Selective inhibition of interleukin 6 receptor decreased inflammatory cytokines and increased proteases in an experimental model of critical calvarial defect.

Considering the lack of consensus related to the impact of selective IL-6 receptor inhibition on bone remodeling and the scarcity of reports, especially on large bone defects, this study proposed to evaluate the biological impact of the selective inhibitor of interleukin-6 receptor (tocilizumab) in an experimental model of critical calvarial defect in rats. In this preclinical and in vivo study, 24 male Wistar rats were randomly divided into two groups (n=12/group): defect treated with collagen sponge (CG) and defect treated with collagen sponge associated with 2 mg/kg tocilizumab (TCZ). The defect in the parietal bone was created using an 8-mm diameter trephine drill. After 90 days, the animals were euthanized, and tissue samples (skull caps) were evaluated through micro-CT, histological, immunohistochemistry, cytokines, and RT-qPCR analyses. Tocilizumab reduced mononuclear inflammatory infiltration (P<0.05) and tumor necrosis factor (TNF)-α levels (P<0.01) and down-regulated tissue gene expression of BMP-2 (P<0.001), RUNX-2 (P<0.05), and interleukin (IL)-6 (P<0.05). Moreover, it promoted a stronger immunostaining of cathepsin and RANKL (P<0.05). Micro-CT and histological analyses revealed no impact on general bone formation (P>0.05). The bone cells (osteoblasts, osteoclasts, and osteocytes) in the defect area were similar in both groups (P>0.05). Tocilizumab reduced inflammatory cytokines, decreased osteogenic protein, and increased proteases in a critical bone defect in rats. Ninety days after the local application of tocilizumab in the cranial defect, we did not find a significant formation of bone tissue compared with a collagen sponge.

Glutamine and alanyl-glutamine accelerate the recovery from 5-fluorouracil-induced experimental oral mucositis in hamster.

INTRODUCTION: Mucositis induced by anti-neoplastic drugs is an important, dose-limiting and costly side effect of cancer therapy. AIM: To evaluate the effect of oral glutamine and alanyl-glutamine, a more stable glutamine derivative, on 5-FU-induced oral mucositis in hamsters. MATERIALS AND METHODS: Oral mucositis was induced by two intraperitoneal (i.p) administrations of 5-FU on the first and second days of the experiment (60 and 40 mg/kg, respectively) followed by mechanical trauma on the fourth day in male hamsters. Animals received saline, glutamine or alanyl-glutamine suspension (100 mM) 1 h before the injections of 5-FU and daily until sacrifice, on the 10th or 14th day. Macroscopic and histopathological analyses were evaluated and graded. Tissues from the cheek pouches were harvested for measurement of myeloperoxidase activity and glutathione stores. For investigation of serum concentration of glutamine, blood was obtained by heart puncture from anesthetized animals before sacrifice, on day 10. RESULTS: Treatment with glutamine and alanyl-glutamine reduced macroscopic and histological parameters of oral mucositis, and reduced the myeloperoxidase activity on day 14, but not on day 10. The 5-FU-induced oral mucositis significantly decreased the serum glutamine levels as well as the cheek pouch glutathione stores observed on day 10. Glutamine or alanyl-glutamine administration reversed the 5-FU effects, restoring serum glutamine levels and cheek pouch glutathione stores, observed on day 10, but did not prevent oral mucositis on the tenth day. CONCLUSION: Glutamine or alanyl-glutamine accelerated the mucosal recovery increasing mucosal tissue glutathione stores, reducing inflammatory parameters and speeding reepithelization.

Role of nitric oxide on pathogenesis of 5-fluorouracil induced experimental oral mucositis in hamster.

INTRODUCTION: Mucositis induced by antineoplastic drugs is an important, dose-limiting, and costly side effect of cancer therapy. AIM: To investigate the role of nitric oxide (NO) on the pathogenesis of 5-fluorouracil (5-FU)-induced oral mucositis. MATERIALS AND METHODS: Oral mucositis was induced by two intraperitoneal (i.p) administrations of 5-FU on the first and second days of the experiment (60 and 40 mg/kg, respectively) in male hamsters. Animals were treated subcutaneously with saline (0.4 ml), 1,400 W (1 mg/kg), aminoguanidine (5 or 10 mg/kg) or Nphi-Nitro-L-Arginine Methyl Ester (L-NAME) (5, 10, or 20 mg/kg) 1 h before the injections of 5-FU and daily until sacrifice, on the tenth day. Macroscopic and histopathological analyses were evaluated and graded. Tissues from the cheek pouches were harvested for measurement of myeloperoxidase (MPO) activity, nitrite level, and immunohistochemistry for induced nitric oxide synthase (iNOS). RESULTS: Treatment with 1,400 W or aminoguanidine reduced macroscopic and histological parameters of oral mucositis, and reduced the inflammatory cell infiltration as detected by histopathology and by MPO activity. In contrast, the administration of L-NAME did not significantly reverse the inflammatory alterations induced by experimental mucositis. Increased NOS activity, nitrite level and immunostaining for iNOS were detected on the check pouch tissue of animals submitted to 5-FU-induced oral mucositis on the tenth day. CONCLUSION: These results suggest an important role of NO produced by iNOS in the pathogenesis of oral mucositis induced by 5-FU.

Nitric oxide synthase inhibition prevents alveolar bone resorption in experimental periodontitis in rats.

BACKGROUND: Periodontitis is the most frequent cause of tooth loss in adults. Nitric oxide (NO) has been linked to bone resorption mechanisms during inflammation processes. The aim of this study was to investigate the effect of NOS (NO synthase) inhibitors in the alveolar bone loss in an experimental periodontitis disease (EPD) model. METHODS: Wistar rats were subjected to a ligature placement around the second upper left molars and were sacrificed at 11 days. Alveolar bone loss was evaluated by the sum of distances between the cusp tips and the alveolar bone along the axis of each molar root, subtracting from the contralateral side. Histopathological analysis was based on cell influx, alveolar bone, and cementum integrity. Leukogram was performed at 6 hours and 1, 7, and 11 days after the EPD induction. Groups were treated with the NOS inhibitors, aminoguanidine (AG) (2.5 to 10 mg/kg/d), or L-arginine methyl ester (L-NAME, 5 to 20 mg/kg/d) intraperitoneally (i.p.), 1 hour before the EPD induction and daily for 11 days. Controls received only saline (EPD group). As controls for L-NAME specificity, groups were co-treated with either L-arginine (150 to 600 mg/kg/d) or D-arginine (600 mg/kg/d) and L-NAME (20 mg/kg/d). Different groups were used for morphometric and histopathological analysis. RESULTS: Both L-NAME and AG significantly and dose-dependently inhibited the alveolar bone loss as compared to EPD group. L-NAME (20 mg/kg/d) reduced the alveolar bone loss by 50%, whereas AG (5 mg/kg/d) reduced it by 47% compared to EPD. This result was coupled to a significant reduction of cell influx to the periodontium, as well as to the preservation of alveolar bone and cementum, seen at histopathology, for both compounds. The co-administration of L-arginine, but not of D-arginine reversed L-NAME effects. CONCLUSION: These data provide evidence that NOS inhibitors prevent inflammatory bone resorption in experimental periodontitis.

Locally applied isosorbide decreases bone resorption in experimental periodontitis in rats.

BACKGROUND: The role of nitric oxide (NO) on bone metabolism is controversial, since it can either stimulate bone formation or resorption. We investigated the effect of local administration of the NO donor isosorbide in an experimental periodontal disease model. METHODS: Wistar rats were subjected to a ligature placement around the cervix of the right second upper molar and were sacrificed after 11 days. Alveolar bone loss was measured in one quadrant as the sum of the distances between the cuspid tip and the alveolar bone along the axis of each molar root, which was subtracted from the contralateral side, used as unligated control. The semiquantitative histopathological scale of the periodontium was based on cell infiltration and alveolar bone and cementum integrity. Groups were treated with a gel containing 1% or 5% isosorbide applied to the vestibular side of the molar gingiva 1 hour before the placement of the ligature and then twice daily until sacrifice. Controls included one group subjected to periodontitis and no treatment (NT) and another that received the gel containing just the vehicle (V). RESULTS: The application of the vehicle gel produced an increase of the alveolar bone resorption, without altering the inflammatory changes, compared to the NT group. The 5% isosorbide produced a significant reduction of the alveolar bone resorption, compared to V and NT. This reduction was confirmed by histological analysis, showing less inflammatory cell infiltration and preservation of the cementum and the alveolar process. CONCLUSION: Local application of isosorbide reduces alveolar bone resorption in experimental periodontal disease in rats, suggesting a local anti-inflammatory effect of isosorbide.

Effect of atorvastatin on 5-fluorouracil-induced experimental oral mucositis.

PURPOSE: Oral mucositis (OM) is a frequent side effect in patients with cancer. We investigate the effect of atorvastatin (ATV), a cholesterol-lowering drug, on OM induced by 5-fluorouracil (5-FU) in hamsters. METHODS: OM was induced by the i.p. administration of 5-FU, with excoriations of the cheek pouch mucosa. The animals were pretreated with i.p. ATV 1, 5 or 10 mg/kg or vehicle (saline and 5% (vol/vol) ethanol) 30 min before 5-FU injection and daily for 5 or 10 days. Samples of cheek pouches and main organs were removed for histopathological analysis, determination of TNF-α, IL-1ß, nitrite, non-protein sulfhydryl group (NP-SH) levels, myeloperoxidase (MPO) assay and immunohistochemistry for induced nitric oxide synthase (iNOS). Blood was collected for a leukogram analysis of biochemical parameters and analysis of bacteremia. RESULTS: ATV at doses of 1 and 5 mg/kg reduced mucosal damage and inflammation, as well as the levels of cytokines, nitrite and myeloperoxidase activity on the 5th and 10th day of OM and immunostaining for iNOS on the 5th day of OM.ATV at 1 mg/kg increased cheek pouch NP-SH when compared to 5-FU groups on the 10th day of OM. The association between ATV 5 mg/kg and 5-FU decreased the survival rate, amplified the leukopenia of animals, increased transaminase serum levels and caused liver lesions. We also detected the presence of Gram-negative bacillus in the blood of 100% of the animals treated with ATV 5 mg/kg + 5-FU. CONCLUSIONS: Atorvastatin prevented mucosal damage and inflammation associated with 5-FU-induced OM, but the association of a higher dose of ATV with 5-FU induced hepatotoxicity and amplified leukopenia.

Polyanionic collagen membranes for guided tissue regeneration: Effect of progressive glutaraldehyde cross-linking on biocompatibility and degradation.

The ultimate goal of periodontal therapy is to control periodontal tissue inflammation and to produce predictable regeneration of that part of the periodontium which has been lost as a result of periodontal disease. In guided tissue regeneration membranes function as mechanical barriers, excluding the epithelium and gingival corium from the root surface and allowing regeneration by periodontal ligament cells. This report aims to study the effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen (PAC) membranes by conducting a histological evaluation of the tissue response (biocompatibility) and by assessing the biodegradation of subcutaneous membrane implants in rats. We studied six different samples: a PAC, a PAC mineralized by alternate soaking processes for either 25 or 75 cycles (PAC 25 and PAC 75, respectively) and these films cross-linked by GA. Inflammatory infiltrate, cytokine dosage, fibrosis capsule thickness, metalloproteinase immunohistochemistry and membrane biodegradation after 1, 7, 15 and 30 days were measured. The inflammatory response was found to be more intense in membranes without cross-linking, while the fibrosis capsules became thicker in cross-linked membranes after 30 days. The membranes without cross-linking suffered intense biodegradation, while the membranes with cross-linking remained intact after 30 days. The cross-linking with GA reduced the inflammatory response and prevented degradation of the membranes over the entire course of the observation period. These membranes are thus an attractive option when the production of new bone depends on the prolonged presence of a mechanical barrier.