Study on transport of small molecule rhodamine B within different layers of cartilage / 生物医学工程学杂志

The small molecule nutrients and cell growth factors required for the normal metabolism of chondrocyte mainly transport into the cartilage through free diffusion. However, the specific mass transfer law in the cartilage remains to be studied. In this study, using small molecule rhodamine B as tracer, the mass transfer models of cartilage were built under different pathways including surface pathway, lateral pathway and composite pathway. Sections of cartilage at different mass transfer times were observed by using laser confocal microscopy and the transport law of small molecules within different layers of cartilage was studied. The results showed that rhodamine B diffused into the whole cartilage layer through surface pathway within 2 h. The fluorescence intensity in the whole cartilage layer increased with the increase of mass transfer time. Compared to mass transfer of 2 h, the mean fluorescence intensity in the superficial, middle, and deep layers of cartilage increased by 1.83, 1.95, and 3.64 times, respectively, after 24 h of mass transfer. Under lateral path condition, rhodamine B was transported along the cartilage width, and the molecular transport distance increased with increasing mass transfer time. It is noted that rhodamine B could be transported to 2 mm away from cartilage side after 24 h of mass transfer. The effect of mass transfer under the composite path was better than those under the surface path and the lateral path, and especially the mass transfer in the deep layer of cartilage was improved. This study may provide a reference for the treatment and repair of cartilage injury.

Enfermedad granulomatosa crónica: diagnóstico mediante el ensayo de dihidrorodamina / Chronic granulomatous disease: diagnosis by the dihydrorhodamine assay

Resumen: Introducción: La enfermedad granulomatosa crónica (EGC) se caracteriza por una alteración de la función oxidativa de neutrófilos, presentando herencia ligada al cromosoma X (EGC LX) y autosómica recesiva (EGC AR). El ensayo de dihidrorodamina (DHR) es utilizado para el diagnóstico y detección de portadoras, además proporciona información sobre patrones de herencia. Objetivo: Detectar casos de EGC en niños con infecciones recurrentes y evaluar a sus familiares femeninos mediante el ensayo de DHR, para identificar portadoras y obtener información acerca de posibles patrones de herencia. Pacientes y Método: Fueron incluidos 107 pacientes (<18 años de edad) con sospecha clínica de EGC como neumonías, linfadenopatías y abscesos, remitidos por médicos de hospitales públicos, del 2014 al 2017. Además, se incluyeron seis mujeres, familiares de los niños con EGC. A las muestras de los pacientes se aplicó el ensayo DHR, expresando los resultados como índice de estimulación de neutrófilos (IE). Resultados: La mediana de edad de los pacientes fue de 3 años y 62/107 fueron varones. El IE promedio fue 39,7 ± 13,8 y 101/107 niños exhibieron un cambio completo de fluorescencia de DHR. En 2/107 niños no se observó dicho cambio (IE = 1,0), lo cual indica posible EGC LX, y un tercer niño mostró un leve cambio (IE = 4,8), compatible con EGC AR. En 5/6 mujeres se encontró un patrón bimodal, indicando un estado de portadora. Conclusiones: Fueron detectados tres casos de EGC y cinco portadoras mediante el ensayo de DHR, realizado por primera vez en Paraguay. También se obtuvo información sobre los posibles patrones de herencia, EGC LX en dos familias y un caso probable de EGC AR.

Abstract: Introduction: Chronic granulomatous disease (CGD) is characterized by an alteration of the neutrophil oxidative function. Its inheritance patterns are linked to the X chromosome (X-linked CGD) and autosomal recessive (AR CGD). The dihydrorhodamine (DHR) assay is used for the diagnosis and detection of carriers and provides information on inheritance patterns. Objective: To detect CGD cases in chil dren with recurrent infections and to evaluate their female relatives through the DHR assay to iden tify carriers and obtain information about possible inheritance patterns. Patients and Method: 107 patients (<18 years of age) with clinical suspicion of CGD such as pneumonia, lymphadenopathies, and abscesses were included, referred by physicians from public hospitals between 2014 and 2017. Six female relatives of children with CGD were also included. The DHR assay was performed on all patient samples and the results were expressed as neutrophils stimulation index (SI). Results: The median age of patients was 3 years and 62/107 of them were male. The average SI was 39.7±13.8 and a complete shift of DHR was found in 101/107 children. In 2/107 children, no DHR shift was observed (SI=1.0) indicating possible X-linked CGD, and a third child showed a slight DHR shift (SI=4.8) compatible with AR CGD. 5/6 female relatives presented a bimodal pattern, showing a carrier status. Conclusions: Three cases of CGD and five female carriers were detected through the DHR assay, being the first time that this technique was used in Paraguay. Information on the most likely inheri tance patterns, two X-linked CGD, and one AR CGD case was also obtained.

Effect of conditioning protocols and ultrasonic application of an infiltrant resin in white spot lesions

Abstract The aim of this study was to evaluate different conditioning protocols and sonic/ultrasonic application of an infiltrant resin (IR) in artificial white spot lesions (AWSL). the V/L surfaces of 48 molars were induced to an AWSL and divided in 6 groups, according to the conditioning protocols and application technique: 15% hydrochloric acid (HA) + manual application of the IR; HA + 37% phosphoric acid (PA) + manual application of the IR; HA + ultrasonic application (U) of the IR; HA + sonic application (S) of the IR; PA+HA+S; and PA+HA+U. For the Penetration Depth (PD), the crowns were etched with HA for 120s. The IR Icon® (DMG) was applied according to the manufacturer`s instructions. The crowns were dye penetrated (0.1% red fluorophore rhodamine B isothiocyanate for 12h) and bleached with 30% hydrogen peroxide for 12 h. The discs were immersed in a 50% ethanol solution, containing 100 µM of sodium fluorescein. The PD (in µm) was measured using confocal laser scanning microscopy (20x). The bond strength (BS) was performed by michoshear test (0.5 mm/min). Data were submitted to 2-way ANOVA and Tukey (α=0.05). For BS, the interaction was not significant (p>0.05). For PD, the main factors were significant (application - p<0.001; conditioning technique - p=0.003). The ultrasonic application showed the highest PD values. PA+HA presented higher results than HA. The sonic/ultrasonic applications and the use of phosphoric acid prior to hydrochloric acid improved PD of the infiltrant resin. Conditioning protocols or application techniques did not influence BS values.

Resumo: O objetivo deste estudo foi avaliar diferentes protocolos de condicionamento e a aplicação sônica/ultrassônica de uma resina infiltrante (RI) em lesões de mancha branca produzidas artificialmente (LMBA). As superfícies vestibulares/linguais de 48 molares foram induzidas à formação de LMBA e divididas em 6 grupos, de acordo com os protocolos de condicionamento e técnica de aplicação da resina infiltrante: ácido hidroclorídrico 15% (AH) + aplicação manual da RI; AH + ácido fosfórico 37% (AF) + aplicação manual da RI; AH + aplicação ultrassônica da RI (U); AH + aplicação sônica da RI (S); AF + AH + S; e AF + AH + U. para o grau de penetração (GP), condicionou-se as coroas com AH por 120 s. A RI Icon® (DMG) foi aplicada de acordo com as instruções do fabricante. As coroas foram coradas (rodamina B 0,1% por 12 h) e clareadas com peróxido de hidrogênio 30% por 12 h. os discos foram imersos em solução de etanol 50%, contendo 100 µM de fluoresceína sódica. O GP (em µm) foi mensurado por meio de microscopia confocal a laser (20´). A resistência de união (RU) foi calculada pelo teste de microcisalhamento (0,5 mm/min). Os dados foram submetidos ao teste ANOVA 2 fatores e Tukey (α=0,05). Para RU, a interação não foi significante (p>0,05). Para GP, os fatores principais foram significantes (técnica de aplicação - p<0,001; protocolos de condicionamento - p=0,003). A aplicação U mostrou os maiores valores de GP. AF+AH demonstrou resultados superiores ao grupo AH. As aplicações sônica/ultrassônica e o uso do ácido fosfórico antes do ácido hidroclorídrico aumentaram o GP da resina infiltrante. Os protocolos de condicionamento ou as técnicas de aplicação não influenciaram os valores de RU.

Influence of size and insertion depth of irrigation needle on debris extrusion and sealer penetration

OBJECTIVES: To determine the effect of size and insertion depth of irrigation needle on the amount of apical extruded debris and the amount of penetration depth of sealer using a confocal laser scanning microscope (CLSM). MATERIALS AND METHODS: Twenty maxillary premolars were assigned to 2 groups (n = 10), according to the size of needle tip, 28 G or 30 G. Buccal roots of samples were irrigated with respective needle type inserted 1 mm short of the working length (WL), while palatal roots were irrigated with respective needle type inserted 3 mm short of the WL. Prepared teeth were removed from the pre-weighed Eppendorf tubes. Canals were filled with F3 gutta-percha cone and rhodamine B dye-labeled AH 26 sealer. Teeth were transversally sectioned at 1 and 3 mm levels from the apex and observed under a CLSM. Eppendorf tubes were incubated to evaporate the irrigant and were weighed again. The difference between pre- and post-weights was calculated, and statistical evaluation was performed. RESULTS: Inserting needles closer to the apex and using needles with wider diameters were associated with significantly more debris extrusion (p < 0.05). The position of needles and level of sections had statistically significant effects on sealer penetration depth (p < 0.05 for both). CONCLUSIONS: Following preparation, inserting narrower needles compatible with the final apical diameter of the prepared root canal at 3 mm short of WL during final irrigation might prevent debris extrusion and improve sealer penetration in the apical third.

Comparative transcriptomic analysis reveals adriamycin-induced apoptosis via p53 signaling pathway in retinal pigment epithelial cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells.@*METHODS@#The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair.@*RESULTS@#ADR could significantly inhibit ARPE-19 cell proliferation and induce caspase-dependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein-protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure.@*CONCLUSIONS@#The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.

Effectiveness of different final irrigation techniques and placement of endodontic sealer into dentinal tubules

Abstract: The aim of this study was to compare two irrigation techniques and four devices for endodontic sealer placement into the dentinal tubules. Ninety-nine single-rooted human teeth were instrumented and allocated to either the control (CO) (n=11) or experimental groups according to the irrigation method: syringe and NaveTip needle (NT) (n=44), and EndoActivator (EA) (n=44). These groups were subdivided according to sealer placement into K-File (KF), lentulo spiral (LS), Easy Clean (EC), and EndoActivator (EA) subgroups. Moreover, the distances of 5 mm and 2 mm from the apex were analyzed. The teeth were obturated with AH Plus and GuttaCore X3. Analyses were performed by scanning electron microscopy associated to cathodoluminescence. The percentage and maximum depth of sealer penetration were measured. Data were evaluated by three-way analysis of variance (ANOVA) and Games-Howell test (p<0.05). EA was superior to NT in percentage of sealer penetration. EC was significantly superior to EA (subgroup) for sealer penetration, and both improved the percentage of sealer penetration when compared to LS. Better sealer penetration was observed at the distance of 5 mm from the apex. Sealer penetration into the dentinal tubules was significantly improved by sonic irrigant activation.

Efeito de adição de rodamina B e fluoresceína sódica a sistemas adesivos não simplificados: aspectos fotofísicos e físico-químicos / Effect of addition of rhodamine B and fluorescein to conventional etch-and-rinse and self-etching adhesive systems: photophysical and physical-chemical aspects

A adição de corantes fluorescentes a adesivos odontológicos possibilita a investigação da distribuição espacial desses materiais na interface dente-restauração, utilizando-se a microscopia confocal de varredura a laser (MCVL). A literatura indica falta de padronização na aplicação de agentes fluorescentes com tal finalidade. Esse estudo sistematizou estratégias para a adição de rodamina B (RB) e fluoresceína sódica (FS) a um sistema adesivo convencional de três passos, Adper Scotchbond Multi-Purpose (MP), e um autocondicionante de dois passos, Clearfil SE Bond (SE), considerados "padrão-ouro" na Odontologia. Os objetivos principais foram (a) determinar a menor faixa de concentrações de RB e FS necessária para produzir imagens satisfatórias da interface dentina-adesivo e (b) avaliar o efeito da adição desses corantes sobre algumas propriedades das resinas. Os adesivos foram marcados com RB ou FS em concentrações decrescentes (0,5, 0,1, 0,02 e 0,004 mg/mL) por meio de um método de dispersão semidireto. O comportamento fotofísico/ fluorescente dos adesivos marcados foi investigado por espectroscopia de fotoluminescência e MCVL. Paralelamente, avaliaram-se os adesivos quanto ao grau de conversão (GC) e ao ângulo de contato (AC). Tanto os resultados de GC como os de AC foram submetidos à análise de variância com dois fatores (adesivo e tratamento) com α = 0,05, seguida de teste post-hoc de Tukey. Os máximos comprimentos de onda de emissão e de excitação da RB e da FS foram influenciados pelo meio polimérico e pela concentração de corante de modo geral. A MCVL preliminar de amostras de adesivo polimerizado, realizada sob condições experimentais padronizadas, mostrou que o comportamento fluorescente da RB em MP e SE foi muito semelhante na mesma concentração de corante, mas o mesmo não pôde ser dito do comportamento da FS, que foi notavelmente inferior no adesivo autocondicionante, SE, na concentração mais alta. Em dentina, os adesivos preparados com RB nas concentrações-alvo de 0,1 e 0,02 mg/mL apresentaram fluorescência ótima; já aqueles preparados com 0,004 mg/mL produziram fraco sinal. Adesivos preparados com FS a 0,5 mg/mL apresentaram ótima fluorescência na interface de adesão, enquanto que concentração menor desse corante não produziu sinal suficiente. Padrões morfológicos aparentemente atípicos foram observados na interface de adesão, quando da associação do adesivo SE com o corante FS. A adição de RB e FS nas quatro concentrações indicadas aos adesivos MP e SE não afetou o GC nem o AC em comparação com os grupos de controle correspondentes. Em suma, a RB mostra-se um corante mais versátil que a FS na avaliação morfológica das interfaces dentina-MP e dentina-SE via MCVL. A menor faixa de concentrações de RB nos adesivos MP e SE, na qual é possível produzir imagens satisfatórias das interfaces, situa-se entre 0,1­0,02 mg/mL. Já o corante FS deve ser adicionado a esses adesivos a pelo menos 0,5 mg/mL para produzir níveis de fluorescência satisfatórios na interface de adesão. A não ocorrência de efeitos deletérios sobre a polimerização e a molhabilidade das resinas estabelece uma margem de segurança para a incorporação desses agentes fluorescentes (em concentração ≤ 0,5 mg/mL) nesses sistemas monoméricos.(AU)

The addition of fluorescent dyes to dental adhesives makes it possible to investigate the spatial distribution of such resin-based materials in the tooth-restoration interface, using confocal laser scanning microscopy (CLSM). Literature indicates a lack of standardization on the application of fluorescent agents for this purpose. This work presents strategies for adding rhodamine B (RB) and fluorescein sodium salt (FS) to a three-step etch-and-rinse adhesive system, Adper Scotchbond Multi-Purpose (MP), and a two-step self-etching one, Clearfil SE Bond (SE), both regarded as "gold standard" in restorative dentistry. The main objectives were (a) to determine the lowest range of RB and FS concentrations required to produce suitable images of the dentin-adhesive interface via CLSM and (b) to investigate potential effects of addition of these dyes on some resin properties. The adhesives were labeled with RB or FS at decreasing concentrations (0.5, 0.1, 0.02 and 0.004 mg/mL) by means of a semi-direct dispersion method. The photophysical/fluorescent behavior of the labeled resins was investigated by photoluminescence spectroscopy and by CLSM. The adhesives were also investigated with regards to the degree of conversion (DC) and contact angle (CA). A two-way ANOVA of "adhesive" and "treatment" was conducted on DC and CA separately, followed by Tukey's test. The maximum emission and excitation wavelengths of RB and FS were influenced by the host polymer and the dye concentration in general. The preliminary CLSM of cured adhesive samples, performed with standardized settings, showed that the fluorescent behavior of RB in MP and SE was very similar in the same dye concentration, unlike the behavior of FS, which was lower in the self-etching adhesive for the highest dye concentration. In dentin, the adhesives prepared with RB at the target concentrations of 0.1 and 0.02 mg/mL presented optimal fluorescence; those with 0.004 mg/mL produced poor signal. Adhesives prepared with FS at 0.5 mg/mL presented optimal fluorescence at the bonding interface, whereas lower concentrations of FS did not produce sufficient signal. Atypical morphological features were observed at the bonding interface, when adhesive SE was used with FS. The addition of RB and FS at the four decreasing concentrations to adhesives MP and SE did not affect DC or CA compared to the corresponding controls. In short, RB is more versatile than FS for the morphological characterization of dentin-MP and dentin-SE interfaces via MCVL. The lowest range of RB concentrations in adhesives MP and SE that can produce suitable images of the bonding interface lies between 0.1­0.02 mg/mL. The dye FS should be added to these adhesives at 0.5 mg/mL at least to produce satisfactory fluorescence levels at the bonding interface. Since negative effects on polymerization and wettability of the resins were not observed, the use of RB and FS (in concentration ≤ 0.5 mg/mL) together with MP and SE should be reliable in terms of resin properties.(AU)

Effects of low-level laser therapy on stem cells from human exfoliated deciduous teeth

ABSTRACT Low-Level Laser Therapy stimulates the proliferation of a variety of types of cells. However, very little is known about its effect on stem cells from human exfoliated deciduous teeth (SHED). Objective This study aimed to evaluate the influence of different laser therapy energy densities on SHED viability and proliferation. Material and Methods SHED were irradiated according to the groups: I (1.2 J/cm2 - 0.5 mW – 10 s), II (2.5 J/cm2 – 10 mW – 10 s), III (3.7 J/cm2 – 15 mW – 10 s), IV (5.0 J/cm2 – 20 mW – 10 s), V (6.2 J/cm2 – 25 mW – 10 s), and VI (not irradiated – control group). Cell viability was assessed 6 and 24 h after irradiation measuring the mitochondrial activity and using the Crystal Violet assay. Cell proliferation was assessed after 24, 48, and 72 h of irradiation by SRB assay. Results MTT assay demonstrated differences from 6 to 24 hours after irradiation. After 24 h, groups I and IV showed higher absorbance values than those of control group. Crystal Violet assay showed statistically differences in the absorbance rate from 6 to 24 h after irradiation for groups III and VI. At 24 h after irradiation, Group III absorbance rate was greater than that of groups I, II, and IV. Group VI absorbance rate was greater than that of groups I and IV. SRB assay showed that the group I had higher rates than those of groups II, III, V, and VI, at 24 h after irradiation. After 48 h, group I exhibited the greatest cell proliferation rate followed by groups III, V, and VI. After 72 h, group III exhibited the lowest cell proliferation rate than those of groups II, IV, and V. Conclusions The Low-Level Laser Therapy energy densities used in this study did not cause loss of cell viability and stimulated SHED proliferation within the parameters described in this study.

Water interaction and bond strength to dentin of dye-labelled adhesive as a function of the addition of rhodamine B

ABSTRACT Objective This study investigated the effect of the fluorescent dye rhodamine B (RB) for interfacial micromorphology analysis of dental composite restorations on water sorption/solubility (WS/WSL) and microtensile bond strength to dentin (µTBS) of a 3-step total etch and a 2-step self-etch adhesive system. Material and Methods The adhesives Adper Scotchbond Multi-Purpose (MP) and Clearfil SE Bond (SE) were mixed with 0.1 mg/mL of RB. For the WS/WSL tests, cured resin disks (5.0 mm in diameter x 0.8 mm thick) were prepared and assigned into four groups (n=10): MP, MP-RB, SE, and SE-RB. For µTBS assessment, extracted human third molars (n=40) had the flat occlusal dentin prepared and assigned into the same experimental groups (n=10). After the bonding and restoration procedures, specimens were sectioned in rectangular beams, stored in water and tested after seven days or after 12 months. The failure mode of fractured specimens was qualitatively evaluated under optical microscope (x40). Data from WS/WSL and µTBS were assessed by one-way and three-way ANOVA, respectively, and Tukey’s test (α=5%). Results RB increased the WSL of MP and SE. On the other hand, WS of both MP and SE was not affected by the addition of RB. No significance in µTBS between MP and MP-RB for seven days or one year was observed, whereas for SE a decrease in the µTBS means occurred in both storage times. Conclusions RB should be incorporated into non-simplified DBSs with caution, as it can interfere with their physical-mechanical properties, leading to a possible misinterpretation of bonded interface.

Push-out bond strength and dentinal tubule penetration of different root canal sealers used with coated core materials

OBJECTIVES: The aim of this study was to compare the push-out bond strength and dentinal tubule penetration of root canal sealers used with coated core materials and conventional gutta-percha. MATERIALS AND METHODS: A total of 72 single-rooted human mandibular incisors were instrumented with NiTi rotary files with irrigation of 2.5% NaOCl. The smear layer was removed with 17% ethylenediaminetetraacetic acid (EDTA). Specimens were assigned into four groups according to the obturation system: Group 1, EndoRez (Ultradent Product Inc.); Group 2, Activ GP (Brasseler); Group 3, SmartSeal (DFRP Ltd. Villa Farm); Group 4, AH 26 (Dentsply de Trey)/gutta-percha (GP). For push-out bond strength measurement, two horizontal slices were obtained from each specimen (n = 20). To compare dentinal tubule penetration, remaining 32 roots assigned to 4 groups as above were obturated with 0.1% Rhodamine B labeled sealers. One horizontal slice was obtained from the middle third of each specimen (n = 8) and scanned under confocal laser scanning electron microscope. Tubule penetration area, depth, and percentage were measured. Kruskall-Wallis test was used for statistical analysis. RESULTS: EndoRez showed significantly lower push-out bond strength than the others (p < 0.05). No significant difference was found amongst the groups in terms of percentage of sealer penetration. SmartSeal showed the least penetration than the others (p < 0.05). CONCLUSIONS: The bond strength and sealer penetration of resin-and glass ionomer-based sealers used with coated core was not superior to resin-based sealer used with conventional GP. Dentinal tubule penetration has limited effect on bond strength. The use of conventional GP with sealer seems to be sufficient in terms of push-out bond strength.

Effect of an Alcohol-Based Caries Detector on the Surface Tension of Sodium Hypochlorite Preparations

The purpose of this study was to evaluate the effect of an alcohol-based caries detector (Kurakay) on the surface tension of a conventional sodium hypochlorite (NaOCl) preparation, and a product containing a surface-active agent (Chlor-XTRA). The surface tensions of the following solutions were tested: NaOCl, a mixture of NaOCl and Kurakay 9:1 w/w, Chlor-XTRA, a mixture of Chlor-XTRA and Kurakay 9:1 w/w. Ten measurements per test solution were made at 20 C, using an optical method called the "Pendant drop method", with a commercially available apparatus. The addition of Kurakay reduced the surface tension for NaOCl (p<0.05) whilst no significant difference was detected for Chlor-XTRA (p>0.05). Statistically significant differences between the NaOCl and Chlor-XTRA groups were found (p<0.05). The addition of an alcohol-based caries detector resulted in a reduction of the original surface tension values for NaOCl only. Taking into account the fact that mixtures of NaOCl and Kurakay have been used to assess the penetration of root canal irrigants in vitro, the related changes in surface tension are a possible source of bias.

O objetivo deste estudo foi avaliar o efeito de um detector de cárie com álcool (Kurakay) sobre a tensão superficial de um preparado convencional de hipoclorito de sódio (NaOCl) e um produto contendo um agente surfactante (Chlor-XTRA). Foram testadas as tensões superficiais das seguintes soluções: NaOCl, uma mistura de NaOCl e Kurakay na proporção de 9:1 em peso, Chlor-XTRA e um mistura de Chlor-XTRA e Kurakay na proporção de 9:1 em peso. Dez medidas foram feitas com cada solução, a 20 °C, utilizando um método óptico chamado "Método da gota pendente" (Pendant drop method) usando aparelhos disponíveis. Adição do Kurakay reduziu a tensão superficial do NaOCl (p<0,05), mas não houve diferença significante para Chlor-XTRA (p>0,05). Foram encontradas diferenças estatisticamente significantes entre os grupos NaOCl e Chlor-XTRA (p<0,05). Adição de um detector de cárie com álcool resultou na diminuição do valor original da tensão superficial apenas para NaOCl. Considerando que as misturas de NaOCl e Kurakay tem sido usadas para estudar a penetração in vitro dos irrigantes radiculares, as diferenças acima são uma possível fonte de desvio nos resultados.

Effect of three different irrigation solutions applied by passive ultrasonic irrigation

OBJECTIVES: This study evaluated the maximum depth and percentage of irrigant penetration into dentinal tubules by passive ultrasonic irrigation (PUI). MATERIALS AND METHODS: Thirty extracted human teeth were instrumented and divided into three groups. According to final irrigation regimen, 5.25% sodium hypochlorite (Group A, NaOCl), 2% chlorhexidine (Group B, CHX) and saline solution (Group C, control group) were applied with Irrisafe 20 tips (Acteon) and PUI. Irrigant was mixed with 0.1% rhodamine B. Sections at 2 mm, 5 mm, and 8 mm from the apex were examined with confocal laser scanning microscopy (CLSM). The percentage and maximum depth of irrigant penetration were measured. Kruskal-Wallis test and Mann-Whitney test were performed for overall comparison between groups at each level and for pairwise comparison, respectively. Within a group, Wilcoxon test was performed among different levels. p values less than 0.05 were considered significant. RESULTS: In all groups, highest penetration depth and percentage of penetration were observed at the 8 mm level. At 2 mm level, Groups A and B had significantly greater depths and percentages in penetration than Group C (p < 0.05), but there were no significant differences between Groups A and B. At 5 mm level, penetration depths and percentage of penetration was not significantly different among the groups. CONCLUSIONS: NaOCl and CHX applied by PUI showed similar depth and percentage of penetration at all evaluated levels.

Effect of three different irrigation solutions applied by passive ultrasonic irrigation

OBJECTIVES: This study evaluated the maximum depth and percentage of irrigant penetration into dentinal tubules by passive ultrasonic irrigation (PUI). MATERIALS AND METHODS: Thirty extracted human teeth were instrumented and divided into three groups. According to final irrigation regimen, 5.25% sodium hypochlorite (Group A, NaOCl), 2% chlorhexidine (Group B, CHX) and saline solution (Group C, control group) were applied with Irrisafe 20 tips (Acteon) and PUI. Irrigant was mixed with 0.1% rhodamine B. Sections at 2 mm, 5 mm, and 8 mm from the apex were examined with confocal laser scanning microscopy (CLSM). The percentage and maximum depth of irrigant penetration were measured. Kruskal-Wallis test and Mann-Whitney test were performed for overall comparison between groups at each level and for pairwise comparison, respectively. Within a group, Wilcoxon test was performed among different levels. p values less than 0.05 were considered significant. RESULTS: In all groups, highest penetration depth and percentage of penetration were observed at the 8 mm level. At 2 mm level, Groups A and B had significantly greater depths and percentages in penetration than Group C (p < 0.05), but there were no significant differences between Groups A and B. At 5 mm level, penetration depths and percentage of penetration was not significantly different among the groups. CONCLUSIONS: NaOCl and CHX applied by PUI showed similar depth and percentage of penetration at all evaluated levels.

Evaluation of penetration depth of 2% chlorhexidine digluconate into root dentinal tubules using confocal laser scanning microscope

OBJECTIVES: This study evaluated the penetration depth of 2% chlorhexidine digluconate (CHX) into root dentinal tubules and the influence of passive ultrasonic irrigation (PUI) using a confocal laser scanning microscope (CLSM). MATERIALS AND METHODS: Twenty freshly extracted anterior teeth were decoronated and instrumented using Mtwo rotary files up to size 40, 4% taper. The samples were randomly divided into two groups (n = 10), that is, conventional syringe irrigation (CSI) and PUI. CHX was mixed with Rhodamine B dye and was used as the final irrigant. The teeth were sectioned at coronal, middle and apical levels and viewed under CLSM to record the penetration depth of CHX. The data were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests. RESULTS: The mean penetration depths of 2% CHX in coronal, middle and apical thirds were 138 microm, 80 microm and 44 microm in CSI group, respectively, whereas the mean penetration depths were 209 microm, 138 microm and 72 microm respectively in PUI group. Statistically significant difference was present between CSI group and PUI group at all three levels (p < 0.01 for coronal third and p < 0.001 for middle and apical thirds. On intragroup analysis, both groups showed statistically significant difference among three levels (p < 0.001). CONCLUSIONS: Penetration depth of 2% CHX into root dentinal tubules is deeper in coronal third when compared to middle and apical third. PUI aided in deeper penetration of 2% CHX into dentinal tubules when compared to conventional syringe irrigation at all three levels.

Evaluation of penetration depth of 2% chlorhexidine digluconate into root dentinal tubules using confocal laser scanning microscope

OBJECTIVES: This study evaluated the penetration depth of 2% chlorhexidine digluconate (CHX) into root dentinal tubules and the influence of passive ultrasonic irrigation (PUI) using a confocal laser scanning microscope (CLSM). MATERIALS AND METHODS: Twenty freshly extracted anterior teeth were decoronated and instrumented using Mtwo rotary files up to size 40, 4% taper. The samples were randomly divided into two groups (n = 10), that is, conventional syringe irrigation (CSI) and PUI. CHX was mixed with Rhodamine B dye and was used as the final irrigant. The teeth were sectioned at coronal, middle and apical levels and viewed under CLSM to record the penetration depth of CHX. The data were statistically analyzed using Kruskal-Wallis and Mann-Whitney U tests. RESULTS: The mean penetration depths of 2% CHX in coronal, middle and apical thirds were 138 microm, 80 microm and 44 microm in CSI group, respectively, whereas the mean penetration depths were 209 microm, 138 microm and 72 microm respectively in PUI group. Statistically significant difference was present between CSI group and PUI group at all three levels (p < 0.01 for coronal third and p < 0.001 for middle and apical thirds. On intragroup analysis, both groups showed statistically significant difference among three levels (p < 0.001). CONCLUSIONS: Penetration depth of 2% CHX into root dentinal tubules is deeper in coronal third when compared to middle and apical third. PUI aided in deeper penetration of 2% CHX into dentinal tubules when compared to conventional syringe irrigation at all three levels.

Sealing Ability of MTA Used as a Root End Filling Material: Effect of the Sonic and Ultrasonic Condensation

Despite the excellent properties of mineral trioxide aggregate (MTA), the condensation technique may have some influence in its sealing ability. The purpose of this study was to compare the sealing ability of sonic and ultrasonic setting of MTA. Thirty-four extracted human teeth had their canals prepared and filled with Sealapex sealer and gutta-percha using the active lateral condensation technique. The teeth were rendered waterproof and apicoectomy performed at 3 mm from the apex. Root-end cavities (3.0 mm deep and 1.4 mm diameter) were prepared with diamond ultrasonic tips. The root-end cavities were filled with Pro-Root MTA® with ultrasonic vibration, sonic vibration or no vibration. The positive control group did not receive any material while the negative control group was totally rendered waterproof. After material set, the specimens were immersed in Rodhamine B for 24 h, under vacuum in the first 15 min, then washed, dried and split longitudinally for evaluating the infiltration at the dentin/material interface. Data were analyzed using ANOVA and Tukey's tests at 5% significance level. Sonic vibration promoted the lowest infiltration values (p<0.05). It was concluded that sonic vibration could be considered an efficient aid to improve the sealing ability of MTA when used as root-end filling material.

Resumo O objetivo deste estudo foi avaliar in vitro, a influência da vibração sônica e ultrassônica no selamento marginal proporcionado pelo MTA. Trinta e quatro dentes humanos tiveram seus canais radiculares instrumentados e obturados com cimento Sealapex® pela técnica da condensação lateral ativa. Os dentes foram impermeabilizados e seccionados os 3 mm apicais. Retrocavidades (3 mm de profundidade e 1,4 mm de diâmetro) foram preparadas com pontas ultrassônicas diamantadas. As retrocavidades foram preenchidas com Pro-Root MTA® com auxílio da vibração ultrassônica, com auxílio da vibração sônica e sem vibração alguma. O grupo controle positivo não recebeu material retrobturador, enquanto que o negativo foi totalmente impermeabilizado. Após a presa do material, os espécimes foram mergulhados em Rodamina B por 15 min em vácuo, permanecendo nesta solução por mais 24 h. Em seguida, foram lavados, secados e clivados longitudinalmente para avaliação da infiltração do corante na interface dentina/material retrobturador. Os dados foram analisados utilizando a análise de variância e o teste de Tukey (p<0,05). Observou-se que apenas a vibração sônica foi diferente apresentando os menores índices de infiltração. Pode-se concluir que vibração sônica contribui com a melhora da capacidade de selamento marginal proporcionado pelo MTA quando empregado como material retrobturador. .

Rapid Determination of Chimerism Status Using Dihydrorhodamine Assay in a Patient with X-linked Chronic Granulomatous Disease Following Hematopoietic Stem Cell Transplantation

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.

Dentine microhardness after different methods for detection and removal of carious dentine tissue

There are several methods for identifying carious dentinal tissue aiming to avoid removal of healthy dentinal tissue. OBJECTIVES: The purpose of this study was to test different methods for the detection of carious dentinal tissue regarding the amount of carious tissue removed and the remaining dentin microhardness after caries removal. MATERIAL AND METHODS: The dentin surfaces of 20 bovine teeth were exposed and half of the surface was protected with nail polish. Cariogenic challenge was performed by immersion in a demineralizing solution for 14 days. After transverse cross-section of the crown, the specimens were divided into four groups (n=10), according to the method used to identify and remove the carious tissue: "Papacárie", Caries-detector dye, DIAGNOdent and Tactile method. After caries removal, the cross-sectional surface was included in acrylic resin and polished. In a microhardness tester, the removed dentin thickness and the Vickers microhardness of the following regions were evaluated: remaining dentin after caries removal and superficial and deep healthy dentin. RESULTS: ANOVA and Tukey's test (α=0.05) were performed, except for DIAGNOdent, which did not detect the presence of caries. Results for removed dentin thickness were: "Papacárie" (424.7±105.0; a), Caries-detector dye (370.5±78.3; ab), Tactile method (322.8±51.5; bc). Results for the remaining dentin microhardness were: "Papacárie" (42.2±10.5; bc), Caries-detector dye (44.6±11.8; abc), Tactile method (24.3±9.0; d). CONCLUSIONS: DIAGNOdent did not detect the presence of carious tissue; Tactile method and "Papacárie" resulted in the least and the most dentinal thickness removal, respectively; Tactile method differed significantly from "Papacárie" and Caries-detector dye in terms of the remaining dentin microhardness, and Tactile method was the one which presented the lowest microhardness values.

Study of internalization and viability of multimodal nanoparticles for labeling of human umbilical cord mesenchymal stem cells / Estudo de internalização e viabilidade de nanopartículas multimodal para marcação de células-tronco mesenquimais de cordão umbilical humano

Objective: To analyze multimodal magnetic nanoparticles-Rhodamine B in culture media for cell labeling, and to establish a study of multimodal magnetic nanoparticles-Rhodamine B detection at labeled cells evaluating they viability at concentrations of 10mug Fe/mL and 100mug Fe/mL. Methods: We performed the analysis of stability of multimodal magnetic nanoparticles-Rhodamine B in different culture media; the mesenchymal stem cells labeling with multimodal magnetic nanoparticles-Rhodamine B; the intracellular detection of multimodal magnetic nanoparticles-Rhodamine B in mesenchymal stem cells, and assessment of the viability of labeled cells by kinetic proliferation. Results: The stability analysis showed that multimodal magnetic nanoparticles-Rhodamine B had good stability in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium. The mesenchymal stem cell with multimodal magnetic nanoparticles-Rhodamine B described location of intracellular nanoparticles, which were shown as blue granules co-localized in fluorescent clusters, thus characterizing magnetic and fluorescent properties of multimodal magnetic nanoparticles-Rhodamine B. Conclusion: The stability of multimodal magnetic nanoparticles-Rhodamine B found in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium assured intracellular mesenchymal stem cells labeling. This cell labeling did not affect viability of labeled mesenchymal stem cells since they continued to proliferate for five days.

Objetivo: Analisar a estabilidade das nanopartículas magnéticas multimodais-Rhodamine B em meios de cultura para marcação celular e, consequentemente, estabelecer o estudo de detecção intracelular de nanopartículas magnéticas multimodais-Rhodamine B nas células marcadas, avaliando a viabilidade celular nas concentrações de 10mig Fe/mL e 100mig Fe/mL. Métodos: Foram realizados: análise da estabilidade das nanopartículas magnéticas multimodais-Rhodamine B em meios de cultura diferentes; marcação das células-tronco mesenquimais com nanopartículas magnéticas multimodais-Rhodamine B; detecção intracelular das nanopartículas magnéticas multimodais-Rhodamine B nas células-tronco mesenquimais e avaliação da viabilidade das células marcadas por meio da cinética de proliferação. Resultados: A análise de estabilidade determinou que as nanopartículas magnéticas multimodais-Rhodamine B presentes nos meios de cultura Dulbecco's Modified Eagle's-Low Glucose e RPMI Medium 1640 apresentaram boa estabilidade. A marcação das células-tronco mesenquimais com nanopartículas magnéticas multimodais-Rhodamine B descreveu localização intracelular das nanopartículas, as quais se mostraram como grânulos azulados colocalizados nos grumos fluorescentes, caracterizando, assim, as propriedades magnéticas e fluorescentes das nanopartículas magnéticas multimodais-Rhodamine B. Conclusão: A estabilidade das nanopartículas magnéticas multimodais-Rhodamine B, presentes nos meios de cultura Dulbecco's Modified Eagle's-Low Glucose e RPMI Medium 1640, garantiu a eficiente marcação intracelular das células-tronco mesenquimais. Esse tipo de marcação não afetou viabilidade das células-tronco mesenquimais marcadas, já que as mesmas continuaram proliferando ao longo de 5 dias.

Análise da expressão e atividade das moléculas de influxo e de efluxo de drogas em células da leucemia mielóide crônica / Analysis of the expression and activity of drug influx and efflux molecules in chronic myeloid leukemia cells

"A leucemia mielóide crônica (LMC) é caracterizada pela presença da translocação t(9:22) que codifica a proteína quimérica oncogênica BCR-ABL. Imatinibe é uma droga alvo-específica que inibe a atividade tirosina quinase (TK) da proteína BCR-ABL. Entretanto, os níveis intracelulares do imatinibe podem ser alterados pelas proteínas transportadoras de efluxo de drogas: glicoproteína P (Pgp), proteína da resistência em câncer de mama (BCRP) e proteína relacionada à resistência à múltiplas drogas (MRP1), assim como a proteína transportadora de influxo de drogas (OCT1). O objetivo do presente estudo foi analisar a participação dessas proteínas, isoladamente ou em associação, na resistência ao imatinibe em linhagens celulares e células de pacientes com LMC. Para análise da atividade das proteínas transportadoras de efluxo, foi utilizado o fluorocromo rodamina-123 associado ao modulador ciclosporina-A (Rho+CSA) e o pheophorbide A associado ao fumitremorgin C (PhA+FTC), ambos através de citometria de fluxo. A análise dos RNAm dos genes ABCB1, ABCG2 e SLC22A1 que codificam as proteínas Pgp, BCRP e OCT1, respectivamente, foi realizada por PCR em tempo real. A inibição da TK BCR-ABL foi mensurada através dos níveis de fosforilação de CrkL (pCrkL), seu principal alvo de ativação. Observamos uma maior positividade para o ensaio Rho+CSA nas amostras que expressavam Pgp comparada com as que expressavam MRP1, sugerindo menor atividade dessa proteína em pacientes com LMC, ou ainda que tal ensaio possa ser menos específico para a atividade da MRP1. O ensaio PhA+FTC foi capaz de identificar a atividade da proteína BCRP em linhagens celulares e células de pacientes. Níveis reduzidos dos RNAm ABCB1 e SLC22A1, mas não do RNAm ABCG2, foram observados quando comparados com as amostras de indivíduos saudáveis. Não houve correlação entre os níveis da proteína Pgp e do RNAm ABCB1. A expressão de Pgp foi detectada na maioria das amostras de LMC, independente da fase da doença, e não foi associada com o prognóstico desfavorável. Variações nos níveis de expressão da Pgp foram observadas durante a evolução da LMC e relacionadas com o tratamento prévio. O imatinibe foi capaz de aumentar a expressão da Pgp, assim como os níveis do RNAm ABCB1 na linhagem K562-Lucena 1, Pgp+. Além disso, observamos uma maior redução de pCrkL e um maior percentual de morte celular nas células K562, Pgp-, quando comparadas à K562-Lucena 1, evidenciando um possível papel do imatinibe como substrato para a Pgp. Este fármaco também demonstrou ter potencial para funcionar como agente modulador da bomba de efluxo, uma vez que impediu o exporte de Rho das células K562-Lucena 1. Amostras de pacientes resistentes ao imatinibe exibiram altos níveis de atividade das proteínas transportadoras de efluxo de drogas (ensaio Rho+CSA). Nossos dados mostram que a atividade e/ou expressão dos transportadores de efluxo e influxo de drogas encontram-se alterados na maioria dos pacientes com LMC, porém não há correlação com a resposta ao imatinibe e o prognóstico na LMC. Entretanto, o conjunto dos resultados sugere um papel para a Pgp na resistência in vitro ao imatinibe"(AU)

"Chronic myeloid leukemia (CML) is characterized by the presence of the t(9:22) encoding the BCR-ABL chimeric oncogenic protein. Imatinib is a target specific drug that inhibits the activity of the tyrosine kinase (TK) protein BCR-ABL. However, imatinib intracellular concentration may be altered by transporter proteins. It was described that efflux proteins, P-glycoprotein (Pgp), breast cancer resistance protein (BCRP) and multidrug resistance related protein (MRP1), and the influx protein, the organic cation transporter protein (OCT1) may contribute for imatinib clinical resistance. The aim of this study was to evaluate the role of these proteins in imatinib resistance in cell lines and leukemic cells from CML patients. To analyze the activity of the efflux transporter proteins, fluorochrome rhodamine-123 associated with the modulator cyclosporin A (Rho+CSA) and pheophorbide A associated with fumitremorgin C (PhA+FTC) were used by flow cytometry. Analysis of ABCG1, ABCG2 and SLC22A1 genes, that encode the Pgp, BCRP and OCT1 proteins, respectively, was performed by real time PCR. The inhibition of BCR-ABL TK was measured by the levels of CrkL phosphorylation (pCrkL), its main target of activation. We observed a higher positivity for Rho+CSA assay in samples expressing Pgp, when compared with the ones expressing MRP1. These results suggest that patients with CML have lower activity of this protein, or this assay might be less specific to indicate the activity of MRP1. The PhA+FTC assay was able to identify BCRP activity in cell lines and cells from patients. Reduced levels of ABCB1 and SLC22A1, but not ABCG2 mRNA were observed when compared with samples from healthy individuals. There was no correlation between the levels of Pgp protein and ABCB1 mRNA. Pgp expression was detected in most samples of CML, regardless of disease stage and was not associated with poor prognosis. Changes in Pgp expression levels have been observed during the development of CML and were related to pretreatment. Imatinib was able to increase Pgp expression as well as ABCB1 mRNA levels in Pgp+ K562Lucena 1 cells. Moreover, we observed a greater pCrkL reduction and a higher percentage of cell death in Pgp- K562 cells compared to K562-Lucena 1, indicating a possible role of imatinib as a Pgp substrate. This drug has also been shown to have potential as an efflux pump modulating agent, once efflux of Rho was prevented in K562-Lucena 1. Imatinib resistant patient samples exhibited high levels of efflux transporter proteins activity (Rho+CSA assay). Our data show that the activity and / or expression of influx and efflux transporters of drugs are altered in most patients with CML, but no correlation with prognosis and response to imatinib in CML was observed. However, our results suggest a role for Pgp in imatinib resistance"(AU)