Solvent-free parallel artificial liquid membrane extraction for drugs of abuse in plasma samples using LC-MS/MS.

BACKGROUND: Parallel artificial liquid membrane extraction (PALME) is a 96-well plate setup variant of liquid-phase microextraction. Basic or acidic analytes are extracted in neutral form from the sample, through a supported liquid membrane (SLM), and into aqueous acceptor. PALME is already considered a green extraction technique, but in the current conceptual work, we sought to make it even greener by replacing the use of organic solvents with essential oils (EO). PALME was combined with LC-MS/MS for analysis of plasma samples and multiple drugs of abuse with toxicological relevance (amphetamines, phenethylamines, synthetic cathinones, designer benzodiazepines, ayahuasca alkaloids, lysergic acid diethylamide, and ketamine). RESULTS: Fourteen EO were compared to organic solvents frequently used in PALME. The EO termed smart & sassy yielded the best analyte recovery for all drugs studied and was thus selected as SLM. Then, factorial screening and Box-Behnken were employed to optimize the technique. The extraction time, concentration of base, sample volume, and percentage of trioctylamine significantly impacted analyte recovery. The optimum values were defined as 120 min, 10 mmol/L of NaOH, 150 µL, and 0%, respectively. Once optimized, validation parameters were 1-100 ng mL-1 as linear range, accuracy ±16.4%, precision >83%, 1 ng mL-1 as limit of quantitation, 0.1-0.75 ng mL-1 as limit of detection, matrix effect <20%, and recovery 20-106%. Additionally, EO purchased from different production batches were tested and achieved acceptable reproducibility. Data were in compliance with requirements set by internationally accepted validation guidelines and the applicability of the technique was proven using authentic samples. SIGNIFICANCE: In this study, the use of an EO provided a solvent-free sample preparation technique suited to extract different classes of drugs of abuse from plasma samples, dismissing the use of hazardous organic solvents. The method also provided excellent sample clean-up, thus being a simple and efficient tool for toxicological applications that is in agreement with the principles of sustainable chemistry.

A new application of the switchable hydrophilicity solvent-based homogenous liquid-liquid microextraction to analyze synthetic cannabinoids in plasma by LC-MS/MS.

Synthetic cannabinoids are still a growing trend among drug users and consist of a group of hundreds of highly potent compounds. To investigate the use of such substances, sample preparation of biological matrices is a crucial step prior to instrumental analysis. Although different efficient extraction techniques have been proposed for that aim, they usually do not fit eco-friendly guidelines that have been gaining popularity in recent years, such as Green Analytical Toxicology. This work uses describes for the first time the use of switchable hydrophilicity solvent-based homogenous liquid-liquid microextraction (SHS-HLLME) for synthetic cannabinoids. This is a green technique that replaces highly toxic organic reagents for switchable hydrophilicity solvents (SHS), substances that can be either water-miscible or immiscible depending on their protonation. Thus, by simply adjusting the pH of the system, these SHS can be used as extraction solvents. A full optimization study including type of SHS, volume of protonated SHS, volume of NaOH, salting-out effect, and extraction time was performed. The optimized procedure consisted of precipitating the proteins of 300 µL of plasma with 300 µL of acetonitrile followed by centrifugation; evaporation of the organic solvent under N2 stream; addition of 500 µL of the protonated DPA, DPA-HCl (6 M) (1:1, v/v); addition of 500 µL of NaOH (10 M); and finally centrifugation and evaporation. Validation results showed determination coefficients ≥ 0.99 for the 0.1-10 ng/mL linear range; 0.01-0.08 ng/mL as limit of detection; 0.1 ng/mL as limit of quantitation; accuracy and imprecision were within acceptable ranges; matrix effect, recovery, and process efficiency ranged from -55.6 to 185.9%, 36-56.7%, and 18.5-148.4%, respectively. The SHS-HLLME herein described was fully optimized providing satisfactory recoveries of 31 synthetic cannabinoids at low concentrations requiring only 300 µL of plasma. In addition, the validation results showed that the technique is a reliable eco-friendly alternative for clinical and toxicological analysis.

The mystery behind the apprehensions of the selective cannabinoid receptor type-2 agonist BZO-HEXOXIZID (MDA-19) as a drug of abuse.

PURPOSE: MDA-19 or BZO-HEXOXIZID (N'-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-benzohydrazide), in a more recent nomenclature, was first synthesized in 2008 as a selective type-2 cannabinoid receptor (CB2) agonist due to its potential to treat neuropathic pain. In Brazil, this substance was identified in a series of 53 apprehensions between September 2021 and February 2022. Nevertheless, what intrigues toxicologists is that BZO-HEXOXIZID does not exert significant type-1 cannabinoid receptor (CB1) agonism-which is responsible for the well-known psychoactivity of Δ-9-tetrahydrocannabinol. Thus, the objective of this work is to report the first apprehension and identification of BZO-HEXOXIZID in Brazil and to discuss pharmacologically the possible reasons why a CB2 agonist has been incorporated to the illicit market. METHODS: Suspected seized samples were sent to the Laboratory of the Scientific Police of the State of Sao Paulo. After the screening, samples were confirmed for the presence of BZO-HEXOXIZID using chromatography gas-mass spectrometry, Fourier-transform infrared spectroscopy and nuclear magnetic resonance techniques. RESULTS: Of the 53 samples analyzed, 25 contained only BZO-HEXOXIZID and 28 with mixtures, of which 11 with the CB1 agonist ADB-BUTINACA. Other substances were found in association such as cocaine and caffeine. CONCLUSIONS: BZO-HEXOXIZID was detected in a series of seized materials for the first time in Brazil. Nevertheless, there are still unanswered questions regarding the use of this selective CB2 agonist as a drug of abuse.

Development of a Dispersive Liquid-Liquid Microextraction for Synthetic Cathinones in Biological Fluids Based on Principles of Green Analytical Toxicology.

The present work describes a practical application of Green Analytical Toxicology (GAT) during the development of an eco-friendly dispersive liquid-liquid microextraction (DLLME) avoiding the use of highly toxic chlorinated solvents that are commonly used in this type of the technique. The purpose was to further consolidate GAT guidelines during method development. Thus, a full method optimization using a multivariate statistical approach and validation were performed. To that end, synthetic cathinones (SCs), one of the major classes of new psychoactive substances, were the target analytes due to their relevance and chemical diversity. Furthermore, whole blood and urine samples were the matrices of choice due to their clinical relevance. The sample preparation step prior to DLLME consisted of protein precipitation of whole blood samples, while urine specimens were centrifuged and diluted with ultrapure water. Then, borate buffer, sodium chloride and ethyl acetate:acetonitrile were added and vortexed. Finally, vials were centrifuged and the organic layer was transferred to autosampler vials, evaporated to dryness and resuspended with mobile phase prior to injection into the ultra-high performance liquid chromatography-tandem mass spectrometry system. Once optimized, the proposed DLLME was fully validated: 0.2 and 1 ng/mL as the limit of detection and 1 and 10 ng/mL as the limit of quantitation for urine and blood samples, respectively. The linear range was established as 1-100 and 10-1,000 ng/mL for urine and blood samples, respectively (r2 > 0.99), while the bias and precision were within acceptable limits (≥80%). The matrix effect was of 1.9-260.2% and -12.3-139.6%; while the recovery was of 27.4-60.0% and 13.0-55.2%; the process efficiency ranged from 45.0% to 192.0% and 17.9% to 58.4% for whole blood and urine, respectively. Finally, the method was applied to real case samples as proof of applicability. Thus, a simple, cheap and fast eco-friendly technique to analyze SCs in two biological specimens was described.

Green Analytical Toxicology for the Determination of Cocaine Metabolites.

Brazil is the third largest contributor to Green Analytical Chemistry, and there is significant participation of toxicologists in the development and improvement of environmental techniques. Currently, toxicologists have their own strategies and guidelines to promote the reduction/replacement or elimination of solvents, reduce the impacts of derivatization and save time, among other objectives, due to the peculiarities of toxicological analysis. Thus, this review aims to propose the concept of Green Analytical Toxicology and conduct a discussion about its relevance and applications specifically in forensic toxicology, using the microextraction methods developed for the determination of cocaine and its metabolites as examples.

Lucy is back in Brazil with a new dress.

Lucy in the Sky with Diamonds, a John Lennon song that was a hit in the 1960s, was born amidst a social context enlightened by lysergic acid diethylamide (LSD). In Brazil, both the drug and the song were very popular at the time, although it gradually mitigated. Nevertheless, while the song remains out of the spotlight, LSD derivatives are currently gaining attention with the rising of the new psychoactive substances (NPS). With this new presentation, the drug is returning to Brazil after a few decades and herein we report and discuss the first cases of an LSD prodrug seized in our country. Nine suspected blotter paper samples were seized by the Sao Paulo State Police in different cities of the State. Gas chromatography-mass spectrometry (GC-MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and liquid chromatography coupled with electrospray ionization-mass spectrometry (LC-ESI-MS) analyses were utilized to confirm the identity of the LSD derivative. The compound was identified as 4-acetyl-N,N-diethyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ALD-52 or 1A-LSD) and no other active substance was detected in all samples. The identity of the unknown compound found in seized blotter papers has been successfully confirmed as an LSD prodrug, ALD-52, which was not controlled by Brazilian legislation. The arrival of a new type of designer drug in Brazil is in support by other reports, although those are still scarce and should not be overlooked. Altogether, these findings indicate the rising of a new NPS strategy that merits proper discussion.

Dried matrix spots in forensic toxicology.

Dried matrix spots (DMS) has gained the attention of different professionals in different fields, including toxicology. Investigations have been carried out in order to assess the potential of using DMS for the analysis of illicit substances, the main interest of forensic toxicologists. This technique uses minimal volumes of samples and solvents, resulting in simple and rapid extraction procedures. Furthermore, it has proved to increase analyte stability, improving storage and transportation. However, DMS presents some limitations: the hematocrit influencing accuracy and inconsistencies regarding the means of spotting samples and adding internal standard on paper. Thus, we provide an overview of analytical methodologies with forensic applications focusing on drugs of abuse and discussing the main particularities, limitations and achievements.

Hydroquinone Exposure Worsens Rheumatoid Arthritis through the Activation of the Aryl Hydrocarbon Receptor and Interleukin-17 Pathways.

Rheumatoid arthritis (RA) development is strongly associated with cigarette smoke exposure, which activates the aryl hydrocarbon receptor (AhR) as a trigger for Th17 inflammatory pathways. We previously demonstrated that the exposure to hydroquinone (HQ), one of the major compounds of cigarette tar, aggravates the arthritis symptomatology in rats. However, the mechanisms related to the HQ-related RA still remain elusive. Cell viability, cytokine secretion, and gene expression were measured in RA human fibroblast-like synoviocytes (RAHFLS) treated with HQ and stimulated or not with TNF-α. Antigen-induced arthritis (AIA) was also elicited in wild type (WT), AhR -/- or IL-17R -/- C57BL/6 mice upon daily exposure to nebulized HQ (25ppm) between days 15 to 21. At day 21, mice were challenged with mBSA and inflammatory parameters were assessed. The in vitro HQ treatment up-regulated TNFR1, TNFR2 expression, and increased ROS production. The co-treatment of HQ and TNF-α enhanced the IL-6 and IL-8 secretion. However, the pre-incubation of RAHFLS with an AhR antagonist inhibited the HQ-mediated cell proliferation and gene expression profile. About the in vivo approach, the HQ exposure worsened the AIA symptoms (edema, pain, cytokines secretion and NETs formation) in WT mice. These AIA effects were abolished in HQ-exposed AhR -/- and IL-17R -/- animals though. Our data demonstrated the harmful HQ influence over the onset of arthritis through the activation and proliferation of synoviocytes. The HQ-related RA severity was also associated with the activation of AhR and IL-17 pathways, highlighting how cigarette smoke compounds can contribute to the RA progression.

Hydroquinone exposure alters the morphology of lymphoid organs in vaccinated C57Bl/6 mice.

The influenza is a common viral infection that can be fatal, especially in high-risk groups such as children, pregnant women, elderly, and immune-deficient individuals. Vaccination is the most efficient approach to prevent the spreading of viral infection and promote individual and public health. In contrast, exposure to environmental pollutants such as cigarette smoke reduces the efficacy of vaccination. We investigated whether chronic exposure to hydroquinone (HQ), the most abundant compound of the tobacco particulate phase, could impair the adaptive immune responses elicited by influenza vaccination. For this, adult male C57BL/6 mice were daily exposed to either nebulized HQ or PBS for 1 h for a total of eight weeks. At weeks 6 and 8, the mice were primed and boosted with the trivalent influenza vaccine via IM respectively. Although the HQ exposure did not alter the body weight of the mice and the biochemical and hematological parameters, the pollutant increased the oxidative stress in splenocytes of immunized animals, modified the morphology of spleen follicles, and augmented the size of their lymph nodes. The lymphoid organs of HQ-exposed mice presented a similar number of vaccine-specific IgG-secreting cells, titers of vaccine-specific total IgG, and respective subclasses. Transcriptome studies with HQ, benzene, or cigarette smoke exposure were also analyzed. The genes up-regulated upon pollutant exposure were associated with neutrophil migration and were shown to be co-expressed with antibody-secreting cell genes. Therefore, these findings suggest that HQ exposure may trigger an immune-compensatory mechanism that enhances the humoral responses induced by influenza vaccination.

Hydroquinone exposure alters the morphology of lymphoid organs in vaccinated C57Bl/6 mice

The influenza is a common viral infection that can be fatal, especially in high-risk groups such as children, pregnant women, elderly, and immune-deficient individuals. Vaccination is the most efficient approach to prevent the spreading of viral infection and promote individual and public health. In contrast, exposure to environmental pollutants such as cigarette smoke reduces the efficacy of vaccination. We investigated whether chronic exposure to hydroquinone (HQ), the most abundant compound of the tobacco particulate phase, could impair the adaptive immune responses elicited by influenza vaccination. For this, adult male C57BL/6 mice were daily exposed to either nebulized HQ or PBS for 1 h for a total of eight weeks. At weeks 6 and 8, the mice were primed and boosted with the trivalent influenza vaccine via IM respectively. Although the HQ exposure did not alter the body weight of the mice and the biochemical and hematological parameters, the pollutant increased the oxidative stress in splenocytes of immunized animals, modified the morphology of spleen follicles, and augmented the size of their lymph nodes. The lymphoid organs of HQ-exposed mice presented a similar number of vaccine-specific IgG-secreting cells, titers of vaccine-specific total IgG, and respective subclasses. Transcriptome studies with HQ, benzene, or cigarette smoke exposure were also analyzed. The genes up-regulated upon pollutant exposure were associated with neutrophil migration and were shown to be co-expressed with antibody-secreting cell genes. Therefore, these findings suggest that HQ exposure may trigger an immune-compensatory mechanism that enhances the humoral responses induced by influenza vaccination.

Hydroquinone exposure alters the morphology of lymphoid organs in vaccinated C57Bl/6 mice

The influenza is a common viral infection that can be fatal, especially in high-risk groups such as children, pregnant women, elderly, and immune-deficient individuals. Vaccination is the most efficient approach to prevent the spreading of viral infection and promote individual and public health. In contrast, exposure to environmental pollutants such as cigarette smoke reduces the efficacy of vaccination. We investigated whether chronic exposure to hydroquinone (HQ), the most abundant compound of the tobacco particulate phase, could impair the adaptive immune responses elicited by influenza vaccination. For this, adult male C57BL/6 mice were daily exposed to either nebulized HQ or PBS for 1 h for a total of eight weeks. At weeks 6 and 8, the mice were primed and boosted with the trivalent influenza vaccine via IM respectively. Although the HQ exposure did not alter the body weight of the mice and the biochemical and hematological parameters, the pollutant increased the oxidative stress in splenocytes of immunized animals, modified the morphology of spleen follicles, and augmented the size of their lymph nodes. The lymphoid organs of HQ-exposed mice presented a similar number of vaccine-specific IgG-secreting cells, titers of vaccine-specific total IgG, and respective subclasses. Transcriptome studies with HQ, benzene, or cigarette smoke exposure were also analyzed. The genes up-regulated upon pollutant exposure were associated with neutrophil migration and were shown to be co-expressed with antibody-secreting cell genes. Therefore, these findings suggest that HQ exposure may trigger an immune-compensatory mechanism that enhances the humoral responses induced by influenza vaccination.

Efeito da exposição à hidroquinona na resposta imune adaptativa induzida pela vacina contra a influenza / Effects of hydroquinone exposure on the adaptive immune response induced by the influenza vaccine

A gripe é causada pelo vírus Influenza e é um problema de saúde pública mundial, que pode levar a problemas sérios em idosos e crianças. O Brasil implantou a vacinação anual contra influenza a partir de 1999, como ação preventiva contra a doença. A vacina é produzida pelo Instituto Butantan e contém três cepas diferentes do vírus Influenza fragmentado para induzir resposta imune adaptativa, com produção de anticorpos específicos e neutralizantes. A literatura tem mostrado que a exposição à xenobióticos com potencial imunossupressor pode comprometer a eficácia de imunizações ativas, como a imunização contra a gripe. Nosso grupo de pesquisa tem mostrado que a exposição à hidroquinona (HQ), um composto tóxico presente em altas concentrações na fumaça do cigarro, prejudica a resposta imune inata e adquirida. Assim, este trabalho avaliou o efeito da exposição à HQ sobre a resposta imune à vacinação contra influenza. Camundongos machos da linhagem C57BL/6 foram diariamente expostos à HQ (2500 ppm) ou PBS, por 1 hora, por nebulização, por um período de 8 semanas. Durante este período, foram imunizados nas semanas 6 e 8 do início das exposições, pela injeção i.m. de 100µL da vacina. Os parâmetros tóxicos e imunológicos foram avaliados 7, 35 e 70 dias após a segunda dose da vacina. A exposição à HQ não alterou o peso corpóreo dos animais e nem causou alterações morfológicas no pulmão, fígado e rins (histologia por H&E); reduziu a frequência de hemácias (11%), hematócrito (14%), hemoglobina (14%) e volume celular (4%); causou estresse oxidativo no baço (citometria de fluxo); aumentou a área dos folículos de células B no baço e linfonodomegalia (histologia por H&E). Em conjunto, os dados aqui obtidos mostram que a exposição à HQ afetou mecanismos envolvidos na gênese da imunidade ativa contra influenza. Assim, os dados deste trabalho mostram mecanismos tóxicos ainda não descritos para a HQ, e ressalta a HQ como um poluente ambiental que deve ser considerado nas avaliações de risco

The flu is a health problem worldwide which is caused by the Influenza virus and may result in severe illness in infants and the elderly. The annually vaccination against influenza was implemented in Brazil in 1999 as a preventive measure. The vaccine is produced by Butantan Institute and contains three different strains of the inactivated Influenza virus which induce the adaptive immune response along with production of specific and neutralizing antibodies. The literature has shown that exposure to immunosuppressive xenobiotics may compromise the efficacy of active immunizations, such as influenza. Our research group has shown that exposure to hydroquinone (HQ), a toxic constituent of cigarette smoke, impairs both innate and adaptive immune response. Thus, the aim of this work was to evaluate the effects of HQ on the immune response induced by the influenza vaccine. Male C57BL/6 mice were daily exposed to HQ (2500 ppm) or PBS by nebulization, for 1 hour, for 8 weeks. During the exposure period, the animals were vaccinated on weeks 6 and 8 with 100µL of the vaccine. Toxicologic and immunological parameters were assessed 7, 35 and 70 days after boost administration. HQ exposure did not alter body weight and did not cause morphological alterations in the lungs, liver and kidneys (H&E staining); reduced the frequency of erythrocytes (11%), hematocrit (14%), hemoglobin (14%) and cellular volume (4%) and caused oxidative stress on the spleen (Flow Cytometry); increased the area of B cell follicles in the spleen and increased the size of draining lymph nodes (H&E staining). Altogether, these data show that HQ exposure affected mechanisms involved in the genesis of the adaptive immune response. Thus, the data presented in this work show toxic mechanisms of HQ that have not yet been described, and it also points out HQ as an environmental pollutant which should be considered on risk assessments

Cisto dentígero: diagnóstico e tratamento / Cisto dentígero: diagnosis and treatment / Cisto dentígero: diagnóstico y tratamiento

O cisto dentígero é um cisto odontogênico que é classificado como de desenvolvimento. Normalmente, está relacionado à coroa de um dente incluso, sendo um dos cistos odontogênicos mais frequentes nos ossos gnáticos. Na maioria das vezes é diagnosticado em pacientes entre a segunda e a terceira década de vida, com grande ocorrência em terceiros molares inferiores e caninos superiores. Clinicamente, apresenta evolução lenta, assintomática e pode causar discreta deformidade facial, deslocamento de dentes e alterações de estruturas na região. Radiograficamente, os cistos dentígeros são descritos como lesões radiolúcidas bem delimitadas e uniloculares. Na maioria dos casos, são observados em exames de rotina ou durante a pesquisa da causa da não erupção de um dente permanente. Apesar da singularidade clínica de cada caso, o prognóstico dessa lesão é favorável. O tratamento para o cisto dentígero pode ser a marsupialização em casos de lesões grandes, enucleação com exodontia do dente incluso ou preservação do elemento dental. Este trabalho visa apresentar um caso clínico de cisto dentígero em região posterior de mandíbula, abordando aspectos clínicos, imaginológicos, histopatológicos e terapêuticos, com a finalidade de familiarizar o cirurgião dentista com tal lesão(AU)

The dentigerous cyst is an odontogenic cyst that is classified as developmental cyst. It is usually related to the crown of an included tooth, being one of the most frequent odontogenic cysts in the gnatic bones. Most of the time it is diagnosed in patients between the second and third decade of life, with great occurrence in third molars and upper canines. Clinically, it presents slow evolution, asymptomatic and can cause discreet facial deformity, tooth dislocation and alterations of structures in the region. Radiographically, dentigerous cysts are described as well delimited and unilocular radiolucent lesions. Normally, they are seen in routine examinations or during the investigation of the cause of non-eruption of a permanent tooth. Despite the clinical singularity of each case, the prognosis of this lesion is favorable. The treatment for dentigerous cyst may be marsupialization in cases of large lesions, enucleation with exodontia of the included tooth or preservation of the dental element. This paper aims to present a case report of a dentigerous cyst in the mandible, posterior region, addressing clinical, imaging, histopathological and therapeutic aspects, in order to familiarize the dentist surgeon with such lesion(AU)

El quiste dentígero es un quiste odontogénico que se clasifica como de desarrollo. Normalmente, esta relacionada con la corona de un diente incluido, siendo uno de los quistes odontogénicos más frecuentes en los huesos gnáticos. La mayoría de las veces se diagnostica en pacientes entre la segunda y la tercera década de vida, con gran ocurrencia en terceros molares inferiores y caninos superiores. Clínicamente, presenta evolución lenta, asintomática y puede causar discreta deformidad facial, desplazamiento de dientes y cambios de estructuras en la región. Los cistos dentígeros se describen como lesiones radiolúcidas bien delimitadas y uniloculares. Normalmente, se observan en exámenes de rutina o durante la investigación de la causa de la no erupción de un diente permanente. A pesar de la singularidad clínica de cada caso, el pronóstico de esta lesión es favorable. El tratamiento para el quiste dentígero puede ser la marsupialización en casos de lesiones grandes, enucleación con exodoncia del diente incluido o preservación del elemento dental. Este trabajo pretende presentar un relato de caso clínico de quiste dentígero en mandíbula, región posterior, abordando aspectos clínicos, imaginológicos, histopatológicos y terapéuticos, con la finalidad de familiarizar al cirujano dentista con tal lesión(AU)

Surgical Treatment of Anterior Sinus Wall Fracture Due to Sports Accident.

Frontal bone fractures represent a low percentage of craniofacial fractures. However, a systematic approach and a correct diagnosis are essential for successful treatment and maintenance of physiology of the frontal sinus and late complications. The purpose of this study was to report a clinical patient with anterior wall fracture of the frontal due to sports accident sinus that was surgically treated.

Immediate Treatment of Complex Mandible Fracture in a Pediatric Patient.

The facial fractures in pediatric patients are rare, and they can need an immediate treatment to recover the function and aesthetic to the young patients. However, the open reduction internal fixation is controversial in pediatrics; it can be effectiveness to treat complex mandible fractures. Therefore, we present a case of a pediatric patient with a complex mandible fracture caused by an auto accident. The immediate treatment was successful with the use of open reduction internal fixation.

Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis.

INTRODUCTION: Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. OBJECTIVES: The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. MATERIAL AND METHODS: Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. RESULTS: Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. CONCLUSION: Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.

Simplified Access for Reduction and Fixation of a Frontal Bone Fracture.

The frontal bone fractures occur very often in service units in oral and maxillofacial surgery. Bicoronal access is the most common for the surgical treatment of frontal bone fractures. However, patients are surprised when they receive notice that such invasive access will be made. This approach allows adequate visualization of the fracture site, but may be associated with complications, which can be avoided by the use of less invasive techniques. We describe an alternative approach that aims to minimize the complications of a coronal incision.

Bone repair access of BoneCeramic™ in 5-mm defects: study on rat calvaria.

OBJECTIVE: The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. MATERIAL AND METHODS: A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in µCT were obtained to calculate bone mineral density (BMD). RESULTS: In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The µCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). CONCLUSION: Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.

Alveolar bone healing in rats: micro-CT, immunohistochemical and molecular analysis

Abstract Alveolar bone healing after upper incisor extraction in rats is a classical model of preclinical studies. The underlying morphometric, cellular and molecular mechanism, however, remains imprecise in a unique study. Objectives The aim of this study was therefore to characterize the alveolar bone healing after upper incisor extraction in rats by micro computed tomographic (Micro-CT), immunohistochemical and real-time polymerase chain reaction (RT-PCR) analysis. Material and Methods Thirty animals (Rattus norvegicus, Albinus Wistar) were divided into three groups after upper incisors extraction at 7, 14, and 28 days. Micro-CT was evaluated based on the morphometric parameters. Subsequently, the histological analyses and immunostaining of osteoprotegerin (OPG), receptor activator of nuclear kappa B ligand (RANKL) and tartrate resistant acid phosphate (TRAP) was performed. In addition, RT-PCR analyses of OPG, RANKL, the runt-related transcription factor 2 (RUNX2), osteocalcin (OC), osteopontin (OPN), osterix (OST) and receptor activator of nuclear kappa B (RANK) were performed to determine the expression of these proteins in the alveolar bone healing. Results Micro-CT: The morphometric parameters of bone volume and trabecular thickness progressively increased over time. Consequently, a gradual decrease in trabecular separation, trabecular space and total bone porosity was observed. Immunohistochemical: There were no differences statistically significant between the positive labeling for OPG, RANKL and TRAP in the different periods. RT-PCR: At 28 days, there was a significant increase in OPG expression, while RANKL expression and the RANKL/OPG ratio both decreased over time. Conclusion Micro-CT showed the newly formed bone had favorable morphometric characteristics of quality and quantity. Beyond the RUNX2, OC, OPN, OST, and RANK proteins expressed in the alveolar bone healing, OPG and RANKL activity showed to be essential for activation of basic multicellular units during the alveolar bone healing.

Bone repair access of BoneCeramic™ in 5-mm defects: study on rat calvaria

Abstract Objective: The aim of this study was to evaluate the osteoconductive potential of BoneCeramic™ on bone healing in rat calvaria 5-mm defects. Material and Methods: A 5-mm calvaria bone defect was induced in three groups and the defect was not filled with biomaterial [Clot Group (CG)], autogenous bone (AG), or Bone Ceramic Group (BCG). Animals were euthanized after 14 or 28 days and the bone tissue within the central area of the bone defect was evaluated. Results were compared using ANOVA and Tukey test (p<0.05). Immunohistochemistry was performed using primary antibodies against osteocalcin, RUNX-2, TRAP, VEGF proteins, and 3-dimensional images of the defects in μCT were obtained to calculate bone mineral density (BMD). Results: In BCG, the defect was completely filled with biomaterial and new bone formation, which was statistically superior to that in the GC group, at both time-points (p<0.001 for 14 days; p=0.002 for 28 days). TRAP protein showed weak, RUNX-2 showed a greater immunolabeling when compared with other groups, VEGF showed moderate immunostaining, while osteocalcin was present at all time-points analyzed. The μCT images showed filling defect by BCG (BMD= 1337 HU at 28 days). Conclusion: Therefore, the biomaterial tested was found to be favorable to fill bone defects for the reporting period analyzed.