35
Bacterial Penetration of Gingiva in the Adult Beagle Dog With Periodontitis L.H. Silverstein,
*
G.S. Schuster, f J.J. Garnick,
*
and B.
Singhf
Invasion of gingival tissues by bacteria is thought to be a major factor in development of periodontal lesions. Morphologic studies have revealed bacteria within the pocket epithelium, gingival connective tissues, alveolar bone, and oral epithelium. The current studies are intended to determine whether they are present in healthy and diseased tissues and to identify the microbial source. Five beagle dogs with naturally occurring Periodontitis were fed a soft diet. Two quadrants of the dentition of each dog were cleaned regularly and health maintained while others were allowed to develop further disease. After 4 weeks, samples of gingiva were removed both prior to and after the animals were sacrificed by perfusion fixation attempting not to introduce microorganisms. The identical samples were examined by light and scanning electron microscopy for numbers and location of organisms. Their numbers were greater in diseased than in healthy tissues. Furthermore, the quantity was greater in samples taken prior to perfusion fixation than after such treatment, especially in the oral epithelium. The results suggest that normally there are some microorganisms present within the periodontal tissues and that their numbers are greater in diseased tissues. However, the data also indicated that significant numbers may be introduced into the tissues during sampling of unfixed tissues. J Periodontol 1990;61:35-41.
Key Words: Gingiva/microbiology, periodontitis/microbiology, periodontitis/etiology.
of periodontal disease is bacterial loss of connective tissue attachment occurs in only 5% of the inflamed sites containing plaque.2'3 One cause of these active periodontal lesions, especially in localized juvenile Periodontitis, is thought to be invasion of the gingival tissues by bacteria.4 In studies of this disease, root planing alone was not considered adequate to remove the etiologic bacteria, and periodontal surgery and/or antibiotics were recommended.5 Manor et al.6 demonstrated bacterial penetration of tissue in 4 of 7 specimens from 4 patients with advanced adult Periodontitis. In other studies of this condition morphologically identifiable microorganisms were observed in the gingiva by light transmission and scanning electron microscopy, reinforcing the concept of a role for intratissue bacteria in the disease process.4-7 Bacteria have been reported to be specifically located in the pocket epithelium,7-11 gingival connective tissue,9'12-14 alveolar bone,8,15 and oral epithelium.16'17 Several studies have implicated different forms and species of bacteria in adult forms of periodontal disease.11-14'16'17 For example, The
primary
cause
plaque,1 however,
•Department of Pcriodontics and Periodontology Section, Dental Research Center, School of Dentistry, Medical College of Georgia, Augusta, Georgia. tDcpartment of Oral Biology/Microbiology. +Dcpartmcnt of Oral Pathology.
Frank and Voegel15 reported a gram-negative flora associated with extensive bone résorption in 6 of 12 patients with adult Periodontitis. However, knowledge of the true prevalence of bacteria in periodontally diseased tissues is
limited.4
Immunocytochemical studies have suggested that bacterial invasion may be an important mechanism in the pathogenesis of the active periodontal lesion.11-14'16,17 While the methods used in the above studies allowed evaluation of the presence of specific antigens in the tissues, they did not indicate whether the antigens represent viable or nonviable bacterial cells or wall fragments. Disruptions of the gingival basement have been reported7,8-18'19 in Periodontitis and may be associated with bacterial invasion. In contrast to these reports, Erickson et al.20 using microscopic observations concluded that at most sites the subgingival microbiota appeared to be confined to the periodontal pocket. Other studies supported these results.21,22 Since bacterial invasion may be an important feature in the destruction of the periodontium during the active phases of Periodontitis,15 and the sources of these organisms are not known, the purpose of the current project was' to determine whether the presence of bacteria in the corium of the lateral wall of the periodontal pocket is a result of 1) past bacterial invasion of the tissue; 2) translocation into
J Periodontol 36
January 1990
BACTERIAL PENETRATION OF GINGIVA
tissues from the pocket during the biopsy procedures; organisms that exist indigenously within the tissues.
or
3)
MATERIALS AND METHODS Five adult female beagle dogs, average weight 13.8 Kg (10.7 to 18.5 Kg), with naturally occurring Periodontitis were fed a standard pellet dog food and water ad libitum. Prior to the experimental procedures, two quadrants of each dog, chosen at random, were treated by scaling and root planing and daily tooth brushing for 4 weeks in order to restore gingival health (control quadrants). During the instrumentation, the dogs were anesthetized with 23 mg/kg intravenous Pentothal. The dentition of the control quadrants was cleaned 5 times a week using a soft tooth brush and a Perio-Aid for the interproximal areas.23 The remaining two quadrants were left untreated (experimental quadrants). During this period, the dogs were placed on a soft diet consisting of the standard pellet which was soaked in water to obtain a mushy consistency24 permitting gross accumulation of plaque and calculus on teeth of the experimental quadrants. In the experimental procedures which immediately followed the 4 week preliminary period, the teeth and the adjacent tissues in the control or experimental quadrants were biopsied either before animals had been sacrificed, or after sacrifice by perfusion fixation. Perfusion fixation techniques were utilized in this study in order to minimize the translocation of microorganisms during the surgical manipulation of the tissues during the biopsy procedures. The perfusion fixation technique was a modification of that reported by McKinney et al.25 The dog was anesthetized with Pentothal (23 mg/kg). Perfusion fixation was accomplished using a carotid cut-down procedure in which the common carotid arteries were isolated, ligated, cannulated, and subsequently perfused with sterile heparinized saline followed by filtration sterilized 2% glutaraldehyde buffered with 0.2 M sodium phosphate at pH 7.2, made 24 hours prior to perfusion. The external jugulars were incised to permit outflow of saline and fixative. The biopsy procedure utilized two vertical incisions made the mesio-buccal and disto-buccal line angles of the specat imen tooth. A horizontal incision was then made 3 mm apical to the previously determined deepest probing depth connecting the vertical incisions. The adjacent tissue at the incision line was bluntly dissected away from the facial gingival tissue of the tooth, being sure not to manipulate that tissue. Following this, a high speed handpiece with a 557 carbide bur was used to make two vertical channels halfway through the alveolar bone in a buccal-lingual direction, then a horizontal channel at the incision line was made to connect the two vertical channels. Next, a mesiodistal occlusal channel was placed through the occlusal groove and extended apically, connecting the two vertical and horizontal channels (Fig. 1). The tooth specimen was then removed with a hemostat, grasping the crown of the tooth and taking care not to disturb the attached soft tissue. The
Figure 1: The appearance of the tissue biopsy just before removal from jaw. Bone channels were made buccal-lingually one-half the distance of the tooth at the mesial, distal, and apical border of the biopsy. A vertical groove made at the occlusal surface connected the three channels and freed the tooth segment and tissue specimen from the jaw. The specimen was then fixed with sterile buffered glutaraldehyde for 24 hours. Afterward, the tissue was removed from the tooth segment and processed.
specimen was then immediately placed into sterile buffered glutaraldehyde fixative for 24 hours. Following fixation, specimens were placed in a 0.2 M phosphate buffer at pH 7.2 and the tissue biopsy was carefully incised free from the tooth segment. A total of 16 biopsies were obtained from each dog: 8 specimens, 4 each of health and disease prior to perfusion fixation; and 8 specimens, 4 each of health and disease after perfusion fixation. Biopsy specimens were dehydrated in alcohol, embedded in paraffin and sectioned in a bucco-lingual plane at
5 µ
intervals. Bacteria
were
located in these sections
by staining using a modification of the gram stain technique described by Garvey et al.,26 substituting Carbol Fuschin for New Fuschin.
Histometric Analysis The analysis included a determination of the location and the numbers of bacterial cells in different compartments of the gingival tissue. The compartments were designated the dental epithelium, oral epithelium, and connective tissue. The base of the compartments was an imaginary line perpendicular to the tooth surface at the most apical extension of the junctional epithelium. The border of the dental and oral epithelium was an imaginary line from the gingival margin perpendicular to the base line (Fig. 2). Upon completion of the gram-staining procedures, the specimens were examined to determine the number of bacteria in the compartments. Using a computer-assisted digitizing system, the area of each of these compartments was
Volume 61 Number 1
SILVERSTEIN, SCHUSTER, GARNICK, SINGH
37
down to fit onto an SEM mounting stub. The cover slip then removed from the glass slide, without disturbing the surface topography of the specimen as recently described by Stefiik and co-workers.29 This specimen was then dehydrated with ethanol, placed in Freon, critical point dried with liquid carbon dioxide, sputter coated with gold, and placed in the SEM. Observations were made at magnifications of 350 x, 1000 x, and 5000 x. The acetate tracing previously obtained from the histologie section at low magnification was placed on the monitor of the SEM. The magnification was adjusted so that the image of the specimen on the monitor matched this acetate tracing. The areas of bacteria indicated on the acetate sheet were located on the SEM monitor, then one or more of these areas were centered on the screen and magnification increased until the image on the monitor matched the higher magnification acetate tracing. This image was further magnified till bacteria could be readily visualized. Once these steps had been completed, these areas were viewed at a series of higher magnifications to confirm that bacteria observed at the light microscopic level were actually bacteria under SEM. Statistical comparisons of bacterial counts were performed using a four way repeated measures analysis of variance taking into consideration the effects of health versus disease, time of biopsy either before or after fixation, tooth type, and tissue compartments. All tests were done at the 95% confidence level. The variability of the histometric methods was assessed by analyzing the differences between duplicate measurements or countings. The standard errors for bacterial cell counts for oral epithelium, connective tissue, and dental epithelium compartments of all groups were 6.86%, 5.95%, and 2.56% respectively. The standard errors for the area measurements of these compartments were 0.05%, 0.04% and 0.03% respectively.
was
um
Figure 2: A diagram illustrating the three compartments used in the histometric analysis. The boundaries were determined with a line perpendicular to the tooth surface at the base of the dental epithelium. The boundary between the dental and oral epithelia was a line perpendicular to the previous line. measured on the histologie sections at a magnification of The measurement system consisted of a Zenith Data 40 Systems computer with the MicroComp M2 Analysis Program* attached to an Olympus microscope and Hipadf digitizing tablet. Then at higher magnification (630 x), the individual bacteria within each of the three compartments were counted. .
Electron Microscopical (SEM) Evaluation In order to corroborate the presence and identification of bacteria seen in light microscopy, three sections from each of the four groups (diseased and healthy specimens and biopsies before and after perfusion) were processed for SEM. Subsequent to histometric analysis, an acetate sheet was placed over the monitor of the computer and the location of the bacteria was traced onto acetate sheets at various magnifications and in relation to the adjacent anatomic structure.27,28 The magnification was then increased and the area with located bacteria was traced onto a different acetate sheet. Upon completion of these tracings, this identical specimen was processed for SEM. Using a thin Flex diamond wafering disk the slide, immersed in xylene, was cut
Scanning
'Southern Microinstruments, Inc., Atlanta, GA. fHouston Electronics, Houston, TX
RESULTS Clinical Appearance of Dogs At the time of sacrifice, the control
(healthy) quadrants of each of the five dogs had plaque present only on a few teeth, calculus was not evident, and there were no signs of gingivitis. One week prior to sacrifice, the mean clinical crevice depth was 2.0 ± 0.44mm in control quadrants. In the experimental (diseased) quadrants, large amounts of plaque could be detected easily and mineralized deposits were regularly present both supragingivally and subgingivally. The periodontal pockets, whose mean probing depth was 5.4 ± 1.3mm, bled upon gentle probing and the tissues were found to be red, bulbous, and displayed clinical signs of gingival inflammation (Fig. 3). The difference in clinical pocket depth between control and experimental quadrants statistically significant (P < 0.05). gingival tissue biopsies sampled from the diseased quadrants of each dog contained periodontal lesions, the major portions of which were confined to the dento-gingival
was
The
J Periodontol 38
BACTERIAL PENETRATION OF GINGIVA
January 1990 smooth in appearance, regularly shaped and about 6 to 8 µ in diameter. This was true for specimens obtained before or after perfusion fixation. The inflammatory infiltrate was present within the epithelium and, to a lesser extent, in the adjacent connective tissue extending apical to the junctional epithelium.
Figure 3: Example of control and experimental quadrants just before the biopsy procedures. The healthy tissues were achieved by scaling, tooth polishing, and daily toothbrushing for 4 weeks.
Figure 4: Photomicrograph demonstrating bacteria in the gingival stroma. The specimen was stained with a modified gram stain. Bacteria are present at (a). Original magnification: x 1000.
region. The dental epithelium of the gingival tissue appeared thin, ulcerated in the marginal portion and in the more apical portions contained pronounced rete pegs. The connective tissue exhibited a fine to moderately dense collagenous stroma supporting a number of chronic inflammatory cells. The bacteria generally were clustered around blood vessels (Fig. 4). For the most part, the organisms appeared round in shape, resembling cocci, were rather
SEM Evaluation Specimens observed under high SEM magnification (5000 ) demonstrated that what we called bacteria under the light microscope actually were bacteria and not stained debris. SEM findings also confirmed that rods or filamentous forms of bacteria were not present in tissues in any of the samples, diseased or healthy (Fig. 5). Histometric Analysis The range of compartmental areas was 2.4 to 52.2 square µ in the oral epithelium, 1.3 to 53.3 square µ in the connective tissue and 1.6 to 43.3 square µ in the dental epithelial tissue compartments; there were no differences between control and experimental groups. The mean number of organisms per square µ in each tissue compartment, combining data for health and disease and before or after perfusion, was 4.40 ± 5.17; 3.79 ± 4.62; and 2.69 ± 4.32 per square µ for oral epithelium, connective tissue and dental epithelium compartments respectively. The differences were not statistically significant (P > 0.05). Table 1 shows the mean number of bacteria per square µ in each tissue compartment for healthy and for diseased tissue sampled before and after perfusion fixation. The results from the histometric measurements of the healthy specimens obtained before perfusion fixation indicated that a significantly greater number of bacteria (P < 0.05) was found in the gingival biopsies obtained before as compared with after perfusion fixation except for the dental epithelium compartment (P > 0.05). Similar results were evident before and after perfusion fixation in diseased specimens except that all compartments of diseased tissue specimens had greater numbers of bacteria per square µ of tissue before perfusion fixation (P< 0.05, Table 1). The diseased oral epithelium before perfusion contained a significantly greater number (P< 0.05) of bacteria per unit as compared to the other diseased tissue compartments. Biopsy after perfusion fixation of diseased tissues decreased the number of bacteria detectable per area to a greater extent within this compartment than in the dental epithelium (P< 0.05, Table 1). Also in a comparison between the diseased and healthy specimens obtained before fixation, the diseased specimens had a greater number of bacteria per area but the differences were not significant when comparable compartments were compared (P> 0.05, Table 1).
DISCUSSION Various studies have suggested indigenous bacteria, bacterial invasion, or penetration with the biopsy procedure as the source of organisms seen in periodontal tissues.17'21 As
Volume 61 Number 1
SILVERSTEIN, SCHUSTER, GARNICK, SINGH
Table 1: Mean Number of Bacteria/µ
2
in Various Tissue
Oral
Compartment Tissue and Time of Sampling
Healthy (before fixation) Healthy (after fixation) Diseased (before fixation) Diseased (after fixation)
Epithelium Number (SD) 5.23 (4.97)*
2.16 7.69 2.50
(2.70) (7.34)t (2.03)
39
Compartments
Connective Tissue Number (SD) 4.61 (5.50)* 1.56 (1.04) 5.95 (6.03)t 3.03 (3.12)
Dental
Epithelium Number (SD) 2.27 (2.28) 1.46 (0.87) 5.54 (7.61)t 1.50 (1.36)
'Healthy tissue sampled prior to perfusion fixation had a significantly greater number of organisms per µ 2 in the respective compartments when compared to tissue sampled after perfusion fixation (.P < 0.05). tDiseased tissue sampled prior to perfusion fixation had a significantly greater number of organisms per µ 2 in the respective compartments when compared to tissue sampled after perfusion fixation (P < 0.05).
indicated, the purpose of the present study was to determine
whether the bacteria seen in the corium of the lateral wall of the periodontal pocket were due to bacterial invasion, translocation of bacteria during biopsy procedures or both. The model involving adult beagle dogs with naturally occurring Periodontitis allowed the investigation of bacterial penetration into the dento-gingival tissues while controlling for the effects of the biopsy procedures. To determine whether bacteria were present in the tissues under both healthy and diseased conditions, the biopsy specimens were obtained both in naturally occurring periodontal disease and after restoring health. They were obtained before perfusion fixation as atraumatically as possible, utilizing sterile solutions. Observations of sections obtained from these specimens indicated that bacteria were found in both the clinically
healthy and inflamed gingiva located in portions of the oral epithelium, connective tissue, and dental epithelium of the dento-gingival tissues. These findings corroborate data from a recent clinical study by Ericsson et al.,20 in which isolated bacteria were found in the ulcerated portion of the pocket epithelium, in some healthy specimens within the intercel-
lular spaces of the oral epithelium, and around the blood vessels. The presence of these microorganisms in the tissues does not explain the source. An additional possibility for this finding in the present study was that the bacteria found in the healthy tissues were actually residual organisms from the previous disease state that was present before root instrumentation. These results conflicted with a recent report by Saghe et al.,17 who concluded that normal gingiva does not contain any intragingival microorganisms. The discrep-
40
J Periodontol 1990
January
BACTERIAL PENETRATION OF GINGIVA
ancy between Saglie's and our conclusions may be due to the different experimental models used; i.e., the human versus the beagle dog model. In comparing the results of sampling before and after perfusion fixation, there was a significantly greater number of bacteria per square µ found in both the healthy and diseased tissue in specimens obtained before than after perfusion fixation (Table 1). The data computed from the specimens obtained after perfusion fixation served as a control for the biopsy procedures that were obtained before perfusion fixation. Since there was a statistical difference in the number of bacteria in the tissues dependent upon time of fixation, bacteria must have penetrated into the tissues during the biopsy procedures. Therefore, studies that investigate bacteria in gingival tissues must have suitable controls for the ingress of bacteria during the biopsy. It must be emphasized that the studies that claimed that bacterial invasion through the dento-gingival attachment area is a consistent finding in advanced Periodontitis have not utilized a controlled model system.6-18 For example, in the studies that used the human model system, the specimens were obtained by a biopsy procedure; eg., punch biopsy,7,8 and according to the results of the present study, this surgical manipulation, regardless of how minimal, causes the translocation of bacteria into tissue biopsy specimens. Even though the beagle model system is perhaps different than the human model system, the evidence that control systems are needed in the beagle must support the review of this need in the human model system. Bacteria were found in all specimens obtained after perfusion fixation; therefore, it was concluded that there is a baseline number of bacteria within the dento-gingival tissues in beagle dogs. This number was not related to compartments or state of health. It is possible that a limited number of bacteria in the tissues are derived from intrinsic sources. The greater number of bacteria in the tissues of diseased gingiva obtained before fixation may be explained by passage through defects in the degenerated basement lamina seen in Periodontitis. This may be accentuated by biopsy procedures which can translocate bacteria into a more "permeable" dento-gingival tissue. Thus, the presence of bacteria in these biopsy specimens was not all necessarily due to bacterial invasion of the intragingival tissues. The greater number of bacteria found in the oral epithelium was also found by Saghe et al.16,17 who gave much importance to this observations in the etiology and severity of periodontal disease. However, these authors did not use any controls in their investigations and the presence of bacteria may be due to the biopsy technique as shown in this study, accentuated by the presence of disease. Thus, we suggest that the microorganisms observed in periodontal tissues are derived from three sources 1) endogenous and likely vascular in origin; 2) exogenous, i.e. from the oral cavity and passing through the tissue, especially diseased tissue; and 3) translocated during tissue manipulation, including the
sampling procedure.
The data from the present study demonstrated that the bacteria present in specimens from the dento-gingival tissues of the beagle dog were predominantly cocci, but not filamentous or rod-shaped bacteria. This finding is in contrast to the reports by Saghe et al.7-11,14 and Ericsson et al.20 who observed rods, filaments, and cocci in cases of advanced Periodontitis. The discrepancy between the present study and these reports could be due to some fault of the modified gram stain used in the present study. However, control specimens from rat intestine, used to develop the stain procedure, demonstrated that filaments, rods, and coccus-shaped microorganisms could be identified with the procedure used. Furthermore, SEM observations showed the presence of cocci without the other forms. Thus, it would appear that the results are a function of the particular model system used. Within the limits of this study in the beagle dog system, the biopsy procedure causes translocation of bacteria into the dento-gingival tissues, and a limited number of bacteria are present in the tissues at all times regardless of the health of the gingiva.
Acknowledgments The authors wish to thank Rollie J. statistical analysis of the data.
Harp for his help in the
REFERENCES 1. 2.
3.
4. 5.
6.
Socransky
SS. Microbiology of periodontal disease—present status and future considerations. J Periodontol 1977;48:497. Goodson JM, Tanner ACR, Haffajee AD, Sornberger GC, Socransky SS. Patterns of progression and regression of advanced destructive periodontal disease. / Clin Periodontol 1982;9:472. Lindhe J, Haffajee AD, Socransky SS. Progression of periodontal disease in adult subjects in the absence of periodontal therapy. / Clin Periodontol, 1983; 10:433. Nisengard R, Bascones A. Bacterial invasion in periodontal disease: A workshop. J Periodontol 1987;58:331. Christersson LA, Slots J, Rosling BG, Genco RJ. Microbiological and clinical effects of surgical treatment of localized juvenile Periodontitis. J Clin Periodontol 1985;12:465. Manor A, Lebendiger A, Shiffer A, Tovel H. Bacterial invasion of periodontal tissues in advanced Periodontitis. J Periodontol
1984;55:567.
7.
Saglie R, Newman MG, Carranza FA, Pattison GL. Bacterial invasion of gingiva in advanced periodontics in humans. J Periodontol
8.
Saglie FR,
1982;53:217.
9.
10.
11.
12.
Carranza FA, Newman MG, Cheng L, Lewin KJ. Identification of tissue-invading bacteria in human periodontal disease. / Periodont Res 1982;17:452. Carranza FA, Jr., Saglie R, Newman MG, Valentin P. Scanning and transmission electron microscope study of tissue invading microorganisms in U?. J Periodontol 1983;54:598. Saglie FR, Carranza FA, Newman MG. The presence of bacteria within the epithelium in periodontal disease. I—A scanning and transmission electron microscope study. J Periodontol 1985;56:'618. Saglie FR, Pertuiset JH, Rezende MT, Sabct MS, Raoufi D, Carranza FA. Bacterial invasion in experimental gingivitis in man. / Periodontol 1987;58:837. Christersson LA, Albini , Zambón J, Wikesjö U, Genco RJ. Tissue localization of Actinobacillus actinomycetemeomitans in human peri-
Volume 61 Number 1
SILVERSTEIN, SCHUSTER, GARNICK, SINGH
I. Light, immunofluoresccnce and electron microscopic odontitis studies. J Perìodontol 1987;58:529. 13. Christersson LA, Albini B, Zambón J, Wikesjo M, Genco RJ. Tissue localization of Actinobacilliis actinomycetemcomitans in human PeriII. Correlation between immunofluoresccnce and culture odontitis techniques. / Perìodontol 1987;58:540. 14. Saglie R, Newman MG, Carranza FA, Jr., et al. Immunohistochemical localization of Actinobacilliis actinomycetemcomitans infections of gingival tissue in localized juvenile Periodontitis. Acta Ódontól —
—
1984;1:41.
15. Frank RM, Voegel JC. Bacterial bone résorption in advanced cases of human Periodontitis. J Periodont Res 1978;13:251. 16. Saglie FR, Smith CT, Newman MG, et al. The presence of bacteria in the oral epithelium in periodontal disease II. Immunohistochemical identification of bacteria. J Perìodontol 1986;57:492. 17. Saglie FR, Pcrtuiset JH, Smith CT, et al. The presence of bacteria in the oral epithelium in periodontal disease III. Correlation with Langerhans cells. J Perìodontol 1987;58:417. 18. Takorada H. Ultrastructural studies of human gingiva. III Changes of the basal lamina in chronic Periodontitis. J Perìodontol 1974;45:288. 19. Slots J, Genco RJ. Black-pigmented Bacteriodes Species, Capnocytophaga species and Actinobacilliis actinomycetemcomitans in human periodontal disease: Virulence factors in colonization, survival and tissue destruction. / Dent Res 1984;63:412. 20. Ericsson I, Lindhe J, Liljenberg B, Persson A-L: Lack of bacterial invasion in experimental Periodontitis. / Clin Perìodontol 1987;14:478. 21. Liakoni H, Barber , Newman . Bacterial penetration of pocket soft tissues in chronic adult and juvenile Periodontitis cases. An ultrastructural study. / Clin Periodontal 1987;14:22.
41
22. Sanavi F, Listgarten MA, Boyd F, Sallcy , Nowotny A. The colonization and establishment of invading bacteria in pcriodontium of ligature-treated immunosupprcssed rats. J Perìodontol 1985;56:273. 23. Tromp JAH, Van Rijn LS, Jansen J. Experimental gingivitis and frequency of toothbrushing in the beagle dog model. Clinical findings. J Clin Periodontal 1986; 13:190. 24. Hamp SE, Lindhe J, Heyden G. Experimental gingivitis in the dog. An enzyme histochemical study. Arch Oral Biol 1972;17:329. 25. McKinncy RV, Koth DL, Stefiik DE. Evidence for a junctional epithelial attachment to ceramic dental implants. A TEM study. J Per-
iodontal 1985;56:579.
Garvey W, Fathi A, Bigelow F, Carpente , Jimenez C. A reliable method for demonstrating gram-positive and gram-negative bacteria. Stain Technology 1986;61:251. 27. Winborn WB, Guerrero B. The use of a single tissue specimen for both transmission and scanning electron microscopy. Cytobios 26.
1974;10:83.
28.
29.
Saglie FR, Ferreira SA, Smith CT, Valentin PL, Carranza FA, Newman MG. Identification of bacteria by studying one section under light microscopy, scanning, and transmission electron microscopy. J Electron Microscopy Tech 1985;2:581. Stefiik DE, McKinncy RV, Singh BB. Retrieval of plastic embedded light microscopy specimens for plasma etched scanning electron microscope analysis. Stain Technology 1983;58:59.
Send reprint requests to: Dr. J. Garnick, Department of Periodontics, School of Dentistry, Medical College of Georgia, Augusta, GA 30912. Accepted for publication July 18, 1989.
Bacterial Penetration of Gingiva in the Adult Beagle Dog With Periodontitis L.H. Silverstein,
*
G.S. Schuster, f J.J. Garnick,
*
and B.
Singhf
Invasion of gingival tissues by bacteria is thought to be a major factor in development of periodontal lesions. Morphologic studies have revealed bacteria within the pocket epithelium, gingival connective tissues, alveolar bone, and oral epithelium. The current studies are intended to determine whether they are present in healthy and diseased tissues and to identify the microbial source. Five beagle dogs with naturally occurring Periodontitis were fed a soft diet. Two quadrants of the dentition of each dog were cleaned regularly and health maintained while others were allowed to develop further disease. After 4 weeks, samples of gingiva were removed both prior to and after the animals were sacrificed by perfusion fixation attempting not to introduce microorganisms. The identical samples were examined by light and scanning electron microscopy for numbers and location of organisms. Their numbers were greater in diseased than in healthy tissues. Furthermore, the quantity was greater in samples taken prior to perfusion fixation than after such treatment, especially in the oral epithelium. The results suggest that normally there are some microorganisms present within the periodontal tissues and that their numbers are greater in diseased tissues. However, the data also indicated that significant numbers may be introduced into the tissues during sampling of unfixed tissues. J Periodontol 1990;61:35-41.
Key Words: Gingiva/microbiology, periodontitis/microbiology, periodontitis/etiology.
of periodontal disease is bacterial loss of connective tissue attachment occurs in only 5% of the inflamed sites containing plaque.2'3 One cause of these active periodontal lesions, especially in localized juvenile Periodontitis, is thought to be invasion of the gingival tissues by bacteria.4 In studies of this disease, root planing alone was not considered adequate to remove the etiologic bacteria, and periodontal surgery and/or antibiotics were recommended.5 Manor et al.6 demonstrated bacterial penetration of tissue in 4 of 7 specimens from 4 patients with advanced adult Periodontitis. In other studies of this condition morphologically identifiable microorganisms were observed in the gingiva by light transmission and scanning electron microscopy, reinforcing the concept of a role for intratissue bacteria in the disease process.4-7 Bacteria have been reported to be specifically located in the pocket epithelium,7-11 gingival connective tissue,9'12-14 alveolar bone,8,15 and oral epithelium.16'17 Several studies have implicated different forms and species of bacteria in adult forms of periodontal disease.11-14'16'17 For example, The
primary
cause
plaque,1 however,
•Department of Pcriodontics and Periodontology Section, Dental Research Center, School of Dentistry, Medical College of Georgia, Augusta, Georgia. tDcpartment of Oral Biology/Microbiology. +Dcpartmcnt of Oral Pathology.
Frank and Voegel15 reported a gram-negative flora associated with extensive bone résorption in 6 of 12 patients with adult Periodontitis. However, knowledge of the true prevalence of bacteria in periodontally diseased tissues is
limited.4
Immunocytochemical studies have suggested that bacterial invasion may be an important mechanism in the pathogenesis of the active periodontal lesion.11-14'16,17 While the methods used in the above studies allowed evaluation of the presence of specific antigens in the tissues, they did not indicate whether the antigens represent viable or nonviable bacterial cells or wall fragments. Disruptions of the gingival basement have been reported7,8-18'19 in Periodontitis and may be associated with bacterial invasion. In contrast to these reports, Erickson et al.20 using microscopic observations concluded that at most sites the subgingival microbiota appeared to be confined to the periodontal pocket. Other studies supported these results.21,22 Since bacterial invasion may be an important feature in the destruction of the periodontium during the active phases of Periodontitis,15 and the sources of these organisms are not known, the purpose of the current project was' to determine whether the presence of bacteria in the corium of the lateral wall of the periodontal pocket is a result of 1) past bacterial invasion of the tissue; 2) translocation into
J Periodontol 36
January 1990
BACTERIAL PENETRATION OF GINGIVA
tissues from the pocket during the biopsy procedures; organisms that exist indigenously within the tissues.
or
3)
MATERIALS AND METHODS Five adult female beagle dogs, average weight 13.8 Kg (10.7 to 18.5 Kg), with naturally occurring Periodontitis were fed a standard pellet dog food and water ad libitum. Prior to the experimental procedures, two quadrants of each dog, chosen at random, were treated by scaling and root planing and daily tooth brushing for 4 weeks in order to restore gingival health (control quadrants). During the instrumentation, the dogs were anesthetized with 23 mg/kg intravenous Pentothal. The dentition of the control quadrants was cleaned 5 times a week using a soft tooth brush and a Perio-Aid for the interproximal areas.23 The remaining two quadrants were left untreated (experimental quadrants). During this period, the dogs were placed on a soft diet consisting of the standard pellet which was soaked in water to obtain a mushy consistency24 permitting gross accumulation of plaque and calculus on teeth of the experimental quadrants. In the experimental procedures which immediately followed the 4 week preliminary period, the teeth and the adjacent tissues in the control or experimental quadrants were biopsied either before animals had been sacrificed, or after sacrifice by perfusion fixation. Perfusion fixation techniques were utilized in this study in order to minimize the translocation of microorganisms during the surgical manipulation of the tissues during the biopsy procedures. The perfusion fixation technique was a modification of that reported by McKinney et al.25 The dog was anesthetized with Pentothal (23 mg/kg). Perfusion fixation was accomplished using a carotid cut-down procedure in which the common carotid arteries were isolated, ligated, cannulated, and subsequently perfused with sterile heparinized saline followed by filtration sterilized 2% glutaraldehyde buffered with 0.2 M sodium phosphate at pH 7.2, made 24 hours prior to perfusion. The external jugulars were incised to permit outflow of saline and fixative. The biopsy procedure utilized two vertical incisions made the mesio-buccal and disto-buccal line angles of the specat imen tooth. A horizontal incision was then made 3 mm apical to the previously determined deepest probing depth connecting the vertical incisions. The adjacent tissue at the incision line was bluntly dissected away from the facial gingival tissue of the tooth, being sure not to manipulate that tissue. Following this, a high speed handpiece with a 557 carbide bur was used to make two vertical channels halfway through the alveolar bone in a buccal-lingual direction, then a horizontal channel at the incision line was made to connect the two vertical channels. Next, a mesiodistal occlusal channel was placed through the occlusal groove and extended apically, connecting the two vertical and horizontal channels (Fig. 1). The tooth specimen was then removed with a hemostat, grasping the crown of the tooth and taking care not to disturb the attached soft tissue. The
Figure 1: The appearance of the tissue biopsy just before removal from jaw. Bone channels were made buccal-lingually one-half the distance of the tooth at the mesial, distal, and apical border of the biopsy. A vertical groove made at the occlusal surface connected the three channels and freed the tooth segment and tissue specimen from the jaw. The specimen was then fixed with sterile buffered glutaraldehyde for 24 hours. Afterward, the tissue was removed from the tooth segment and processed.
specimen was then immediately placed into sterile buffered glutaraldehyde fixative for 24 hours. Following fixation, specimens were placed in a 0.2 M phosphate buffer at pH 7.2 and the tissue biopsy was carefully incised free from the tooth segment. A total of 16 biopsies were obtained from each dog: 8 specimens, 4 each of health and disease prior to perfusion fixation; and 8 specimens, 4 each of health and disease after perfusion fixation. Biopsy specimens were dehydrated in alcohol, embedded in paraffin and sectioned in a bucco-lingual plane at
5 µ
intervals. Bacteria
were
located in these sections
by staining using a modification of the gram stain technique described by Garvey et al.,26 substituting Carbol Fuschin for New Fuschin.
Histometric Analysis The analysis included a determination of the location and the numbers of bacterial cells in different compartments of the gingival tissue. The compartments were designated the dental epithelium, oral epithelium, and connective tissue. The base of the compartments was an imaginary line perpendicular to the tooth surface at the most apical extension of the junctional epithelium. The border of the dental and oral epithelium was an imaginary line from the gingival margin perpendicular to the base line (Fig. 2). Upon completion of the gram-staining procedures, the specimens were examined to determine the number of bacteria in the compartments. Using a computer-assisted digitizing system, the area of each of these compartments was
Volume 61 Number 1
SILVERSTEIN, SCHUSTER, GARNICK, SINGH
37
down to fit onto an SEM mounting stub. The cover slip then removed from the glass slide, without disturbing the surface topography of the specimen as recently described by Stefiik and co-workers.29 This specimen was then dehydrated with ethanol, placed in Freon, critical point dried with liquid carbon dioxide, sputter coated with gold, and placed in the SEM. Observations were made at magnifications of 350 x, 1000 x, and 5000 x. The acetate tracing previously obtained from the histologie section at low magnification was placed on the monitor of the SEM. The magnification was adjusted so that the image of the specimen on the monitor matched this acetate tracing. The areas of bacteria indicated on the acetate sheet were located on the SEM monitor, then one or more of these areas were centered on the screen and magnification increased until the image on the monitor matched the higher magnification acetate tracing. This image was further magnified till bacteria could be readily visualized. Once these steps had been completed, these areas were viewed at a series of higher magnifications to confirm that bacteria observed at the light microscopic level were actually bacteria under SEM. Statistical comparisons of bacterial counts were performed using a four way repeated measures analysis of variance taking into consideration the effects of health versus disease, time of biopsy either before or after fixation, tooth type, and tissue compartments. All tests were done at the 95% confidence level. The variability of the histometric methods was assessed by analyzing the differences between duplicate measurements or countings. The standard errors for bacterial cell counts for oral epithelium, connective tissue, and dental epithelium compartments of all groups were 6.86%, 5.95%, and 2.56% respectively. The standard errors for the area measurements of these compartments were 0.05%, 0.04% and 0.03% respectively.
was
um
Figure 2: A diagram illustrating the three compartments used in the histometric analysis. The boundaries were determined with a line perpendicular to the tooth surface at the base of the dental epithelium. The boundary between the dental and oral epithelia was a line perpendicular to the previous line. measured on the histologie sections at a magnification of The measurement system consisted of a Zenith Data 40 Systems computer with the MicroComp M2 Analysis Program* attached to an Olympus microscope and Hipadf digitizing tablet. Then at higher magnification (630 x), the individual bacteria within each of the three compartments were counted. .
Electron Microscopical (SEM) Evaluation In order to corroborate the presence and identification of bacteria seen in light microscopy, three sections from each of the four groups (diseased and healthy specimens and biopsies before and after perfusion) were processed for SEM. Subsequent to histometric analysis, an acetate sheet was placed over the monitor of the computer and the location of the bacteria was traced onto acetate sheets at various magnifications and in relation to the adjacent anatomic structure.27,28 The magnification was then increased and the area with located bacteria was traced onto a different acetate sheet. Upon completion of these tracings, this identical specimen was processed for SEM. Using a thin Flex diamond wafering disk the slide, immersed in xylene, was cut
Scanning
'Southern Microinstruments, Inc., Atlanta, GA. fHouston Electronics, Houston, TX
RESULTS Clinical Appearance of Dogs At the time of sacrifice, the control
(healthy) quadrants of each of the five dogs had plaque present only on a few teeth, calculus was not evident, and there were no signs of gingivitis. One week prior to sacrifice, the mean clinical crevice depth was 2.0 ± 0.44mm in control quadrants. In the experimental (diseased) quadrants, large amounts of plaque could be detected easily and mineralized deposits were regularly present both supragingivally and subgingivally. The periodontal pockets, whose mean probing depth was 5.4 ± 1.3mm, bled upon gentle probing and the tissues were found to be red, bulbous, and displayed clinical signs of gingival inflammation (Fig. 3). The difference in clinical pocket depth between control and experimental quadrants statistically significant (P < 0.05). gingival tissue biopsies sampled from the diseased quadrants of each dog contained periodontal lesions, the major portions of which were confined to the dento-gingival
was
The
J Periodontol 38
BACTERIAL PENETRATION OF GINGIVA
January 1990 smooth in appearance, regularly shaped and about 6 to 8 µ in diameter. This was true for specimens obtained before or after perfusion fixation. The inflammatory infiltrate was present within the epithelium and, to a lesser extent, in the adjacent connective tissue extending apical to the junctional epithelium.
Figure 3: Example of control and experimental quadrants just before the biopsy procedures. The healthy tissues were achieved by scaling, tooth polishing, and daily toothbrushing for 4 weeks.
Figure 4: Photomicrograph demonstrating bacteria in the gingival stroma. The specimen was stained with a modified gram stain. Bacteria are present at (a). Original magnification: x 1000.
region. The dental epithelium of the gingival tissue appeared thin, ulcerated in the marginal portion and in the more apical portions contained pronounced rete pegs. The connective tissue exhibited a fine to moderately dense collagenous stroma supporting a number of chronic inflammatory cells. The bacteria generally were clustered around blood vessels (Fig. 4). For the most part, the organisms appeared round in shape, resembling cocci, were rather
SEM Evaluation Specimens observed under high SEM magnification (5000 ) demonstrated that what we called bacteria under the light microscope actually were bacteria and not stained debris. SEM findings also confirmed that rods or filamentous forms of bacteria were not present in tissues in any of the samples, diseased or healthy (Fig. 5). Histometric Analysis The range of compartmental areas was 2.4 to 52.2 square µ in the oral epithelium, 1.3 to 53.3 square µ in the connective tissue and 1.6 to 43.3 square µ in the dental epithelial tissue compartments; there were no differences between control and experimental groups. The mean number of organisms per square µ in each tissue compartment, combining data for health and disease and before or after perfusion, was 4.40 ± 5.17; 3.79 ± 4.62; and 2.69 ± 4.32 per square µ for oral epithelium, connective tissue and dental epithelium compartments respectively. The differences were not statistically significant (P > 0.05). Table 1 shows the mean number of bacteria per square µ in each tissue compartment for healthy and for diseased tissue sampled before and after perfusion fixation. The results from the histometric measurements of the healthy specimens obtained before perfusion fixation indicated that a significantly greater number of bacteria (P < 0.05) was found in the gingival biopsies obtained before as compared with after perfusion fixation except for the dental epithelium compartment (P > 0.05). Similar results were evident before and after perfusion fixation in diseased specimens except that all compartments of diseased tissue specimens had greater numbers of bacteria per square µ of tissue before perfusion fixation (P< 0.05, Table 1). The diseased oral epithelium before perfusion contained a significantly greater number (P< 0.05) of bacteria per unit as compared to the other diseased tissue compartments. Biopsy after perfusion fixation of diseased tissues decreased the number of bacteria detectable per area to a greater extent within this compartment than in the dental epithelium (P< 0.05, Table 1). Also in a comparison between the diseased and healthy specimens obtained before fixation, the diseased specimens had a greater number of bacteria per area but the differences were not significant when comparable compartments were compared (P> 0.05, Table 1).
DISCUSSION Various studies have suggested indigenous bacteria, bacterial invasion, or penetration with the biopsy procedure as the source of organisms seen in periodontal tissues.17'21 As
Volume 61 Number 1
SILVERSTEIN, SCHUSTER, GARNICK, SINGH
Table 1: Mean Number of Bacteria/µ
2
in Various Tissue
Oral
Compartment Tissue and Time of Sampling
Healthy (before fixation) Healthy (after fixation) Diseased (before fixation) Diseased (after fixation)
Epithelium Number (SD) 5.23 (4.97)*
2.16 7.69 2.50
(2.70) (7.34)t (2.03)
39
Compartments
Connective Tissue Number (SD) 4.61 (5.50)* 1.56 (1.04) 5.95 (6.03)t 3.03 (3.12)
Dental
Epithelium Number (SD) 2.27 (2.28) 1.46 (0.87) 5.54 (7.61)t 1.50 (1.36)
'Healthy tissue sampled prior to perfusion fixation had a significantly greater number of organisms per µ 2 in the respective compartments when compared to tissue sampled after perfusion fixation (.P < 0.05). tDiseased tissue sampled prior to perfusion fixation had a significantly greater number of organisms per µ 2 in the respective compartments when compared to tissue sampled after perfusion fixation (P < 0.05).
indicated, the purpose of the present study was to determine
whether the bacteria seen in the corium of the lateral wall of the periodontal pocket were due to bacterial invasion, translocation of bacteria during biopsy procedures or both. The model involving adult beagle dogs with naturally occurring Periodontitis allowed the investigation of bacterial penetration into the dento-gingival tissues while controlling for the effects of the biopsy procedures. To determine whether bacteria were present in the tissues under both healthy and diseased conditions, the biopsy specimens were obtained both in naturally occurring periodontal disease and after restoring health. They were obtained before perfusion fixation as atraumatically as possible, utilizing sterile solutions. Observations of sections obtained from these specimens indicated that bacteria were found in both the clinically
healthy and inflamed gingiva located in portions of the oral epithelium, connective tissue, and dental epithelium of the dento-gingival tissues. These findings corroborate data from a recent clinical study by Ericsson et al.,20 in which isolated bacteria were found in the ulcerated portion of the pocket epithelium, in some healthy specimens within the intercel-
lular spaces of the oral epithelium, and around the blood vessels. The presence of these microorganisms in the tissues does not explain the source. An additional possibility for this finding in the present study was that the bacteria found in the healthy tissues were actually residual organisms from the previous disease state that was present before root instrumentation. These results conflicted with a recent report by Saghe et al.,17 who concluded that normal gingiva does not contain any intragingival microorganisms. The discrep-
40
J Periodontol 1990
January
BACTERIAL PENETRATION OF GINGIVA
ancy between Saglie's and our conclusions may be due to the different experimental models used; i.e., the human versus the beagle dog model. In comparing the results of sampling before and after perfusion fixation, there was a significantly greater number of bacteria per square µ found in both the healthy and diseased tissue in specimens obtained before than after perfusion fixation (Table 1). The data computed from the specimens obtained after perfusion fixation served as a control for the biopsy procedures that were obtained before perfusion fixation. Since there was a statistical difference in the number of bacteria in the tissues dependent upon time of fixation, bacteria must have penetrated into the tissues during the biopsy procedures. Therefore, studies that investigate bacteria in gingival tissues must have suitable controls for the ingress of bacteria during the biopsy. It must be emphasized that the studies that claimed that bacterial invasion through the dento-gingival attachment area is a consistent finding in advanced Periodontitis have not utilized a controlled model system.6-18 For example, in the studies that used the human model system, the specimens were obtained by a biopsy procedure; eg., punch biopsy,7,8 and according to the results of the present study, this surgical manipulation, regardless of how minimal, causes the translocation of bacteria into tissue biopsy specimens. Even though the beagle model system is perhaps different than the human model system, the evidence that control systems are needed in the beagle must support the review of this need in the human model system. Bacteria were found in all specimens obtained after perfusion fixation; therefore, it was concluded that there is a baseline number of bacteria within the dento-gingival tissues in beagle dogs. This number was not related to compartments or state of health. It is possible that a limited number of bacteria in the tissues are derived from intrinsic sources. The greater number of bacteria in the tissues of diseased gingiva obtained before fixation may be explained by passage through defects in the degenerated basement lamina seen in Periodontitis. This may be accentuated by biopsy procedures which can translocate bacteria into a more "permeable" dento-gingival tissue. Thus, the presence of bacteria in these biopsy specimens was not all necessarily due to bacterial invasion of the intragingival tissues. The greater number of bacteria found in the oral epithelium was also found by Saghe et al.16,17 who gave much importance to this observations in the etiology and severity of periodontal disease. However, these authors did not use any controls in their investigations and the presence of bacteria may be due to the biopsy technique as shown in this study, accentuated by the presence of disease. Thus, we suggest that the microorganisms observed in periodontal tissues are derived from three sources 1) endogenous and likely vascular in origin; 2) exogenous, i.e. from the oral cavity and passing through the tissue, especially diseased tissue; and 3) translocated during tissue manipulation, including the
sampling procedure.
The data from the present study demonstrated that the bacteria present in specimens from the dento-gingival tissues of the beagle dog were predominantly cocci, but not filamentous or rod-shaped bacteria. This finding is in contrast to the reports by Saghe et al.7-11,14 and Ericsson et al.20 who observed rods, filaments, and cocci in cases of advanced Periodontitis. The discrepancy between the present study and these reports could be due to some fault of the modified gram stain used in the present study. However, control specimens from rat intestine, used to develop the stain procedure, demonstrated that filaments, rods, and coccus-shaped microorganisms could be identified with the procedure used. Furthermore, SEM observations showed the presence of cocci without the other forms. Thus, it would appear that the results are a function of the particular model system used. Within the limits of this study in the beagle dog system, the biopsy procedure causes translocation of bacteria into the dento-gingival tissues, and a limited number of bacteria are present in the tissues at all times regardless of the health of the gingiva.
Acknowledgments The authors wish to thank Rollie J. statistical analysis of the data.
Harp for his help in the
REFERENCES 1. 2.
3.
4. 5.
6.
Socransky
SS. Microbiology of periodontal disease—present status and future considerations. J Periodontol 1977;48:497. Goodson JM, Tanner ACR, Haffajee AD, Sornberger GC, Socransky SS. Patterns of progression and regression of advanced destructive periodontal disease. / Clin Periodontol 1982;9:472. Lindhe J, Haffajee AD, Socransky SS. Progression of periodontal disease in adult subjects in the absence of periodontal therapy. / Clin Periodontol, 1983; 10:433. Nisengard R, Bascones A. Bacterial invasion in periodontal disease: A workshop. J Periodontol 1987;58:331. Christersson LA, Slots J, Rosling BG, Genco RJ. Microbiological and clinical effects of surgical treatment of localized juvenile Periodontitis. J Clin Periodontol 1985;12:465. Manor A, Lebendiger A, Shiffer A, Tovel H. Bacterial invasion of periodontal tissues in advanced Periodontitis. J Periodontol
1984;55:567.
7.
Saglie R, Newman MG, Carranza FA, Pattison GL. Bacterial invasion of gingiva in advanced periodontics in humans. J Periodontol
8.
Saglie FR,
1982;53:217.
9.
10.
11.
12.
Carranza FA, Newman MG, Cheng L, Lewin KJ. Identification of tissue-invading bacteria in human periodontal disease. / Periodont Res 1982;17:452. Carranza FA, Jr., Saglie R, Newman MG, Valentin P. Scanning and transmission electron microscope study of tissue invading microorganisms in U?. J Periodontol 1983;54:598. Saglie FR, Carranza FA, Newman MG. The presence of bacteria within the epithelium in periodontal disease. I—A scanning and transmission electron microscope study. J Periodontol 1985;56:'618. Saglie FR, Pertuiset JH, Rezende MT, Sabct MS, Raoufi D, Carranza FA. Bacterial invasion in experimental gingivitis in man. / Periodontol 1987;58:837. Christersson LA, Albini , Zambón J, Wikesjö U, Genco RJ. Tissue localization of Actinobacillus actinomycetemeomitans in human peri-
Volume 61 Number 1
SILVERSTEIN, SCHUSTER, GARNICK, SINGH
I. Light, immunofluoresccnce and electron microscopic odontitis studies. J Perìodontol 1987;58:529. 13. Christersson LA, Albini B, Zambón J, Wikesjo M, Genco RJ. Tissue localization of Actinobacilliis actinomycetemcomitans in human PeriII. Correlation between immunofluoresccnce and culture odontitis techniques. / Perìodontol 1987;58:540. 14. Saglie R, Newman MG, Carranza FA, Jr., et al. Immunohistochemical localization of Actinobacilliis actinomycetemcomitans infections of gingival tissue in localized juvenile Periodontitis. Acta Ódontól —
—
1984;1:41.
15. Frank RM, Voegel JC. Bacterial bone résorption in advanced cases of human Periodontitis. J Periodont Res 1978;13:251. 16. Saglie FR, Smith CT, Newman MG, et al. The presence of bacteria in the oral epithelium in periodontal disease II. Immunohistochemical identification of bacteria. J Perìodontol 1986;57:492. 17. Saglie FR, Pcrtuiset JH, Smith CT, et al. The presence of bacteria in the oral epithelium in periodontal disease III. Correlation with Langerhans cells. J Perìodontol 1987;58:417. 18. Takorada H. Ultrastructural studies of human gingiva. III Changes of the basal lamina in chronic Periodontitis. J Perìodontol 1974;45:288. 19. Slots J, Genco RJ. Black-pigmented Bacteriodes Species, Capnocytophaga species and Actinobacilliis actinomycetemcomitans in human periodontal disease: Virulence factors in colonization, survival and tissue destruction. / Dent Res 1984;63:412. 20. Ericsson I, Lindhe J, Liljenberg B, Persson A-L: Lack of bacterial invasion in experimental Periodontitis. / Clin Perìodontol 1987;14:478. 21. Liakoni H, Barber , Newman . Bacterial penetration of pocket soft tissues in chronic adult and juvenile Periodontitis cases. An ultrastructural study. / Clin Periodontal 1987;14:22.
41
22. Sanavi F, Listgarten MA, Boyd F, Sallcy , Nowotny A. The colonization and establishment of invading bacteria in pcriodontium of ligature-treated immunosupprcssed rats. J Perìodontol 1985;56:273. 23. Tromp JAH, Van Rijn LS, Jansen J. Experimental gingivitis and frequency of toothbrushing in the beagle dog model. Clinical findings. J Clin Periodontal 1986; 13:190. 24. Hamp SE, Lindhe J, Heyden G. Experimental gingivitis in the dog. An enzyme histochemical study. Arch Oral Biol 1972;17:329. 25. McKinncy RV, Koth DL, Stefiik DE. Evidence for a junctional epithelial attachment to ceramic dental implants. A TEM study. J Per-
iodontal 1985;56:579.
Garvey W, Fathi A, Bigelow F, Carpente , Jimenez C. A reliable method for demonstrating gram-positive and gram-negative bacteria. Stain Technology 1986;61:251. 27. Winborn WB, Guerrero B. The use of a single tissue specimen for both transmission and scanning electron microscopy. Cytobios 26.
1974;10:83.
28.
29.
Saglie FR, Ferreira SA, Smith CT, Valentin PL, Carranza FA, Newman MG. Identification of bacteria by studying one section under light microscopy, scanning, and transmission electron microscopy. J Electron Microscopy Tech 1985;2:581. Stefiik DE, McKinncy RV, Singh BB. Retrieval of plastic embedded light microscopy specimens for plasma etched scanning electron microscope analysis. Stain Technology 1983;58:59.
Send reprint requests to: Dr. J. Garnick, Department of Periodontics, School of Dentistry, Medical College of Georgia, Augusta, GA 30912. Accepted for publication July 18, 1989.
