Liquid Chromatography-Tandem Mass Spectrometry Method for Detection and Quantification of Meloxicam and 5'-Carboxymeloxicam in Oral Fluid Samples.

A sensitive, selective and particularly fast method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated for the determination of meloxicam and its main metabolite, 5'-carboxymeloxicam, in oral fluid samples. Meloxicam and its major metabolite were separated using a Shim-Pack XR-ODS 75 L × 2.0 column and C18 pre-column at 40 °C using a mixture of methanol and 10 mM ammonium acetate (80:20, v/v) with an injection flow rate of 0.3 mL/min. The total time of the analytical run was 5 min. Sixteen volunteers had oral fluid samples collected sequentially before and after taking a meloxicam tablet (15 mg) for up to 96 h. With the concentrations obtained, the pharmacokinetic parameters were determined using the Phoenix WinNonlin software. The parameters evaluated for meloxicam and 5'-carboxymeloxicam in the oral fluid samples showed linearity, accuracy, precision, medium-quality control (MQC-78.12 ng/mL), high-quality control (HQC-156.25 ng/mL), lower limits of quantification (LLOQ-0.6103 ng/mL), low-quality control (LQC-2.44 ng/mL), stability and dilution. Prostaglandin E2 (PGE2) was also detected and quantified in the oral fluid samples, demonstrating the possibility of a pharmacokinetic/pharmacodynamic (PK/PD) study with this methodology. All the parameters evaluated in the validation of the methodology in the oral fluid samples proved to be stable and within the possible variations in each of the described parameters. Through the data presented, the possibility of a PK/PD study was demonstrated, detecting and quantifying meloxicam, its main metabolite and PGE2 in oral fluid samples using LC-MS/MS.

CYP2C9 Polymorphism Influence in PK/PD Model of Naproxen and 6-O-Desmethylnaproxen in Oral Fluid.

Polymorphisms in CYP2C9 can significantly interfere with the pharmacokinetic (PK) and pharmacodynamic (PD) parameters of nonsteroidal anti-inflammatory drugs (NSAIDs), including naproxen. The present research aimed to study the PK/PD parameters of naproxen and its metabolite, 6-O-desmethylnaproxen, associated with allelic variations of CYP2C9. In our study, a rapid, selective, and sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method was developed and validated for the determination of naproxen and its main metabolite, 6-O-desmethylnaproxen, in oral fluid. Naproxen and its main metabolite were separated using a Shim-Pack XR-ODS 75L × 2.0 column and C18 pre-column at 40 °C using a mixture of methanol and 10 mM ammonium acetate (70:30, v/v), with an injection flow of 0.3 mL/min. The total analytical run time was 3 min. The volunteers, previously genotyped for CYP2C9 (16 ancestral­CYP2C9 *1 and 12 with the presence of polymorphism­CYP2C9 *2 or *3), had their oral fluids collected sequentially before and after taking a naproxen tablet (500 mg) at the following times: 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 8, 11, 24, 48, 72 and 96 h. Significant differences in the PK parameters (* p < 0.05) of naproxen in the oral fluid were: Vd/F (L): 98.86 (55.58−322.07) and 380.22 (261.84−1097.99); Kel (1/h): 0.84 (0.69−1.34) and 1.86 (1.09−4.06), in ancestral and mutated CYP2C9 *2 and/or *3, respectively. For 6-O-desmethylnaproxen, no PK parameters were significantly different between groups. The analysis of prostaglandin E2 (PGE2) proved to be effective and sensitive for PD parameters analysis and showed higher levels in the mutated group (p < 0.05). Both naproxen and its main metabolite, 6-O-desmethylnaproxen, and PGE2 in oral fluid can be effectively quantified using LC-MS/MS after a 500 mg oral dose of naproxen. Our method proved to be effective and sensitive to determine the lower limit of quantification of naproxen and its metabolite, 6-O-desmethylnaproxen, in oral fluid (2.4 ng/mL). All validation data, such as accuracy, precision, and repeatability intra- and inter-assay, were less than 15%. Allelic variations of CYP2C9 may be considered relevant in the PK of naproxen and its main metabolite, 6-O-desmethylnaproxen.

Salivary proteomic profile of dogs with and without dental calculus.

BACKGROUND: Dogs' saliva is a complex mixture of inorganic and organic constituents, rich in proteins. Therefore, knowing the saliva composition of these animals is extremely important to identify the presence of proteins that may be involved in physiological and pathological mechanisms of their oral cavity. The present study aimed to characterize the proteomic profile of saliva from dogs with and without dental calculus. RESULTS: Saliva samples were collected from 20 dogs. Before the collection, a visual clinical examination was performed and 8 subjects (40%) did not present any signs of dental calculus, while 12 (60%) presented dental calculus. After saliva collection, the samples were submitted to protein quantification (mBCA), and then they were prepared for analysis by nLC-ESI-MS/MS. A total of 658 unique proteins were identified, of which 225 were specific to dogs without dental calculus, 300 were specific to dogs with dental calculus, and 133 were common to all subjects. These proteins presented functions including transportation, immune response, structural, enzymatic regulation, signal transduction, transcription, metabolism, and some proteins perform functions as yet unknown. Several salivary proteins in dogs with dental calculus differed from those found in the group without dental calculus. Among the abundant proteins detected in periodontal affected cases, can be highlighting calcium-sensing receptor and transforming growth factor beta. Enrichment analysis reveled the presence of Rho GTPases signaling pathway. CONCLUSIONS: This research identified salivary proteins, that should be further investigated as potencial biomarkers of chronic periodontits with dental calculus formation in dogs.

Molecular Response of Pulp Fibroblasts after Stimulation with Pulp Capping Materials.

This in vitro study evaluated cell viability and metabolism, nitric oxide release and production of two chemokines and one cytokine by cultured human dental pulp fibroblasts (HDPF) in contact with two glass ionomer cements (Ketac Molar-KM and Vitrebond-VB), Single Bond (SB) and calcium hydroxide (Dycal-DY). Cultures of HDPF were established by means of an explant technique. The specimens were prepared under sterile conditions and in disks measuring 5 mm x 2 mm obtained from a prefabricated mold and placed on a permeable membrane to avoid direct contact with the cells. Cytotoxicity was assessed by Trypan Blue exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide release in cell supernatant was detected by the Griess Method whereas stromal derived factor-1 alpha (SDF-1α or CXCL12), chemokine (C-X-C motif) ligand 8 [Interleukin 8 (IL-8 or CXCL8)] and interleukin-6 (IL-6) were detected by ELISA. RT-qPCR was employed for gene expression analysis. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test for materials independent of the time, and Two-way ANOVA followed by Bonferroni correction test for the comparisons between materials and experimental time (p<0.05). Cytotoxic tests showed significant differences only for DY. Protein levels and mRNA expression were significantly increased for IL-8 for both periods of time. IL-6 production increased when fibroblasts were stimulated by KM. SDF-1α protein production and mRNA expression were not affected by any of the materials. There was a decrease in nitrate/nitrite levels only for KM. Although DY caused intense cell death and did not stimulate the production of the inflammatory mediators evaluated in this work, it is known that this event seems to be fundamental for the process of repair of the pulp tissue and formation of mineralized barrier. KM and VB increased production of proteins related to the inflammatory process, thus favoring tissue repair. Therefore, although these glass ionomer cements did not lead to large cell death, they should be used with caution.

What is the response profile of deciduous pulp fibroblasts stimulated with E. coli LPS and E. faecalis LTA?

BACKGROUND: Oral fibroblast immunological responses to bacterial stimuli are well known. However, there are few studies about pulp fibroblasts from deciduous teeth (HDPF) responses, which are important for the treatment of pulp infections in children. The aim of this study was to evaluate expression and production of inflammatory cytokines and chemokines by HDPF when challenged with bacterial antigens normally present in advanced caries lesions. METHODS: Triplicate HDPF from 4 children (n = 4; 2 boys and 2 girls) were cultured by explant technique and challenged or not with Escherichia coli lipopolysaccharide/1 µg/mL (EcLPS) or Enterococcus faecalis lipoteichoic acid/1 µg/mL (EfLTA) for 6 and 24 h. Most of published studies employed immortalized cells, i.e., without checking possible gender and genetic variables. mRNA expression and protein production were evaluated by RT-qPCR and ELISA MILLIPLEX®, respectively, for Interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, Chemokine C-C motif ligand 2/monocyte chemoattractant protein 1 (CCL2/MCP-1), Chemokine C-C motif ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP1-α), Chemokine C-C motif ligand 5/ regulated on activation, normal T cell expressed and secreted (CCL5/RANTES), C-X-C motif chemokine 12/ stromal cell-derived factor 1 (CXCL12/SDF-1), Tumor Necrosis Factor-alpha (TNF-α), Interferon-gamma (IFN γ), Vascular Endothelial Growth Factor (VEGF), Colony stimulating factor 1 (CSF-1) and Macrophage colony-stimulating factor (M-CSF). RESULTS: EcLPS increased IL-1α, IL-1ß, IL-8, CCL2, CCL5, TNF-α and CSF-1 mRNA and protein levels while EfLTA was only able to positively regulate gene expression and protein production of IL-8. CONCLUSION: The results of the present study confirmed our hypothesis, since pulp fibroblasts from deciduous teeth are capable of increasing gene expression and protein production after being stimulated with EcLPS and EfLTA.

Molecular response of pulp fibroblasts after stimulation with pulp capping materials

Abstract This in vitro study evaluated cell viability and metabolism, nitric oxide release and production of two chemokines and one cytokine by cultured human dental pulp fibroblasts (HDPF) in contact with two glass ionomer cements (Ketac Molar-KM and Vitrebond-VB), Single Bond (SB) and calcium hydroxide (Dycal-DY). Cultures of HDPF were established by means of an explant technique. The specimens were prepared under sterile conditions and in disks measuring 5 mm x 2 mm obtained from a prefabricated mold and placed on a permeable membrane to avoid direct contact with the cells. Cytotoxicity was assessed by Trypan Blue exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide release in cell supernatant was detected by the Griess Method whereas stromal derived factor-1 alpha (SDF-1α or CXCL12), chemokine (C-X-C motif) ligand 8 [Interleukin 8 (IL-8 or CXCL8)] and interleukin-6 (IL-6) were detected by ELISA. RT-qPCR was employed for gene expression analysis. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test for materials independent of the time, and Two-way ANOVA followed by Bonferroni correction test for the comparisons between materials and experimental time (p<0.05). Cytotoxic tests showed significant differences only for DY. Protein levels and mRNA expression were significantly increased for IL-8 for both periods of time. IL-6 production increased when fibroblasts were stimulated by KM. SDF-1α protein production and mRNA expression were not affected by any of the materials. There was a decrease in nitrate/nitrite levels only for KM. Although DY caused intense cell death and did not stimulate the production of the inflammatory mediators evaluated in this work, it is known that this event seems to be fundamental for the process of repair of the pulp tissue and formation of mineralized barrier. KM and VB increased production of proteins related to the inflammatory process, thus favoring tissue repair. Therefore, although these glass ionomer cements did not lead to large cell death, they should be used with caution.

Resumo Este estudo avalia in vitro a viabilidade e metabolismo celular, a liberação de óxido nítrico e a produção de duas quimiocinas e uma citocina por fibroblastos de polpa dentária humana em cultura (FPDH) em contato com dois cimentos de ionômero de vidro (Ketac Molar-KM e Vitrebond-VB), Single Bond (SB) e hidróxido de cálcio (Dycal-DY). As culturas de FPDH foram estabelecidas por meio de uma técnica de explante. As amostras foram preparadas em condições estéreis e em discos de 5 mm x 2 mm, obtidas de um molde pré-fabricado e colocadas em uma membrana permeável (Maxicell 24 W 0,4 µm) para evitar o contato direto com as células. A citotoxicidade foi avaliada pelo método de exclusão de Trypan Blue e pelo ensaio de MTT. A liberação de óxido nítrico no sobrenadante celular foi detectada pelo método Griess, enquanto fator 1 derivado do estroma (SDF-1α ou CXCL12), interleucina-8 (IL-8 ou CXCL8) and interleucina-6 (IL-6) foram detectados por ELISA. RT-qPCR foi empregada para análise de expressão gênica. As análises estatísticas foram realizadas por ANOVA a 1 critério, seguida pelo pós-teste de Tukey para os materiais independentes do tempo, e ANOVA a 2 critérios, seguida pelo teste de correção de Bonferroni para comparações entre materiais e tempo experimental (p<0,05). Os testes citotóxicos mostraram diferenças significativas apenas para DY. Os níveis da proteína e a expressão de RNAm para IL-8 aumentaram significativamente para ambos os tempos estudados. A produção de IL-6 aumentou quando os fibroblastos foram estimulados por KM. A produção da proteína e a expressão de RNAm para SDF-1α não foram afetadas por nenhum dos materiais. Houve uma diminuição nos níveis de nitrato/nitrito apenas para KM. Embora o DY tenha causado intensa morte celular e não tenha estimulado a produção dos mediadores inflamatórios avaliados neste trabalho, sabe-se que esse evento parece ser fundamental para o processo de reparo do tecido pulpar e formação de barreira mineralizada. Os cimentos de ionômero de vidro utilizados aumentaram a produção de proteínas relacionadas ao processo inflamatório, favorecendo a reparação tecidual e, portanto, esses materiais, embora não causem grande morte celular, devem ser utilizados com cautela.

Losartan and isoproterenol promote alterations in the local renin-angiotensin system of rat salivary glands.

Renin-angiotensin system (RAS) systemically or locally collaborates with tissue homeostasis, growth and development, which has been extensively studied for its pharmacological implications. This study was primarily aimed at finding and characterizing local RAS in rat parotid, sublingual and submandibular glands. It was also hypothesized that vasoactive drugs could affect the expression of RAS targets, as well as saliva flow and its composition. Therefore, another objective of this study was to compare the effects of losartan (angiotensin II receptor blocker) and isoproterenol (ß-adrenergic receptor agonist). Forty-one Wistar rats were divided into three groups and administered a daily intraperitoneal dose of saline, losartan or isoproterenol solutions for one week. The following RAS targets were studied using qPCR: renin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), ACE-2, elastase-2 (ELA-2), AT1-a and MAS receptors, using RPL-13 as a reference gene. Morphology of glands was analyzed by immunohistochemistry using REN, ACE, ACE-2, AT1, AT2 and MAS antibodies. The volume and total protein content of saliva were measured. Our results revealed that ACE, ACE-2, AT1-a, AT2 and MAS receptors were expressed in all salivary gland samples, but REN and ELA-2 were absent. Losartan decreased mRNA expression of RAS targets in parotid (MAS) and submandibular glands (ACE and both AT receptors), without affecting morphological alterations, and significantly decreased saliva and total protein secretions. Isoproterenol treatment affected gene expression profiles in parotid (ACE, ACE-2, AT1-a, MAS, AGT), and submandibular (ACE, AT2, AGT) glands, thus promoting acinar hypertrophy in serous acini, without significant changes in salivary flow or total protein content. These drugs affected mainly acini, followed by duct systems and myoepithelial cells, whereas blood vessels were not affected. In conclusion, there is a local RAS in major rat salivary glands and losartan, an angiotensin II receptor blocker, affected not only the RAS-target gene expression but also decreased salivary flow and total protein content.

Multidisciplinary esthetic approach for multiple anterior diastema: 36-month clinical performance / Abordagem estética multidisciplinar para múltiplos diastemas anteriores: desempenho clínico de 36 meses

The presence of diastema, between anterior teeth, distorts a pleasing smile by concentrating the observer’s attention on the spaces. The patient’s needs and expectations must be considered in the process of treatment planning to ensure satisfaction with the treatment out comes. There are many forms of therapy that can be used for diastema closure. A carefully developed diagnosis, which includes a determination of the causal element sand advanced treatment planning, allows the mostappropriate treatment to be selected for each case. The aim of this paper is to present a multidisciplinary approach as a solution to multiple diastemata in the anterior region using gingival tissue recontouring and direct adhesive restorations, with minimumwear of the dental structure, after the orthodontic intervention, discussing the minimal intervention toobtain imperceptive and aesthetic final restoration. Thirty-six months after the treatment was carriedout, the final aesthetic was maintained with all dental element details and gingival tissue harmony, without recurrence of periodontal pockets and the preservation of the tooth color and shape...

A presença de diastemas, entre dentes anteriores, distorce um sorriso agradável, concentrando a atenção do observador para os espaços. As necessidades e expectativas do paciente devem ser consideradas no processo de planejamento para garantir a satisfação no resultado do tratamento. Existem muitas formas de tratamento que podem ser utilizadas para o fechamento de diastemas. Um diagnóstico desenvolvido cuidadosamente, que inclui a determinação dos elementos causais e planejamento avançado do tratamento, permite as eleção do tratamento mais adequado para cada caso. O objetivo deste artigo é apresentar uma abordagem multidisciplinar como solução para diastemas multiplos na região anterior usando o recontorno gengival e restaurações adesivas diretas, com mínimo desgaste da estrutura dental, após a intervenção ortodôntica discutindo a intervenção mínima para a obtenção de restaurações finais estéticas e imperceptiveis. Trinta e seis meses após o tratamento foi avaliado, a estética final foi mantida com os detalhes do elemento dental e harmonia tecido gengival, sem recorrência de bolsas periodontais e preservando a cor e forma dos dentes...

O uso de implantes, enxerto ósseo e condicionamento do tecido gengival perimplantar na reabilitação estética de área anterior de maxila / The use of dental implants, bone graft and peri-implant gingival tissue modeling in the esthetic rehabilitation of the maxillary anterior area

A reabilitação protética implanto-suportada de espaços edêntulos na região anterior da maxila é um desafio para o cirurgião-dentista. Um adequado posicionamento dos implantes e sua relação harmoniosa com os tecidos duros e moles são fundamentais para uma alta demanda estética. Esse caso relata a correção de um severo defeito estético no qual foi necessário associar vários procedimentos, como a realização de enxerto ósseo prévio, a instalação de implantes osseointegráveis, a manipulação dos tecidos peri-implantares e a escolha de componentes protéticos estéticos, para se alcançar um resultado estético satisfatório.

Implant-supported prosthetic rehabilitation of anterior edentulous spaces is a challenge to the dentist performing the implant surgery. Proper positioning of the implant and its harmonious relationship with the hard and soft tissues are fundamental for a high aesthetic demand. This case report a correction of a serious esthetic defect in that was necessary associate some procedures, like a previous bone graft, installation of osseointegrated implants, soft tissue manipulation and the use of esthetic prosthetic components to reach a satisfactory result.

Peri-implantite: alternativas de tratamento / Peri-implantitis: treatment alternatives: a literature review

A peri-implantite é um processo inflamatório que ocorre nos tecidos adjacentes aos implantes osseointegrados e pode resultar em perda de suporte ósseo e consequentemente na perda do implante. O tratamento da peri-implantite visa eliminar a causa e restabelecer a condição original de saúde dos tecidos peri-implantares. Diversas terapias têm sido propostas com esta finalidade, dentre as quais se incluem debridamento mecânico, uso de antimicrobianos tópicos e/ou sistêmicos, terapias cirúrgicas, além de diferentes tipos de laser. A seleção da terapia a ser aplicada depende do tipo e do tamanho do defeito ósseo apresentado. No entanto, em virtude da variedade de morfologia, extensão e severidade das lesões peri-implantares, nenhuma terapia foi estabelecida, ainda, como alternativa totalmente segura e eficaz. Ainda que não haja um consenso de tratamento a ser estabelecido, todas as modalidades terapêuticas se mostram benéficas no tratamento da peri-implantite, dentro de suas limitações. Portanto, utilizadas de forma individual ou associadas, as terapias podem ser aplicadas visando o sucesso no tratamento das lesões peri-implantares. Diante disso, este trabalho teve como objetivo revisar na literatura as diversas alternativas utilizadas para o tratamento da peri-implantite.

Peri-implantitis is an inflammatory process in the adjacent tissues of osseointegrated dental implants, which can result in bone loss support and consequently in implant failure. The treatment for peri-implantitis aims to eliminate the cause and to reestablish the original health condition. Several therapies have been proposed to restore peri-implant tissues, among these mechanical debridement, conditioning with chemical agents, flap surgery, bone regeneration procedures, use of antiseptic agents, as well as topic and antibiotic systemic therapy, besides different laser therapies. The selection of the therapeutic technique to be employed depends on bone defect type and size. Due to the variety of peri-implant lesion morphology, measurement, and severity, none of these therapies have been established as totally safe and efficient. The aim of this study was to review the literature on the several alternatives used for peri-implantitis treatment. Although there has not been a treatment consensus established, all therapeutic modalities showed benefits for peri-implantitis treatment within the limitations of each study. Therefore, either individually or associated, therapies could be applied aiming the treatment success of peri-implant lesions.