Time of clerkship rotations' interruption during COVID-19 and differences on Progress Test's scores

SUMMARY OBJECTIVES: The transition from face-to-face to remote teaching is yet to be fully understood. In clinical training, traditional teaching must prevail because it is essential for the acquisition of skills and professionalism. However, the responses of each school to the pandemic and the decision on when to resume clerkship rotations were mixed. In this study, we aimed to analyze whether the time to resume clerkship rotations was associated with the performance of the students by using a multi-institutional Progress Test. METHODS: This is a cross-sectional study conducted at nine different Brazilian medical schools that administer the same annual Progress Test for all students. We included information from 1,470 clerkship medical students and analyzed the time of clinical training interruption as the independent variable and the student's scores as the dependent variable. RESULTS: The comparisons of the students' scores between the schools showed that there are differences; however, they cannot be attributed to the time the clerkship rotations were paused. The correlation between the schools' average scores and the time to resume clerkship rotations was not significant for the fifth year (r= -0.298, p=0.436) and for the sixth year (r= -0.440, p=0.240). By using a cubic regression model, the time to resume clerkship rotations could explain 3.4% of the 5-year students' scores (p<0.001) and 0.9% of the 6-year students, without statistical difference (p=0.085). CONCLUSIONS: The differences between the students' scores cannot be attributed to the time when the schools paused the clerkship rotations.

Antimicrobial potential of plant extracts from the Brazilian Cerrado

Abstract Bacteria are related do different oral diseases, such as dental caries and periodontal disease. Therefore, the control or/and eradication of microorganisms and their by-products is primordial for the success of their treatment. An alternative for decrease bacterial load is the use of plant extracts used in popular medicine. The cytotoxicity and antimicrobial action of extracts of Cariniana rubra Gardiner ex Miers, Senna martiniana, Anadenanthera colubrina (Vell.) Brenan and Spiranthera odoratissima St. Hil. against strains of Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Aggregatibacter actinomyces- tencomitans and Candida albicans were investigated. Cytotoxicity was assessed at concentrations of 1, 10, 40, 80, 100 and 1000 μg/mL by means of the MTT test and compared to a control group with untreated cells. Those with acceptable cytotoxicity had the antimicrobial action measured by the XTT test. As a positive control, sodium hypochlorite was used. Cariniana rubra Gardiner ex Miers had the highest citototoxicity results while Spiranthera odoratissima St. Hil. had the best results, but all extracts showed acceptable cytotoxicity at different concentrations. The plant extracts showed higher activity against A. actinomycetencomitans: Anadenanthera columbrina (Vell.) Brenan (80.52%) at 40 μg/mL, Spiranthera odoratissima St. Hil (78.48%) in 1 μg/mL, Senna martiniana (73.28%) in the concentration of 40 μg/mL and Cariniana rubra Gardiner ex Miers (70.50%) in 10 μg/mL. All extracts analyzed showed acceptable cytotoxicity at different concentrations and were promising for inhibition of the pathogenic microorganisms studied.

Resumo Bactérias estão relacionadas a diferentes doenças bucais, como a cárie dentária e a doença periodontal. Assim, o controle e/ou erradicação de microrganismos e seus subprodutos é primordial para o sucesso dos tratamentos. Uma alternativa para diminuir a carga bacteriana é a utilização de extratos vegetais utilizados na medicina popular. A citotoxicidade e ação antimicrobiana de extratos de Cariniana rubra Gardinerex Miers, Senna martiniana H.S. Irwin & Barneby, Anadenanthera colubrina (Vell.) Brenan e Spiranthera odoratissima St. Hil. contra cepas de Streptococcus mutans, Enterococcusfaecalis, Staphylococcus aureus, Escherichia coli, Agartibacter actinomycetencomitans e Candida albicans foram investigados. A citotoxicidade foi avaliada nas concentrações de 1, 10, 40, 80, 100 e 1000 μg/mL por meio do teste MTT. Aqueles com citotoxicidade aceitável tiveram a ação antimicrobiana medida pelo teste XTT. Cariniana rubra Gardinerex Miers apresentou os maiores resultados de citototoxicidade, enquanto Spiranthera odoratissima St. Hil. obteve os melhores resultados, mas todos os extratos apresentaram citotoxicidade aceitável em diferentes concentrações. Os extratos vegetais apresentaram maior atividade contra A. actinomycetencomitans: Anadenanthera columbrina (Vell.) Brenan (80,52%) a 40 μg/mL, Spiranthera odoratissima St. Hil (78,48%) em 1 μg/mL, Senna martiniana H.S. Irwin & Barneby (73,28%) na concentração de 40 μg/mL e Cariniana rubra Gardinerex Miers (70,50%) em 10 μg/mL. Todos os extratos analisados apresentaram citotoxicidade aceitável em diferentes concentrações e foram promissores na inibição dos microrganismos patogênicos estudados.

Profile of host cell responses to exposure to stressed bacteria in planktonic; dislodged, and intact biofilm mode

Abstract The host defense response to microbial challenge emerging from the root canal system leads to apical periodontitis. The aim of this study was to evaluate the expression of inflammatory cytokines and Nitric Oxide (NO) by macrophages after interaction with Enterococcus faecalis in the: plankton and dislodged biofilm mode; intact biofilm mode stimulated by calcium hydroxide (CH), CH and chlorhexidine (CHX) or Triple Antibiotic Paste (TAP). For this purpose, culture of macrophages from monocytes in human peripheral blood (N=8) were exposed to the different modes of bacteria for 24 hours. Subsequently, the cytokines, such as, Tumor Necrotic Factor- alfa (TNF-α), interleukin (IL)-1β, IL-6, IL-10; and NO were quantified by Luminex xMAP and Greiss reaction, respectively. In addition to the potential therapeutic effects of the intracanal medication, their antimicrobial activity against Enterococcus faecalis biofilm were also tested in vitro by confocal microscopy. The experiments` data were analyzed by the Kruskal-Wallis test with the Dunn post hoc test (α < 0.05). Bacteria in dislodged biofilm mode were shown to be more aggressive to the immune system than bacteria in plankton mode and negative control, inducing greater expression of NO and TNF-α. Relative to bacteria in intact biofilm mode, the weakest antimicrobial activity occurred in Group CH. In Groups CH/CHX and TAP the percentage of dead bacteria was significantly increased to the same extent. Interestingly, the biofilm itself did not induce the release of pro-inflammatory cytokines - except for NO - while the biofilm treated with TAP and CH based pastes enhanced the levels of IL-6 and TNF-α; and IL-1 β, respectively. In contrast, the levels of a potent anti-inflammatory (IL-10) were increased in Group TAP.

Resumo A resposta de defesa do hospedeiro ao desafio microbiano que emerge do sistema de canais radiculares leva à periodontite apical. Os objetivos deste estudo foram avaliar a expressão de citocinas pró e anti-inflamatórias e Óxido Nítrico (NO) por macrófagos após interação com Enterococcus faecalis no modo: planctônio e de biofilme desalojado; biofilme intacto estimulado por hidróxido de cálcio (CH), CH e clorexidina ou Pasta Tri Antibiótica (TAP). Para isto, a cultura de macrófagos originados de monócitos do sangue periférico de humanos (N=8) foi exposta aos diferentes tipos de bactéria por 24 horas. Então, a quantificação da produção de of Fator de Necrose Tumoral- alfa (TNF-α), interleucina (IL)-1β, IL-6, IL-10 e NO por macrófagos se deu por meio do Luminex xMAP e reação de Greiss, respectivamente. Além dos potenciais efeitos terapêuticos desses compostos, sua atividade antimicrobiana contra E. faecalis também foi testada através microscopia confocal. Os dados dos experimentos foram analisados através do teste de Kruskal-Wallis com Dunn`s post hoc (α < 0.05). Bactéria em modo de biofilme desalojado se mostrou mais agressivo ao sistema imune que as bactérias planctônicas e controle negativo induzindo a maior excreção de NO e TNF-α. Em relação ao biofilme intacto, a atividade antimicrobiana mais fraca ocorreu no grupo de CH. Os grupos CHX e TAP aumentaram significativamente a porcentagem de bactérias mortas na mesma extensão. Interessantemente, o biofilme por ele mesmo não induziu a liberação de citocinas pro-inflamatórias - exceto por NO - enquanto que o biofilme tratado com TAP ou pastas a base de CH aumentaram os níveis de IL-6; e TNF-α e IL-1 β respectivamente. Em contraste, os níveis da potente citocina anti-inflamatória (IL-10) foram aumentados pelo grupo TAP.

Effects of Qualea grandiflora Extract on the Expression of MMP-14 and HIF-1α in Cultured Fibroblasts and Preosteoblasts

Abstract Background: Qualea grandiflora (QG) (Vochysiaceae), also known as "pau-ferro", "pau-terra" or "pau-de-tucano", is a very common deciduous tree in the Brazilian Cerrado used in traditional medicine to treat inflammations, ulcers, diarrhea, and infections. There are reports in the scientific literature that demonstrate the medicinal effects of the bark and leaf of the QG. However, studies involving this plant are rather imited. Aim of the study: To perform the phytochemical analysis of the QG hydroalcoholic extract (HAE) of leaves, and to investigate it effects on fibroblast and preosteoblasts. Methods: Phytochemical analysis was done by HPLC-DAD. Murine NIH/3T3 fibroblasts and MC3T3-E1 preosteoblasts cell lines (ATCC) were used for the experiments. Cell viability was assessed by the MTT colorimetric assay and the expression of MMP-14 and HIF-1α by immunofluorescence. Results and conclusion: The following compounds were identified by HPLC-DAD, such as quinic acid, ethyl galate, ellagic acid derivatives as O-methylellagic acid O-galloyl, O-methylellagic acid O-deoxyhexoside, galloyl derivatives, flavonol glycoside as kaempferol-O-deoxyhexoside, quercetin-O-deoxyhexoside, myricetin-O-deoxyhexoside and the pentacyclic triterpene arjunglucoside. Cell viability results demonstrated no cytotoxic effects in the studied concentrations. We found in QG HAE some compounds with therapeutic properties that can increase the expression of MMP-14 and HIF-1α, in fibroblasts and preosteoblasts. These data suggest that QG HAE has an action on these two molecules widely involved in physiological conditions, such as collagen remodeling, bone development and growth and pathological processes as HIF signaling in cancer metastasis.

Biochemistry and Renal Function: Use of Inquiry-Based Teaching Sequences for Retrieval of Specific Concepts / Bioquímica e Função Renal: Utilizações de Sequências Didáticas com Enfoque Investigativo para Reaproximação de Conceitos Específicos

ABSTRACT Current teaching proposals involve teaching strategies that seek to assist and encourage the construction of knowledge by the students, turning them into active participants during the learning process and these have been the subject of several researches. This article reports a didactic sequence carried out with students of applied training for the first year Medical Sciences USP - Bauru, and proposes, as part of its pedagogical design, Problem Based Learning (PBL) as the main teaching strategy. In this didactic sequence we used experimental investigative activities. A didactic sequence that includes investigative activities can be characterized as a gradual evolutionary process, with the objective of intertwining the scientific perspective and the students' conceptions, through well-planned teaching and learning activities contextualized and empirically adapted to the student's reasoning. The objective of this work is to share an assertive experience of the application of a contextualized research didactic sequence that involved concepts ranging from the simplest chemical properties of biomolecules and ions to the association and discussion of a hypothetical clinical case involving proteinuria. Its pathophysiology consists of the excretion of protein in the urine, mainly albumin, and occurs when there is some damage to the kidneys. Therefore, the dosage of the protein fraction in urine (albuminuria) is mainly used for the early detection of chronic or acute kidney disease and can also be an instrument for the diagnosis of cardiovascular diseases. Therefore, one should be aware of the possible interferences and the various causes of errors inherent to this examination. In this way, through a didactic resource involving contextualized research experimental activities, having proteinuria as the key problem, we were able to re-approximate specific concepts and to value procedural and attitudinal knowledge, which is important for students in this training phase. In this proposal, the students were protagonists of the learning process, where they were able to raise and test their hypotheses, interconnecting knowledge, acquiring specific skills and competences, allowing reflection on the importance of fundamentals and applications of the basic sciences. The purpose of the investigative and contextualized didactic sequences is to form autonomous subjects, who know how to make decisions and work in teams and have a sound and critical understanding of how scientific knowledge evolves and is related.

RESUMO As propostas de ensino atuais envolvem estratégias de ensino que auxiliam e incentivam a construção do conhecimento, fazendo com que o aluno seja um participante ativo do processo de aprendizagem, e têm sido alvo de diversas pesquisas. Este artigo relata, aula a aula, uma sequência didática realizada com alunos em fase inicial de formação, aplicada à primeira turma do curso de Ciências Médicas da USP - Bauru, que propõe em seu projeto pedagógico o Aprendizado Baseado em Problemas (PBL) como principal estratégia de ensino. Nesta sequência didática, utilizamos atividades experimentais investigativas. A característica principal de uma sequência didática que inclui atividades investigativas é seu processo evolutivo gradual, com o objetivo de entrelaçar a perspectiva científica e as concepções dos estudantes por meio de atividades de ensino-aprendizagem bem planejadas, contextualizadas e empiricamente adaptadas ao raciocínio do aluno. O objetivo deste trabalho é compartilhar uma vivência assertiva da aplicação de uma sequência didática de caráter investigativo contextualizada, que envolveu conceitos que foram desde as propriedades químicas mais simples das biomoléculas/íons até a associação e discussão de um caso clínico hipotético envolvendo proteinúria. Essa fisiopatologia consiste na excreção de proteína na urina, principalmente albumina, e ocorre quando há algum dano nos rins. Sendo assim, a dosagem da fração proteica na urina (albuminúria) é utilizada principalmente para detecção precoce de doença renal crônica e pode ser também um instrumento para o diagnóstico de doenças cardiovasculares. Portanto, deve-se estar atento aos possíveis elementos interferentes e às variadas causas de erros inerentes a esse exame. Desta maneira, por meio de um recurso didático que envolve atividades experimentais investigativas contextualizadas, tendo como questão problematizadora um caso de proteinúria, pudemos nos reaproximar de conceitos específicos e valorizar os saberes procedimentais e atitudinais, o que é importante para os alunos nesta fase de formação. Nesta proposta, os alunos foram protagonistas do processo de aprendizagem, no qual puderam levantar e testar suas hipóteses, interligando conhecimentos e adquirindo habilidades e competências específicas, o que possibilitou uma reflexão sobre a importância dos fundamentos e aplicações das ciências básicas. O propósito das sequências didáticas investigativas e contextualizadas é formar sujeitos autônomos, que saibam tomar decisões e trabalhar emequipe, seguros e críticos, compreendendo como os saberes científicos evoluem e estão relacionados.

The Dynamics of Subcutaneous Tissue Response to Microorganisms Associated with the Extract of Araçá (Psidium cattleianum): An Edemogenic and Microscopic Analysis / A Dinâmica da Resposta do Tecido Subcutâneo para Associação de Microorganismos com o Extrato de Araçá (Psidium cattleianum): Uma Análise Microscópica e Edemogênica

Objective: To evaluate in vivo tissue reaction to the extract of araçá (Psidium cattleianum) associated with inactivated microorganisms. Material and Methods: A 0.1 mL suspension was used containing Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Enterococcus faecalis, Peptostreptococcus micros, and Porphyromonas endodontalis, which were inactivated by heat and mixed into a 1.0 mL saline (control group), an aqueous solution, or a hydroalcoholic extract of araçá. Eighteen male rats (Rattus norvegiccus) under general anesthesia received 0.2 mL of 1% intravenous Evans blue. Thirty minutes later, 0.1 mL of one of the associations was injected into the animals' dorsal region. The animals were euthanized after 3 and 6 hours, and the materials obtained were placed in formamide for 72 hours then analyzed in a spectrophotometer (λ=630 ηm). For the morphological analysis, 30 rats received polyethylene tubes implants with the extracts or the saline with the associations in the dorsal region and euthanized after 7 and 30 days to be analyzed according to an inflammation cell score. Results: No significant difference (p>0.05) was observed in the edema among groups. The optical microscopy results showed a repair in the 30-day-period, which was higher when compared to the 7-day-period (p< 0.0001). Nevertheless, in the 7-day-period, the hydroalcoholic extract presented a significant response compared to the aqueous extract (p=0.05) and a trend for better results than the control group. Conclusion: The aqueous and hydroalcoholic araçá extracts associated with inactivated microorganisms showed similar responses to control, indicating no interference on the toxic effects of the bacterial components in tissue repair. (AU)

Objetivo: Avaliar in vivo a reação tecidual do extrato de araçá (Psidium cattleianum) associado com microorganismos inativados. Material e Métodos: Uma suspensão de 0.1mL foi usada contendo Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Enterococcus faecalis, Peptostreptococcus micros e Porphyromonas endodontalis dos quais foram inativos por aquecimento e misturados a 1,0 mL de soro fisiológico (grupo controle), uma solução aquosa ou hidroalcoólica de araçá. Dezoito ratos machos (Rattus norvegiccus) sob anestesia geral receberam 0,2mL de Azul de Evans a 1% intravenoso. Após trinta minutos, 0,1mL de um dos extratos (associado com microorganismos inativos) foi injetado nos animais na região dorsal. Os animais foram eutanasiados após 3 e 6 horas, e os materiais obtidos colocados em formamida por 72 horas para análise em espectrofotômetro (λ=630 ηm). Para análise morfológica, 30 ratos receberam implante subcutâneo de tubo de polietileno com as associações na região dorsal, eutanasiados após 7 e 30 dias para serem analisados de acordo com um escore de células inflamatórias. Resultados: Não houve diferença significativa (p>0,05) no edema entre os grupos. Os resultados obtidos em microscópio óptico apontaram reparo em 30 dias superior ao de 7 dias (p< 0,0001). No período de 7 dias a solução hidroalcoólica apresentou resposta superior a solução aquosa (p=0,05) e uma tendência de melhor resultado que o controle. Conclusão: A solução aquosa e hidroalcoólica de extrato de araçá associadas a microrganismos inativados apresentaram respostas biológicas semelhantes ao controle, indicando que não há interferência sobre os efeitos tóxicos advindos dos componentes bacterianos, no sentido de favorecer o reparo(AU)

Storage protocol of dental pulp cells from human exfoliated deciduous teeth / Protocolo de armazenamento de células pulpares de dentes decíduos humanos

Objetivo: O objetivo deste estudo foi isolar células do tecido pulpar de dentes decíduos humanos, avaliar a capacidade de proliferação, caracterizá-las e normatizar as técnicas de cultivo e expansão celular destas para a criação de um banco de células. Material e métodos: Dentes decíduos sem cárie e com indicação ortodôntica de para extração foram utilizados como doadores de tecido para a pesquisa. As células foram extraídas de tecidos pulpares, isoladas e cultivadas em condições ideais até alcançarem confluência. Resultados: Após consecutivas passagens, as células cultivadas foram caracterizadas por meio de técnicas de imunofluorescência e congeladas entre a 2ª e a 6ª passagem, criando-se um biorrepositório de células da polpa de dentes decíduos humanos. Conclusão: A criação de bancos de células pulpares de dentes decíduos humanos permite uma aplicação mais ágil nas pesquisas laboratoriais, reduzindo o tempo e o custo da obtenção de novas amostras. Evita necessidade de triagem e obtenção de novos doadores de dentes e tecidos, e permite maior rapidez nas repetições de protocolos de pesquisas. (AU)

Objective: This study aimed to isolate the cells from the dental pulp tissue of human primary teeth, study the capacity of proliferation, characterize the cells and standardize the technique of culture and expansion to create a cell banking. Material and Methods: Primary teeth with no caries and orthodontic reasons were extracted for pulp tissue obtainment. The cells were extracted from the pulp cells, isolated and cultured under ideal conditions until full expansion. Results: After consecutive passages, the cultured cells were characterized using immunofluorescence technique and frozen between the 2nd and 6th passage, thus creating a biorepository of dental pulp cells from human primary teeth. Conclusion: The creation of a cell banking from dental pulp cells from human primary teeth enables the easy application of cells in laboratorial studies, reducing the cost and time for obtaining the samples, avoid the involvement of new subjects and allow a fast reproducibility of the researches. (AU)

Liver proteome of mice with different genetic susceptibilities to the effects of fluoride

ABSTRACT A/J and 129P3/J mice strains have been widely studied over the last few years because they respond quite differently to fluoride (F) exposure. 129P3/J mice are remarkably resistant to the development of dental fluorosis, despite excreting less F in urine and having higher circulating F levels. These two strains also present different characteristics regardless of F exposure. Objective In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. Material and Methods Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). Results Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. Conclusion This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress.

Viability of Human Gingival Fibroblast (FGH) Treated with Ethanolic "Aroeira" Extract (Myracrodruon urundeuva Allemão)

ABSTRACT The aim of this study was to evaluate the effect of ethanolic "aroeira" (Myracrodruon urundeuva) extract on the viability of human gingival fibroblast. For this, fibroblasts (2x103 cells/well) were plated in a 96-well plate and incubated for 24 h; the medium (Eagle's medium modified by Dulbecco - DMEM) supplemented with 10% fetal bovine serum was replaced by DMEM with different ethanolic extract concentration (0, 0.1, 1, 10, 100, and 1000μg / mL). The fibroblast viability was analyzed after 48,72, and 96 h by the neutral red capture test and violet crystal. The "aroeira" extract, at high concentrations (100 and 1000 µg/mL) caused decrease in both cellular viability tests (p<0.05). However, dilutions between 0.1 and 10 µg/mL did not affect the viability of the cells. It was concluded that "aroeira" extract was able to change the gingival fibroblast viability, and this effect was concentration dependent.

Experimental Calcium Silicate-Based Cement with and without Zirconium Oxide Modulates Fibroblasts Viability

The aim of this study was to verify whether the use of zirconium oxide as a radiopacifier of an experimental calcium silicate-based cement (WPCZO) leads to cytotoxicity. Fibroblasts were treated with different concentrations (10 mg/mL, 1 mg/mL, and 0.1 mg/mL) of the cements diluted in Dulbecco's modified Eagle's medium (DMEM) for periods of 12, 24, and 48 h. Groups tested were white Portland cement (WPC), white Portland cement with zirconium oxide (WPCZO), and white mineral trioxide aggregate Angelus (MTA). Control group cells were not treated. The cytotoxicity was evaluated through mitochondrial-activity (MTT) and cell-density (crystal violet) assays. All cements showed low cytotoxicity. In general, at the concentration of 10 mg/mL there was an increase in viability of those groups treated with WPC and WPCZO when compared to the control group (p<0.05). A similar profile for the absorbance values was noted among the groups: 10 mg/mL presented an increase in viability compared to the control group. On the other hand, smaller concentrations presented a similar or lower viability compared to the control group, in general. A new dental material composed of calcium silicate-based cement with 20% zirconium oxide as the radiopacifier showed low cytotoxicity as a promising material to be exploited for root-end filling.

Resumo O objetivo deste estudo foi verificar se o uso do óxido de zircônia como radiopacificador de um cimento experimental à base de silicato de cálcio (WPCZO) levou a citotoxicidade. Os fibroblastos foram tratados com diferentes concentrações (10 mg/mL, 1 mg/mL e 0,1 mg/mL) dos cimento diluídos em meio Eagle modificado por Dulbecco (DMEM) durante períodos de 12, 24, e 48 horas. Os grupos testados foram: cimento Portland (WPC), cimento Portland branco com óxido de zircônio (WPCZO) e MTA Angelus branco (MTA). No grupo controle as células não foram tratadas. A citotoxicidade foi avaliada por meio da mitocondrial-atividade (MTT) e ensaio de densidade celular (cristal violeta). Todos os cimentos apresentaram baixa citotoxicidade. Em geral, na concentração de 10 mg/mL, houve um aumento na viabilidade desses grupos tratados com WPC e WPCZO quando comparado com o grupo controle (p <0,05). Um perfil semelhante para os valores de absorvância foi observado entre os grupos: 10 mg/mL apresentaram um aumento da viabilidade em relação ao grupo controle. Por outro lado, as concentrações menores apresentaram uma viabilidade semelhante ou inferior em comparação com o grupo controle, em geral. Um novo material odontológico composto de cimento à base de silicato de cálcio com 20% de óxido de zircônio, como o radiopacificador, apresentou baixa citotoxicidade e pode ser explorado como um material promissor para retrobturações.

Cell density and solvent are critical parameters affecting formazan evaluation in MTT assay

The aim of this study was to establish the more accurate protocol for fibroblast cell viability using MTT assay. NIH/3T3 fibroblasts were seeded at the following cell densities: 3.125x10³; 1.156x10(4); 3.125x10(4); 1.156x10(5) and 3.125x10(5) cells/cm². Following 24h of seeding, MTT was added to the wells. After 4h of the MTT addition, different solvents were added to solubilize the formazan crystals: 1) HCl/SDS group- 20% SDS and 0.01 M HCl; 2) EtOH/ HAc group-50% ethanol and 1% acetic acid; 3) DMSO group- 99.5% dimethyl sulfoxide; and 4) PropOH group- 99.5% isopropanol. The absorbance values were measured using a spectrophotometer at 570 nm. The data were analyzed by 2-way ANOVA (p<0.05) and showed that the absorbance average varied according to the number of cells and solvents: HCl/SDS (0 to 0.13), EtOH/HAc (0 to 0.22), DMSO (0.76 to 1.31) and PropOH (0.66 to 1.04). The DMSO and PropOH groups presented the most appropriate protocols for NIH/3T3 fibroblasts cell viability, especially at the density of 1.156x10(4) cells/cm².

Seven years of external control of fluoride levels in the public water supply in Bauru, São Paulo, Brazil

Fluoridation of the public water supplies is recognized as among the top ten public health achievements of the twentieth century. However, the positive aspects of this measure depend on the maintenance of fluoride concentrations within adequate levels. Objective: To report the results of seven years of external control of the fluoride (F) concentrations in the public water supply in Bauru, SP, Brazil in an attempt to verify, on the basis of risk/benefit balance, whether the levels are appropriate. Material and Methods: From March 2004 to February 2011, 60 samples were collected every month from the 19 supply sectors of the city, totaling 4,641 samples. F concentrations in water samples were determined in duplicate, using an ion-specific electrode (Orion 9609) coupled to a potentiometer after buffering with TISAB II. After the analysis, the samples were classified according to the best risk-benefit adjustment. Results: Means (±standard deviation) of F concentrations ranged between 0.73±0.06 and 0.81±0.10 mg/L for the different sectors during the seven years. The individual values ranged between 0.03 and 2.63 mg/L. The percentages of the samples considered “low risk” for dental fluorosis development and of “maximum benefit” for dental caries prevention (0.55-0.84 mg F/L) in the first, second, third, fourth, fifth, sixth, and seventh years of the study were 82.0, 58.5, 37.4, 61.0, 89.9, 77.3, and 72.4%, respectively, and 69.0% for the entire period. Conclusions: Fluctuations of F levels were found in the public water supply in Bauru during the seven years of evaluation. These results suggest that external monitoring of water fluoridation by an independent assessor should be implemented in cities where there is adjusted fluoridation. This measure should be continued in order to verify that fluoride levels are suitable and, if not, to provide support for the appropriate adjustments.

Fluoride effect on the process of alveolar bone repair in rats: evaluation of activity of MMP-2 and 9

Objective: The aim of this study was to evaluate comparatively the effect of fluoride (F) on the activity of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) involved in process of alveolar bone repair. Material and methods: This study used 4 groups of Wistar rats with 80 days of life (n = 160) which received drinking water containing different doses of fluoride (NaF): 5, 15, 50 ppm and deionized water (control) throughout the experiment. These animals had their right upper incisors extracted. After extraction, the animals were euthanized at 7, 14, 21 and 30 days and the hemi-maxillae were collected for microscopic analysis (Hematoxylin and Eosin and immunohistochemistry for MMP-9) and zymography (MMP-2 and 9).Results: Microscopically the process of bone repair was similar in all groups, being noted only a delay of the blood clot resorption and bone formation in the group of 50 ppm F. The expression for MMP-9 showed differences betweengroups only during the initial repair (7 days). However, the zymography showed no significant differences between treated and control groups. Conclusion: Ours results suggest an effect of fluoride on the activity of MMPs 2 and 9 at the initial period of alveolar repair which could be associated to the process of blood clot remission and delay in bone repair. Further studies are needed to establish the relationship between the initial process of resorption of the blood clot, and the involvement of MMPs 2 and 9 and its regulators/tissue inhibitors.

Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects

It is known that current trends on bone bioengineering seek ideal scaffolds and explore innovative methods to restore tissue function. In this way, the objective of this study was to evaluate the behavior of anorganic bovine bone as osteoblast carrier in critical-size calvarial defects. MC3T3-E1 osteoblast cells (1x10(5) cells/well) were cultured on granules of anorganic bovine bone in 24-well plates and after 24 h these granules were implanted into rat critical-size calvarial defects (group Biomaterial + Cells). In addition, other groups were established with different fillings of the defect: Blood Clot (negative control); Autogenous Bone (positive control); Biomaterial (only granules) and Cells (only MC3T3-E1 cells). After 30 days, the animals were euthanized and the calvaria were technically processed in order to allow histological and morphometric analysis. It was possible to detect blood vessels, connective tissue and newly formed bone in all groups. Particularly in the Biomaterial + Cells group, it was possible to observe a profile of biological events between the positive control group (autogenous bone) and the group in which only anorganic bovine granules were implanted. Altogether, the results of the present study showed that granules of anorganic bovine bone can be used as carrier to osteoblasts and that adding growth factors at the moment of implantation should maximize these results.

Sabe-se que uma das atuais tendências na bioengenharia óssea é procurar um carreador ideal e explorar métodos inovadores para restaurar a função do tecido. Desta forma, nosso objetivo foi avaliar o comportamento do osso bovino inorgânico como carreador de osteoblastos em defeitos ósseos de tamanho crítico em calvária de ratos. Osteoblastos da linhagem MC3T3-E1 (1x10(5) células/poço) foram cultivadas em grânulos de osso bovino inorgânico sob placas de 24 poços e após 24 h esses grânulos foram implantados em defeitos ósseos de tamanho crítico em calvária de ratos. Além deste grupo experimental (Biomaterial + Células), foram estabelecidos outros grupos com diferentes preenchimentos do defeito crítico: coágulo sanguíneo (controle negativo); osso autógeno (controle positivo); Biomaterial (apenas grânulos) e Células (apenas células MC3T3-E1). Após 30 dias, os animais foram eutanasiados e as calvárias foram processadas histotecnicamente, a fim de permitir a análise histológica e morfometria. Nossos resultados mostraram que em todos os grupos avaliados foi possível detectar vasos sanguíneos, tecido conjuntivo e osso neoformado. Em especial para o grupo tratado com Biomaterial + Células, foi possível observar um perfil de eventos biológicos intermediário ao grupo controle positivo (osso autógeno) e o grupo de biomaterial (apenas grânulos inorgânico bovino). Ao todo, nossos resultados mostraram que os grânulos de osso bovino inorgânico podem ser usados como carreador de osteoblastos e que a adição de fatores de crescimento no momento em que ocorre o implante deve maximizar os resultados.

Alkaline phosphatase activity in plasma and liver of rats submitted to chronic exposure to fluoride

The aim of this study was to compare the effect of fluoride (F) on alkaline phosphatase activity in the liver and plasma of the rats. Four groups of male Wistar rats (n=6), which received drinking water containing 5, 15 or 50 ppm F or deionized water (control) throughout the experiment were included in the study. The animals were euthanized and had their tissues and blood plasma collected for the analysis of fluoride and alkaline phosphatase. There was an increase in F concentration in most tissues in the animals treated with higher F concentrations, except for the heart. The alkaline phosphatase assay showed an increase in the activity in the liver and blood plasma of the animals treated with fluoride concentrations of 15 and 50 ppm (p<0.05). This study suggested that F at a concentration of 50 ppm in drinking water promotes increased the activity of alkaline phosphatase in the liver and blood plasma.

Matriz colagênica de tendão bovino como potencial biomaterial para bioengenharia de tecidos / Collagenic matrix from bovine tendon as a potential biomaterial for tissue bioengineering

A Engenharia de Tecidos é um campo interdisciplinar que busca preservar, restaurar ou criar um tecido funcional, apoiando- se em três elementos fundamentais: células, fatores tróficos e carreadores. Um desses elementos, que ainda permanece sob intensa investigação, é o carreador. O objetivo do trabalho foi avaliar o comportamento tecidual ao implante de membrana colagênica derivada de tendão bovino em subcutâneo de camundongos. Nos animais do grupo controle foi feita apenas a incisão, divulsão e sutura. Depois de 3, 7, 15, 30 e 60 dias, os camundongos foram eutanasiados por dose excessiva de anestésico, sendo os tecidos reacionais coletados para análise histológica. Foram observados os seguintes parâmetros: biodegradação em relação ao tempo, vascularização, integração tecidual e reação de corpo estranho. O tecido adjacente ao material implantado apresentou infiltrado inflamatório nos períodos iniciais, com angiogênese e proliferação fibroblástica. No grupo experimental constatamos uma moderada reabsorção da membrana nos períodos de 15 e 30 dias e absorção completa aos 60 dias. A absorção foi mediada por células tipo macrófagos, sem a necessidade de células gigantes. Concomitantemente, houve a regeneração tecidual. No grupo controle observamos resultados compatíveis com o procedimento operatório, mostrando formação de coágulo e rede de fibrina nos primeiros períodos, proliferação angioblástica e fibroblástica nos períodos seguintes e regeneração tecidual nos 2 últimos períodos analisados. Diante dos resultados obtidos, podemos concluir que a membrana de tendão bovino é biocompatível e reabsorvível, posicionando- se como um promissor material a ser explorado pela medicina regenerativa.

Tissue Engineering is an interdisciplinary field that seeks to preserve, restore or create a functional tissue, relying on three key elements: cells, growth factors and carriers. Thus, our objective was to evaluate the reactional tissue induced by collagenic matrices derived from bovine tendon in the subcutaneous tissue of mice. Thereafter, the animals were killed at 3, 7, 15, 30 and 60 days post-surgery of implantation and tissues collected for histological analysis for analyzing: biodegradation, angiogenesis, tissue integration and foreign body reaction. The reactional tissue showed a moderate inflammatory infiltrate, with angiogenesis and fibroblast- like cells proliferation, while a moderate resorption of the membrane was found at 15 and 30 days and it being complete at 60 days. Our results suggest that the absorption was mediated by mononuclear cells such as macrophages, without giant cells involvement. Based on these results, we conclude that the membrane of bovine tendon is biocompatible and absorbable, it being a promising material to be exploited for regenerative medicine.

Análise morfométrica da resposta tecidual ao osso bovino misto medular implantado em defeito ósseo na calvária de rato / Morphometric analysis of tissue response to mixed bovine bone marrow implanted in bone defects in rat calvaria

O propósito deste trabalho foi avaliar o reparo ósseo de defeito de tamanho crítico em crânio de ratos tratados com disco de osso bovino misto (OBM) medular poroso. Foram utilizados 30 ratos Wistar machos adultos submetidos à cirurgia para confecção de um defeito circular de 8 mm de diâmetro removendo toda a díploe da calvaria. Os defeitos foram preenchidos com um disco de OBM de 8 mm de diâmetro por 3 mm de espessura, enquanto no grupo controle o defeito ósseo foi preenchido com coágulo sanguíneo do próprio animal. Os animais foram mortos após 30, 60 e 120 dias. As peças foram removidas, fixadas em formol 10% tamponado, desmineralizadas e processadas pela técnica histotécnica padrão para coloração em hematoxilina e eosina. Os cortes foram submetidos à análise histológica descritiva e morfometria (densidade de volume). Os resultados demonstraram uma pequena diminuição do material implantado com o decorrer dos períodos experimentais, sugerindo uma reabsorção lenta e gradual do OBM. Em contrapartida foi observado uma pequena neoformação óssea nos dois grupos (teste e controle) e formação de tecido conjuntivo denso nas áreas do defeito ósseo aos 120 dias também nos dois grupos. O tecido conjuntivo, no grupo teste, foi capaz de penetrar nos poros do material e ocupar esses espaços com o passar dos períodos. Dentro dos limites desse estudo, pode-se afirmar que o OBM é biocompativel, no entanto, não apresenta capacidade osteocondutora ou osteoindutora em defeitos críticos na calvária de ratos Wistar.

The purpose of this study was to evaluate bone repair of critical size defects in the calvarias of rats treated with bovine bone mixed disk (OBM) porous medullary. We used 30 adult male Wistar rats that underwent surgery to confection a bone defect of 8 mm diameter circular removing diploe calvaria completely. The defects were filled with a disc of OBM 8 mm diameter and 3 mm thick, while the control group the bone defect was filled with blood clot. The animals were killed after 30, 60 and 120 days. The pieces were removed, fixed in 10% buffered formalin, demineralized and processed by standard technique to staining with hematoxylin and eosin. The sections were submitted to descriptive histology and morphometry (volume density). The results showed a small decrease of the implanted material in the course of the experimental periods, suggesting a slow and gradual resorption of the OBM. On the other hand, there was a small new bone formation observed in both groups (test and control) and formation of dense connective tissue areas of the bone defect at 120 days also in the two groups. The connective tissue in the test group was able to penetrate the pores of the material and occupy these spaces over the periods. Within the limits of this study, it can be said that the OBM is biocompatible, however, does not have osteoinductive or osteoconductive capacity in critical defects in the calvaria of rats.

Recovery of silver residues from dental amalgam

Dental amalgam residues are probably the most important chemical residues generated from clinical dental practice because of the presence of heavy metals among its constituents, mainly mercury and silver. OBJECTIVE: The purpose of this study was to develop an alternative method for the recovery of silver residues from dental amalgam. MATERIAL AND METHODS: The residue generated after vacuum distillation of dental amalgam for the separation of mercury was initially diluted with 32.5 percent HNO3, followed by precipitation with 20 percent NaCl. Sequentially, under constant heating and agitation with NaOH and sucrose, the sample was reduced to metallic silver. However, the processing time was too long, which turned this procedure not viable. In another sequence of experiments, the dilution was accomplished with concentrated HNO3 at 90ºC, followed by precipitation with 20 percent NaCl. After washing, the pellet was diluted with concentrated NH4OH, water and more NaCl in order to facilitate the reaction with the reducer. RESULTS: Ascorbic acid was efficiently used as reducer, allowing a fast reduction, thus making the procedure viable. CONCLUSIONS: The proposed methodology is of easy application and does not require sophisticated equipment or expensive reagents.

Biocompatibility evaluation of a new bioresorbable pin for membrane fixation

The aim of this study was to morphometrically analyze the tissue response to a customized pin obtained from devitalized bovine cortical bone (DBCB-pin) implanted in the subcutaneous tissue of rats, as well as to assess its microstructural aspect by scanning electron microscopy (SEM). The pins were implanted in the subcutaneous tissue of 20 rats, which were killed at 7, 14, 28 and 60 days (5 rats/period) after implantation. In the subcutaneous tissue, DBCB-pin promoted the formation of a fibrous capsule. At 7 days, capsule showed thickness of 70 ± 3.2 µm with higher density of newly formed capillaries and smaller density of collagen fibers. Between 14 and 60 days, more organized fibrous capsule exhibited smaller thickness (53 ± 5.5 µm) with higher density of fibroblasts and collagen fibers. In this period, a small and slow bioresorption of the DBCB-pin by macrophages and rare multinucleated giant cells without tissue damage was observed. The thickness of DBCB-pin resorbed was in mean only of 9.3 µm. During all experimental periods not occurred presence of immune reaction cells as lymphocytes and plasma cells. It was concluded that the pin derived from cortical bovine bone was well tolerated by subcutaneous tissue of rats and slowly resorbed could be an alternative material for membrane fixation in the guided tissue regeneration procedures.

O objetivo deste estudo foi analisar morfometricamente a resposta tecidual a um pino obtido a partir de osso bovino desvitalizado cortical (DBCB pinos) implantado no tecido subcutâneo de ratos, bem como para avaliar o seu aspecto microestrutural por microscopia eletrônica de varredura (MEV). Os pinos foram implantados no tecido subcutâneo de 20 ratos, que foram sacrificados aos 7, 14, 28 e 60 dias (5 animais / período) após a implantação. No tecido subcutâneo, o pino DBCB promoveu a formação de uma cápsula fibrosa. Aos 7 dias, a cápsula apresentou espessura de 70 ± 3,2 μm com maior densidade de capilares neoformados e menor densidade de fibras colágenas. Entre 14 e 60 dias, a cápsula fibrosa apresentava-se mais organizada e exibiram menor espessura (53 ± 5,5 μm) com maior densidade de fibroblastos e fibras colágenas. Nesse período, foi observada uma bioreabsorção pequena e lenta dos pinos DBCB por macrófagos e raras células gigantes multinucleadas, sem dano tecidual. A espessura dos pinos DBCB reabsorvidos foi em média de apenas 9,3 µm. Durante todos os períodos experimentais não ocorreu presença de células como linfócitos e células plasmáticas. Concluiu-se que o pino derivado de osso bovino cortical foi bem tolerado pelo tecido subcutâneo de ratos e reabsorvido lentamente, sendo um potencial material alternativo para fixação da membrana nos procedimentos de regeneração tecidual guiada.