Light and Electron Microscopic
resorption. For example bone resorption due to osteo clastic activity has been shown to occur in sensitized oral tissues that were repeatedly challenged with antigen. Endotoxin and plaque extracts stimulate osteoclastic bone resorption in vitro. Sensitized lymphocytes release an osteoclast activating factor when challenged with antigen. It has been shown also that sera will stimulate bone resorption in tissue culture and that antigen-antibody complexes increase the effect. Recent scanning electron microscopic studies of hamsters fed Keyes high carbohydrate diet 2000 have demonstrated that the alveolar bone surface of these animals is charac terized by numerous Howship's lacunae. These obser vations suggest that the disappearance of alveolar bone was due to osteoclastic bone resorption. In view of the increasing evidence implicating osteo clast activation as an important and perhaps key phe nomenon leading to bone destruction in periodontal disease, the conclusion drawn by Irving, et al. that bone loss in periodontal disease might occur via some previ ously undetermined process, is rather surprising. On the basis of ultrastructural studies of the progres sion of periodontal disease in gnotobiotic rats monoin fected with Actinomyces naeslundii this author does not agree with the findings of Irving et al. The present paper reports the presence of osteoclastic activity along crestal alveolar bone in severely inflamed interdental periodon tal tissues of 2- to 4-month-old monoinfected rats and in addition a widespread appearance of osteoclasts in the later stages of the disease. Several temporal and morpho logical features which could account for the findings of Irving et al. are discussed. 10-12
Observations of Osteoclastic
13-14
Alveolar Bone Resorption in
15
Rats Monoinfected with
16
Actinomyces Naeslundii* by
17
PHILIAS R .
GARANT†
W H E N GNOTOBIOTIC RATS and hamsters are monoin
fected with Actinomyces naeslundii they rapidly de velop a severe periodontitis characterized by extensive alveolar bone destruction and extensive root caries. Innoculation of capsule forming streptococci in gnoto biotic rats and in conventional rats also causes exten sive bone loss. Although periodontal disease can be a naturally occur ring phenomenon in rodents as exemplified by the observations that epithelial migration and alveolar bone resorption increase with age in both germ free and conventional mice, there is no comparison to the massive and rapid bone destruction seen in gnotobiotic rats monoinfected with Actinomyces naeslundii. The precise cause and the mechanism whereby the alveolar bone is destroyed in the monoinfected rats is still a source of contention. Undoubtedly, the plaque is the important factor since a common feature of most studies was the association of massive plaque accumulation and resorp tion of the adjacent alveolar bone. Jordan, Keyes and Bellack have reported that osteo clastic activity was responsible for the bone resorption adjacent to surfaces coated with plaque in hamsters and gnotobiotic rats monoinfected with Actinomyces. More recently Irving, Socransky and Heeley in reporting on the histologic changes seen in periodontal disease in gnotobiotic rats have stated that alveolar bone loss was not accompanied by the presence of osteoclasts. They concluded on the basis of autoradiography (to label bone formation) and histology that the bone loss was due to a gradual cessation of bone formation instead of active osteoclastic resorption. Furthermore, they stated that if some active resorption did occur it must have been brought about by an as yet undescribed mechanism. There is considerable evidence that inflammatory processes can generate stimuli for osteoclastic bone 1,2
3
4
5-8
9
9
9
1-4
2
9
* This research was supported by U.S. Public Health Service grant No. 2 R01 D E 03745. † Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, N . Y . 11794.
717
MATERIAL AND M E T H O D S
Five-week-old germ free rats were inoculated with Actinomyces naeslundii and reared in isolators on Diet 2000. Cultures were taken 1 week after inoculation to insure that an infection had been established. A total of 16 monoinfected rats were used in this study. The rats were sacrificed at 4, 10, 24, 28, 40, 60, 65 and 180 days after initial introduction of the bacteria. Two germ free rats sacrificed at each of the above times, plus two at 300 days, were examined as controls. In order to insure that proper tissue relationships in the critical interdental areas were maintained during the processing and sectioning, fixation by whole body perfu sion followed by decalcification of entire jaws was employed. The primary fixative consisted of 1% glutaraldehyde in a Tyrode solution with an osmolarity of about 350 mOsm. The rats were anesthetized with Nembutal (5 to 7 mg/100 gm of body weight) prior to the start of the procedure. The perfusate was administered through a cannula inserted into the ascending aorta via an inci sion into the left ventricle. Two to three hundred milli liters of fixative were delivered over a period of about 20 minutes at a pressure equivalent to five feet of water. Upon completion of the perfusion the maxillae and
J. Periodontol. December, 1976
7 1 8 Garant mandibles were dissected free of surrounding tissues and placed in a 0.1 M solution of ethylenediaminetetraacetate (EDTA) adjusted to pH 7.4 with sodium hydroxide and containing 4.0% glutaraldehyde. The jaws were allowed to undergo decalcification in this solution for about 3 to 4 weeks at 4°C. Upon completion of the de calcification the jaws were cut into small blocks, each block to contain one interdental area. These blocks were then rinsed in several changes of buffer and were postfixed for 1½ hours at 4°C in 2% solution of osmium tetroxide buffered to pH 7.4 with collidine. Dehydration was carried out in a graded series of cold ethanol followed by several changes of propylene oxide. The tissue blocks were then embedded in Epon 812. Flat embedding trays were utilized in order to insure the correct tissue orientation required to obtain sections through the interdental area in a mesiodistal plane. One micron sections through the interdental region were stained with toluidine blue for light microscopic exami nation. Thin sections, ranging from 600 to 1000 Angstroms were cut on a Porter-Blum MT2 equipped with a second diamond knife. The thin sections were stained with uranyl acetate and lead citrate prior to examination in a JEM-100B electron microscope. RESULTS
Light microscopic examination of 1µthick sections of germ free interdental areas with toluidine blue revealed
intact junctional and col epithelium overlying a narrow lamina propria and a well defined zone of transseptal fibers. The junctional epithelium was usually terminated at the cemento-enamel junction. Dense, round inflamma tory cells, some readily classified as polymorphonuclear neutrophilic leukocytes (PMNLs) on the basis of nuclear morphology, were present in moderate numbers within the lamina propria and junctional epithelium. The dense connective tissue of the transseptal fibers and the perio dontal ligament was nearly free of inflammatory cells. The crest of the alveolar bone in the germ free animals was usually covered by an osteoblastic layer. Osteoclastic activity was rarely noted at the crest of the alveolar bone in the germ free rats. Examination of the monoinfected rats revealed greater infiltration of the lamina propria and overlying epithe lium by inflammatory cells. Although the P M N L pre dominated, numerous macrophages and monocytes were also present in the lamina propria. Large adherent plaque deposits were present in the interdental spaces. Migration of the epithelial attachment and an increased incidence of rete peg formation were commonly observed. A distinct layer of transseptal fibers could be identified to form a barrier between the enlarged lamina propria and the underlying alveolar bone. Inflammatory cells were con fined for the most part to the lamina propria and epithelial tissue. In approximately 10% of the interdental areas that were examined from the monoinfected group
FIGURE 1. Light micrograph of an interdental area of a monoinfected rat. A dense inflammatory infiltrate is present in the lamina propria and has spread throughout the transseptal fiber region. Note the many osteoclasts (OC) against the alveolar crest as well as within the adjacent connective tissue. (Original magnification, x 200.) FIGURE 2. Light micrograph depicting osteoclastic resorption of alveolar crest in a monoinfected rat. Note the relatively small size of the osteoclasts (OC). (Original magnification, x 450.)
Volume 47 Number 12
Osteoclastic Alveolar Bone Resorption7 1 9
FIGURE 3. Electron micrograph of binucleated osteoclast situated in a small Howship's lacuna at the crest of the alveolar bone. Note the typical central ruffled border region (RB) flanked by clear zones (CZ). Numerous vacuoles (V) fill the cytoplasm situated nearest to the bone surface. The distal region of the cell is occupied by nuclei (N), mitochondria (M) and small profiles of granular endoplasmic reticulum and Golgi saccules best seen in figure 4. x 3500. (Original magnification, x 3500.) aged 10 to 65 days postinfection a more severe inflamma tory reaction was noted (Fig. 1). In these specimens a dense P M N L infiltration extended into and disrupted the dense connective tissue of the transseptal fibers. Close scrutiny of the epithelium revealed small regions of ulceration. The alveolar bone of these interdental areas
invariably demonstrated scalloping of the surface and several small osteoclasts (Fig. 1). These small osteoclasts frequently contained no more than two or three nuclei, and in some cases only one nucleus was observed in their sectioned profile (Fig. 2). Cells of similar staining characteristics (gray with toluidine blue) containing one
7 2 0 Garant or more nuclei were frequently noted in the connective tissue adjacent to the bone. Because this degree of osteoclastic activity was observed in but a small percent of the interdental areas, it is felt that alveolar bone destruction in the early phase of the disease is the result of repeated short bursts of osteoclastic activity associ ated with periods of acute inflammation. Epithelial ulceration in the presence of the plaque seemed to play an important role in inducing the more acute inflamma tory reaction. In the older monoinfected rats (i.e. 65 days postinfec tion) great masses of plaque were routinely observed especially in the maxillary regions. Histologic examina tion of these specimens revealed that in spite of a continuous epithelial lining and a generally more granu lomatous connective tissue the remaining alveolar and basal bone was frequently covered by osteoclasts. At the ultrastructural level the osteoclasts were charac terized by numerous mitochondria, several well devel oped Golgi apparatuses, granular endoplasmic reticu lum, cytoplasmic vacuoles, ruffled borders and clear zones (Figs. 3, 4 and 5). The nuclei which were concentrated in the distal cytoplasm, i.e. away from the bone surface, contained prominent nucleoli. A uniformly narrow nuclear enve
J. Periodontol. December, 1976
lope interrupted by the presence of several nuclear pores surrounded the nucleus (Fig. 4). The outer membrane of the nuclear envelope was not in association with ribosomes as is the case in osteoblasts and osteocytes. Many short profiles of granular endoplasmic reticulum were present, especially in the more distal and peripheral areas of the cytoplasm (Figs. 4 and 6). Free ribosomes were present in large numbers, aggregated in the form of small polyribosomes (Fig. 6). The Golgi complex consisted of numerous distinct concentrations of flattened saccules and many vesicles, usually situated in the perinuclear areas of the cytoplasm (Fig. 4). Larger membrane-bound granules containing a substance of moderate electron opacity were also present near the Golgi apparatuses and also in the cytoplasm adjacent to the ruffled border. The cell membrane along the lateral and distal surfaces of the osteoclast was characterized by several small microvilli. The proximal surface, abutting directly upon the alveolar bone, was specialized into two regions which have been shown to be of prime importance in bone resorption. A central region consisting of numerous cytoplasmic processes was juxtaposed against the region of bone breakdown (Figs. 3 and 5). This central zone, called the ruffled border, is the hallmark of the active
FIGURE 4. Distal cytoplasm of an osteoclast depicting the mitochondria (M), Golgi saccules (G) and the granular endoplasmic reticulum (GER). The nuclei (N) are encircled by a narrow nuclear envelope (NE) devoid of any attached ribosomes. (Original magnification, x 7000.) FIGURE 5. Enlarged view ofpart of an osteoclast juxtaposed to the alveolar bone. Typical ruffled border (RB) regions characterized by numerous microvilli and cytoplasmic infoldings were noted. Clear zones (CZ) are filled with densely packed cytoplasmic filaments. These cytoplasmic specializations provide morphological evidence of active osteoclastic activity. (Original magnification, x 34,000.)
Volume 47 Number 12
Osteoclastic Alveolar Bone Resorption7 2 1
FIGURE 6. Osteoclast-like cell in the connective tissue adjacent to alveolar bone. Note the modified ruffled border consisting of broad cytoplasmic processes (CP). G E R , granular endoplasmic reticulum; M , mitochondria; N , nucleus. (Original magnification, x 4600.)
osteoclast. A peripheral clear zone containing numerous cytoplasmic filaments, to the exclusion of all other cytoplasmic components, formed a rim around the centrally located ruffled border (Figs. 3 and 5). The bone surface underneath the osteoclast did not have a lamina
limitans suggesting that the surface was in a dynamic state. Osteoclast-like cells were also present in the connective tissue adjacent to the bone surface (Figs. 1 and 6). Although these cells were usually multinucleated, some
722
J. Periodontol. December, 1976
Garant
uninucleated cells with similar cytoplasmic features were observed. Characteristically they demonstrated a modi fied ruffled border zone and numerous short segments of granular endoplasmic reticulum, free ribosomes and mitochondria (Fig. 6). DISCUSSION
These observations of the progressive development of periodontal disease in rats monoinfected with Actinomyces naeslundii suggests that alveolar bone de struction in periodontitis is the result of osteoclastic activity. The ultrastructural appearance of osteoclasts has been amply documented. The cells observed in this study manifested the same features reported in previous publi cations and in addition their well developed ruffled borders, clear zones, mitochondria and abundant Golgi apparatuses, suggest that they are actively engaged in bone resorption. The presence of osteoclasts on the crest of the alveolar bone in the early phases of the disease (10 to 65 days postinfection) was associated with a concur rent dense inflammatory cell infiltration of the lamina propria and ulceration of the interdental col epithelium. Since osteoclasts and the associated micropathological changes were seen only in about 10% of the interdental areas examined, it is further suggested that osteoclastic activity leading to reduction in the height of the alveolar process is a discontinuous phenomenon characterized by short periods of vigorous osteoclastic activity followed by longer periods of inactivity. During the periods of inactivity the local stimuli for osteoclast differentiation are reduced and the crestal surfaces of the alveolar bone are covered by osteoblast-like cells or in some cases appear to be free of any bone cells. The findings of Irving et al. that alveolar bone formation is markedly reduced in the monoinfected rats could account for the observed net loss of bone in spite of the fact that osteoclastic activity occurs during only a small percent of the time. 18-21
9
The additional finding that osteoclasts seen at the alveolar crest are usually quite small, many appearing uninuclear in profile, could be an indication that most are immature in the sense that the acquisition of multiple nuclei represents a maturation of the osteoclast cell line. It has been postulated on the basis of E M autoradio graphic studies of tritiated thymidine labelled cells that a separate osteoprogenitor cell line for osteoclasts exists in periosteal tissue and that the osteoclast precursor cell is a round cell rich in mitochondria and Golgi membranes. Although the small osteoclast may represent a newly differentiated osteoclast, its histologic (in Epon embed ded material) and ultrastructural features indicate a physiologic capability for bone resorption. These cells are present up against the bone surface forming small cup-like Howship's lacunae. They contain a ruffle border area, numerous mitochondria and an ample system of Golgi membranes. A decrease in local osteoclaststimulating factors might favor the break-up of multinu 22
cleated cells back to the uninuclear cell type and a concurrent movement away from the bone surface. The action of osteoclast stimulating factors for brief periods of time might favor small osteoclasts on the bone surface and the presence of uninuclear osteoclast-like cells in the connective tissue adjacent to the bone. Osteoclasts stimulated by parathyroid hormone (PTH) have large ruffled borders in contact with the bone surface during periods of active bone resorption, but when PTH is depleted or when calcitonin is added to the culture media the osteoclasts migrate away from the bone and their ruffled border regions undergo atro phy. It has been suggested that osteoclasts modulate to and from the active multinucleated giant cell jux taposed to bone and an inactive cell type situated away from bone and that this modulation is under the control of several hormones, chiefly P T H and calcitonin. Conceivably the osteoclastic activating factors generated in plaque and/or the inflammatory infiltrate might act in a similar fashion to favor the modulation of activated osteoclasts. In the advanced stage of the disease (65 days postinfec tion and older) osteoclasts were observed in greater frequency along the remaining alveolar bone. Large masses of plaque and advanced root caries characterized the lesion at this stage. In spite of intact epithelium the concentration of plaque derived osteoclast activating stimuli could reach high levels within the connective tissue simply because of the greater concentration of bacteria in the interdental spaces. The smaller size of the osteoclasts seen at the alveolar crest and their infrequent presence in the early phases of the disease might account for the apparent differences between this report and the findings presented by Irving et al. 23-25
24
9
SUMMARY
Alveolar bone destruction in rats monoinfected with Actinomyces naeslundii proceeds via osteoclastic resorp tion. The osteoclastic activity is discontinuous, i.e. short periods of vigorous osteoclastic activity are followed by longer periods of inactivity during which the alveolar crest may be covered with osteoblastic cells or in many cases devoid of bone cells. The net effect in the monoin fected rats is rapid bone loss. Periods of osteoclastic activity were associated with dense inflammatory infil tration of the interdental tissue, ulceration of col epi thelium and the presence of large adherent plaques of Actinomyces. Ultrastructural examination revealed uninuclear as well as multinulear osteoclasts. These cells contained nu merous mitochondria, abundant Golgi apparatuses, and well developed ruffled borders indicating that they were physiologically active in bone resorption. REFERENCES
1. Socransky, S. S., Hubersak, C , and Propas, D.: Induc tion of periodontal destruction in gnotobiotic rats by a human
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Osteoclastic Alveolar Bone Resorption7 2 3
oral strain of Actinomyces naeslundii. Arch Oral Biol 15: 993, 1970. 2. Jordan, H . V., Keyes, P. H., and Bellack, S.: Periodontal lesions in hamsters and gnotobiotic rats infected with actinomyces of human origin. J Periodon Res 7: 21, 1972. 3. Gibbons R. J., Berman, K. S., Knoettner, P., and Kapsimalis, B.: Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Arch Oral Biol 11: 549, 1966. 4. Sharawy, A . M . , and Socransky, S. S.: Effect of human streptococcus strain GS-5 on caries and alveolar bone loss in conventional mice and rats. J Dent Res 46: 1385, 1967. 5. Baer, P. N . , and Bernick, S.: Age changes in the periodontium of the mouse. Oral Surg 10: 430, 1957. 6. Baer, P. N . , and Newton, W. L.: The occurrence of periodontal disease in germfree mice. J Dent Res 38: 1238, 1959. 7. Baer, P. N . and Newton, W. L.: Studies of periodontal disease in the mouse. Oral Surg 13: 1134, 1960. 8. Baer, P. N . , Newton, W. L., and White, C. L.: Studies on periodontal disease in the mouse. J Periodontol 35: 388, 1964. 9. Irving, J. T., Socransky, S. S., and Heeley, J. D.: Histological changes in experimental periodontal disease in gnotobiotic rats and conventional hamsters. J. Periodont Res 9: 73, 1974. 10. Rizzo, A . A., and Mergenhagen, S. E.: Studies on the significance of local hypersensitivity in periodontal disease. Periodontics 3: 271, 1965. 11. Ranney, R. R., and Zander, H . A.: Allergic periodontal disease in sensitized squirrel monkeys. J Periodontol 41: 12, 1970. 12. Thonard, J. C , and Aladjem, R.: Local cellular anti bodies. Response in splenectomized rats following oral mu cosa immunization with sheep erythrocytes. Aust J Exp Biol Med 5c/48: 501, 1970.
13. Hausmann, E., Raisz, L. G., and Miller, W. A.: Endotoxin: Stimulation of bone resorption in tissue culture. Science 168: 862, 1970.
14. Hausmann, E., and Weinfeld, N . : Human dental plaque: Stimulation of bone resorption in tissue culture. Arch Oral Biol 18: 1509, 1973. 15. Horton, J. E., Raisz, L. G., Simmons, H . A., Oppenheim, J. J., and Mergenhagen, S. F.: Bone resorbing activity in supernatant fluid from cultured human peripheral blood leuko cytes. Science 111: 793, 1972. 16. Hausmann, E., Genco, R., Weinfeld, N . , and Sacco, R.: Effects of sera on bone resorption in tissue culture. Calcif Tis sue Res 13: 311, 1973. 17. Kerebel, B., Clergeau-Guerithault, S. and Brion, M . : A scanning electron microscope study of experimental periodon tal disease. Its induction and inhibition. J Periodontol 46: 27, 1975. 18. Dudley, H . R., and Spiro, D.: The fine structure of bone cells. J Biophys Biochem Cytol 11: 627, 1961. 19. Gonzales, F., and Karnovsky, M . J.: Electron micros copy of osteoclasts in healing fractures of rat bone. J Biophys Biochem Cytol 9: 299, 1961. 20. Lucht, V.: Osteoclasts and their relationship to bone as studied by electron microscopy. Z Zellforsch Microsk Anat 135: 211, 1973. 21. Yaeger, J. A. and Kraucunas, E.: Fine structure of the resorptive cells in the teeth of frogs. Anat Rec 164: 1, 1969. 22. Scott, B. L.: Thymidine- H electron microscope radioautography of osteogenic cells in the fetal rat. J Cell Biol 35: 115, 1967. 23. Kallio, D. M . , Garant, P. R., and Minkin, C : Ultrastructural effects of calcitonin on osteoclasts in tissue culture. J Ultras true Res 24. Holtrop, M . E., Raisz, L. G., and Simmons, H . A.: The effects of parathyroid hormone, colchicine, and calcitonin on the ultrastructure and the activity of osteoclasts in organ culture. J Cell Biol 60: 346, 1974. 25. Lucht, V.: 1973. Effects of calcitonin on osteoclasts in vivo. An ultrastructural and histochemical study. Z Zellforsch Microsk Anat 145: 75, 1973. 3
Announcements T H E A M E R I C A N BOARD OF ORAL MEDICINE The American Board of Oral Medicine will examine candidates for certification in conjunction with the annual meeting of the American Academy of Oral Medicine in Toronto, Canada. The date of the examination is May 25, 1977. Further details can be obtained from the Secretary, Dr. Joseph L . Bernier, 6905 Hillmead Road, Bethesda, Maryland 20034. T H E SEVENTH A N N U A L CONFERENCE ON T H E I M M U N O P A T H O L O G Y O F T H E SKIN The Seventh Annual Conference on the Immunopathology of the Skin organized by Dr. Ernst H . Beutner and his associates at the State University of New York at Buffalo, by Drs. Stefania Jablonska and Tadeusz P. Chorzelski of Warsaw, Poland and by Dr. Beno Michel of Case Western Reserve University in Cleveland will be given on May 26 to 28, 1977 by the Departments of Microbiology
and Dermatology through Continuing Medical Education of S U N Y / B at the Sheraton East in Buffalo, New York, immediately following the Second Annual Westwood Conference organized by Dr. Richard L. Dobson and his associates. The first part of this conference on May 26th and 27th on "Im munopathology of the Skin for Practicing Clinicians" is designed primarily to afford practitioners a basic understanding of recent advances in the field and to enable them to utilize present knowl edge of this subject in their practice. Registration fee is $95.00. For further information on registration write to Mrs. Gloria Griffin, 219 Sherman Hall, S U N Y / B , Buffalo, New York 14214. The second part of this conference which is scheduled for May 28, 1977 will be a workshop for laboratory workers interested in pre senting their own findings on the immunopathology of the skin and related problems. Titles for this workshop should be submitted be fore January 31, 1977 to Dr. Ernst H . Beutner at the Department of Microbiology, Sherman Hall, S U N Y / B , Buffalo, New York 14214.
resorption. For example bone resorption due to osteo clastic activity has been shown to occur in sensitized oral tissues that were repeatedly challenged with antigen. Endotoxin and plaque extracts stimulate osteoclastic bone resorption in vitro. Sensitized lymphocytes release an osteoclast activating factor when challenged with antigen. It has been shown also that sera will stimulate bone resorption in tissue culture and that antigen-antibody complexes increase the effect. Recent scanning electron microscopic studies of hamsters fed Keyes high carbohydrate diet 2000 have demonstrated that the alveolar bone surface of these animals is charac terized by numerous Howship's lacunae. These obser vations suggest that the disappearance of alveolar bone was due to osteoclastic bone resorption. In view of the increasing evidence implicating osteo clast activation as an important and perhaps key phe nomenon leading to bone destruction in periodontal disease, the conclusion drawn by Irving, et al. that bone loss in periodontal disease might occur via some previ ously undetermined process, is rather surprising. On the basis of ultrastructural studies of the progres sion of periodontal disease in gnotobiotic rats monoin fected with Actinomyces naeslundii this author does not agree with the findings of Irving et al. The present paper reports the presence of osteoclastic activity along crestal alveolar bone in severely inflamed interdental periodon tal tissues of 2- to 4-month-old monoinfected rats and in addition a widespread appearance of osteoclasts in the later stages of the disease. Several temporal and morpho logical features which could account for the findings of Irving et al. are discussed. 10-12
Observations of Osteoclastic
13-14
Alveolar Bone Resorption in
15
Rats Monoinfected with
16
Actinomyces Naeslundii* by
17
PHILIAS R .
GARANT†
W H E N GNOTOBIOTIC RATS and hamsters are monoin
fected with Actinomyces naeslundii they rapidly de velop a severe periodontitis characterized by extensive alveolar bone destruction and extensive root caries. Innoculation of capsule forming streptococci in gnoto biotic rats and in conventional rats also causes exten sive bone loss. Although periodontal disease can be a naturally occur ring phenomenon in rodents as exemplified by the observations that epithelial migration and alveolar bone resorption increase with age in both germ free and conventional mice, there is no comparison to the massive and rapid bone destruction seen in gnotobiotic rats monoinfected with Actinomyces naeslundii. The precise cause and the mechanism whereby the alveolar bone is destroyed in the monoinfected rats is still a source of contention. Undoubtedly, the plaque is the important factor since a common feature of most studies was the association of massive plaque accumulation and resorp tion of the adjacent alveolar bone. Jordan, Keyes and Bellack have reported that osteo clastic activity was responsible for the bone resorption adjacent to surfaces coated with plaque in hamsters and gnotobiotic rats monoinfected with Actinomyces. More recently Irving, Socransky and Heeley in reporting on the histologic changes seen in periodontal disease in gnotobiotic rats have stated that alveolar bone loss was not accompanied by the presence of osteoclasts. They concluded on the basis of autoradiography (to label bone formation) and histology that the bone loss was due to a gradual cessation of bone formation instead of active osteoclastic resorption. Furthermore, they stated that if some active resorption did occur it must have been brought about by an as yet undescribed mechanism. There is considerable evidence that inflammatory processes can generate stimuli for osteoclastic bone 1,2
3
4
5-8
9
9
9
1-4
2
9
* This research was supported by U.S. Public Health Service grant No. 2 R01 D E 03745. † Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, N . Y . 11794.
717
MATERIAL AND M E T H O D S
Five-week-old germ free rats were inoculated with Actinomyces naeslundii and reared in isolators on Diet 2000. Cultures were taken 1 week after inoculation to insure that an infection had been established. A total of 16 monoinfected rats were used in this study. The rats were sacrificed at 4, 10, 24, 28, 40, 60, 65 and 180 days after initial introduction of the bacteria. Two germ free rats sacrificed at each of the above times, plus two at 300 days, were examined as controls. In order to insure that proper tissue relationships in the critical interdental areas were maintained during the processing and sectioning, fixation by whole body perfu sion followed by decalcification of entire jaws was employed. The primary fixative consisted of 1% glutaraldehyde in a Tyrode solution with an osmolarity of about 350 mOsm. The rats were anesthetized with Nembutal (5 to 7 mg/100 gm of body weight) prior to the start of the procedure. The perfusate was administered through a cannula inserted into the ascending aorta via an inci sion into the left ventricle. Two to three hundred milli liters of fixative were delivered over a period of about 20 minutes at a pressure equivalent to five feet of water. Upon completion of the perfusion the maxillae and
J. Periodontol. December, 1976
7 1 8 Garant mandibles were dissected free of surrounding tissues and placed in a 0.1 M solution of ethylenediaminetetraacetate (EDTA) adjusted to pH 7.4 with sodium hydroxide and containing 4.0% glutaraldehyde. The jaws were allowed to undergo decalcification in this solution for about 3 to 4 weeks at 4°C. Upon completion of the de calcification the jaws were cut into small blocks, each block to contain one interdental area. These blocks were then rinsed in several changes of buffer and were postfixed for 1½ hours at 4°C in 2% solution of osmium tetroxide buffered to pH 7.4 with collidine. Dehydration was carried out in a graded series of cold ethanol followed by several changes of propylene oxide. The tissue blocks were then embedded in Epon 812. Flat embedding trays were utilized in order to insure the correct tissue orientation required to obtain sections through the interdental area in a mesiodistal plane. One micron sections through the interdental region were stained with toluidine blue for light microscopic exami nation. Thin sections, ranging from 600 to 1000 Angstroms were cut on a Porter-Blum MT2 equipped with a second diamond knife. The thin sections were stained with uranyl acetate and lead citrate prior to examination in a JEM-100B electron microscope. RESULTS
Light microscopic examination of 1µthick sections of germ free interdental areas with toluidine blue revealed
intact junctional and col epithelium overlying a narrow lamina propria and a well defined zone of transseptal fibers. The junctional epithelium was usually terminated at the cemento-enamel junction. Dense, round inflamma tory cells, some readily classified as polymorphonuclear neutrophilic leukocytes (PMNLs) on the basis of nuclear morphology, were present in moderate numbers within the lamina propria and junctional epithelium. The dense connective tissue of the transseptal fibers and the perio dontal ligament was nearly free of inflammatory cells. The crest of the alveolar bone in the germ free animals was usually covered by an osteoblastic layer. Osteoclastic activity was rarely noted at the crest of the alveolar bone in the germ free rats. Examination of the monoinfected rats revealed greater infiltration of the lamina propria and overlying epithe lium by inflammatory cells. Although the P M N L pre dominated, numerous macrophages and monocytes were also present in the lamina propria. Large adherent plaque deposits were present in the interdental spaces. Migration of the epithelial attachment and an increased incidence of rete peg formation were commonly observed. A distinct layer of transseptal fibers could be identified to form a barrier between the enlarged lamina propria and the underlying alveolar bone. Inflammatory cells were con fined for the most part to the lamina propria and epithelial tissue. In approximately 10% of the interdental areas that were examined from the monoinfected group
FIGURE 1. Light micrograph of an interdental area of a monoinfected rat. A dense inflammatory infiltrate is present in the lamina propria and has spread throughout the transseptal fiber region. Note the many osteoclasts (OC) against the alveolar crest as well as within the adjacent connective tissue. (Original magnification, x 200.) FIGURE 2. Light micrograph depicting osteoclastic resorption of alveolar crest in a monoinfected rat. Note the relatively small size of the osteoclasts (OC). (Original magnification, x 450.)
Volume 47 Number 12
Osteoclastic Alveolar Bone Resorption7 1 9
FIGURE 3. Electron micrograph of binucleated osteoclast situated in a small Howship's lacuna at the crest of the alveolar bone. Note the typical central ruffled border region (RB) flanked by clear zones (CZ). Numerous vacuoles (V) fill the cytoplasm situated nearest to the bone surface. The distal region of the cell is occupied by nuclei (N), mitochondria (M) and small profiles of granular endoplasmic reticulum and Golgi saccules best seen in figure 4. x 3500. (Original magnification, x 3500.) aged 10 to 65 days postinfection a more severe inflamma tory reaction was noted (Fig. 1). In these specimens a dense P M N L infiltration extended into and disrupted the dense connective tissue of the transseptal fibers. Close scrutiny of the epithelium revealed small regions of ulceration. The alveolar bone of these interdental areas
invariably demonstrated scalloping of the surface and several small osteoclasts (Fig. 1). These small osteoclasts frequently contained no more than two or three nuclei, and in some cases only one nucleus was observed in their sectioned profile (Fig. 2). Cells of similar staining characteristics (gray with toluidine blue) containing one
7 2 0 Garant or more nuclei were frequently noted in the connective tissue adjacent to the bone. Because this degree of osteoclastic activity was observed in but a small percent of the interdental areas, it is felt that alveolar bone destruction in the early phase of the disease is the result of repeated short bursts of osteoclastic activity associ ated with periods of acute inflammation. Epithelial ulceration in the presence of the plaque seemed to play an important role in inducing the more acute inflamma tory reaction. In the older monoinfected rats (i.e. 65 days postinfec tion) great masses of plaque were routinely observed especially in the maxillary regions. Histologic examina tion of these specimens revealed that in spite of a continuous epithelial lining and a generally more granu lomatous connective tissue the remaining alveolar and basal bone was frequently covered by osteoclasts. At the ultrastructural level the osteoclasts were charac terized by numerous mitochondria, several well devel oped Golgi apparatuses, granular endoplasmic reticu lum, cytoplasmic vacuoles, ruffled borders and clear zones (Figs. 3, 4 and 5). The nuclei which were concentrated in the distal cytoplasm, i.e. away from the bone surface, contained prominent nucleoli. A uniformly narrow nuclear enve
J. Periodontol. December, 1976
lope interrupted by the presence of several nuclear pores surrounded the nucleus (Fig. 4). The outer membrane of the nuclear envelope was not in association with ribosomes as is the case in osteoblasts and osteocytes. Many short profiles of granular endoplasmic reticulum were present, especially in the more distal and peripheral areas of the cytoplasm (Figs. 4 and 6). Free ribosomes were present in large numbers, aggregated in the form of small polyribosomes (Fig. 6). The Golgi complex consisted of numerous distinct concentrations of flattened saccules and many vesicles, usually situated in the perinuclear areas of the cytoplasm (Fig. 4). Larger membrane-bound granules containing a substance of moderate electron opacity were also present near the Golgi apparatuses and also in the cytoplasm adjacent to the ruffled border. The cell membrane along the lateral and distal surfaces of the osteoclast was characterized by several small microvilli. The proximal surface, abutting directly upon the alveolar bone, was specialized into two regions which have been shown to be of prime importance in bone resorption. A central region consisting of numerous cytoplasmic processes was juxtaposed against the region of bone breakdown (Figs. 3 and 5). This central zone, called the ruffled border, is the hallmark of the active
FIGURE 4. Distal cytoplasm of an osteoclast depicting the mitochondria (M), Golgi saccules (G) and the granular endoplasmic reticulum (GER). The nuclei (N) are encircled by a narrow nuclear envelope (NE) devoid of any attached ribosomes. (Original magnification, x 7000.) FIGURE 5. Enlarged view ofpart of an osteoclast juxtaposed to the alveolar bone. Typical ruffled border (RB) regions characterized by numerous microvilli and cytoplasmic infoldings were noted. Clear zones (CZ) are filled with densely packed cytoplasmic filaments. These cytoplasmic specializations provide morphological evidence of active osteoclastic activity. (Original magnification, x 34,000.)
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FIGURE 6. Osteoclast-like cell in the connective tissue adjacent to alveolar bone. Note the modified ruffled border consisting of broad cytoplasmic processes (CP). G E R , granular endoplasmic reticulum; M , mitochondria; N , nucleus. (Original magnification, x 4600.)
osteoclast. A peripheral clear zone containing numerous cytoplasmic filaments, to the exclusion of all other cytoplasmic components, formed a rim around the centrally located ruffled border (Figs. 3 and 5). The bone surface underneath the osteoclast did not have a lamina
limitans suggesting that the surface was in a dynamic state. Osteoclast-like cells were also present in the connective tissue adjacent to the bone surface (Figs. 1 and 6). Although these cells were usually multinucleated, some
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Garant
uninucleated cells with similar cytoplasmic features were observed. Characteristically they demonstrated a modi fied ruffled border zone and numerous short segments of granular endoplasmic reticulum, free ribosomes and mitochondria (Fig. 6). DISCUSSION
These observations of the progressive development of periodontal disease in rats monoinfected with Actinomyces naeslundii suggests that alveolar bone de struction in periodontitis is the result of osteoclastic activity. The ultrastructural appearance of osteoclasts has been amply documented. The cells observed in this study manifested the same features reported in previous publi cations and in addition their well developed ruffled borders, clear zones, mitochondria and abundant Golgi apparatuses, suggest that they are actively engaged in bone resorption. The presence of osteoclasts on the crest of the alveolar bone in the early phases of the disease (10 to 65 days postinfection) was associated with a concur rent dense inflammatory cell infiltration of the lamina propria and ulceration of the interdental col epithelium. Since osteoclasts and the associated micropathological changes were seen only in about 10% of the interdental areas examined, it is further suggested that osteoclastic activity leading to reduction in the height of the alveolar process is a discontinuous phenomenon characterized by short periods of vigorous osteoclastic activity followed by longer periods of inactivity. During the periods of inactivity the local stimuli for osteoclast differentiation are reduced and the crestal surfaces of the alveolar bone are covered by osteoblast-like cells or in some cases appear to be free of any bone cells. The findings of Irving et al. that alveolar bone formation is markedly reduced in the monoinfected rats could account for the observed net loss of bone in spite of the fact that osteoclastic activity occurs during only a small percent of the time. 18-21
9
The additional finding that osteoclasts seen at the alveolar crest are usually quite small, many appearing uninuclear in profile, could be an indication that most are immature in the sense that the acquisition of multiple nuclei represents a maturation of the osteoclast cell line. It has been postulated on the basis of E M autoradio graphic studies of tritiated thymidine labelled cells that a separate osteoprogenitor cell line for osteoclasts exists in periosteal tissue and that the osteoclast precursor cell is a round cell rich in mitochondria and Golgi membranes. Although the small osteoclast may represent a newly differentiated osteoclast, its histologic (in Epon embed ded material) and ultrastructural features indicate a physiologic capability for bone resorption. These cells are present up against the bone surface forming small cup-like Howship's lacunae. They contain a ruffle border area, numerous mitochondria and an ample system of Golgi membranes. A decrease in local osteoclaststimulating factors might favor the break-up of multinu 22
cleated cells back to the uninuclear cell type and a concurrent movement away from the bone surface. The action of osteoclast stimulating factors for brief periods of time might favor small osteoclasts on the bone surface and the presence of uninuclear osteoclast-like cells in the connective tissue adjacent to the bone. Osteoclasts stimulated by parathyroid hormone (PTH) have large ruffled borders in contact with the bone surface during periods of active bone resorption, but when PTH is depleted or when calcitonin is added to the culture media the osteoclasts migrate away from the bone and their ruffled border regions undergo atro phy. It has been suggested that osteoclasts modulate to and from the active multinucleated giant cell jux taposed to bone and an inactive cell type situated away from bone and that this modulation is under the control of several hormones, chiefly P T H and calcitonin. Conceivably the osteoclastic activating factors generated in plaque and/or the inflammatory infiltrate might act in a similar fashion to favor the modulation of activated osteoclasts. In the advanced stage of the disease (65 days postinfec tion and older) osteoclasts were observed in greater frequency along the remaining alveolar bone. Large masses of plaque and advanced root caries characterized the lesion at this stage. In spite of intact epithelium the concentration of plaque derived osteoclast activating stimuli could reach high levels within the connective tissue simply because of the greater concentration of bacteria in the interdental spaces. The smaller size of the osteoclasts seen at the alveolar crest and their infrequent presence in the early phases of the disease might account for the apparent differences between this report and the findings presented by Irving et al. 23-25
24
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SUMMARY
Alveolar bone destruction in rats monoinfected with Actinomyces naeslundii proceeds via osteoclastic resorp tion. The osteoclastic activity is discontinuous, i.e. short periods of vigorous osteoclastic activity are followed by longer periods of inactivity during which the alveolar crest may be covered with osteoblastic cells or in many cases devoid of bone cells. The net effect in the monoin fected rats is rapid bone loss. Periods of osteoclastic activity were associated with dense inflammatory infil tration of the interdental tissue, ulceration of col epi thelium and the presence of large adherent plaques of Actinomyces. Ultrastructural examination revealed uninuclear as well as multinulear osteoclasts. These cells contained nu merous mitochondria, abundant Golgi apparatuses, and well developed ruffled borders indicating that they were physiologically active in bone resorption. REFERENCES
1. Socransky, S. S., Hubersak, C , and Propas, D.: Induc tion of periodontal destruction in gnotobiotic rats by a human
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oral strain of Actinomyces naeslundii. Arch Oral Biol 15: 993, 1970. 2. Jordan, H . V., Keyes, P. H., and Bellack, S.: Periodontal lesions in hamsters and gnotobiotic rats infected with actinomyces of human origin. J Periodon Res 7: 21, 1972. 3. Gibbons R. J., Berman, K. S., Knoettner, P., and Kapsimalis, B.: Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Arch Oral Biol 11: 549, 1966. 4. Sharawy, A . M . , and Socransky, S. S.: Effect of human streptococcus strain GS-5 on caries and alveolar bone loss in conventional mice and rats. J Dent Res 46: 1385, 1967. 5. Baer, P. N . , and Bernick, S.: Age changes in the periodontium of the mouse. Oral Surg 10: 430, 1957. 6. Baer, P. N . , and Newton, W. L.: The occurrence of periodontal disease in germfree mice. J Dent Res 38: 1238, 1959. 7. Baer, P. N . and Newton, W. L.: Studies of periodontal disease in the mouse. Oral Surg 13: 1134, 1960. 8. Baer, P. N . , Newton, W. L., and White, C. L.: Studies on periodontal disease in the mouse. J Periodontol 35: 388, 1964. 9. Irving, J. T., Socransky, S. S., and Heeley, J. D.: Histological changes in experimental periodontal disease in gnotobiotic rats and conventional hamsters. J. Periodont Res 9: 73, 1974. 10. Rizzo, A . A., and Mergenhagen, S. E.: Studies on the significance of local hypersensitivity in periodontal disease. Periodontics 3: 271, 1965. 11. Ranney, R. R., and Zander, H . A.: Allergic periodontal disease in sensitized squirrel monkeys. J Periodontol 41: 12, 1970. 12. Thonard, J. C , and Aladjem, R.: Local cellular anti bodies. Response in splenectomized rats following oral mu cosa immunization with sheep erythrocytes. Aust J Exp Biol Med 5c/48: 501, 1970.
13. Hausmann, E., Raisz, L. G., and Miller, W. A.: Endotoxin: Stimulation of bone resorption in tissue culture. Science 168: 862, 1970.
14. Hausmann, E., and Weinfeld, N . : Human dental plaque: Stimulation of bone resorption in tissue culture. Arch Oral Biol 18: 1509, 1973. 15. Horton, J. E., Raisz, L. G., Simmons, H . A., Oppenheim, J. J., and Mergenhagen, S. F.: Bone resorbing activity in supernatant fluid from cultured human peripheral blood leuko cytes. Science 111: 793, 1972. 16. Hausmann, E., Genco, R., Weinfeld, N . , and Sacco, R.: Effects of sera on bone resorption in tissue culture. Calcif Tis sue Res 13: 311, 1973. 17. Kerebel, B., Clergeau-Guerithault, S. and Brion, M . : A scanning electron microscope study of experimental periodon tal disease. Its induction and inhibition. J Periodontol 46: 27, 1975. 18. Dudley, H . R., and Spiro, D.: The fine structure of bone cells. J Biophys Biochem Cytol 11: 627, 1961. 19. Gonzales, F., and Karnovsky, M . J.: Electron micros copy of osteoclasts in healing fractures of rat bone. J Biophys Biochem Cytol 9: 299, 1961. 20. Lucht, V.: Osteoclasts and their relationship to bone as studied by electron microscopy. Z Zellforsch Microsk Anat 135: 211, 1973. 21. Yaeger, J. A. and Kraucunas, E.: Fine structure of the resorptive cells in the teeth of frogs. Anat Rec 164: 1, 1969. 22. Scott, B. L.: Thymidine- H electron microscope radioautography of osteogenic cells in the fetal rat. J Cell Biol 35: 115, 1967. 23. Kallio, D. M . , Garant, P. R., and Minkin, C : Ultrastructural effects of calcitonin on osteoclasts in tissue culture. J Ultras true Res 24. Holtrop, M . E., Raisz, L. G., and Simmons, H . A.: The effects of parathyroid hormone, colchicine, and calcitonin on the ultrastructure and the activity of osteoclasts in organ culture. J Cell Biol 60: 346, 1974. 25. Lucht, V.: 1973. Effects of calcitonin on osteoclasts in vivo. An ultrastructural and histochemical study. Z Zellforsch Microsk Anat 145: 75, 1973. 3
Announcements T H E A M E R I C A N BOARD OF ORAL MEDICINE The American Board of Oral Medicine will examine candidates for certification in conjunction with the annual meeting of the American Academy of Oral Medicine in Toronto, Canada. The date of the examination is May 25, 1977. Further details can be obtained from the Secretary, Dr. Joseph L . Bernier, 6905 Hillmead Road, Bethesda, Maryland 20034. T H E SEVENTH A N N U A L CONFERENCE ON T H E I M M U N O P A T H O L O G Y O F T H E SKIN The Seventh Annual Conference on the Immunopathology of the Skin organized by Dr. Ernst H . Beutner and his associates at the State University of New York at Buffalo, by Drs. Stefania Jablonska and Tadeusz P. Chorzelski of Warsaw, Poland and by Dr. Beno Michel of Case Western Reserve University in Cleveland will be given on May 26 to 28, 1977 by the Departments of Microbiology
and Dermatology through Continuing Medical Education of S U N Y / B at the Sheraton East in Buffalo, New York, immediately following the Second Annual Westwood Conference organized by Dr. Richard L. Dobson and his associates. The first part of this conference on May 26th and 27th on "Im munopathology of the Skin for Practicing Clinicians" is designed primarily to afford practitioners a basic understanding of recent advances in the field and to enable them to utilize present knowl edge of this subject in their practice. Registration fee is $95.00. For further information on registration write to Mrs. Gloria Griffin, 219 Sherman Hall, S U N Y / B , Buffalo, New York 14214. The second part of this conference which is scheduled for May 28, 1977 will be a workshop for laboratory workers interested in pre senting their own findings on the immunopathology of the skin and related problems. Titles for this workshop should be submitted be fore January 31, 1977 to Dr. Ernst H . Beutner at the Department of Microbiology, Sherman Hall, S U N Y / B , Buffalo, New York 14214.
