Effect of nicotine on the viability and morphology of fibroblasts: in vitro study

O objetivo do estudo foi avaliar in vitro o efeito da nicotina sobre a viabilidade e a morfologia celular utilizando-se uma linhagem contínua de fibroblastos. Para tal, foram formados dois grupos experimentais segundo a dose (0 - controle, 10 mig, 100 mig, 0,5 mg, 1 mg) e o tempo de condicionamento (1 e 24 horas). Cada um dos 12 orifícios de uma placa para cultura celular recebeu 2 ml de meio de Eagle, e 1 ml de suspensão de meio de cultura contendo aproximadamente 1 × 10(5) células/ml. Foi, então, acrescentada a solução de nicotina nas diferentes concentrações. Após o condicionamento com a droga, nos dois períodos testados, as células foram coradas com azul de trypan 0,4%, e observadas em microscópio invertido por um examinador cego para os grupos experimentais, que avaliou a viabilidade e a morfologia segundo o índice de Gamal. Os experimentos foram repetidos 5 vezes. Quanto à morfologia, os resultados obtidos demonstraram, no grupo condicionado por 1 h, que os controles apresentaram diferenças estatisticamente significantes em relação apenas à maior dose de nicotina; no entanto, foram encontradas diferenças estatisticamente significantes entre o controle e todas as concentrações após 24 horas de condicionamento. Na viabilidade celular, um maior número de células não viáveis foi observado nas diferentes concentrações de nicotina em comparação aos controles tanto após 1 quanto 24 horas de condicionamento (p < 0,05). Em ambos períodos existiu uma tendência significativa de aumento do número de células não viáveis com o aumento da dose de nicotina (p = 0,0053; p = 0,00001 após 1 e 24 h respectivamente). Portanto, conclui-se que a nicotina pode alterar, in vitro, a viabilidade e a morfologia de fibroblastos de forma proporcional à dose e ao tempo de exposição.The aim of this study was to evaluate, in vitro, the effect of nicotine on the viability and morphology of fibroblasts from a continuous lineage. Two experimental groups were prepared, with different drug dosages (0 - control, 10 mug, 100 mug, 0.5 mg, 1 mg) and conditioning time (1 and 24 hours). Twelve-well microplates were utilized. Each well received 2 ml of fresh culture medium and 1 ml of a solution containing 1 × 10(5) cells/ml. Nicotine was then added to the wells, at the tested concentrations. After the incubation period, cell viability was assessed by means of 0.4% trypan blue staining. Cell viability and morphology were assessed in an inverted microscope, by a single examiner, who was blind as to the experimental groups. The experiment was repeated 5 times. Regarding morphology, in the 1-hour conditioned group there was statistically significant difference between the control group and the group with the greatest dose of nicotine. These differences were also observed between the control group and all nicotine groups after 24 hours. The results of the Kruskal-Wallis test revealed that more unviable cells were found in the groups exposed to nicotine, in comparison with the control group, both after 1 and 24 hours of conditioning (p < 0.05). Moreover, with increasing doses of nicotine there was a directly proportional increase in the number of unviable cells, both after 1 and 24 hours of exposure (p = 0.0053 and p = 0.00001, respectively). The conclusion of this study is that nicotine can alter, in vitro, the viability and morphology of fibroblasts in a manner proportional to the dose and time of exposure

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide fromEscherichia coli (EcLPS). Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR.PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities232FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPNão te

Exposure of periodontal ligament progenitor cells to lipopolysaccharide from Escherichia coli changes osteoblast differentiation pattern

Periodontal ligament mesenchymal stem cells (PDLMSCs) are an important alternative source of adult stem cells and may be applied for periodontal tissue regeneration, neuroregenerative medicine, and heart valve tissue engineering. However, little is known about the impact of bacterial toxins on the biological properties of PDLSMSCs, including self-renewal, differentiation, and synthesis of extracellular matrix. Objective : This study investigated whether proliferation, expression of pro-inflammatory cytokines, and osteogenic differentiation of CD105-enriched PDL progenitor cell populations (PDL-CD105+ cells) would be affected by exposure to bacterial lipopolysaccharide from Escherichia coli (EcLPS). Material and Methods : Toll-like receptor 4 (TLR4) expression was assessed in PDL-CD105+ cells by the immunostaining technique and confirmed using Western blotting assay. Afterwards, these cells were exposed to EcLPS, and the following assays were carried out: (i) cell viability using MTS; (ii) expression of the interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) genes; (iii) osteoblast differentiation assessed by mineralization in vitro, and by mRNA levels of run-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) determined by quantitative PCR. Results : PDL-CD105+ cells were identified as positive for TLR4. EcLPS did not affect cell viability, but induced a significant increase of transcripts for IL-6 and IL-8. Under osteogenic condition, PDL-CD105+ cells exposed to EcLPS presented an increase of mineralized matrix deposition and higher RUNX2 and ALP mRNA levels when compared to the control group. Conclusions : These results provide evidence that CD105-enriched PDL progenitor cells are able to adapt to continuous Escherichia coli endotoxin challenge, leading to an upregulation of osteogenic activities

Transcriptome profile of highly osteoblastic/cementoblastic periodontal ligament cell clones

Heterogeneous cell populations of osteo/cementoblastic (O/C) or fibroblastic phenotypes constitute the periodontal dental ligament (PDL). A better understanding of these PDL cell subpopulations is essential to propose regenerative approaches based on a sound biological rationale. Objective: Our study aimed to clarify the differential transcriptome profile of PDL cells poised to differentiate into the O/C cell lineage. Methodology: To characterize periodontal-derived cells with distinct differentiation capacities, single-cell-derived clones were isolated from adult human PDL progenitor cells and their potential to differentiate into osteo/cementoblastic (O/C) phenotype (C-O clones) or fibroblastic phenotype (C-F clones) was assessed in vitro. The transcriptome profile of the clonal cell lines in standard medium cultivation was evaluated using next-generation sequencing technology (RNA-seq). Over 230 differentially expressed genes (DEG) were identified, in which C-O clones showed a higher number of upregulated genes (193) and 42 downregulated genes. Results: The upregulated genes were associated with the Cadherin and Wnt signaling pathways as well as annotated biological processes, including “anatomical structure development” and “cell adhesion.” Both transcriptome and RT-qPCR showed up-regulation of WNT2, WNT16, and WIF1 in C-O clones. Conclusions: This comprehensive transcriptomic assessment of human PDL progenitor cells revealed that expression of transcripts related to the biological process “anatomical structure development,” Cadherin signaling, and Wnt signaling can identify PDL cells with a higher potential to commit to the O/C phenotype. A better understanding of these pathways and their function in O/C differentiation will help to improve protocols for periodontal regenerative therapies

O efeito do fator de crescimento de fibroblastos básico aplicado em superfícies radiculares condicionadas com cloridrato de tetraciclina ou EDTA na morfologia e densidade de fibroblastos. Estudo in vitro

O objetivo do presente estudo foi avaliar in vitro o efeito do condicionamento radicular com fator de crescimento de fibroblastos básico (b-FGF) sobre a morfologia e densidade de fibroblastos. Para tal, blocos de dentina com 4 mm2 de área de superfície foram obtidos de raízes de dentes humanos extraídos devido severo envolvimento periodontal, sendo instrumentados manualmente e autoclavados. Noventa amostras foram selecionadas aleatoriamente e distribuídas em 3 grupos segundo o tratamento de superfície prévio ao condicionamento com o b-FGF: sem tratamento - controle; 50 mg/mL de cloridrato de tetraciclina e EDTA a 24%. As 30 amostras de cada um destes 3 grupos foram distribuídas em 3 subgrupos quanto à dose de b-FGF: 0 æg/mL - controle; 50 æg/mL e 125 æg/mL. Após os tratamentos, as amostras foram incubadas a 37º C e 98% de umidade com 2mL de meio Eagle, sendo 1mL com fibroblastos de linhagem contínua (células McCoy) na concentração de 1 x 105 células/mL e 1mL meio sem células, por 24 h. Após as 24 h, as amostras foram submetidas a preparo de rotina para MEV e então, fotomicrografadas nos aumentos de 500X (densidade celular) e 1000X (morfologia celular). Em seguida, as fotomicrografias foram avaliadas por 3 examinadores treinados, calibrados, independentes e cegos, os quais verificaram morfologia e densidade celular segundo os escores propostos por Gamal et al. (1998) e Jenkins et al. (1988), respectivamente. A aplicação da Análise de Regressão pela Técnica da Árvore demonstrou haver diferenças estatisticamente significantes para a densidade celular (p<0,0001) entre os grupos EDTA, tetraciclina e controle, sendo que também houve diferenças entre as doses de 0/50 æg e 125 æg de b-FGF nas amostras condicionadas com EDTA (p<0,0001) e entre as doses de 0 e 50/125 æg de b-FGF nas amostras condicionadas com tetraciclina... .The aim of this study was to evaluate in vitro the effect of the root surface conditioning with basic fibroblast growth factor (b-FGF) about morphology and density of fibroblasts. Dentin slices of with 4 mm2 of surface area were obtained from roots of teeth extracted due to severe periodontal involvment. These were scaled and sterilized. Ninety samples were randomly distributed into 3 groups according to treatment before application of b-FGF: non-treated - control; 50mg/mL of tetracycline HCl and EDTA 24%. The thirty samples of each group were distributed into 3 subgroups according to the concentration of b-FGF: 0 æg/mL - control; 50 æg/mL and 125 æg/mL. After treatments, the samples were incubated at 37ºC and 98% humidity with 1mL of Eagle Medium with 1 x 105 cells/mL of fibroblast from continuos lineage (McCoy Cells) plus 1mL this solution without cells during 24 hours. The samples were submitted to routine preparation for SEM and photographed at 500x (density celular) and 1000x (morphology celular). Three independent and blind examiners evaluated the fibroblast`s morphology and density, according to Gamam et al. (1998) and Jenkins et al. (1998), respectively. Classification and Regression Trees test results indicated significant differences on the density (p<0,0001) among EDTA, tetracycline and control groups with also differences between concentrations of 0/50æg and 125æg of b-FGF at the samples conditioning with EDTA (p<0,0001) and between concentrations of 0 and 50/125 æg of b-FGF at the samples conditioning with tetracycline(p<0,0001). The results of this test to morphology indicated significant differences between treatment or non-treatment with b-FGF, and that concentration of 125 æg demonstrated to be more favorable than the concentration of 50 æg. In conclusion, the treatment of root surfaces with b-FGF influenced the density... (Complete abstract, click electronic address below)

Efeitos da combinação do fator de crescimento de fibroblastos básico e fator de transformação de crescimento beta na proliferação, expressão de genes para colágeno tipos I e III, metaloproteases-1 e -2 e TIMPs 1,2 e 3, e na modulação da síntese destes próprios fatores de crescimento pelas células do ligamento periodontal de humanos

O objetivo do presente estudo foi avaliar o efeito da combinação do fator de crescimento de fibroblastos básico (b-FGF) e fator de transformação de crescimento beta (TGF- beta) na proliferação, expressão de genes para colágeno tipos I e III, metaloproteases -1 e -2 e TIMPs 1, 2 e 3, e na síntese destes próprios fatores de crescimento pelas células do ligamento periodontal de humanos. A avaliação da proliferação celular foi realizada após os períodos de 24 e 48h na presença dos fatores de crescimento, através da mensuração do nível de MTS reduzido a formazan pelas células viáveis, e a síntese de b-FGF e TGF-b pelo teste ELISA empregando a técnica sandwich. Conclui-se que as associações do bFGF e do TGF-b atuaram de maneira dose-dependente no estímulo à proliferação celular, o aumento na expressão de genes para colágeno e TIMPs e redução para as metalproteases e modulação da síntese deles próprios.The aim of this study was to evaluate the effect of association of basic fibroblast growth factor and transforming growth factor beta on the proliferation, expression of colagen type I e III, matrix metalloproteinases-1 e -2 e TIMPs 1, 2 e 3, and on the modulation of the syntheses oh these growth factors by humans periodontal ligament cells. The cellular proliferation was evaluated to 24 and 48 hours of incubation by the colorimetric method. The synthese of bFGF and TGF-ß was verificated by ELISA method, using a sandwich techinique, and mRNA expression by Real Time - PCR. In conlusion, the associations of bFGF e TGF-ß influenced of dose-dependent manner on the cellular proliferation, on the increasing of the expression to collagen and TIMPs, and on the reduction to metaloproteinases and, the modulation of these own synthesis.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Processo de reparo em feridas de extração dentária em camundongos tratados com o complexo Symphytum officinale e Calendula officinallis

Medicamentos homeopáticos como o Symphytum officinalle e a Calendula officinallis são dotados de propriedades anti-sépticas, antiinflamatória, cicatrizantes e também agem como promotores da consolidação de fraturas ósseas. Neste trabalho, uniram-se esses dois medicamentos similares em um complexo para verificar o seu efeito no reparo em feridas de extração dentária em camundongos. O complexo Symphytum officinalle e Calendula officinallis nas potências de 6CH e 3CH, respectivamente, foi ministrado por via oral ao grupo tratado durante 5 dias antes e após a extração do incisivo superior direito. No grupo controle, administraram-se 5ml de álcool etílico a 70% diluídos em 30 ml de soro fisiológico. Após a proservação, os animais foram sacrificados, a maxila direita separada da esquerda, fixada e processada para inclusão em parafina. Após a microtomia, os cortes obtidos foram corados pela H/E. A análise histológica mostrou que, tanto no grupo controle como no tratado, o alvéolo dentário estava preenchido por tecido de granulação e tecido ósseo neoformado, com graus variáveis de maturação, rico em osteócitos. No entanto, nos animais tratados, o processo de reparo em feridas após extração dentária do incisivo superior direito mostrou um avanço progressivo de neoformação óssea mais acentuado quando comparado ao grupo controle, em tempos equivalentes. Estes resultados enfatizam as propriedades biológicas do complexo Symphytum officinalle e Calendula officinallis e sua possível utilização como recurso terapêutico na Odontologia.Homeopathic medicines as Symphytum officinale and Calendula officinallis are endowed with antiseptic, antiphlogistic and cicatrizant properties and promoter of the consolidation of bone fracture. This research combined these two similar medicines in a compound to examine its action in the repair of tooth extraction sores in mice. The compound Symphytum offic. and Calendula offic. at the respective potencys of 6CH and 3CH was orally administered to the treated group during 5 days before and after the extraction of the rigth upper incisor. To the control group were administered 5 ml of ethylic alchol 70% diluted in 30 ml of physiologic serum. After a period of expectation, the animals were sacrificed, the right maxila was separated of the left maxila, this was fixed and the laboratories techniques were realized for inclusion in paraffin. After that, the piece was cut in the microtome, and the laminas were dyed by H/E. The analysis showed that the control and treated group exhibited the dental alveolus fulfilled with granulation tissue and neoformed bone tissue with variable degrees of maturation, abundant in osteocites. However, at the treated animal the healing process of the sore after the extraction of the rigth upper incisor showed a bone neoformation very pronounced when compared with the control group at equivalent times. Those results showed the biological properties of the compound Symphytum offic. and Calendula offic. and its utilization as a therapeutical help in Odontology

Isotretinoin: action on mice tooth germs and palate

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Periodontal conditions of teeth presenting pathologic migration.

The aim of the present study was to evaluate the periodontal conditions of anterior teeth that presented pathologic migration in patients with chronic periodontitis and to compare periodontal destruction in migrated versus non-migrated teeth. The sample included 32 patients of both sexes (mean age: 46.0 +/- 11.6 years) diagnosed with generalized chronic periodontitis and selected on the basis of the presence of pathologic migration in one or more anterior teeth. This migration was classified according to the following categories: facial flaring, diastema, proximal tilting, rotation or extrusion. The periodontal parameters recorded were clinical attachment loss (CAL) and percentage of radiographic bone loss (BL). Mean CAL of 5.50 +/- 2.20 mm and mean BL of 41.90 +/- 15.40% were found in 115 teeth assessed. The most frequent type of migration was facial flaring (34.80%), followed by diastema (27.00%). Extrusion was hardly observed in the sample (4.30%). However, greater severity of BL and CAL were observed in teeth with this type of migration (59.44% and 8.42 mm, respectively), and in teeth with facial flaring (45.17% of BL and 6.07 mm of CAL). Kruskal-Wallis test indicated that BL presented by teeth with extrusion or facial flaring was greater than that observed in rotated or tilted teeth (p < 0.05), while there was no difference between groups regarding CAL (p = 0.11). It was observed that anterior teeth with pathologic migration presented greater CAL and BL (5.1 mm and 40%) than non-migrated teeth (4.1 and 31%). The study indicated that the most prevalent kind of pathologic migration is facial flaring, which was associated to higher level of bone loss