Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions

Abstract Simulating a bacterial-induced pulpitis environment in vitro may contribute to exploring mechanisms and bioactive molecules to counteract these adverse effects. Objective To investigate the chronic exposure of human dental pulp cells (HDPCs) to lipopolysaccharides (LPS) aiming to establish a cell culture protocol to simulate the impaired odontogenic potential under pulpitis conditions. Methodology HDPCs were isolated from four healthy molars of different donors and seeded in culture plates in a growth medium. After 24 h, the medium was changed to an odontogenic differentiation medium (DM) supplemented or not with E. coli LPS (0 - control, 0.1, 1, or 10 µg/mL) (n=8). The medium was renewed every two days for up to seven days, then replaced with LPS-free DM for up to 21 days. The activation of NF-κB and F-actin expression were assessed (immunofluorescence) after one and seven days. On day 7, cells were evaluated for both the gene expression (RT-qPCR) of odontogenic markers (COL1A1, ALPL, DSPP, and DMP1) and cytokines (TNF, IL1B, IL8, and IL6) and the production of reactive nitrogen (Griess) and oxygen species (Carboxy-H2DCFDA). Cell viability (alamarBlue) was evaluated weekly, and mineralization was assessed (Alizarin Red) at 14 and 21 days. Data were analyzed with ANOVA and post-hoc tests (α=5%). Results After one and seven days of exposure to LPS, NF-κB was activated in a dose-dependent fashion. LPS at 1 and 10 µg/mL concentrations down-regulated the gene expression of odontogenic markers and up-regulated cytokines. LPS at 10 µg/mL increased both the production of reactive nitrogen and oxygen species. LPS decreased cell viability seven days after the end of exposure. LPS at 1 and 10 µg/mL decreased hDPCs mineralization in a dose-dependent fashion. Conclusion The exposure to 10 µg/mL LPS for seven days creates an inflammatory environment that is able to impair by more than half the odontogenic potential of HDPCs in vitro, simulating a pulpitis-like condition.

Effect of guarana seed powder on cholesterol absorption in vitro and in Caco-2 cells.

Cardiovascular disease risk is related to oxidative stress and hypercholesterolemia. Guarana seed powder contains flavanols that possess antioxidant properties and cholesterol-lowering effects. However, the molecular mechanism through which guarana seed powder may decrease cholesterol uptake from the intestinal lumen remains unclear. Thus, this study aimed to investigate the effect of guarana powder aqueous extract on cholesterol absorption mechanisms. After simulated gastrointestinal digestion, we performed assays to determine enzymatic inhibitory capacity, bile acid binding capacity, and cholesterol micellar solubilization. Caco-2 cells were used for permeation and protein identification assays. Digested guarana powder extract inhibited pancreatic lipase in a dose-dependent manner (half-maximal inhibitory capacity: 1.033 µg/mL) and, at a concentration of 1 mg/mL, bound 45.63 % of sodium taurocholate and decreased cholesterol micellar solubilization by 10.14 %. Moreover, incubation with the extract reduced cholesterol absorption by Caco-2 cells and decreased intracellular cholesterol transporter levels. These results indicate that guarana seed powder have potential applications for blood cholesterol management, presenting hypocholesterolemic effects owing to the presence of bioacessible polyphenols.

Chitosan-Calcium-Simvastatin Scaffold as an Inductive Cell-Free Platform.

The development of biomaterials based on the combination of biopolymers with bioactive compounds to develop delivery systems capable of modulating dentin regeneration mediated by resident cells is the goal of current biology-based strategies for regenerative dentistry. In this article, the bioactive potential of a simvastatin (SV)-releasing chitosan-calcium-hydroxide (CH-Ca) scaffold was assessed. After the incorporation of SV into CH-Ca, characterization of the scaffold was performed. Dental pulp cells (DPCs) were seeded onto scaffolds for the assessment of cytocompatibility, and odontoblastic differentiation was evaluated in a microenvironment surrounded by dentin. Thereafter, the cell-free scaffold was adapted to dentin discs positioned in artificial pulp chambers in direct contact with a 3-dimensional (3D) culture of DPCs, and the system was sealed to simulate internal pressure at 20 cm/H2O. In vivo experiments with cell-free scaffolds were performed in rats' calvaria defects. Fourier-transform infrared spectroscopy spectra proved incorporation of Ca and SV into the scaffold structure. Ca and SV were released upon immersion in a neutral environment. Viable DPCs were able to spread and proliferate on the scaffold over 14 d. Odontoblastic differentiation occurred in the DPC/scaffold constructs in contact with dentin, in which SV supplementation promoted odontoblastic marker overexpression and enhanced mineralized matrix deposition. The chemoattractant potential of the CH-Ca scaffold was improved by SV, with numerous viable and dentin sialoprotein-positive cells from the 3D culture being observed on its surface. Cells at 3D culture featured increased gene expression of odontoblastic markers in contact with the SV-enriched CH-Ca scaffold. CH-Ca-SV led to intense mineralization in vivo, presenting mineralization foci inside its structure. In conclusion, the CH-Ca-SV scaffold induces differentiation of DPCs into a highly mineralizing phenotype in the presence of dentin, creating a microenvironment capable of attracting pulp cells to its surface and inducing the overexpression of odontoblastic markers in a cell-homing strategy.

Cartilage tissue formation from human adipose-derived stem cells via herbal component (Avocado/soybean unsaponifiables) in scaffold-free culture system.

BACKGROUND: The use of stem cells, growth factors, and scaffolds to repair damaged tissues is a new idea in tissue engineering. The aim of the present study is the investigation of Avocado/soybean (A/S) effects on chondrogenic differentiation of human adipose-derived stem cells (hADSCs) in micromass culture to access cartilage tissue with high quality. MATERIALS AND METHODS: In this an experimental study After hADSCs characterization, chondrogenic differentiation was induced using transforming growth factor beta 1 (TGF-ß1) (10 ng/ml) and different concentrations (5, 10, and 20 µg/ml) of A/S in micromass culture. The efficiency of A/S on specific gene expression (types I, II, and X collagens, SOX9, and aggrecan) was evaluated using quantitative polymerase chain reaction. In addition, histological study was done using hematoxylin and eosin and toluidine blue staining all data were analyzed using one-way analysis of variance (ANOVA) and P ≤ 0.05 was considered to be statistically significant. RESULTS: The results of this study indicated that A/S can promote chondrogenic differentiation in a dose-dependent manner. In particular, 5 ng/ml A/S showed the highest expression of type II collagen, SOX9, and aggrecan which are effective and important markers in chondrogenic differentiation. In addition, the expression of types I and X collagens which are hypertrophic and fibrous factors in chondrogenesis is lower in present of 5 ng/ml A/S compared with TGF-ß1 group (P ≤ 0.05). Moreover, the sulfated glycosaminoglycans in the extracellular matrix and the presence of chondrocytes within lacuna were more prominent in 5 ng/ml A/S group than other groups. CONCLUSION: It can be concluded that A/S similar to TGF-ß1 is able to facilitate the chondrogenic differentiation of hADSCs and do not have adverse effects of TGF-ß1. Thus, TGF-ß1 can be replaced by A/S in the field of tissue engineering.

Innovative preclinical models for pulmonary drug delivery research.

Introduction: Pulmonary drug delivery is a complex field of research combining physics which drive aerosol transport and deposition and biology which underpins efficacy and toxicity of inhaled drugs. A myriad of preclinical methods, ranging from in-silico to in-vitro, ex-vivo and in-vivo, can be implemented.Areas covered: The present review covers in-silico mathematical and computational fluid dynamics modelization of aerosol deposition, cascade impactor technology to estimated drug delivery and deposition, advanced in-vitro cell culture methods and associated aerosol exposure, lung-on-chip technology, ex-vivo modeling, in-vivo inhaled drug delivery, lung imaging, and longitudinal pharmacokinetic analysis.Expert opinion: No single preclinical model can be advocated; all methods are fundamentally complementary and should be implemented based on benefits and drawbacks to answer specific scientific questions. The overall best scientific strategy depends, among others, on the product under investigations, inhalation device design, disease of interest, clinical patient population, previous knowledge. Preclinical testing is not to be separated from clinical evaluation, as small proof-of-concept clinical studies or conversely large-scale clinical big data may inform preclinical testing. The extend of expertise required for such translational research is unlikely to be found in one single laboratory calling for the setup of multinational large-scale research consortiums.

Effect of single and multiple doses of low-level laser therapy on viability and proliferation of stem cells from human exfoliated deciduous teeth (SHED).

The present study aimed to evaluate in vitro whether the low-level laser (LLL) delivering fractionated total energy (multiple irradiation) or single irradiation stimulates regeneration-associated events (viability and proliferation) in stem cells from human exfoliated deciduous teeth (SHED). Cells received LLL irradiation (InGaAlP-660 nm), according to the following experimental groups: G1 (single irradiation 2.5 J/cm2, 10 mW, 10 s, 0.10 J), G2 (single irradiation 5.0 J/cm2, 10 mW, 20 s, 0.20 J), G3 (single irradiation 7.5 J/cm2, 10 mW, 30 s, 0.30 J), G4 (two irradiations 2.5 J/cm2, 10 mW, 10 s; total energy 0.20 J), G5 (three irradiations 2.5 J/cm2, 10 mW, 10 s; total energy 0.30 J), and G6 (non-irradiated). Cell viability was assessed by MTT and trypan blue exclusion (TBE) methods, while cell proliferation was evaluated by crystal violet (CV) and sulforhodamine B (SRB) assays after 24, 48, and 72 h after the first irradiation. By MTT, there was no difference between groups at 24 and 72 h. At 48 h, the groups subjected to multiple irradiation (G4 and G5) presented higher cell viability rates. The average percentages of viable cells for all groups by TBE method were 91.04%, 96.63%, and 97.48% at 24, 48, and 72 h, respectively. By CV, there was no significant difference between groups at 24 and 48 h; at 72 h, G2, G3, and G4 presented higher cell proliferation. By SRB, G1 and G4 presented lower proliferation rates in all the periods. When the groups presenting the same total energy were compared, G2 (0.20 J) presented lower cell viability rates and higher cell proliferation rates in comparison with G4; G3 (0.30 J) presented similar results to those of G5, with higher cell viability and proliferation. The application of laser delivering fractionated total energy (two or three applications of 2.5 J/cm2) induced higher cell viability at 48 h, while the single irradiation with 2.5 J/cm2 did not stimulate metabolic activity in such period and the proliferation over time. The 5.0 and 7.5 J/cm2 single doses and the three applications of 2.5 J/cm2 maintained cell viability and stimulated proliferation of SHED at 72 h.

Laser treatment contributes to maintain membrane integrity in stem cells from human exfoliated deciduous teeth (shed) under nutritional deficit.

This study aimed to analyze the effects of laser irradiation on the membrane integrity and viability of stem cells from human exfoliated deciduous teeth (SHED) that were kept in serum starvation. Nutritional deficit was used to mimic the cellular stress conditions of SHED isolation for regenerative dental approaches, where laser therapy could be beneficial. SHED were cultured under serum starvation (MEMα + 1%FBS) for 1 or 24 h pre-irradiation (protocols A and B, respectively). Then, cells received low-level laser therapy (LLLT; 660 nm) at 2.5 J/cm2 (0.10 W; groups I and V), 5.0 J/cm2 (0.20 W; groups II and VI), 7.5 J/cm2 (0.30 W; groups III and VII), or remained non-irradiated (groups IV and VIII). During irradiation, cells were maintained in 1% FBS (groups I-IV) or 10% FBS (normal culture conditions; groups V-VIII). Membrane integrity was evaluated by quantifying lactate dehydrogenase (LDH) release (immediately after irradiation), and cell viability was assessed by the MTT assay (24, 48, and 72 h post-irradiation). Serum starvation did not alter LDH release by non-irradiated SHED, while LDH release decreased significantly in groups irradiated in 1% FBS (I and III), but not in groups irradiated in 10% FBS (V-VII), regardless the pre-irradiation conditions (protocols A/B). Cell viability was significantly higher 24 h after irradiation, in most protocol A groups. In contrast, cell viability remained mostly unaltered in protocol B groups. LLLT contributed to maintain membrane integrity in SHED subjected to nutritional deficit before and during irradiation with 0.10 or 0.30 W. Short serum starvation before irradiation improved SHED viability at 24 h post-irradiation.

Expressão de DMP-1, DSPP, VEGF e FGF2 em células derivadas da polpa de dentes decíduos após o uso do laser de baixa intensidade / FGF2, VEGF, DSPP, and DMP-1 expression in cells of the pulp deciduous teeth with a low intensity laser

O objetivo do presente trabalho foi avaliar o efeito de diferentes densidades de energia do Laser de Baixa Intensidade na viabilidade e proliferação celular de fibroblastos derivados da polpa de dentes decíduos humanos e na expressão de RNAm para DMP- 1, DSPP, VEGF e FGF-2. Amostras de fibroblastos pulpares da polpa de dentes decíduos humanos foram obtidas de um Biorrepositório. Foram utilizadas células entre a 4ª e a 7ª passagem, irradiadas com Laser de Baixa Intensidade (InGaAlP) de acordo com os seguintes grupos experimentais: Grupo 1: 1,2 J/cm2 - 05 mW - 10s; Grupo 2: 2,5 J/cm2 - 05 mW - 20s; Grupo 3: 3,7 J/cm2 - 05 mW - 30s; Grupo 4: 5,0 J/cm2 - 05 mW - 40s; Grupo 5: 6,2 J/cm2 - 05 mW - 50s; Grupo 6: 2,5 J/cm2 - 10 mW - 10s; Grupo 7: 3,7 J/cm2 - 15 mW - 10s; Grupo 8: 5,0 J/cm2 - 20 mW - 10s; Grupo 9: 6,2 J/cm2 - 25 mW - 10s; Controle Negativo: DMEM 1% SFB ­ não irradiado; Controle Positivo: DMEM 10% SFB ­ não irradiado. As técnicas utilizadas para as análises de viabilidade e proliferação celular foram MTT e CV. A técnica utilizada para avaliação da expressão de RNAm para os alvos DMP-1, DSPP, VEGF e FGF-2 foi RT-PCR. Os resultados foram analisados pelo método ANOVA a dois critérios, seguido pelo teste de Tukey (p<0,05). Para o teste MTT, na comparação intragrupos observou-se que houve diferença estatisticamente significativa entre os períodos 6h, 12h e 24h, diminuindo a viabilidade com o passar do tempo, exceto para o Grupo 1. Na comparação intergrupos, o MTT mostrou menor viabilidade para o controle negativo em comparação com os outros grupos (p<0,05), exceto com grupo 5 (5mW/50 seg). Observou-se que os grupos com maiores potências (10mW, 15mW, 20mW e 25mW), menores tempos de aplicação (10 segundos) e densidades de energia entre 2,5 J/cm2 e 6,2 J/cm2, apresentaram estatisticamente maior viabilidade que o grupo com menor potência (5mW), maior tempo de aplicação (50 segundos) e densidade de energia de 6,2 J/cm2. Para o teste CV não houve diferença intragrupos, mas houve diferença intergrupos entre os controles positivo e negativo. Para a expressão de RNAm por RTPCR, os fatores de crescimento VEGF e FGF-2 foram expressos em grande quantidade no primeiro período experimental, enquanto que DMP-1 e DSPP não foram expressos de maneira significativa. De acordo com os resultados obtidos, frente as diferentes densidades de energia, sugere-se que a terapia a laser de baixa intensidade manteve os fibroblastos viáveis e aumentou a expressão de RNAm para VEGF e FGF-2.(AU)

This study aimed to evaluate the effect of different energy densities of Low Level Laser (LLL) on cell viability and proliferation of fibroblasts from the pulp of human primary teeth (DHPF) and on the RNAm expression of DMP-1, DSPP, VEGF and FGF-2. DHPF were obtained from a biorepository and used at passages 4th to 7th. The cells were irradiated with LLL (InGaAlP) according to the following experimental groups: Group 1: 1.2 J/cm2 - 05 mW - 10s; Group 2: 2.5 J/cm2 - 05 mW - 20s; Group 3: 3.7 J/cm2 - 05 mW - 30s; Group 4: 5.0 J/cm2 - 05 mW - 40s; Group 5: 6.2 J/cm2 - 05 mW - 50s; Group 6: 2.5 J/cm2 - 10 mW - 10s; Group 7: 3,7 J/cm2 - 15 mW - 10s; Group 8: 5.0 J/cm2 - 20 mW - 10s; Group 9: 6.2 J/cm2 - 25 mW - 10s; Negative Control: DMEM 1% SFB ­ not irradiated; Positive Control: DMEM 10% SFB ­ not irradiated. The techniques used to evaluate the cell viability/proliferation were MTT and Crystal Violet (CV) assays. RT-PCR was used to verify the RNAm expression of DMP-1, DSPP, VEGF, and FGF-2. Two-way ANOVA, followed by Tukey test (p<0.05) was used to analyze the results. In the intragroup comparison, MTT assay revealed statistically significant differences among the periods of 6h, 12h, and 24h, with viability reduction as time went by, except for Group 1. In the intergroup comparison, the MTT assay showed that the negative control had statistically lower viability than that of the other groups (p<0.05), except for Group 5 (5mW/50 s). The groups with higher powers (10mW, 15mW, 20mW, and 25mW), shortest application periods (10 s), and energy densities between 2.5 J/cm2 and 6.2 J/cm2 exhibited statistically higher viability than that of the group with small power (5mW), longer application period (50 s), and energy density of 6.2 J/cm2 . CV assay did not show intergroup statistically differences. In the intragroup comparison, CV assay revealed statistically significant differences between positive and negative controls (p<0.05). RT-PCR revealed increased RNAm expression of the growth factors VEGF and FGF-2 at the first experimental period, while DMP-1 and DSPP was not significant. Based on the results and different energy densities used, LLL maintained DHPF viability and increased the RNAm expression of VEGF and FGF-2.(AU)

Influência de diferentes densidades de energia do laser de baixa intensidade em fibroblastos derivados da polpa de dentes decíduos / Influence of low-level laser with different energy densities on pulp fibroblasts from human primary teeth

O objetivo deste trabalho foi comparar os efeitos de diferentes densidades de energia e irradiâncias do Laser de Baixa Intensidade (LBI), variando em função do tempo de irradiação e potência, na viabilidade e proliferação de fibroblastos derivados da polpa de dentes decíduos humanos (HPF). HPF foram cultivados em DMEM e usados entre a 4ª e 8ª passagem. Os grupos foram divididos de acordo com diferentes densidades de energia, variando: Tempo de irradiação - Grupo I Ia (1,2 J/cm2 - 5 mW - 10 s), Ib (2,5 J/cm2 - 5 mW - 20 s), Ic (3,7 J/cm2 - 5 mW - 30 s), Id (5,0 J/cm2 - 5 mW - 40 s), e Ie (6,2 J/cm2 - 5 mW - 50 s); ou potência - Grupo II IIa (1,2 J/cm2 - 5 mW - 10 s), IIb (2,5 J/cm2 - 10 mW - 10 s), IIc (3,7 J/cm2 - 15 mW - 10 s), IId (5,0 J/cm2 - 20 mW - 10 s), e IIe (6,2 J/cm2 - 25 mW - 10 s). Células não irradiadas - cultivadas em condições nutricionais regulares - 10% Soro Fetal Bovino (SFB) (If e IIf) e células não irradiadas - cultivadas em déficit nutricional - 1% SFB (Ig e IIg), foram consideradas controles positivos e negativos, respectivamente. A viabilidade e proliferação celular foram avaliadas, repesctivamente, pelas técnicas MTT e Cristal violeta (CV), nos períodos de 24, 48 e 72 horas após a irradiação. Os dados obtidos foram submetidos à análise estatística por ANOVA 2 critérios, seguido pelo teste de Tukey (P<0,05). No ensaio MTT, os controles negativos, Ig e IIg, apresentaram significativamente menor viabilidade em relação aos correspondentes grupos experimentais: IIa e IIb, 24 horas após a irradiação; Ia, Ib, Ie, If e IIf no período de 48 horas; e Ib-If, assim como, IIa-IIf após 72 horas. Nos diferentes períodos de avaliação do ensaio CV, todos os grupos, exceto Ie, IIe e If, exibiram significativamente maior proliferação em comparação aos respectivos controles negativos. Dentro de um mesmo grupo nos diferentes períodos, os grupos If e IIe apresentaram menor viabilidade durante o período de 24 horas em comparação ao período de 72 horas pelo ensaio MTT. Na avaliação intragrupos, o ensaio CV revelou menor proliferação no período de 24 horas em comparação aos períodos de 48 e 72 horas, independente do grupo avaliado. Os diferentes protocolos de irradiação, grupos I e II, não apresentaram diferença estatisticamente significativa na viabilidade e proliferação celular entre densidades de energia iguais com irradiâncias diferentes durante os períodos avaliados. De acordo com os resultados obtidos, as diferentes densidades de energia e irradiâncias propostas não prejudicaram a viabilidade e proliferação de fibroblastos pulpares de dentes decíduos humanos. A variação do protocolo de irradiação LBI, em função do tempo ou da potência, não interferiram nas respostas celulares após a aplicação da mesma densidade de energia com irradiâncias diferentes.(AU)

The aim of this study was to compare the effects of Low-level laser (LLL) with different energy densities and irradiances, varying according to the irradiation time and power, on cell viability and proliferation of pulp fibloblasts from human primary teeth (HPF). HPF were culture in DMEM and used between 4th and 8th passages. Groups were divided according to different energy densities, varying: Time of irradiation Ia (1.2 J/cm2 - 5 mW - 10 s), Ib (2.5 J/cm2 - 5 mW - 20 s), Ic (3.7 J/cm2 - 5 mW - 30 s), Id (5.0 J/cm2 - 5 mW - 40 s), and Ie (6.2 J/cm2 - 5 mW - 50 s); or output power - Grupo II IIa (1.2 J/cm2 - 5 mW - 10 s), IIb (2.5 J/cm2 - 10 mW - 10 s), IIc (3.7 J/cm2 - 15 mW - 10 s), IId (5.0 J/cm2 - 20 mW - 10 s), e IIe (6.2 J/cm2 - 25 mW - 10 s). Non-irradiated cells - grown in regular nutritional conditions - 10% Fetal Bovine Serum (FSB) (If and IIf) and non-irradiated cells - grown in nutritional deficit - 1% FBS (Ig and IIg) were considered positive and negative controls, respectively. Cell viability and proliferation were respectively assessed through MTT and Crystal violet (CV) assays at 24, 48 and 72h after irradiation. Data were submitted to statistical analysis by ANOVA 2 criteria, followed by Tukey test (P<0.05). In the MTT assay, the negative controls, Ig and IIg, showed significantly lower viability in relation to the corresponding groups: IIa and IIb 24 hours after irradiation; Ia, Ib, Ie, If and IIf at 48 hours period; and Ib-If, as IIa-IIf, after 72 hours. At different periods of evaluation of CV assay, all groups, except Ie, IIe and If, exhibited significantly higher proliferation compared to the respective negative controls. Within the same group at different periods, groups If and IIe showed lower viability during 24 hours compared to 72 hours period by MTT assay. In the intragroup evaluation, CV assay revealed lower proliferation at 24 hours compared to 48 and 72 hours periods, regardless of the evaluated group. Different irradiation protocols, groups I and II, showed no statistically significant differences on cell viability and proliferation among equals energy densities with different irradiances at the evaluated periods. According to these findings, different LLL energy densities and irradiances proposed did not impair viability and proliferation of pulp fibloblasts from human primary teeth. The variation of the LLL irradiation protocol, by the time or power, did not interfere in cellular responses after the application of the same energy density with different irradiances.(AU)

Efeitos dos tratamentos mecânico, químico e fotodinâmico na proliferação de células da granulação óssea humana sobre raízes dentárias / The effects of mechanical, chemical and photodynamic treatment on the proliferation of osseous granulation cells on dental roots

A técnica do enxerto de granulação óssea tem demonstrado bons resultados na recuperação do periodonto e na melhora dos parâmetros clínicos dos dentes com comprometimento periodontal. Pouco se sabe porém, a respeito de qual tipo de tratamento de superfície radicular se faz mais condizente com o emprego dessa técnica. O objetivo dessa pesquisa foi avaliar a proliferação de células de granulação óssea sobre fragmentos radiculares com os seguintes tratamentos de superfície: Controle - somente raspagem, EDTA, terapia fotodinâmica (PDT- laser InGaAIP - 30mW, 30s, 45J/cm², 660nm + azul de toluidina), e ácido cítrico com tetraciclina. Todos os grupos teste receberam previamente tratamento com raspagem e alisamento com 20 golpes de cureta. Células de granulação óssea foram cultivadas em quadruplicata sobre os fragmentos por um período de 24, 48 e 72 horas. Após esse período de cultivo os fragmentos foram fixados para análise em microscópio eletrônico de varredura (MEV). Cinco campos por fragmento foram usados para a visualização e contagem de células aderidas a superfície radicular (centro, campo superior direito e esquerdo e campo inferior direito e esquerdo). A análise da calibração do examinador foi feita através de uma combinação de testes estatísticos como erro casual de Dahlberg, erro sistemático e correlação de Pearson (p<0,05). A análise da amostra foi realizada através do ANOVA de medidas repetidas complementado por Tukey, com nível de significância de 5% (p<0,05). Os resultados demostraram diferenças estatisticamente significantes, quanto ao numero de células, para as superfícies tratadas com terapia fotodinâmica no período de 72 h (p<0,05). Através de nossos resultados concluímos que o tratamento radicular com terapia fotodinâmica favorece a proliferação de células de granulação óssea humanas in vitro.(AU)

The osseous granulation graft has been demonstrating good results on the periodontal healing, resulting the improvement of clinical periodontal parameters. There are very few knowledge about what kind of dental surface would be more proper for the application of this technique. The aim of this study was to evaluate the proliferation of osseous granulation cells on human root fragments treated by different techniques as scaling and root planning (control), citric acid plus tetracycline, EDTA and photodynamic therapy (PDT InGaAIP, 45J/cm², 30mW, 30s, 660nm, toluidine blue O). All test groups were previously treated which 20 curette strikes. Osseous granulation cells was culture in quadruplicate on these fragments for 24h, 48h and 72h. After that, all fragments were fixed and prepared for analysis in Scanning Electron Microscopy (SEM). Aiming to counting the cells adhered on the roots, we obtained electron micrographs of 5 areas (center, upper right and left field, lower right and left field). The examiner calibration was analyzed by Dahlberg Casual Error measurement, systematic error test and Pearson correlation test (p<0.05). Statistical analysis was performed by ANOVA, followed by Tukey test, with a 5% level of significance (p<0.05). There were significant differences in cell number after 72h culture in favor of PDT group (p<0,05). We can conclude that the surface treatment of roots which PDT favor the proliferation of osseous granulation cells in vitro.(AU)

Spermatogonial stem cells as a therapeutic alternative for fertility preservation of prepubertal boys / Espermatogônias-tronco como uma alternativa para a preservação da fertilidade de meninos pré-púberes

ABSTRACT Spermatogonial stem cells, which exist in the testicles since birth, are progenitors cells of male gametes. These cells are critical for the process of spermatogenesis, and not able to produce mature sperm cells before puberty due to their dependency of hormonal stimuli. This characteristic of the reproductive system limits the preservation of fertility only to males who are able to produce an ejaculate. This fact puts some light on the increase in survival rates of childhood cancer over the past decades because of improvements in the diagnosis and effective treatment in pediatric cancer patients. Therefore, we highlight one of the most important challenges concerning male fertility preservation that is the toxic effect of cancer therapy on reproductive function, especially the spermatogenesis. Currently, the experimental alternative for fertility preservation of prepubertal boys is the testicular tissue cryopreservationfor, for future isolation and spermatogonial stem cells transplantation, in order to restore the spermatogenesis. We present a brief review on isolation, characterization and culture conditions for the in vitro proliferation of spermatogonial stem cells, as well as the future perspectives as an alternative for fertility preservation in prepubertal boys. The possibility of restoring male fertility constitutes a research tool with an huge potential in basic and applied science. The development of these techniques may be a hope for the future of fertility preservation in cases that no other options exist, e.g, pediatric cancer patients.

RESUMO As espermatogônias-tronco, presentes nos testículos desde o nascimento, são as células progenitoras dos gametas masculinos, e, desse modo, críticas para o processo de espermatogênese. Antes da puberdade, essas células não são capazes de produzir espermatozoides maduros, o que só ocorrerá após o estímulo hormonal. Essa característica do sistema reprodutivo limita a possibilidade de preservação da fertilidade apenas para homens capazes de produzir um ejaculado. Tal fato coloca em evidência o aumento nas taxas de sobrevivência de crianças com câncer nas últimas décadas, devido principalmente à melhora no diagnóstico e ao tratamento dos pacientes pediátricos. Dessa forma, destaca-se um dos mais importantes desafios relativos à preservação da fertilidade masculina, que é o efeito tóxico das terapias anticâncer para o sistema reprodutivo, especialmente a espermatogênese. Tendo isso em vista, a alternativa experimental atualmente estudada para a preservação da fertilidade de pacientes pré-púberes é a criopreservação de tecido testicular para futuro isolamento e transplante de espermatogônias-tronco, a fim de restabelecer a espermatogênese. Apresentamos aqui uma breve revisão sobre isolamento, caracterização e condições de cultivo para a proliferação de espermatogônias-tronco, bem como as futuras perspectivas, como alternativa para preservação da fertilidade de meninos pré-púberes. A possibilidade de restabelecer a fertilidade masculina é uma ferramenta de pesquisa com potencial enorme de uso na pesquisa básica e aplicada. O desenvolvimento dessas técnicas pode fornecer uma esperança futura de preservação de fertilidade nos casos em que não há nenhuma outra opção, como para os pacientes pediátricos de câncer.

HCV culture system and its application in antiviral research on Chinese herb extracts in vitro / 临床肝胆病杂志

Many studies have shown that Chinese herb extracts could treat patients who suffer hepatitis C. However, the clinical efficacy is limited due to the use of traditional Chinese medicine (TCM) compounds. Because each drug contains multiple active substances, some of which may even act against each other in vivo, it is hard to clarify the mechanism of treatment. The current research on pseudotyped hepatitis C virus (HCV), replicon, and cell culture system is summarized, and the advances in antiviral research on the monomers or active components of Chinese herbs in vitro based on HCV RNA models are reviewed, thus providing new ideas for TCM treatment of hepatitis C.

Research advances in cell models of hepatitis B virus / 临床肝胆病杂志

Hepatitis B virus (HBV) infection has become the main cause of hepatocellular carcinoma. A series of cell models for HBV infection are established to lay a good foundation for the research on the pathogenesis and treatment of HBV. In this review, we summarize culture models for a single type of cells, co-culture models for multiple types of cells, and other animal models, which are mainly for HBV transfection and infection. We also discuss the methods of cell model construction for HBV infection and their virological characteristics. It provides reliable evidence of models for scientific interpretation of advantages of traditional Chinese medicine in the prevention and treatment of chronic hepatitis B.

Efeito do composto natural Yo Jyo Hen Shi Ko (YHK) no ciclo de replicação do vírus da hepatite C (VHC) / Effects of the natural compound Yo Jyo Hen Shi Ko (YHK) on the replication cycle of the hepatitis C virus

Estima-se que 170 milhões de pessoas no mundo estejam infectadas com o vírus da hepatite C (VHC), o que está altamente relacionado à ocorrência de hepatite crônica e carcinoma hepatocelular. A prevalência de esteatose hepática em doentes com hepatite C crônica é muito maior do que na população geral variando entre 40 a 75%. A associação entre a infecção pelo VHC e esteatose hepática é multifatorial. Duas formas de esteatose hepática são encontradas em pacientes com hepatite C crônica: esteatose metabólica (fatores de risco) e citopática relacionada ao genótipo 3a. Os lipídios são essenciais para o ciclo de replicação do VHC, eles podem exercer seu efeito em diferentes níveis como: grupos prostéticos em proteínas virais e/ou cofatores celulares na replicação de VHC, componentes especializados na estrutura do VHC onde ocorre a replicação ou como constituinte das partículas lipovirais. Trabalhos experimentais realizados anteriormente por nosso grupo relataram que a administração do composto natural Yo Jyo Hen Shi Ko (YHK) promove a inibição do desenvolvimento da esteatose, redução dos marcadores de estresse oxidativo, menor escore de inflamação, melhora nas concentrações de aminotransferases e diminuição da gordura visceral em um modelo animal de esteato-hepatite não alcoólica. A terapia padrão da hepatite C consiste em uma combinação de interferon peguilado alfa (PEG-IFN-alfa) que estimula o sistema imunológico do hospedeiro para combater a infecção e o composto antiviral ribavirina. Atualmente foram aprovados pelas agências de saúde os inibidores de protease Boceprevir, Telaprevir, Daclatasvir e Simeprevir. No entanto, sua eficiência varia entre os genótipos e as constantes mutações do vírus podem levar a resistência. A falta de uma vacina ou uma terapia definitiva faz com que diversos compostos com diferentes mecanismos de ação sejam testados como possíveis alternativas de tratamento. Tendo em vista a capacidade do YHK de reduzir a esteatose e a importância...

Worldwide is estimated that nearly 170 million people are infected with hepatitis C virus (HCV), highly correlated with the occurrence of chronic hepatitis and hepatocellular carcinoma. Hepatitis C patients present higher prevalence of steatosis when compared with the general population, ranging between 40% and 75%. There are two forms of steatosis in HCV infected patients: metabolic steatosis (risk factors) and cytopathic associated with genotype 3. Lipids are essential for the HCV replication cycle. It acts on different functions: as prosthetic groups into viral proteins and / or cellular cofactors in the HCV replication, as specific HCV components or as a constituent of lipovirals particles. Our group previously reported that the administration of the natural compound Yo Jyo Hen Shi Ko (YHK) inhibits steatosis development, decreases markers of oxidative stress and inflammation, improves aminotransferases concentration and decreases the visceral fat. Standard therapy for hepatitis C is a combination of pegylated interferon alpha (PEG-IFN-alfa), stimulating the host immune system to fight infection and the antiviral compound named ribavirin. Nowadays, Telaprevir, Boceprevir, Sofosbovir and Simeprevir are approved as new anti-HCV drugs; they act as protease inhibitors. Its efficiency, however, varies between genotypes, and the constant mutations of the virus can lead to resistance. The lack of vaccines, or a definitive therapy, stimulates the research of new compounds and alternative treatments. In this study, we evaluated the effect of YHK in HCV replication cycle due to the effect of YHK and the importance of lipid metabolism for HCV. For this purpose we used cell culture techniques allowing the study of different stages of HCV replication cycle: entry (HCVpp), replication - replicons JFH1 NS3-5B and Con1, also replication and infection-JC1-Fluc. We also used active compounds of its ingredients: Panax pseudo ginseng - Notoginsenoside R1...

Resposta de células odontoblastóides MDPC-23 irradiadas com LED de 630nm / Response of odontoblast-like cells to 630 nm LED irradiation

Na biofotônica, lasers e LEDs (light-emitting diodes) têm sido empregados na bioestimulação de células e tecidos. LED é um diodo semicondutor que, quando energizado, produz luz de espectro estreito, em forma de eletroluminescência. Experimentos in vitro utilizando LEDs com diferentes comprimentos de onda demonstraram a ocorrência de significativo estímulo no crescimento celular, efeito antiinflamatório e antimicrobiano, além do metabolismo celular aumentado. Na odontologia, a aplicação clínica de lasers e LEDs em terapias objetivando a redução da hipersensibilidade dentinária tem se mostrado efetiva, através de aparente síntese e deposição de dentina reacional. Entretanto, não há trabalhos na literatura que demonstrem o efeito do LED sobre a cultura de odontoblastos, tampouco dados científicos caracterizando a relação entre LED e redução da hipersensibilidade dentinária. Assim, o objetivo desta pesquisa foi investigar a ação do LED em 630 nm sobre o metabolismo de células de linhagem odontoblástica MDPC-23. Para isto, as células foram descongeladas, cultivadas e plaqueadas. Então, o LED foi aplicado diretamente sobre estas células, em diferentes tempos (20, 40, 80 e 240'') e condições de estresse (2 ou 10% de SFB), de acordo com cada grupo experimental, por três dias consecutivos, através de um dispositivo de irradiação denominado "LEDTable". Posteriormente, foram avaliados a viabilidade celular, através do teste MTT, e a morfologia celular, por microscopia eletrônica de varredura. Os dados obtidos nos testes de MTT foram submetidos ao teste de Mann-Whitney para a comparação das concentrações de soro fetal bovino em cada dose de energia individualmente. Foi utilizado também o teste de Kruskal-Wallis para comparar as diferentes doses de energia em cada concentração de soro fetal bovino. Os dados foram analisados estatisticamente em nível de significância pré-determinado de 5%. Numa avaliação comparativa entre os grupos foi possível demonstrar que células MDPC-23 mantidas em condições normais são bioetimuladas através de sua irradiação com baixa dose de LED, tal como 1 J/cm2. Porém, em condições de estresse por restrição de soro fetal bovino (2% SFB), as células MDPC-23 apenas são bioestimuladasa através de doses mais elevadas de irradiação com LED, tal como com 4 J/cm2. Por outro lado, a aplicação de uma dose de 8 J/cm2 não influencia o metabolismo desta linhagem de célula odontoblastóide. A análise de MEV demonstrou um maior número de células aderidas ao substrato após irradiação com 4 J/cm2. De acordo com as condições experimentais deste estudo in vitro, foi possível concluir, tal como anteriormente demonstrado para outras linhagens celulares, que a terapia com LED no comprimento de onda em torno de 630 nm sob fluência de 4 J/cm2 foi efetiva no processo de bioestimulação das células odontoblastóides MDPC-23

Lasers and LEDs (light-emitting diodes) have been used for biostimulation of cells and tissues. LED is a semiconductor diode which produces limited spectrum visible light. In vitro experiments using LEDs at different wavelengths have shown an enhancement of cell growth, anti-inflammatory and antibacterial effects and increased cell metabolism. In dentistry, the use of lasers and LEDs in therapies to reduce dental hypersensitivity has been proved to be clinically effective, through the synthesis and deposition of reactionary dentin. However, there are no studies that demonstrate the effect of LED therapy on odontoblast-like cells and there is no scientific data linking LED irradiation to dental hypersensitivity reduction. For this reason, the aim of this study was to investigate the effect of LED 630 nm irradiation on MDPC-23 (odontoblast-like) cells metabolism. Cells were seeded on 24-wells plates and cultured. Then the LED light was directly applied to these cells under different experimental conditions (time and % of BFS), according to each experimental group, for three following days. A device named LEDTable provided red LED irradiation. Then, cell viability (MTT Assay) and cell morphology (SEM) were evaluated. The cell viability results were first submitted to Mann-Whitney tests in order to compare the fetal bovine serum concentrations and energy dose, and then Kruskal-Wallis test was performed to compare different energy doses in every serum concentration. Data were statistically analyzed (p=0,05). Results show a biostimulation of cells kept under normal culture conditions and submitted to low LED irradiation dose (1 J/cm2). However, under nutritional stress, cells required higher energy dose to be stimulated, such as 4 J/cm2. On the other hand, a 8 J/cm2 dose did not affect the metabolism of this immortalized cell line. The SEM analysis showed a higher number of cells attached to the glass substrate after 4 J/cm2 dose irradiation. According to the experimental conditions, it was possible to conclude that LED therapy under fluence of 4 J/cm2 biostimulates MDPC-23 cells, such as previously demonstrated by other studies in which different cell lines were submitted to similar LED irradiation