Inhibitory effect of distilled water on viability of human lens epithelial cells in vitro / 中华实验眼科杂志

Objective:To investigate the effect of distilled water on the viability of human lens epithelial cells (LECs) cultured in vitro. Methods:A total of 156 anterior capsule specimens were collected from 156 patients (156 eyes) who were diagnosed with age-related cataract during phacoemulsification combined with intraocular lens implantation from May to December 2020 in Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School.The 156 specimens were divided into 312 small pieces.Of the 312 pieces, 157 pieces were divided into normal control group (23 pieces), positive control group (10 pieces), balanced salt solution (BSS) immersion group (61 pieces) and distilled water immersion group (63 pieces) using computer-generated random numbers.Normal control group received no treatment.Positive control group was directly fixed with a mass fraction of 4% histiocytes fixative solution.For the 61 pieces in BSS immersion group, 20 pieces were soaked for 1 minute, 21 pieces for 2 minutes, and 20 pieces for 3 minutes.For the 63 pieces in distilled water immersion group, 20 pieces were soaked for 1 minute, 23 pieces for 2 minutes, and 20 pieces for 3 minutes.Another 125 pieces were selected to simulate the cataract aspiration-irrigation according to the treatment in BSS immersion group and distilled water immersion group respectively, plus rinse in a bottle containing BSS at a height of 70 cm for 1 minute.Cell viability was detected by trypan blue-eosin staining.LECs density, dead cell count, cell death rate and percentage of shedding (%) were calculated.Of the remaining 30 pieces, every 15 pieces were divided into normal control group, BSS immersion group, and distilled water immersion for 1, 2 and 3 minutes groups, with 3 pieces in each group.BSS immersion group was immersed for 3 minutes, and the other four groups were treated as mentioned above, and the LECs structure of the four groups was observed by light microscopy and transmission electron microscopy.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School (No.2019-248-01). Written informed consent was obtained from each subject.Results:The boundaries of LECs in BSS treatment groups were clear, and there was no significant difference in morphology compared with normal control group.With time increasing, LECs in distilled water treatment groups gradually swelled, and the boundaries of dead cells were not clear.There were significant differences in LECs density, dead LECs count and LECs mortality ( F=13.459, 98.918, 130.600; all at P<0.001). The LECs density was lower in 2-minute and 3-minute distilled water treatment groups than in normal control group, showing statistically significant differences (both at P<0.05). The dead LECs count and LECs mortality were higher in 1-minute, 2-minute and 3-minute distilled water treatment groups than in normal control group and BSS treatment groups for the same time, and the differences were statistically significant (all at P<0.05). Only a few shed LECs were seen in normal control group, 1-minute, 2-minute and 3-minute BSS treatment groups, and BSS immersion combined rinse group.After different time of soaking, there were more shed LECs in distilled water immersion combined rinse group, and the range of LECs shedding increased with the extension of distilled water immersion.There was a significant difference in the shedding percentage of LECs among different groups ( F=123.670, P<0.001). The shedding percentages of LECs at different time points were higher in distilled water immersion groups and distilled water immersion combined rinse groups than in normal control group, and the difference was statistically significant (all at P<0.05). The shedding percentage of LECs increased significantly in distilled water immersion groups with the extension of immersion.Light microscopy showed that the cells were destroyed in 1-minute, 2-minute and 3-minute distilled water treatment groups, and some LECs shed in the 2-minute and 3-minute treatment groups.Transmission electron microscopy showed cell lysis and destruction, suborganelles swelling, disruption of intercellular junctions in 1-minute, 2-minute and 3-minute distilled water treatment groups, loose attachment between cells and capsule in the 2-minute and 3-minute treatment groups, and cell detachment from capsule in the 3-minute treatment group. Conclusions:Distilled water immersion leads to LECs death in a time-dependent manner, and distilled water immersion combined with rinse can remove LECs on the lens capsule.

Therapeutic Properties of Flavonoids in Treatment of Cancer through Autophagic Modulation: A Systematic Review / 中国结合医学杂志

Cancers have high morbidity and mortality rates worldwide. Current anticancer therapies have demonstrated specific signaling pathways as a target in the involvement of carcinogenesis. Autophagy is a quality control system for proteins and plays a fundamental role in cancer carcinogenesis, exerting an anticarcinogenic role in normal cells and can inhibit the transformation of malignant cells. Therefore, drugs aimed at autophagy can function as antitumor agents. Flavonoids are a class of polyphenolic secondary metabolites commonly found in plants and, consequently, consumed in diets. In this review, the systematic search strategy was used, which included the search for descriptors "flavonoids" AND "mTOR pathway" AND "cancer" AND "autophagy", in the electronic databases of PubMed, Cochrane Library, Web of Science and Scopus, from January 2011 to January 2021. The current literature demonstrates that flavonoids have anticarcinogenic properties, including inhibition of cell proliferation, induction of apoptosis, autophagy, necrosis, cell cycle arrest, senescence, impaired cell migration, invasion, tumor angiogenesis and reduced resistance to multiple drugs in tumor cells. We demonstrate the available evidence on the roles of flavonoids and autophagy in cancer progression and inhibition. (Registration No. CRD42021243071 at PROSPERO).

Fibra de carbono ativada: avaliação das interações biológicas in vitro / Activated carbon fiber: evaluation of biological interactions in vitro

Devido a constante necessidade de desenvolver materiais biocompatíveis com propriedades osteocondutores e osteoindutoras, a presente tese conta com o desenvolvimento de dois estudos in vitro com fibra de carbono obtida a partir de fibra PAN têxtil, incorporada com diferentes íons de metais, na osteogênese com vistas à compreensão das necessidades da engenharia tecidual no desenvolvimento desse biomaterial com adequadas propriedades biológicas. As células foram obtidas dos fêmures de 09 ratos machos adultos (Wistar) pesando 300g, com 90 dias.Estudo 1: A partir da preparação da fibras foram obtidos corpos de prova de 4 mm de diâmetro e 2 mm de altura, dos seguintes grupos: fibra de carbono não ativada (FCNA), fibra carbono ativada (FCA) e fibra carbono ativada com prata (FCAAg). Após plaqueamento (n=5) em meio suplementado (MTS) e meio suplementado osteogênico (MTSO) foram analisados: viabilidade celular, conteúdo de proteína total (PT), atividade de fosfatase alcalina (ALP), interaçãocelular e formação de nódulos de mineralização. Foi avaliada a formação de biofilme nos corpos de prova, utilizando cepas de S. aureus, P. aeruginosa e E. coli. Na viabilidade celular, houve diferença estatística entre grupo controle celular (C) e FCA-MTS, FCAAg-MTS e FCAAg-MTSO. Em PT, não houvediferença, na ALP houve diferença entre C-MTS e as fibras, C-MTSO se mostrou semelhante. Em nódulos, houve diferença entre C-MTS e C-MTSO e as fibras do MTSO. Houve redução de formação de biofilme do S. aureus na FCAAg.Estudo 2: Foram obtidos corpos de prova da mesma dimensão do estudo 1 (n=5) dos seguintes grupos: fibra carbono ativada com prata (FCAAg), fibra carbono ativada com ouro (FCAAu), fibra carbono ativada com cobre (FCACu), fibra carbono ativada com paládio (FCAPd) e fibra carbono ativada com platina (FCAPt). Foram quantificadas a proliferação celular, viabilidade celular, formação de nódulos de mineralização, conteúdo de PT e ALP. Todas as amostras mostraram-se semelhantes quanto a proliferação celular, com exceção do grupo FCAAg comparado ao grupo controle (C). Sobre viabilidade celular, C obteve maior viabilidade que os outros grupos, e FCA obteve maior taxa que os grupos FCAAg, FCACu, FCAPt, sendo semelhante aos grupos FCAAu e FCAPd. Já os grupos FCAAu e FCAPd apresentaram diferença aos grupos FCAAg e FCACu. Na análise de expressão de PT apenas houve diferença entre FCA e FCAAu, sendo FCAAu com menor expressão de produção de PT. Na avaliação da ALP os grupos FCAAg e FACu mostraram diferença estatística e inferior com os grupos C, FCAAu, FCAPd e FCAPt, além disso, o grupo FCA mostrou menor taxa que C.Conclusões: As fibras utilizadas de base para a incorporação dos íons demonstraram grande potencial para uso como scaffold para reparação óssea, isso porque em ambos os estudos, na forma ativada e não ativada, as fibras apresentaram viabilidade celular e quantificação de cálcio satisfatórias. Sendo a versão não ativada mais econômica no que diz respeito ao tempo e custo de preparação. Mais estudos devem ser empregados a fim de assegurar sua segurança clínica em relação à citotoxicidade da incorporação de íons de ouro e paládio.(AU)

Due to the constant need to develop biocompatible materials with osteoconductive and osteoinductive properties, this thesis involves the development of two in vitro studies with carbon fiber obtained from textile PAN fiber, incorporated with different metal ions, in osteogenesis with a view to understanding the needs of tissue engineering in the development of this biomaterial with adequate biological properties. The cells were obtained from the femurs of 9 adult male rats (Wistar) weighing 300g, aged 90 days. Study 1: From the fiber preparation, specimens measuring 4 mm in diameter and 2 mm in height were obtained from the following groups: non-activated carbon fiber (FCNA), activated carbon fiber (FCA) and silver-activated carbon fiber (FCAAg). After plating (n=5) in supplemented medium (MTS) and supplemented osteogenic medium (MTSO), cell viability, total protein content (PT), alkaline phosphatase (ALP) activity, cell interaction and formation of mineralization nodules were analyzed. . Biofilm formation was evaluated in the specimens, using strains of S. aureus, P. aeruginosa and E. coli. In cell viability, there was a statistical difference between the cell control group (C) and FCAMTS, FCAAg-MTS and FCAAg-MTSO. In PT, there was no difference, in ALP there was a difference between C-MTS and fibers, C-MTSO was similar. In nodules, there was a difference between C-MTS and C-MTSO and MTSO fibers. There was a reduction in S. aureus biofilm formation on FCAAg. Study 2: Specimens of the same size as in study 1 (n=5) were obtained from the following groups: carbon fiber activated with silver (FCAAg), carbon fiber activated with gold (FCAAu), carbon fiber activated with copper (FCACu), palladium-activated carbon fiber (FCAPd) and platinum-activated carbon fiber (FCAPt). Cell proliferation, cell viability, formation of mineralization nodules, PT and ALP content were quantified. All samples were similar in terms of cell proliferation, with the exception of the FCAAg group compared to the control group (C). Regarding cell viability, C obtained higher viability than the other groups, and FCA obtained a higher rate than the FCAAg, FCACu, FCAPt groups, being similar to the FCAAu and FCAPd groups. The FCAAu and FCAPd groups showed differences to the FCAAg and FCACu groups. In the analysis of PT expression, there was only a difference between FCA and FCAAu, with FCAAu having lower expression of PT production. In the ALP assessment, the FCAAg and FACu groups showed a lower statistical difference compared to the C, FCAAu, FCAPd and FCAPt groups, in addition, the FCA group showed a lower rate than C. Conclusions: The fibers used as the basis for the incorporation of ions demonstrated great potential for use as a scaffold for bone repair, because in both studies, in activated and non-activated form, the fibers showed satisfactory cell viability and calcium quantification. The non-activated version is moreeconomical in terms of preparation time and cost. More studies must be carried out to ensure its clinical safety in relation to the cytotoxicity of the incorporation of gold and palladium ions. (AU)

The effects of thymoquinone on pancreatic cancer and immune cells

SUMMARY OBJECTIVES: Black cumin is widely used as a spice and as a traditional treatment. The active ingredient in black cumin seeds is thymoquinone. Thymoquinone has shown anticancer effects in some cancers. We planned to investigate its anticancer effect on pancreatic cancer cell lines. METHODS: Thymoquinone chemical component in various doses was prepared and inoculated on pancreatic cancer cell culture, healthy mesenchymal stem cells, and peripheral blood mononuclear cell culture. IC50 values were calculated by absorbance data and measuring cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide staining of cells incubated with thymoquinone at 24, 48, and 72 h. RESULTS: There was dose-related cytotoxicity. Maximal cytotoxicity was observed at 24 h and 100 μM thymoquinone concentrations in pancreatic cancer cell culture and mesenchymal stem cells. Any concentration of thymoquinone was not cytotoxic to peripheral blood mononuclear cell. Thymoquinone even caused proliferation at a concentration of 6.25 μM. CONCLUSIONS: Since the cytotoxic concentration of thymoquinone on pancreatic cancer cell culture and mesenchymal stem cells is the same, it is not appropriate to use thymoquinone to achieve cytotoxicity in pancreatic cancer. However, since thymoquinone provides proliferation in peripheral blood mononuclear cell at a noncytotoxic dose, it may have an immune activator effect. Therefore, in vivo studies are needed to investigate the effect of thymoquinone on the immune system.

Dynamic changes in and time sequence of ultraviolet B-induced apoptosis in rat corneal epithelial cells / Alterações dinâmicas na sequência temporal da apoptose induzida por radiação ultravioleta B em células epiteliais da córnea de ratos

ABSTRACT Purpose: To systematically examine the dynamic changes and time sequence in corneal epithelial cell apoptosis after excessive ultraviolet B irradiation. Methods: Ultraviolet B (144 mJ/cm2) was used to irradiate rat corneal epithelial cells for 2 h. Cell morphology was observed on differential interference contrast microscopy, and the numbers of the different kinds of apoptotic cells were counted using the ImageJ software. Cell viability was measured with the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide method. Cell apoptotic rate and loss of mitochondrial membrane potential were detected using flow cytometric analyses. The expression levels of 3 apoptotic genes were measured with real-time quantitative polymerase chain reaction at different time points within 0-24 h after irradiation. Results: After 144-mJ/cm2 ultraviolet B irradiation for 2 h, the expression levels of caspase-8 and Bax were highest at 0 h; furthermore, the mitochondrial membrane potential decreased at 0 h and remained constant for 6 h in a subsequent culture. At 6 h, caspase-3 was activated. The decrease in cell viability and increase in apoptotic rate peaked at 6 h. The caspase-3 expression level decreased within 12-24 h, which led to a decline in apoptotic rate and change in apoptotic stage. Conclusions: The corneal epithelial cells exhibited rapid apoptosis after ultraviolet B irradiation, which was associated with both extrinsic and intrinsic pathways.

RESUMO Objetivos: Explorar sistematicamente as mudanças dinâmicas e a sequência temporal no processo de apoptose de células epiteliais corneanas após excesso de irradiação com ultravioleta B. Métodos: A radiação ultravioleta B (144 mJ/cm2) foi utilizada para irradiar células epiteliais da córnea de rato durante 2h. A morfologia celular foi observada por meio de microscópio de contraste de interferência diferencial, e os números de diferentes tipos de células apoptóticas foram contados e registrados pelo software ImageJ. A viabilidade celular foi medida pelo método brometo de 3- (4, 5-dimetil-2-tiazolil) -2, 5-difenil-2-H-tetrazólio. A taxa apoptótica celular e a perda do potencial da membrana mitocondrial foram detectadas por meio de análises citométricas de fluxo. Os níveis de expressão de três genes apoptóticos foram medidos por reação em cadeia da polimerase quantitativa em tempo real em diferentes momentos dentro de 0-24 h após a irradiação. Resultados: Após 144 mJ/cm2 de irradiação com ultravioleta B por 2h, os níveis de expressão de caspase-8 e Bax foram maiores em 0h; o potencial da membrana mitocondrial diminuiu a 0h e permaneceu constante por 6h na cultura subsequente. Às 6h, a caspase-3 foi ativada. A diminuição da viabilidade celular e o aumento da taxa apoptótica atingiu o pico em 6h. A expressão de caspase-3 diminuiu dentro de 12 - 24 h, levando a um declínio na taxa apoptótica e alteração no estágio apoptótico. Conclusões: As células epiteliais da córnea apresentaram uma apoptose rápida após excesso de irradiação com ultravioleta B, e esse processo foi associado tanto à via extrínseca como à via intrínseca.

Effect of alpha-humulene incorporation on the properties of experimental light-cured periodontal dressings

Abstract The objective of this study was to formulate an experimental light-cured periodontal dressing containing alpha-humulene and to compare its physical, antimicrobial, and cytotoxicity properties with commercial gold standards (Barricaid® and Periobond®). Two periodontal dressing formulations were developed (a and b). The formulations were divided into 5 groups according to the alpha-humulene concentration as follows: Ea - control group, Ea1 - 1%, Ea5 - 5%, Ea10 - 10%, and Ea20 - 20%; Eb - control group, Eb1 - 1%, Eb5 - 5%, Eb10 - 10%, and Eb20 - 20%. Materials characterization was performed using the degree of conversion, cohesive strength, sorption, and solubility assays. Antimicrobial assay was performed using the modified direct contact test against E. faecalis and S. aureus. Cytotoxicity was assessed by the cell viability experiment using L929 fibroblasts. In general, the cohesive strength values of materials decreased as the alpha-humulene concentration increased. All the experimental dressings showed antimicrobial activity against both bacteria tested. Cell viability results for the Ea, Ea1, Eb, and Eb1 groups showed moderate cytotoxic effect. The formulations containing alpha-humulene showed similar behavior to the commercial references. Thus, formulations containing alpha-humulene have potential to be used as periodontal dressing.

Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells

Abstract: Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.

Bioprocess optimization of interferon ß-1-a in Pichia pastoris and its improved inhibitory effect against hepatocellular carcinoma cells

Interferon-ß-1a (INF-ß-1a) has gained significant attention due to its emerging applications in the treatment of different human diseases. Therefore, many researchers have attempted to produce it in large quantities and also in a biologically active form using different expression systems. In the present study, we aimed to improve the expression level of INF-ß-1a by Pichia pastoris using optimization of culture conditions. The codon-optimized INF-ß- 1a gene was cloned into pPICZαA plasmid under the control of alcohol oxidase I (AOX1) promoter. The protein expression was induced using different concentrations of methanol at different pHs and temperatures. The biological activity of produced protein was evaluated by anti-proliferative assay. The ideal culture conditions for the expression of INF-ß-1a by P. pastoris were found to be induction with 2% methanol at pH 7.0 culture medium at 30 C which yielded a concentration of 15.5 mg/L INF-ß-1a in a shake flask. Our results indicate that differences in glycosylation pattern could result in different biological activities as INF- ß-1a produced by P. pastoris could significantly more reduce the cell viability of HepG-2 cells, a hepatocellular carcinoma cell line, than a commercially available form of this protein produced by CHO

Skin fibroblasts derived from Leber's hereditary optic neuropathy patients present mitochondrial dysfunction / 中华眼底病杂志

Objective:To investigate the feasibility of immoribund skin fibroblast cell line derived from Leber's hereditary optic neuropathy (LHON) patients as a cell model.Methods:A basic research. Two LHON patients and 2 healthy volunteers were recruited from Department of Ophthalmology of Genetic Clinic of Henan Provincial Eye Hospital. The skin tissue of participants was obtained, and the 4 immortalized skin fibroblasts were constructed by SV40 virus infection, including 2 LHON patient cells (LHON-1 and LHON-2 cells) and 2 healthy volunteers cells (NC-1 and NC-2 cells). Mitochondrial morphology in cells was observed by electron microscope. The levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide-oxidation state (NAD +), nicotinamide adenine dinucleotide-reduction state (NADH) and adenosine triphosphate (ATP) in fibroblasts were detected. Cellular oxygen consumption was measured by seahorse mitochondrial pressure assay. Cell viability was detected using cell counting kit-8 (CCK8). One-way ANOVA was performed to compare the levels of ROS, NAD +, NADH and ATP in LHON and NC cells, as well as basal oxygen consumption, maximal oxygen consumption, ATP-coupled oxygen consumption, and cell viability. Results:Compared with NC-1 and NC-2, the number of mitochondrial crest in LHON fibroblasts was significantly reduced, indicating abnormal mitochondrial morphology. Biochemical analysis showed that ROS levels in LHON cells increased, but NAD +/NADH and ATP levels decreased, and the oxygen consumption was significantly inhibited, indicating the presence of mitochondrial damage and respiratory dysfunction. The results of CCK-8 detection showed that the survival ability of LHON-1 and LHON-2 cells was worse under stress conditions. Conclusion:Immortalized skin fibroblast cell lines from LHON patients presented mitochondrial dysfunction.

Cinamaldeído e terpineol como inibidores de biofilmes de Candida albicans e Enterococcus faecalis / Cinamaldehído y terpineol como inhibidores de biopelículas de Candida albicans y Enterococcus faecalis / Cinnamaldehyde and terpineol as inhibitors of Candida albicans and Enterococcus faecalis biofilms

Introdução: Os fitoconstituintes são moléculas naturais que apresentam atividade antimicrobiana satisfatória e devem ser estudados quanto ao seu uso como novas substâncias para irrigação dos canais radiculares. Objetivo: Avaliar o efeito inibitório dos fitoconstituintes cinamaldeído e α-terpineol frente a biofilmes monoespécie e duoespécie de microrganismos envolvidos na infecção endodôntica. Métodos: Trata-se de um estudo experimental na área de microbiologia aplicada, in vitro, cego quanto às análises e randomizado. Foram selecionados os fitoconstituintes cinamaldeído e α-terpineol. A atividade antimicrobiana frente Candida albicans e Enterococcus faecalis foi avaliada por meio da análise da capacidade metabólica com o uso da resazurina e análise da viabilidade celular pelo plaqueamento. O meio de cultura e a clorexidina 1 porcento serviram de controle negativo e positivo, respectivamente. Resultados: Observou-se ausência de crescimento para exposição dos biofilmes nas concentrações de 10 e 5 mg/mL de ambos os fitoconstituintes. Na concentração de 2,5 mg/mL de terpineol, constatou-se crescimento somente nos biofilmes monoespécie de C. albicans e duoespécie. Já na concentração de 1mg/mL de terpineol e cinamaldeído, verificou-se crescimento para todos os biofilmes. Conclusão: O cinamaldeído e α-terpineol apresentaram atividade inibitória frente biofilmes monoespécie e duoespécie de Candida albicans e Enterococcus faecalis, nas concentrações de 10 e 5 mg/mL(AU)

Introducción: Los fitoconstituyentes son moléculas naturales que presentan actividad antimicrobiana satisfactoria y deben ser estudiados en cuanto a su uso como nuevas sustancias para irrigación de los canales radiculares. Objetivo: Evaluar el efecto inhibitorio de fitoconstituyentes cinamaldehído y α-terpineol frente a biopelículas monoespecies y duoespecies de microorganismos involucrados en la infección endodóntica. Métodos: Estudio experimental en el campo de la microbiología aplicada, in vitro, ciego al análisis y aleatorizado. Se seleccionaron los fitoconstituyentes cinamaldehído y α-terpineol. La actividad antimicrobiana frente Candida albicans y Enterococcus faecalis fue evaluada por medio del análisis de la capacidad metabólica con el uso de la resazurina y análisis de la viabilidad celular por el plaqueamiento. El medio de cultivo y la clorexidina 1 por ciento sirvieron de control negativo y positivo, respectivamente. Resultados: Se observó ausencia de crecimiento para exposición de las biopelículas en las concentraciones de 10 y 5 mg/mL de ambos fitoconstituyentes. En la concentración de 2,5 mg/mL de terpineol se constató crecimiento solo en los biofilmios monoespecies de C. albicans y duoespecies. En la concentración de 1 mg/mL de terpineol y cinamaldehído se verificó crecimiento para todas las biopelículas. Conclusiones: Cinamaldehído y α-terpineol presentaron actividad inhibitoria frente a biofilmes monoespecies y duoespecies de Candida albicans y Enterococcus faecalis, en las concentraciones de 10 y 5 mg/mL(AU)

Introduction: Phytoconstituents are natural molecules displaying satisfactory antimicrobial activity. Studies should be conducted about their use as new root canal irrigants. Objective: Evaluate the inhibitory effect of the phytoconstituents cinnamaldehyde and α-terpineol against mono- and duo-species biofilms of microorganisms involved in endodontic infection. Methods: An experimental applied microbiology blind randomized in vitro study was conducted. The phytoconstituents selected were cinnamaldehyde and α-terpineol. Antimicrobial activity against Candida albicans and Enterococcus faecalis was evaluated by metabolic capacity analysis with resazurin and cell viability analysis by the plaque. The culture medium and 1 percent chlorhexidine served as negative and positive controls, respectively. Results: An absence of growth was observed for exposure of the biofilms at concentrations of 10 and 5 mg/ml of both phytoconstituents. At a concentration of 2.5 mg/ml terpineol displayed growth only in the mono-species biofilms of C. albicans and duo-species biofilms. At a concentration of 1 mg/ml terpineol and cinnamaldehyde displayed growth in all biofilms. Conclusions: Cinnamaldehyde and α-terpineol displayed inhibitory activity against mono- and duo-species biofilms of Candida albicans and Enterococcus faecalis at concentrations of 10 and 5 mg/ml(AU)

Cytotoxicity and Elemental Release of Dental Acrylic Resin Modified with Silver and Vanadium Based Antimicrobial Nanomaterial / Citotoxicidade e Liberação Elementar de Resina Acrílica Odontológica Modificada com Nanomaterial Antimicrobiano a Base de Prata e Vanádio

The acrylic resin used for the prosthesis base accumulates biofilm, causing diseases such as stomatitis. The addition of some nanoparticles promotes antimicrobial action. This study incorporated the nanostructured silver vanadate decorated with silver nanoparticles (AgVO3) to the acrylic resin by two methods and evaluated the cytotoxicity for human gingival fibroblasts (HGF) and the released silver and vanadium ions. The concentrations of 0.5, 1, 2.5, and 5% of AgVO3 was incorporated by vacuum spatulation and polymeric film. The vacuum spatulation was performed for 60 s using the Turbomix equipment, and the polymeric film was obtained from the polymer solubilization in chloroform, the film was subjected to a cryogenic grinding, and the powder obtained was manually mixed at the monomer. HGF cell viability was assessed after 24 hours, 7 and 14 days by the MTT assay. The release of silver (Ag) and vanadium (V) ions were quantified by inductively coupled plasma mass spectrometry after 30 days. Kruskal-Wallis and Dunn's test were applied (α = 0.05). The HGF viability was inversely proportional to the incubation time. Both incorporation techniques and the negative and positive control groups presented significant statistical differences (p<0.05). The experimental groups presented no statistical difference compared to the negative control (p>0.05), except the vacuum spatulation group with 5% of AgVO3 that showed greater viability than the negative control (p=0.013) in 24 hours. The release of Ag and V ions was proportional to the concentration of AgVO3 The 5% group presented a significant difference compared to the other groups (p<0.05). In conclusion, the acrylic resin with and without the AgVO3 incorporation had a small cytotoxic potential for HGF in 24 hours, with a lower viability in longer contact times; the release of Ag and V ions was proportional to the concentration of AgVO3, not influencing cell viability. (AU)

A resina acrílica utilizada para a base da prótese acumula biofilme, causando doenças como a estomatite. A adição de algumas nanopartículas promove ação antimicrobiana. Este estudo incorporou o vanadato de prata nanoestruturado decorado com nanopartículas de prata (AgVO3) à resina acrílica por dois métodos e avaliou a citotoxicidade para fibroblastos gengivais humanos (HGF) e os íons prata e vanádio liberados. As concentrações de 0,5%, 1%, 2,5% e 5% de AgVO3 foram incorporadas por espatulação a vácuo e filme polimérico. A espatulação a vácuo foi realizada por 60 s no equipamento Turbomix, e o filme polimérico foi obtido a partir da solubilização do polímero em clorofórmio, o filme foi submetido a uma moagem criogênica e o pó obtido foi misturado manualmente ao monômero. A viabilidade celular de HGF foi avaliada após 24 horas, 7 e 14 dias pelo ensaio de MTT. A liberação de íons prata (Ag) e vanádio (V) foi quantificada por espectrometria de massa com plasma indutivamente acoplado após 30 dias. Os testes de Kruskal-Wallis e Dunn foram aplicados (α=0,05). A viabilidade de HGF foi inversamente proporcional ao tempo de incubação. As técnicas de incorporação e os grupos controle negativo e positivo apresentaram diferença estatisticamente significante (p<0,05). Os grupos experimentais não apresentaram diferença estatística em relação ao controle negativo (p>0,05), exceto o grupo de espatulação a vácuo com 5% de AgVO3 que apresentou maior viabilidade que o controle negativo (p = 0,013) em 24 horas. A liberação de íons Ag e V foi proporcional à concentração de AgVO3. O grupo 5% apresentou diferença significativa em relação aos demais grupos (p <0,05). Em conclusão, a resina acrílica com e sem a incorporação de AgVO3 apresentou um pequeno potencial citotóxico para o HGF em 24 horas, com menor viabilidade nos tempos de maior contato, e a liberação de íons Ag e V foi proporcional à concentração de AgVO3, não influenciando na viabilidade celular. (AU)

Biological effects of formulation containing curcuminoids and Bidens Pilosa L. in oral carcinoma cell line

Abstract: FITOPROT, which contains curcuminoids and Bidens pilosa L. extract, is an innovative mucoadhesive formulation indicated for the topical treatment of chemoradiotherapy-induced oral mucositis (OM) in patients with advanced and visible oral squamous cell carcinoma. The formulation is used as a mouthwash directly on tumor tissue of patients with advanced neoplasms, without triggering cancer cell proliferation or tumor invasiveness. Thus, the aim of this study was to evaluate the biological effects of FITOPROT on an oral squamous cell carcinoma cell line (SCC-4). The viability of SCC-4 cells was assessed after exposure to FITOPROT using MTT reduction assay. The effects of the mucoadhesive formulation on cell cycle progression and cell death parameters were evaluated using flow cytometry. In addition, the inflammatory profile of the tumor cells was evaluated using the cytometric bead array (CBA) assay. FITOPROT promoted a concentration-dependent decrease in cell viability and cell cycle arrest at the G2/M phase (p < 0.05). Mitochondrial membrane potential was also altered after exposure to the formulation (p < 0.05), in parallel with a reduction in VEGF and IL-8 production (p = 0.01 and p = 0.05, respectively). In summary, the results indicate that FITOPROT reduces SCC-4 cell viability, promotes cell cycle arrest, modulates mitochondrial membrane potential, and exhibits antiangiogenic and anti-inflammatory properties, thus indicating its potential for topical use in patients with OM and visible tumors in the mouth.

A Preliminary Research Study on the Cytotoxicity of Expired and Non-expired Composite Resins: In Vitro Study / Estudio preliminar in vitro de la citotoxicidad de resinas compuestas caducadas y no caducadas

Astracts Objective: Studies have focused on use of non-expired composites. Unfortunately some clinicians still use expired composite resins without considering their effects. The objective of this in vitro preliminary research was to investigate cytotoxicity of expired(6-months) and non-expired composite resins. Materials and methods: Expired (E) and non-expired (NE) samples of one bulk-fill (Tetric N-Ceram Bulk-fill (TNB), Ivoclar Vivadent), two nano-hybrid (Tetric N-Ceram (TN), Ivoclar Vivadent; Clearfil Majesty ES-2 (CM), Kuraray) composite resins were tested on L929 fibroblast cells. Medium covering cells was removed then plastic rings (2-mm height) were filled with non-polymerized composite resins, placed in direct contact with cells and polymerized with LED light curing unit (LCU). Three samples were prepared for each group. After polymerization, removed medium was added to the cells. Cells that were left without medium (WOM) and cells that were exposed to LCU were used as positive control groups. Cells without any treatment were used as negative control group (C). Cells were incubated with tested materials for 7-days to evaluate cytotoxicity. Cell viability was calculated by sulforhodamine B test as a percentage (%). One-way ANOVA and post-hoc Tukey tests were used for statistical analyses (p0.05), except between TN NE and TN E (p0.05). All experimental groups compared with C group showed statistically significant cytotoxicity (p<0.05). A statistically significant difference existed between LCU and C groups (p<0.05). Conclusions: In clinical practice, expired composite resins should never be used. Although a correlation was found between expiration dates of nano-hybrid composite resins and cell viability, opposite data were obtained for bulk- fill composite resin. Researches are still required to evaluate biocompatibility of bulk- fill composite resins at various thicknesses with current LCUs.

Resumen Objetivo: Los estudios se han concentrado en el uso de resinas compuestas no vencidos. Desafortunadamente, algunos clínicos aún usan resinas caducadas sin considerar sus efectos. El objetivo de este estudio preliminar in vitro fue investigar la citotoxicidad de resinas compuestas caducadas (6 meses) y no caducadas. Materiales y métodos: muestras caducadas (E) y no caducadas (NE) de una resina bulk-fill (Tetric N-Ceram Bulk-fill (TNB), Ivoclar Vivadent) y dos resinas nanohíbridas (Tetric N-Ceram (TN) Ivoclar Vivadent) (Clearfil Majesty ES-2 (CM), Kuraray), se probaron en células de fibroblastos L929. Se retiraron las células que cubrían el medio, luego se llenaron anillos de plástico (2 mm de altura) con resinas no polimerizadas, se colocaron en contacto directo con las células y se polimerizaron con una unidad de fotocurado LED (LCU). Se prepararon tres muestras para cada grupo. Después de la polimerización, se añadió el medio eliminado a las células. Las células que quedaron sin medio (WOM) y las células que se expusieron a LCU se usaron como grupos de control positivo. Las células sin ningún tratamiento se utilizaron como grupo de control negativo (C). Las células se incubaron con las resinas durante 7 días para evaluar la citotoxicidad. La viabilidad celular se calculó mediante la prueba de sulforodamina B como un porcentaje (%). ANOVA unidireccional y pruebas post-hoc de Tukey se utilizaron para los análisis estadísticos (p 0.05), excepto entre TN NE y TN E (p 0.05). Todos los grupos experimentales en comparación con el grupo C mostraron citotoxicidad estadísticamente significativa (p <0,05). Existió una diferencia estadísticamente significativa entre LCU y grupos C (p <0.05). Conclusiones: En la práctica clínica, las resinas compuestas caducadas nunca deben usarse. Aunque se encontró una correlación entre las fechas de vencimiento de las resinas compuestas nano-híbridas y la viabilidad celular, se obtuvieron datos opuestos para la resina bulk-fill. Se requieren nuevas investigaciones para evaluar la biocompatibilidad de las resinas bulk-fill en distintos espesores con las LCU actuales.

Effect of preheating on cytotoxicity and physicochemical properties of light-cured calcium-based cements / Efeito do pré-aquecimento na citotoxicidade e propriedades físico-químicas de cimentos à base de cálcio fotopolimerizáveis

ABSTRACT The aim of this study was to evaluate the degree of conversion, cytotoxicity, solubility and pH of photopolymerizable calciumbased cements submitted to preheating. The degree of conversion was analyzed by Fourier transform infrared, cytotoxicity by the MTT test and solubility through loss of mass. The data were subjected to statistical tests (ANOVA / Tukey's, p<0.05). The photopolymerizable materials showed a low degree of conversion, regardless of preheating. All materials caused a reduction in cell viability at 24 hours and 7 days, with the Dycal (control) being more cytotoxic. Heat had a positive effect on Biocal at 7 days. Dycal is the most soluble material. Heat had no effect on the solubility or pH of the polymerizable materials. It is concluded that photopolymerizable calcium-based cements have a low degree of conversion and are soluble, which results in mild to moderate cytotoxicity.

RESUMO O objetivo do presente estudo foi avaliar o grau de conversão, citotoxicidade, solubilidade e pH de cimentos à base de cálcio fotopolimerizáveis submetidos a pré-aquecimento. O grau de conversão foi analisado por espectroscopia no infravermelho com transformada de Fourier, a citotoxicidade pelo teste de MTT e a solubilidade através da perda de massa. Os dados foram submetidos a testes estatísticos (ANOVA/Tukey, p<0,05). Os materiais fotopolimerizáveis apresentaram baixo grau de conversão, independente do pré-aquecimento. Todos os materiais causaram redução da viabilidade celular nas análises de 24 horas e 7 dias, sendo que o Dycal (controle) apresentouse mais citotóxico e o calor apresentou efeito positivo sobre o Biocal na análise de 7 dias. O Dycal é o material mais solúvel e o calor não causou efeito na solubilidade e pH dos materiais polimerizáveis. Assim, conclui-se que os cimentos à base de cálcio fotopolimerizáveis apresentam baixo grau de conversão e são solúveis, que resulta em citotoxicidade suave e moderada.

Development of a new protocol for freeze-drying preservation of Pseudoalteromonas nigrifaciens and its protective effect on other marine bacteria

Background: Freeze-drying is known as one of the best methods to preserve bacterial strains. Protectant is the key factor affecting the survival rate of freeze-dried strains. In addition, salinity, bacterial suspension concentration, drying time, and other factors can also affect the survival rate of strains to varying degrees. At present, there are relatively few studies on freeze-drying preservation of marine bacteria. In the present study, we performed the freeze-drying protectant screening and optimized the preservation conditions for Pseudoalteromonas nigrifaciens, which is widely distributed in marine environment. The protective effects of the screened protectants were verified by 18 other marine bacterial strains. Results: The results indicated that the combination of 5.0% (w/v) lactose, 5.0% (w/v) mannitol, 5.0% (w/v) trehalose, 10.0% (w/v) skim milk powder, 0.5% (w/v) ascorbic acid and 0.5% (w/v) gelatin was the best choice for the preservation of P. nigrifaciens. The suggested salinity and concentration of initial cell suspension were 10 g/L NaCl and 1.0 × 109 CFU/mL, respectively. Furthermore, stationary-phase cells were the best choice for the freeze-drying process. The highest survival rate of P. nigrifaciens reached 52.8% when using 5­10% (w/v) skim milk as rehydration medium. Moreover, the other 18 marine strains belonging to Pseudoalteromonas, Vibrio, Photobacterium, Planomicrobium, Edwardsiella, Enterococcus, Bacillus, and Saccharomyces were freezedried under the abovementioned conditions. Their survival rates were 2.3­95.1%. Conclusion: Collectively, our results supported that the protectant mixture and parameters were beneficial for lyophilization of marine bacteria

Cytotoxicity of Calcium Hydroxide on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To examine the cytotoxicity of calcium hydroxide on human umbilical cord mesenchymal stem cells (HUCMSC) to understand the characteristics for use in regenerative dentistry procedures especially regenerative endodontics. Material and Methods: HUCMSC was isolated, cultured, and confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSC was cultured and divided into two groups, the control group (cultured in minimum essential medium (MEM) alpha) and calcium hydroxide group (cultured in MEM alpha and calcium hydroxide). Methyl-thiazole-tetrazolium (MTT) assay was done on different concentrations of calcium hydroxide (0.39 to 25 µg/mL) and the cells were observed and counted. One-way ANOVA test was used with a significance level set at 5%. Results: Flow cytometric analysis confirmed positive of CD73, CD90, CD105, negative of CD45 and CD34. A significant difference was found between the concentration of 6.25 and 3.125 µg/mL (p=0.004). There was no significant difference among 6.25, 12.5 and 25 µg/mL concentrations. There was also no significant difference among 0.39, 0.78, 1.56, and 3.125 µg/mL concentrations. Conclusion: Even though calcium hydroxide is a medicament of choice in clinical endodontics, it decreases the viability of HUCMSC. The lower the concentration of calcium hydroxide, the higher the viability of HUCMSC.

Effect of ScLL and 15d-PGJ2 on viability and cytokine release in LPS-stimulated fibroblasts: an in vitro study

Abstract This study evaluated the effect of a cyclopentenone-type PG, 15-Deoxy-Δ12,14-PG J2 (15d-PGJ2), and lectin (ScLL) on the viability of human gingival fibroblasts (HGFs), and on IL-6 and TGFβ-1 release by these fibroblasts, stimulated with lipopolysaccharide (LPS). HGFs were stimulated with LPS 10 μg/ml and treated with 15d-PGJ2 1 and 2 μg/ml, and ScLL 2 and 5 μg/ml, for 1 and 3h, and then evaluated for viability by MTT assay. Supernatant was collected to detect IL-6 and TGFβ-1 release, by ELISA. Positive control was cells kept in Dulbecco's Modified Eagle's Medium, and negative control was those kept in LPS. Data were analyzed by ANOVA and Dunnett's test (α = 0.05). No significant difference was found in viability among experimental groups at 1h (p > 0.05). Percentage of ScLL 5 µg/ml viable cells was similar to that of positive control at evaluated periods (p > 0.05), whereas the other groups had lower levels than the positive control (p < 0.05). IL-6 release was statistically higher for ScLL 5 μg/ml and 15d-PGJ2 2 µg/ml at 1h, compared with the other treated groups and positive control (p < 0.05). No significant differences were found among the groups at 3h (p > 0.05), except for ScLL 2 µg/ml and 15d-PGJ2 1 µg/ml, which showed lower IL-6 release compared with that of negative control (p < 0.05). No significant difference was found among the groups for TGFβ-1 release (p > 0.05). Results indicated that ScLL 5 μg/ml did not interfere in viability, and ScLL 2 µg/ml and 15d-PGJ2 1 µg/ml demonstrated reduced IL-6 release. Tested substances had no effect on TGFβ-1 release.

Difference in the culture of dorsal root ganglion cells in serum-free medium and serum medium / 中国组织工程研究

BACKGROUND: Both serum-free and serum media have been used to culture dorsal root ganglion cells, but the difference between the two remains unclear. OBJECTIVE: To explore whether serum-free medium can completely replace serum medium for culture of dorsal root ganglion cells. METHODS: The dorsal root ganglion of ICR mice at 8-10 weeks was taken and treated with collagenase and trypsin. After that, the mice were divided into the electroporation + serum group, electroporation + serum-free group, non-electroporation + serum group and non-electroporation + serum-free group. In the electroporation groups, the dorsal root ganglion cells were transfected with electroporation buffer and enhanced green fluorescent protein particles. Cells were cultured for three days. After Tuj1 antibody staining, in the non-electroporation + serum group and non-electroporation + serum-free group, axon branches, axon regeneration length, number of cell survival and the expression of proteins related to axon regeneration were counted. In the electroporation + serum group and electroporation + serum-free group, axon branches, length of axon regeneration, number of cell survival, and electroporation efficiency were measured. This study was approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Soochow University. RESULTS AND CONCLUSION: (1) In the non-electroporation + serum group and non-electroporation + serum-free group, there was no significant difference in axon branches, axon regeneration length, number of cell survival and the expression of axon regeneration related proteins (P > 0.05). (2) In the electroporation + serum group and electroporation + serum-free group, there was no significant difference in axon branches, axon regeneration length and electroporation efficiency (P > 0.05). Compared with electroporation + serum group, the number of cell survival of the electroporation + serum-free group was significantly lower (P 0.05). The number of cell survival of the non-electroporation + serum group was significantly higher than that of the electroporation + serum group (P < 0.05). (4) The results showed that, in the condition of non-electroporation, the absence of serum does not affect the culture of dorsal root ganglion in vitro, and serum-free medium can replace serum medium. However, under the condition of electroporation, the number of cell survival would be decreased without serum medium, suggesting that serum plays an important role in the culture of dorsal root ganglion in vitro under the condition of electroporation. Therefore, serum-free media cannot replace serum media.

Inhibitory effects of iridoid glycosides from Boschniakia rossica combined with 5-fluorouracil on human hepatoma HepG2 and SK-Hep1 cells epithelial-mesenchymal transition / 中草药

Objective: To study the inhibitory effect of iridoid glycosides from Boschniakia rossica (IGBR) combined with 5-Fu on epithelial-mesenchymal transition induced by TGF-β1 in human hepatoma SK-Hep1 and HepG2 cells, and compare the efficacy of drugs. Methods: The survival ability of HepG2 and SK-Hep1 cells was detected by MTT and the combination index (Q value) was calculated to judge the interaction of combined drugs. The EMT model of HepG2 and SK-Hep1 cells was established. The cell adhesion rate was detected by MTT. The expression of matrix metalloproteinase (MMP) MMP2, MMP7, MMP9, Snail, and Slug was detected by Western blotting. The localization and expression intensity of E-cadherin and Vimentin was detected by immunofluorescence. Results: MTT showed that compared with the control group, the 5-FU group, IGBR group and combination group cell survival ability were decreased (P < 0.05) at 48 h after administration; IGBR and 5-Fu had an additive or synergistic effect. Compared with the model group, the adhesion rate of 5-FU group, IGBR group and combination group was reduced (P < 0.05). Western blotting results showed that compared with the control group, the expression of MMP2, MMP7, MMP9, Snail, Slug were up-regulated (P < 0.05) in the model group. Compared with the model group, the expression of MMP2, MMP7, MMP9, Snail and Slug were down-regulated (P < 0.05) in 5-FU group, IGBR group and combination group. Compared with the control group, immunofluorescence showed that the E-cadherin fluorescence intensity was decreased in the model group, but the Vimentin fluorescence intensity was increased. Compared with the model group, the E-cadherin fluorescence intensity was increased in 5-FU group, IGBR group and combined group, but the Vimentin fluorescence intensity was decreased. Conclusion: IGBR and 5-Fu can inhibit human hepatoma EMT. The combined drugs have the combined effect on HepG2 cells and synergistic effect on SK-Hep1 cells. The therapeutic effect on SK-Hep1 cells is better than HepG2 cells.

Microglia-induced Neurotoxicity: A Review of in Vitro Co-culture Models

@#Microglia-induced neurotoxicity occurs when inflammation mediated by microglia causes loss of neuronal structures or functions in the central nervous system implicated in stroke, spinal cord injury, sepsis, neurodegenerative diseases and even psychiatric illnesses. Various co-culture in vitro microglia-induced neurotoxicity (MINT) models have been established to enable an in-depth study of this process and yet there is a dearth of information regarding usages, advantages and limitations of each of the components of this model. In this review, we examined 56 MINTs for the cells, stimuli, parameters, methods of neurotoxicity measurement and formats of co-culture used in their construction. We aim to provide foundational information, overall guideline and framework for the novice researcher to develop his/her own model and for the advancement of improved, novel and more representative MINT models.