Comparação in vitro da efetividade de vários meios de conservação na manutenção da viabilidade de fibroblastos do ligamento periodontal de dentes humanos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-graduação em OdontologiaOs objetivos deste estudo foram: estabelecer uma linhagem de fibroblastos oriundos de ligamento periodontal de dentes humanos; avaliar a efetividade de diferentes meios de conservação em manter a viabilidade desses fibroblastos; e verificar o efeito de diferentes temperaturas sobre a efetividade dos meios estudados. Uma vez estabelecida a linhagem de fibroblastos, a viabilidade celular foi avaliada pelo método colorimétrico MTT, após a conservação a 37°C por 3, 6, 24, 48 e 72h em diferentes meios: Meio Essencial Mínimo (MEM- controle-positivo), solução salina balanceada de Hank (HBSS) estéril, HBSS não estéril, leite desnatado, Save-A-Tooth® e água (controle-negativo). Para verificar o efeito da temperatura, o experimento foi repetido utilizando-se os mesmos meios e períodos, porém cultivando-se as células em temperatura ambiente (20°C). A viabilidade celular também foi avaliada pelo método de exclusão do Azul de Tripano. As células foram coradas após 6, 24, 48 e 72h de contato com MEM, HBSS estéril, HBSS não estéril, leite desnatado e Save-A-Tooth® a 20°C. Todos os experimentos foram realizados em triplicata. Calculadas as médias dos valores de absorbância (MTT) e do percentual de viabilidade celular (Azul de Tripano), a comparação dos resultados foi realizada pelo teste de Kruskal-Wallis. Posteriormente, foi aplicado o teste de comparações múltiplas de Scheffé para localizar as diferenças significativas entre as médias de absorbância e o percentual de células viáveis nos diferentes meios e períodos, num nível de significância de 5%. O teste de Mann-Whitney foi utilizado para comparar os resultados obtidos com o ensaio MTT à temperatura ambiente e a 37°C. Os resultados do MTT realizado à temperatura ambiente demonstraram que o controle-positivo (MEM-37) foi o melhor meio de conservação, seguido pelo leite e depois pela HBSS não estéril e estéril. Após 72h, o desempenho do leite e da HBSS estéril e não estéril foi semelhante. No experimento a 37°C, o leite foi o melhor meio de conservação até 24h. Após 48 e 72h, o desempenho do leite diminuiu e o da HBSS aumentou, apresentando resultados similares aos do grupo-controle. Independentemente da temperatura de cultivo, a água foi o pior meio de conservação e o Save-A-Tooth® demonstrou resultados semelhantes aos da água a partir de 24h. Em relação ao efeito da temperatura, no geral o cultivo a 37°C reduziu a viabilidade das células conservadas em água e em Save-A-Tooth®, mas aumentou a das mantidas em leite, HBSS estéril e HBSS não estéril, pelo menos até 24h. Pelo método do corante Azul de Tripano, os melhores meios de conservação foram o leite e a HBSS estéril e não estéril, não havendo diferença estatística entre eles, em qualquer período de tempo. Foi concluído que: a técnica de cultivo celular utilizada permitiu a obtenção de uma linhagem de fibroblastos periodontais; a temperatura interfere na efetividade do meio de conservação; leite desnatado, à temperatura ambiente, pode ser utilizado como meio de conservação até 48h. The aims of this study were to establish a fibroblast lineage from human periodontal ligament cells, to evaluate the effectiveness of different storage media for maintaining the viability of fibroblasts, and to verify the effect of different temperatures on the effectiveness of these media. After the determination of the fibroblasts lineage, the cell viability was evaluated by the MTT colorimetric method, after preservation at 37°C for 3, 6, 24, 48, and 72h in the following media: Minimal Essential Medium (MEM - positive-control), sterile and non-sterile Hank's balanced salt solution (HBSS), non-fat milk, Save-A-Tooth®, and water (negative-control). In order to verify the effect of the temperature, the experiment was repeated using the same storage media and periods, but with cell culturing at room temperature (20°C). The cell viability was also evaluated by Trypan Blue exclusion method. The cells were stained after 6, 24, 48, and 72h of contact with MEM, sterile and non-sterile HBSS, non-fat milk, and Save-A-Tooth® at 20°C. All experiments were performed in triplicate. After calculating the mean absorbance values (MTT) and the mean percent cell viability (Trypan Blue), the results were compared using the Kruskal-Wallis test. Scheffé's multiple comparisons test was used to determine the differences between absorbance means and the percent of viable cells in the different media and periods (p = 0.05). Mann-Whitney's test was used to compare the results obtained from MTT at room temperature and 37o C. MTT results at room temperature revealed that the positive-control (MEM-37) was the best storage medium, followed by milk, non-sterile HBSS, and sterile HBSS. After 72h, the performance of milk, sterile and HBSS non-sterile was similar. Milk was the best storage medium at 37o C for up to 24h. After 48 and 72h, milk performed poorer and HBSS performed better, exhibiting similar results to the positive-control. Regardless of the culture temperature, water was the poorer preservation medium, while Save-A-Tooth® showed similar results to the water after 24h. The culture at 37o C reduced the viability of cells preserved in water or Save-A-Tooth®, but increased the viability of cells maintained in milk, sterile and non-sterile HBSS, for up to 24h. The Trypan Blue staining method showed that the best storage media were milk, sterile and non-sterile HBSS, without statistical difference between them. Based on these results, it was concluded that the cell culture used allowed to obtain a periodontal fibroblasts lineage; the temperature interferes in the storage medium effect; non-fat milk at room temperature can be used for preservation for up to 48h

Meios de conservação de dentes avulsionados: estudo em cultura de células

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Odontologia, Florianópolis, 2009.Diversas pesquisas clínicas ou com cultura de células têm sido realizadas com o intuito de identificar o meio mais adequado para a conservação de dentes avulsionados ou de fibroblastos periodontais. Embora, na maioria das vezes, essas pesquisas confirmem ou refutem uma hipótese experimental, normalmente elas dão origem a algumas dúvidas. Esta tese de doutorado é composta de 4 pesquisas desenvolvidas com os objetivos de: 1) verificar se a renovação do leite, em intervalos regulares de 12, 24 ou 48h, é capaz de prolongar sua capacidade de manter a viabilidade de fibroblastos do ligamento periodontal humano (FLPH); 2) verificar se o tempo de estocagem da solução salina balanceada de Hank (HBSS) exerce influência sobre a sua capacidade de manter a viabilidade de FLPH; 3) comparar o efeito de diferentes meios de conservação sobre a viabilidade de FLPH; e 4) comparar o efeito de diferentes meios de conservação sobre a viabilidade e a capacidade proliferativa de FLPH. Para executá-las, as células foram conservadas, a 5 e/ou a 20°C, em própolis, clara de ovo, água de coco, HBSS, Save-A-Tooth®, leite desnatado e leite integral por períodos variáveis. Células armazenadas em Meio Essencial Mínimo (MEM) a 37°C e em água de torneira (5 e/ou a 20°C) serviram de controle-positivo e negativo, respectivamente. A viabilidade e a capacidade de proliferação celular foram determinadas pelo ensaio MTT. Os dados foram analisados pelos testes de Kruskal-Wallis, Scheffé e Mann-Whitney (a=5%). Com base nos resultados obtidos nos diferentes experimentos foi possível concluir que: 1) a renovação do leite foi capaz de prolongar a sua capacidade de manter a viabilidade de FLPH somente quando realizada, a cada 48h, no leite mantido a 5ºC; 2) o tempo de estocagem da HBSS influenciou negativamente a sua capacidade de manter a viabilidade de FLPH; 3) em ambas as temperaturas, o leite desnatado foi o melhor meio de conservação, seguido pelo leite integral e HBSS. O própolis, a clara de ovo e a água de coco podem ser indicados para a conservação de fibroblastos periodontais por um período máximo de 3h; 4) em ambas as temperaturas, os meios mais efetivos em manter a viabilidade celular (0h) foram o leite desnatado e o leite integral. Quando os meios foram mantidos a 5°C, o leite desnatado e o leite integral proporcionaram maior capacidade proliferativa aos FLPH. Quando mantidos a 20°C, a HBSS revelou os melhores resultados.Several clinical trials or cell culture studies have been carried out in order to identify the most appropriate storage medium for the maintenance of avulsed teeth or periodontal fibroblasts. Although in most cases, these studies confirm or refute an experimental hypothesis, they often give rise to some uncertainties. This doctoral thesis is composed of 4 studies developed with the following purposes: 1) to analyze whether the replacement of milk at 12, 24 or 48 h, can extend its ability to maintain human periodontal ligament fibroblasts (HPDLF) viability; 2) to verify if the storage time influences the effectiveness of Hank's balanced salt solution (HBSS) in maintaining HPDLF viability; 3) to compare the effect of different storage media to preserve HPDLF viability; and 4) to evaluate the effect of different storage media on the maintenance of the viability and proliferative capacity of HPDLF. The cells were stored at 5 and / or 20°C in propolis, egg white, coconut water, HBSS, Save-A-Tooth ®, skimmed and whole milk for different periods of time. Cells stored at 37°C in Minimum Essential Medium (MEM) and tap water (5 and / or 20°C) served, respectively, as positive and negative controls. The viability and cellular proliferation capacity was determined by MTT assay. Data were analyzed by Kruskal-Wallis, Scheffe and Mann-Whitney tests (á = 5%). Based on the results obtained in the different experiments, it was concluded that: 1) milk replacement was able to extend its ability to maintain HPDLF viability only when carried out every 48 hours at 5°C, 2) the storage time of HBSS negatively influenced its effectiveness, 3) at both temperatures, skim milk, followed by whole milk and HBSS preserved significantly more viable cells than any other tested solution. Propolis, egg white and coconut water can be indicated to preserve HPDLF for up to 3 hours, 4) at both temperatures, the most effective mediums to maintain cell viability (0 h) were skimmed and whole milk. When the media were kept at 5° C, skimmed and whole milk provided higher proliferative capacity to HPDLF. When kept at 20°C, HBSS showed the best results

Crown discoloration promoted by materials used in regenerative endodontic procedures and effect of dental bleaching: spectrophotometric analysis

Regenerative endodontic procedure (REP) has been proposed as a new approach to treat immature permanent teeth. However, materials used in REP for root canal disinfection or cervical sealing may induce tooth discoloration. Objectives To assess tooth crown’s color after intracanal treatment with triple antibiotic paste (TAP) or calcium hydroxide (CH); cervical sealing with glass ionomer cement (GIC) or mineral trioxide aggregate (MTA); and bleaching with carbamide peroxide. Material and Methods After pulp removal and color spectrophotometer measurement, 50 bovine incisors were divided into 4 experimental groups and one control (untreated). Experiments were performed in phases (Ph). Ph1: TAP (ciprofloxacin, metronidazole, minocycline), TAPM (ciprofloxacin, metronidazole, amoxicillin), DAP (ciprofloxacin, metronidazole), or CH treatment groups. After 1 and 3 days (d); 1, 2, 3 weeks (w); and 1, 2, 3 and 4 months (m), color was measured and medications were removed. Ph2: GIC or MTA cervical sealing, each using half of the specimens from each group. Color was assessed after 1d, 3d; 1w, 2w, 3w; 1m and 2m. Ph3: Two bleaching sessions, each followed by color measurement. Data were analyzed with ANOVA and post-hoc Holm-Sidak method. Results Ph1: Specimens of TAP group presented higher color alteration (ΔE) mean than those of TAPM group. No significant difference was found among TAP or TAPM and CH, DAP or Control groups. Ph2: cervical sealing materials showed no influence on color alteration. Ph3: Different ΔE means (from different groups), prior to bleaching, became equivalent after one bleaching session. Conclusions TAP induces higher color alteration than TAPM; color alteration increases over time; cervical sealing material has no influence on color alteration; and, dental bleaching was able to recover, at least partially, the tooth crown’s color

Prevalence of temporomandibular disorder in patients with fibromyalgia: a systematic review / Prevalência de distúrbio temporomandibular em pacientes com fibromialgia: uma revisão sistemática

INTRODUCTION: Temporomandibular Disorders (TMDs) and Fibromyalgia (FM) may share similar signs and symptoms. Among them, muscle pain may be involved and significantly reduce the quality of life of these patients. AIM: The aim of this systematic review was to determine the prevalence of TMD in patients with FM.MATERIALS AND METHODS: In this systematic review six electronic databases (LILACS, LIVIVO, PubMed, ScienceDirect, PsycINFO, and Web of Science), as well as three grey literature databases (Google Scholar, Open Grey, and ProQuest) were searched. Cross-sectional studies were selected by two independent reviewers and analyzed in two-phases, following the PRISMA statement. Risk of bias was assessed through the MASTARI (Meta-Analysis of Statistics Assessment and Review Instrument for observational studies from the Joana Briggs Institute).RESULTS: From 660 articles, 51 were eligible for full-text reading and six were finally included. None of the articles met all quality methodological criteria. Therefore, considering the overall risk of bias, one article was judged with moderate risk and five with low risk of bias. A heterogeneity was considered high; thus, a meta-analysis was not performed. From the qualitative analysis it was possible to determine that between 13% to 87.1% of patients with FM can present TMD.CONCLUSION: The prevalence of TMD in patients with FM ranged from 13% to 87.1%. It is suggested that further studies be carried out, mainly with longitudinal design and better quality methodology to help answer whether fibromyalgia is a risk factor for the development of TMDs

Crown discoloration promoted by materials used in regenerative endodontic procedures and effect of dental bleaching: spectrophotometric analysis

Abstract Regenerative endodontic procedure (REP) has been proposed as a new approach to treat immature permanent teeth. However, materials used in REP for root canal disinfection or cervical sealing may induce tooth discoloration. Objectives To assess tooth crown’s color after intracanal treatment with triple antibiotic paste (TAP) or calcium hydroxide (CH); cervical sealing with glass ionomer cement (GIC) or mineral trioxide aggregate (MTA); and bleaching with carbamide peroxide. Material and Methods After pulp removal and color spectrophotometer measurement, 50 bovine incisors were divided into 4 experimental groups and one control (untreated). Experiments were performed in phases (Ph). Ph1: TAP (ciprofloxacin, metronidazole, minocycline), TAPM (ciprofloxacin, metronidazole, amoxicillin), DAP (ciprofloxacin, metronidazole), or CH treatment groups. After 1 and 3 days (d); 1, 2, 3 weeks (w); and 1, 2, 3 and 4 months (m), color was measured and medications were removed. Ph2: GIC or MTA cervical sealing, each using half of the specimens from each group. Color was assessed after 1d, 3d; 1w, 2w, 3w; 1m and 2m. Ph3: Two bleaching sessions, each followed by color measurement. Data were analyzed with ANOVA and post-hoc Holm-Sidak method. Results Ph1: Specimens of TAP group presented higher color alteration (ΔE) mean than those of TAPM group. No significant difference was found among TAP or TAPM and CH, DAP or Control groups. Ph2: cervical sealing materials showed no influence on color alteration. Ph3: Different ΔE means (from different groups), prior to bleaching, became equivalent after one bleaching session. Conclusions TAP induces higher color alteration than TAPM; color alteration increases over time; cervical sealing material has no influence on color alteration; and, dental bleaching was able to recover, at least partially, the tooth crown’s color

Effect of Milk Renewal on Cell Viability In Vitro at Different Time Frames

<div><p>Abstract The purpose of this study was to evaluate if the renewal of milk as a storage medium, every 12, 24 and 48 h, is able to increase its ability to maintain human periodontal ligament fibroblasts (PDLF) viability over time. PDLF were soaked in Minimum Essential Medium at 37 °C (MEM-37) (positive control), tap water (Water) (negative control) and in skimmed milk (44 wells) at 5 °C and 20 °C. The skimmed milk was renewed every 12 h (Milk-12), 24 h (Milk-24) and 48 h (Milk-48) in 11 wells of each plate, and the milk in the remaining 11 wells of each plate was maintained in situ (not renewed milk) (NRM). After 24, 48, 72, 96 and 120 h, cell viability was determined by the tetrazolium salt-based colorimetric (MTT) assay. Data were statistically analyzed by Kruskal-Wallis, Scheffé and Mann-Whitney tests (a=5%). At 5 °C, only Milk-48 was significantly better than NRM. At 20 °C, NRM was more effective than Milk-12 and Milk-24 in all time periods. In relation to the temperature (5 °C or 20 °C), renewal of milk at 5 °C was better in maintaining cell viability than the renewal at 20 °C. In conclusion, the renewal of milk was able to increase its ability to maintain cell viability only when performed every 48 h in milk maintained at 5 °C.</p></div