Periodontal status and mandibular biomechanics in rats subjected to hyposalivation and periodontitis.

Xerostomia emerges as a consequence of salivary gland hypofunction, and seriously compromises the integrity of hard and soft oral tissues, whileperiodontitis is an infectious disease characterized by biofilm accumulation, inflammation and alveolar bone resorption. AIM: The aim this study was to compare the deleterious effects caused by experimental hyposalivation, periodontitis, and the combination of both on periodontal tissues and mandibular biomechanics in rats. MATERIALS AND METHOD: Hyposalivation (group H) was induced through bilateral submandibulectomy. Periodontitis (group EP) was induced by injecting LPS (1 mg/ml) into the gingiva of the first lower molars. A third group was subjected to both conditions (group H+EP). Alveolar bone loss was evaluated by micro-computed tomography and histomorphometric analysis, and gingival inflammatory mediators were assessed by specific techniques. Biomechanical properties were evaluated in mandible. RESULTS: Alveolar bone loss increased similarly in groups H, EP and H+EP compared to control. Metalloproteinase (MMP2 and MMP9) activity was similar in H and control, but higher in groups EP and H+EP (MMP2: C 9644+2214, EP 34441+3336, H 5818+1532, H+EP 42673+3184; MMP9: C 5792+961, EP 14807+861, H 9295+520, H+EP 4838+1531). The rest of the inflammatory mediators evaluated increased in groups H, EP and H+EP to a greater or lesser extent with respect to the control, although in most cases, they were higher in groups EP and H+EP than in group H. The biomechanical properties of the mandible increased in group H compared to the other three groups. CONCLUSIONS: Both hyposalivation and periodontitis cause periodontal damage, but hyposalivation also produces biomechanical alterations, causing more extensive deleterious effects than periodontitis.

La xerostomía surge como consecuencia de la hipofunción de las glándulas salivales y compromete seriamente la integridad de los tejidos orales duros y blandos, mientras que la periodontitis es una enfermedad infecciosa caracterizada por la acumulación de biofilm, inflamación y reabsorción ósea alveolar. OBJETIVO: El objetivo del presente estudio fue comparar los efectos deletéreos causados por la hiposalivación y la periodontitis experimental, y la combinación de ambas sobre los tejidos periodontales y la biomecánica mandibular en ratas. MATERIALES Y MÉTODOS: La hiposalivación (H) se indujo mediante una submandibulectomía bilateral. Por otra parte, la periodontitis (PE) se indujo mediante la inyección de LPS (1 mg/ml) en la encía de los primeros molares inferiores. Otro grupo se sometió a ambas condiciones (H+PE). La pérdida ósea alveolar se evaluó mediante tomografia microcomputarizada y análisis histomorfométrico, mientras que los mediadores inflamatorios gingivales fueron determinados mediante técnicas específicas. Se evaluaron las propiedades biomecánicas en la mandíbula. RESULTADOS: La hiposalivación aumentó la pérdida ósea alveolar en comparación con el control de forma similar a la PE y H+PE. La actividad de las metaloproteinasas (MMP2 y MMP9) fue similar en los grupos H y control, pero resultó mayor en los grupos PE y H+PE (MMP2: C 9644+2214, PE 34441+3336, H 5818+1532, H+PE 42673+3184; MMP9: C 5792+961, PE 14807+861, H 9295+520, H+PE 24838+1531). El resto de los mediadores inflamatorios evaluados aumentaron en mayor o menor medida en los grupos H, PE y H+PE respecto al control, aunque en la mayoría de los casos fueron superiores en los grupos PE y H+PE respecto al grupo H. Sin embargo, las propiedades biomecánicas de la mandíbula aumentaron en el grupo H con respecto a los otros grupos. CONCLUSIONES: Tanto la hiposalivación como la periodontitis causan daño periodontal, pero la hiposalivación también produce alteraciones biomecánicas, provocando efectos deletéreos más extensos que la periodontitis.

Signs of alveolar bone damage in early stages of periodontitis and its prevention by stimulation of cannabinoid receptor 2. Model in rats.

The aims of the present study were, first, to identify signs of alveolar bone damage in early stages of experimental periodontitis (EP) and, second, to assess its possible prevention by treatment with cannabinoid receptor 2 agonist HU 308. Experimental periodontitis was induced by injections of lipopolysaccharide (LPS) (1mg/ml) in gums surrounding maxillary and mandibular first molar, 3 days per week, and untreated controls were kept for comparison. Then, a 3-week study was conducted including eighteen new rats (six rats per group): 1) controls; 2) experimental periodontitis rats; and 3) experimental periodontitis rats treated daily with HU 308 (500 ng/ml). After euthanasia, alveolar bone loss was assessed by morphometric and histomorphometric techniques, and the content of prostaglandin E2 (PGE2) in gingival tissue was evaluated by radioimmunoassay. The first signs of alveolar bone loss were apparent at 3 weeks of experimental periodontitis (ρ<0.05) in the mandibular first molar, but there was no detectable change at 1 week, leading us to establish 3 weeks as an early stage of experimental periodontitis. Rats subjected to 3-week experimental periodontitis showed less interradicular bone volume, less whole bone perimeter and fewer bone formation areas, and higher periodontal space height, bone resorption areas, number of osteoclasts and gingival content of prostaglandin E2 than controls, while HU 308 prevented, at least partially, the deleterious effects (ρ<0.001). We can conclude that a 3-week term of lipopolysaccharide-induced periodontitis in rats provides a valid model of the early stage of the disease, as emerging damage is observed in bone tissue. Furthermore, harmful effects at 3 weeks could be prevented by local stimulation of cannabinoid receptor 2, before greater damage is produced.

El objetivo del presente trabajo fue, en primer lugar, identificar signos de daño óseo alveolar en estadios tempranos de periodontitis experimental y, en segundo lugar, evaluar su posible prevención mediante el tratamiento con el agonista del receptor cannabinoide 2, HU 308. La periodontitis experimental fue inducida por inyecciones de lipopolisacárido (1mg/ml) en la encía circundante al primer molar maxilar y mandibular, 3 días por semana, en tanto que controles no tratados fueron mantenidos para la comparación. Posteriormente, un estudio de 3 semanas con dieciocho nuevas ratas (seis por grupo) fue desarrollado: 1) controles; 2) ratas con periodontitis experimental, y 3) ratas con periodontitis experimental tratadas diariamente con HU 308 (500ng/ml). Luego de la euthanasia, la pérdida ósea alveolar fue evaluada por técnicas morfométricas e histomorfométricas, y el contenido de prostaglandina E2 en el tejido gingival fue determinado por radioinmunoensayo. Los primeros signos de pérdida ósea alveolar fueron evidentes a las 3 semanas de inducción de periodontitis experimental (ρ<0.05) en el primer molar mandibular, mientras que no hubo cambios detectables luego de 1 semana de inducción, hecho que nos condujo a establecer a las 3 semanas como un estadio temprano de periodontitis experimental, Las ratas sometidas a perdiodontitis experimental de 3 semanas mostraron menor volumen óseo interradicular, menor perímetro óseo y menos áreas de formación ósea, y mayor altura del espacio periodontal, más áreas de reabsorción ósea, mayor número de osteoclastos y mayor contenido gingival de prostaglandina E2, en comparación a los controles, mientras que el tratamiento con HU 308 previno, al menos parcialmente, los efectos deletéreos (ρ<0.001). Podemos concluir que el término de 3 semanas de periodontitis inducida por lipopolisacárido es un modelo válido de estadio inicial de la enfermedad experimental, dado que se evidencia daño emergente en el tejido óseo. Asimismo, los efectos deletéreos de 3 semanas podrían ser prevenidos por la estimulación local del receptor cannabinoide 2, antes que un daño mayor sea producido.

Signs of alveolar bone damage in early stages of periodontitis and its prevention by stimulation of cannabinoid receptor 2. Model in rats / Signos de daño óseo alveolar en estadios tempranos de periodontitis y su prevención por estimulación del receptor cannabinoide 2. Modelo en ratas microcisalhamento do cimento resinoso

ABSTRACT The aims of the present study were, first, to identify signs of alveolar bone damage in early stages of experimental periodontitis (EP) and, second, to assess its possible prevention by treatment with cannabinoid receptor 2 agonist HU 308. Experimental periodontitis was induced by injections of lipopolysaccharide (LPS) (1mg/ml) in gums surrounding maxillary and mandibular first molar, 3 days per week, and untreated controls were kept for comparison. Then, a 3-week study was conducted including eighteen new rats (six rats per group): 1) controls; 2) experimental periodontitis rats; and 3) experimental periodontitis rats treated daily with HU 308 (500 ng/ml). After euthanasia, alveolar bone loss was assessed by morphometric and histomorphometric techniques, and the content of prostaglandin E2 (PGE2) in gingival tissue was evaluated by radioimmunoassay. The first signs of alveolar bone loss were apparent at 3 weeks of experimental periodontitis (ρ<0.05) in the mandibular first molar, but there was no detectable change at 1 week, leading us to establish 3 weeks as an early stage of experimental periodontitis. Rats subjected to 3-week experimental periodontitis showed less interradicular bone volume, less whole bone perimeter and fewer bone formation areas, and higher periodontal space height, bone resorption areas, number of osteoclasts and gingival content of prostaglandin E2 than controls, while HU 308 prevented, at least partially, the deleterious effects (ρ<0.001). We can conclude that a 3-week term of lipopolysaccharide-induced periodontitis in rats provides a valid model of the early stage of the disease, as emerging damage is observed in bone tissue. Furthermore, harmful effects at 3 weeks could be prevented by local stimulation of cannabinoid receptor 2, before greater damage is produced.

RESUMEN El objetivo del presente trabajo fue, en primer lugar, identificar signos de daño óseo alveolar en estadios tempranos de periodontitis experimental y, en segundo lugar, evaluar su posible prevención mediante el tratamiento con el agonista del receptor cannabinoide 2, HU 308. La periodontitis experimental fue inducida por inyecciones de lipopolisacárido (1mg/ml) en la encía circundante al primer molar maxilar y mandibular, 3 días por semana, en tanto que controles no tratados fueron mantenidos para la comparación. Posteriormente, un estudio de 3 semanas con dieciocho nuevas ratas (seis por grupo) fue desarrollado: 1) controles; 2) ratas con periodontitis experimental, y 3) ratas con periodontitis experimental tratadas diariamente con HU 308 (500ng/ml). Luego de la euthanasia, la pérdida ósea alveolar fue evaluada por técnicas morfométricas e histomorfométricas, y el contenido de prostaglandina E2 en el tejido gingival fue determinado por radioinmunoensayo. Los primeros signos de pérdida ósea alveolar fueron evidentes a las 3 semanas de inducción de periodontitis experimental (ρ<0.05) en el primer molar mandibular, mientras que no hubo cambios detectables luego de 1 semana de inducción, hecho que nos condujo a establecer a las 3 semanas como un estadio temprano de periodontitis experimental, Las ratas sometidas a perdiodontitis experimental de 3 semanas mostraron menor volumen óseo interradicular, menor perímetro óseo y menos áreas de formación ósea, y mayor altura del espacio periodontal, más áreas de reabsorción ósea, mayor número de osteoclastos y mayor contenido gingival de prostaglandina E2, en comparación a los controles, mientras que el tratamiento con HU 308 previno, al menos parcialmente, los efectos deletéreos (ρ<0.001). Podemos concluir que el término de 3 semanas de periodontitis inducida por lipopolisacárido es un modelo válido de estadio inicial de la enfermedad experimental, dado que se evidencia daño emergente en el tejido óseo. Asimismo, los efectos deletéreos de 3 semanas podrían ser prevenidos por la estimulación local del receptor cannabinoide 2, antes que un daño mayor sea producido.

A new target to ameliorate the damage of periodontal disease: The role of transient receptor potential vanilloid type-1 in contrast to that of specific cannabinoid receptors in rats.

BACKGROUND: Transient receptor potential vanilloid type-1 (TRPV1) is expressed in oral tissues cells and its activity can be regulated by inflammation products and anandamide. The aim of the present study was to evaluate the effects of blocking TRPV1 or specific cannabinoid receptors 1 (CB1r) and 2 (CB2r) on periodontal status of rats subjected to experimental periodontitis (EP). METHODS: Male rats were distributed in groups 1) control, 2) lipopolysaccharide-induced EP (LPS), and 3) LPS plus capsazepine (Capz, TRPV1 antagonist) application (LPS+Capz). EP was induced by injections of LPS (1 mg/mL) around first molars and treatment was performed with Capz (2 µg/mL) applied locally during 6 weeks. Additional experiment was performed by applying CB1r and CB2r antagonists (AM251 and AM630) to rats with EP. RESULTS: Capz prevented alveolar bone loss (ABL) on the external crests and in the interradicular bone of the first molars (periodontal space height: LPS, 270.7 ± 33.5µm versus LPS+Capz, 216.4 ± 19.9 µm; P <0.01). Inflammatory mediators, like tumor necrosis factor-alpha and prostaglandin E2 , increased by LPS-induced EP, were diminished in gingival tissue of rats treated with Capz. In contrast, application of AM251 and AM630 exacerbated ABL and gingival inflammatory mediators, increased by LPS, altering also biomechanical properties. CONCLUSIONS: TRPV1 blockade attenuates periodontal impairment in EP rats, since it reduces local inflammation, unlike CB1r and CB2r blockade. This work lays the foundation for developing therapeutics in humans based on the pharmacological manipulation of these receptors to treat periodontal disease.

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.

Chronic alcohol consumption alters periodontal health in rats.

BACKGROUND: The aim of this study was to assess the effects of chronic alcohol consumption on periodontitis development in rats. METHODS: Periodontal disease was experimentally induced by lipopolysaccharide (LPS; 2 mg/ml) injections into the gingival tissue around first upper and lower molar's neck, and into the interdental space between first and second molars. This protocol was repeated for 6 weeks on days 1, 3, and 5 of each week. Chronic alcohol consumption was induced by 20% ethanol (EtOH) as the only liquid source during 4 months. RESULTS: Chronic alcohol consumption by itself increased alveolar bone loss and biological mediators of periodontal disease such as prostaglandin E2 (PGE2 ) content on gingival tissue, and inducible nitric oxide synthase activity plus PGE2 content in submandibular gland. Unexpectedly, alcohol consumption did not increase the damage evoked by the proved model of LPS injections for periodontitis induction. CONCLUSIONS: Results suggest 20% alcohol consumption during 4 months generates differential effects on oral health of rats, depending on its pathophysiological state: It would exacerbate the inflammatory condition when periodontal damage is absent, but it would not when damage is installed.

Long-term treatment with methanandamide attenuates LPS-induced periodontitis in rats.

OBJECTIVE: Evidence exists of the anti-inflammatory and immunological properties of endocannabinoids in various tissues; the aim of the present study was therefore to assess the effect of long-term treatment with the synthetic cannabinoid methanandamide (Meth-AEA) on the progression of periodontitis in rats. MATERIALS AND METHODS: Periodontitis was induced by injecting LPS (1 mg/ml) into the gingiva around the neck of the first upper and lower molars, and into the inter-dental space between the first and second molars. This protocol was repeated for 6 weeks on days 1, 3, and 5 of each week. RESULTS: Long-term treatment with topical Meth-AEA (500 ng/ml), applied daily to gingival tissue of rats induced with periodontitis, significantly diminished the alveolar bone loss, measured as the distance between the cemento-enamel junction and the alveolar crest, in both maxillary and mandibular first molars, compared to rats without treatment (P < 0.05). The treatment also reduced the production of some biological mediators of periodontal disease augmented by LPS, such as tumor necrosis factor alpha (from 119.4 ± 9.9 pg/mg protein to 75.1 ± 10.8, P < 0.05) and nitric oxide produced by inducible nitric oxide synthase (from 507.7 ± 107.1 pmol/min/mg protein to 163.1 ± 53.9, P < 0.01). CONCLUSION: These results demonstrate the beneficial effects of treatment with Meth-AEA on gingival tissue of rats with periodontitis.