Arck oralBiol.Vol. 37, No. 6, pp. 435-438.1992 Printed in Great

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PERIODONTAL BONE LOSS IN MICE INDUCED BY DIFFERENT PERIODONTOPATHIC ORGANISMS D. WRAY and L. GRAHAME Department of Oral Medicine and Oral Pathology, University of Edinburgh, Edinburgh EHl INR, U.K. (Received 8 October 1991; accepted I8 February 1992) Summary-Periodontal bone loss in mice orally inoculated with Peptostreptococcusunaerobius, Pept. magnus and Actinobacilfusacrinomycefemcomita was compared to that in sham-inoculated mice. Six-to-8-week-old BALB/c mice were inoculated with I x IO’, 1 x IO’ or I x 10’ colony-forming units (c.f.u.) of bacteria in 50~1 of medium. Ten mice received each concentration of bacteria and 10 sham-inoculated mice acted as controls. Five mice from each of the groups were killed 6 weeks after inoculation and the remaining five mice at I2 weeks. Right hemimandibles were defleshed, stained and bone loss was measured using an image analyser. All the organisms tested were associated with bone loss. Animals that had received Pept. anaerobiusand Pepr. mugnus had up to 18% more bone loss than those sham inoculated. In contrast, mice inoculated with A. actinomycetemcomitans had up to 38% more bone loss than the sham-inoculated animals, this amount of loss occurring at the lowest inoculation of I x IO’ c.f.u. These data demonstrate a differential ability of micro-organisms to cause periodontal bone loss in mice. Key words: periodontal bone loss, mice, micro-organisms.

1990) found in association with periodontal bone loss. We here examine the potential of various orally inoculated bacteria to cause murine periodontal bone loss.

INTRODUCTION The role of specific Gram-negative

bacteria in the pathogenesis of human periodontal disease has been increasingly appreciated in recent years (Slots, 1979). Porphyromonas gingivalis (Slots, 1982a) has been implicated in many cases of severe adult periodontitis, Actinobacillus actinomycetemcomitans in localized juvenile periodontitis (Slots and Rosling, 1983; Zambon, Christersson and Slots, 19831, and Prerotella intermedius and spirochaetes of intermediate size in acute necrotizing ulcerative gingivitis (Chung et al., 1983; Loesche et af., 1982). P. intermedius has been associated with pregnancy gingivitis (Komman and Loesche, 1980), Cupnocytophuga spp. with advanced periodontitis in juvenile diabetics (Mashimo et al., 1983a), granulocytopenic and immunocompromised hosts (Mashimo et al., 1983b). Other organisms, including Eubacterium spp., Haemophilus spp., Wolinelia spp., Fusobacterium spp., Selenomonas sputigena and Eikenella corrodens, may also participate in some forms of destructive periodontal disease. The pathogenicity of these putative periodontopathic organisms is difficult to assess in man and the roles of host reactions and parasitic effects in leading to the destruction of the periodontium cannot be reliably separated. For these reasons, the microbiology of periodontal disease remains controversial. Previous studies in this laboratory have shown that BALB/c mice can be successfully orally inoculated with Actinomyces viscosus, and that, with appropriate inoculation dosages, they develop 50% more periodontal bone loss than sham-inoculated mice within 12 weeks (Gilbert, Wray and Charon, 1990). The increased bone loss is associated with increased specific IgA responses, making this murine model suitable for the study of both genetic factors (Gilbert and Sofaer, 1988) and host responses (Gilbert et al.,

MATERIALS AND METHODS Selection of micro-organisms

Pilot studies were carried out to establish the infectivity of various human periodontopathic organisms in mice. The organisms studied included Bacteroides oralis, P. intermedius, B. melaninogenicus, Porph. gingivalis, Pept. anaerobius, Pept. magnus, Act. actinomycetemcomitans and Capnocytophaga sputigena. Organisms were cultured on appropriate media and adjusted to 1 x IO’, 1 x IO6 or 1 x 10’ colony

forming units (c.f.u.) in 50 ~1 of medium. Six- to 8-week-old BALB/c mice were orally inoculated on three consecutive afternoons and fluids withheld overnight to establish the minimum infective inoculum. Four mice were inoculated with each concentration of bacteria and four sham-inoculated mice acted as controls. All mice were killed 2 weeks after inoculation, their molar teeth ground in medium, and the slurry plated out on relevant semi selective media and blood agar. Colonial appearance and Gram staining confirmed positive identification. Pept. spp., Act. actinomycetemcomitans and C. sputigeno but no black pigmenting organisms could be successfully recovered. Organisms successfully infecting the mice were all recovered from the ground molars at a concentration of 1 x 104 c.f.u. per mouse regardless of the initial inoculum dose. Pept. magnus, Pept. anaerobius and Act. actinomycetemcomitans were selected for further study on the basis of these preliminary studies of infectivity.

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and L. GRAHA.HE

Micro-organisms Pept. magnus was an isolate from a wound and was obtained from the Microbiology Department of the Western General Hospital, Edinburgh, U.K. Strain 27317 of Pept. anaerobius was obtained as a lyophilized culture from the American Type Culture Collection. Strain 9710 of Act. actinomycetemcomitans was obtained as lyophilized cultures from the National Collection of Type Cultures, London, U.K. These cultures were reconstituted with cooked meat broth and transferred to the appropriate medium. Peptostreptococcus spp. were cultured anaerobically for 24-48 h in pre-reduced PPY medium at 37’C. Act. actinomycetemcomitans was cultured in an anaerobic atmosphere containing 5% CO: at 37” for X-48 h in tryptic soy serum, bacitracin, vancomycin agar (Slots, 1982b). Mice

BALB,‘c mice were obtained from the breeding colony in Edinburgh University. All mice were conventionally reared and not pathogen free. They were maintained at 21°C and allowed food and water ad libitum except as otherwise stated in the experimental schedule. All experimental and sham-inoculated mice were housed under identical conditions. Diet

Mice were weaned at 3 weeks of age and initially fed a standard laboratory diet (Oxoid mouse diet, Oxoid, Basingstoke, U.K.). All mice were then transferred to a special soft diet that was high in carbohydrate and low in fat. consisting of 40 g of sucrose, I5 g wheat flour, 32 g skimmed milk, 5 g brewers’ yeast and 2 g vitamin-mineral-protein supplement (MacKay & Lynn, Edinburgh, U.K.). Experimental

Mean periodontal bone loss for the different inoculum groups is shown in Tables 1-3, together with the percentage increase in bone loss seen in association with the various micro-organisms compared to that in the sham-inoculated animals. Mean bone loss is shown for the groups killed 6 and 12 weeks after inoculation. Tables 1 and 2 show that 6 weeks after inoculation with Peptostreptococcus spp., bone loss was increased only with the higher inoculum doses, with up to 18% increased bone loss for Pept. anaerobius. This pattern of enhanced bone loss in association with higher doses of Peptostreptococcus spp. remained substantially unchanged 12 weeks after inoculation. At 12 weeks, bone loss among sham-inoculated animals had continued but inoculation with high doses of Peptostreptococcus spp. was persistently associated with increased bone loss. Table 3 shows the mean bone loss in the groups inoculated with Act. actinomycetemcomitans. These data show a different pattern of bone loss. At 6 weeks the smallest dose of I x 1O’c.f.u. Act. actinomycetemcomitans was associated with 38% more bone loss than in the sham-inoculated animals. This is more than twice the increase in bone loss seen after inoculation with the Peptostreptococcus spp. Higher doses of Act. actinomycetemcomitans were associated with a relative decrease in bone loss, and inoculation with 1 x IO9c.f.u. produced no increase in bone loss over that in sham-inoculated animals after 6 weeks. Twelve weeks after inoculation the enhanced bone loss seen in association with Act. actinomycetemcomitans was less dramatic. Even the most pathogenic dose produced only 8% increased bone loss compared to the sham-inoculated animals. DISCUSSION

schedule

After 1 week on the special soft diet, 6-8-rveek-old BALB/c mice were orally inoculated with 1 x IO’, 1 x IO’ or 1 x IO9 c.f.u. of each bacteria in 50 ~1 of medium. Ten mice were inoculated with each concentration and 10 sham-inoculated mice acted as controls. After inoculation, fluid was withheld overnight. Inoculation took place on three consecutive days. Five mice from each group were killed by ether inhalation 6 weeks after inoculation and the remaining five mice 12 weeks after inoculation. Assessment

RESULB

of bone loss

After death, right hemimandibles were defleshed in a 5% solution of papain and stained with van Gieson stain for approx. 10min (Luther, 1949). Stain was taken up by the cementum but not by the enamel. The lingual surfaces of the mandibles were then photographed in a standardized fashion and the area of bone loss measured on a Micro Magiscan image analyser (Joyce Loebl, Gateshead U.K.). Periodontal bone loss was measured as the area of exposed molar root surfaces on the lingual side bounded by the anterior border of the first molar, the amelocemental junctions, the posterior border of the third molar and the alveolar bone crest.

We demonstrate different patterns of periodontal bone loss in mice after oral inoculation with different organisms. For both Pept. magnus and Pept. anaerobius, higher doses of inoculation were associated with an early and sustained increase in bone loss compared to that in sham-inoculated animals, indicating that these micro-organisms are indeed periodontopathic in mice. The pattern of bone loss associated with Act. actinomycetemcomitans inoculations was quite differ-

ent. There was a 38% increase in bone loss after 6 weeks at the lowest dose but the increase was not sustained to 12 weeks. In addition, higher doses were Table I. Mean bone loss in mm* 6 and 12 weeks after oral inoculation with Pepr. magnus

Inoculum Sham 105 10’ 109

6 weeks 0.356’ f 0.014 (O%)t 0.353 & 0.010 (0%) 0.410 f 0.014 (15%): 0.394 f 0.022 (11%)

12 weeks 0.407 + 0.397 k 0.449 + 0.450 +

0.012 (0%) 0.016 (0%) 0.017 (lo%): 0.012 (I 1%):

*Mean f SE in mm*. tpercentage increase compared to bone loss in sham-inoculated animals. :Bone loss significantly increased compared to sham-inoculated animals (p < 0.05 r-test).

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Microbial induced periodontitis in mice Table 2. Mean bone loss in mm’ 6 and 12 weeks after oral inoculation with Pepr. onaerobius Inoculum Sham IO’ IO’ IO9

6 weeks

12 weeks

0.356, f 0.014 (O%)t 0.358+0.018(1%) 0.400 + 0.017 (12%): 0.420 f 0.014 (18%):

0.407 * 0.399 * 0.419 + 0.457 *

0.012 (0%) 0.004 (0%) 0.011 (3%) 0.01 I (12%)$

*Mean + SE in mm’. TPercentage increase compared to bone loss in sham-inoculated animals. IBone loss significantly increased compared to sham-inoculated animals @ < 0.0s r-test).

not associated with such marked bone loss. The lack of sustained bone loss at 12 weeks may reflect a reduction in colonization, although higher doses were consistently less pathogenic, and it seems more likely that host responses are protective. The inoculated mice in general had less increased bone loss at 12 weeks than at 6 weeks and perhaps this implies age-associated changes in mouse responses, as described with A. viscosus (Fitzgerald, Gebhardt and Birdsell, 1981). Regardless, Acf. acfinomycetemcomifuns at the lowest inoculum dose was associated with twice as much increased bone loss at 6 weeks as any other dose or organism. There were profound differences in the isotypespecific immunoglobulin responses in this mouse model when inoculated with A. viscosus (Gilbert er al., 1990) but here all our attempts to measure serum and salivary antibodies to the specific organisms failed through technical difficulties. Increased periodontal bone loss with A. riscosus was associated with elevated specific IgA levels at an inoculum dose of 10’ c.f.u. rather than with elevated IgG levels, which were found after inoculation of lo6 or lo9 c.f.u. Non-complement-fixing IgA may preferentially encourage perpetuation of the local inflammatory response by sequestering foreign antigen in the gingival crevice rather than the more cytotoxic IgG, which would more readily recruit phagocytes and eliminate the cause of the periodontal inflammation. Perpetuation of the inflammatory response in the gingiva would promote periodontal bone loss by osteoclast-activating factor. Inoculation with the lowest number of A. viscosus used (i.e. 1 x 10’ c.f.u.) may allow colonization of the gingival crevice only, which as it is in the domain of systemic immunity would stimulate an IgG response without significant stimulation of gut antibody. Increasing the inoculum 100 times (10’ c.f.u.) may still cause successful colonization of the gingival crevice Table 3. Mean bone loss in mm2 6 and 12 weeks after oral inoculation with ACI. ac~inomyceremcomitans Inoculum Sham IO5 IO’

IO9

12 weeks 0.407 + 0.012 (0%) 0.439 + 0.017 (8%) 0.416 +_0.013 (2%j 0.366 + 0.014 10%) -

A. viscosus.

Inoculation with Peptosrreptococcus spp. did not produce such severe bone loss as with an inoculum dose of 10’ c.f.u. of Acf. ucfinomycetemcomifans, the bone loss generally increasing with increasing dose despite similar levels of infectivity or colonization of the molar teeth. This could be explained by suggesting that the immune responses to these bacteria were different from those obtained with A. viscosus; the variation in systemic and gut-associated immune responses not being stimulated in a similar way and, in particular, an IgA response not being produced. It is unfortunate that for technical reasons it was not possible to measure the serum and salivary immunoglobulin levels to the specific organisms used in this study. Regardless,

our findings clearly show a varying periodontopathic potential. albeit in the mouse, for different putative human periodontopathogens and thus lend credence to the infection theory of periodontal disease. Alternatively, different organisms may elicit different host responses that in themselves may be harmful or protective. REFERESCES Chung C. P., Nisengard R. J.. Slots J. and Genco R. J. (1983) Bacterial IgG and IgM antibody titers in acute necrotizing ulcerative gingivitis. J. Periodonr. 54, 557-562. Fitzgerald J. E., Gebhardt B. M. and Birdsell D. E. (1981) Murine model for analysis of the immune response to oral colonisation. J. periodonf. Res. 16, 564583. Gilbert A. D. and Sofaer J. A. (1988) Host genotype, pathogenic challenge and periodontal bone loss in the mouse-, Archs oral kol. 33,. 855-861.

Gilbert A. D.., Wrav. D. and Charon J. (1990) Resnonse . immunitaire humorale au tours des parodonutes e?cperimentales chez la souris. J. fr. Parodonr. 9, 175-178. Kornman K. S. and Loesche W. J. (1980) The subgingival microbial flora during pregnancy. J. periodont. Res. 15, 111-122.

Loesche W. J., Syed S. A., Laughon B. G. and Stall J. (1982) The bacteriology of acute necrotizing ulcerative gingivitis. J. Periodon!. 53, 223-230.

6 weeks 0.356* + 0.014 (O%)t 0.491 + 0.007 (38%): 0.420 kO.024 (18%)1 0.344 -+ 0.010 (0%) \ I

but simultaneously the gut-associated lymphoid tissue could be stimulated to produce IgA preferentially. When the dose is increased 10,000 times (lo9 c.f.u.) the gut-associated lymphoid tissue may be overwhelmed and the systemic immune system stimulated once again to produce an IgG response. Our studies demonstrate a variable periodontal response to different inoculum doses of bacteria despite similar levels of molar tooth colonization or infection with all inoculation regimes. Inoculation with the lowest number of Act. acfinomycefemcomifuns used (1 x IO’) produced the maximum bone loss. In terms of immune outcome this suggests that this inoculum for Act. ucfinomycefemcomifuns was equivalent to one of 1 x 10’ for

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*Mean k SE in mm?. tpercentage increase compared to bone loss in sham-inoculated animals. $Bone loss significantly increased compared to sham-inoculated animals (p c 0.05 t-test).

Luther P. G. (1949) Enzymatic maceration of skeletons. Proc. Linn. Sot. 161, 146-147. Mashimo P. A., Yamamoto Y., Slots J., Park B. H. and Genco R. J. (1983a) The periodontal microflora of juvenile diabetics: culture, immunofluorescence and serum antibody studies. J. Periodonf. 54, 420-430. Mashimo P. A., Yamamoto Y., Nakamura M. and Slots J. (1983b) Selective recovery of oral Capnocytophaga spp. with sheep blood agar containing bacitracin and polymyxin B. J. clin. Microbial. 17, 189-191.

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Slots J. (1979) Subgingival microflora and periodontal disease. J. clin. Periodont. 6, 351-382. Slots J. (1982a) Importance of black-pigmented bacteroides. In Human Periodontal Diseases (Eds Genco R. J. and Mergenhagen S. E.), pp. 27-45. American Society for Microbial., Washington, DC. Slots J. (1982b) Selective medium for isolation of Actinobacillus accinomycetemcomirans. J. clin. Microbial. IS, 606609.

Slots J. and Rosling B. G. (1983) Suppression of the periodontopathic microflora in localized juvenile periodontitis by systemic tetracycline. J. &I. Periodont. 10, 465-486. Zambon J. J., Christenson L. A. and Slots J. (1983) Actinobacillus actinomycetemcomitans in human periodontal disease: prevalence in patient groups and distribution of biotypes and serotypes within families. J. Periodonr. 54, 707-71 I.

Periodontal bone loss in mice induced by different periodontopathic organisms.

Periodontal bone loss in mice orally inoculated with Peptostreptococcus anaerobius, Pept. magnus and Actinobacillus actinomycetemcomitans was compared...
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