Deficiency of iNOS contributes to Porphyromonas gingivalis‐induced tissue damage

Periodontitis is a chronic inflammatory disease that results in extensive soft and hard tissue destruction of the periodontium. Porphyromonas gingivalis possesses an array of virulence factors and has been shown to induce expression of inducible nitric oxide synthase (iNOS) in inflammatory cells. The aim of this study was to investigate the effect of eliminating iNOS in a murine model of P. gingivalis infection. This was achieved by utilizing a P. gingivalis‐induced skin abscess model, and an alveolar bone loss model employing an oral infection of P. gingivalis in iNOS knockout mice. The results indicated that iNOS knockout mice exhibit more extensive soft tissue damage and alveolar bone loss in response to P. gingivalis infection compared to wild‐type mice. The local immune response to P. gingivalis in iNOS knockout mice was characterized by increased numbers of polymorphonuclear monocytes, while the systemic immune response was characterized by high levels of interleukin‐12. The iNOS is required for an appropriate response to P. gingivalis infection.     

Mouse model of experimental periodontitis induced by Porphyromonas gingivalis/Fusobacterium nucleatum infection: bone loss and host response

Aim: To compare the effect of oral infection with Porphyromonas gingivalis or Fusobacterium nucleatum versus infection with both bacteria on mouse periodontal tissues, and to characterize the inflammatory response.
Materials and Methods: Mice were orally infected with P. gingivalis, F. nucleatum or both. At 42 days post-infection, alveolar bone loss was quantified using micro-computerized tomography. Tumour necrosis factor- α (TNF- α ) and interleukin (IL)-1 β levels induced by the infection were quantified using the subcutaneous chamber model.
Results: Mice orally infected with F. nucleatum / P. gingivalis exhibited significantly more bone loss compared with that of mono-infected and sham-infected mice. F. nucleatum / P. gingivalis infection also increased the levels of TNF- α and IL1 β compared with the levels found in the mono-infected groups.
Conclusions: Polymicrobial infection with P. gingivalis / F. nucleatum aggravates alveolar bone loss and induces a stronger inflammatory response compared with that observed upon infection with either bacterium alone. The results suggest that oral infection of mice with a mixture of P. gingivalis and F. nucleatum may be superior to mono-infection models of experimental periodontitis.     

Heterogeneity of Porphyromonas gingivalis strains in the induction of alveolar bone loss in mice

These experiments examine alveolar bone loss in a model in which specific pathogen-free mice are exposed orally with Porphyromonas gingivalis. Alveolar bone loss was induced as a result of a specific infection with P. gingivalis, rather than other environmental antigens. Infection with live P. gingivalis was required, as significant bone loss did not follow gavage with formalin-killed P. gingivalis. The virulence of different strains of P. gingivalis was compared. Two laboratory strains of the bacteria (ATCC 53977 and W50) and a mutant strain lacking the 43-kDa fimbrillin (strain DPG3) induced bone loss. P. gingivalis 381, however, did not induce bone loss. There was a strong immunoglobulin G (IgG) antibody response to infection with each strain but a significant serum IgA response only to strain 381. These studies show that in mice with a background oral microflora bone loss is induced by a specific infection with P. gingivalis and that bacterial strain variation is important in determining whether alveolar bone loss will ensue.     

Morphometric, histomorphometric, and microcomputed tomographic analysis of periodontal inflammatory lesions in a murine model

BACKGROUND: Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in chronic periodontitis, a common infectious disease characterized by inflammation and destruction of periodontal tissues. Several animal models with P. gingivalis have been used in periodontitis studies. Additionally, multiple approaches have also been applied to measuring alveolar bone loss in periodontitis models, including histomorphometry, morphometry, and radiography. The aims of this study were to assess periodontal inflammatory lesions after P. gingivalis-induced periodontitis and use this model to compare three approaches for assessing alveolar bone loss. METHODS: Twelve-week-old male C57BL/6 mice were divided into two groups: 48 P. gingivalis-infected and 52 untreated control mice. Periodontitis was induced by wrapping P. gingivalis-soaked ligatures around the left maxillary second molar and changing the ligatures every other day. Mice were euthanized on days 0, 3, 7, and 10 after ligature placement, for a total of 12 experimental and 13 control mice per time point. Epithelial downgrowth, inflammation, and osteoclast activity were evaluated; alveolar bone loss was determined by histomorphometry, morphometry, and microcomputed tomography. RESULTS: The P. gingivalis-infected group showed significantly increased epithelial downgrowth (P <0.05), inflammation (P <0.05), alveolar bone loss (P <0.05), and osteoclast activity (P <0.05) throughout the experimental period compared to the controls. All three methods yielded efficient evaluation of alveolar bone loss. CONCLUSIONS: Our results show evidence that the P. gingivalis-soaked ligature-induced murine model mounts an adequate inflammatory response and exhibits periodontal tissue breakdown compatible with other models of periodontal disease. In addition, alveolar bone loss can accurately be quantified using any of the three alveolar bone analyses presented in this article.     

Effect of dietary omega-3 polyunsaturated fatty acids on experimental periodontitis in the mouse

Background and Objective:  Periodontitis is an infective disease caused predominantly by gram-negative anerobes. The host inflammatory response to these bacteria causes alveolar bone loss, which characterizes periodontitis. Omega-3 polyunsaturated fatty acids have recognized anti-inflammatory effects; their oxygenated derivatives are key mediators in reducing inflammation. In this study we tested the hypothesis that dietary supplementation with tuna fish oil rich in the n-3 polyunsaturated fatty acid, docosahexaenoic acid, would reduce alveolar bone loss in mice inoculated with periodontopathic bacteria.
Material and Methods:  Adult mice were fed experimental diets containing either 10% tuna oil or Sunola oil for 57 d. After 14 d, 35 mice on each diet were inoculated orally with Porphyromonas gingivalis , with a mixture of P. gingivalis and Fusobacterium nucleatum , with carboxymethylcellulose or remained untreated. The mice were killed, and soft tissue biopsies from the oral cavity of treated mice were used to determine the polyunsaturated fatty acid concentrations. The maxilla was removed, stained and digitally imaged to assess bone loss around the upper molars.
Results:  n-3 polyunsaturated fatty acid levels were significantly higher in oral soft tissues of mice fed tuna oil compared with the control group. Mice fed tuna oil and inoculated with P. gingivalis or with the combination of F. nucleatum and P. gingivalis exhibited 72% and 54% less alveolar bone loss respectively, compared with the treatment control group.
Conclusion:  Alveolar bone loss was inversely related to n-3 polyunsaturated fatty acid tissue levels. In conclusion, fish oil dietary supplementation may have potential benefits as a host modulatory agent in the prevention and/or adjunctive management of periodontitis.     

Synergistic virulence of Porphyromonas gingivalis and Treponema denticola in a murine periodontitis model

Chronic periodontitis is characterized by the destruction of the tissues supporting the teeth and has been associated with the presence of a subgingival polymicrobial biofilm containing Porphyromonas gingivalis and Treponema denticola. We have investigated the potential synergistic virulence of P. gingivalis and T. denticola using a murine experimental model of periodontitis. An inoculation regime of four intra-oral doses of 1 × 10(10) P. gingivalis cells induced significant periodontal bone loss compared with loss in sham-inoculated mice, whereas doses of 1 × 10(9) cells or lower did not induce bone loss. Inoculation with T. denticola with up to eight doses of 1 × 10(10) cells failed to induce bone loss in this model. However, four doses of a co-inoculum of a 1 : 1 ratio of P. gingivalis and T. denticola at 5 × 10(8) or 1 × 10(9) total bacterial cells induced the same level of bone loss as four doses of 1 × 10(10) P. gingivalis cells. Co-inoculation induced strong P. gingivalis-specific T-cell proliferative and interferon-γ-dominant cytokine responses, and induced a strong T. denticola-specific interferon-γ dominant cytokine response. Only at the higher co-inoculum dose of 1 × 10(10) total cells was a T. denticola-specific T-cell proliferative response observed. These data show that P. gingivalis and T. denticola act synergistically to stimulate the host immune response and to induce alveolar bone loss in a murine experimental periodontitis model.     

Induction of experimental periodontitis in mice with Porphyromonas gingivalis-adhered ligatures

BACKGROUND: Little information is available on the colonization of periodontopathic bacteria and alveolar bone loss in a mouse system, because of the difficulty in establishing bacteria in the oral cavity. The aim of this study was to establish experimental periodontitis in mice by applying a Porphyromonas gingivalis-adhered ligature onto the molars. METHODS: Specific pathogen-free C3H/HeN mice were divided into 3 groups: 80 infected, 80 sham-infected, and 48 non-treated control mice. Sterile silk ligatures were preincubated with and without P. gingivalis 381 in vitro and then physically tied on the right maxillary first molar of infected and sham-infected mice, respectively. Ten mice from the infected and sham-infected groups and 6 from the control group were sacrificed at 2-week intervals for up to 15 weeks after infection. RESULTS: Plaque samples were collected at the time of sacrifice and alveolar bone loss was examined. The results indicated that P. gingivalis was recovered from the plaque samples in 95% of the infected mice after 1 week and then gradually dropped to 58% after 15 weeks of infection, whereas P. gingivalis was not isolated in either sham-infected or control mice throughout the experimental period. The infected mice showed significant P. gingivalis-induced bone loss at the sites where the ligature was tied weeks 13 to 15. A linear regression analysis revealed a significant positive correlation between the number of P. gingivalis recovered and alveolar bone loss at 15 weeks after infection (P <0.01). CONCLUSIONS: The use of a P. gingivalis-adhered ligature supported a long-lasting infection of P. gingivalis in mice, resulting in P. gingivalis-induced alveolar bone breakdown.     

Serum antibody response to oral infection precedes but does not prevent Porphyromonas gingivalis–induced alveolar bone loss in mice

The purpose of this study was to determine whether humoral immunity prevents bacterially induced alveolar bone loss. BALB/cByJ mice were orally infected with the human periodontopathic bacterium Porphyromonas gingivalis, and were compared with sham-infected mice. Specific serum antibody titers to P. gingivalis were measured by enzyme-linked immunosorbent assay. Alveolar bone levels were measured as the distance from the cementoenamel junction to the alveolar bone crest and bone loss was defined as a change in bone levels over time or between infected and sham-infected animals. The specific humoral response was predominantly of the IgG isotype, although low levels of specific serum IgA were also present. Antibody titers in the infected animals were significantly different from those in the sham-infected animals by 18 days and remained at maximal levels at 47 days. Bone loss became significant by 26 days and continued to progress at 47 days. Thus the serum antibody response to oral infection with P. gingivalis preceded detectable bone loss and remained elevated while bone loss increased. The presence of specific antibody did not prevent the onset or progression of bone loss.     

Arg-gingipain A DNA vaccine prevents alveolar bone loss in mice

One major pathogenic factor of Porphyromonas gingivalis is Arg-gingipain (Rgp), an arginine-specific cysteine proteinase. To clarify the effect of rgpA DNA vaccine, we immunized BALB/c mice via the abdomen with a Gene Gun or via the nasal cavity weekly for 6 weeks. After immunization, the mice were challenged orally with P. gingivalis. Immunization elicited IgG responses against P. gingivalis in both groups. Nasal immunization also induced sIgA against P. gingivalis, although Gene Gun immunization did not. Reduction of alveolar bone loss was observed in both groups at 42 days following initial infection. This effect was more pronounced in the intranasal immunization group than in the Gene Gun group. The results of this study suggest that immunization with rgpA DNA vaccine via the nasal cavity is an effective method for preventing alveolar bone loss incurred by infection with P. gingivalis.     

Virulence of major periodontal pathogens and lack of humoral immune protection in a rat model of periodontal disease

Oral Diseases (2010) 16 , 686–695 Objective: This study was designed to test the hypothesis that periodontal pathogens Tannerella forsythia and Porphyromonas gingivalis are synergistic in terms of virulence potential using a model of mixed‐microbial infection in rats. Materials and methods: Three groups of rats were infected orally with either T. forsythia or P. gingivalis in mono‐bacterial infections or as mixed‐microbial infections for 12 weeks and a sham‐infected group were used as a control. This study examined bacterial infection, inflammation, immunity, and alveolar bone loss changes with disease progression. Results: Tannerella forsythia and P. gingivalis genomic DNA was detected in microbial samples from infected rats by PCR indicating their colonization in the rat oral cavity. Primary infection induced significantly high IgG, IgG2b, IgG1, and IgG2a antibody levels indicating activation of mixed Th1 and Th2 immune responses. Rats infected with the mixed‐microbial consortium exhibited significantly increased palatal horizontal and interproximal alveolar bone loss. Histological examinations indicated significant hyperplasia of the gingival epithelium with moderate inflammatory infiltration and apical migration of junctional epithelium. The results observed differ compared to uninfected controls. Conclusion: Our results indicated that T. forsythia and P. gingivalis exhibit virulence, but not virulence synergy, resulting in the immuno‐inflammatory responses and lack of humoral immune protection during periodontitis in rats.     

T-cell contributions to alveolar bone loss in response to oral infection with Porphyromonas gingivalis

We have previously shown that mice lacking CD4+, but not CD8+, T cells lose less alveolar bone loss in response to oral infection with Porphyromonas gingivalis than do immunocompetent mice of the same genetic background, indicating that CD4+ T cells contribute to bone resorption. The CD4+ and CD8+ T-cell knockouts were produced by targeted deletions of, respectively, major histocompatibility complex II (MHCII) or beta2-microglobulin (producing non-expression of MHCI). Because MHC deletions can have other effects in addition to those on T-cell selection, we wanted to confirm that the lessened bone loss was truly an effect of the lack of T cells. Consequently, we repeated our experiments with C57B1 /6J-Tcra mice that have a targeted deletion of the alpha chain of the T-cell receptor (Tcra). Six weeks after oral infection with P. gingivalis ATCC 53977 the total bone loss at buccal maxillary sites was 0.28 mm in infected immunocompetent mice (P=0.002 compared with sham-infected mice), whereas in Tcra knockouts the bone loss was only 0.08 mm (P=0.04 compared with shams). The T-cell-deficient mice thus lost 70% less bone after infection than did genetically matched immunocompetent mice (P =0.003). These experiments confirm that T cells, and their responses to oral infection with P. gingivalis, help to push bone remodeling in the direction of net loss of bone.     

Effects of Toll-like receptor 4 on Porphyromonas gingivalis-induced bone loss in mice

Background and Objective:  Toll-like receptor 4 (TLR-4)/myeloid differentiation protein-2 complex ligation by lipopolysaccharide induces production of pro-inflammatory cytokines and co-stimulatory molecules on antigen presenting cells. The aim of this study was to determine the role of the TLR-4 in bone loss-resistant C57BL mice and in bone loss-susceptible BALB/c mice after infection with Porphyromonas gingivalis .
Material and Methods:  The BALB/c and C57BL/10 mice, either normal or TLR-4 deficient, were infected or sham-infected orally four times, at 4 day intervals, with 10⁹ colony forming units of P. gingivalis . At 47 days, defleshed jaws were stained and photographed in a standardized position. We measured the surface area of the root trunk to assess the alveolar bone loss.
Results:  Porphyromonas gingivalis -infected wild-type BALB/c mice lost 13.8% more bone than P. gingivalis -infected wild-type C57BL/10 mice. In contrast, P. gingivalis -infected TLR-4-deficient C57BL/10 mice lost 12.7% more bone than P. gingivalis -infected TLR-4-deficient BALB/c mice. Porphyromonas gingivalis -infected wild-type C57BL/6 and TLR-2 knockout C57BL/6 mice had similar bone levels to sham-infected control mice.
Conclusion:  Toll-like receptor 4 is protective for C57BL/10 but detrimental to BALB/c mice, since its absence allowed C57BL/10 but not BALB/c mice to lose alveolar bone. Toll-like receptor 2 does not contribute to this protection in genetically similar C57BL/6 mice.     

The role of cytokines in a Porphyromonas gingivalis-induced murine abscess model

INTRODUCTION: Porphyromonas gingivalis is an important periodontopathic bacterium that is strongly associated with periodontal disease and is part of human dental plaque. Periodontal disease results from the interaction of the host with bacterial products, and T-cell-derived cytokines remain critical in the immunoregulation of periodontal disease. METHODS: The aim of this study was to examine the role of T helper type 1 [interleukin-12p40 (IL-12p40), interferon-gamma, tumour necrosis factor (TNF)] and type 2 (IL-4, IL-10) cytokines in the immune response to a subcutaneous challenge with P. gingivalis using a well-established murine abscess model, in genetically modified cytokine-specific knockout mice. RESULTS: IL-12p40(-/-) mice exhibited more advanced tissue destruction and a reduced inflammatory cell infiltrate after subcutaneous P. gingivalis challenge. Deficiency of IL-4 or IL-10 did not result in increased susceptibility to P. gingivalis-mediated tissue destruction. Furthermore, TNF deficiency appeared to reduce local tissue destruction. Interestingly, serum-specific antibodies suggested a strong T helper type 2 response. CONCLUSION: The results of our study indicate an important role for IL-12 in a primary P. gingivalis subcutaneous challenge.     

Induction of IL-10-producing CD4+ T-cells in chronic periodontitis

Precise immunological aspects of inflamed gingival mucosa remain to be elucidated in the murine experimental periodontitis model; therefore, we have characterized the mucosal immune cells in the inflamed gingiva of mice with alveolar bone reduction. Mice were orally infected with Porphyromonas gingivalis 15 times over 2 weeks. Gingival mononuclear cells (GMCs) were isolated from P. gingivalis- and sham-infected mice 1, 7, 15, and 30 days after the last infection. Although the greatest degree of periodontitis was seen in P. gingivalis-infected mice at 30 days after infection, the highest levels of IL-6 and TNF-α production were noted in the GMCs isolated 7 days after infection. Further, the frequency of RANKL(+)CD4(+) T-cells in GMCs of inflamed gingiva peaked 15 days after infection. Importantly, the number of Foxp3(+)CD4(+) CD25(+) regulatory T (Treg)-cells was increased only in the experimental group 30 days after infection. Thus, intracellular cytokine analysis revealed an increased number of IL-10-producing CD4(+) T-cells in inflamed gingiva when compared with the control group. These results suggest that there are potential roles for Treg cells during the chronic stage of periodontitis in the regulation of gingival inflammation and alveolar bone loss.     

iNOS-derived nitric oxide stimulates osteoclast activity and alveolar bone loss in ligature-induced periodontitis in rats

Background: Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long‐term iNOS inhibition, and the differentiation and activity of OC from iNOS‐knockout (KO) mice in vitro. Methods: Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild‐type C57BL/6 mice. OC were counted 6 days later after tartrate‐resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. Results: Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature‐induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild‐type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. Conclusion: Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature‐induced periodontitis, thus confirming that iNOS‐derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.     

靶向牙周炎DNA疫苗免疫原性及免疫效能的实验研究

目的：检测靶向牙周炎DNA疫苗pCTLA4-FimA的免疫反应性,并评价其免疫保护能力。方法：分3组免疫BALB/c小鼠：pCTLA4-FimA鼻黏膜免疫组,非靶向牙周炎DNA疫苗pFimA鼻黏膜免疫组,pCI载体鼻黏膜免疫组。酶联免疫吸附实验检测血清及唾液中特异性抗体水平。建立小鼠牙周炎模型,检测牙槽骨水平吸收程度。结果：与pFimA免疫组相比,pCTLA4-FimA免疫组诱导了显著增强的血清特异性IgG抗体和唾液特异性IgA抗体水平（P〈0.01）。与免疫前相比,3实验组牙槽骨均有吸收,其中pCTLA4-FimA免疫组牙槽骨水平吸收程度最低。结论：靶向牙周炎DNA疫苗pCTLA4-FimA能有效诱导机体的免疫反应,并抑制牙周炎的发展,效果优于非靶向牙周炎DNA疫苗pFimA。     

The r40-kDa outer membrane protein human monoclonal antibody protects against Porphyromonas gingivalis-induced bone loss in rats

BACKGROUND: Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We recently reported the construction and characterization of human immunoglobulin G isotype clones, which were specifically reactive with recombinant (r) 40-kDa outer membrane protein (OMP) of P. gingivalis. The aim of this study was to investigate the efficacy of human monoclonal antibody (hMAb) against r40-kDa OMP of P. gingivalis to the protection alveolar bone loss by P. gingivalis in rats. METHODS: The role of 40-kDa OMP in the adherence of P. gingivalis to human gingival epithelial cells (HGECs) was examined by preincubating with r40-kDa OMP hMAb before adding the HGECs. Moreover, we used a rat model to examine the effect of the anti-r40-kDa OMP hMAb in alveolar bone loss by oral infection. Forty-six days after the last infection, the periodontal bone level was assessed morphometrically on defleshed rat jaws. RESULTS: The adherence to HGECs was reduced by 84% compared to adherence levels without the antibody. P. gingivalis could not be detected from rats in a P. gingivalis-non-infected group and a group that was administered the anti-r40-kDa OMP hMAb. The bone loss in P. gingivalis-infected animals that were administered the anti-r40-kDa OMP hMAb was significantly lower than that of P. gingivalis-infected rats. CONCLUSIONS: Our results suggest that transchromosomic mouse-derived hMAb against r40-kDa OMP of P. gingivalis protects against periodontal bone loss. This newly constructed anti-r40-kDa OMP hMAb was used to protect against periodontal diseases caused by P. gingivalis infection.     

Strain-dependent activation of the mouse immune response is correlated with Porphyromonas gingivalis-induced experimental periodontitis

Aims: To evaluate the effect of oral infection with three Porphyromonas gingivalis strains on alveolar bone loss (ABL) and its correlation with the mouse immune response.
Materials and Methods: Mice were orally infected with P. gingivalis strains 381, 33277 and 53977. After 42 days, maxillae were analysed for ABL using micro-computed tomography and the serum for anti- P.gingivalis IgG1 and IgG2a levels. The cytokine response to P. gingivalis was tested using the subcutaneous chamber model.
Results: The P. gingivalis 53977-infected group showed the highest ABL, which was significantly different from all other groups ( p <0.001). In addition, the humoral response to P. gingivalis 53977 was significantly lower than the response to P. gingivalis 381 and 33277 ( p 0.01). The IgG2a/IgG1 ratio was higher in the P. gingivalis 33277-infected group (1.6) compared with the P. gingivalis 381-infected group (0.51). Four days post-infection, interleukin (IL)-1 β levels remained significantly higher in the P. gingivalis 53977-infected group only (1198.2±260.0, p <0.05), while IL-4 levels remained significantly higher in the P. gingivalis 381-infected group (265.8±131.6, p <0.05).
Conclusions: The high levels of ABL induced by P. gingivalis 53977 were inversely correlated with the humoral response to this bacterium. In addition, ABL was correlated with an elevated pro-inflammatory response.     

Three-dimensional quantification of alveolar bone loss in Porphyromonas gingivalis-infected mice using micro-computed tomography

BACKGROUND: Animal models are routinely used for the study of the pathogenesis of periodontal disease. However, some of the methods used for evaluating alveolar bone loss are limited to linear or two dimension measurements, while other methods, such as histology, are labor consuming. The present study was designed to evaluate a novel method for assessing the volume of alveolar bone loss in mice and to compare it to the traditional morphometric (linear) technique. METHODS: Seven- to 8-week-old BALB/c mice were divided into three equal groups of six each; two groups were infected orally with Porphyromonas gingivalis (P. gingivalis) 381 or 53,977, while the third group was used as non-infected control. Forty-two days following infection, serum samples were obtained and maxillae were harvested. Bone loss was evaluated by micro-computed tomography (micro-CT) and by the morphometric technique. RESULTS: Significant decrease in residual supportive bone volumes (RSBV) was observed in both P. gingivalis-infected groups compared to the control group (P<0.0001). The P. gingivalis 53,977-infected group showed less residual supportive bone volume compared to the P. gingivalis 381-infected group, but there were no significant differences between these two groups. Using the morphometric technique, the differences between the three groups were not found to be statistically significant (P>0.05). CONCLUSIONS: The present study describes a novel micro-CT technique for volumetric evaluation of alveolar bone loss in mice. This technique is relatively simple and accurate, and due to its high sensitivity, enables the investigator to reduce the number of animals needed in each experimental group.