely adherent plaque D. H. EiNE, L. TABAK, H . OSHRAIN, A. SALKIND AND K. SIEGEL Division of Preventive Dentistry School of Dental and Oral Surgery Columbia University New York, New York, U.S.A. A study was undertaken to assay and compare the pathological potential of loosely adherent plaque (LAP) with that of adherent plaque (AP) using the limulus lysate assay (LLA). A technique was devised for collecting both AP and LAP from supra- as well as subgingival tooth areas under pyrogen-free conditions. Eive supragingival samples (AP & LAP) were collected from teeth (G.I. = < 1) after subjects had abstained from tooth cleaning for two weeks. Eive subgingival samples were collected from mylar strips one week after placement in periodontal pockets not less than 4 mm and not more than 7 mm in depth. All samples were lyophilized, reconstituted and analyzed for plaque mass by the quantitative ninhydrin reaction (Moore and Stein, 1954). Samples were analyzed for limulus lysate activity by a modification of the LLA of Levin and Bang (1964). Results from the five supragingival samples indicate that plaque mass, as determined by the ninhydrin reaction, was 4-5 fold greater in AP than in LAP. In the five subgingival samples, ninhydrin values for AP were two times higher than LAP. In spite of these differences in plaque mass, limulus activity in all LAP samples was consistently higher than in comparable AP samples when expressed as limulus activity per mole of amino acid. These preliminary findings suggest that subgingival LAP, situated in a position of strategic importance, is biologically active and deserves further study. (Accepted for publication March 4, 1977)
Introduction
Although it is generally accepted that adherent plaque plays an important role in the etiology of periodontal disease, little is known about the role of loosely adherent plaque in relation to this disease. Recently, several papers have suggested that various subgingival plaque bacteria, possessing characteristics of minimal adherence, could be involved in gingivitis Listgarten, Mayo & Tremblay 1975, Soames & Davies 1975) and periodontosis (Newman et al. 1973). On the basis of these
observations, a series of studies was undertaken to determine the pathological potential of loosely adherent plaque (LAP) and to compare this potential with that of adherent plaque (AP). To make this comparison it was necessary: 1) to develop a collection technique that would allow for the separation of AP from LAP; and 2) to develop an assay system to allow comparison of the pathological activity or potential of these materials. This paper will present the techniques
18
F I N E ,
T A B A K ,
O S H R A I N ,
Fig. 1. Photograph of the pyrogen-free maxillary mouthguard (M) with anterior window exposing four anterior teeth, Pyrogen-free funnel is used to collect wash of LAP.
used for collection of AP and LAP and the assay used to screen biological activity of collected samples. A technique for the specific determination of endotoxin in various plaque fractions and the chemotactic activity of plaque fractions will be presented in subsequent reports. Materials and Methods
All laboratory glassware was rendered pyrogen-free by heating to 180°C for three hours or by sterilization with ethylene oxide. All dilutions were made with sterile, pyrogen-free water, obtained from Abbott Laboratories (Chicago, 111.). 1) Sample collection from supragingival area Supragingival samples were taken primarily
S A L K I N D
Fig. 2. of the herent (LAP),
A N D S I E G E L
Diagram depicting the subgingivai placement mylar strip (S) and the accumulation of adplaque (AP) and loosely adherent plaque In vivo strip rests against root surface.
to determine the feasibility of the collection system and the sensitivity of the lysate assay to plaque samples. Five subjects with normal gingival health and having a gingival index of less than one (Loe & Silness 1963) were given a thorough prophylaxis to achieve a plaque index (Silness & Loe 1964) of zero. Custom mouthguards with a window exposing the labial surfaces of the maxillary incisors were constructed from study models (Fig. 1). Subjects abstained from all tooth cleaning procedures for two weeks. On the day of sampling the mouthguards were carefully inserted and the exposed four anterior teeth were washed with a gentle stream of pyrogen-free water delivered by syringe. Samples of loosely adherent plaque were collected in a test tube below a funnel held under the maxillary incisors (Fig. 1). Adherent plaque was
PLAOUE
a
•^9
PATHOGENICITY
19
0
Fig. 3. Diagram illustrating the strip removed with its adherent plaque (AP depicted as lines perpendicular to the strip) and loosely adherent plaque (LAP depicted as circles adjacent to the AP) intact (a); the strip being washed to remove the LAP (b); and, the strip being scraped to remove the AP (c).
collected with a curet from one of the maxillary incisors. 2) Sample collection from subgingival area Three subjects with periodontal pockets of less than 7 mm and greater than 4 mm were given a thorough prophylaxis and subgingival scaling and curettage to achieve a supragingival plaque index of zero. Five subgingival samples were collected from each of the three subjects by means of a mylar strip inserted subgingivally for one week (Oshrain, Salkind & Mandel 1968) (Fig. 2). The strip was carefully removed from the tooth and washed with pyrogenfree water for thirty seconds to collect nonadherent material (Fig. 3). 3) Sample processing a) Plaque mass measurement: All collected materials were frozen at — 20°C and then lyophilized. The lyophilized materials were then resuspended in 0.5 ml water, and boiled for no more than one minute. A
Fig. 4. Photograph of a positive (-F) and a negative
(-) limulus lysate test.
0.1 ml aliquot was hydrolyzed in 6 N HCl at 110°C for 18 hours in sealed tubes. The hydrolysate was dried in vacuo over solid NaOH pellets. The quantitative ninhydrin reaction measuring total amino acid content colorimetrically (Moore & Stein 1954) was used as an estimate of plaque mass. b) Limulus Lysate Activity Testing: A second 0.1 ml aliquot was tested with the limulus lysate assay, as described by Levin & Bang (1964) and modified by Wildfeuer et al. (1974). A 0.1 ml volume of limulus lysate (obtained from Associates of Cape Cod, Inc., Woods Hole, Mass.) was added to each 0.1 ml sample and allowed to incubate at 37°C in a water bath for one hour, and then for 15 minutes at 4°C. Tubes were inverted 180° and results were recorded as follows: + , firm adherent clot; ±, incomplete clot with a definite opacity, granularity and viscosity; —, no change in opacity, granularity and viscosity (Fig. 4). Appropriate controls of standard endotoxin (Klebsiella pneumoniae, obtained from the
20
F I N E ,
T A B A K ,
O S H R A I N ,
S A L K I N D
A N D S I E G E L
Table 1 Ninhydrin values and LLA from supragingival AP and LAP Limulus lysate activity' (LLA)
AA X 10-'
1:100
1:1000
ri8* Li9**
25,85 88.70
-1-
Li3**
146,00 33.75
M4* Ii6**
552,00 84,35
+
-1- -t-
r24*
635,00 51,70
-1-
4-
-t-
+
4-
MO*
1:100,000
4-
-
' - Standard - K. pneumoniae endotoxin active at 10.0 ng/ml * AP ** LAP 1 X 10* *«* 1 1 A\/
11 e
— niA
1.4 2.3 ,45 1,13
4-/-
-1- -1-
L22**
1 :10,000
++
-1-
++
Li5**
70,85 43,85
-1- +
ri2*
1:10
LLA value*** = LLA/moles AA X 10"
_
.6 2.9
++
Moles
Sample ^
4-
.18 11.8
4-
44-
1.6 19
V ini2
70,85 X 10-» [ = samples from the same strip
Food and Drug Administration), and water blanks were run with each assay. All assays were performed in triplicate and read double blind. Results
1) Plaque mass measurement All plaque mass-ninhydrin values that follow are expressed in moles of amino acid times 10-8. In the supragingival region, adherent plaque collected from the coronal surface after two weeks of abstaining from tooth cleaning had a mean relative plaque mass of 285.94 per tooth, as determined by the quantitative ninhydrin reaction. Supragingival loosely adherent plaque had a mean ninhydrin value of 60.47 for four anterior teeth, or 15.12 per tooth surface (Tables 1 and 3). In the subgingival region adherent plaque collected from a mylar strip placed subgingivally in a 7-mm pocket for one week had a mean relative cell mass of 140.99
per tooth, approximately half the value obtained after two weeks supragingivally. On the other hand, subgingival loosely adherent plaque collected after one week had a mean ninhydrin value of 95.31 per tooth, approximately six times the value obtained after two weeks from the supragingival region (Tables 2 and 3). 2) Limulus lysate activity testing The limulus lysate activity value (LLAV) was determined by dividing the highest dilution of the sample that had yielded a positive limulus test by the moles of amino acid of that sample. Sample number twelve (Table 1) provides an example of the calculation used. In this case, LLAV = LLA/ 1 X 104
mole A.A. =
- = .014 X 1012
70.85 X 10-8 or 1.4 X 1010 units of activity/mole. Values determined in this manner show that LAP activity levels were consistently higher thar those of AP and ranged from approximate
PLAQUE
21
PATHOGENICITY
Table 2 Ninhydrin values and LLA from subgingival AP and LAP
1:100
1:1,000
1:10,000
1:100,000
LLA value*** LLA/moles AA X 10"
4-
222
Moles Sample AA X 10-«
8**
238,50 45,00
44-
44-
44-
44-
r32* L30**
84,35 47,25
44-
44-
44-
+/
r36* L34**
123,65 217,00
44-
44-
44-
+/
r55*
44-
44-
44-
_
[56**
146,00 71.70
.07 .14
r52* [51**
112,45 95,60
4-
44-
4-/-
—
,009 ,11
.42
.12 .21
4-/
.08 .46
4-
4-
' Standard - K. pneumoniae endotoxin active at 10,0 ng/ml * AP ** LAP 1 X
*** LLAV = LLA/moles AA; e,g, sample* 7;
-^'
,0042 X
238,50 X
[ = samples from the same strip
ly two to sixty times higher in the five supragingival and five subgingival samples (Tables 1 and 2). Discussion
Dramatic differences appear to exist between the supragingival adherent and loosely adherent samples in terms of relative plaque mass/tooth surface. This result could be expected, in view of the constant ex-
posure of supragingival plaque to the mechanical cleansing afforded by the tongue, muscle action, and salivary flow. The higher levels of plaque mass found in the subgingival region for loosely adherent plaque as compared to that collected from the supragingival region (Table 3) is consistent with this view. In the subgingival area, as a result of the protected confines of the sulcular or pocket environment, many more motile non-adherent organisms can survive.
Table 3 Total and mean ninhydrin values for AP and LAP Supragingival
AP LAP
Subgingival
Total'
Mean/tooth surface^
Total'
Mean/tooth surface'
1429,70 302.35
285,94 15.12"
704,95 476,53
140,99 95,31
addition of all AP* and LAP** ninhydrin values from Table 1 addition of all AP* and LAP** ninhydrin values from Table 2 Total divided by the number of samples (5) (* = supragingival LAP divided by 20 (5 x 4) to achieve mean/tooth surface score)
22
F I N E ,
T A B / V K ,
O S H R A I N ,
sheltered from mechanical oral cleansing mechanisms. Loosely adherent plaque is most probably a collection of motile, anaerobic, amino acid utilizing organisms (Listgarten, Mayo & Tremblay 1975). The sulcus or pocket provides shelter, a reduced atmosphere with little or no oxygen and the amino acids necessary for growth of these fastidious microorganisms (Loesche 1968). In general, it appears that plaque mass does not correlate with the level of limulus lysate activity obtained (see Table 2, samples 55 and 56). Comparison of adherent with loosely adherent supragingival plaque reveals an equivalent limulus lysate activity despite a four times greater plaque mass in adherent plaque (Tables 1 and 3). Limulus lysate activity has been shown to have a statistically significant correlation with B-cell mitogenic and/or pyrogenic activity (Elin, Sandberg & Rosenstreich 1976; Johnson et al. 1976). Results of our study suggest that LAP may possess higher levels of mitogenic and pyrogenic activity than AP, as indicated by limulus activity per mole of amino acid of plaque. Previous claims that the limulus lysate assay was a specific assay for endotoxin presence (Levin et al. 1972) or that plaque stimulation of limulus lysate activity was the exclusive property of endotoxin (Shapiro et al. 1972) are inaccurate. In addition to endotoxin, substances such as ribonucleic acid (Elin & Wolff 1973) and peptidoglycans (Wildfeuer et al. 1975) have been shown to provoke a positive limulus test. Our results support the work of Cobb & Brown (1968), Powell (1969), Baboolal, Mlinek & Powell (1970), Simon et al. (1970) and Shapiro et al. (1972), and add the suggestion that material from loosely adherent as well as adherent plaque could play a role in the pathogenesis of periodontal inflammation. Loosely adherent material may have a
S A L K I N D
A N D
S I E G E L
significant pathological potential in the subgingival region. This is especially important when one takes into consideration the positional relationship of loosely adherent plaque to the sulcular epithelium. Whereas adherent plaque, by definition, adheres to the tooth and root surface, the bulk of loosely adherent plaque lies adjacent to the vulnerable sulcular epithelium.. A pathologically active material in this position could be responsible for tissue destruction. Therefore, one could speculate that removal of this less adherent plaque could have a clinical effect. A water irrigation device, capable of removing loosely adherent plaque, could conceivably have both beneficial and destructive effects. If the sulcular epithelium is ulcerated, the loosely adherent plaque could be forced subgingivally. If the sulcular epithelium is intact, removal of LAP could significantly decrease the susceptibility of the sulcular tissue to its inflammatory potential. A recent study has suggested that the use of a water irrigation device will reduce levels of gingival inflammation in an experimental gingivitis design (Wheatcroft & Sciantarelli 1974). Since adherent plaque is resistant to removal by water spray, it is possible that removal of this loosely adherent plaque could be responsible for the improvement reported.
Acknowledgements
This study was supported in part by Public Health Service Grant #DE 01554-15 from the National Institute of Dental Research and by a grant from the Colgate-Palmolive Company. The authors are indebted to Professor I. D. Mandel for his assistance throughout the study, to Jeanne Cole for her help editing the manuscript, and to Mrs. Zoila Noguerole for her typing help.
PLAOUE
PATHOGENICITY
References
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Address: Division of Preventive Dentistry School of Dental and Oral Surgery Columbia University New York, N.Y. 10032 U.S.A.
