Animal Model Of Periodontitis Research Articles

Periodontitis induced by Porphyromonas gingivalis drives periodontal microbiota dysbiosis and insulin resistance via an impaired adaptive immune response.

ObjectiveTo identify a causal mechanism responsible for the enhancement of insulin resistance and hyperglycaemia following periodontitis in mice fed a fat-enriched diet.DesignWe set-up a unique animal model of periodontitis in C57Bl/6 female mice by infecting the periodontal tissue with specific and alive pathogens like Porphyromonas gingivalis (Pg), Fusobacterium nucleatum and Prevotella intermedia. The mice were then fed with a diabetogenic/non-obesogenic fat-enriched diet for up to 3 months. Alveolar bone loss, periodontal microbiota dysbiosis and features of glucose metabolism were quantified. Eventually, adoptive transfer of cervical (regional) and systemic immune cells was performed to demonstrate the causal role of the cervical immune system.ResultsPeriodontitis induced a periodontal microbiota dysbiosis without mainly affecting gut microbiota. The disease concomitantly impacted on the regional and systemic immune response impairing glucose metabolism. The transfer of cervical lymph-node cells from infected mice to naive recipients guarded against periodontitis-aggravated metabolic disease. A treatment with inactivated Pg prior to the periodontal infection induced specific antibodies against Pg and protected the mouse from periodontitis-induced dysmetabolism. Finally, a 1-month subcutaneous chronic infusion of low rates of lipopolysaccharides from Pg mimicked the impact of periodontitis on immune and metabolic parameters.ConclusionsWe identified that insulin resistance in the high-fat fed mouse is enhanced by pathogen-induced periodontitis. This is caused by an adaptive immune response specifically directed against pathogens and associated with a periodontal dysbiosis.

Serpine1 Mediates Porphyromonas gingivalis Induced Insulin Secretion in the Pancreatic Beta Cell Line MIN6.

Periodontitis is an inflammatory disease resulting in destruction of gingiva and alveolar bone caused by an exuberant host immunological response to periodontal pathogens. Results from a number of epidemiological studies indicate a close association between diabetes and periodontitis. Results from cross-sectional studies indicate that subjects with periodontitis have a higher odds ratio of developing insulin resistance (IR). However, the mechanisms by which periodontitis influences the development of diabetes are not known. Results from our previous studies using an animal model of periodontitis suggest that periodontitis accelerates the onset of hyperinsulinemia and IR. In addition, LPS from a periodontal pathogen, Porphyromonas gingivalis (Pg), stimulates Serpine1 expression in the pancreatic beta cell line MIN6. Based on these observations, we hypothesized that a periodontal pathogen induces hyperinsulinemia and Serpine1 may be involved in this process. To test this hypothesis, we co-incubated Pg with the pancreatic beta cell line MIN6 and measured the effect on insulin secretion by MIN6 cells. We further determined the involvement of Serpine1 in insulin secretion by downregulating Serpine1 expression. Our results indicated that Pg stimulated insulin secretion by approximately 3.0 fold under normoglycemic conditions. In a hyperglycemic state, Pg increased insulin secretion by 1.5 fold. Pg significantly upregulated expression of the Serpine1 gene and this was associated with increased secretion of insulin by MIN6 cells. However, cells with downregulated Serpine1 expression were resistant to Pg stimulated insulin secretion under normoglycemic conditions. We conclude that the periodontal pathogen, Pg, induced insulin secretion by MIN6 cells and this induction was, in part, Serpine1 dependent. Thus, Serpine1 may play a pivotal role in insulin secretion during the accelerated development of hyperinsulinemia and the resulting IR in the setting of periodontitis.

Chronic stress enhances progression of periodontitis via α1-adrenergic signaling: a potential target for periodontal disease therapy.

This study assessed the roles of chronic stress (CS) in the stimulation of the sympathetic nervous system and explored the underlying mechanisms of periodontitis. Using an animal model of periodontitis and CS, the expression of tyrosine hydroxylase (TH) and the protein levels of the α1-adrenergic receptor (α1-AR) and β2-adrenergic receptor (β2-AR) were assessed. Furthermore, human periodontal ligament fibroblasts (HPDLFs) were stimulated with lipopolysaccharide (LPS) to mimic the process of inflammation. The proliferation of the HPDLFs and the expression of α1-AR and β2-AR were assessed. The inflammatory-related cytokines interleukin (IL)-1β, IL-6 and IL-8 were detected after pretreatment with the α1/β2-AR blockers phentolamine/propranolol, both in vitro and in vivo. Results show that periodontitis under CS conditions enhanced the expression of TH, α1-AR and β2-AR. Phentolamine significantly reduced the inflammatory cytokine levels. Furthermore, we observed a marked decrease in HPDLF proliferation and the increased expression of α1-ARfollowing LPS pretreatment. Pretreatment with phentolamine dramatically ameliorated LPS-inhibited cell proliferation. In addition, the blocking of α1-ARsignaling also hindered the upregulation of the inflammatory-related cytokines IL-1β, IL-6 and IL-8. These results suggest that CS can significantly enhance the pathological progression of periodontitis by an α1-adrenergic signaling-mediated inflammatory response. We have identified a potential therapeutic target for the treatment of periodontal disease, particularly in those patients suffering from concurrent CS.

The inflammophilic character of the periodontitis‐associated microbiota

In periodontitis, dysbiotic microbial communities exhibit synergistic interactions for enhanced protection from host defenses, nutrient acquisition, and persistence in an inflammatory environment. This review discusses evidence that periodontitis-associated communities are 'inflammo-philic' (=loving or attracted to inflammation) in that they have evolved to not only endure inflammation but also to take advantage of it. In this regard, inflammation can drive the selection and enrichment of these pathogenic communities by providing a source of nutrients in the form of tissue breakdown products (e.g. degraded collagen peptides and heme-containing compounds). In contrast, those species that cannot benefit from the altered ecological conditions of the inflammatory environment, or for which host inflammation is detrimental, are likely to be outcompeted. Consistent with the concept that inflammation fosters the growth of dysbiotic microbial communities, the bacterial biomass of human periodontitis-associated biofilms was shown to increase with increasing periodontal inflammation. Conversely, anti-inflammatory treatments in animal models of periodontitis were shown to diminish the periodontal bacterial load, in addition to protecting from bone loss. The selective flourishing of inflammophilic bacteria can perpetuate inflammatory tissue destruction by setting off a 'vicious cycle' for disease progression, in which dysbiosis and inflammation reinforce each other. Therefore, the control of inflammation appears to be central to the treatment of periodontitis, as it is likely to control both dysbiosis and disease progression.

Preparation and characteristics of thermoresponsive gel of minocycline hydrochloride and evaluation of its effect on experimental periodontitis models

In this study, a thermoresponsive gel for minocycline (MCL) with chitosan/β-glycerophosphate (C/β-GP) was formulated and its characterization, in vitro release, stability, toxicity and pharmacodynamics were investigated. The formulation containing MCL was prepared by pouring the chitosan solution directly onto the sterilized drug powder and stirring before mixing with the β-glycerophosphate (β-GP) solution. The final preparations contained 0.5% (w/v) chitosan, 1.8% (w/v) β-GP and 2% (w/v) MCL. The drug content of prepared gels was in the range of 92–99%, and the pH value of the optimized formulation was found to be 5.6–6.2. The gelation temperature of the prepared C/β-GP thermogelling solutions was 37 °C. Color, consistency, pH, viscosity and drug content of the in situ gels were found to be consistent, and no signs of separation and deterioration were observed over a period of 90 d. In vivo studies showed that rats' liver and kidney tissue sections were normal, with no structural damage. The constituents of the in situ gels formulation had a well-sustained release efficacy on the animal model of periodontitis.

Allogeneic Stem Cells From Deciduous Teeth in Treatment for Periodontitis in Miniature Swine

Regeneration of lost periodontium in periodontitis is a challenge in that alveolar bone, cementum, and periodontal ligament need to be restored to their original architecture. Stem cells from exfoliated deciduous teeth (SHEDs) appear to be an attractive candidate for periodontium tissue regeneration. Previously, the authors successfully regenerated periodontal defects using autologous and allogeneic periodontal ligament stem cells (PDLSCs). The purpose of the present study is to investigate the ability of allogeneic SHEDs to regenerate lost periodontium in a swine periodontitis model. Animal models of periodontitis were established in miniature pigs, and allogeneic stem cells were isolated from miniature pig deciduous teeth (SPDs). The animal models were treated with SPDs plus hydroxyapatite/tricalcium phosphate (HA/TCP). Allogeneic PDLSCs plus HA/TCP or HA/TCP alone were set as positive control or control, respectively. Clinical assessments, computed tomography (CT) scanning, and histologic examination were used to evaluate the outcome of tissue regeneration. Clinical indices including probing depth, gingival recession, and attachment loss showed significant restoration in the SPD and PDLSC treatment groups, compared to the HA/TCP group 12 weeks post-transplantation. Meanwhile, CT scans showed that 75% of the samples had successful hard-tissue regeneration in both PDLSC and SPD groups, compared to the HA/TCP group, where the success rate was only 25%. In addition, histologic examination demonstrated that SPD and PDLSC treatment brought about remarkable regeneration of periodontal tissues, whereas periodontal regeneration was rare in the HA/TCP group. Allogeneic SPDs can effectively repair hard and soft tissue loss brought about by periodontitis in a swine model. Allogeneic SHEDs, which are easily accessible, may be applied to treat periodontitis in clinics in the future.

Experimental periodontitis promotes transient vascular inflammation and endothelial dysfunction

ObjectivesThis study aimed to evaluate the systemic inflammatory response and cardiovascular changes induced by experimental periodontitis in rats. DesignExperimental periodontitis was induced by placing a cotton ligature around the cervix of both sides of mandibular first molars and maxillary second molars in each male rat. Sham-operated rats had the ligature removed immediately after the procedure. Seven, 14 or 28 days after procedure, the effects of acetylcholine, sodium nitroprusside and phenylephrine were evaluated on blood pressure, aortic rings and isolated and perfused mesenteric bed. The blood was obtained for plasma Interleukin-6 (IL-6), C-reactive protein (CRP) and lipid evaluation. The mesenteric vessels were obtained to evaluate superoxide production and nitric oxide synthase 3 (NOS-3) expression. ResultsLigature induced periodontitis reduced endothelium-dependent vasodilatation, a hallmark of endothelial dysfunction. This effect was associated with an increase in systemic inflammatory markers (IL-6 and CRP), worsens on lipid profile, increased vascular superoxide production and reduced NOS-3 expression. It is interesting to note that many of these effects were transitory. ConclusionPeriodontitis induced a transient systemic and vascular inflammation which leads to endothelial dysfunction, an initial step for cardiovascular diseases. Moreover, the animal model of periodontitis used here may represent a valuable tool for studying the relationship between periodontitis and endothelial dysfunction.

Isorhamnetin inhibits Prevotella intermedia lipopolysaccharide‐induced production of interleukin‐6 in murine macrophages via anti‐inflammatory heme oxygenase‐1 induction and inhibition of nuclear factor‐κB and signal transducer and activator of transcription 1 activation

Interleukin-6 (IL-6) is a key proinflammatory cytokine that has been considered to be important in the pathogenesis of periodontal disease. Therefore, host-modulatory agents directed at inhibiting IL-6 appear to be beneficial in terms of attenuating periodontal disease progression and potentially improving disease susceptibility. In the current study, we investigated the effect of the flavonoid isorhamnetin on the production of IL-6 in murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. Lipopolysaccharide from P.intermedia ATCC 25611 was isolated using the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time PCR to quantify IL-6 and heme oxygenase-1 (HO-1) mRNA expression. The expression of HO-1 protein and the levels of signaling proteins were monitored using immunoblot analyses. The DNA-binding activity of nuclear factor-κB (NF-κB) was analyzed using ELISA-based assay kits. Isorhamnetin significantly down-regulated P.intermedia LPS-induced production of IL-6 as well as its mRNA expression in RAW264.7 cells. Isorhamnetin up-regulated the expression of HO-1 at both gene transcription and translation levels in cells stimulated with P.intermedia LPS. In addition, inhibition of HO-1 activity by tin protoporphyrin IX blocked the inhibitory effect of isorhamnetin on IL-6 production. Isorhamnetin failed to prevent LPS from activating either c-Jun N-terminal kinase or p38 pathways. Isorhamnetin did not inhibit NF-κB transcriptional activity at the level of inhibitory κB-α degradation. Isorhamnetin suppressed NF-κB signaling through inhibition of nuclear translocation and DNA binding activity of NF-κB p50 subunit and attenuated signal transducer and activator of transcription 1 signaling. Although further research is required to clarify the detailed mechanism of action, we propose that isorhamnetin may contribute to blockade of the host-destructive processes mediated by IL-6 and could be a highly efficient modulator of the host response in the treatment of inflammatory periodontal disease. Further research in animal models of periodontitis is required to better evaluate, the potential of isorhamnetin as a novel agent for treating periodontal disease.

Involvement of nitrosative stress in experimental periodontitis in diabetic rats

Periodontal disease is highly prevalent and severe in diabetic patients, and is considered one of the diabetic complications. To elucidate how periodontitis progresses in diabetes, we examined an animal model of periodontitis in diabetic rats. Two weeks after the induction of diabetes by streptozotocin, surgical nylon thread was ligated around the cervical portion of the unilateral maxillary second molar to induce periodontitis. Periodontitis was evaluated 2weeks after the ligation by gingival blood flow, mRNA expressions, Western blot analysis, histological examination and micro CT. Ligation-induced severe periodontitis in the diabetic rats, which was apparently shown by the increase of TNF-α and iNOS mRNA expressions and inflammatory cell infiltration in the gingiva and alveolar bone loss. The number of nitrotyrosine, a footprint of nitrosative stress, -positive cells was significantly higher in the periodontitis of the diabetic rats compared with that in the normal rats. Western blot analysis confirmed that the nitrotyrosine was increased in the periodontitis of the diabetic rats. This is the first study to confirm increased nitrosative stress due to periodontitis in diabetic rats. Nitrosative stress may play a crucial role in the exacerbation of periodontitis in diabetic patients.

Anti‐inflammatory effects and the underlying mechanisms of action of daidzein in murine macrophages stimulated with Prevotella intermedia lipopolysaccharide

Host modulatory agents directed at inhibiting specific proinflammatory mediators could be beneficial in terms of attenuating periodontal disease progression and potentially enhancing therapeutic responses. The aim of this study was to investigate whether daidzein could modulate the production inflammatory mediators in macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and to delineate underlying mechanisms of action. LPS was extracted from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The amounts of nitric oxide (NO) and interleukin-6 (IL-6) secreted into the culture medium were assayed. A real-time PCR was performed to quantify inducible nitric oxide synthase (iNOS) and IL-6 mRNA expression. We used immunoblot analysis to characterize iNOS protein expression, phosphrylation of c-Jun N-terminal kinase (JNK) and p38, degradation of inhibitory κB-α (IκB-α), nuclear translocation of nuclear factor-κB (NF-κB) subunits and phosphorylation of signal transducer and activator of transcription 1 (STAT1). The DNA-binding activity of NF-κB was assessed by using ELISA-based kits. Daidzein significantly inhibited the production of NO and IL-6, as well as their mRNA expression, in P. intermedia LPS-treated RAW264.7 cells. The JNK and p38 pathways were not involved in the regulation of LPS-induced NO and IL-6 release by daidzein. Daidzein inhibited the degradation of IκB-α induced by P. intermedia LPS. In addition, daidzein suppressed NF-κB transcriptional activity via regulation of the nuclear translocation and DNA-binding activity of NF-κB p50 subunit and blocked STAT1 phosphorylation. Although additional studies are required to dissect the molecular mechanism of action, our results suggest that daidzein could be a promising agent for treating inflammatory periodontal disease. Further research in animal models of periodontitis is necessary to better evaluate the potential of daidzein as a novel therapeutic agent to treat periodontal disease.

Emerging Role of PPAR-β/δin Inflammatory Process Associated to Experimental Periodontitis

The aim of the present study was to evaluate the contribution of peroxisome proliferator-activated receptor (PPAR-β/δ) in animal model of periodontitis. Male Sprague-Dawley rats were lightly anaesthetized with pentobarbitone (35 mg/kg). Sterile, 2-0 black braided silk thread was placed around the cervix of the lower left first molar and knotted medially. Animals received GW0742 (0.3 mg/kg, 10% DMSO, i.p. after the ligature placement and daily for eight days). At day 8, the gingivomucosal tissue encircling the mandibular first molar was removed. One the eighth day after placement of the ligature, we evaluated (1) NF-κB expression, (2) cytokines expression, (3) iNOS expression, (5) the nitration of tyrosine, (6) apoptosis, and (8) the degree of gingivomucosal tissues injury. Administration of GW0742 significantly decreased all of the parameters of inflammation as described above. Taken together, these results demonstrate that GW0742 exerts an anti-inflammatory role during experimental periodontitis and is able to ameliorate the tissue damage.

Bacteria and the pathogenesis of periodontal diseases: vaccine development

Periodontitis is a chronic inflammatory disease of the supporting tissues of the teeth leading to tissue destruction and tooth loss. Advances in the techniques used to identify microbial pathogens linked with periodontal tissue destruction, particularly in prospective clinical studies, have led to the identification of <i>Porphyromonas gingivalis</i> as a major aetiological agent in disease initiation and progression. Detailed molecular studies of the pathogen have shown that extracellular proteinases are a key virulence factor for this bacterium. The proteinases are expressed and secreted by a novel secretion system as polyproteins with C-terminal adhesins. Once secreted the polyproteins are attached to a novel surface LPS anchor and proteolytically processed to form a non-covalent complex of proteinases and adhesins. This surface complex can be released into the host tissue where it dysregulates the host immune response and induces vascular disruption and tissue destruction. The purified proteinase-adhesin complex protects against disease when used as a vaccine in animal models of period-ontitis. Novel recombinant chimeric forms of the proteinase and adhesins when used as a vaccine in animals produced antibodies to both the proteinase active site and major adhesin and provided better protection against periodontal tissue destruction than the native complex or killed whole cells. This chimeric vaccine shows potential for the development of a vaccine against human disease.

Resolution of inflammation-unraveling mechanistic links between periodontitis and cardiovascular disease

Resolution of inflammation-unraveling mechanistic links between periodontitis and cardiovascular disease

The Management of Inflammation in Periodontal Disease

It has become clear in recent years that periodontitis is an inflammatory disease initiated by oral microbial biofilm. This distinction implies that it is the host response to the biofilm that destroys the periodontium in the pathogenesis of the disease. As our understanding of pathways of inflammation has matured, a better understanding of the molecular basis of resolution of inflammation has emerged. Resolution of inflammation is an active, agonist-mediated, well-orchestrated return of tissue homeostasis. There is an important distinction between anti-inflammation and resolution; anti-inflammation is pharmacologic intervention in inflammatory pathways, whereas resolution is biologic pathways restoring homeostasis. A growing body of research suggests that chronic inflammatory periodontal disease involves a failure of resolution pathways to restore homeostasis. This article reviews the resolution of inflammation in the context of periodontal disease and the potential for the modification of resolution pathways for the prevention and treatment of periodontal diseases. Proof-of-concept studies in the 1980s demonstrated that pharmacologic anti-inflammation prevented and slowed the progression of periodontal diseases in animals and man. However, the side-effect profile of such therapies precluded the use of non-steroidal anti-inflammatory drugs or other enzyme inhibitors or receptor antagonists in periodontal therapy. The isolation and characterization of resolving agonist molecules has opened a new area of research using endogenous lipid mediators of resolution as potential therapeutic agents for the management of inflammatory periodontitis. Work in animal models of periodontitis has revealed the potential of this therapeutic approach for its prevention and treatment and forced the reconsideration of our understanding of the pathogenesis of human periodontal diseases.

The Effects of a Calorie‐Reduced Diet on Periodontal Inflammation and Disease in a Non‐Human Primate Model

Low-calorie diets are commonplace for reducing body weight. However, no information is available on the effects of a reduced-calorie diet on periodontal inflammation and disease. The purpose of this study was to evaluate the clinical effects of a long-term calorie-restriction (CR) diet on periodontitis in an animal model of periodontitis. Periodontitis was induced in 55 young, healthy, adult rhesus monkeys (Macaca mulatta) by tying 2.0 silk ligatures at the gingival margins of maxillary premolar/molar teeth. Animals on a CR diet (30% CR; N = 23) were compared to ad libitum diet controls (N = 32). Clinical measures, including the plaque index (PI), probing depth (PD), clinical attachment level (CAL), modified gingival index (GI), and bleeding on probing (BOP) were recorded at baseline and 1, 2, and 3 months after ligature placement. Significant effects of CR were observed on the development of inflammation and the progression of periodontal destruction in this model. Compared to controls, CR resulted in a significant reduction in ligature-induced GI (P <0.0001), BOP (P <0.0015), PD (P <0.0016), and CAL (P <0.0038). Periodontal destruction, as measured by CAL, progressed significantly more slowly in the CR animals than in the controls (P <0.001). These clinical findings are consistent with available evidence that CR has anti-inflammatory effects. Moreover, these experimental findings are the first observations, to the best of our knowledge, that CR dampens the inflammatory response and reduces active periodontal breakdown associated with an acute microbial challenge.

Alveolar bone resorption in animal models of periodontitis

Periodontitis is a chronic inflammatory disease caused by infection of periodontopathic bacteriae, which induces alveolar bone resorption, tooth mobility, and finally tooth loss. In order to clarify the pathogenesis and to develop prevention and treatment of periodontitis, it is important to establish animal models of periodontitis. In this article, we showed several types of experimental models previously reported, and also showed a rat model using ligature and a hamster model using periodontopathic bacteria, both we used in our laboratory. Micro computed tomography (CT) analysis and histological observation revealed the inflammatory localization and osteoclast appearance in periodontal tissues. The usefulness of animal models of periodontitis is demonstrated by exhibiting an inhibitory effect of bisphosphonate and prostaglandin E(2) on alveolar bone resorption in our experimental systems.

Fimbriated Porphyromonas gingivalis is more efficient than fimbria-deficient P. gingivalis in entering human dendritic cells in vitro and induces an inflammatory Th1 effector response.

Porphyromonas gingivalis is a fimbriated mucosal pathogen implicated in chronic periodontitis (CP). The fimbriae are required for invasion of the gingival mucosa and for induction of CP in animal models of periodontitis. CP is associated with infection of immature dendritic cells (DCs) by P. gingivalis in situ and with increased numbers of dermal DCs (DDCs) and mature DCs in the lamina propria. The role of fimbriae in gaining entry into human DCs and how this modulates the inflammatory and effector immune responses, however, have not been explored. To address this, we generated monocyte-derived DCs (MDDCs) in vitro which phenotypically and functionally resemble DDCs. We show here that virulent fimbriated P. gingivalis 381, in contrast to its fimbria-deficient mutant, P. gingivalis DPG3, efficiently gains entry to MDDCs in a manner dependent on active cell metabolism and cytoskeletal rearrangement. In addition, uptake of 381, unlike DPG3, induces DCs to undergo maturation, upregulate costimulatory molecules, and secrete inflammation cytokines interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, IL-10, and IL-12. Moreover, MDDCs pulsed with 381 also stimulated a higher autologous mixed lymphocyte reaction and induced a Th1-type response, with gamma interferon (IFN-gamma) being the main cytokine. Monocytes used as controls demonstrated fimbria-dependent uptake of 381 as well but produced low levels of inflammatory cytokines compared to MDDCs. When MDDCs were pulsed with recombinant fimbrillin of P. gingivalis (10 micro g/ml), maturation of MDDCs was also induced; moreover, matured MDDCs induced proliferation of autologous CD4(+) T cells and release of IFN-gamma. Thus, these results establish the significance of P. gingivalis fimbriae in the uptake of P. gingivalis by MDDCs and in induction of immunostimulatory Th1 responses.

A combination of a chemically modified doxycycline and a bisphosphonate synergistically inhibits endotoxin-induced periodontal breakdown in rats.

Chemically modified non-antimicrobial tetracyclines (CMTs) have been shown to inhibit pathologically elevated collagenase (and other matrix metalloproteinase, MMP) activity and bone resorption in vivo and in vitro. In the current study, suboptimal doses of CMT-8 (a non-antimicrobial chemically modified doxycycline) and a bisphosphonate (clodronate, an anti-bone resorption compound) were administered daily, either as a single agent or as a combination therapy, to rats with experimental periodontitis induced by repeated injection of bacterial endotoxin (LPS) into the gingiva. At the end of the 1-week protocol, the gingival tissues were dissected, extracted, and the extracts analyzed for MMPs (collagenases and gelatinases) and for elastase, and the defleshed jaws were morphometrically analyzed for alveolar bone loss. LPS injection significantly (P<0.001) increased alveolar bone loss and increased collagenase (MMP-8), gelatinase (MMP-9), and elastase activities. Treatment of the LPS-injected rats with suboptimal CMT-8 alone or suboptimal clodronate alone produced slight reductions in the tissue-destructive proteinases and no significant reductions in alveolar bone loss. However, a combination of suboptimal CMT-8 and clodronate "normalized" the pathologically elevated levels of MMPs, elastase, and alveolar bone loss, indicating synergistic inhibition of tissue breakdown in this animal model of periodontitis. Combination of a CMT and a bisphosphonate may be a useful treatment to optimally suppress periodontal destruction and tooth loss and in other tissue-destructive inflammatory diseases such as arthritis.