KLF6 facilitates differentiation of odontoblasts through modulating the expression of P21 in vitro / 国际口腔科学杂志·英文版

Multiple signaling pathways are involved in the regulation of cell proliferation and differentiation in odontogenesis and dental tissue renewal, but the details of these mechanisms remain unknown. Here, we investigated the expression patterns of a transcription factor, Krüppel-like factor 6 (KLF6), during the development of murine tooth germ and its function in odontoblastic differentiation. KLF6 was almost ubiquitously expressed in odontoblasts at various stages, and it was co-expressed with P21 (to varying degrees) in mouse dental germ. To determine the function of Klf6, overexpression and knockdown experiments were performed in a mouse dental papilla cell line (iMDP-3). Klf6 functioned as a promoter of odontoblastic differentiation and inhibited the proliferation and cell cycle progression of iMDP-3 through p21 upregulation. Dual-luciferase reporter assay and chromatin immunoprecipitation showed that Klf6 directly activates p21 transcription. Additionally, the in vivo study showed that KLF6 and P21 were also co-expressed in odontoblasts around the reparative dentin. In conclusion, Klf6 regulates the transcriptional activity of p21, thus promoting the cell proliferation to odontoblastic differentiation transition in vitro. This study provides a theoretical basis for odontoblast differentiation and the formation of reparative dentine regeneration.

Aberrant NF-κB activation in odontoblasts orchestrates inflammatory matrix degradation and mineral resorption / 国际口腔科学杂志·英文版

Inflammation-associated proteinase functions are key determinants of inflammatory stromal tissues deconstruction. As a specialized inflammatory pathological process, dental internal resorption (IR) includes both soft and hard tissues deconstruction within the dentin-pulp complex, which has been one of the main reasons for inflammatory tooth loss. Mechanisms of inflammatory matrix degradation and tissue resorption in IR are largely unclear. In this study, we used a combination of Cre-loxP reporter, flow cytometry, cell transplantation, and enzyme activities assay to mechanistically investigate the role of regenerative cells, odontoblasts (ODs), in inflammatory mineral resorption and matrices degradation. We report that inflamed ODs have strong capabilities of matrix degradation and tissue resorption. Traditionally, ODs are regarded as hard-tissue regenerative cells; however, our data unexpectedly present ODs as a crucial population that participates in IR-associated tissue deconstruction. Specifically, we uncovered that nuclear factor-kappa b (NF-κB) signaling orchestrated Tumor necrosis factor α (TNF-α)-induced matrix metalloproteinases (Mmps) and Cathepsin K (Ctsk) functions in ODs to enhance matrix degradation and tissue resorption. Furthermore, TNF-α increases Rankl/Opg ratio in ODs via NF-κB signaling by impairing Opg expression but increasing Rankl level, which utterly makes ODs cell line 17IIA11 (A11) become Trap+ and Ctsk+ multinucleated cells to perform resorptive actions. Blocking of NF-κB signaling significantly rescues matrix degradation and resorptive functions of inflamed ODs via repressing vital inflammatory proteinases Mmps and Ctsk. Utterly, via utilizing NF-κB specific small molecule inhibitors we satisfactorily attenuated inflammatory ODs-associated human dental IR in vivo. Our data reveal the underlying mechanisms of inflammatory matrix degradation and resorption via proteinase activities in IR-related pathological conditions.

Role of autophagy in lipopolysaccharide-induced apoptosis of odontoblasts / 南方医科大学学报

OBJECTIVE@#To investigate the role of autophagy in lipopolysaccharide (LPS)-induced apoptosis of murine odontoblasts.@*METHODS@#Murine odontoblasts (mDPC-23 cells) were treated with 5 μg/mL LPS for 6, 12 and 24 h, and the changes in cell viability was examined using CCK8 kit and cell apoptosis was detected by TUNEL staining. The changes in the protein levels of LC3, Beclin1, Atg5, AKT, p-AKT, mTOR and p-mTOR were detected using Western blotting. The effect of 3-MA treatment for 24 h on LPS-induced apoptosis of mDPC-23 cells was evaluated by detecting the expressions of apoptosis-related proteins caspase-3 and Bax using Western blotting.@*RESULTS@#Stimulation with LPS for 6 and 12 h did not cause significant changes in the proliferation or apoptosis of mDPC-23 cells, but LPS treatment for 24 h significantly suppressed cell proliferation (@*CONCLUSIONS@#LPS stimulation induces autophagy to promote apoptosis of mDPC-23 cells, and suppression of autophagy attenuates LPS-induced apoptosis. Autophagy may play an important role in the injury of inflamed pulp tissues.

Red LED Photobiomodulates the Metabolic Activity of Odontoblast-Like Cells

Abstract Phototherapy has been indicated as an adjunctive treatment for tissue repair, including the pulp tissue. However, there are no defined irradiation parameters, which is a great challenge to the clinical use of phototherapy. The aim of this study was to evaluate the effect of phototherapy with red LED on odontoblast-like MDPC-23 cells, using different parameter settings. Cells were seeded (104 cells/cm²), incubated for 12 h in complete DMEM and then the culture medium was replaced by DMEM supplemented with 0.5% FBS. After 12 h incubation, irradiations were performed (630±10 nm) using a LEDTable device with a 20 or 40 mW/cm² power density and 2 J/cm² energy dose. The cells were irradiated 1 or 3 times, at 1 min intervals. Non-irradiated cells served as control. The cells were evaluated for viability (MTT assay), total protein dosage (Lowry method) and number of viable cells (Trypan blue). The data (n=12 per group) were submitted to Kruskal-Wallis and Mann-Whitney tests (p=0.05). A single irradiation with 20 or 40 mW/cm² enhanced cell viability, which was negatively affected after 3 consecutive irradiations. Cells irradiated only once with 20 mW/cm² produced more proteins compared with those irradiated with 40 mW/cm². Reduction in the number of viable cells occurred only after 3 consecutive irradiations with 40 mW/cm². In conclusion, red LED was capable of biomodulating the metabolic activities of cultured MDPC-23 odontoblast-like cells. The best cell biostimulation was obtained when a single irradiation with 2 J/cm2 energy dose and 20 mW/cm2 power density was delivered to the pulp cells.

Resumo Fototerapia tem sido indicada como um tratamento adjuvante para o reparo de tecidos, incluindo o tecido pulpar. Entretanto, não há parâmetros de irradiação definidos, o que representa um grande desafio para o uso clínico da fototerapia. O objetivo deste estudo foi avaliar o efeito da fototerapia com LED vermelho em células MDPC-23 com fenótipo odontoblastóide, usando vários parâmetros. As células foram semeadas (104 células/cm2), incubadas por 12 h em DMEM completo e então o meio de cultura foi trocado por DMEM com 0,5% SFB. Após 12 h de incubação, as irradiações foram realizadas (630±10 nm) usando um dispositivo com densidade de potência de 20 ou 40 mW/cm2 e dose de energia de 2 J/cm2. As células foram irradiadas 1 ou 3 vezes, com intervalos de 1 min. Células não irradiadas serviram como controle. Foram avaliadas a viabilidade (ensaio de MTT), dosagem de proteína total (método de Lowry) e número de células viáveis (ensaio de Trypan blue). Os dados (n=12 por grupo) foram submetidos aos testes de Kruskal-Wallis e Mann-Whitney (p=0,05). Uma única irradiação com 20 ou 40 mW/cm2 aumentou a viabilidade celular, a qual foi negativamente afetada após 3 irradiações. Células irradiadas apenas uma vez com 20 mW/cm2 produziram mais proteínas comparadas com aquelas irradiadas com 40 mW/cm2. Redução no número de células viáveis ocorreu apenas após 3 irradiações com 40 mw/cm2. Em conclusão, o LED vermelho foi capaz de biomodular a atividade metabólica de células MDPC-23. A melhor bioestimulação celular foi obtida quando uma única irradiação com dose de energia de 2 J/cm2 e densidade de potência de 20 mW/cm2 foi administrada às células pulpares.

Effect of Fluoride-Treated Enamel on Indirect Cytotoxicity of a 16% Carbamide Peroxide Bleaching Gel to Pulp Cells

The aim of this study was to evaluate the possibility of fluoride solutions applied to enamel to protect pulp cells against the trans-enamel and transdentinal cytotoxicity of a 16% carbamide peroxide (CP) bleaching gel. The CP gel was applied to enamel/dentin discs adapted to aicial pulp chambers (8 h/day) during 1, 7 or 14 days, followed by fluoride (0.05% or 0.2%) application for 1 min. The extracts (culture medium in contact with dentin) were applied to MDPC-23 cells for 1 h, and cell metabolism (MTT assay), alkaline phosphatase (ALP) activity and cell membrane damage (flow cytometry) were analyzed. Knoop microhardness of enamel was also evaluated. Data were analyzed statistically by ANOVA and Kruskal-Wallis tests (α=0.05). For the MTT assay and ALP activity, significant reductions between the control and the bleached groups were observed (p<0.05). No statistically significant difference occurred among bleached groups (p>0.05), regardless of fluoride application or treatment days. Flow cytometry analysis demonstrated 30% of cell membrane damage in all bleached groups. After 14 days of treatment, the fluoride-treated enamel presented significantly higher microhardness values than the bleached-only group (p<0.05). It was concluded that, regardless of the increase in enamel hardness due to the application of fluoride solutions, the treated enamel surface did not prevent the toxic effects caused by the 16% CP gel to odontoblast-like cells.

Resumo O objetivo do presente estudo foi avaliar o possível efeito protetor de soluções fluoretadas aplicadas sobre o esmalte dentário frente à citotoxicidade trans-amelodentinária de um gel clareador com 16% de peróxido de carbamida (PC). O gel de PC foi aplicado sobre discos de esmalte/dentina adaptados a câmaras pulpares aiciais (8 h/dia) durante períodos de 1, 7 ou 14 dias, seguido de aplicação de soluções fluoretadas (0,05% ou 0,2%) durante 1 min. Os extratos (meio de cultura em contato com a dentina) foram aplicados sobre células MDPC-23 durante 1 h, seguido de análise do metabolismo celular (teste do MTT), atividade de fosfatase alcalina (ALP) e danos à membrana celular (citometria de fluxo). A microdureza Knoop do esmalte dental foi avaliada. Os dados foram analisados pelos testes de ANOVA e Kruskal-Wallis. Para o teste do MTT e atividade de ALP, redução significante entre os grupos controle e clareados foram observados (p<0,05). Nenhuma diferença entre os grupos clareados foi observada (p>0,05), independente da aplicação das soluções fluoretadas ou tempo de tratamento. A análise por citometria de fluxo demonstrou lesão à membrana celular em torno de 30% para todos os grupos clareados. Após 14 dias de tratamento, os espécimes clareados e fluoretados apresentaram aumento significante na microdureza do esmalte (p<0,05). Pôde-se concluir que apesar do aumento na dureza do esmalte decorrente da aplicação das soluções fluoretadas, este tratamento não preveniu os efeitos tóxicos causados pelo gel com 16% de PC sobre as células odontoblastóides. .

Micrometric measurement of the density of stained odontoblast processes

The embryological, structural and functional unit of the dentine-pulp complex shares the odontoblast, located in the border of the dentine pulp, with basal nuclei and organelles. The odontoblast process emerges from its apical pole. It is formed by microtubules, microfilaments and vesicles covered by membranes penetrating the dentinal tubules, isolated from the inter-tubular matrix, along the extent of the dentine. The objective of this study was to evaluate the efficacy of three staining techniques: hematoxylin-eosin, periodic acid-Schiff and Schmorl, by staining the process, from beginning to end, and compare the results with the erosion technique. Thirty human teeth were employed in the trial; after their extraction the pulp was fixated, the pieces demineralized in nitric acid at 8%, the collagen filaments eliminated with Type II Collage-nase, the tissue was stained, and the measurements were made. The portions with no pulp were prepared with the erosion technique. Results: Comparing the best results obtained by staining with the values obtained with the erosion technique, the former showed lower values. Conclusion: Staining techniques show lower density of the staining processes compared with the dentinal tubules in the erosion technique.

Effects of light-curing time on the cytotoxicity of a restorative composite resin on odontoblast-like cells

This in vitro study evaluated the cytotoxicity of an experimental restorative composite resin subjected to different light-curing regimens. METHODS: Forty round-shaped specimens were prepared and randomly assigned to four experimental groups (n=10), as follows: in Group 1, no light-curing; in Groups 2, 3 and 4, the composite resin specimens were light-cured for 20, 40 or 60 s, respectively. In Group 5, filter paper discs soaked in 5 µL PBS were used as negative controls. The resin specimens and paper discs were placed in wells of 24-well plates in which the odontoblast-like cells MDPC-23 (30,000 cells/cm²) were plated and incubated in a humidified incubator with 5 percent CO2 and 95 percent air at 37ºC for 72 h. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). The data were analyzed statistically by Kruskal-Wallis and Mann-Whitney tests (p<0.05). RESULTS: In G1, cell metabolism decreased by 86.2 percent, indicating a severe cytotoxicity of the non-light-cured composite resin. On the other hand, cell metabolism decreased by only 13.3 percent and 13.5 percent in G2 and G3, respectively. No cytotoxic effects were observed in G4 and G5. In G1, only a few round-shaped cells with short processes on their cytoplasmic membrane were observed. In the other experimental groups as well as in control group, a number of spindle-shaped cells with long cytoplasmic processes were found. CONCLUSION: Regardless of the photoactivation time used in the present investigation, the experimental composite resin presented mild to no toxic effects to the odontoblast-like MDPC-23 cells. However, intense cytotoxic effects occurred when no light-curing was performed.

Toxicity of chlorhexidine on odontoblast-like cells

Chlorhexidine gluconate (CHX) is recommended for a number of clinical procedures and it has been pointed out as a potential cavity cleanser to be applied before adhesive restoration of dental cavities. OBJECTIVE: As CHX may diffuse through the dentinal tubules to reach a monolayer of odontoblasts that underlies the dentin substrate, this study evaluated the cytotoxic effects of different concentrations of CHX on cultured odontoblast-like cells (MDPC-23). MATERIAL AND METHODS: Cells were cultured and exposed to CHX solutions at concentrations of 0.06 percent, 0.12 percent, 0.2 percent, 1 percent and 2 percent. Pure culture medium (á-MEM) and 3 percent hydrogen peroxide were used as negative and positive control, respectively. After exposing the cultured cells to the controls and CHX solutions for 60 s, 2 h or 60 s with a 24-h recovery period, cell metabolism (MTT assay) and total protein concentration were evaluated. Cell morphology was assessed under scanning electron microscopy. CHX had a dose-dependent toxic effect on the MDPC-23 cells. RESULTS: Statistically significant difference was observed when the cells were exposed to CHX in all periods (p<0.05). Significant difference was also determined for all CHX concentrations (p<0.05). The 60-s exposure time was the least cytotoxic (p<0.05), while exposure to CHX for 60 s with a 24-h recovery period was the most toxic to the cells (p<0.05). CONCLUSION: Regardless of the exposure time, all CHX concentrations had a high direct cytotoxic effect to cultured MDPC-23 cells.

Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC-23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cel ls, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.

Dentin matrix protein 1 (DMP1) expression in developing human teeth

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein that plays an important role in mineralized tissue formation by initiation of nucleation and modulation of mineral phase morphology. The purpose of the present study was to examine the immunoexpression of DMP1 in tooth germs of 7 human fetuses at different gestational ages (14, 16, 19, 20, 21, 23 and 24 weeks) comparing with completed tooth formation erupted teeth. The results showed the presence of DMP1 in the dental lamina, as well as in the cells of the external epithelium, stellate reticulum and stratum intermedium of the enamel organ. However, in the internal dental epithelium, cervical loop region and dental papilla some cells have not labeled for DMP1. In the crown stage, DMP1 was expressed in the ameloblast and odontoblast layer, as well as in the dentinal tubules of coronal dentin near the odontoblast area. Erupted teeth with complete tooth formation exhibited immunolabeling for DMP1 only in the dentinal tubules mainly close to the dental pulp. No staining was observed in the enamel, predentin or dental pulp matrix. DMP1 is present in all developing dental structures (dental lamina, enamel organ, dental papilla) presenting few immunoexpression variations, with no staining in mineralized enamel and dentin.

A proteína da matriz dentinária 1 (DMP1) é uma fosfoproteína ácida que tem sido relacionada diretamente ao processo de mineralização dos tecidos em formação sendo iniciadora do processo de nucleação e modulação da fase mineral. O objetivo desse trabalho foi avaliar a imunoexpressão da DMP1 em germes dentários em diferentes fases da odontogênese, obtidos de 7 fetos humanos em diversos estágios gestacionais (14, 16, 19, 20, 21, 23 e 24 semanas), comparando-se com dentes com rizogênese completa. Os resultados mostraram que a DMP1 esteve expressa na lâmina dentária, bem como, nas células do epitélio externo, retículo estrelado e estrato intermediário do órgão do esmalte. Diferentemente, no epitélio interno do órgão do esmalte, alça cervical e papila dentária algumas células não apresentaram a DMP1. Nas fases de coroa, os ameloblastos e odontoblastos apresentaram marcação positiva para a DMP1, bem como os túbulos dentinários da dentina coronária próximos à região odontoblástica. Os dentes com rizogênese completa exibiram marcação para a DMP1 apenas nos túbulos dentinários principalmente próximos à polpa dentária. Nenhuma marcação foi observada na matriz de esmalte ou pré-dentina, nem na polpa dentária. Concluímos que a DMP1 está presente em todas as fases da odontogênese, tanto na lâmina dentária, órgão do esmalte, bem como na papila dentária, com pequenas variações de nuances de expressão, estando ausente na dentina e esmalte mineralizados.

Efeito da sialoadenectomia sobre a incorporaçäo de 3H-prolina nas células e matriz do esmalte jovem e dentina de incisivos de camundongos, estudados através de radioautografia / Sialoadenectomy effect on 3H-proline incorporation in cells and matrix of young enamel and incisors dentin of mice, studied by radioautography

Estudaram-se os efeitos da remoçäo das glândulas salivares maiores sobre a incorporaçäo de 3H-prolina pelos ameloblastos secretores e odontoblastos de incisivos de camundongos. Animais sialoadenectomizados e controles foram injetados com 3H-prolina, sendo sacrificados e perfundidos 30 min, 2h, 12h e 24h após. Cortes de 1*m obtidos de incisivos inferiores incluídos em Polybed foram radioautografados. A distribuiçäo percentual de gräos de prata reduzida/100*m* sobre as referidas células, assim como em suas respectivas matrizes, foi sempre maior nos animais sialoadenectomizadas, nos diferentes intervalos de tempo. Duas hipóteses poderiam explicar esses resultados: 1. um aumento na biossíntese protéica provavelmente devida à falta do fator submandibular inibidor da insulina (SII) associada a um relativo aumento do pool de 3H-prolina devido à remoçäo das glândulas salivares; 2. um decrécimo na degradaçäo protéica devido ao hipotireoidismo que eventualmente ocorre nos animais sialoadenectomizados

Dentinogênese: papel dos microtúbulos e açäo da colchicina / Dentinogenesis: role of microtubules and action of colchicine

Nesta revisäo foram apresentados conhecimentos recentes sobre o papel dos microtúbulos nos processos secretórios, dando especial ênfase á sua participaçäo na secreçäo do colágeno pelos odontoblastos. A utilizaçäo de drogas antimicrotubulares, como a colchicina, tem evidenciado a importância destes componentes citoplásmaticos na dentinogênese