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.

Treatment of oral myiasis in a patient with implant-supported fixed prosthesis.

Oral myiasis is a rare parasitic disease that requires immediate treatment once diagnosed. However, no standard treatment protocol can be found in the literature. Through a clinical-surgical report, we present the case of an 82-year-old man with lesions extending through the vestibule and alveolar ridge of the maxilla on both sides, in addition to occupying a large part of the palate, with a considerable number of larvae. The patient was initially treated with a single dose of systemic ivermectin (6 mg orally) and topical application of a tampon soaked in ether. The larvae were then surgically removed and debridement of the wound was performed. A crushed tablet of ivermectin 6 mg was applied topically for 2 days, the remaining larvae were again mechanically removed, and the patient received intravenous antimicrobial therapy. Treatment with systemic and topical ivermectin combined with antibiotic therapy and debridement proved to be effective in treating oral myiasis.

Treatment of oral myiasis in a patient with implant-supported fixed prosthesis

ABSTRACT Oral myiasis is a rare parasitic disease that requires immediate treatment once diagnosed. However, no standard treatment protocol can be found in the literature. Through a clinical-surgical report, we present the case of an 82-year-old man with lesions extending through the vestibule and alveolar ridge of the maxilla on both sides, in addition to occupying a large part of the palate, with a considerable number of larvae. The patient was initially treated with a single dose of systemic ivermectin (6 mg orally) and topical application of a tampon soaked in ether. The larvae were then surgically removed and debridement of the wound was performed. A crushed tablet of ivermectin 6 mg was applied topically for 2 days, the remaining larvae were again mechanically removed, and the patient received intravenous antimicrobial therapy. Treatment with systemic and topical ivermectin combined with antibiotic therapy and debridement proved to be effective in treating oral myiasis.

Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography-tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazol-Results.

After performing liquid-liquid extraction with ethyl acetate and HCl, samples from 12 volunteers who performed sequential collections after taking a tablet of naproxen alone (n = 6) or associated with esomeprazole (n = 6) were analyzed in a triple quadrupole mass spectrometer 8040 LC MS/MS Shimadzu. Separation of naproxen and its main metabolite 6-O-desmethylnaproxen was performed in a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column at 40°C using a mixture of methanol and ammonium acetate 10 mM (70:30, v/v) with an injection rate of 0.3 ml/min. The total analytical run time for each sample was 5 min. The association of naproxen with esomeprazole take considerably longer time to reach the maximum concentration [Tmax 0.17 h (interquartile range, 0.13-1.95) for naproxen alone and 13.18*h (interquartile range, 10.12-27.15) for naproxen with esomeprazole, p = 0.002], also to be eliminated [T1/2 0.12 h (interquartile range, 0.09-1.35) for naproxen alone and 9.16*h (interquartile range, 7.16-41.40) for naproxen with esomeprazole, p = 0.002] and lower maximum concentrations (Cmax 4.6 ± 2.5 ug/mL for naproxen alone and 2.04 ± 0.78* µg/mL, p = 0.038). The association of naproxen with esomeprazole showed increased values of AUC0-t [82.06* h*µg/mL (interquartile range, 51.90-157.00) with esomeprazole and 2.97 h*µg/mL (interquartile range, 1.82-7.84) naproxen alone, p = 0.002] in drug concentrations in relation to the naproxen tablet alone, probably, such differences are due to the delay in the absorption of naproxen when it is associated with the drug proton pump inhibitor, esomeprazole. As well as reduced values of full clearance when naproxen is combined with esomeprazole (0.07* µg/h (interquartile range, 0.005-0.01) with esomeprazole and 7.29 µg/h (interquartile range, 3.17-16.23) in naproxen alone, p = 0.002). Both naproxen and 6-O-desmethylnaproxen in saliva samples can be effectively quantified using LC-MS/MS, this methodology proved to be rapid, sensitive, accurate and selective for each drug and allows for the analysis of their pharmacokinetic parameters, in both situations.

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.

Detection and quantification of prostaglandin E2 in saliva by liquid chromatography-tandem mass spectrometry using microextraction by packed sorbent.

The detection of eicosanoids in saliva samples can assist pharmacokinetic/pharmacodynamic studies due to the facility of obtaining samples, minimal discomfort and high adherence of volunteers to the study. The present study enabled determine prostaglandin E2 concentrations in saliva samples, using a microextraction by packed sorbent methodology and subsequent detection in liquid chromatography-tandem mass spectrometry. Twelve volunteers underwent scaling and coronary-radicular polishing of the upper molars and sequential saliva collections: 0.25-96 h after ingestion of a 600 mg ibuprofen tablet, to quantify prostaglandin E2 concentrations. There was an increase in the level of prostaglandin E2 with a significant difference after the dental procedure (0.25 h) compared to 11, 24, 48 and 72 h (*p < 0.05). After taking the drug, these levels begin to decrease up to 5 h, returning to normal in the subsequent hours. The method was developed and validated with linearity between 2.4 and 1250 ng/mL and r2 above 0.9932. The limit of quantitation was about 2.4 ng/mL. The coefficients of variation and the relative standard errors of the accuracy and precision analyzes were < 15%. The proposed extraction and analysis methodology proved to be efficient, fast and promising for pharmacokinetic/pharmacodynamic assays after using anti-inflammatory drugs.

Analysis of Different Methods of Extracting NSAIDs in Biological Fluid Samples for LC-MS/MS Assays: Scoping Review.

The aim of this study was to carry out a systematic investigation and analysis of different drug extraction methods, specifically non-steroidal anti-inflammatory drugs in biological fluid samples, for Liquid Chromatography in Mass Spectrometry assays (LC-MS/MS). A search was carried out in the main databases between 1999 and 2021, following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) checklist. Data were obtained through PubMed, Lilacs, Embase, Scopus, and Web of Science databases using the Boolean operators AND and OR. Studies were pre-selected by title and abstract by two independent reviewers. The selected texts were read in full, and only those that were complete and compatible with the inclusion and exclusion criteria were eligible for this research. A total of 248 references were obtained in the databases. After removing the duplicates and analyzing the titles and abstracts, 79 references were evaluated and passed to the next phase, which comprised the complete reading of the article. A total of 39 publications were eligible for this study. In 52% of the studies, the authors used the liquid-liquid extraction method (LLE), while in 41%, the solid-phase extraction method (SPE) was used. A total of 5% used microextraction methods and 2% used less-conventional techniques. The literature on the main methods used, the LLE and SPE methods, is extensive and consolidated; however, we found other studies that reported modifications of these traditional techniques, which were equally validated for use in LC-MS/MS. From this review, it is concluded that the diversity of techniques, reliability, and practical information about each analytical method used in this study can be adapted to advances in LC-MS/MS techniques; however, more ecological, economic, and sustainable approaches should be explored in the future.

Multifocal Analysis of Acute Pain After Third Molar Removal.

Background: To analyze the pain modulation capacity profile in a Brazilian population, the relationship between opioid receptor (OPRM1) and Catechol-O-methyltransferase (COMT) 1polymorphisms and pain modulation capacity was determined through preoperative pain modulation tests and acute postoperative pain control evaluation, swelling, and trismus in 200 volunteers undergoing lower third molar removal. Methods: Psychologic and clinical parameters were measured. Patient DNA was sequenced for single nucleotide polymorphisms in OPRM1 and COMT, and the salivary concentration of interleukin (IL)-2 (IL)-6, interferon (IFN)-γ and tumor necrosis factor (TNF)-α was evaluated. Primary outcomes were the influence of all predictors on the fluctuation of pain intensity using a visual analogue scale (VAS), and swelling and trismus on the 2nd and 7th postoperative days. Preoperative pain modulation capacity (CPM), pain catastrophizing scale (PCS), body mass index (BMI), and surgery duration and difficulty were evaluated. Results: Salivary concentration of IFN-γ and IL-2 as well as the duration of surgery influenced the fluctuation of postoperative pain in the VAS, and in the sum of the differences in pain intensity test at 8, 48, and 96 h. BMI influenced swelling, while both BMI and COMT haplotype influenced trismus on the 2nd postoperative day. Conclusion: Polymorphisms in COMT, salivary concentrations of IL-2 and IFN-γ, BMI, and duration of surgery were predictors for pain fluctuation, swelling, and trismus on the 2nd day after lower third molar extraction. This therapy was effective in controlling inflammatory symptomatology after lower third molar extraction and ibuprofen was well tolerated by patients. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03169127.

Pharmacogenetic and Pharmacokinetic Assays from Saliva Samples Can Guarantee Personalized Drug Prescription.

Saliva is widely used for clinical and laboratory analysis. This study proposed to use DNA extracted from saliva for genotyping and pharmacokinetics of piroxicam. A fast and efficient genotyping method was used to determine relevant allelic variants of CYP2C9 (*2 and *3), since genetic factors can influence in non-steroidal anti-inflammatory drugs (NSAIDs) metabolization. DNA Extract All Reagents Kit® was used for DNA extraction and genotyping was performed using TaqMan® GTXpress™ Master Mix, SNP genotyping assays and a Viia7 Real-Time PCR system. Volunteers performed sequential collections of saliva samples before and after taking a single dose of piroxicam (0.25 to 72 h) which were used for pharmacokinetics assays. Piroxicam concentrations were analyzed using LC-MS/MS. Sixty-six percent of volunteers were ancestral homozygous (CYP2C9*1/*1), and 34% showed one or both polymorphisms. Of these 34%, 22 individuals showed CYP2C9*2 polymorphism, 8 CYP2C9*3, and 4 CYP2C9*2/*3. Piroxicam pharmacokinetics were performed in 5 subjects. Areas under the curve (AUC0-t(h*ng/mL)) for CYP2C9*1/*1, *1/*2 and *1/*3 were, respectively, 194.33±70.93, 166 and 303. Maximum concentrations (Cmax(ng/mL)) for these genotypes were respectively 6.46±2.56, 4.3 and 10.2. Saliva sampling was a very effective matrix for both pharmacogenetic and pharmacokinetic tests, ensuring the speed of the procedure and the well-being and agreement of the participants. Once having the knowledge about the slow and fast metabolizers, it is possible to make an adequate prescription in order to avoid the adverse effects of the medication and to guarantee greater analgesic comfort to the patients respectively.

Simultaneous separation of naproxen and 6-O-desmethylnaproxen metabolite in saliva samples by liquid chromatography-tandem mass spectrometry: Pharmacokinetic study of naproxen alone and associated with esomeprazole.

Naproxen is a widely used non-steroidal anti-inflammatory drug for the control of postoperative inflammatory signs and symptoms in dentistry. Its association with esomeprazole has been widely studied and has yielded good results for the control of acute pain, even with the delayed absorption of naproxen owing to the presence of esomeprazole. To further understand the absorption, distribution, and metabolism of this drug alone and in combination with esomeprazole, we will analyze the pharmacokinetic parameters of naproxen and its major metabolite, 6-O-desmethylnaproxen, in saliva samples. A rapid, sensitive, and selective liquid chromatography-tandem mass spectrometric method for the simultaneous determination of naproxen and 6-O-desmethylnaproxen in saliva will be developed and validated. Sequential saliva samples from six patients will be analyzed before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 8, 11, 24, 48, 72, and 96 h after the ingestion of one naproxen tablet (500 mg) and esomeprazole-associated naproxen tablets (500 + 20 mg), at two different times. After liquid-liquid extraction with ethyl acetate and HCl, the samples will be analyzed using an 8040 Triple Quadrupole Mass Spectrometer (Shimadzu, Kyoto, Japan). Separation of naproxen and its major metabolic products will be performed using a Shim-Pack XR-ODS 75Lx2.0 column and C18 pre-column (Shimadzu, Kyoto, Japan) 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 will be 5 min. The detection and quantification of naproxen and its metabolite will be validated, which elucidate the pharmacokinetics of this drug, thereby contributing to its proper prescription for the medical and dental interventions that cause acute pain.

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.

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.

Dental Pulp Fibroblasts Response after Stimulation with HEMA and Adhesive System.

This study evaluated in vitro cell viability and metabolism, nitric oxide release and production of chemokines by cultured human dental pulp fibroblasts (DPF) under contact with HEMA and Single Bond. Cultures of DPF were established by means of an explant technique. Once plated, cells were kept under contact with increasing concentrations of HEMA (10, 100 and 1000 nM) or Single Bond (SB) [10-fold serially diluted in culture medium (10-4, 10-3 and 10-2 v/v)] and also with polymerized SB components. Cytotoxicity was assessed by Trypan Blue exclusion method and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide release on cell supernatant was detected by Griess Method whereas chemokines (CXCL12 and CXCL8) were detected by ELISA. RT-qPCR was employed for chemokines gene expression analysis. Cytotoxic tests showed significant differences for SB 10-2. None of the tested materials significantly altered NO levels. Protein levels of CXCL12 were significantly decreased only by HEMA. On the other hand, while CXCL12 mRNA remained unaltered, gene expression of CXCL8 had significant decrease with all materials, except for polymerized SB. In conclusion, Single Bond and HEMA at various concentrations, decreased expression and production of molecules involved in inflammatory processes and, therefore, the use of adhesive systems such as pulp capping materials must be viewed with caution due to its large cytotoxic effect when in close contact with the pulp.

Dental pulp fibroblasts response after stimulation with hema and adhesive system

Abstract This study evaluated in vitro cell viability and metabolism, nitric oxide release and production of chemokines by cultured human dental pulp fibroblasts (DPF) under contact with HEMA and Single Bond. Cultures of DPF were established by means of an explant technique. Once plated, cells were kept under contact with increasing concentrations of HEMA (10, 100 and 1000 nM) or Single Bond (SB) [10-fold serially diluted in culture medium (10-4, 10-3 and 10-2 v/v)] and also with polymerized SB components. Cytotoxicity was assessed by Trypan Blue exclusion method and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide release on cell supernatant was detected by Griess Method whereas chemokines (CXCL12 and CXCL8) were detected by ELISA. RT-qPCR was employed for chemokines gene expression analysis. Cytotoxic tests showed significant differences for SB 10-2. None of the tested materials significantly altered NO levels. Protein levels of CXCL12 were significantly decreased only by HEMA. On the other hand, while CXCL12 mRNA remained unaltered, gene expression of CXCL8 had significant decrease with all materials, except for polymerized SB. In conclusion, Single Bond and HEMA at various concentrations, decreased expression and production of molecules involved in inflammatory processes and, therefore, the use of adhesive systems such as pulp capping materials must be viewed with caution due to its large cytotoxic effect when in close contact with the pulp.

Resumo Este estudo avaliou in vitro a viabilidade e metabolismo celular, liberação de óxido nítrico e produção de quimiocinas em cultura de fibroblastos de polpa dental humana (DPF) em contato com HEMA e Single Bond. Culturas de DPF foram estabelecidas por meio de uma técnica de explante. Uma vez plaqueadas, as células foram mantidas em contato com concentrações crescentes de HEMA (10, 100 e 1000 nM) ou Single Bond (SB) [10 vezes diluídas em série em meio de cultura (10-4, 10-3 e 10-2 v/v)] e também com SB polimerizado. A citotoxicidade foi avaliada pelo método de exclusão de Trypan Blue e pelo ensaio de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio brometo (MTT). A liberação de óxido nítrico no sobrenadante celular foi detectada pelo método de Griess, enquanto as quimiocinas (CXCL12 e CXCL8) foram detectadas por ELISA. RT-qPCR foi empregada para análise de expressão gênica de quimiocinas. Testes citotóxicos mostraram diferenças significativas para SB 10-2. Nenhum dos materiais testados alterou significativamente os níveis de NO. Os níveis de proteína de CXCL12 foram significativamente diminuídos apenas pelo HEMA. Por outro lado, enquanto o RNAm de CXCL12 permaneceu inalterado, a expressão gênica de CXCL8 teve redução significativa com todos os materiais, com exceção do SB polimerizado. Em conclusão, Single Bond e HEMA, em várias concentrações, diminuíram a expressão e produção de moléculas envolvidas em processos inflamatórios e, portanto, o uso de sistemas adesivos, como o material protetor da polpa, deve ser visto com cautela devido ao seu grande efeito citotóxico quando em contato com a polpa.

Efficacy of Ketoprofen With or Without Omeprazole for Pain And Inflammation Control After Third Molar Removal.

In view of the gastrointestinal problems generated by the ketoprofen use, the ketoprofen association with omeprazole is available on the market. However, this association efficacy in acute pain control has not been established. Bilateral extraction of lower third molars in similar positions is currently the most used model for the evaluation and investigation of the efficacy and pharmacological effects of new compounds for the treatment of acute postoperative pain. The randomized and crossover study consisted in evaluating the clinical efficacy of therapy performed by ketoprofen 100 mg (twice daily-b.i.d.) versus ketoprofen 200 mg + omeprazole 20 mg (once daily-q.d.) to pain, swelling and trismus control in the bilateral extraction model of lower third molars in similar positions in two different appointments, in 50 volunteers. Volunteers reported significantly less postoperative pain at various post-operative periods and consumed less rescue analgesic medication (acetaminophen 750 mg) throughout the study when they took the combination of ketoprofen 200 mg + omeprazole 20 mg (q.d.). Following administration of both study drugs, no gastrointestinal adverse reactions were reported by volunteers. Furthermore, the evaluations of the drugs in pain control by the volunteers were significantly favorable to ketoprofen 200 mg + omeprazole 20 mg (q.d.). For swelling and trismus control, the treatments presented similar results. In conclusion, when volunteers took ketoprofen 200 mg + omeprazole 20 mg (q.d.), they reported significantly less postoperative pain at various post-surgical periods and consumed less rescue analgesic medication throughout the study compared with ketoprofen 100 mg (b.i.d).

Ideal Versus Late Secondary Alveolar Bone Graft Surgery: A Bone-Thickness Cone-Beam Computed Tomographic Assessment.

OBJECTIVE: To compare the bone morphology after secondary alveolar bone graft surgery (SABG) performed before and after permanent canine eruption. DESIGN: Cross-sectional study. SETTING: Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo, Bauru, SP, Brazil. PATIENTS: 25 cone-beam computed tomography (CBCT) scans of complete unilateral cleft lip and palate (CLP) individuals who underwent SABG before or after eruption of the permanent canine taken 2 and 6 months (T1 and T2) after SAGB, resulting in 50 CBCT scans. Two groups were assessed, Ideal Group (IG; n = 10) and Late Group (LG; n = 15), according to the time of the SABG. INTERVENTIONS: SABG buccal-palatal thicknesses were measured in 3 different root levels: cement-enamel junction (cervical slice), middle point of the root (intermediate slice), and apex of the central incisor (apical slice). Thickness measurements were assessed in the mesial, distal, and intermediate aspects of the alveolar bone graft. Clinical long-term follow-up was also done. RESULTS: The IG showed significantly greater bone thickness, especially in the intermediate and apical slices, when compared to LG, in T1 and T2. Bone thickness was maintained over time. Clinically, all the IG individuals completed orthodontics, and no major complications were observed. In contrast, 27% of the LG individuals had failures, and rehabilitation was achieved through prosthesis. CONCLUSION: Ideal SABG presents with better results compared with late ABG. When it is not possible to perform SABG at the ideal time, acceptable outcomes still can be expected for late bone grafting.

Efficacy of piroxicam for postoperative pain after lower third molar surgery associated with CYP2C8*3 and CYP2C9.

OBJECTIVE: Nonsteroidal anti-inflammatory drugs (NSAIDs) are metabolized by the cytochrome P450 enzymes (CYPs), predominantly CYP2C8 and CYP2C9. The aim of this study was to evaluate the possible association of polymorphisms in the CYP2C8*3 and CYP2C9 genes with the clinical efficacy of oral piroxicam (20 mg daily for 4 days) after lower third molar surgeries with regard to postoperative pain, swelling, trismus, adverse reactions, need for rescue medication and the volunteer's overall satisfaction. MATERIALS AND METHODS: For this purpose, 102 volunteers were genotyped for CYP2C8*3 and CYP2C9 polymorphisms. Briefly, genomic DNA was isolated from saliva collected from volunteers subjected to invasive lower third molar surgeries, and the preoperative, intraoperative and postoperative parameters were collected and analyzed. RESULTS: An equal amount of piroxicam sufficiently managed postoperative pain and inflammatory symptoms, with visual analog pain scores typically <40 mm for all genotypes investigated. Furthermore, only two out of 102 volunteers heterozygous for CYP2C8*3 and CYP2C9*3 reported adverse side effects. CONCLUSION: In general, slow metabolizers of piroxicam, who were volunteers with mutant alleles, were indifferent from normal metabolizers with the wild-type alleles and therefore did not require specialized piroxicam doses to manage postoperative pain and inflammation.

Effective method for the detection of piroxicam in human plasma using HPLC.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used by the general population to alleviate inflammation and pain after oral surgeries. Piroxicam is among the most commonly used NSAIDs and excels in controlling pain, swelling, trismus and other common symptoms of inflammation. This study aimed to evaluate different concentrations of piroxicam and its major metabolite, 5'-hydroxypiroxicam, in human plasma samples over time using high performance liquid chromatography (HPLC) after liquid-liquid extraction. Briefly, 10 volunteers participated in this study after approval by the Ethics Committee of Bauru School of Dentistry, Universidade de São Paulo - USP, Brazil. Volunteers received a single dose oral of piroxicam (20 mg) and had blood collected at various times following an established protocol. The methodology of liquid-liquid extraction was effective for determining concentrations of piroxicam in plasma using HPLC in 10 out of 10 volunteers while 5'-hydroxypiroxicam was only detected in 2 out of 10 volunteers.

Quantification of piroxicam and 5'-hydroxypiroxicam in human plasma and saliva using liquid chromatography-tandem mass spectrometry following oral administration.

Saliva sampling used to quantify piroxicam and 5'-hydroxypiroxicam is a noninvasive and painless method when compared to sequential blood sampling. For that, a rapid, selective and sensitive liquid chromatography-tandem mass spectrometric method for simultaneous determination of piroxicam and 5'-hydroxypiroxicam in saliva and human plasma was developed and validated. Piroxicam and its major metabolite were separated using a LiChroCART 125-4 RP Select-B Sorbent C18 column using a mixture of methanol and 2% phosphoric acid (pH 2.7) (70:30, v/v) for the mobile phase with a flow injection of 1mL/min. The run time was 4min. Volunteers had saliva and blood sampled before, 1, 2, 3, 4, 5, 6, 8, 11, 24, 48 and 72h after taking a 20mg oral dose of piroxicam. The pharmacokinetic parameters of piroxicam in plasma samples were as follows: AUC0-72 (64819hng/mL), predicted clearance (0.2L/h), distribution volume (14.8L), elimination half-life (50.7h) and saliva/plasma concentration ratio (0.003). The estimation of all pharmacokinetic parameters for 5'-hydroxypiroxicam would require collections beyond 72h; however, it was possible to quantify the mean maximum concentration (133ng/mL), time to peak concentration (53.6h), mean AUC0-72 (6213hng/mL), predicted clearance (110.3L/h) and saliva/plasma concentration ratio (0.04). The developed methods proved effective and sensitive for determining the lower quantification limit of piroxicam in plasma (6.1ng/mL) and saliva (0.15ng/mL) and of 5'-hydroxypiroxicam in plasma (1.2ng/mL) and saliva (0.15ng/mL).