Resumo Este estudo demonstra o efeito da modificação da superfície de titânio por alcalinização sobre o metabolismo de osteoblastos humanos. Discos de titânio polidos foram submetidos ao protocolo de alcalinização com NaOH (5M) a 60°C ou 120°C. A topografia e rugosidade superficial foram avaliados por microscopia eletrônica de varredura (MEV). Osteoblastos foram cultivados sobre discos de titânio, seguido de análise da adesão e viabilidade celular, síntese de proteína total e colágeno, além de atividade de fosfatase alcalina (ALP). A expressão genica do fator de necrose tumoral-alfa (TNF-α) e beta-defensina (HBD-3) foram avaliadas após estimulo inflamatório com lipopolissacarídeos (LPS) de Porphyromonas gingivalis (1 μg/mL) por 4 h. Os discos submetidos à modificação com NaOH demonstraram maiores irregularidades, especialmente para o protocolo de 120°C. Aumento da adesão celular foi observada para as superfícies modificadas por NaOH. Os osteoblastos cultivados sobre as superfícies modificadas apresentaram maior viabilidade, síntese de proteína total e de colágeno, além de maior atividade de ALP quando comparadas às células cultivadas sobre os discos polidos. Os osteoblastos apresentaram aumento da síntese de TNF-α em resposta ao LPS para as células cultivadas sobre os discos polidos, enquanto aumento da expressão de HBD-3 foi detectado para todos os grupos na presença de LPS. A modificação dos discos de titânio por NaOH a 60°C ou 120°C promoveu aumento da adesão e metabolismo de osteoblastos e favoreceu a resposta a estímulo inflamatório. humanos 5M M (5M 60C C 60 120C 120 MEV. MEV . (MEV) colágeno ALP. (ALP) tumoralalfa tumoral alfa TNFα TNF α (TNF-α betadefensina beta defensina HBD3 HBD 3 (HBD-3 (LPS 1 ( μg/mL μgmL μg mL h irregularidades HBD- 6 12 (MEV (ALP (HBD- (HBD

Abstract: This investigation demonstrates the effect of alkali modification of titanium on the metabolism of human osteoblasts. Polished titanium discs were subjected to alkalinization protocols with NaOH (5M) at 60°C or 120°C. Surface topography and roughness were evaluated using scanning electron microscopy (SEM). Osteoblasts were seeded onto titanium discs, followed by cell adhesion and viability analysis, total protein and collagen production, and alkaline phosphatase (ALP) activity. Gene expression of tumor necrosis factor-alpha (TNF-α) and beta-defensin 3 (HBD3) was evaluated after inflammatory stimulus with lipopolysaccharides (LPS) of Porphyromonas gingivalis (1 μg/mL) for 4 h. Discs subjected to modification with NaOH showed major irregularities, especially for 120°C-protocol. Increased adhered cell number was observed for surfaces modified by NaOH. Osteoblasts cultured on modified surfaces showed higher cell viability, total protein and collagen synthesis, and ALP activity than that of cells cultured on the polished discs. Osteoblast response to LPS exposure showed increased TNF-α gene expression by these cells when cultured on the polished discs, while increased expression of HBD3 was detected for all groups in the presence of LPS. Modification of titanium discs by NaOH at 60°C or 120°C promoted an increase in adhesion and metabolism of osteoblasts and favored the response to inflammatory stimulus. Abstract 5M M (5M 60C C 60 120C 120 SEM. SEM . (SEM) analysis production (ALP factoralpha factor alpha TNFα TNF α (TNF-α betadefensin beta defensin HBD (HBD3 (LPS 1 ( μg/mL μgmL μg mL h irregularities 120°Cprotocol. 120Cprotocol Cprotocol protocol. protocol 120°C-protocol synthesis 6 12 (SEM (HBD 120°Cprotocol

Resumo O objetivo deste estudo foi o desenvolvimento de um recobrimento de vidro bioativo sobre a zircônia (Zr) para modular o fenótipo de fibroblastos gengivais. Para este propósito, partículas de Biosilicato® (6 mg/mL) em um veículo a base de água/álcool isopropílico (1:1) foram aplicadas sobre discos de zircônia seguido por tratamento térmico a 1100 °C por 20 min. A topografia de superfície (MEV), composição química (EDX), rugosidade de supefície (Ra; microscopia confocal), energia livre de superfície (goniômetro) e alteração de cor (Espectrofotometria UV-vis) foram avaliadas (n=6). A seguir, fibroblastos L929 foram semeados sobre discos de Zr e BS+Zr e a proliferação (Alamar Blue; n=6), morfologia (MEV; n=2), migração celular (wound healing; n=4) e a síntese de colágeno (Sirius Red; n=6) foram avaliados até 7 dias. Os dados foram analisados pelos testes ANOVA/Tukey (a=5%). Um recobrimento homogêneo consistindo de Si, Na, O e Ca foi detectado na superfície da Zr após o tratamento térmico com BS, o qual promoveu um aumento significante na rugosidade e energia livre de superfície (p<0,05). Nenhuma mudança nos parâmetros de cor foi observada (p>0,05). Células semeadas na superfície de Zr+BS apresentaram maior proliferação, expressão de colágeno e capacidade de migração em comparação com aquelas cultivadas sobre a superfície de Zr (p<0,05). Imagens de MEV revelaram que o espalhamento celular ocorreu mais rápido na presença de BS. Assim, conclui-se que o tratamento térmico da superfície da Zr com BS levou a deposição de um recobrimento bioativo, o qual induziu in vitro o espalhamento, proliferação e migração de fibroblastos gengivais e expressão de colágeno.

Abstract The objective of this study was the development of a bioactive glass coating on zirconia (Zr) to modulate the gingival fibroblast phenotype. For this purpose, Biosilicate® (BS) particles in a water/isopropyl alcohol (1:1) vehicle (6 mg/mL) were applied to zirconia discs followed by thermal treatment at 1100 °C for 20 min. The surface topography (SEM), chemical composition (EDX), surface roughness (Ra; confocal microscopy), surface free energy (goniometry), and color alteration (UV-vis spectrophotometry) were assessed (n=6). Thereafter, L929 fibroblasts were seeded onto Zr and Zr+BS discs, and cell proliferation (Alamar Blue; n=6), morphology (SEM; n=2), migration (wound healing; n=4), and collagen synthesis (Sirius Red; n=6) were evaluated up to 7 days. Data were analyzed by ANOVA/Tukey tests (a=5%). A homogeneous coating consisting of Si, Na, O, and Ca was detected on the Zr surface after thermal treatment with BS, which led to a significant increase in surface roughness and free energy (p<0.05). No change in color parameters was observed (p>0.05). Cells seeded on the Zr+BS surface featured increased proliferation, collagen expression, and migration capability in comparison with those cultured on plain Zr (p<0.05). SEM images revealed that cell spreading occurred faster in the presence of BS. Therefore, it was concluded that thermal treatment of the Zr surface with BS led to the deposition of a bioactive coating, which induced gingival fibroblast spread, proliferation, migration, and collagen expression in vitro.

Abstract Objective This study was designed for the chemical activation of a 35% hydrogen peroxide (H2O2) bleaching gel to increase its whitening effectiveness and reduce its toxicity. Methodology First, the bleaching gel - associated or not with ferrous sulfate (FS), manganese chloride (MC), peroxidase (PR), or catalase (CT) - was applied (3x 15 min) to enamel/dentin discs adapted to artificial pulp chambers. Then, odontoblast-like MDPC-23 cells were exposed for 1 h to the extracts (culture medium + components released from the product), for the assessment of viability (MTT assay) and oxidative stress (H2DCFDA). Residual H2O2 and bleaching effectiveness (DE) were also evaluated. Data were analyzed with one-way ANOVA complemented with Tukey’s test (n=8. p<0.05). Results All chemically activated groups minimized MDPC-23 oxidative stress generation; however, significantly higher cell viability was detected for MC, PR, and CT than for plain 35% H2O2 gel. Nevertheless, FS, MC, PR, and CT reduced the amount of residual H2O2 and increased bleaching effectiveness. Conclusion Chemical activation of 35% H2O2 gel with MC, PR, and CT minimized residual H2O2 and pulp cell toxicity; but PR duplicated the whitening potential of the bleaching gel after a single 45-minute session.

Resumo Biomateriais inovadores podem fornecer uma promissora nova direção para o tratamento de defeitos ósseos, estimulando um processo de reparo adequado, sem danos aos tecidos adjacentes. O objetivo deste estudo in vivo foi avaliar a biocompatibilidade e a capacidade osteoindutora de uma biomembrana e um scaffold compostos por colágeno e quitosana, contendo cimento de aluminato de cálcio. Dezoito coelhos (New Zealand White, Oryctolagus cuniculus) foram distribuídos de acordo com os períodos experimentais de análise (7, 15 e 30 dias). Quatro defeitos foram criados na calvaria dos coelhos, que foram individualmente preenchidos com a biomembrana, scaffold, coágulo (controle negativo) e osso autólogo (controle positivo). A avaliação histopatológica foi realizada em microscópio óptico em aumentos de 32´, 64´, 125´ e 320´. A resposta celular à inflamação e à formação de novo tecido ósseo foi quantificada utilizando um sistema de escore. O grupo da biomembrana apresentou maior resposta inflamatória no período de 15 dias, com diferença significativa para o grupo do osso autólogo (p<0,05). Não houve diferença estatística significante para a reação do tipo corpo estranho entre os grupos (p>0,05). Em relação à neoformação óssea, observou-se fechamento linear da área do defeito, que foi mais evidente no grupo em que se utilizou o osso autólogo. O grupo scaffold apresentou resultados semelhantes ao grupo do osso autólogo no período de 30 dias (p>0,05). Ambos os biomateriais testados apresentaram biocompatibilidade similar em comparação com os grupos controle. Além disso, a biomembrana e o scaffold apresentaram capacidade osteoindutora similar, estimulando o reparo ósseo ao longo dos intervalos de tempo experimentais.

Abstract Innovative biomaterials can provide a promising new direction for the treatment of bone defects, stimulating a proper repair process, with no damage to adjacent tissues. The purpose of this in vivo study was to evaluate the biocompatibility and the osteoinductive capacity of chitosan-collagen biomembrane and scaffold containing calcium aluminate cement. Eighteen New Zealand white rabbits (Oryctolagus cuniculus) were distributed according to the experimental times of analysis (7, 15 and 30 days). Four bone defects were created in the rabbits calvaria, which were individually filled with the biomembrane, scaffold, blood clot (negative control) and autologous bone (positive control). Histopathological analysis was performed using optical microscope at 32´, 64´, 125´ and 320´ magnifications. Cell response to inflammation and new bone tissue formation was quantified using a score system. The biomembrane group presented greater inflammatory response at 15 days, with significant difference to autologous bone group (p<0.05). There was no statistically significant difference for foreign body type reaction among groups (p>0.05). Concerning new bone formation, linear closure of the defect area was observed more evidently in the group with autologous bone. The scaffold group presented similar results compared with the autologous bone group at 30 days (p>0.05). Both tested biomaterials presented similar biocompatibility compared with the control groups. In addition, the biomembrane and scaffold presented similar osteoinductive capacity, stimulating bone repair process in the course of the experimental time intervals.

Resumo O objetivo do presente estudo foi avaliar a citotoxicidade dos cimentos dos canais radiculares em condições próximas à realidade clinica. Uma linhagem primária de fibrolastos humanos foi semeada em placas acrílicas de 24-poços com meio de cultura Dulbecco’s modified Eagle’s medium suplementado com 10% de soro fetal bovino e incubados por 24 h. Os canais radiculares de pré-molares foram obturados e individualmente adaptados aos dispositivos de nylon para serem estabilizados nos poços com as células já semeadas. Amostras foram dividas de acordo com os grupos: Controle: cones de gutta-percha (CGP); AH Plus+CGP; Sealapex+CGP; MTA Fillapex+CGP e Endofill+CGP. Após 24 e 48 h, a viabilidade e a morfologia celular foram avaliadas pelo ensaio de MTT e microscopia eletrônica de varredura (MEV), respectivamente. Análises estatísticas foram realizadas pelo teste de Mann-Whitney, complementadas por Kruskal Wallis (p<0,05). Apenas o Endofill apresentou citotoxicidade após 24 h. MTA Fillapex e Endofill reduziram a produção da enzima desidrogenase succinica após 48 h. AH Plus não apresentou toxicidade em nenhum momento. MEV mostrou que os grupos AH Plus e o MTA Fillapex apresentaram fibroblastos com morfologia próxima ao grupo controle, enquanto que o grupo do Endofill apresentou poucas células com finos prolongamentos. O presente estudo demonstrou que resultados satisfatórios foram apresentados nos grupos AH Plus e Sealapex, mas não para o Endofill após 48 h. O método utilizado permitiu avaliar a citotoxicidade dos cimentos que se difundem pelo ápice radicular.

Abstract The aim of the present study was to evaluate the cytotoxicity of root canal sealers under conditions closely resembling a clinical reality. A primary human fibroblast cell line was seeded in 24-well acrylic plates with Dulbecco’s modified Eagle’s medium supplemented with 10% serum fetal bovine (SFB) and incubated for 24 h. Root canals from premolars were filled and individually attached to nylon devices to be stabilized in the wells with the already seeded cells. Specimens were divided into groups as follows: Control: gutta-percha cones (GPC); AH Plus+GPC; Sealapex+GPC; MTA Fillapex+GPC and Endofill+GPC. After 24 and 48 h, cell viability and morphology were evaluated by MTT assay and scanning electron microscopy (SEM), respectively. Statistical analysis was performed by Mann-Whitney test, complemented by Kruskal Wallis test (p<0.05). Only Endofill presented cytotoxicity after 24 h. MTA Fillapex and Endofill reduced the production of succinic desidrogenase after 48 h. AH Plus was non-toxic at any time point. SEM showed that the AH Plus and MTA Fillapex groups presented fibroblasts with morphology close to the control group, while the Endofill group presented few cells with thin extensions cells. The present study showed that good results were present in AH Plus and Sealapex, but not the Endofill group after 48 h. The method used enabled evaluation of the cytotoxicity of the studied sealers that diffused through the root apex.

Resumo Este estudo avaliou a citotoxicidade de um cimento de aluminato de cálcio (EndoBinder) contendo diferentes radiopacificadores, Bi2O3, ZnO ou ZrO2, comparativamente ao trióxido mineral agregado (MTA). Seguindo a norma ISO 10993-12:2012 (E), 0,2 g de cada cimento foi aplicada em insertos transwell, que foram colocados em placas de cultura de 24 wells contendo 1 mL de meio de cultura (DMEM). Após 24 h de incubação, os extratos (DMEM contendo componentes liberados dos cimentos) foram aplicados sobre células pulpares imortalizadas MDPC-23. Viabilidade celular (teste de MTT), atividade da fosfatase alcalina (ALP), produção de proteína total e a morfologia das células (Microscópio Eletrônico de Varredura - MEV) foram avaliadas. Um volume de 50 µL do extrato foi utilizado para determinar, através de Espectroscopia de Energia Dispersiva (EDS), os elementos químicos liberados pelos cimentos. Os seguintes grupos foram estabelecidos (n=6): NC - controle negativo (sem tratamento); EB - EndoBinder sem radiopacificador; EBBO - EndoBinder+Bi2O3; EBZnO - EndoBinder+ZnO; EBZrO - EndoBinder+ZrO2 e WMTA - MTA branco. Os dados foram submetidos à análise estatística (teste de Kruskal-Wallis, nível de significância=5%). Células expostas às diferentes versões de EndoBinder apresentaram pequena redução na viabilidade, produção de proteína total e atividade da ALP, com valores semelhantes aos grupos NC e WMTA (p>0,05). Diversos elementos (C, O, Na, Al, P, Si, Cl, Bi, K) liberados pelos cimentos foram detectados nos extratos. Entretanto, as células não apresentaram alterações significativas em sua morfologia. EndoBinder e MTA, não afetaram negativamente o metabolismo das células odontoblastóides, mostrando-se citocompatíveis, independente do radiopacificador utilizado.

Abstract This study aimed to evaluate the cytotoxicity of a calcium aluminate cement (EndoBinder) containing different radiopacifiers, Bi2O3, ZnO or ZrO2, compared with Mineral Trioxide Aggregate (MTA). According to ISO 10993-12:2012 (E) recommendations, 0.2 g of each cement were applied in transwell inserts and placed in 24-well culture plates containing 1 mL of culture medium (DMEM). After 24 h of incubation, the extracts (DMEM containing components released from the cements) were applied to immortalized odontoblast-like MDPC-23 cells. Cell viability (MTT test), alkaline phosphatase activity (ALP), total protein production and cell morphology (Scanning Electron Microscopy - SEM) were evaluated. The volume of 50 µL of extract was used to determine the chemical elements released by the cements using Energy Dispersive Spectroscopy (EDS). The following groups were established (n=6): NC - negative control (without treatment); EB - EndoBinder without radiopacifier; EBBO - EndoBinder+Bi2O3; EBZnO - EndoBinder+ZnO; EBZrO - EndoBinder+ZrO2 and WMTA - White MTA. Data were subjected to statistical analysis (Kruskal-Wallis test, level of significance=5%). Cells exposed to the different versions of EndoBinder presented small reduction in viability, total protein production and ALP activity, with values similar to the NC and WMTA groups (p>0.05). Different elements (C, O, Na, Al, P, Si, Cl, Bi, K) released by the cements were detected in the extracts. However, the cells had no significant changes in their morphology. EndoBinder and MTA did not affect negatively the metabolism of the odontoblastic-like cells, showing it to be cytocompatible, irrespective of the used radiopacifier.

Abstract: The aim of this study was to evaluate the bioactivity and cytocompatibility of simvastatin (SV) applied to MDPC-23 odontoblast-like cells. For this purpose, MDPC-23 cells were seeded in 96-well plates and submitted to treatments with 0.01 or 0.1 μM of SV for 24 h, 72 h or continuously throughout the experimental protocol. The negative control group (NC) was maintained in DMEM. Cell viability (MTT), ALP activity (thymolphthalein monophosphate), and mineralized matrix deposition (alizarin red) were analyzed at several time points. The data were submitted to ANOVA and Tukey's test (α = 0.05). Although cell viability was observed in the groups treated with SV, these groups did not differ from the NC up to 7 days. There was a reduction in cell viability for the groups treated with 0.1 μM of SV for 72 h, and submitted to continuous mode after 14 days. A significant increase in ALP activity occurred in the group treated with 0.01 μM of SV for 24 h, compared with the NC; however, only the group treated with 0.1 μM of SV in continuous mode reduced the ALP activity, in comparison with the NC. After 14 days, only continuous treatment with 0.1 μM of SV did not differ from NC, whereas the other experimental groups showed increased mineralized matrix deposition. Thus, it was concluded that low concentrations of simvastatin were bioactive and cytocompatible when applied for short periods to cultured MDPC-23 odontoblast-like cells.

Resumo Fibroblastos participam no processo de reparação de ferida através da proliferação e migração, bem como a síntese de fatores de crescimento e moléculas da matriz extracelular. No entanto, o envelhecimento celular e o próprio indivíduo podem levar à redução de funções celulares e, consequentemente, a capacidade de reparação de tecidos. Este estudo avaliou a atividade dos fibroblastos gengivais de pacientes jovens (J) e idosos (I) e sua capacidade de resposta frente ao fator de necrose tumoral alfa (TNF-a). Fibroblastos gengivais foram isolados de seis pacientes (3J e 3I) e semeados em meio de cultura completo (DMEM). Para a análise de viabilidade celular, a produção de proteína total e a síntese de colágeno, fibroblastos foram cultivados em placas de 96 poços, durante 24, 48 ou 72 h (n = 36). Respostas celulares frente ao TNF-a, foram avaliadas por aplicação desta citocina (100 ng/mL) nas células cultivadas durante 24 h, seguida por avaliação de espécies reativas de oxigênio (EROs), produção de óxido nítrico (NO) e produção CCL5 (n= 36). Os dados foram analisados por testes de Kruskal-Wallis e Mann-Whitney U (a = 0,05). A viabilidade de fibroblastos I foi mais elevada do que os fibroblastos J para 24 e 48 h, mas estas células mostraram uma redução gradual de viabilidade ao longo do tempo. Para as células de J, foi observada redução da síntese de colágeno em 48 h. Não foi observada diferença na produção de EROs para ambas as células após exposição ao TNF-a. No entanto, ambas as culturas apresentaram aumento da produção de NO e CCL5 na presença de TNF-a. Diferenças funcionais e alteração na capacidade de resposta ao TNF-a foram observadas de acordo com a idade do paciente.

Abstract Fibroblasts participate in the wound repair process through proliferation and migration as well as the synthesis of factors growth and extracellular matrix molecules. However, cell aging and the individual himself can lead to reduction of cell functions and consequently, the ability of tissue repair. This study evaluated the activity of gingival fibroblasts from young (Y) and elderly (Y) patients and their responsiveness to tumor necrosis factor alpha (TNF-a). Gingival fibroblasts were isolated from six patients (3Y; and 3E) and seeded in complete culture medium (DMEM). For cell viability analysis, total protein production and collagen synthesis, fibroblasts were cultured in 96-well plates for 24, 48 or 72 h (n=36). Cell responses to TNF-a, was evaluated by application of this cytokine to cultured cells (100 ng/mL) for 24 h, followed by evaluation of reactive oxygen species (ROS), nitric oxide (NO) and CCL5 production (n=36). Data were analyzed by Kruskal-Wallis and the Mann-Whitney U tests (a = 0.05). Viability of E fibroblasts was higher than Y fibroblasts for 24 and 48 h, but these cells showed gradual reduction of viability over the course of time. For Y cells, reduced collagen synthesis was observed at 48 h. No difference was observed in ROS production for both cells after TNF-a exposure. However, both cultures showed increased production of NO and CCL5 in the presence of TNF-a. Functional differences and distinct responsiveness to TNF-a were observed according to patient's age.

Resumo Fototerapia tem sido indicada como um tratamento adjuvante para o reparo de tecidos, incluindo o tecido pulpar. Entretanto, não há parâmetros de irradiação definidos, o que representa um grande desafio para o uso clínico da fototerapia. O objetivo deste estudo foi avaliar o efeito da fototerapia com LED vermelho em células MDPC-23 com fenótipo odontoblastóide, usando vários parâmetros. As células foram semeadas (104 células/cm2), incubadas por 12 h em DMEM completo e então o meio de cultura foi trocado por DMEM com 0,5% SFB. Após 12 h de incubação, as irradiações foram realizadas (630±10 nm) usando um dispositivo com densidade de potência de 20 ou 40 mW/cm2 e dose de energia de 2 J/cm2. As células foram irradiadas 1 ou 3 vezes, com intervalos de 1 min. Células não irradiadas serviram como controle. Foram avaliadas a viabilidade (ensaio de MTT), dosagem de proteína total (método de Lowry) e número de células viáveis (ensaio de Trypan blue). Os dados (n=12 por grupo) foram submetidos aos testes de Kruskal-Wallis e Mann-Whitney (p=0,05). Uma única irradiação com 20 ou 40 mW/cm2 aumentou a viabilidade celular, a qual foi negativamente afetada após 3 irradiações. Células irradiadas apenas uma vez com 20 mW/cm2 produziram mais proteínas comparadas com aquelas irradiadas com 40 mW/cm2. Redução no número de células viáveis ocorreu apenas após 3 irradiações com 40 mw/cm2. Em conclusão, o LED vermelho foi capaz de biomodular a atividade metabólica de células MDPC-23. A melhor bioestimulação celular foi obtida quando uma única irradiação com dose de energia de 2 J/cm2 e densidade de potência de 20 mW/cm2 foi administrada às células pulpares.

Abstract Phototherapy has been indicated as an adjunctive treatment for tissue repair, including the pulp tissue. However, there are no defined irradiation parameters, which is a great challenge to the clinical use of phototherapy. The aim of this study was to evaluate the effect of phototherapy with red LED on odontoblast-like MDPC-23 cells, using different parameter settings. Cells were seeded (104 cells/cm²), incubated for 12 h in complete DMEM and then the culture medium was replaced by DMEM supplemented with 0.5% FBS. After 12 h incubation, irradiations were performed (630±10 nm) using a LEDTable device with a 20 or 40 mW/cm² power density and 2 J/cm² energy dose. The cells were irradiated 1 or 3 times, at 1 min intervals. Non-irradiated cells served as control. The cells were evaluated for viability (MTT assay), total protein dosage (Lowry method) and number of viable cells (Trypan blue). The data (n=12 per group) were submitted to Kruskal-Wallis and Mann-Whitney tests (p=0.05). A single irradiation with 20 or 40 mW/cm² enhanced cell viability, which was negatively affected after 3 consecutive irradiations. Cells irradiated only once with 20 mW/cm² produced more proteins compared with those irradiated with 40 mW/cm². Reduction in the number of viable cells occurred only after 3 consecutive irradiations with 40 mW/cm². In conclusion, red LED was capable of biomodulating the metabolic activities of cultured MDPC-23 odontoblast-like cells. The best cell biostimulation was obtained when a single irradiation with 2 J/cm2 energy dose and 20 mW/cm2 power density was delivered to the pulp cells.

Abstract The development of biomaterials capable of driving dental pulp stem cell differentiation into odontoblast-like cells able to secrete reparative dentin is the goal of current conservative dentistry. In the present investigation, a biomembrane (BM) composed of a chitosan/collagen matrix embedded with calcium-aluminate microparticles was tested. The BM was produced by mixing collagen gel with a chitosan solution (2:1), and then adding bioactive calcium-aluminate cement as the mineral phase. An inert material (polystyrene) was used as the negative control. Human dental pulp cells were seeded onto the surface of certain materials, and the cytocompatibility was evaluated by cell proliferation and cell morphology, assessed after 1, 7, 14 and 28 days in culture. The odontoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, total protein production, gene expression of DMP-1/DSPP and mineralized nodule deposition. The pulp cells were able to attach onto the BM surface and spread, displaying a faster proliferative rate at initial periods than that of the control cells. The BM also acted on the cells to induce more intense ALP activity, protein production at 14 days, and higher gene expression of DSPP and DMP-1 at 28 days, leading to the deposition of about five times more mineralized matrix than the cells in the control group. Therefore, the experimental biomembrane induced the differentiation of pulp cells into odontoblast-like cells featuring a highly secretory phenotype. This innovative bioactive material can drive other protocols for dental pulp exposure treatment by inducing the regeneration of dentin tissue mediated by resident cells.

Abstract Osteonecrosis of the jaw is an adverse effect of bisphosphonates. While the etiopathogenesis of this condition has been investigated, the interactions and effects of bisphosphonates on oral mucosa cells remain unclear. It is hypothesized that cell culture models, such as co-culture or three-dimensional cell culture models, can provide valuable insight. Therefore, the aim of this study was to evaluate the effects of zoledronic acid (ZA) on epithelial cells and gingival fibroblasts in a co-culture model. Briefly, epithelial cells were seeded on transwell inserts and gingival fibroblasts were seeded in the lower well of 24-well plates. The latter were treated with ZA (5 μM) for 24 or 48 h. Cell viability and synthesis of the inflammatory chemokine, CCL2, were subsequently assessed. Data were subjected to statistical analysis with a 5% significance level. In the presence of ZA, the epithelial cells exhibited significant toxicity in both cell culture models and at both time points. However, greater cytotoxicity was observed in the co-culture model. Greater viability for the gingival fibroblasts was also associated with the co-culture model, and ZA-mediated toxicity was observed for the 48 h time point. ZA promoted a significant increase in CCL2 synthesis in both sets of cells, with greater CCL2 synthesis detected in the gingival fibroblasts. However, this effect was diminished in the co-culture model. Taken together, these results confirm the specific response patterns of the cells seeded in the co-culture model and also demonstrate the protective mechanism that is mediated by epithelial/mesenchymal cell interactions upon exposure to ZA.

Resumo Os resíduos gerados na indústria de sucos têm sido subaproveitados, desperdiçando um produto que pode apresentar potencial para ser incluído na alimentação humana. Assim, o objetivo do estudo foi realizar análise microbiológica e da composição nutricional das farinhas do resíduo de suco de uva, maçã, laranja e acerola, com diferentes granulometrias. Os resíduos da produção de suco foram obtidos através de doação de uma indústria do município de Bento Gonçalves-RS, sendo então secos e moídos para obter a farinha em diferentes granulometrias (>0,600 mm; 0,600 mm; 0,425 mm; 0,300 mm; ≤0,250 mm). As amostras de farinha foram submetidas à análise de composição centesimal, acidez, pH, Brix e polifenóis totais. As análises microbiológicas incluíram contagem de coliformes termotolerantes e pesquisa de Salmonella spp. Os resultados indicaram qualidade microbiológica satisfatória. Quanto à composição centesimal, os resultados mostraram que a granulometria influenciou o valor nutricional das farinhas e que os valores energéticos das farinhas do resíduo de acerola, laranja e uva foram inversamente proporcionais ao tamanho da partícula. Todas as farinhas analisadas podem ser consideradas ricas em fibras, cujos valores variaram de 11,2% a 54,5%. As farinhas de uva e acerola apresentaram maiores teores de proteínas, 8,2% a 15,6% e 8,3% a 12,1%, respectivamente. As farinhas do resíduo de laranja e uva apresentaram elevados teores de polifenóis totais. Portanto, o uso destes resíduos na alimentação humana pode ser recomendado, melhorando o valor nutricional de preparações.

Summary Residues obtained from the juice industry have been underused, wasting a product that might present potential to be included in food for humans. Thus the objective of the present study was to carry out microbiological and nutritional compositional analyses of the flours made from grape, apple, orange and acerola juice residues, with different granulometries. The juice residues were obtained by donation from an industry in Bento Gonçalves (RS/Brazil), dried and ground in order to obtain flours with different granulometries (>0.600, 0.600 mm, 0.425 mm, 0.300 mm and ≤0.250 mm). The flour samples were submitted to an analysis of the proximate composition, acidity, pH, °Brix and total polyphenols. The microbiological analysis consisted of enumerating heat tolerant coliforms and testing for the presence of Salmonella spp. The results indicated satisfactory microbiological quality. The proximate composition showed that granulometry influenced the nutritional values of the flours and that the energy values of flours obtained from acerola, orange and grape residues were inversely proportional to the particle size. All the flours analysed could be considered rich in fibres, with values varying from 11.2% to 54.5%. The grape and acerola flours presented higher protein levels – from 8.2% to 15.6% and from 8.3% to 12.1%, respectively – and the orange and grape residue flours presented high levels of total polyphenols. Thus the use of such residues in human nutrition can be recommended, improving the nutritional value of food preparations.

<p>Apesar de diversos estudos envolvendo regeneração tecidual, incluindo o reparo pulpar induzido por diferentes fontes de luz, dados limitados têm sido reportados a respeito dos efeitos da irradiação com diodos emissores de luz (LED) sobre células-tronco de dentes decíduos esfoliados (SHEDs). O objetivo do presente estudo foi avaliar os efeitos de diferentes doses de energia (DE) do LED infravermelho sobre a viabilidade celular, número de células viáveis e produção de nódulos mineralizados (NM) por SHEDs. As células foram obtidas a partir de dentes decíduos próximos ao período de esfoliação (n=3), semeadas em DMEM completo (10⁴ células/cm2) e irradiadas utilizando um protótipo de LED (LEDTable 850 nm, 40 mW/cm²⁾ com as doses de 0 (controle), 2, 4, 8, 15 ou 30 J/cm² (n=9). A viabilidade celular (MTT), o número de células viáveis (trypan blue assay) e a formação de NM (alizarin red stain) foram realizados 12 e 72 h após a irradiação. Os dados foram avaliados utilizando os testes Kruskal-Wallis e Mann-Whitney (α=0,05). As células irradiadas com 2 ou 4 J/cm² exibiram uma maior viabilidade em 72 h, e todas as DE aumentaram o número de células viáveis após 12 h. Para a formação de NM, os melhores resultados foram observados 72 h após a irradição das SHEDs, com as doses de 8 e 15 J/cm². Concluiu-se que a viabilidade celular, o número de células e a formação de NM por células pulpares são aumentados após exposição ao LED infravermelho. De um modo geral, a melhor bioestimulação celular (SHEDs) foi obtida com 4 e 8 J/cm².</p>

<p>Despite several reports regarding tissue regeneration, including pulp repair induced by different light sources, only limited data have been reported concerning the effects of light-emitting diodes (LED) on stem cells from human exfoliated deciduous teeth (SHEDs). The aim of this study was to evaluate the effects of different energy densities of infrared LED on the cell viability, number of cells and mineralized tissue production by SHEDs. SHEDs were obtained from near-exfoliation primary teeth (n=3), seeded in plain DMEM (104 cells/cm2), and irradiated by a LED prototype (LEDTable 850 nm, 40 mW/cm2) delivering 0 (control), 2, 4, 8, 15 or 30 J/cm2 (n=9). Cell viability (MTT assay), cell proliferation (trypan blue assay), and mineralized nodule (MN) formation (alizarin red stain) were assessed 12 and 72 h post-irradiation. Data were subjected to Kruskal-Wallis and Mann-Whitney tests (α=0.05). Cells irradiated with 2 or 4 J/cm2 exhibited higher metabolism at 72 h, and all energy densities provided increase in cell proliferation after 12 h. Regarding MN formation, the best results were observed at 72 h after SHED irradiation with 8 and 15 J/cm2. It was concluded that the cell viability, cell number and MN formation by pulp cells are enhanced after exposure to infrared LED irradiation. Overall, the greatest SHED biostimulation was obtained with 4 and 8 J/cm2.</p>

Neste estudo, foi avaliado o potencial protetor do isômero alfa-tocoferol da vitamina E (-T) contra a ação tóxica do peróxido de hidrogênio (PH) aplicado sobre células pulpares. Células odontoblastóides MDPC-23 foram semeadas em placas de 96 compartimentos por 72 h, tratadas com diferentes concentrações de α-T (1, 3, 5 e 10 mM) por diferentes períodos (1, 4, 8 e 24 h) e, então, expostas ou não a uma solução com 0,0018% de PH por 30 min. Nos controles positivo e negativo, as células foram expostas ao PH ou meio de cultura (DMEM contendo 5% de DMSO), respectivamente. A viabilidade celular foi avaliada pelo teste do MTT e os valores numéricos de absorbância, expressos em porcentagem, foram submetidos a análise estatística pelos testes de Kruskal-Wallis e Mann-Whitney (α=5%). Considerando as células do grupo controle negativo como apresentando 100% de viabilidade celular, todas as combinações de α-T, nas diferentes concentrações, e tempos de pré-tratamento demonstraram um efeito protetor contra a citotoxicidade do PH. Redução significativa da viabilidade (59%) foi observada para o grupo controle positivo comparado ao controle negativo. Os maiores valores de viabilidade celular foram obtidos após pré-tratamento com 1 e 3 mM de α-T por 24 h seguido de exposição ao PH (126% e 97% de viabilidade celular, respectivamente). Assim, de acordo com as condições experimentais, o efeito protetor mais efetivo contra os efeitos tóxicos do PH foi observado para as menores concentrações de α-T (1 e 3 mM) aplicado por 24 h.

This in vitro study evaluated the potential protective effect of vitamin E alpha-tocopherol (α-T) isomer against the toxicity of hydrogen peroxide (HP) applied on dental pulp cells. Odontoblast-like MDPC-23 cells were seeded on 96-well plates for 72 h, treated with different concentrations of α-T (1, 3, 5, and 10 mM) for different times (1, 4, 8, and 24 h) and then exposed or not to a 0.018% HP solution for 30 min. In positive and negative control groups, cells were exposed to HP or culture medium (DMEM containing 5% DMSO), respectively. Cell viability was assessed by the MTT assay and the absorbance numeric data, expressed as percentage values, were subjected to the statistical analysis by Kruskal-Wallis and Mann-Whitney tests (α=5%). Considering the cells in the negative control as having 100% of cell viability, all combinations of α-T concentrations and pretreatment times showed a protective effect against HP cytotoxicity. Significant reduction of cell viability (59%) was observed in the positive control compared with the negative control. The highest values of pulp cell viability were obtained after pretreatment with 1 and 3 mM α-T concentrations for 24 h followed by exposure to HP (126% and 97% of cell viability, respectively). Under the tested conditions, the most effective cell protection against the cytotoxic effects of HP was provided by the lowest concentrations of α-T (1 and 3 mM) applied for 24 h.

A osteonecrose induzida por bisfosfonatos tem sido associada a um efeito citotóxico destes medicamentos sobre as células da mucosa oral. Um estudo recente demonstrou que 5 µM de ácido zoledrônico (AZ), um potente bisfosfonato nitrogenado, foi a maior concentração encontrada na cavidade oral da pacientes em tratamento com este medicamento. Portanto, para simular esta condição in vivo, o objetivo deste estudo foi avaliar o efeito da aplicação desta concentração de AZ sobre células epiteliais (HaCaT) e fibroblasto de gengiva. As células foram semeadas (3×104 células/cm2) e incubadas por 48 h em placas de 24 compartimentos, utilizando meio de cultura completo (cDMEM). Após permanecer por 24 h em DMEM sem soro fetal bovino (DMEM-SFB), 5 µM do AZ foram adicionados a este meio de cultura, o qual foi incubado em contato com as células por 48 h. Após este período, foram avaliados o número de células viáveis (trypan blue), viabilidade celular (teste de MTT), produção de proteína total e a morfologia celular (MEV). Os dados obtidos foram submetidos aos testes estatísticos de Mann-Whitney e ANOVA complementada por testes de Tukey (p>0,05). Foi demonstrado que o AZ causou diminuição significativa no número de células viáveis, além de redução do metabolismo celular para ambos os tipos celulares avaliados. Porém, redução na produção de proteína total ocorreu apenas para as células epiteliais. Alterações morfológicas foram observadas em ambos os tipos celulares tratados com AZ. Estes dados científicos indicam que a concentração de AZ avaliada neste estudo (5 µM) apresenta ação citotóxica sobre células epiteliais e fibroblastos de gengiva, o que poderia estar associado, clinicamente, ao desenvolvimento da osteonecrose induzida por bisfosfonatos.

Bisphosphonate-induced osteonecrosis has been related to the cytotoxicity of these drugs on oral mucosa cells. A previous study showed that 5 µM of zoledronic acid (ZA), a nitrogen-containing bisphosphonate, is the highest concentration of this drug found in the oral cavity of patients under treatment. Therefore, in order to simulate an osteonecrosis clinical condition, the aim of this study was to evaluate the highest concentration of ZA applied on human epithelial cells (HaCaT) and gingival fibroblasts. For this purpose, cells (3×104 cells/cm2) were seeded in wells for 48 h using complete culture medium (cDMEM). After 48 h incubation, the cDMEM was replaced by fresh serum-free culture medium (DMEM-FBS) in which the cells were maintained for additional 24 h. Then, 5 µM ZA were added to the DMEM–FBS and the cells incubated in contact with the drug for 48 h. After this period, the number of viable cells (trypan blue), cell viability (MTT assay), total protein (TP) production and cell morphology (SEM analysis) were assessed. Data were analyzed statistically by Mann-Whitney, ANOVA and Tukey's test (α=0.05). ZA caused a significant reduction in the number of viable cells and decreased the metabolic activity of both cell lines. However, decrease of TP production occurred only in the epithelial cell cultures. Morphological alterations were observed in both cell types treated with ZA. In conclusion, ZA (5 µM) was cytotoxic to human epithelial cells and gingival fibroblast cultures, which could be associated, clinically, with the development of bisphosphonate-induced osteonecrosis.