Chalcone T4, a novel chalconic compound, inhibits inflammatory bone resorption in vivo and suppresses osteoclastogenesis in vitro.

OBJECTIVE: This study aimed to assess the effect of a novel synthetic chalcone, Chalcone T4, on a murine model of periodontitis and on RANKL-induced osteoclastogenesis in vitro. BACKGROUND: Chalcones are natural compounds with anti-inflammatory properties, and its synthetic analogs with enhanced biological effects have potential as therapeutic agents. Periodontitis is characterized by chronic inflammation of the periodontium and alveolar bone resorption. Safe and effective anti-inflammatory agents can have an important additive effect in the treatment in this disease. METHODS: Periodontitis was induced via the installation of a ligature around the first molar. Rats (n = 32) received Chalcone T4 (5 and 50 mg/kg) or distilled water by gavage daily for 15 days. Outcomes assessed were bone resorption (µCT), TNF-α production (ELISA), cellular infiltrate, and collagen content (stereometric analysis, CD45+ cells by immunohistochemistry), and activation of NFATc1 and NF-kB (immunohistochemistry). In vitro, RAW 264.7 were treated with Chalcone T4 and stimulated with RANKL for assessment of osteoclast differentiation (actin ring staining) and activity (pit assay). RESULTS: Chalcone T4 significantly reduced periodontitis-associated bone resorption, as well as the cellular infiltrate, while increasing the collagen content. Production of TNF-α, infiltration of CD45-positive cells, and NF-kB activation were markedly reduced. In vitro, chalcone T4 inhibited both osteoclast differentiation and activity. CONCLUSION: Chalcone T4 significantly inhibited alveolar bone resorption and inflammation in vivo and RANKL-induced osteoclastogenesis in vitro, suggesting a therapeutic role for this compound in the treatment of periodontitis.

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes.

Molecular mechanisms responsible for periodontal disease (PD) and its worsening in type 1 Diabetes Mellitus (DM1) remain unknown. Cytokine profile and expression levels of collagenases, Mmp14, and tissue inhibitors were determined, as were the numbers of neutrophils and macrophages in combined streptozotocin-induced DM1 and ligature-induced PD models. Increased IL-23 (80-fold) and Mmp8 expression (25-fold) was found in DM1. Ligature resulted in an IL-1ß/IL-6 profile, increased expression of Mmp8, Mmp13, and Mmp14 (but not Mmp1), and transient expression of Timp1 and Reck in non-diabetics. PD in DM1 involved IL-1ß (but not IL-6) and IL-23/IL-17, reduced IL-6 and IL-10, sustained Mmp8 and Mmp14, increased Mmp13 and reduced Reck expression in association with 20-fold higher counts of neutrophils and macrophages. IL-23 and Mmp8 expression are hallmarks of DM1. In association with the IL-1/IL-6 (Th1) response in PD, one found a secondary IL-17 (Th17) pathway in non-diabetic rats. Low IL-6/TNF-α suggest that the Th1 response was compromised in DM1, while IL-17 indicates a prevalence of the Th17 pathway, resulting in high neutrophil recruitment. Mmp8, Mmp13, and Mmp14 expression seems important in the tissue destruction during PD in DM1. PD-associated IL-1/IL-6 (Th1), IL-10, and Reck expression are associated with the acute-to-chronic inflammation transition, which is lost in DM1. In conclusion, IL-23/IL-17 are associated with the PD progression in DM1.

Periodontal disease-associated compensatory expression of osteoprotegerin is lost in type 1 diabetes mellitus and correlates with alveolar bone destruction by regulating osteoclastogenesis.

Alveolar bone resorption results from the inflammatory response to periodontal pathogens. Systemic diseases that affect the host response, such as type 1 diabetes mellitus (DM1), can potentiate the severity of periodontal disease (PD) and accelerate bone resorption. However, the biological mechanisms by which DM1 modulates PD are not fully understood. The aim of this study was to determine the influence of DM1 on alveolar bone resorption and to evaluate the role of receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) in osteoclastogenesis in rats. PD was induced by means of ligature in nondiabetic and in streptozotocyn-induced DM1 rats. Morphological and morphometric analyses, stereology and osteoclast counting were performed. RANKL and OPG mRNA levels, protein content, and location were determined. PD caused alveolar bone resorption, increased the number of osteoclasts in the alveolar bone crest and also promoted changes in RANKL/OPG mRNA expression. DM1 alone showed alveolar bone destruction and an increased number of osteoclasts at the periapical and furcal regions. DM1 exacerbated these characteristics, with a greater impact on bone structure, resulting in a low OPG content and a higher RANKL/OPG ratio, which correlated with prominent osteoclastogenesis. This work demonstrates that the effects of PD and DM1 enhance bone destruction, confirms the importance of the RANKL signaling pathway in bone destruction in DM1 in animal models and suggests the existence of alternative mechanisms potentiating bone degradation in PD.

Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells.

Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.

Relevance of Caspase-1 and Nlrp3 Inflammasome on Inflammatory Bone Resorption in A Murine Model of Periodontitis.

This study investigates the role of NLRP3 inflammasome and its main effector Caspase-1 in inflammation and alveolar bone resorption associated with periodontitis. Heat-killed Aggregatibacter actinomycetemcomitans (Aa) was injected 3x/week (4 weeks) into gingival tissues of wild-type (WT), Nlrp3-KO and Caspase1-KO mice. Bone resorption was measured by µCT and osteoclast number was determined by tartrate-resistant acid phosphatase (TRAP) staining. Inflammation was assessed histologically (H/E staining and immunofluorescence of CD45 and Ly6G). In vitro studies determined the influence of Nlrp3 and Caspase-1 in Rankl-induced osteoclast differentiation and activity and on LPS-induced expression of inflammation-associated genes. Bone resorption was significantly reduced in Casp1-KO but not in Nlrp3-KO mice. Casp1-KO mice had increased in osteoclast numbers, whereas the inflammatory infiltrate or on gene expression were similar to those of WT and Nlrp3-KO mice. Strikingly, osteoclasts differentiated from Nlrp3-deficient macrophages had increased resorbing activity in vitro. LPS-induced expression of Il-10, Il-12 and Tnf-α was significantly reduced in Nlrp3- and Casp1-deficient macrophages. As an inceptive study, these results suggest that Nlrp3 inflammasome does not play a significant role in inflammation and bone resorption in vivo and that Caspase-1 has a pro-resorptive role in experimental periodontal disease.

Functionality of the Interleukin 8 haplotypes in lymphocytes and macrophages in response to gram-negative periodontopathogens.

Individuals carrying the ATC/TTC haplotype (Hap-1) in the interleukin 8 (IL8) gene were reported as more susceptible to chronic periodontitis (CP), an infectious disease associated with Gram-negative bacteria, in comparison to patients with the ATT/TTC haplotype (Hap-2). This study investigated the functionality of the IL8 haplotypes in lymphocytes and monocytes of individuals carrying the Hap-1 or Hap-2 IL8 haplotypes in the response to CP-associated Gram-negative bacteria (periodontopathogens). Peripheral blood was collected from 6 subjects carrying each haplotype, and their immune cells were challenged with periodontopathogens or phorbol 12-myristate 13-acetate (PMA) plus Ionomycin. Depending on the immune cell type (lymphocytes or monocyte-derived macrophages) the assessed outcomes were: phenotypical polarization, gene expression, phagocytic activity, chemotaxis and production of reactive oxygen species (ROS). Subjects carrying the Hap-1 haplotype showed increased expression of IL8 and TNFA and significantly skewing towards pro-inflammatory Th1/M1/Th17 phenotypes. There was increased percentage of ROS-producing monocyte-derived macrophages from individuals carrying the Hap-1 haplotype. Cells from individuals presenting the Hap-2 haplotype had an overall attenuated response to periodontopathogens, with a significant shift towards the Treg phenotype. In conclusion, the IL8 haplotypes showed to be functional both in monocyte-derived macrophages and lymphocytes. The Hap-1 haplotype previously associated with increased susceptibility to CP demonstrated greater skewing to pro-inflammatory Th1/M1/Th17 phenotypes and production of ROS.

The ATC/TTC haplotype in the Interleukin 8 gene in response to Gram-negative bacteria: A pilot study.

OBJECTIVE: The aim of this study was to investigate the functionality of ATC/TTC (Hap-1) and ATT/TTC (Hap-2) Interleukin (IL) 8 gene haplotypes in the response of neutrophils to Gram-negative bacteria associated with periodontitis. DESIGN: Neutrophils were isolated by gradient centrifugation from whole peripheral blood of systemically healthy individuals presenting the two IL8 gene haplotypes. Neutrophils were stimulated with P. gingivalis, A. actinomycetemcomitans and PMA/ionomycin. Cytokine gene expression (RT-qPCR) and migration/chemotaxis (boyden chamber assay) were compared according to the presence of Hap-1 or Hap-2 haplotypes. Protein production was also evaluted in the multiplex assay using the mixed population of leukocytes present in the whole blood from the same individuals. The influence of these two haplotypes on the IL8 promoter activity was assessed in gene-reporter experiments. RESULTS: Hap-1 haplotype in neutrophils and leukocytes exacerbated the response to stimulation with Gram-negative bacteria, with higher levels of TNF-α (mRNA and protein), IL-1ß, IL-2R and IFN-γ (protein) and with increased chemotaxis. Presence of the T allele at the rs4071 polymorphism (alias -251) was associated with increased activity of IL8 proximal promoter. CONCLUSIONS: Neutrophils and leukocytes carrying the Hap-1 haplotype (ATC/TTC) in the IL8 gene present an enhanced response to stimulation with Gram-negative bacteria associated with periodontitis. Presence of the T allele (rs4073) in the IL8 proximal promoter increases transcription activity.

Local administration of curcumin-loaded nanoparticles effectively inhibits inflammation and bone resorption associated with experimental periodontal disease.

There is evidence indicating that curcumin has multiple biological activities, including anti-inflammatory properties. In vitro and in vivo studies demonstrate that curcumin may attenuate inflammation and the connective tissue destruction associated with periodontal disease. Most of these studies use systemic administration, and considering the site-specific nature of periodontal disease and also the poor pharmacodynamic properties of curcumin, we conducted this proof of principle study to assess the biological effect of the local administration of curcumin in a nanoparticle vehicle on experimental periodontal disease. We used 16 rats divided into two groups of 8 animals according to the induction of experimental periodontal disease by bilateral injections of LPS or of the vehicle control directly into the gingival tissues 3×/week for 4 weeks. The same volume of curcumin-loaded nanoparticles or of nanoparticle vehicle was injected into the same sites 2×/week. µCT analysis showed that local administration of curcumin resulted in a complete inhibition of inflammatory bone resorption and in a significant decrease of both osteoclast counts and of the inflammatory infiltrate; as well as a marked attenuation of p38 MAPK and NF-kB activation. We conclude that local administration of curcumin-loaded nanoparticles effectively inhibited inflammation and bone resorption associated with experimental periodontal disease.

Inhibition of 5-lipoxygenase attenuates inflammation and BONE resorption in lipopolysaccharide-induced periodontal disease.

BACKGROUND: Arachidonate-5-lipoxygenase (5-LO) activity and increased leukotriene B4 (LTB4) production have been implicated in various inflammatory conditions. Increased production of leukotrienes has been associated with periodontal diseases; however, their relative contribution to tissue destruction is unknown. In this study, an orally active specific 5-LO inhibitor is used to assess its role in inflammation and bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontal disease. METHODS: Periodontal disease was induced in Balb/c mice by direct injections of LPS into the palatal gingival tissues adjacent to the maxillary first molars three times per week for 4 weeks. Animals were treated with biochemical inhibitor (2 mg/kg/daily) or the same volume of the vehicle by oral gavage. Microcomputed tomography analysis was used to assess bone resorption. Enzyme immunoassay determined LTB4, and enzyme-linked immunosorbent assays quantified tumor necrosis factor (TNF), interleukin (IL)-12, and IL-10 in gingival tissues. Histologic sections were used for the morphometric analysis (number of neutrophils and mononuclear cells). Osteoclasts were counted in tartrate-resistant acid phosphatase-stained sections. RESULTS: Administration of 5-LO inhibitor effectively reduced production of LTB4 (23.7% decrease) and significantly reduced TNF and IL-12 levels in gingival tissues. Moreover, reduction of LTB4 levels in gingival tissues was associated with a significant decrease in bone resorption and a marked reduction in number of osteoclasts and inflammatory cells. CONCLUSION: 5-LO activity plays a relevant role in inflammation and bone resorption associated with the LPS model of experimental periodontal disease.

Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells

Abstract: Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.

The long-term administration of calcineurin inhibitors decreases antioxidant enzyme activity in the rat parotid and submandibular salivary glands.

AIMS: Calcineurin inhibitors are widely used for prevention of graft rejection and treatment of autoimmune disorders, which result in increased longevity and enhanced quality of life for patients. Unfortunately, the toxic side effects of these drugs (mainly renal, hepatic and cardiac) limit their use. In this work, we studied the effects of long-term treatment of rats with the immunosuppressant cyclosporin (CsA) or tacrolimus (Tac) on salivation, saliva composition and on the major salivary glands (parotid and submandibular) in terms of histological alterations and oxidative stress, evaluated as lipoperoxidation (thiobarbituric acid reactive species--TBARS) and antioxidant enzyme activity contents (superoxide dismutase--SOD, catalase--CAT and glutathione peroxidase--GPx). MAIN METHODS: Male adult rats were treated with either CsA (10 mg/kg/day) or Tac (1 mg/kg/day) subcutaneously for 30 or 60 days. At the end of the experimental periods, pilocarpine-stimulated salivary flow rate was measured, saliva samples were collected and the salivary glands were dissected for morphological and biochemical analyses. KEY FINDINGS: After a 60-day treatment with any of the immunosuppressants, the total protein, Ca(2+) and Na(+) saliva concentrations were decreased but salivary flow rates were unaffected. In addition, both parotid and submandibular glands showed decreased SOD, CAT and GPx activities, increased TBARS contents and histomorphological alterations involving the epithelium and acini. SIGNIFICANCE: Based on these results, we suggest that the systemic long-term administration of the calcineurin inhibitor CsA or Tac induces an impairment of the antioxidant enzymatic defense in the rat major salivary glands, which may, in turn, lead to altered saliva composition.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Effect of selective cyclooxygenase-2 inhibition on the development of ligature-induced periodontitis in rats.

BACKGROUND: The purpose of this study was to evaluate the effect of a selective cyclooxygenase-2 inhibitor on the progression of alveolar bone loss in an experimental periodontitis model in rats. METHODS: One hundred eighty (180) Wistar rats were separated into 3 experimental groups. Cotton ligatures were placed at the gingival margin level of lower right first molars. The rats were randomly assigned to one of the following groups that received: a daily oral dose of 10 mg/kg body weight of celecoxib (Ce1); 20 mg/kg body weight of celecoxib (Ce2); or 10 ml/kg of saline solution (C). Serum levels of celecoxib and white blood cell count were determined. Standardized digital radiographs were taken after sacrifice at 3, 5, 10, 18, and 30 days to measure the amount of bone loss around the mesial root surface of the first molar tooth in each rat. RESULTS: Two-way analysis of variance (ANOVA) indicated that groups treated with celecoxib had significantly less bone loss compared to controls (P < 0.0001) and that there was a significant interaction between treatment with celecoxib and time (P < 0.03). Post-hoc comparisons showed that in both groups treated with celecoxib, the bone loss became significant only after 10 days of ligature placement, while in the control group it was already significant after 5 days. However, differences in mean bone loss between control and Ce1 were significant only at 18 days and, between control and Ce2, at 5 and 18 days. There was no significant difference in bone loss among experimental groups at the end of the experimental period. CONCLUSION: These data provide evidence that systemic therapy with celecoxib can modify the progression of experimentally induced periodontitis in rats.

Influence of natural fruit juices in removing the smear layer from root surfaces--an in vitro study.

Certain elements of a patient's diet may be associated with dentin hypersensitivity. The intent of this study was to evaluate the degree of removal of the smear layer from dentin surfaces by various fruit juices. A smear layer was created on extracted human teeth by manual scaling. The roots were reduced and distributed into 8 experimental groups. Distilled water was the negative control. The juices were applied by 2 methods: topical application and topical application with friction. Specimens were photomicrographed and graded according to an index of smear layer removal. With topical application, all but 2 of the tested substances resulted in significantly greater removal of the smear layer and opening of dentinal tubules than was the case with the negative control (p = 0.05); the exceptions were Gala apple and Italian grape juices, which were no different from the control. For the active application (with friction), most substances removed more smear layer than the control (p < 0.05); Gala apple, Italian grape and orange juices were similar to the control. For each of the tested substances, removal of the smear layer did not differ with the method of application (topical vs. friction; p > 0.05). It is concluded that natural fruit juices can remove the smear layer from dentin surfaces, and the efficacy of this removal varies with the type of juice.

Magnesium deficiency results in an increased formation of osteoclasts.

Magnesium (Mg(2+)) deficiency is a frequently occurring disorder that leads to loss of bone mass, abnormal bone growth and skeletal weakness. It is not clear whether Mg(2+) deficiency affects the formation and/or activity of osteoclasts. We evaluated the effect of Mg(2+) restriction on these parameters. Bone marrow cells from long bone and jaw of mice were seeded on plastic and on bone in medium containing different concentrations of Mg(2+) (0.8 mM which is 100% of the normal value, 0.4, 0.08 and 0 mM). The effect of Mg(2+) deficiency was evaluated on osteoclast precursors for their viability after 3 days and proliferation rate after 3 and 6 days, as was mRNA expression of osteoclastogenesis-related genes and Mg(2+)-related genes. After 6 days of incubation, the number of tartrate resistant acid phosphatase-positive (TRACP(+)) multinucleated cells was determined, and the TRACP activity of the medium was measured. Osteoclastic activity was assessed at 8 days by resorption pit analysis. Mg(2+) deficiency resulted in increased numbers of osteoclast-like cells, a phenomenon found for both types of marrow. Mg(2+) deficiency had no effect on cell viability and proliferation. Increased osteoclastogenesis due to Mg(2+) deficiency was reflected in higher expression of osteoclast-related genes. However, resorption per osteoclast and TRACP activity were lower in the absence of Mg(2+). In conclusion, Mg(2+) deficiency augmented osteoclastogenesis but appeared to inhibit the activity of these cells. Together, our in vitro data suggest that altered osteoclast numbers and activity may contribute to the skeletal phenotype as seen in Mg(2+) deficient patients.

Topical application of bFGF on acid-conditioned and non-conditioned dentin: effect on cell proliferation and gene expression in cells relevant for periodontal regeneration

Abstract Periodontal regeneration is still a challenge in terms of predictability and magnitude of effect. In this study we assess the biological effects of combining chemical root conditioning and biological mediators on three relevant cell types for periodontal regeneration. Material and Methods: Bovine dentin slices were conditioned with 25% citric acid followed by topical application of basic fibroblast growth factor (bFGF, 10 and 50 ng). We used ELISA to assess the dynamics of bFGF release from the dentin surface and RT-qPCR to study the expression of Runx2, Col1a1, Bglap and fibronectin by periodontal ligament (PDL) fibroblasts, cementoblasts and bone marrow stromal cells (BMSC) grown onto these dentin slices. We also assessed the effects of topical application of bFGF on cell proliferation by quantification of genomic DNA. Results: Acid conditioning significantly increased the release of bFGF from dentin slices. Overall, bFGF application significantly (p<0.05) increased cell proliferation, except for BMSC grown on non-conditioned dentin slices. Dentin substrate discretely increased expression of Col1a1 in all cell types. Expression of Runx2, Col1a1 and Fn was either unaffected or inhibited by bFGF application in all cell types. We could not detect expression of the target genes on BMSC grown onto conditioned dentin. Conclusion: Acid conditioning of dentin improves the release of topically-applied bFGF. Topical application of bFGF had a stimulatory effect on proliferation of PDL fibroblasts, cementoblasts and BMSC, but did not affect expression of Runx2, Col1a1, Bglap and fibronectin by these cells.

Mineral trioxide aggregate-based endodontic sealer stimulates hydroxyapatite nucleation in human osteoblast-like cell culture.

INTRODUCTION: The main purpose of this study was to evaluate the biocompatibility and bioactivity of a new mineral trioxide aggregate (MTA)-based endodontic sealer, MTA Fillapex (MTA-F; Angelus, Londrina, Brazil), in human cell culture. METHODS: Human osteoblast-like cells (Saos-2) were exposed for 1, 2, 3, and 7 days to MTA-F, Epiphany SE (EP-SE; SybronEndo, Orange, CA), and zinc oxide-eugenol sealer (ZOE). Unexposed cultures were the control group (CT). The viability of the cells was assessed by MTT assay and the morphology by scanning electron microscopy (SEM). The bioactivity of MTA-F was evaluated by alkaline phosphatase activity (ALP) and the detection of calcium deposits in the culture with alizarin red stain (ARS). Energy-dispersive X-ray spectroscopy (EDS) was used to chemically characterize the hydroxyapatite crystallites (HAP). Saos-2 cells were cultured for 21 days for ARS and SEM/EDS. ARS results were expressed as the number of stained nodules per area. Statistical analysis was performed with analysis of variance and Bonferroni tests (P < .01). RESULTS: MTA-F exposure for 1, 2, and 3 days resulted in increased cytotoxicity. In contrast, viability increased after 7 days of exposure to MTA-F. Exposure to EP-SE and ZOE was cytotoxic at all time points. At day 7, ALP activity increase was significant in the MTA-F group. MTA-F presented the highest percentage of ARS-stained nodules (MTA-F > CT > EP-SE > ZOE). SEM/EDS analysis showed hydroxyapatite crystals only in the MTA-F and CT groups. In the MTA-F group, crystallite morphology and chemical composition were different from CT. CONCLUSIONS: After setting, the cytotoxicity of MTA-F decreases and the sealer presents suitable bioactivity to stimulate HAP crystal nucleation.

Lipid peroxidation is associated with the severity of periodontal disease and local inflammatory markers in patients with type 2 diabetes.

CONTEXT: Periodontitis is the most common lytic disease of bone and is recognized as a common complication of diabetes. Lipid peroxidation (LPO) is increased in diabetes and may be related to modulation of the inflammatory response. LPO levels in patients with diabetes and periodontal disease have not been evaluated. OBJECTIVE: The aim of this study was to evaluate the levels of LPO and its correlation with periodontal status and inflammatory cytokines in type 2 diabetic and nondiabetic patients. DESIGN AND SETTING: This is a cross-sectional study involving Brazilian patients recruited at the State University of São Paulo. PATIENTS: The sample comprised 120 patients divided into four groups based upon diabetic and dyslipidemic status: poorly controlled diabetics with dyslipidemia, well-controlled diabetics with dyslipidemia, normoglycemic individuals with dyslipidemia, and healthy individuals. MAIN OUTCOME MEASURES: Blood analyses were carried out for fasting plasma glucose, glycated hemoglobin, and lipid profile. Periodontal examinations were performed, and gingival crevicular fluid was collected. LPO levels were evaluated by measuring oxidized low-density lipoprotein (ELISA) and malondialdehyde (HPLC). Cytokines were evaluated by the multiplex bead technique. RESULTS: LPO evaluated by malondialdehyde in plasma and gingival crevicular fluid was significantly increased in diabetes groups. Significant correlations between LPO markers and periodontal parameters indicate a direct relationship between these levels and the severity of inflammation and secretion of inflammatory cytokines, particularly in diabetic patients. CONCLUSION: These findings suggest an important association for LPO with the severity of the local inflammatory response to bacteria and the susceptibility to periodontal disease in diabetic patients.

Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats

Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

Cytotoxicity and genotoxicity of calcium silicate-based cements on an osteoblast lineage

Abstract Several calcium silicate-based biomaterials have been developed in recent years, in addition to Mineral Trioxide Aggregate (MTA). The aim of this study was to evaluate the cytotoxicity, genotoxicity and apoptosis/necrosis in human osteoblast cells (SAOS-2) of pure calcium silicate-based cements (CSC) and modified formulations: modified calcium silicate-based cements (CSCM) and three resin-based calcium silicate cements (CSCR1) (CSCR 2) (CSCR3). The following tests were performed after 24 hours of cement extract exposure: methyl-thiazolyl tetrazolium (MTT), apoptosis/necrosis assay and comet assay. The negative control (CT-) was performed with untreated cells, and the positive control (CT+) used hydrogen peroxide. The data for MTT and apoptosis were submitted to analysis of variance and Bonferroni's posttest (p < 0.05), and the data for the comet assay analysis, to the Kruskal-Wallis and Dunn tests (p < 0.05). The MTT test showed no significant difference among the materials in 2 mg/mL and 10 mg/mL concentrations. CSCR3 showed lower cell viability at 10 mg/mL. Only CSC showed lower cell viability at 50 mg/mL. CSCR1, CSCR2 and CSCR3 showed a higher percentage of initial apoptosis than the control in the apoptosis test, after 24 hours exposure. The same cements showed no genotoxicity in the concentration of 2 mg/mL, with the comet assay. CSC and CSCR2 were also not genotoxic at 10 mg/mL. All experimental materials showed viability with MTT. CSC and CSCR2 presented a better response to apoptosis and genotoxicity evaluation in the 10 mg/mL concentration, and demonstrated a considerable potential for use as reparative materials.