Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis

Background and Objective: Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. Our objective was to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. Material and Methods: Ligature‐induced periodontitis was applied in periodontitis‐susceptible Fischer 344 rats. The animals were given daily intraperiotonal (i.p.) injections of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous (s.c.) injections in the neck skin with nicotine (0.8 mg/kg), or treated with the same amount of saline i.p. and nicotine s.c., or with mecamylamine and saline. Control rats received i.p. and s.c. injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 weeks. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 μg/kg, i.p.) to induce a robust immune and stress response. Results: Compared with saline/saline‐treated control rats, saline/nicotine‐treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor α (TNF‐α), transforming growth factor 1β (TGF‐1β) and interleukin‐10 (IL‐10). Mecamylamine pretreatment of nicotine‐treated rats abrogated the increased periodontal bone loss and the LPS‐induced TNF‐α decrease, but had no significant effects on the levels of TGF‐1β and IL‐10, or the stress hormone corticosterone. Conclusion: The results indicate that nicotine enhances susceptibility to periodontitis via nAChRs, which may act via suppressing protective immune responses through the cholinergic anti‐inflammatory pathway.     

Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis

Background and Objective: Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. This study was designed to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. Material and Methods: Ligature‐induced periodontitis was applied in periodontitis‐susceptible Fischer 344 rats. The animals were either given daily intraperitoneal injections of the nicotinic acetylcholine receptor antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous injections in the neck skin of nicotine (0.8 mg/kg), or treated with the same amount of saline intraperitoneally and nicotine subcutaneously, or treated with mecamylamine and saline. Control animals received intraperitoneal and subcutaneous injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 wk. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally) to induce a robust immune and stress response. Results: Compared with saline/saline‐treated control animals, saline/nicotine‐treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor‐α, transforming growth factor‐1β and interleukin‐10. Mecamylamine pretreatment of nicotine‐treated rats abrogated the increased periodontal bone loss and the LPS‐induced decrease in tumour necrosis factor‐α, but had no significant effects on the levels of transforming growth factor‐1β and interleukin‐10, or the stress hormone corticosterone. Conclusion: The results indicate that nicotine enhances the susceptibility to periodontitis via nicotinic acetylcholine receptors, which may act by suppressing protective immune responses through the cholinergic anti‐inflammatory pathway.     

Efficacy of structurally diverse aldose reductase inhibitors on experimental periodontitis in rats

Background: To study aldose reductase and the sorbitol pathway in periodontitis and diabetes, rats with experimental periodontitis with or without diabetes were treated with three structurally diverse aldose reductase inhibitors (ARIs). Methods: Periodontitis was induced with three consecutive palatal injections of Porphyromonas gingivalis lipopolysaccharide (LPS) at 48‐hour intervals between the first and second molars on the right side in young, age‐matched, streptozotocin‐induced rats with and without diabetes 44 days after initiation of diets with and without the ARIs tolrestat, imirestat, and quercetin. As an internal control, phosphate‐buffered saline (PBS) was similarly injected on the left side. Twenty‐four days after the final injection, all rats were euthanized. Defleshed samples were stained with 5% toluidine blue and palatal digital images were traced to include the enamel crown and exposed root. The root/enamel ratios (to estimate alveolar bone loss) were analyzed with repeated measures analysis of variance. Results: LPS injections resulted in significantly more bone loss versus PBS injections in both the rats with and without diabetes on normal diets (P <0.0001). All three ARIs significantly reduced LPS‐induced periodontitis in the animals with and without diabetes (P ≤0.003) to the level where they were not different from PBS‐injected sites in normal diet controls. Conclusion: All ARIs demonstrated efficacy in preventing alveolar bone loss because of periodontitis in both animals with and without diabetes, suggesting a role for the sorbitol pathway and the potential for ARIs to reduce inflammatory responses downstream from aldose reductase.     

Protective Mechanisms of Simvastatin in Experimental Periodontal Disease

Background: Simvastatin is a cholesterol‐lowering drug whose pleiotropic effects may have a therapeutic impact on bone. This study evaluates the effect of simvastatin on rats subjected to experimental periodontal disease. Methods: Periodontitis was induced by ligature placement around the maxillary left second molar of rats for 11 days. Groups of six animals received oral saline or simvastatin (3, 10, and 30 mg/kg/day) until sacrifice on day 11. Alveolar bone loss was determined by macroscopic and histologic examination. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total alkaline phosphatase (TAP) were evaluated. Gingival myeloperoxidase activity and gingival levels of interleukin‐1β (IL‐1β), tumor necrosis factor‐α, IL‐10, reduced glutathione, malonaldehyde, and nitrate/nitrite were analyzed to investigate oxidative stress and inflammation. Expression of inducible nitric oxide synthase (iNOS), matrix metalloproteinases 1 and 8 (MMP‐1 and ‐8), bone morphogenetic protein‐2 (BMP‐2), receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) were also investigated by immunohistochemistry to assess bone turnover and metabolism. Immunofluorescence microscopy was used to confirm the expression of RANKL in rats’ maxillae. Results: Treatment with simvastatin improved alveolar bone loss within all of the parameters studied, thus demonstrating anti‐inflammatory and antioxidant activity. Simvastatin reduced expression of iNOS, MMP‐1 and ‐8, RANK, and RANKL and increased BMP‐2 and OPG levels in the periodontal tissue. Simvastatin (30 mg/kg) increased TAP activity on day 11 compared with the saline group. No differences were found in the levels of AST and ALT in any of the groups studied. Conclusion: The present data suggest that simvastatin prevents inflammatory bone resorption in experimental periodontitis, which may be mediated by its anti‐inflammatory and antioxidant properties.     

Protective effects of baicalin on ligature-induced periodontitis in rats

Background and Objective: Baicalin is a flavonoid compound purified from the medicinal plant, Scutellaria baicalensis Georgi, and has been reported to possess anti‐inflammatory and antioxidant activities. The purpose of this study was to test the ability of baicalin to influence the progression of experimental periodontitis in rats, as well as the expression of cyclooxygenase‐2 and inducible nitric oxide synthase. Material and Methods: Adult male Sprague–Dawley rats were subjected to placement of a nylon thread around the bilateral lower first molars and killed after 7 d. Baicalin (50, 100 or 200 mg/kg) was supplied to the animals by oral gavage, starting 1 d before the induction of periodontitis. The ligature group consisted of rats subjected to periodontitis and receiving vehicle (0.5% carboxymethylcellulose) alone. The alveolar bone loss and the area fraction occupied by collagen fibers were assessed. The expression of cyclooxygenase‐2 and inducible nitric oxide synthase protein in the gingiva were detected by immunohistochemistry and western blotting. Results: Baicalin‐treated groups presented with lower alveolar bone loss than that of the ligature group, reaching statistical significance at the dose of 200 mg/kg (p = 0.009). The area fraction of collagen fibers was significantly higher in the baicalin (200 mg/kg)‐treated group than in the ligature group (p = 0.047). Baicalin treatment significantly down‐regulated the protein expression for cyclooxygenase‐2 (p = 0.000) and inducible nitric oxide synthase (p = 0.003), compared with the ligature group. Conclusion: Baicalin protects against tissue damage in ligature‐induced periodontitis in rats, which might be mediated, in part, by its inhibitory effect on the expression of cyclooxygenase‐2 and inducible nitric oxide synthase. These activities could support the continued investigation of baicalin as a potential therapeutic agent in periodontal disease.     

Effects of Polycan,a β‐glucan,on experimental periodontitis and alveolar bone loss in Sprague‐Dawley rats

Kim YS, Kang SJ, Kim JW, Cho HR, Moon SB, Kim KY, Lee HS, Han CH, Ku SK, Lee YJ. Effects of Polycan, a β‐glucan, on experimental periodontitis and alveolar bone loss in Sprague‐Dawley rats. J Periodont Res 2012; 47: 800–810. © 2012 John Wiley & Sons A/S Background and Objective: Polycan is a promising candidate for the treatment of periodontal disease. This study was undertaken to examine whether Polycan, a type of β‐glucan, has a protective effect on ligature‐induced experimental periodontitis and related alveolar bone loss in Sprague‐Dawley rats. Material and Methods: Polycan was orally administered, daily, for 10 d, at 21.25, 42.5 or 85 mg/kg, beginning 1 d after ligation. Changes in body weight and alveolar bone loss were monitored, and the anti‐inflammatory effects of Polycan were determined by measuring the levels of myeloperoxidase (MPO), interleukin‐1beta (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α) in gingival tissue. We also evaluated inducible nitric oxide synthase (iNOS) activity and malondialdehyde (MDA) concentrations as a measure of the antioxidant effect. Results: Ligature placement led to a marked decrease in body weight, increased alveolar bone loss and increased concentrations of MPO, IL‐1β, TNF‐α and MDA, as well as increased iNOS activity and inflammatory cell infiltration and decreased collagen‐fiber content. Histological examination revealed increases in the number and activity of osteoclast cells, decreases in alveolar bone volume and elevated percentages of osteclasts on the alveolar bone surface. Daily oral treatment with 42.5 or 85 mg/kg of Polycan for 10 d led to significant, dose‐dependent inhibition of the effect of ligature placement. Conclusion: Taken together, these results suggest that 10 d of oral treatment with Polycan effectively inhibits ligature placement‐induced periodontitis and related alveolar bone loss via an antioxidant effect.     

Hypertension favors the inflammatory process in rats with experimentally induced periodontitis

Bonato CF, do‐Amaral CCF, Belini L, Salzedas LMP, Oliveira SHP. Hypertension favors the inflammatory process in rats with experimentally induced periodontitis. J Periodont Res 2012; 47: 783–792. © 2012 John Wiley & Sons A/S Background and Objective: Cardiovascular diseases are significantly correlated with chronic periodontitis. The aim of this study was to evaluate bone‐loss level, neutrophil migration, CXCL2/CINC‐2α, CXCL5/LIX, CCL20/MIP‐3α and tumor necrosis factor‐α (TNF‐α) production, inducible nitric oxide synthase (iNOS) expression and C‐reactive protein (CRP) release in spontaneously hypertensive rats (SHRs) and normotensive (WTK) rats after experimental induction of periodontal disease. Material and Methods: Periodontitis was induced by placement of silk yarn ligatures around the first molar counterparts. The levels of CRP, CCL20/MIP‐3α and CXCL5/LIX were evaluated in the peripheral blood, and bone‐loss level, neutrophil recruitment, the production of myeloperoxidase, CXCL2, CXCL5, CCL20 and TNF‐α, and the expression of iNOS were evaluated in the gingival tissue. Histological sections were taken to evaluate and measure bone resorption and neutrophil recruitment in the furcation region. Results: Rats with periodontitis had alveolar bone resorption. SHRs with periodontitis showed marked bone loss and increased neutrophil infiltration in comparison with WTK rats. SHRs with periodontitis showed increased levels of TNF‐α and CXCL2, and a slight tendency for increased levels of CXCL5, in the gingival tissue but no increase in the level of CCL20. In SHRs, even without periodontitis, the levels of TNF‐α, CXCL2, CXCL5 and CCL20 showed a slight tendency to increase. In the WTK rats, TNF‐α, CXCL2 and CXCL5 levels were increased with periodontitis, but the level of CCL20 was not. iNOS was expressed in the gingival tissue of WTK rats and SHRs with periodontitis; however, SHRs appeared to express a higher level of iNOS than did WKT rats. The CRP level was elevated in both types of rats with periodontitis; however, the CRP level was higher in SHRs with periodontitis than in WTK rats with periodontitis. Conclusion: In SHRs, the hypertensive condition per se seems to favor the inflammatory processes that become potentiated with periodontitis, when compared with WKT rats.     

Effect of Simvastatin Prodrug on Experimental Periodontitis

Background: Local application of statins has shown potential in preventing and regenerating bone loss associated with experimental periodontitis. This study evaluates the effect of a novel simvastatin (SIM) prodrug (capable of delivering high doses to periodontitis inflammatory lesion and cells) on experimental periodontitis bone loss and inflammation. Methods: Forty mature female Sprague Dawley rats were subjected to ligature‐induced experimental periodontitis between maxillary first and second molars (M1‐M2). Equal groups were treated with three weekly doses of: 1) prodrug carrier alone (mPEG); 2) 0.5 mg SIM dose equivalent in carrier (SIM/SIM‐mPEG); 3) 1.0 mg SIM/SIM‐mPEG; 4) 1.5 mg SIM/SIM‐mPEG; or 5) ligature alone. Contralateral molars served as unmanipulated controls. Four weeks after initiation of periodontitis, animals were euthanized, the M1‐M2 interproximal was evaluated with microcomputed tomography and histology, and data were analyzed with one‐way analysis of variance. Results: Ligature alone caused a mean bone loss of 1.01 ± 0.06 mm from the cemento‐enamel junction, whereas all doses of SIM/SIM‐mPEG reduced bone loss, especially 1.5 mg SIM/SIM‐mPEG (0.68 ± 0.05 mm, P <0.001), which was not statistically different from contralateral control (0.47 ± 0.06 mm). A dose of 1.5 mg SIM/SIM‐mPEG also reduced percentage of neutrophils compared with carrier alone (2.0% ± 1.0% versus 5.7% ± 1.1%; P <0.05), and increased amount of uninflamed connective tissue in the M1‐M2 interproximal area (65.2% ± 3.3% versus 46.3% ± 3.3%; P <0.001). The mPEG carrier alone did not have bone‐sparing or anti‐inflammatory properties. Conclusion: Multiple local 1.5‐mg doses of a macromolecular SIM prodrug decreases amount of experimental periodontitis bone loss and inflammation in rats.     

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.     

Macrophage‐mediated nanoparticle delivery to the periodontal lesions in established murine model via Pg‐LPS induction

We established a murine periodontitis model by local injection of lipopolysaccharide of Porphyromonas gingivalis (Pg‐LPS) into the gingival sulcus of mandibular left incisor four times with 48‐h interval. The histological examination of the periodontal tissues demonstrated that significant loss of periodontal bone and ligaments was observed in the lesion side with abundant inflammatory cell infiltration. Two days after the last injection, Cy5‐labelled siRNA/chitosan particles were injected intraperitoneally (ip). The chitosan/siRNA particles were taken up by peritoneal macrophages, which subsequently migrated to the inflamed gingival area evaluated by in vivo imaging. The localization of macrophages in the inflamed region was further confirmed by immunofluorescent staining. The present report demonstrates that intragingival injection of Pg‐LPS can be used to create an experimental model of periodontal inflammation in mice and that recruitment of macrophages with chitosan/siRNA nanoparticles to the inflamed area opens the possibility of an RNAi‐based therapeutic approach using chitosan as a carrier in periodontitis.     

Anti‐Inflammatory and Osteoprotective Effects of Cannabinoid‐2 Receptor Agonist HU‐308 in a Rat Model of Lipopolysaccharide‐Induced Periodontitis

Background: Anti‐inflammatory and immunologic properties of cannabinoids have been reported in several tissues. Expression of cannabinoid receptor Type 2 was reported in osteoblasts and osteoclasts, suggesting a key role in bone metabolism. The aim of this study is to assess the effect of treatment with cannabinoid‐2 receptor agonist HU‐308 in the oral health of rats subjected to lipopolysaccharide (LPS)‐induced periodontitis. Methods: Twenty‐four rats were distributed in four groups (six rats per group): 1) control rats; 2) sham rats; 3) rats submitted to experimental periodontitis (LPS); and 4) rats submitted to experimental periodontitis and treated with HU‐308 (LPS+HU). In groups LPS and LPS+HU, periodontitis was induced by LPS (1 mg/mL) injected into the gingival tissue (GT) of maxillary and mandibular first molars and into the interdental space between the first and second molars, 3 days per week for 6 weeks. In group LPS+HU, HU‐308 (500 ng/mL) was applied topically to the GT daily. Results: Alveolar bone loss resulting from LPS‐induced periodontitis was significantly attenuated with HU‐308 treatment (LPS+HU), measured by macroscopic and histologic examination. Treatment also reduced gingival production of inflammatory mediators augmented in LPS‐injected rats, such as: 1) inducible nitric oxide (iNOS) activity (LPS: 90.18 ± 36.51 pmol/minute/mg protein versus LPS+HU: 16.37 ± 4.73 pmol/minute/mg protein; P <0.05); 2) tumor necrosis factor alpha (LPS: 185.70 ± 25.63 pg/mg protein versus LPS+HU: 95.89 ± 17.47 pg/mg protein; P <0.05); and 3) prostaglandin E₂ (PGE₂) (LPS: 159.20 ± 38.70 pg/mg wet weight versus LPS+HU: 71.25 ± 17.75 pg/mg wet weight; P <0.05). Additionally, HU‐308 treatment prevented the inhibitory effect of LPS‐induced periodontitis on the salivary secretory response to pilocarpine. Moreover, iNOS activity and PGE₂ content, which were increased by LPS‐induced periodontitis in the submandibular gland, returned to control values after HU‐308 treatment. Conclusion: This study demonstrates anti‐inflammatory, osteoprotective, and prohomeostatic effects of HU‐308 in oral tissues of rats with LPS‐induced periodontitis.     

Effects of tumor necrosis factor-alpha inhibitors pentoxifylline and thalidomide on alveolar bone loss in short-term experimental periodontal disease in rats

BACKGROUND: Pentoxifylline (PTX) and thalidomide (TLD) have been shown to inhibit cytokine synthesis, mainly tumor necrosis factor (TNF)-alpha, in different inflammatory conditions. We studied the effects of these cytokine inhibitors in an experimental model of periodontitis. METHODS: Wistar rats were subjected to a ligature placement around the second upper right molars. Alveolar bone loss was evaluated by the sum of the distances between the cusp tip and the alveolar bone along the axis of each molar root, subtracting from the contralateral side. Histopathological analysis was based on cell influx, amount of alveolar bone, and cementum integrity. Animals were weighed daily, and total and differential peripheral white blood cell counts were performed at 6 hours and 1, 7, and 11 days after induction of periodontitis. Groups were treated with saline (positive control), PTX, or TLD 1 hour before and daily up to 11 days after induction of periodontitis. RESULTS: Alveolar bone loss was inhibited 42%, 54%, and 69% by PTX at 5, 15, and 45 mg/kg, and 25%, 25%, 42%, and 54% by TLD at 5, 15, 45, and 90 mg/kg, respectively, as compared to the control (P < 0.05; analysis of variance). Histological analysis showed that PTX and TLD reduced cell influx and alveolar bone and cementum destruction. PTX and TLD also reversed peripheral lymphomonocytosis but not weight loss, as compared to controls. These data showed that both PTX and TLD reduced alveolar bone loss in periodontitis. CONCLUSION: The data showed a protective effect of PTX and TLD on experimental periodontitis, suggesting a role for TNF-alpha in the pathophysiology of periodontitis.     

Disodium chlodronate prevents bone resorption in experimental periodontitis in rats

BACKGROUND: Periodontitis is a chronic disease characterized by alveolar bone loss and inflammatory changes. We studied the effect of disodium chlodronate (CD), a bisphosphonate used in metastatic and metabolic bone disease as a bone resorbing drug, in an experimental periodontitis model (EPD) focusing on anti-resorptive and anti-inflammatory parameters. METHODS: A nylon thread ligature was placed around the left maxillary molars of 72 male Wistar rats who were sacrificed after 7 or 11 days. Groups were treated daily with CD (1, 5, or 25 mg/kg/sc) starting at day 0 until day 7 (prophylactic CD) or from day 5 until day 11 (curative CD) after periodontitis induction. Non-treated group (NT) consisted of rats subjected to periodontitis that received no pharmacological treatment. Alveolar bone loss (ABL) was measured as the distance between the cuspid tips and the alveolar bone. The right jaw was used as control. The hemiarcades were processed for histopathologic analysis. RESULTS: In NT group there was significant ABL, severe mononuclear cells influx, and increase in osteoclast numbers. Prophylactic CD treatment decreased the ABL 25.8%, 61.6%, and 75.5% as compared to NT for the 1, 5, and 25 mg/kg CD doses, respectively. Curative CD treatment decreased the ABL 20%, 62%, and 69% as compared to NT for the 1, 5 and 25 mg/kg CD doses, respectively. Both prophylactic and curative CD decreased histological changes, as compared to NT rats (P <0.01). CONCLUSIONS: CD has both bone sparing and anti-inflammatory activity in EPD in rats when administered as a pretreatment or in an ongoing process. The possibility of using CD as an alternative treatment in human periodontitis should be considered.     

Exenatide and Sitagliptin Decrease Interleukin 1β, Matrix Metalloproteinase 9, and Nitric Oxide Synthase 2 Gene Expression But Does Not Reduce Alveolar Bone Loss in Rats With Periodontitis

Background: New drugs for the treatment of diabetes, glucagon‐like peptide‐1 (GLP‐1) receptor agonists and inhibitors of dipeptidyl peptidase‐4 (DPP‐4) have shown pleiotropic effects on bone metabolism and anti‐inflammatory properties. The aim of this study is to evaluate the effects of exenatide (GLP‐1 agonist) and sitagliptin (DPP‐4 inhibitor) during periodontitis induction by ligature insertion in rats. Methods: Forty rats were divided into four groups: 1) animals with induced periodontitis that received exenatide (EG); 2) animals with induced periodontitis that received sitagliptin (SG); 3) animals with induced periodontitis and without drug treatment (LG); and 4) animals without induced periodontitis and without drug treatment (controls). The drugs were administered for 28 days. On the day the animals were sacrificed, blood was collected for analysis of glucose and DPP‐4 levels. The gene expressions of prostaglandin‐endoperoxide synthase 2, tissue inhibitor of metalloproteinase 1, Dpp4, nitric oxide synthase 2 (Nos2), interleukin 1β (Il1b), and matrix metalloproteinase 9 (Mmp9) in the gingiva; support and alveolar bone loss; connective tissue attachment; and the quantity of gingival collagen were evaluated. Results: Exenatide and sitagliptin treatments have led to a lower percentage of weight gain but did not influence glycemia. Sitagliptin reduced the serum concentration of DPP‐4. Interestingly, although the gene expression profile has revealed a downregulation of Mmp9, Nos2, and Il1b in both EG and SG compared to LG, a significant protective effect was not observed on alveolar bone and collagen tissue in this model. Conclusion: Regardless of the reduction of the expression of Il1b, Nos2, and Mmp9, the drugs were not effective in the stabilization or reduction of alveolar bone loss and collagen degradation in rats.     

Experimental periodontitis in mice selected for maximal or minimal inflammatory reactions: increased inflammatory immune responsiveness drives increased alveolar bone loss without enhancing the control of periodontal infection

Background and Objective: Inflammatory immune reactions that occur in response to periodontopathogens are thought to protect the host against infection, but may trigger periodontal destruction. However, the molecular and genetic mechanisms underlying host susceptibility to periodontal infection and to periodontitis development have still not been established in detail. Material and Methods: In this study, we examined the mechanisms that modulate the outcome of Aggregatibacter (Actinobacillus) actinomycetemcomitans‐induced periodontal disease in mice mouse strains selected for maximal (AIRmax) or minimal (AIRmin) inflammatory reactions. Results: Our results showed that AIRmax mice developed a more severe periodontitis than AIRmin mice in response to A. actinomycetemcomitans infection, and this periodontitis was characterized by increased alveolar bone loss and inflammatory cell migration to periodontal tissues. In addition, enzyme‐linked immunosorbent assays demonstrated that the levels of the cytokines interleukin‐1β, tumor necrosis factor‐α and interleukin‐17 were higher in AIRmax mice, as were the levels of matrix metalloproteinase (MMP)‐2, MMP‐13 and receptor activator of nuclear factor‐κB ligand (RANKL) mRNA levels. However, the more intense inflammatory immune reaction raised by the AIRmax strain, in spite of the higher levels of antimicrobial mediators myeloperoxidase and inducible nitric oxide synthase, did not enhance the protective immunity to A. actinomycetemcomitans infection, because both AIRmax and AIRmin strains presented similar bacterial loads in periodontal tissues. In addition, the AIRmax strain presented a trend towards higher levels of serum C‐reactive protein during the course of disease. Conclusion: Our results demonstrate that the intensity of the inflammatory immune reaction is associated with the severity of experimental periodontitis, but not with the control of A. actinomycetemcomitans periodontal infection, suggesting that the occurrence of hyperinflammatory genotypes may not be an evolutionary advantage in the complex host–pathogen interaction observed in periodontal diseases.     

Micro‐computerized tomography analysis of alveolar bone loss in ligature‐ and nicotine‐induced experimental periodontitis in rats

Liu Y‐F, Wu L‐A, Wang J, Wen L‐Y, Wang X‐J. Micro‐computerized tomography analysis of alveolar bone loss in ligature‐ and nicotine‐induced experimental periodontitis in rats. J Periodont Res 2010; 45: 714–719. © 2010 John Wiley & Sons A/S Background and Objective: Nicotine reportedly is a risk factor for periodontitis, but accurate data regarding nicotine‐induced alveolar bone loss is lacking. The aim of this study was to quantitatively assess alveolar bone loss in ligature‐ and nicotine‐induced periodontitis in rats using micro‐computerized tomography (micro‐CT). Material and Methods: Thirty‐six adult male rats were treated by placing silk ligatures around the cervixes of the right second maxillary molar; the contralateral tooth was untreated. After ligation, the animals were randomly divided into three groups: group A received intraperitoneal injections of saline solution, group B received 0.83 mg of nicotine/kg/d, and group C received 1.67 mg of nicotine/kg/d. Six animals in each group were killed on days 14 and 28 after ligature placement, and then micro‐CT examinations were conducted. Results: In all groups, bone mineral density (BMD), bone volume fraction (BVF), trabecular number (Tb.N) and trabecular thickness (Tb.Th) values of the ligated sides were significantly lower than those of the unligated sides (p < 0.001), whereas alveolar bone height loss (ABHL) and trabecular separation (Tb.Sp) of the ligated sides were significantly higher than those of the unligated sides (p < 0.001). Compared with the control group, nicotine administration increased the ABHL value and decreased the BMD, BVF and Tb.Th values of both sides in a dose‐dependent manner (p < 0.05). Conclusion: Our results confirmed that ligature could cause significant loss in the trabecula of alveolar bone, and daily administration of nicotine resulted in further bone loss and microstructure deterioration.     

Odanacatib,A Cathepsin K‐Specific Inhibitor,Inhibits Inflammation and Bone Loss Caused by Periodontal Diseases

Background: Periodontitis is a bacteria‐induced inflammatory disease mainly affecting periodontal tissues, leading to periodontal inflammation, bone breakdown, and loss of the tooth. The main obstacle for treating periodontitis effectively is the difficulty in finding a target that can inhibit bone loss and inflammation simultaneously. Recent studies showed that cathepsin K (CTSK) might have functions in the immune system besides its role in osteoclasts. Thus, targeting CTSK would have a potential therapeutic effect in both the bone system and the immune system during the progression of periodontitis. Methods: In the current study, a small molecular inhibitor (odanacatib [ODN]) is explored to inhibit the function of CTSK in a bacteria‐induced periodontitis mouse model. Results: The application of ODN decreased the number of osteoclasts, macrophages, and T cells, as well as the expression of Toll‐like receptors (TLRs) in the periodontitis lesion area. Furthermore, lack of CTSK inhibited the expression of TLR4, TLR5, and TLR9 and their downstream cytokine signaling in the gingival epithelial cells in periodontitis lesions, demonstrating that the innate immune response was inhibited in periodontitis. Conclusion: The present results show that inhibition of CTSK can prevent bone loss and the immune response during the progression of periodontitis, indicating that CTSK is a promising target for treating inflammatory diseases such as periodontitis by affecting both osteoclasts and the immune system.     

Inhibition of matrix metalloproteinase-mediated periodontal bone loss in rats: a comparison of 6 chemically modified tetracyclines

BACKGROUND: Chemically modified tetracyclines (CMTs), devoid of antimicrobial activity, inhibit pathologically elevated collagenase activity both in vivo and in vitro. In the current study, doxycycline and 5 different CMTs were tested to prevent matrix metalloproteinase (MMP)-dependent periodontal tissue breakdown in an animal model of periodontitis. METHODS: Adult male rats received intragingival injections with either 10 microl of physiologic saline or Escherichia coli endotoxin (1 mg/ml) every other day for 6 days and were distributed into 8 treatment groups (12 rats/group): saline (S), endotoxin alone (E), E + CMT-1, E + CMT-3, E + CMT-4, E + CMT-7, E + CMT-8, and doxycycline. All animals were treated daily with 1 ml of 2% carboxymethyl cellulose (CMC) alone or containing one of the above-mentioned CMTs (2 mg/day) orally. The gingival tissues were removed, extracted, and assayed for gelatinase (GLSE). Some rat maxillary jaws from each treatment group were fixed in buffered formalin and processed for histology and immunohistochemistry for the cytokines tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6, and MMP-2 and MMP-9. RESULTS: Endotoxin injection induced elevated GLSE activity (functional assay and osteoclast-mediated bone resorption), the former identified as predominantly MMP-9 (92 kDa GLSE) by gelatin zymography. All 6 tetracyclines (2 mg/day) inhibited periodontal breakdown in the following order of efficacy: CMT-8 > CMT- 1 > CMT-3 > doxycycline > CMT-4 > CMT-7. Immunohistochemistry was positive for TNF, IL-1, and IL-6 in the inflammatory cells from untreated endotoxin rat tissues, whereas treatment with CMTs decreased the number of immuno-positive stained cells for cytokines and MMPs. The in vivo efficacy of these drugs varied with CMT structure and was significantly correlated with bone resorption: r2 = -0.77, P<0.01; gelatinase inhibitory activity: r2 = -0.84, P <0.01; and serum drug concentrations. CONCLUSION: Since both conventional (antimicrobial) and non-antimicrobial tetracyclines inhibited periodontal bone resorption induced by endotoxin injection, MMP-mediated bone loss in this model can be prevented by inhibition of MMPs.