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.

Collection and culture of human connective tissue cells from gingival explant technique for oral tissue bioengineering / Colección de células de tejido conectivo humano a partir de la técnica de explante gingival para bioingeniería de tejidos orales

SUMMARY: Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. Gingival fibroblasts present structural function, being able to modulate their metabolic capacity, which is reflected in the tissue morphology. The possibility of culturing fibroblasts in vitro, in monolayer or on three-dimensional scaffolds, for subsequent transplants in vivo opens important perspectives for the periodontal surgical clinic. The objective of the present article is to present a method of obtaining and cultivating viable human gingival fibroblasts for in vitro research. Explants derived from periodontal surgical discards were used, grown in 25 cm2 bottles to obtain a primary cell culture. After observing the proliferation and growth of the fibroblasts that interconnected and formed a monolayer network, involving the periphery of the explants, it was possible to remove the explants, to make the passage and the new subcultures were obtained in a ratio of 1:1. After 7 days, the amount of viable cells was analyzed in triplicate, using the Neubauer chamber technique, in cell culture bottles of 25 mm2 (T25) and 75 mm2 (T75). Fibroblasts were described and subclassified morphologically. The results showed a growth pattern in both bottles, but with a larger number in bottles of 75 cm2. Cells with fibroblastic morphology were subclassified into reticular and fusiform, being predominant those with fusiform morphology. In conclusion, culture of explant of human gingival connective tissue is a viable method for obtaining gingival connective tissue cells suitable for laboratory tests in cell culture, aiming at obtaining constructs for gingival tissue engineering.

RESUMEN: El cultivo celular es una herramienta importante en los laboratorios de investigación médica, odontológica y biológica, que apoyan las terapias celulares y las estrategias de bioingeniería de tejidos. Los fibroblastos gingivales presentan una función estructural, pudiendo modular su capacidad metabólica, que se refleja en la morfología tisular. La posibilidad de cultivar fibroblastos in vitro, en monocapa o en andamios tridimensionales, para trasplantes posteriores in vivo abre perspectivas importantes para la clínica de cirugía periodontal. El objetivo del presente artículo es presentar un método para obtener y cultivar fibroblastos gingivales humanos viables para investigación in vitro. Se utilizaron explantes derivados de los descartes quirúrgicos periodontales, crecidos en frascos de 25 cm2 para obtener un cultivo de células primarias. Después de observar la proliferación y el crecimiento de los fibroblastos que se interconectaron y formaron una red de monocapa, que involucraba la periferia de los explantes, fue posible eliminar los explantes, hacer el pasaje y los nuevos subcultivos se obtuvieron en una proporción de 1:1. Después de 7 días, la cantidad de células viables se analizó por triplicado, utilizando la técnica de cámara de Neubauer, en botellas de cultivo celular de 25 mm2 (T25) y 75 mm2 (T75). Los fibroblastos fueron descritos y sub-clasificados morfológicamente. Los resultados mostraron un patrón de crecimiento en ambas botellas, pero con un número mayor en botellas de 75 cm2. Las células con morfología fibroblástica se subclasificaron en reticulares y fusiformes, predominando aquellas con morfología fusiforme. En conclusión, el cultivo de explante de tejido conectivo gingival humano es un método viable para obtener células de tejido conectivo gingival adecuadas para pruebas de laboratorio en cultivos celulares, con el objetivo de obtener construcciones para la ingeniería del tejido gingival.

Leucocyte Platelet Rich Fibrin with Autologous Gingival Fibroblasts in the Treatment of Adjacent Recession Defects / Fibrina Rica en Plaquetas Leucocitarias con Fibroblastos Gingivales Autólogos en el Tratamiento de Defectos de Recesión Adyacentes

ABSTRACT: Periodontium can submit changes that lead to loss of integrity, such as periodontal disease, immune disorders or traumatic brushing. One of the most common consequences resulting from these events is the apical migration of gingival marginal tissue. Among biomaterials used for periodontal tissue regeneration, fibrin matrices have received significant attention to correct gingival recessions. Five oral mucosa biopsies were extracted, fibroblasts were in vitro cultured and frozen in liquid nitrogen. Three 10 mL glass sterile tubes were filled with patient blood and centrifuged immediately; clots were extracted and compressed to obtain L-PRF membranes. Autologous oral mucosa fibroblasts were added to the membranes and surgical procedures were performed in five patients. L-PRF fibrin network pore size was too small to allow human fibroblasts penetration but they were firmly attached to membrane surface. Gingival fibroblasts from fresh cell culture and recently thawed were used to attach on the L-PRF membranes. It was possible to establish a protocol for blood collection, centrifugation, fibrin clot compression, fibroblast adhesion to the membrane surface and patient application in a relatively short time (1 hour-1 hour and 30 minutes). Two patients expressed pain symptoms and the other ones presented light swelling without pain. In the first week, adjacent tissue showed few inflammation signs. Research efforts are being conducted to develop more conservative surgical techniques and new biomaterials that can promote cellular proliferation. Because of its properties, L-PRF membranes represent a tempting alternative. A combined technique to treat adjacent recession defects with L-PRF membranes and autologous oral mucosa fibroblasts in a coronal displaced flap did not show initial advantage compared with a gold standard surgery that includes an autologous soft tissue graft. Nevertheless, it could be an alternative for clinical application as a new functional cell biomaterial. More clinical evidence is needed.

RESUMEN: El periodonto puede presentar cambios que conducen a la pérdida de integridad, como la enfermedad periodontal, los trastornos inmunes o el cepillado traumático. Una de las consecuencias más comunes que resultan de estos eventos es la migración apical del tejido marginal gingival. Entre los biomateriales utilizados para la regeneración del tejido periodontal, las matrices de fibrina han recibido una atención significativa para corregir las recesiones gingivales. Se extrajeron cinco biopsias de mucosa oral, los fibroblastos se cultivaron in vitro y se congelaron en nitrógeno líquido. Tres tubos de vidrio estériles de 10 ml se llenaron con sangre del paciente y se centrifugaron inmediatamente. Los coágulos fueron extraídos y comprimidos para obtener membranas de L-PRF. Se agregaron fibroblastos autólogos de mucosa oral a las membranas y se realizaron procedimientos quirúrgicos en cinco pacientes. El tamaño de poro de la red de fibrina L-PRF era demasiado pequeño para permitir la penetración de los fibroblastos humanos, pero estaban firmemente unidos a la superficie de la membrana. Se usaron fibroblastos gingivales de cultivos de células frescas y recientemente descongelados para unirlos a las membranas L-PRF. Fue posible establecer un protocolo para la extracción de sangre, centrifugación, compresión de coágulos de fibrina, adhesión de fibroblastos a la superficie de la membrana y aplicación al paciente en un tiempo relativamente corto (1 hora, 1 hora y 30 minutos). Dos pacientes expresaron síntomas de dolor y los otros presentaron hinchazón leve sin dolor. En la primera semana, el tejido adyacente mostró pocos signos de inflamación. Se están realizando esfuerzos de investigación para desarrollar técnicas quirúrgicas más conservadoras y nuevos biomateriales que puedan promover la proliferación celular. Debido a sus propiedades, las membranas L-PRF representan una alternativa tentadora. Una técnica combinada para tratar los defectos de recesión adyacentes con membranas de L-PRF y fibroblastos de mucosa oral autóloga en un colgajo coronal desplazado no mostró una ventaja inicial en comparación con una cirugía estándar que incluye un injerto de tejido blando autólogo. Sin embargo, podría ser una alternativa para la aplicación clínica como un nuevo biomaterial de células funcionales. Se necesita más evidencia clínica.

Effects of cyclosporin, nifedipine and phenytoin on gingival myofibroblast transdifferentiation in monkeys

Abstract Objective: Myofibroblasts have been associated with the development of several pathologic fibrotic conditions. This longitudinal study aims to assess the proliferative and antiapoptotic effects of cyclosporin, nifedipine and phenytoin on gingival connective tissue cells of nonhuman primate, as well as to analyze a possible role of myofibroblasts in gingival overgrowth. Materials and Methods: Gingival samples from the right superior canine area were obtained from 12 male monkeys ( Sapajus spp ) to comprise the control group. After one week, the animals were randomly assigned to three groups, which received daily oral doses of cyclosporin, nifedipine or phenytoin for 120 days. Gingival samples were collected from the left superior canine area of two animals of each group at 52 and 120 days. Histological sections were stained with hematoxylin and eosin, and immunoreacted against α-SMA, Ki- 67 and bcl-2. Results: α-SMA immunoreaction was negative in the control and experimental groups. Similarly, no difference between groups concerning immunostaining against Ki-67 and bcl-2 was observed in connective tissue cells. Conclusion: Based on this methodology, it may be concluded that gingival overgrowths induced by cyclosporin, nifedipine and phenytoin are not associated with neither myofibroblast transdifferentiation, proliferation nor apoptosis of gingival connective cells in monkeys.

Effect of titanium tetrafluoride varnish on fibroblasts (NIH/3T3): viability assay, morphological analysis and cell signals for apoptotic pathways / Efeito do verniz de tetrafluoreto de titânio sobre fibroblastos (NIH/3T3): ensaios de viabilidade, morfologia e sinalização celular para apoptose

Current knowledge supports the application of TiF4 varnish to protect against tooth caries and erosion; however, it is indispensable to know its cytotoxic potential and the mechanism involved on it before applying in patients. Therefore, this study aimed to evaluate 1) The cytotoxic effect of titanium tetrafluoride (TiF4) varnish compared with sodium fluoride (NaF) varnish on murine fibroblast (NIH/3T3), varying the fluoride concentration and time of treatment and 2) The percentage of apoptosis and its mechanism (both mitochondrial mediated by the Bcl-2 family- and death receptorpathways) in human gingival fibroblasts (HGF) and murine fibroblasts (NIH/3T3) treated with TiF4 varnish compared to NaF varnish for 6 h. Step 1) NIH/3T3 were exposed to NaF or TiF4 varnishes containing 0.95, 1.95 or 2.45% F, for 6, 12 or 24 h. MTT viability (n=6) and Hoescht/PI stain assays (n=3) as well as the cells morphology (HE, only for 24 h, n=3) and stiffness (AFM, only for 2.45% F, 6 or 12 h) were analyzed. Both varnishes, at 1.90 and 2.45% F, reduced cells viability by similar extent (33-86% at 6 h, 35-93% at 12 h, and 87-98% at 24 h) compared to control, regardless of the type of fluoride. TiF4 and NaF (2.45% F) reduced cell stiffness to a similar extent, but only TiF4 differed from control. Step 2) HGF and NIH/3T3 were exposed to NaF or TiF4 (2.45% F) varnishes for 6 h. Cells were examined by the TUNEL method using fluorescence microscope. The caspases-3, -8 and -9 activities were assessed. The cDNA for cytocrome c, Bax, Bad, Bcl-2, VDAC-1 and Fas-L was amplified by quantitative PCR (qPCR). Bax, Bcl-2 and Fas-L were further detected by western blot. Both fluorides similarly increased the percentage of apoptosis, while they failed in activating caspases-3, -8 and -9 for both types of cells. Bax/Bcl-2 ratio, cytochrome C and VDAC-1 gene expressions were not altered by both fluoride treatments. However, NaF varnish increased the amplification of Fas-L gene for NIH/3T3 and HGF, while TiF4 varnish induced lower Bad/Bcl-2 ratio expression compared to control for NIH/3T3, but not for HGF. No effect of the fluorides was detected in the proteins analysis. TiF4 and NaF have similar cytotoxicity on NIH/3T3, which is dependent on the F concentration and the exposure time. Both fluorides, at the studied conditions, similarly induce a low percentage of apoptosis, with consequent modest activation of Bcl-2 and Fas-L-dependent signaling pathways.(AU)

Conhecimento atual suporta a aplicação de verniz de TiF4 para proteção contra cárie e erosão dentárias; entretanto, é indispensável conhecer o seu potencial citotóxico e o mecanismo envolvido antes de aplicá-lo em pacientes. Portanto, o objetivo deste estudo foi avaliar 1) o efeito citotóxico do verniz de tetrafluoreto de Titânio (TiF4) comparado ao fluoreto de sódio (NaF), em fibroblastos NIH/3T3, variando a concentração de fluoreto e o tempo de tratamento 2) a porcentagem de apoptose e seus mecanismos (ambos mitocondrial mediado pela família Bcl-2 e pelo receptor de morte celular) em fibroblastos gengivais humanos (FGH) e fibroblastos murinos (NIH/3T3) tratados com verniz de TiF4 comparado com verniz de NaF por 6 h. Etapa 1) NIH/3T3 foram expostos a vernizes de NaF e TiF4 contendo 0,95, 1,95 ou 2,45% F, por 6, 12 ou 24 h. Ensaios de viabilidade por MTT (n=6) e Hoechst 33342/iodeto de propídeo (n=3) bem como a morfologia (HE, apenas para 24 h, n = 3) e a rigidez celular (MFA, apenas para 2,45% F, 6 ou 12 h) foram realizados. Ambos os vernizes com 1,90 e 2,45% F reduziram a viabilidade das células de forma semelhante (33-86% em 6 h, 35-93% em 12 h e 87-98% em 24 h) em comparação com o controle, independentemente do tipo de fluoreto. TiF4 e NaF (2,45%) reduziram de forma similar a rigidez celular, mas somente TiF4 diferiu do controle no período de 6 h. Etapa 2) FGH e NIH/3T3 foram tratadas com verniz de NaF ou TiF4 por 6h. As células foram examinadas pelo método de TUNEL, usando microscopia de fluorescência. A atividade das caspases -3, -8 e -9 foram avaliadas. O cDNA para citocromo C, Bax, Bad, Bcl-2, VDAC-1 e Fas-L foi amplificado e quantificado por PCR em tempo real (qPCR). A expressão das proteínas Bax, Bcl-2 e Fas-L foi quantificada por western blot. Ambos os fluoretos aumentaram de forma semelhante a porcentagem de apoptose, enquanto falharam na ativação de caspases-3, -8 e -9 para ambos tipos celulares. A expressão gênica da relação Bax/Bcl-2, do citocromo C e do VDAC-1 não foram alteradas por ambos fluoretos. No entanto, o verniz NaF aumentou a amplificação do gene Fas-L para ambas as células, enquanto que o verniz TiF4 induziu menor expressão da razão Bad/Bcl-2 em comparação com o controle para NIH/3T3, mas não para FGH. Nenhum efeito foi detectado na análise de proteínas. TiF4 e NaF apresentam citotoxicidade similar em NIH/3T3, a qual é dependente da concentração de F e do tempo de exposição. Ambos os fluoretos, nas condições estudadas, induzem uma baixa porcentagem de apoptose, com consequente modesta ativação das vias de sinalização dependentes de Bcl-2 e Fas-L.(AU)

Assessment of toxicity and oxidative DNA damage of sodium hypochlorite, chitosan and propolis on fibroblast cells

Abstract The objective of this study was to evaluate and compare the cytotoxicity and genotoxicity on human fibroblast cell lines of sodium hypochlorite (NaOCl), chitosan and propolis as root canal irrigating solutions. Human fibroblast cells were exposed to chitosan, propolis and NaOCl for 4 and 24 h. Cell viability was assessed by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, and oxidative DNA damage was assessed by determination of 8-hydroxydeoxyguanosine (8-OHdG) level with an ELISA kit. The data of cell cytotoxicity were analysed statistically using a test of one-way analysis of variance at a significance level of p < 0.05. In the NaOCI group, the 8-OHdG level was higher than in the chitosan group, but there was no statistical difference when compared with the other groups (p < 0.05). It was determined that the irrigation solutions were cytotoxic, depending on the dose and time. NaOCl was the most toxic solution after both 4 and 24 h of exposure (p < 0.05). Chitosan and propolis may be alternatives to NaOCl for irrigation solutions, because they are both less toxic and produce less oxidative DNA damage.

Effects of platelet-rich plasma on human gingival fibroblast proliferation and migration in vitro

Abstract Objective This study evaluated the influence of platelet-rich plasma (PRP) on the behaviour of human gingival fibroblasts (hGFs), including fibroblast proliferation, migration and colony formation. Methods PRP was obtained from the human peripheral blood of a healthy volunteer and then was diluted into platelet concentrations of 1%, 2% and 5%. The proliferation of hGFs was determined by two methods: (1) Cell-number counting with a haemocytometer method at days 1, 3, 5 and 7; (2) Colony-forming unit-fibroblast (CFU-F) assay at 2 weeks. The migration of hGFs was evaluated with scratch assay, then recorded digital images were analysed by Image-Analysis J 1.51j8 software to compare the remaining artificial wound areas between PRP groups at 0, 24 and 48 hours. Results All hGFs that were cultivated in media with 1%, 2% and 5% PRP showed their ability to proliferate and migrate. Cell numbers incubated with 1% PRP increased significantly during the first three days and peaked at day 5, tending to be similar to their proliferation in complete medium. With concentrations of 2% and 5% PRP, hGFs outgrew and peaked at day 3, which was faster than with those in medium with 1% PRP. Especially, hGFs in the group 5% PRP proliferated with higher cell numbers than those in the other remaining groups at day 3. The hGF colony number that was formed in the group 5% PRP was significantly higher than those in the groups 1% and 2% PRP. Scratch assay showed hGFs in the groups 2% and 5% PRP almost filled the artificial wound and migrated more effectively than in the group 1% PRP at 24 hours, which was significant. Conclusion In this study, perhaps the medium with 5% PRP is the dominant option, promoting the abilities of hGFs to heal wounds, because of its fast and effective impact on cell proliferation, colony formation and migration.

Efficacy of relacin combined with sodium hypochlorite against Enterococcus faecalis biofilms

Abstract Objective Relacin is a synthetic molecule that targets RelA, an essential protein in a conserved bacterial stress response system. It was shown to inhibit bacterial growth. The aims of this study were to evaluate the antimicrobial effect of relacin combined with sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms and to evaluate the cytotoxicity of relacin. Material and Methods 48-h E. faecalis OG1RF biofilms were treated by various concentrations of relacin in order to determine its inhibitory concentration. Then, the 48-h biofilms were treated either with 1-min NaOCl (0.01%, 0.05%) alone, or in combination of relacin. As a means of comparison, the biofilms of ΔrelA were also treated by 1-min NaOCl (0.01%, 0.05%, 0.25%). The treatment efficacy was determined by agar plate count assays. The cytotoxicity of relacin was examined on human gingival epithelial cells Ca9-22 and murine fibroblasts NIH-3T3 by a methyl thiazolyltetrazolium (MTT) assay and a lactate dehydrogenase assay. Statistical analysis was performed by one-way or two-way analysis of variance (ANOVA) with Bonferroni's post-hoc test and an independent Student's t-test. A significance level of p<0.05 was used. Results Relacin inhibited the growth of OG1RF biofilms partially at 8 mM and fully at 14 mM. The relacin (14 mM) and NaOCl combined treatment resulted in significantly higher treatment efficacy than NaOCl treatment alone. At 0.05% NaOCl, the combined treatment resulted in 5.65 (±0.19) log reduction in biofilm viability. The ΔrelA biofilms were more susceptible to NaOCl treatment than the wild type biofilms at 0.25% NaOCl. Relacin at 14 mM was not toxic to host epithelial cells and fibroblasts. Conclusions The combination of relacin with a low concentration of NaOCl was effective and not cytotoxic.

Comparison of primary human gingival fibroblasts from an older and a young donor on the evaluation of cytotoxicity of denture adhesives

Abstract Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. Objective To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). Material and Methods Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. Results While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. Conclusions All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.

Cytotoxicity Evaluation of Two Bis-Acryl Composite Resins Using Human Gingival Fibroblasts

Abstract Bis-acryl resins are used for temporary dental restorations and have shown advantages over other materials. The aim of this work was to evaluate the in vitro cytotoxicity of two bis-acryl composite resins (Protemp 4 and Luxatemp Star), obtained at 1, 7 and 40 days after mixing the resin components, using a standardized assay employing human primary cells closely related to oral tissues. Human gingival fibroblast cell cultures were exposed for 24 h to either bis-acryl composite resins, polystyrene beads (negative control) and latex (positive control) extracts obtained after incubation by the different periods, at 37 °C under 5% CO2. Cell viability was evaluated using a multiparametric procedure involving sequential assessment (using the same cells) of mitochondrial activity (XTT assay), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). The cells exposed to the resin extracts showed cell viability indexes exceeding 75% after 24 h. Even when cells were exposed to extracts prepared with longer conditioning times, the bis-acryl composite resins showed no significant cytotoxic effects (p>0.05), compared to the control group or in relation to the first 24 h of contact with the products. There were no differences among the results obtained for the bis-acryl composite resins evaluated 24 h, 7 days and 40 days after mixing. It may be concluded that the bis-acryl resins Protemp 4 and Luxatemp Star were cytocompatible with human gingival fibroblasts, suggesting that both materials are suitable for use in contact with human tissues.

Resumo Resinas bisacrílicas são usadas em restaurações dentárias provisórias e têm mostrado vantagens em relação a outros materiais. O objetivo deste trabalho foi avaliar a citotoxicidade in vitro de duas resinas compostas bisacrílicas (Protemp 4 e Luxatemp Star), obtidas após 1, 7 e 40 dias da mistura com os componentes da resina, usando um ensaio padronizado empregando células primárias humanas fortemente relacionadas aos tecidos orais. Culturas de células de fibroblastos gengivais humanos foram expostas por 24 h aos extratos das resinas bisacrílicas, esferas de poliestireno (controle negativo) e látex (controle positivo), obtidos após diferentes períodos de incubação, a 37 °C e com 5% CO2. A viabilidade celular foi avaliada usando procedimentos multiparamétricos que envolvem a avaliação sequencial (usando as mesmas células) da atividade mitocondrial (ensaio XTT), a integridade de membrana (teste do vermelho neutro) e a densidade celular total (teste de exclusão do corante cristal violeta). As células expostas aos extratos mostraram viabilidade celular acima de 75% depois de 24 h. Mesmo quando as células foram expostas aos extratos com aumento do tempo de condicionamento, as resinas bisacrílicas não apresentaram efeitos citotóxicos significativos (p<0,05), comparadas ao grupo controle ou em relação às primeiras 24 h de contato com os produtos. Não houve diferença entre os resultados obtidos para as resinas bisacrílicas avaliadas entre as 24 h, 7 e 40 dias depois da mistura. Concluímos que as resinas bisacrílicas Protemp 4 e Luxatemp Star foram citocompatíveis com os fibroblastos gengivais humanos, sugerindo que ambos materiais são adequados para uso em contato com tecidos humanos.

Functional Differences In Gingival Fibroblasts Obtained from Young and Elderly Individuals

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

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

MatrigelBD, a biocompatible scaffold that improves gingival mesenchymal stem cells proliferation / MatrigelBD, un andamiaje biocompatible que estimula la proliferación de celulas troncales mesenquimales derivadas del tejido gingival

MatrigelBD is a hydrogel scaffold with three-dimensional intercrossed networks of hydrophilic polymers with high water content. Human gingival tissue might represent a better source of MSCs, allowing these cells to be easily obtained in a relatively non-invasive way. The objective of this study was to evaluate the biocompatibility of MatrigelBD with GMSCs in vitro. Gingival connective tissue samples were obtained from healthy donors. Fresh tissue was minced and cultured during two weeks, after which cells at passage fourth were analyzed for their immune phenotype by flow cytometry. Differentiation into osteogenic, chondrogenic, and adipogenic lineages was induced and evaluated by culture staining. The "construct" was made of MatrigelBD with GMSC. To assess the biocompatibility, an MTT cellular proliferation assay was performed. The differentiation potential of the cells toward the osteogenic, adipogenic, and chondrogenic lineages was analyzed after 21 days of growth in MatrigelBD with induction differentiation media. The MTT analysis showed that MatrigelBD stimulated cell proliferation; the GMSCs maintained the expression of MSC markers. Importantly, the growth of GMSCs within the MatrigelBD did not interfere with the cell differentiation potential. These findings indicate that MatrigelBD is biocompatible with GMSCs, and this matrix improves cell proliferation in vitro.

MatrigelBD es un andamiaje de hidrogel con redes tridimensionales entrecruzadas de polímeros hidrófilos con un alto contenido de agua. El tejido gingival humano podría representar una mejor fuente de MSCs, estas células pueden obtenerse fácilmente de una manera relativamente no invasiva. El objetivo de este estudio fue evaluar la biocompatibilidad de MatrigelBD con GMSCs in vitro. Muestras gingivales de tejido conectivo se obtuvieron de donantes sanos. El tejido se trituró y se cultivó durante dos semanas, y cuando las células se encontraban en el cuarto pasaje se les analizó su fenotipo inmunológico utilizando citometría de flujo. Se indujo la diferenciación hacia los linajes osteogénico, condrogénico y adipogénico, evaluandose con tinciones. El "constructo" se hizo de MatrigelBD con GMSC. Para evaluar la biocompatibilidad, se realizó un ensayo de proliferación celular: MTT. Se analizó el potencial de diferenciación de las células hacia los linajes osteogénico, adipogénico y condrogénico después de 21 días de cultivo en MatrigelBD con medio de diferenciación de inducción. El análisis de MTT mostró que MatrigelBD estimula la proliferación celular; GMSCs mantiene la expresión de marcadores de MSC. Es importante destacar que el crecimiento de GMSCs en MatrigelBD no interfirió con el potencial de diferenciación celular. Estos hallazgos indican que MatrigelBD es biocompatible con GMSCs, y esta matriz mejora la proliferación celular in vitro.

Avaliação dos eixos ECA / ANG II / AT1 e ECA-2 / ANG (1-7) / MAS em fibroblastos de gengiva e ligamento periodontal humanos estimulados por IL-1ß e sua contribuição na produção e regulação de citocinas e quimiocinas / Evaluation ACE /ANG II /AT1 and ACE-2 /ANG (1-7) /MAS axis in gingival and periodontal ligament human fibroblasts stimulated with IL-1ß and its contribution in the production of cytokines and chemokines

O Sistema renina-angiotensina (SRA) tem sido relatado como um importante modulador de processos inflamatórios e imunológicos, incluindo a doença periodontal (DP). Estudos sugerem neste sistema um eixo alternativo (ECA-2 /ANG(1-7) /MAS) que atuaria como um contra-regulador de efeitos mediados pelo clássico eixo (ECA /ANGII /AT1). Sabe-se que bactérias periodontopatogênicas, como a Porphyromonas gingivalis (Pg), possuem componentes bioativos de membrana (ex. lipopolissacarídeos-LPS) capazes de induzir uma forte resposta imune no hospedeiro devido à liberação de citocinas nas células, entre elas Interleucina (IL)- 1ß. Neste contexto, fibroblastos são as células mais abundantes nos tecidos periodontais e possuem em sua superfície celular receptores necessários para o reconhecimento da invasão bacteriana, ativando cascatas intracelulares, que levam à produção de citocinas. O objetivo deste estudo foi verificar se os eixos ECA/ ANGII/ AT1 e ECA-2/ ANG(1-7)/ MAS contribuem para a produção e/ ou regulação de citocinas inflamatórias (CI) por fibroblastos de gengiva humana (HGF) e ligamento periodontal humano (HPLF) estimulados por IL-1ß. Após o pré-tratamento com Losartan e Ang (1-7) ou silenciamento mediado por RNA de interferência (RNAi) de AT1, HGF e HPLF foram estimulados por IL-1ß por 3 horas (RNAm) ou 24 horas (proteína). Expressão de RNAm para AT1, MAS, ECA, ECA-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L e OPG foram avaliados por RT-qPCR e das proteínas IL-6, IL-8, ECA e ECA-2 por ELISA. Foi realizado também Western Blot para detecção de AT1 e ECA nos extratos celulares e dosagem de nitrito no sobrenadante das culturas. Ambos os subtipos de fibroblastos mostraram aumento da expressão de RNAm para AT1, IL-1ß, IL-6, IL-8, TNF-α e OPG, quando estimulados por IL-1ß. No entanto, apenas em HPLF foi observado aumento para MAS, ECA e TGF-ß. Losartan e Ang (1-7) não modularam o transcrito, a secreção de CI e nem a produção de nitrito no sobrenadante das culturas, tanto em HGF como em HPLF. O silenciamento do receptor AT1 reduziu a secreção de IL-6 e IL-8 induzida por IL-1ß em cultura de HGF e HPLF e aumentou a expressão gênica de OPG somente em HGF. Estes resultados sugerem que o silenciamento de AT1, mas não o bloqueio farmacológico deste receptor pelo antagonista Losartan, em HGF e HPLF, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal.(AU)

The renin-angiotensin system (RAS) has been reported as an important modulator of inflammatory and immune responses, including periodontal disease (PD). Studies suggest an alternative axis as part of this system (ACE-2 / ANG (1-7) / MAS) that would act as counter-regulatory to the classical axis (ECA / ANGII / AT1). It is known that periodontal bacteria such as Porphyromonas gingivalis (Pg) have bioactive components in their membrane (such as lipopolysaccharide-LPS) capable of inducing a strong immune response in the host due to the release of cytokines in cells, including interleukin (IL) - 1ß. In this regard, fibroblasts are the most abundant cells in periodontal tissues and receptors needed for the recognition of bacterial invasion by activating intracellular cascades that lead to cytokine production. The aim of this study was to determine whether the axes ACE / ANGII / AT1 and ACE-2 / ANG (1-7) / MAS contribute to the production and / or regulation of inflammatory cytokines (IC) by fibroblasts of human gingiva (HGF) and human periodontal ligament (HPLF) stimulated IL-1ß. After pre-treatment with Losartan, Ang (1-7) or silencing mediated by RNA interference (RNAi) of AT1, HGF and HPLF were stimulated by IL-1ß for 3 hours (RNAm) or 24 hours (protein). Expression mRNA for AT1, MAS, ACE, ACE-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L and OPG was assessed by RT- qPCR and proteins IL-6, IL-8, ACE and ACE-2 by ELISA. Western Blot for the detection of AT1 and ECA and dosage of nitrite was also performed. Experiments stimulated by IL-1ß showed a positive control for gene expression AT1, IL-1ß, IL-6, IL-8, TNF-α and OPG in HGF and HPLF and MAS, ACE and TGF-ß only HPLF. Losartan and Ang (1-7) did not modulate the transcription and secretion of IC and no nitrite production in the culture supernatant of HGF and HPLF. The silencing AT1 reduced IL-6 secretion and IL-8 induced by IL- ß in cultured HGF and HPLF and increased OPG gene expression only HGF. These results suggest that silencing AT1, but not pharmacological blockade of this receptor by Losartan in HPLF and HGF, can control the production of IL-6 and IL-8, which in turn contribute to the pathogenesis of periodontal disease.(AU)

Efeito de cianoacrilatos e adesivo tissular a base de veneno de cobra na citotoxicidade sobre fibroblastos gengivais e estresse oxidativo sobre células do biofilme de C. albicans / Effect cyanoacrylate and snake venom tissue adhesive on cytotoxicity of gingival fibroblasts and oxidative stress of C. albicans biofilms

A estomatite protética é uma patologia que acomete muitos usuários de prótese total. Um dos principais fatores da sua etiologia é a fácil adesão de microrganismos, como a Candida albicans, à resina acrílica, principalmente devido às características de superfície facilitadoras deste material. Por esta razão, estuda-se materiais que possam modificar as características de superfície da resina acrílica sem alterar as suas propriedades mecânicas. A investigação das propriedades desses materiais, como a biocompatibilidade e como eles influenciam as células de C. albicans, é de extrema importância para uma futura aplicação clínica. Dessa forma, a proposta desta pesquisa foi avaliar a citotoxicidade sobre fibroblastos gengivais humanos (FGH) e o estresse oxidativo causado em células de C. albicans por 6 produtos (etil-cianoacrilato convencional -ECAc; etil-cianoacrilato em forma de gel formulação a -ECAga; etil-cianoacrilato em forma de gel formulação b -ECAgb; butil-cianoacrilato - BCA; octil-cianoacrilato OCA; selante tissular a base de veneno de cobra-VC;), que quando aplicados na superfície da resina acrílica, modificam suas características de superfície, visando diminuir a formação do biofilme de C. albicans. Espécimes de resina acrílica foram confeccionados e duas camadas de cada produto foram aplicadas nas superfícies. Cada espécime foi deixado em 1, 066 mL de meio de cultura durante 24 horas para extração e utilização do meio condicionado para os dois ensaios. Para a investigação da citotoxicidade, os FGH foram cultivados em placas de 96 poços, meios condicionado de cada grupo foram depositados em cada poço onde permaneceram em contato por 24, 48 e 72 h. Após o processamento para o ensaio de MTT as placas foram analisadas em espectrofotômetro a 500 nm. Foram realizados quatro experimentos independentes em triplicata (n=12). Para a investigação de estresse oxidativo (ERO) causado em C. albicans, biofilme foi desenvolvido em placas de 96 poços durante 24 h. Então, os meios condicionados foram adicionados aos poços e a placa foi mantida em agitadora durante 1 h. Após a retirada dos meios condicionados, foi adicionado a cada poço o reagente CellRox® Deep Red e leituras foram realizadas a cada 30 min durante 3 h em fluorímetro a 640/665nm. Amostras foram coletadas e ensaio de cultura microbiológica foi executado para a realização da normalização dos valores. Foram realizados 3 experimentos independentes em triplicata (n=9). Os resultados obtidos pelo ensaio MTT foram analisados por meio de ANOVA a 2 critérios, seguido pelo teste Tukey para a comparação entre grupos (p<0,05). Para o ensaio ROS, a análise estatística realizada foi ANOVA a 1 critério, seguida, igualmente, por Tukey (p<0,05). Os resultados mostraram que todos os grupos apresentaram certo grau de citotoxicidade quando comparados ao controle, com exceção do VC (ECAc

Denture stomatitis is a disease that affects many denture wearers. One of the main factors of its cause is the easy adhesion of microorganisms, such as Candida albicans, to acrylic resin, mainly due to its permissive surface characteristics. For this reason, materials that can modify surface characteristics of acrylic resin without changing its mechanical properties are being studied. The investigation of these materials features, such as biocompatibility and how they affect Candida albicans, is of utmost importance for future clinical application. Thus, the purpose of this study was to evaluate the cytotoxicity of human gingival fibroblasts (HGF) and oxidative stress in C. albicans cells after contact with 6 experimental products (conventional ethyl-cyanoacrylate -ECAc; ethyl-cyanoacrylate in gel formulation "a" -ECAga; ethyl-cyanoacrylate in gel formulation "b" -ECAgb; butyl- cyanoacrylate - BCA; octyl-cyanoacrylate -OCA; tissue adhesive based on snake venom -VC) applied to the surface of the resin acrylic, modifying its surface characteristics, in order to decrease C. albicans biofilm formation. Acrylic resin specimens were manufactured and two layers of each product were applied to the surfaces. Each specimen was immersed in 1,066 mL of growth media for 24 hours for extraction and the resulting conditioned media were used for both tests. To investigate cytotoxicity, HGF were cultured in 96-well plates and conditioned media from each group were deposited into each well, where they remained in contact for 24, 48 and 72h. After the MTT assay processing, plates were analyzed in a spectrophotometer at 500 nm. Four independent experiments were performed in triplicate (n=12). To investigate oxidative stress (ROS), C. albicans biofilm was developed in 96-well plates for 24 h, conditioned media were added to the wells and the plate was allowed to stir for 1 h. Then, conditioned media were removed and CellRox® Deep Red reagent was added to each well. Readings were performed every 30 min for 3 h in fluorometer at 640/665nm. Samples were collected and microbiological culture test was executed to values normalization. Three independent experiments were performed in triplicate (n=9). Results obtained in MTT assay were analyzed by two-way ANOVA and Tukey's test for comparison between groups (p<0.05). for comparison between groups (p<0.05). For ROS results, one-way ANOVA was performed followed by Tukey test, as well (p<0.05). The results showed that all groups presented different degrees of cytotoxicity when compared to control group, except for VC (ECAc

Response of a co-culture model of epithelial cells and gingival fibroblasts to zoledronic acid

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

Evaluation of the Biological Behavior of Mucograft® in Human Gingival Fibroblasts: An In Vitro Study

Mucograft(r) is a resorbing porcine matrix composed of type I and type III collagen, used for soft tissue augmentation in guided tissue bony regeneration procedures. This in vitro study aimed to evaluate the biological behavior of Mucograft(r) in human gingival fibroblasts, as well as the ability of the matrix to induce production of extracellular matrix. Six resorbing Mucograft(r) matrices (MCG) were cut into 3 x 2 mm rectangles and 5 x 5 mm squares and were placed in 96- and 24-well plates, respectively. The control group (CTRL) consisted of cells plated on polystyrene without the MCG. After one, two, three and seven days, cell proliferation and viability were assessed using the Trypan exclusion method and MTT test, respectively. Type III collagen (COL 3A1) and vimentin (VIM) expression were also evaluated at 10 and 14 days, using Western blotting. Statistical analysis, using ANOVA with post hoc Bonferroni test, revealed that human gingival fibroblasts from MCG showed similar results (p>0.05) for proliferation and viability as the cells cultured on CTRL. After 14 days, a significant decrease in COL 3A1 expression (p<0.05) was observed when cultured with the MCG. VIM expression showed no significant difference at any time period (p>0.05). Although no increase in extracellular matrix secretion was observed in this in vitro study, Mucograft(r) presented cellular compatibility, being an option for a scaffold whenever it is required.

Resumo A Mucograft(r) é uma matriz reabsorvível, de origem suína, composta de colágenos do tipo I e III, utilizada para aumento de tecido mole em regeneração óssea guiada. Este estudo in vitro teve como objetivo avaliar o comportamento biológico da Mucograft(r), em fibroblastos gengivais humanos, bem como a indução da síntese de matriz extracelular. Seis matrizes reabsorvíveis de Mucograft(r) (MCG) foram cortadas em retângulos e quadrados medindo 3 x 2 mm e 5 x 5 mm e alocadas em placas de 96 e 24 poços, respectivamente. O grupo controle (CTRL) consistiu no plaqueamento celular em poliestireno, sem MCG. Após um, dois, três e sete dias, a proliferação e a viabilidade celular foram avaliadas utilizando o corante vital azul de Trypan e o teste MTT, respectivamente. Além disso, a expressão de colágeno tipo III (COL 3A1) e vimentina (VIM) foi avaliada após 10 e 14 dias, por meio de Western-blotting. Após análise estatística (Anova e pós teste de Bonferroni), pode-se observar que os fibroblastos gengivais humanos, cultivados sobre MCG, apresentaram proliferação e viabilidade semelhantes em comparação às células que foram cultivadas apenas no poliestireno (CTRL). Após 14 dias, notou-se uma diminuição significativa da expressão de COL 3A1 (p<0,05) quando as células foram cultivadas sobre a MCG. A expressão da VIM não mostrou diferença significativa em nenhum dos períodos estudados (p>0,05). No presente estudo in vitro pode-se concluir que apesar de não ter sido observado aumento da síntese de matriz extracelular, a Mucograft(r) apresentou compatibilidade celular, sendo uma opção de biomaterial em casos que o arcabouço é necessário.

Novel biological activity of ameloblastin in enamel matrix derivative

Objective Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells. Material and Methods Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein. Conclusion The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues. .

Biocompatibilidad de células madres mesenquimales de tejido gingival humano en cultivoconun andamiaje de polímero sintético de ácido poliláctico (OPLA) / Biocompatibility of mesenchymal stem cells derived from human gingival tissue in culture with a scaffold of a synthetic polymer open porosity polylactic acid polymer (OPLA)

La ingeniería tisular se plantea como tratamiento ideal para la regeneración de tejidos con la utilización de andamiajes, células madres y factores de crecimiento. Las células madres de origen gingival plantean ventajas de obtención, mientras que el OPLA 3D permite cultivos de alta densidad celular. El objetivo de esta investigación fue evaluar la biocompatibilidad de células madres de origen gingival en OPLA. Las células se obtuvieron de tejido gingival y fueron caracterizadas fenotípica y funcionalmente. La biocompatibilidad se evaluó mediante la proliferación celular, prueba de viabilidad con azul tripán y diferenciación celular a linaje condrogénico y osteogénico. El recultivo del constructo se utilizó para evaluar la capacidad de transporte. Las células al interior del OPLA se visualizaron mediante cortes teñidos con H-E. Las células madres mesenquimales en OPLA proliferaron, 80% de confluencia a la cuarta semana. La viabilidad celular en OPLA fue de 83,32%. En el recultivo, las células comienzan a proliferar a la semana. El OPLA permite la diferenciación celular a linaje condrogénico y osteogénico. Se observan células al interior del OPLA, permite la proliferación, viabilidad y diferenciación celular. El OPLA podría ser utilizado como andamiaje celular para la ingeniería de tejidos.

Tissue engineering arises as the ideal treatment for tissue regeneration with the use of scaffolds, stem cells and growth factors. Stem cells derived from gingival tissue present benefits in its objection. 3D OPLA allow high cell density cultures. The objective of this study was to evaluate the biocompatibility of gingival stem cells in OPLA. Cells were obtained from gingival tissue and were characterized phenotypically and functionally. The biocompatibility was evaluated through cell proliferation, viability test with trypan blue and cell differentiation to chondrogenic and osteogenic lineage. Recultivation of the construct was used to evaluate transportability. Cells inside OPLA were visualized by stained sections with H&E. Mesenchymal stem cells proliferated in OPLA, 80% confluence at the fourth week. Cell viability in OPLA was 83.32%. In recultivation, cells start proliferating in a week. OPLA allows cell differentiation to chondrogenic and osteogenic lineage. Cells were observed within OPLA. In conclusion OPLA allows proliferation, viability and cell differentiation. OPLA could be used as scaffolds for cells in tissue engineering.

Cytotoxic effect of Salvadora persica extracts on human gingival fibroblast cells

To assess the cytotoxic potential of Salvadora persica [S. persica] extracts on human gingival fibroblast [HGF] cells. This study was conducted between January and May 2012 in collaboration with Dental Caries Research Chair, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. Extracts of S. persica using hexane, ethylacetate, and ethanol as solvents at concentrations of 0.5 mg/ml and 1 mg/ml were evaluated for their cytotoxic activity against HGFs using the 3 cytotoxic assays: [3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, a tetrazole] [MTS], lactic dehydrogenase [LDH], and crystal violet [CV]. International standards for the evaluation of medical materials recommended cut-off value of cell survival >70% was used for interpretation of the results. Ethanol extract of S. persica at 0.5 mg/ml and 1 mg/ml and hexane extract of S. persica at 0.5 mg/ml were completely devoid of cytotoxic activity, hexane extract at 1 mg/ml in comparison with controls demonstrated some cytotoxicity with cell survival of 88% [p=0.045] in MTS, 86% [p=0.01] in LDH, and 88% [p=0.002] in CV assays. Similarly, ethyl acetate extract of S. persica at 0.5 mg/ml maintained cell viability of 91% in MTS, 81% in LDH, and 80% in CV assays. Maximum cytotoxicity against HGFs was observed with ethyl acetate extract of S. persica at 1 mg/ml with cell survival of 60% in MTS, 40% in LDH, and 66% CV assays [p=0.0001]. The acceptable level of cytotoxicity associated with S. persica ethanol and hexane extracts requires further evaluation to be used as irrigation solutions in endodontic treatment

Cytotoxic Effects of Zoledronic Acid on Human Epithelial Cells and Gingival Fibroblasts

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

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