Pursuing new periodontal pathogens with an improved RNA-oligonucleotide quantification technique (ROQT).

OBJECTIVE: The aim of this study was to optimize the sensitivity, specificity and cost-effectiveness of the RNA-Oligonucleotide Quantification Technique (ROQT) in order to identify periodontal pathogens that remain unrecognized or uncultured in the oral microbiome. DESIGN: Total nucleic acids (TNA) were extracted from subgingival biofilm samples using an automated process. RNA, DNA and Locked Nucleic Acid (LNA) digoxigenin-labeled oligonucleotide probes targeting 5 cultivated/named species and 16 uncultivated or unnamed bacterial taxa were synthesized. Probe specificity was determined by targeting 96 oral bacterial species; sensitivity was assessed using serial dilutions of reference bacterial strains. Different stringency temperatures were compared and new standards were tested. The tested conditions were evaluated analyzing samples from periodontally healthy individuals, and patients with moderate or severe periodontitis. RESULTS: The automated extraction method at 63°C along with LNA-oligunucleotides probes, and use of reverse RNA sequences for standards yielded stronger signals without cross-reactions. In the pilot clinical study, the most commonly detected uncultivated/unrecognized species were Selenomonas sp. HMT 134, Prevotella sp. HMT 306, Desulfobulbus sp. HMT 041, Synergistetes sp. HMT 360 and Bacteroidetes HMT 274. In the cultivated segment of the microbiota, the most abundant taxa were T. forsythia HMT 613 and Fretibacterium fastidiosum (formerly Synergistetes) HMT 363. CONCLUSIONS: In general, samples from severe patients had the greatest levels of organisms. Classic (T. forsythia, P. gingivalis) and newly proposed (F. alocis and Desulfobulbus sp. HMT 041) pathogens were present in greater amounts in samples from severe periodontitis sites, followed by moderate periodontitis sites.

HPV knowledge and vaccine acceptability: a survey-based study among parents of adolescents (KAPPAS study).

BACKGROUND: Human papillomavirus (HPV) infection is recognized as one of the major causes of infection-related cancer worldwide. In Spain, the HPV vaccination program started in 2007 and until 2022, it targeted 12-year-old girls. METHODS: This was a cross-sectional, multicenter survey-based research carried out at 24 pediatric offices to describe HPV knowledge and vaccine acceptability in parents of children aged between 9 and 14 years-old in Spain. Parents were randomly selected from the medical records following specific quotas to ensure representativeness. The survey included five sections that aim to collect information about sociodemographic characteristics, knowledge of HPV, knowledge and acceptability of vaccines in general, HPV vaccination knowledge and HPV vaccine acceptability. Each section was constituted by a number of close questions with different answer options. Specific scores were assigned to each possible answer to these questions. Based on these scores, four composite variables were created to assess HPV knowledge, HPV vaccine knowledge, HPV vaccine acceptability and vaccines knowledge and acceptability in general. A latent class analysis was performed to identify different group of respondents according to their HPV vaccine acceptability. RESULTS: A total of 1405 valid surveys were included, with 86.19% of the respondents being mothers. The mean score of HPV knowledge was 28.92 out of 40 (maximum value) (95% CI 28.70-29.20) and the mean score of HPV vaccine acceptability was 3.37 out of 5 (maximum value). One third of parents still need more information to take a final decision about HPV vaccination in their children. Parents perceived that females were more likely to become infected than males and tended to associate HPV infection mainly with cervical cancer, showing a. a lack of information about other HPV-related diseases affecting males. CONCLUSIONS: This study results highlight the need for future actions and educational initiatives to raise awareness of HPV consequences in both genders and to contribute to achieving the elimination of HPV-related diseases beyond cervical cancer.

Survey based assessment of diagnosis through periapical radiograph and CBCT and treatment of root resorption with Brazilian and American dentists and endodontists.

BACKGROUND: This study assesses and compares the knowledge level of endodontists (ENDs) and general dental practitioners (GPs) from Brazil and United States of America (USA) in the diagnosis and treatment of internal and external inflammatory root resorptions through periapical radiographic (PA) and cone beam computed tomography (CBCT) examinations. MATERIAL AND METHODS: A cross-sectional online questionnaire-based survey was presented to the volunteers containing questions regarding personal and professional profile, as well as three clinical cases of internal and external inflammatory root resorption. A series of multiple-choice questions about the diagnosis and treatment options were surveyed. The data collected was analysed by the Chi-square test with Yates correction with a significance level of 5 %. RESULTS: Most answers were considered adequate when all three questions about the diagnosis and all two questions relating to the treatment were answered accurately. A total of 374 dentists answered the survey (n: 229 from Brazil vs. 145 from USA) being 41% END and 59% GP. END presented higher level of knowledge than GP regarding to diagnosis and treatment of inflammatory root resorptions both in Brazil and USA (p<0.05); USA presented higher level of adequate responses than Brazil (p<0.05). CONCLUSIONS: END achieved a level of knowledge of the diagnosis and treatment of root resorption superior to the GP. Comparing the results obtained in both countries, it was observed that the USA had a higher correct response rate than Brazil. Key words:Internal root resorption, external root resorption, management, diagnosis, treatment.

[How to prepare an oral presentation†?] / Comment se préparer à une présentation orale?

Oral presentations are an important means of transmitting knowledge. Good preparation is essential to succeed in this exercise. Some steps are necessary, such as defining the main objective, focusing on the audience, managing the time, choosing the first and last words, or anticipating the audience's questions, while using the visual aids appropriately. The purpose of this article is to lead young doctors in the process of creating an oral presentation.

Les présentations orales sont un moyen important de transmission du savoir. Une bonne préparation est essentielle pour réussir cet exercice. Certaines étapes sont nécessaires, comme définir l'objectif principal, s'intéresser à son audience, gérer le temps, choisir les premiers et derniers mots ou encore anticiper les questions du public, le tout en utilisant de manière adaptée les supports visuels. Cet article a pour objectif d'accompagner les jeunes médecins dans le processus de création d'une présentation orale.

Estimación de costos de hospitalización por lesiones asociadas al transito en un hospital publico de Tucumán, Argentina, 2017 / Estimation of Costs of Hospitalized Patients due to Traffic Related Injuries in a Public Hospital of Tucumán, Argentina, 2017

INTRODUCCIÓN: Las lesiones asociadas al tránsito(LAT) constituyen un serio problema en hospitales públicos.Para mejorar la administración y distribución de recursos,es importante conocer el costo de hospitalización por esta causa. El objetivo del estudio fue caracterizar a los pacientes hospitalizados por LAT y estimar los costos directos de la asistencia entre enero y junio de 2017 en un hospital público de referencia de la provincia de Tucumán. MÉTODOS: Se realizó un estudio de estimación de costos de hospitalización por LAT, con seguimiento retrospectivo y sumatoria de variables contempladas. Se estimaron promedios, se compararon proporciones. Se consideró significativo p<0,05. Se empleó el programa estadístico Stata 11.2. RESULTADOS: Hubo 458 pacientes hospitalizados por LAT, el 79% fueron varones y y la mediana de edad fue de 27 años en ambos sexos. Las motos representaron el 77% de los vehículos utilizados. Los pacientes graves (25%) permanecieron más de 30 días en servicios quirúrgicos (costo promedio: USD 7.252). El costo promedio de estudios complementarios por paciente fue de USD 180, el de cirugías USD 823 y el de internación USD 1320. CONCLUSIONES: Las hospitalizaciones por LAT fueron principalmente en varones, jóvenes adultos y motociclistas. El mayor costo se observó en pacientes graves, que requirieron cirugía y un promedio de 35 días de internación en servicios quirúrgico

NTRODUCTION: Traffic-related injuries (TRI) are a major problem in public hospitals. For better resource management and distribution, it is important to know the cost of hospitalizations. The objective of this study was to characterize patients hospitalized due to TRI and to estimate the direct costs of the care provided between January and June 2017in a public hospital ofTucumán Province in Argentina. METHODS: A study of costs of patients hospitalized due to TRI was conducted, with retrospective follow-up and summation of the variables considered for the analysis. Averages were estimated and proportions were compared. A p value <0.05 was considered significant. Statistical software Stata 11.2 was used. RESULTS: A total of 458 patients were hospitalized due to TRI, 79% were men, and the median age was 27 for both sexes. Motorcycles accounted for 77% of all vehicles used. Severely injured patients (25%) stayed more than 30 days in surgical facilities (average cost: USD 7252). The average cost per patient of complementary studies, in surgeries and hospitalization was USD 180, USD 823 and USD 1320, respectively. CONCLUSIONS: Patients hospitalized due to TRI were mainly males, young adults and motorcyclists. The highest cost was in the group of patients with serious injuries, who required surgery, with an average stay of 35 days of hospitalization in surgical facilitie

Detecting illegal intra-corporeal cocaine containers: Which factors influence their density?

PURPOSE: To determine parameters related to hyperdensity (>40 HU) of intra-corporeal cocaine packets on low-dose CT (LDCT); hyperdensity increases detectability on abdominal radiographs. METHODS: LDCT showing drug packets (n = 46) were analyzed for mean radiological density and packets volume. Following expulsion, packets weight and cocaine concentration were measured. Hypercompaction was defined as >0.9 g/cm3. RESULTS: Packets were hyperdense in 33 cases (72%). Mean compaction was 1.0 g/cm3, mean density 118.5 HU and mean cocaine concentration 44.2%. On multivariate analysis, only high compaction remained significantly related to hyperdensity (p = 0.001). CONCLUSION: Compaction >0.9 g/cm3 is the only parameter significantly associated with hyperdense packets.

Estudio de costos de pacientes hospitalizados por lesiones de tránsito en un hospital de referencia de Tucumán. 1° semestre 2017 / Study of costs of patients hospitalized for traffic injuries in a reference hospital in Tucumán. 1st semester 2017

INTRODUCCIÓN Las lesiones de tránsito (LT) constituyen uno de los principales problemas que enfrentan hospitales públicos. Conocer el costo total de las hospitalizaciones por estas causas ayudará a realizar una mejor administración y distribución de recursos. OBJETIVO Conocer el perfil epidemiológico de los pacientes hospitalizados por LT y estimar los costos directos de la atención realizada por el Hospital de Referencia cuyo egreso fue entre enero y junio de 2017. METODOLOGÍA Estudio de costos directos de pacientes que ingresaron a la institución y se hospitalizaron por LT en el período de estudio. Se realizó una trazabilidad retrospectiva. Se hizo una descripción del perfil epidemiológico de los pacientes. Para el análisis de costos se realizó una sumatoria de variables contempladas; luego de ajustar por estándares del hospital. Se estimaron promedios y se realizaron comparaciones mediante proporciones. Se consideró significativo un valor de p<0,05. Para el análisis se empleó el software estadístico Stata 11.2. RESULTADOS Se registraron 458 pacientes hospitalizados por LT, el 79% fueron varones y la mediana de edad fue de 27 años para ambos sexos. Las motos representaron el 77% del total de los vehículos utilizados. Los pacientes graves (25%), permanecieron más de 30 días en servicios quirúrgicos (costo promedio por paciente usd 7.252). El 38% de los lesionados, recibió intervención quirúrgica (costo promedio usd 588), de ellos el 65% requirió prótesis o implantes (costo promedio usd 2350). El costo promedio de estudios complementarios por paciente fue de usd 180; mientras que en cirugías fue de usd 823 y el de internación fue de usd1320. DISCUSIÓN Los pacientes hospitalizados por LT fueron principalmente varones, jóvenes adultos, motociclistas. El costo más elevado fue en el grupo de pacientes con lesiones graves, que requirieron cirugía, con una estadía promedio de 35 días de internación en servicios quirúrgicos

Endothelial dysfunction in rats with ligature-induced periodontitis: Participation of nitric oxide and cycloxygenase-2-derived products.

OBJECTIVES: Considering the evident relationship between periodontitis and cardiovascular diseases in humans, we aimed to study the in vitro vascular reactivity of aorta rings prepared from rats with ligature-induced periodontitis. METHODS: Seven days after the induction of unilateral periodontitis, the animals were euthanised; rings were prepared from the descending abdominal aortas and mounted in tissue baths for the in vitro measurement of the isometric force responses to norepinephrine (NE) and acetylcholine (ACh), as well as in the presence of inhibitors of nitric oxide synthase (NOS) and cycloxygenase (COX) isoenzymes. Aortic COX and NOS gene expressions were analysed by RT-PCR, as well as protein COX-2 expression by Western blot. RESULTS: Periodontitis resulted in significant alveolar bone loss and did not affect arterial pressure. However, both NE-induced contraction and ACh-induced relaxation were significantly decreased and related to the presence of endothelium. Diminished eNOS and augmented COX-2 and iNOS expressions were found in the aortas from rats with periodontitis, and the pharmacological inhibition of COX-2 or iNOS improved the observed vasomotor deficiencies. CONCLUSIONS: We can thus conclude that periodontitis induces significant endothelial dysfunction in rat aorta which is characterized by decreased eNOS expression and mediated by upregulated iNOS and COX-2 products.

LXA4 actions direct fibroblast function and wound closure.

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A4 (LXA4), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-ß1, to induce fibroblast activation. The impact of exogenous TGF-ß1 (1 ng/mL) on LXA4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a "scratch" assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-ß1 up-regulates LXA4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-ß1 alone at 48 and 72 h. LXA4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-ß1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function.

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.

The H2S-releasing naproxen derivative, ATB-346, inhibits alveolar bone loss and inflammation in rats with ligature-induced periodontitis.

BACKGROUND: In experimental periodontitis, non-steroidal antiinflammatory drugs (NSAIDs) effectively inhibit the resultant alveolar bone loss. However, their deleterious gastric effects, observed in both animals and humans, dramatically limit their long-term use. It has been proven that the addition of a hydrogen sulfide (H2S)-releasing moiety to classical NSAID structures results in antiinflammatory compounds with improved gastric safeness. In this way, we decided to compare the effects of naproxen with its H2S-releasing derivative ATB-346 on ligature-induced periodontitis in rats. METHODS: Male Holtzman rats had a cotton ligature placed subgingivally around the lower right first molar during 7 days. During this period, groups of animals were daily treated with Na2S (a spontaneous H2S donor) or equimolar oral doses of naproxen (10 mg/kg) or ATB-346 (16 mg/kg). The mandibles were finally collected for histological analysis, radiographical measurements of alveolar bone loss and micro-computed tomography (µCT) analysis. Interleukin (IL)-1ß, IL-6 and IL-10 were quantified in gingiva samples, and the stomachs were also collected for scoring of tissue damage and measurement of myeloperoxidase (MPO, a marker of granulocyte infiltration). RESULTS: Ligature-induced bone loss was significantly inhibited by all the treatments, although only ATB-346 treatment resulted in significant inhibition of bone defect and other histological characteristics (such as flatness of the gingival epithelium, chronic inflammatory cell infiltration and loss of connective tissue in the gingival papillae). Both naproxen and ATB-346 inhibited the increase of gingival IL-1ß and IL-6 secondary to periodontitis, but IL-10 was unaffected. Significant damage and increased MPO contents were only found in the stomachs of the naproxen-treated animals. CONCLUSION: The H2S-releasing moiety in the ATB-346 compound not only does not impair the effects of the parent naproxen on periodontitis, but also improves bone quality and prevents the gastric mucosa damage due to prostaglandin inhibition, thus configuring a potentially new adjuvant therapy for periodontal diseases.

Impact of resolvin E1 on murine neutrophil phagocytosis in type 2 diabetes.

Diabetic complications involve inflammation-mediated microvascular and macrovascular damage, disruption of lipid metabolism, glycosylation of proteins, and abnormalities of neutrophil-mediated events. Resolution of inflamed tissues to health and homeostasis is an active process mediated by endogenous lipid agonists, including lipoxins and resolvins. This proresolution system appears to be compromised in type 2 diabetes (T2D). The goal of this study was to investigate unresolved inflammation in T2D. Wild-type (WT) and genetically engineered mice, including T2D mice (db/db), transgenic mice overexpressing the human resolvin E1 (RvE1) receptor (ERV1), and a newly bred strain of db/ERV1 mice, were used to determine the impact of RvE1 on the phagocytosis of Porphyromonas gingivalis in T2D. Neutrophils were isolated and incubated with fluorescein isothiocyanate-labeled P. gingivalis, and phagocytosis was measured in a fluorochrome-based assay by flow cytometry. Mitogen-activated protein kinase (MAPK) (p42 and p44) and Akt (Thr308 and Ser473) phosphorylation was analyzed by Western blotting. The mouse dorsal air pouch model was used to evaluate the in vivo impact of RvE1. Results revealed that RvE1 increased the neutrophil phagocytosis of P. gingivalis in WT animals but had no impact in db/db animals. In ERV1-transgenic and ERV1-transgenic diabetic mice, phagocytosis was significantly increased. RvE1 decreased Akt and MAPK phosphorylation in the transgenic animals. In vivo dorsal air pouch studies revealed that RvE1 decreases neutrophil influx into the pouch and increases neutrophil phagocytosis of P. gingivalis in the transgenic animals; cutaneous fat deposition was reduced, as was macrophage infiltration. The results suggest that RvE1 rescues impaired neutrophil phagocytosis in obese T2D mice overexpressing ERV1.

Peripheral blood mononuclear phagocytes from patients with chronic periodontitis are primed for osteoclast formation.

BACKGROUND: During inflammatory periodontal disease, peripheral blood mononuclear cells (PBMCs) are attracted to bone and differentiate into active bone-resorbing osteoclasts (OCs), thus providing evidence that the impact of chronic periodontitis (CP) on the activity of circulating mononuclear cells is of central importance. The authors test the hypothesis that peripheral blood mononuclear phagocytes (PBMPs) from patients with CP are activated and more susceptible to differentiation into OCs, which in turn would lead to more intense bone resorption. METHODS: In vitro cytokine production by both unstimulated and lipopolysaccharide-stimulated PBMCs from individuals with (n = 10) or without (n = 12) periodontitis was determined by cytokine array. OC differentiation from CD14(+) PBMCs was induced by receptor activator of nuclear factor-kappa B ligand (RANKL), either alone or in the presence of macrophage colony-stimulating factor (M-CSF). PBMC differentiation to OCs was confirmed by tartrate-resistant acid phosphatase staining; bone resorbing activity was assessed by using an osteologic plate assay (bone resorption pit formation). RESULTS: PBMCs from patients with CP produced tumor necrosis factor-α and higher amounts of interferon-γ, interleukin (IL)-1α, IL-1ß, IL-1rα, CXC motif chemokine 10, macrophage migration inhibitory factor, macrophage inflammatory protein (MIP)-1α, and MIP-1ß than the control cells. OC differentiation was induced by RANKL alone in PBMCs from patients with CP, but not in PBMCs from the healthy controls, which required the addition of M-CSF. In addition, PBMC-derived OCs from patients with CP showed significantly higher resorption activity than that observed in the healthy controls. Also, the circulating concentrations of M-CSF were significantly higher in patients with CP than in the control participants. CONCLUSIONS: These data indicate that in patients with CP, circulating PBMCs are primed for increased proinflammatory activity and that M-CSF plays a central role in this process by increasing OC formation and the consequent bone resorption activity.

Correction: A critical role for suppressors of cytokine signaling 3 in regulating LPS-induced transcriptional activation of matrix metalloproteinase-13 in osteoblasts.

[This corrects the article DOI: 10.7717/peerj.51.].

A critical role for suppressors of cytokine signaling 3 in regulating LPS-induced transcriptional activation of matrix metalloproteinase-13 in osteoblasts.

Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of cytokine signaling in macrophages and T cells. Although SOCS3 seems to contribute to the balance between the pro-inflammatory actions of IL-6 family of cytokines and anti-inflammatory signaling of IL-10 by negatively regulating gp130/Jak/Stat3 signal transduction, how and the molecular mechanisms whereby SOCS3 controls the downstream impact of TLR4 are largely unknown and current data are controversial. Furthermore, very little is known regarding SOCS3 function in cells other than myeloid cells and T cells. Our previous study demonstrates that SOCS3 is expressed in osteoblasts and functions as a critical inhibitor of LPS-induced IL-6 expression. However, the function of SOCS3 in osteoblasts remains largely unknown. In the current study, we report for the first time that LPS stimulation of osteoblasts induces the transcriptional activation of matrix metalloproteinase (MMP)-13, a central regulator of bone resorption. Importantly, we demonstrate that SOCS3 overexpression leads to a significant decrease of LPS-induced MMP-13 expression in both primary murine calvariae osteoblasts and a mouse osteoblast-like cell line, MC3T3-E1. Our findings implicate SOCS3 as an important regulatory mediator in bone inflammatory diseases by targeting MMP-13.

Resolvin E1 and chemokine-like receptor 1 mediate bone preservation.

The polyunsaturated ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss, and induces bone regeneration. Although the inflammation-resolving actions of RvE1 are characterized, the molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice overexpressing the RvE1 receptor chemokine-like receptor 1 (chemR23) on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in receptor activator of NF-κB ligand levels, whereas the osteogenic markers alkaline phosphatase, bone sialoprotein, and Runt-related transcription factor 2 remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable receptor activator of NF-κB ligand/OPG ratio, in addition to known anti-inflammatory and proresolving actions.

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.

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells.

OBJECTIVE: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. DESIGN: Cell line SAOS-2 (1×10(5)cells/mL) were grown in culture medium α-MEM with 10% FBS for 24h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10g/mL) and IL-1ß (1mg/mL) for 24h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO(2)(-) with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. RESULT: There were no significant differences amongst the groups in terms of NO(2)(-), protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p<0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1ß caused up-regulation of this cytokine. CONCLUSIONS: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation.

iNOS-derived nitric oxide stimulates osteoclast activity and alveolar bone loss in ligature-induced periodontitis in rats.

BACKGROUND: Inflammatory stimuli activate inducible nitric oxide synthase (iNOS) in a variety of cell types, including osteoclasts (OC) and osteoblasts, resulting in sustained NO production. In this study, we evaluate the alveolar bone loss in rats with periodontitis under long-term iNOS inhibition, and the differentiation and activity of OC from iNOS-knockout (KO) mice in vitro. METHODS: Oral aminoguanidine (an iNOS inhibitor) or water treatment was started 2 weeks before induction of periodontitis. Rats were sacrificed 3, 7, or 14 days after ligature placement, and alveolar bone loss was evaluated. In vitro OC culture experiments were also performed to study the differentiation of freshly isolated bone marrow cells from both iNOS KO and wild-type C57BL/6 mice. OC were counted 6 days later after tartrate-resistant acid phosphatase staining (a marker of osteoclast identity), and bone resorption activity was assessed by counting the number of resorption pits on dentin disks. RESULTS: Rats with ligature showed progressive and significant alveolar bone loss compared to sham animals, and aminoguanidine treatment significantly inhibited ligature-induced bone loss at 7 and 14 days after the induction. In comparison to bone marrow cells from wild-type mice, cells from iNOS KO mice showed decreased OC growth and the resulting OC covered a smaller culture dish area and generated fewer resorption pit counts. CONCLUSION: Our results demonstrate that iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating OC differentiation and activity.

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats.

OBJECTIVE: in this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. MATERIALS AND METHODS: oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. RESULTS: in NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. CONCLUSIONS: in addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney.