Polysacharide of Agaricus blazei gel mitigates bone necrosis in model of the jaws related to bisphosphonate via Wnt signaling.

To investigate de effect of PAb gel on the bone tissue of rats submitted to Bisphosphonate-related osteonecrosis of the jaws (BRONJ). Initially, 54 animals were submitted to BRONJ model by Zoledronic Acid (ZA) (0.1 mg/kg 3x/wk for 9 wk, ip), followed by the 1st upper left molar extraction at the 8th wk. After tooth removal, the animals were divided into 3 groups, ZA that received placebo gel or PAb gel that received 1% PAb gel, inside the dental alveolus. The control Group (CONTROL) received 0.1 mg/kg of 0.9% saline and then placebo gel. Three weeks after tooth extraction, the animals were euthanized, and maxillae were colleted for macroscopic, radiographic, histological and Raman spectomery assays. Additionally, GSK3b, beta-catenin, and Runx2 mRNA expressions were determined. Blood samples were collected for the analysis of Bone-specific alkaline phosphatase (BALP) levels. PAb gel improved mucosal healing, increased the number of viable osteocytes, while it reduced the number of empty lacunae, as well as the amount of bone sequestration. Furthermore, PAb gel positively influenced the number and functionality of osteoblasts by stimulating Wnt signaling, thereby inducing bone remodeling. Additionally, PAb gel contributed to improved bone quality, as evidenced by an increase in bone mineral content, a decrease in bone solubility, and an enhancement in the quality of collagen, particularly type I collagen. PAb gel mitigated bone necrosis by stimulating of bone remodeling through Wnt signaling and concurrently improved bone quality. PAb gel emerges as a promising pharmacological tool for aiding in BRONJ therapy or potentially preventing the development of BRONJ.

Chenopodium Ambrosioides Linn Mitigates Bone Loss in Rats with Periodontitis.

Statement of the Problem: Periodontitis is an inflammatory disease that causes bone loss. Some patients do not respond well to the classic treatment and need therapies that minimize bone loss, the main sequel of the disease. Chenopodium ambrosioides L. has stood out due to its anti-inflammatory and anti-oxidative activities. However, no study has yet investigated its effect on periodontitis. Purpose: This study aimed to evaluate the bone protective effect of Chenopodium ambrosioides L. (CAL) extract on ligature-induced periodontitis model in rats. Materials and Method: For this, a pre-clinical assay was performed, using male Wistar rats divided into 3 groups: Naive (N) (n=6), not submitted to any procedure; Saline (SAL) (n=6), submitted to ligature-induced periodontitis and receiving 2 ml/kg of 0.9% saline solution; and CAL extract, which was subdivided into 3 subgroups (n=6/subgroup) receiving the CAL at 3 (CAL3), 10 (CAL10) or 30 mg/kg (CAL30). All agents were given, by oral gavage, 30 min before periodontitis induction and daily until euthanasia (11th day). By then, maxillae were removed for macroscopic, histological, and histometric analyses. Kidneys, liver, and stomach were collected to evaluate the safety of CAL extract. High-performance liquid chromatography (HPLC) assay was used to investigate the flavonoid content in the extract. Results: Chenopodium ambrosioides L. extract at 30mg/kg showed a reduction by 58% in bone loss marked by an increase (+35%) in the number of osteoblasts and a reduction (-51%) on the number of osteoclasts (p< 0.05). No significant alteration in the liver, kidney, or stomach was seen. Rutin was the main flavonoid found. Conclusion: In summary, it was observed that Chenopodium ambrosioides L. extract has shown important anti-inflammatory and bone anabolic and anti-resorptive properties without causing toxicity in the main organs. Rutin, as the main flavonoid of the extract, seems to be responsible for the beneficial effect of this agent.

Temporomandibular joint arthritis increases canonical Wnt pathway expression in the articular cartilage and trigeminal ganglion in rats.

The canonical Wnt pathway participates in inflammatory diseases and it is involved in neuropathic pain. This study evaluated the immunoexpression of the canonical Wnt signaling pathway in the articular cartilage of the temporomandibular joint (TMJ) and along the nociceptive trigeminal pathway in arthritic rats. For this, male Wistar rats were divided into Control (C) and Arthritic (RA) groups. Arthritis induction was performed through subcutaneous injection of methylated bovine serum albumin (mBSA) and complete Freund Adjuvant (CFA)/ Incomplete Freund Adjuvant (IFA) on the first 14 days (once a week), followed by 3 weekly intra-articular injections of mBSA (10 µl/joint; left TMJ). The following parameters were evaluated: nociceptive threshold, inflammatory infiltrate, type I and III collagen birefringence, immunohistochemistry for IL-1ß, TNF-α, IL-6, Wnt10b, ß-catenin, cyclin-D1 in articular cartilage, c-Myc in synovial membrane, and immunofluorescence analysis for c-Fos, Wnt-10b and ß-catenin in the trigeminal ganglion and the trigeminal subnucleus caudalis. The RA group showed intense articular cartilage damage with proliferation of type III collagen, increased immunoexpression of proinflammatory cytokines and Wnt-10b, ß-catenin and cyclin-D1 in the articular cartilage and c-Myc in the synovial membrane. In the RA group, a reduction in the nociceptive threshold was observed, followed by a significant increase in the expression of Wnt-10b in neurons and ß-catenin in satellite cells of the trigeminal ganglion. c-Fos immunoexpression was observed in neurons, peripherally and centrally, in arthritic rats. Our data demonstrated that TMJ arthritis in rats causes articular cartilage damage and nociceptive behavior, with increased immunoexpression of canonical Wnt pathway in the articular cartilage and trigeminal ganglion.

Milk kefir therapy improves the skeletal response to resistance exercise in rats submitted to glucocorticoid-induced osteoporosis.

Glucocorticoid-induced osteoporosis (GIO) has emerged as a challenge after long-term glucocorticoids (GCs) administration. Exercise has been an important non-pharmacological option, while medications modulate bone remodeling despite adverse effects. In this way, milk Kefir (MK) therapy stands out as a safe alternative to improve bone metabolism. Our study aimed to investigate the effect of MK associated to resistance exercise on bone loss in rats with GIO. For this, sixty male Wistar rats were divided into 2 groups: normal (N) and subjected to GIO, which was subdivided into 4 groups: control (C), milk kefir therapy (K), Exercise (Ex), and Exercise+K (ExK). GIO was induced by dexamethasone (7 mg/kg - i.m.; 1×/wk, 5 wk). MK was administered daily (1×/day; 0.7 ml/animal) and the climb exercise with load was performed 3×/wk; both for 16 wk. Femur was collected for assessment of bone microarchitecture, quality and metabolism. GIO markedly reduced trabecular bone volume density (BV/TV) (-35 %), trabecular thickness (Tb.Th) (-33 %), mineral content of femur (-26 %) as well as bone collagen content (-56 %). Bone strength and its biomechanical properties given by flexural strength (-81 %), fracture load (-80 %), and the number of osteocytes (-84 %) were lowered after GIO. GCs reduced osteoblast number and function while increased osteoclast number, altering bone remodeling (p < 0.05). On the other hand, ExK significantly improved bone microarchitecture and quality, marked by fractal dimension increase (+38 %), cortical volume (+34 %), BV/TV (+34 %), Tb.Th (+33 %), mineral content and collagen maturity, while reduced the space between trabecula (-34 %). The Ex and ExK increased the number of osteocytes (p < 0.05) and they were able to reverse the lower osteoblast number. Both treatments used alone significantly enhanced bone biomechanical properties, but the ExK showed a more significant improvement. ExK ameliorated bone strength and biomechanics (p < 0.05) and stimulated bone formation and modulated bone remodeling (p < 0.05). MK and exercise administered isolated or in association increased the percentage of collagen bone filling after GIO (p < 0.05), but only ExK improved collagen maturity. Our results showed that MK associated to resistance exercise enhanced bone microarchitecture, quality and metabolism, being therefore an interesting tool to improve skeletal response during GIO.

Molecular docking study and antireabsorptive activity of a semi-synthetic coumarin derivative from Platymiscium floribundum in the ligature-induced periodontitis in rats: the involvement of heme oxygenase-1.

OBJETIVE: This study aimed to evaluate the anti-resorptive activity of a semi-synthetic coumarin derivative from Platymiscium floribundum, named 6,7-dimethoxy-3-nitrocoumarin. MATERIAL AND METHODS: Molecular docking studies were performed to test the binding performance of the derivative against targets associated with alveolar bone loss (TNF-α, IL-1ß, and catalase) and a target considered an antioxidant defense (HO-1) during periodontitis. Periodontitis was induced by placing a nylon ligature around the second molars. The rats received for 11 days 6,7-dimethoxy-3-nitrocoumarin (0.01, 0.1, or 1 mg/kg) or vehicle. We investigated by RT-qPCR analysis (TNF-α, IL-1ß, and HO-1 mRNA expression levels) and by colorimetric assay (catalase activity) the mechanism of action mediated by 6,7-dimethoxy-3-nitrocoumarin. The in vivo toxicity of 6,7-dimethoxy-3-nitrocoumarin was evaluated. RESULTS: 6,7-Dimethoxy-3-nitrocoumarin (0.1 or 1 mg/kg) reduced alveolar bone loss (1.05 ± 0.24), when compared to vehicle-treated group (3.05 ± 0.30). The interactions of 6,7-dimethoxy-3-nitrocoumarin and the four targets (TNF-α, IL-1ß, catalase, and HO-1) showed firm bonds above 6.0 kcal/mol. 6,7-dimethoxy-3-nitrocoumarin (1 mg/kg) lowered mRNA expression levels of TNF-α (2.33 ± 0.56) and IL-1ß (19.87 ± 2.9), while it increased both the mRNA expression levels of HO-1 (43.40 ± 1.05) and the catalase activity (46.42 ± 4.59), when compared to vehicle-treated group (46.29 ± 8.43; 37.83 ± 4.38; 1.58 ± 0.11; 8.93 ± 1.86, respectively). The animals did not show any signs of toxicity. CONCLUSION: 6,7-Dimethoxy-3-nitrocoumarin decreased inflammatory bone loss in the ligature-induced periodontitis in rats, and the activation of the HO-1 pathway may contribute, at least partially, to its protective effects by reducing TNF-α and IL-1ß mRNA levels and increasing catalase activity. CLINICAL RELEVANCE: 6,7-Dimethoxy-3-nitrocumarin could be used as an adjunct to subgingival instrumentation during active and supportive periodontal treatment.

Anti-inflammatory and Hepatoprotective Effects of Quercetin in an Experimental Model of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation in the joints. Although methotrexate (MX) is the first-line treatment, side effects are common. This study aimed to investigate the effects of quercetin (QT) and/or MX on inflammation and systemic toxicity in a rat model of RA. Male Wistar rats were divided into control (C), RA, QT, MX, and QT + MX groups (n=6). The RA induction consisted of three intra-articular injections of methylated bovine serum albumin (1×/week) in the temporomandibular joint (TMJ). QT (25 mg/kg) and/or MX (0.75 mg) administration occurred by oral gavage daily. We performed mechanical hyperalgesia in TMJ, leukocyte recruitment in synovial fluid, histopathology, and immunohistochemistry (TNF-α, IL-17, and IL-10) in synovial membrane and toxicity parameters. The RA showed a reduction in the nociceptive threshold (p<0.001), increase in leukocyte recruitment in synovial fluid (p<0.001), intense inflammatory infiltrate (p<0.001), and intense immunoexpression of TNF-α, IL-17, and IL-10 in the synovial membrane (p<0.001) compared to C (p<0.001). QT and/or MX therapy reduced inflammatory parameters (p<0.001). However, downregulation of IL-10 was observed only in the groups that received MX (p<0.001). Leukocytosis was seen in RA (p<0.05), but QT and/or MX reversed it (p<0.05). MX was associated with pathological changes in the liver and higher levels of transaminases when compared to the other groups (p<0.05). QT co-administered with MX reversed this hepatotoxicity (p<0.05). There were no alterations in the kidney between the groups (p>0.05). QT has potential to support MX therapy, showing anti-inflammatory and hepatoprotective effects in this model.

Milk Kefir therapy reduces inflammation and alveolar bone loss on periodontitis in rats.

Periodontitis is a chronic inflammatory disease that affects the tooth-supporting tissues. This study evaluated the anti-inflammatory and antiresorptive effects of milk kefir (MK) on periodontitis in rats. Micro-Raman spectroscopy was performed on MK at different fermentation times to verify the presence of Lactobacillus kefiri. From these results, Wistar rats were divided into the following groups: C (Control); EP (experimental periodontitis); K1 (animals that received MK with one day of fermentation); K1+EP; K4 (animals without EP using MK with four days of fermentation) and K4+EP. MK was administered 28 days before EP induction and during the disease development period (11 days). On day 28, in the EP groups, periodontitis was induced. The animals were euthanized on day 39. The hemimaxillae were removed and the following parameters were evaluated: micro-Raman analysis of the presence of inflammation; histomorphometric analysis to quantify alveolar bone loss and immunohistochemistry for IL-6, TNF-α, IL-Iß and IL-10 in the periodontal ligament. Micro-Raman analysis showed that four days fermentation MK has a higher intensity spectrum of L. kefiri. Furthermore, the administration of this probiotic reduced the intensity of the inflammation spectrum when compared to one day fermentation MK. It was observed that the animals from the K4+EP group showed significant reduction of alveolar bone loss, as well as a lower IL-6, TNF-α and IL-Iß immunoexpression and a higher IL-10 immunoexpression, when compared to EP groups. We conclude that MK has anti-inflammatory and antiresorptive effects on periodontitis in rats and that these effects are fermentation time dependent.

The effect of high concentration of zoledronic acid on tooth induced movement and its repercussion on root, periodontal ligament and alveolar bone tissues in rats.

Zoledronic acid (ZA) is often prescribed for osteoporosis or resorptive metabolic bone disease. This study aims to evaluate the effect of ZA on orthodontic tooth movement (OTM) and root and bone resorption and its repercussion on root, periodontal ligament and alveolar bone tissues. The experimental group consisted of 72 Wistar rats divided in four subgroups: Naive, Saline and Zoledronic Acid groups at the concentration of 0.2 mg/kg [ZA (0.2)] or 1.0 mg/kg [ZA (1.0)]. The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, on days 0, 7, 14 and 42. Under anesthesia, NiTi springs were installed in the first left maxillary molar with 50gf allowing the OTM, except for the negative control group (N) for mesial movement of the left first maxillary teeth. The animals were sacrificed and maxillae were removed for macroscopic and histopathological analyzes, scanning electron microscopy, computerized microtomography and confocal microscopy. Treatment with ZA decreased the OTM and the number of osteoclasts and loss of alveolar bone when compared to the naive and saline groups. Reduction of radicular resorption, increased necrotic areas and reduced vascularization in the periodontal ligament were observed in the ZA groups. ZA interferes with OTM and presents anti-resorptive effects on bone and dental tissues associated with a decreased vascularization, without osteonecrosis.

Role of osteogenic Dickkopf-1 in bone remodeling and bone healing in mice with type I diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is associated with low bone mass and a higher risk for fractures. Dickkopf-1 (Dkk1), which inhibits Wnt signaling, osteoblast function, and bone formation, has been found to be increased in the serum of patients with T1DM. Here, we investigated the functional role of Dkk1 in T1DM-induced bone loss in mice. T1DM was induced in 10-week-old male mice with Dkk1-deficiency in late osteoblasts/osteocytes (Dkk1f/f;Dmp1-Cre, cKO) and littermate control mice by 5 subsequent injections of streptozotocin (40 mg/kg). Age-matched, non-diabetic control groups received citrate buffer instead. At week 12, calvarial defects were created in subgroups of each cohort. After a total of 16 weeks, weight, fat, the femoral bone phenotype and the area of the bone defect were analyzed using µCT and dynamic histomorphometry. During the experiment, diabetic WT and cKO mice did not gain body weight compared to control mice. Further they lost their perigonadal and subcutaneous fat pads. Diabetic mice had highly elevated serum glucose levels and impaired glucose tolerance, regardless of their Dkk1 levels. T1DM led to a 36% decrease in trabecular bone volume in Cre- negative control animals, whereas Dkk1 cKO mice only lost 16%. Of note, Dkk1 cKO mice were completely protected from T1DM-induced cortical bone loss. T1DM suppressed the bone formation rate, the number of osteoblasts at trabecular bone, serum levels of P1NP and bone defect healing in both, Dkk1-deficient and sufficient, mice. This may be explained by increased serum sclerostin levels in both genotypes and the strict dependence on bone formation for bone defect healing. In contrast, the number of osteoclasts and TRACP 5b serum levels only increased in diabetic control mice, but not in Dkk1 cKO mice. In summary, Dkk1 derived from osteogenic cells does not influence the development of T1DM but plays a crucial role in T1DM-induced bone loss in male mice by regulating osteoclast numbers.

Bisphosphonate-related osteonecrosis induced change in alveolar bone architecture in rats with participation of Wnt signaling.

OBJECTIVE: This work aimed to study the role of inflammation in medication-related osteonecrosis of the jaw (MRONJ) in rats with focus on Wnt signaling. METHODS: A total of 36 female Wistar rats (12 weeks ± 200 g) were divided into 2 groups (n = 6) in 3 experiments: saline (SAL) and zoledronic acid (ZOL). For MRONJ induction, rats received 0.1 mg/kg of ZOL (ip) 3×/week for 9 weeks. Animals from the SAL group received 0.1 mg/kg of 0.9% SAL, ip 3×/week for 9 weeks. On the 8th week, 3 left upper molars were extracted, and on the 11th week, they were euthanized. Maxillae were evaluated by macroscopic and histopathological analyses; scanning electron microscopy (SEM); immunohistochemistry for DKK-1, Wnt 10b, and caspase-3; and Raman spectrometry. Gingiva was also collected for TNF-α e IL-1ß quantification. RESULTS: Bone necrosis was confirmed by healing impairment, reduced number of viable osteocytes, increased caspase-3 immunoexpression, and increased number of empty lacunae (p < 0.05). ZOL enhanced inflammation and increased gingival levels of IL-1ß and TNF-α (p < 0.05). Irregular indentations were seen on bone after ZOL administration. Bone necrosis was marked by reduced amount of total and type I collagen. ZOL reduced the mineral/matrix ratio and increased carbonate/phosphate ratio. It was observed a significant reduction on Wnt10b and beta-catenin immunolabeling in the bone tissue of ZOL group. CONCLUSION: In summary, MRONJ model caused bone necrosis due to intense inflammation. Wnt signaling seems to play an important role in this process. CLINICAL RELEVANCE: New therapeutic strategies focusing on Wnt pathway can provide an interesting approach for future treatments.

Immunoexpression of canonical Wnt and NF-κB signaling pathways in the temporomandibular joint of arthritic rats.

OBJECTIVE: To investigate the participation of canonical Wnt and NF-κB signaling pathways in an experimental model of chronic arthritis induced by methylated bovine serum albumin (mBSA) in rat temporomandibular joint (TMJ). MATERIALS AND METHODS: Wistar rats were sensitized by mBSA+Complete Freund Adjuvant (CFA)/Incomplete Freund Adjuvant (IFA) on the first 14 days (1 ×/week). Subsequently, they received 1, 2 or 3 mBSA or saline solution injections into the TMJ (1 ×/week). Hypernociceptive threshold was assessed during the whole experimental period. 24 h after the mBSA injections, the TMJs were removed for histopathological and immunohistochemical analyses for TNF-α, IL-1ß, NF-κB, RANKL, Wnt-10b, ß-catenin and DKK1. RESULTS: The nociceptive threshold was significantly reduced after mBSA injections. An inflammatory infiltrate and thickening of the synovial membrane were observed only after mBSA booster injections. Immunolabeling of TNF-α, IL-1ß and Wnt-10b was increased in the synovial membrane in arthritic groups. The immunoexpression of nuclear ß-catenin was significantly higher only in the group that received 2 booster TMJ injections. However, NF-κB, RANKL and DKK1 immunoexpression were increased only in animals with 3 mBSA intra-articular injections. CONCLUSION: Our results suggest that canonical Wnt and NF-κB signaling pathways participate in the hypernociception and inflammatory response in TMJ synovial membrane during the development of rheumatoid arthritis in rats.

Loss of Dkk-1 in Osteocytes Mitigates Alveolar Bone Loss in Mice With Periodontitis.

Background: Periodontitis is a highly prevalent infection-triggered inflammatory disease that results in bone loss. Inflammation causes bone resorption by osteoclasts, and also by suppression of bone formation via increase of Dickkopf-1 (Dkk-1), an inhibitor of Wnt signaling. Here, we tested the hypothesis that osteocytic Dkk-1 is a key factor in the pathogenesis of periodontitis-induced alveolar bone loss (ABL). Methods: Twelve-week-old female mice with a constitutive deletion of Dkk-1 specifically in osteocytes (Dkk-1fl/fl;Dmp1:Cre) were subjected to experimental periodontitis (EP). Cre-negative littermates served as controls. EP was induced by placing a ligature around the upper 2nd left molar, the contralateral side was used as control. Mice were killed after 11 days and maxillae removed for micro-CT and histological analyses. The mRNA expression of Dkk-1, Runx2, Osteocalcin, OPG, RANKL, RANKL/OPG ratio, LEF-1, and TCF-7 were assessed in maxillae, while mRNA expressions of TNF and IL-1 were evaluated on gingiva using real-time PCR. Blood samples were collected for Dkk-1, CTX, and P1NP measurement by ELISA. Results: The deletion of Dkk-1 in osteocytes prevented ABL in mice with EP, compared to Cre-negative control mice with EP. Micro-CT analysis showed a significant reduction of bone loss (-28.5%) in EP Dkk-1fl/fl;Dmp1:Cre-positive mice compared to their littermate controls. These mice showed a greater alveolar bone volume, bone mineral density, trabecular number, and trabecular thickness after EP when compared to the Cre-negative controls. The local expression in maxillae as well as the serum levels of Dkk-1 were reduced in Dkk-1fl/fl;Dmp1:Cre-positive mice with EP. The transgenic mice submitted to EP showed increase of P1NP and reduction of CTX-I serum levels, and increase of TCF-7 expression. Histological analysis displayed less inflammatory infiltrates, a reduction of TNF and IL-1 expressions in the gingiva and fewer osteoclasts in Cre-positive animals with EP. Moreover, in mice with EP, the osteocytic deletion of Dkk-1 enhanced bone formation due to increased expressions of Runx2 and Osteocalcin and decreased expression of RANKL in maxillae. Conclusion: In summary, Dkk-1 derived from osteocytes plays a crucial role in ABL in periodontitis.

Tocoyena sellowiana extract decreases bone loss in an experimental model of periodontitis in rats: Putative role for cyclooxygenase-2 and IL-1? inhibition.

Tocoyena sellowiana (Cham. & Schltdl.) K.Schum is one of the most important families of Brazilian medicinal plants. This study aimed to evaluate the effect of Tocoyena sellowiana (Cham. & Schltdl.) K.Schum ethanolic extract in a pre-clinical trial of periodontitis and to investigate possible mechanisms underlying such effects. Periodontitis was induced in Wistar rats by placing a nylon thread ligature around second upper left molars for 11 days. Rats received (per os) Tocoyena sellowiana (0.1, 1 or 10?mg?kg) or vehicle 1?h before ligature and daily until day 11. Macroscopic, histopathological, and COX-2 immunohistochemical analyses were performed to evaluate the periodontium. The gingival tissue was used to quantify the myeloperoxidase (MPO) activity and interleukin (IL)-1? levels by ELISA. Blood samples were collected to evaluate bone-specific alkaline phosphatase (BALP), the dosage of creatinine, aspartate and alanine transaminases. The liver, kidneys, spleen, and body mass variations were also evaluated. Tocoyena sellowiana decreased bone loss, reduced MPO, IL-1? levels as well as COX-2 immunostaining, and increased BALP activity. Moreover, Tocoyena sellowiana did not alter organs nor body weight. Tocoyena sellowiana reduced bone loss in rats and its efficacy was at least partially dependent upon both IL-1? and cyclooxygenase-2 inhibition.

Anti-inflammatory and antiresorptive effects of Calendula officinalis on inflammatory bone loss in rats.

OBJECTIVE: The aim of this work was to evaluate the anti-inflammatory and antiresorptive effects of Calendula officinalis (CLO) on alveolar bone loss (ABL) in rats. MATERIAL AND METHODS: Male Wistar rats were subjected to ABL by ligature with nylon thread around the second upper left molar. The contralateral hemimaxillae were used as control. Rats received saline solution (SAL) or CLO (10, 30, or 90 mg/kg) 30 min before ligature and daily until the 11th day. The maxillae were removed and prepared for macroscopic, radiographic, micro-tomographic, histopathologic, histometric analysis, and immunohistochemical localization of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). The gingival tissues were used to quantify the myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-α), and interleukin-1ß (IL-1ß) concentrations by ELISA. Blood samples were collected for leukogram and to evaluate the bone-specific alkaline phosphatase (BALP) activity and serum levels of aspartate and alanine transaminases (AST/ALT). RESULTS: The bone loss induced by 11 days of ligature induced bone loss, reduced levels of BALP, leukocyte infiltration, increased MPO activity, gingival concentrations of TNF-α and IL-1ß, and RANKL while reduced OPG immunoexpressions in the periodontal tissue and leukocytosis. Of the CLO, 90 mg/kg reduced bone loss, neutrophilia, the levels of pro-inflammatory mediators, and RANKL expression, while it increased OPG immunopositive cells and BALP serum levels, when compared to SAL. CLO did not affect either kidney or liver function, indicated by serum AST/ALT levels. CONCLUSION: The present data suggests that CLO reduced inflammatory bone resorption in experimental periodontitis, which may be mediated by its anti-inflammatory properties and its effects on bone metabolism. CLINICAL RELEVANCE: CLO can be a potential therapeutical adjuvant in the treatment of periodontitis.

Stemodia maritima L. Extract Decreases Inflammation, Oxidative Stress, and Alveolar Bone Loss in an Experimental Periodontitis Rat Model.

Periodontitis is very prevalent worldwide and is one of the major causes of tooth loss in adults. About 80% of the worldwide population use medicinal plants for their health care. Stemodia maritima L. (S. maritima) antioxidant and antimicrobial effects in vitro as well as anti-inflammatory properties. Herein, the potential therapeutic effect of S. maritima was assessed in rats subjected to experimental periodontitis (EP). EP was induced in female Wistar rats by nylon thread ligature around 2nd upper left molars for 11 days. Animals received (per os) S. maritima (0.2; 1 or 5 mg/kg) or vehicle (saline + DMSO) 1 h before ligature and then once daily for 11 days. The naive group had no manipulation. After this time-point, the animals were terminally anesthetized, and the maxillae were removed for morphometric and histological analyzes (HE). Gingival tissues were dissected to cytokine levels detection (TNF-α, IL1-ß, CINC-1, and IL-10), enzymes superoxide dismutase (SOD), and catalase (CAT) analysis, as well as gene expression (TNF-α, IL-1ß, RANK, and iNOS) by qRT-PCR. Systemic parameters (weight variation, plasma levels of hepatic enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT), creatinine, total alkaline phosphatase (TALP), and bone alkaline phosphatase (BALP) were performed. Histological analysis of the stomach, liver, kidney, and heart was also performed. S. maritima (5 mg/kg) decreased alveolar bone loss, TNF-α and CINC-1 gingival levels, oxidative stress, and transcription of TNF-α, IL1-ß, RANK, and iNOS genes. It elevated both BALP activity and IL-10 gingival levels. The animals showed no any signs of toxicity. In conclusion, S. maritima reduced pro-inflammatory cytokine production, oxidative stress, and alveolar bone loss in a pre-clinical trial of periodontitis. S. maritima is a potential tool for controlling the development of periodontitis.

The Effect of Calendula officinalis on Oxidative Stress and Bone Loss in Experimental Periodontitis.

Periodontitis is associated with reduced antioxidant capacity and increased oxidative damage. Oxidative stress induces inflammation and bone loss contributing to the pathological progression of periodontal disease. Calendula officinalis (CLO) has demonstrated anti-inflammatory and anti-oxidant activities. Therefore, the aim of this study was to evaluate the effect of CLO on oxidative stress and bone loss in rats subjected to experimental periodontitis (EP). For this, 72 male Wistar rats were divided into groups: Naïve, Saline (SAL) and CLO. Rats received SAL or CLO (90 mg/kg) 30 min before ligature and daily until the 11th day. Naïve group experienced no manipulation. After 11 days, the animals were euthanized and left maxillae collected for macroscopic analysis of alveolar bone loss (ABL). Periodontium was analyzed by macroscopy, scanning electron microscopy; confocal and light polarized microscopy. Immunohistochemical examination of DKK1, WNT 10b and ß-catenin was performed. The gingival tissue was collected to reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) analyses. The 11 days of ligature induced bone loss, breakdown of collagen fibers, increased the immunostaining DKK-1 while reduced WNT 10b and ß-catenin expressions. Periodontitis reduced GSH, SOD, CAT and increase MDA. All findings were reversed by 90 mg/kg of CLO. In summary our findings demonstrated that CLO reduced oxidative stress and bone loss and preserved collagen fibers in rats with EP, with participation of WNT signaling pathway.

Effects of glucocorticoid-induced osteoporosis on bone tissue of rats with experimental periodontitis.

OBJECTIVE: To evaluate the effects of osteoporosis induced by glucocorticoid (GIOP) on bone tissue of rats with experimental periodontitis (EP). DESIGN: 48 male Wistar rats divided into groups: Naïve, EP, GIOP and GIOP+EP. Rats of GIOP and GIOP+EP groups received 7mg/kg of dexamethasone intramuscularly once a week for 5 weeks. Following, EP and GIOP+EP groups were subjected to ligature-induced periodontitis. Naïve group experienced no manipulation. After 11 days, the animals were euthanized and left maxillae collected for macroscopic, radiographic, micro-tomographic and microscopic analysis of alveolar bone loss (ABL). Blood samples were collected for determination of bone-specific alkaline phosphatase (BALP) levels and the right femurs were removed for radiographic and biomechanical analysis. RESULTS: EP caused ABL and reduced BALP levels (p<0,05), but it did not change the architecture or biomechanics of femur, compared to Naïve. GIOP did not cause ABL, but it significantly decreased alveolar bone mineral density (ABMD), bone percentage and trabecular thickness (Tb.Th) and increased alveolar bone porosity (p<0.05) and significantly reduced BALP serum levels, as well as radiographic density and Young's module of femur, compared to Naïve. There was a greater ABL in group GIOP+EP when compared to EP (p<0.05). GIOP+EP caused a greater decrease on ABMD, Tb.Th, bone percentage and increased bone porosity (p<0.05) and also presented a significant reduction in BALP levels (p<0.05), in radiographic density and in Young's module of femur compared to EP (p<0.05). CONCLUSIONS: GIOP can potentiate the destructive effects of EP on alveolar bone and alter the systemic bone loss, by promoting bone resorption and reducing osteoblast activity.

Clinical efficacy of a 1% Matricaria chamomile L. mouthwash and 0.12% chlorhexidine for gingivitis control in patients undergoing orthodontic treatment with fixed appliances.

This pilot study evaluated the clinical efficacy of a mouthwash containing 1% Matricaria chamomilla L. (MTC) extract in reducing gingival inflammation and plaque formation in patients undergoing orthodontic treatment with fixed appliances. This randomized, double-blind, placebo-controlled study enrolled a total of 30 males and females (age, 10-40 years) with fixed orthodontic appliances and a minimum of 20 natural teeth. The participants were allocated to three groups (n = 10 each) and asked to rinse with 15 mL of a placebo, 0.12% chlorhexidine (CHX), or 1% MTC mouthwash, immediately after brushing for 1 min, in the morning and evening, for 15 days. Data (mean ± SD) on visible plaque index (VPI) and gingival bleeding index (GBI) were recorded on days 1 and 15. The placebo group exhibited increases in VPI and GBI (10.2% and 23.1%, respectively) from day 1 to day 15. As compared with placebo, VPI and GBI significantly decreased in the MTC group (-25.6% and -29.9%, respectively) and the CHX group (-39.9% and -32.0%, respectively). In summary, MTC reduced biofilm accumulation and gingival bleeding in patients with gingivitis, probably because of its antimicrobial and anti-inflammatory activities.(J Oral Sci 58, 569-574, 2016).

Effects of Atorvastatin on Periodontitis of Rats Subjected to Glucocorticoid-Induced Osteoporosis.

BACKGROUND: Atorvastatin (ATV) has shown pleiotropic effects on bone tissue, and osteoporosis can aggravate periodontitis. Thus, the effects of ATV on experimental periodontitis (EP) in rats subjected to glucocorticoid-induced osteoporosis (GIOP) was assessed. METHODS: Male Wistar rats were divided into the following groups: 1) naive; 2) EP; 3) GIOP + EP; and 4) ATV. Groups GIOP + EP and ATV received 7 mg/kg dexamethasone intramuscularly once per week for 5 weeks, and the others received saline (SAL). Groups EP, GIOP + EP, and ATV were submitted to EP by ligature around the maxillary left second molars for 11 days. Group ATV received 27 mg/kg ATV orally, and the others received SAL 30 minutes before EP. Periodontium was analyzed by macroscopy, microtomography, and histopathology; by immunohistochemical examination of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), wingless (WNT) 10b, dickkopf-related protein 1 (DKK-1), and ß-catenin; and by enzyme-linked immunosorbent assay analysis of myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL10, reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). Leukogram, liver and kidney enzymes, and bone-specific alkaline phosphatase (BALP) serum levels were evaluated. RESULTS: ATV decreased bone loss, reduced MPO, TNF-α, IL-1ß, IL-6, and IL-8, and increased IL-10, GSH, SOD, and CAT levels. ATV reduced RANKL and DKK-1 and increased OPG, WNT10b, and ß-catenin expressions and BALP activity. CONCLUSION: ATV reduced inflammation, oxidative stress, and bone loss in rats with EP and GIOP, with participation of the WNT signaling pathway.