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Successful treatment for any type of carcinoma largely depends on understanding the patterns of invasion and migration. For oral squamous cell carcinoma (OSCC), these processes are not entirely understood as of now. Invadopodia and podosomes, called invadosomes, play an important role in cancer cell invasion and migration. Previous research has established that cortactin (CTTN) is a major inducer of invadosome formation. However, less is known about the expression patterns of CTTN and other genes related to it or invadopodia formation in OSCC during tumor progression in particular. In this study, gene expression patterns of CTTN and various genes (n = 36) associated with invadopodia formation were analyzed to reveal relevant expression patterns and give a comprehensive overview of them. The genes were analyzed from a whole genome dataset of 83 OSCC samples relating to tumor size, grading, lymph node status, and UICC (Union for Internatioanl Cancer Control). The data revealed significant overexpression of 18 genes, most notably CTTN, SRC (SRC proto-onocogene, non-receptor tyrosine kinase), EGFR (epidermal growth factor receptor), SYK (spleen associated tyrosine kinase), WASL (WASP like actin nucleation promotion factor), and ARPC2 (arrestin beta 1) due to their significant correlation with further tumor parameters. This study is one of the first to summarize the expression patterns of CTTN and related genes in a complex group of OSCC samples.
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Background and Objectives: This study aims to identify the minor allele of the single nucleotide polymorphisms (SNPs) DAB2IP rs7025486, IL6R rs2228145, CDKN2BAS rs10757278, LPA rs3798220, LRP1 rs1466535, and SORT1 rs599839 in order to assess the risk of abdominal aortic aneurysm (AAA) formation and define the linkage among these SNPs. Materials and Methods: A case-control study with AAA patients (AAA group) and non-AAA controls (control group) was carried out in a study population. DNA was isolated from whole blood samples; the SNPs were amplified using PCR and sequenced. Results: In the AAA group of 148 patients, 87.2% of the patients were male, 64.2% had a history of smoking, and 18.2% had relatives with AAA. The mean ± SD of age, BMI, and aneurysmal diameter in the AAA group were 74.8 ± 8.3 years, 27.6 ± 4.6 kg/m2, and 56.2 ± 11.8 mm, respectively. In comparison with 50 non-AAA patients, there was a significantly elevated presence of the SNPs DAB2IP rs7025486[A], CDKN2BAS rs10757278[G], and SORT1 rs599839[G] in the AAA group (p-values 0.040, 0.024, 0.035, respectively), while LPA rs3798220[C] was significantly higher in the control group (p = 0.049). A haplotype investigation showed that the SNPs DAB2IP, CDKN2BAS, and IL6R rs2228145[C] were significantly elevated in the AAA group (p = 0.037, 0.037, and 0.046) with minor allele frequencies (MAF) of 25.5%, 10.6%, and 15.4%, respectively. Only DAB2IP and CDKN2BAS showed significantly higher occurrences of a mutation (p = 0.028 and 0.047). Except for LPA, all SNPs were associated with a large aortic diameter in AAA (p < 0.001). Linkage disequilibrium detection showed that LPA to DAB2IP, to IL6R, to CDKN2BAS, and to LRP1 rs1466535[T] had D' values of 70.9%, 80.4%, 100%, and 100%, respectively. IL6R to LRP1 and to SORT1 had values for the coefficient of determination (r2) of 3.9% and 2.2%, respectively. Conclusions: In the investigated study population, the SNPs CDKN2BAS rs10757278, LPA rs3798220, SORT1 rs599839, DAB2IP rs7025486, and IL6R rs2228145 were associated with the development of abdominal aortic aneurysms. Individuals with risk factors for atherosclerosis and/or a family history of AAA should be evaluated using genetic analysis.
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Aneurisma da Aorta Abdominal , Predisposição Genética para Doença , Humanos , Masculino , Idoso , Idoso de 80 Anos ou mais , Feminino , Estudos de Casos e Controles , Polimorfismo de Nucleotídeo Único/genética , Aneurisma da Aorta Abdominal/genética , Fatores de Risco , Inflamação , Apoptose , Colesterol , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Bone regeneration after oral and maxillofacial surgery is a long-term process, which involves various mechanisms. Recently, cold atmospheric plasma (CAP) has become known to accelerate wound healing and have an antimicrobial effect. Since the use of CAP in dentistry is not yet established, the aim of the present study was to investigate the effect of CAP on human calvaria osteoblasts (HCO). HCO were treated with CAP for different durations of time and distances to the cells. Cell proliferation was determined by MTT assay and cell toxicity by LDH assay. Additionally, RT-qPCR was used to investigate effects on osteogenic markers, such as alkaline phosphatase (ALP), bone morphogenic protein (BMP)2, collagen (COL)1A1, osteonectin (SPARC), osteoprotegerin (OPG), osterix (OSX), receptor activator of NF-κB (RANK), RANK Ligand (RANKL), and Runt-related transcription factor (RUNX)2. There were small differences in cell proliferation and LDH release regarding treatment duration and distance to the cells. However, an increase in the expression of RANK and RANKL was observed at longer treatment times. Additionally, CAP caused a significant increase in mRNA expression of genes relevant to osteogenesis. In conclusion, CAP has a stimulating effect on osteoblasts and may thus represent a potential therapeutic approach in the regeneration of hard tissue defects.
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Osteogênese , Gases em Plasma , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Osteoblastos/metabolismo , Osteogênese/genética , Osteoprotegerina/metabolismo , Gases em Plasma/metabolismo , Gases em Plasma/farmacologia , Ligante RANK/metabolismoRESUMO
A fundamental step for cell growth and differentiation is the cell adhesion. The purpose of this study was to determine the adhesion of different cell lineages, adipose derived stromal cells, osteoblasts, and gingival fibroblast to titanium and zirconia dental implants with different surface treatments. Primary cells were cultured on smooth/polished surfaces (titanium with a smooth surface texture (Ti-PT) and machined zirconia (ZrO2-M)) and on rough surfaces (titanium with a rough surface texture (Ti-SLA) and zirconia material (ZrO2-ZLA)). Alterations in cell morphology (f-actin staining and SEM) and in expression of the focal adhesion marker were analysed after 1, 7, and 14 days. Statistical analysis was performed by one-way ANOVA with a statistical significance at p = 0.05. Cell morphology and cytoskeleton were strongly affected by surface texture. Actin beta and vimentin expressions were higher on rough surfaces (p < 0.01). Vinculin and FAK expressions were significant (p < 0.05) and increased over time. Fibronectin and laminin expressions were significant (p < 0.01) and did not alter over time. Strength of cell/material binding is influenced by surface structure and not by material. Meanwhile, the kind of cell/material binding is regulated by cell type and implant material.
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Tecido Adiposo/citologia , Implantes Dentários , Células Estromais/citologia , Células Cultivadas , Fibroblastos/citologia , Fibronectinas/genética , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/fisiologia , Gengiva/citologia , Humanos , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Propriedades de Superfície , Titânio/química , Vinculina/genética , Vinculina/metabolismo , Zircônio/químicaRESUMO
The aim of this study was to evaluate the biocompatibility of two comparatively new calcium silicate containing sealers (MTA-Fillapex and BioRoot-RCS) with that of two established sealers (AH-Plus, epoxy resin-based; Pulp-Canal-Sealer, zinc oxide eugenol containing). Human periodontal ligament cells (PDL-cells) were brought in contact with eluates from freshly mixed and set sealer. The sealers were mixed strictly according to the manufacturers' instructions and identically samples were produced. 1:1, 1:2, and 1:10 dilutions of sealers extract were used. Extracts from freshly mixed sealer were added to the PDL-cells on day one to simulate a clinical scenario. Subsequently, at 24 h, 7, 14, and 21 days extracts form set sealers were used for PDL-cell culturing. PDL-cell viability was analyzed by living-cell-count, MTT-assay, and living/dead-staining, cytotoxicity by LDH-assay, and changes by Richardson-staining. All data were statistically evaluated by one way ANOVA and a posthoc analysis with Bonferroni-Holm testing (p < 0.05). In contact with BioRoot-RCS a regeneration of the PDL-cells were observed over time. This sealer showed the lowest toxicity in a freshly mixed and set state (p < 0.05). MTA-Fillapex and Pulp-Canal-Sealer were cytotoxic in a fresh as well as in a set state, whereas AH-Plus was cytotoxic in a freshly mixed state, but not when the sealer was set. BioRoot-RCS is biocompatible and bioactive because it seems to have a positive influence on the PDL-cell metabolism. Pulp Canal Sealer and MTA-Fillapex showed no biocompatibility in contact with PDL-cells at all. Freshly mixed AH Plus is less biocompatible on PDL than in a set state.
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Ligamento Periodontal/citologia , Materiais Restauradores do Canal Radicular/farmacologia , Compostos de Alumínio/farmacologia , Materiais Biocompatíveis , Compostos de Cálcio/farmacologia , Sobrevivência Celular , Células Cultivadas , Combinação de Medicamentos , Resinas Epóxi/farmacologia , Humanos , Técnicas In Vitro , Teste de Materiais , Dente Serotino , Óxidos/farmacologia , Cimento de Policarboxilato/farmacologia , Povidona/farmacologia , Silicatos/farmacologia , Cimento de Óxido de Zinco e Eugenol/farmacologiaRESUMO
For the guided regeneration of periimplant hard and soft tissues, human adipose-derived stromal cells (hADSC) seem to be a promising source for mesenchymal stromal cells. For this, the proliferation and differentiation of hADSC were evaluated on titanium and zirconia dental implants with different surface treatments. Results were compared to edaphic cells as human osteoblasts (hOB) and human gingival fibroblasts (HGF). Primary cells were cultured on (1) titanium implants with a polished surface (Ti-PT), (2) sandblasted and acid-etched titanium (Ti-SLA), (3) sandblasted and alkaline etched zirconia (ZrO2-ZLA) and (4) machined zirconia (ZrO2-M). The cell proliferation and differentiation on osteogenic lineage were assessed after 1, 7 and 14 days. Statistical analysis was performed by one-way ANOVA and a modified Levene test with a statistical significance at p = 0.05. PostHoc tests were performed by Bonferroni-Holm. Zirconia dental implants with rough surface (ZrO2-ZLA) showed the highest proliferation rates (p = 0.048). The osteogenic differentiation occurred early for zirconia and later for titanium implants, and it was enhanced for rough surfaces in comparison to polished/machined surfaces. Zirconia was more effective to promote the proliferation and differentiation of hADSCs in comparison to titanium. Rough surfaces were able to improve the biological response for both zirconia and titanium.
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Adipócitos/citologia , Implantes Dentários , Células Estromais/citologia , Titânio/farmacologia , Zircônio/farmacologia , Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibroblastos/citologia , Gengiva/citologia , Humanos , Osteoblastos/citologia , Cultura Primária de Células , Células Estromais/efeitos dos fármacos , Propriedades de Superfície , Titânio/química , Zircônio/químicaRESUMO
Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer development and metastasis, supporting the idea that other developmental markers may have application as biomarkers for BC.
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Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Transcriptoma , Fatores Etários , Animais , Neoplasias da Mama/genética , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismo , Fator 4 Semelhante a Kruppel , Lactação/genética , Lactação/metabolismo , Macaca fascicularis , Menopausa/genética , Menopausa/metabolismo , Gravidez , Receptores de Esteroides/metabolismo , Maturidade Sexual/genética , Transdução de SinaisRESUMO
The circulating low-density lipoprotein concentration in blood can be reduced by the administration of statins. Frequently simvastatin (SV) is prescribed. Due to the reported pleiotropic effects of SV the aim of this study was to evaluate mineralization effects on human adipose tissue-derived stromal cells upon administration of SV. After informed consent human adipose tissue-derived stromal cells were obtained from tissue surplus of regular treatments of 14 individuals. According to established protocols after adding various SV concentrations (0.01 µM, 0.1 µM, 1.0 µM, 2.0 µM), alkaline phosphate (osteoblastic marker), mineralization capability and viability were determined at day 18, 21 and 28. The Kruskal-Wallis test was performed for statistical analysis. After adding SV a dose-dependent significant decreased viability and levels of alkaline phosphatase (p < 0.01) and a significantly increased mineralization (p < 0.01) of the primary cultures was recognized during the late mineralization stage. Mineralization of the human adipose tissue-derived stromal cells was induced by SV, possibly originated from alternative pathways than the alkaline phosphatase pathway. Further investigations should be performed regarding switching into the osteoblastic differentiation and as a possible source of cells that can be used as the basis for a potential bone graft substitute, which may allow an extension of the field of application.
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Fosfatase Alcalina , Sinvastatina , Tecido Adiposo , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Osteoblastos/metabolismo , Osteogênese , Sinvastatina/metabolismo , Sinvastatina/farmacologia , Células Estromais/metabolismoRESUMO
Simvastatin (SV) is an often prescribed statin reducing the LDL-concentration in circulating blood. The aim of this study was to evaluate the pleiotropic effects of SV to primary human odontoblast-like cells. Twenty four wisdom teeth of different subjects were extracted and the pulp tissue was removed and minced under sterile conditions. After mincing, the requested cells were passaged according to established protocols. Osteoblastic marker (ALP conversion), viability and mineralization were determined at days 14, 17 and 21 after simvastatin exposition (0.01 µM, 0.1 µM, 1.0 µM, 2.0 µM). The sample size per group was 24 cultures with three replicates per culture for ALP-conversion and mineralization and 6 replicates for viability. A Kruskal-Wallis test was used for statistical analysis. After adding SV, viability was significantly (p < 0.01) decreased in a time- and dose-dependent manner, whereas after 21 days, mineralization was significant (p < 0.01). ALP-conversion in groups with SV concentrations of 1 and 2 µM SV was significantly (p < 0.01) increased. Pleiotropic effects regarding mineralization in higher SV concentrations were possibly induced via alternative mineralization pathways as almost equal elevations of ALP conversion were not evident in the control and experimental groups.
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BACKGROUND: Frequently statins were administered to reduce the LDL-concentration in circulating blood. Especially simvastatin (SV) is an often prescribed statin. Pleiotropic effects of these drugs were reported. Thus, the aim of this study was to evaluate effects of SV on osteoblastic mineralization. METHODS: After informed consent primary osteoblasts were collected from tissue surplus after treatment of 14 individuals in the Department of Cranio-Maxillofacial Surgery, University Hospital Münster. The cells were passaged according to established protocols. Viability, mineralization capability and osteoblastic marker (alkaline phosphatase) were determined at day 9, 13 and 16 after adding various SV concentrations (0.05 µM, 0.1 µM, 0.5 µM, 1.0 µM). Statistical analysis was performed using the Kruskal-Wallis-test. RESULTS: The cell cultures showed a time and dose-dependent significantly decreased viability (p < 0.01) and a significantly increased mineralization (p < 0.01) in a late mineralization stage after adding SV. The typical alteration of the alkaline phosphatase (ALP) levels during osteogenic differentiation was not recognizable. CONCLUSIONS: The pleiotropic effects found for different SV concentrations were possibly originated from other mineralization pathways beside the ALP induced one. Additionally, possible alterations of protein expression levels during mineralization and investigation of possible deviating application of SV in other treatment fields can be considered after gaining a deeper insight in the affected mechanisms.
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Anticolesterolemiantes , Osteoblastos , Osteogênese , Sinvastatina , Adulto , Anticolesterolemiantes/efeitos adversos , Diferenciação Celular , Proliferação de Células , Feminino , Humanos , Masculino , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Sinvastatina/efeitos adversosRESUMO
BACKGROUND: Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDAC) are well known. Genetic and epigenetic data were correlated with tumor biology to find specific alterations responsible for invasion and metastasis in pancreatic ductal adenocarcinomas. METHODS: A total of 16 human PDAC cell lines were used in murine orthotopic PDAC models. By means of standardized dissemination scores, local invasion and metastatic spread were assessed. mRNA and microRNA expression were studied by microarray and TaqMan low-density array. Quantitative real-time-polymerase chain reaction and flow cytometry were used for expression validation. RESULTS: CD40 was detected as a relevant target gene for differentially expressed miRNAs observed in highly invasive and metastatic PDAC only. A significant overexpression (P < .05) of CD40-related miRNAs miR-224 and miR-486 was detected in highly invasive and metastatic PDAC, whereas CD40 mRNA expression was not significantly altered. Instead, CD40 protein expression at cell surfaces of these highly invasive and metastatic PDAC was significantly reduced (P < .01). CONCLUSIONS: Epigenetic alterations with upregulated CD40-targeting miR-224 and miR-486 are related to downregulated CD40 protein expression at cell surfaces in highly invasive and metastatic PDAC. Thus, miRNA-regulated CD40 expression seems to play an important role in progression of PDAC. These data suggest a diagnostic and therapeutic potential for CD40 and/or its targeting miRNAs in PDAC.
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Adenocarcinoma/genética , Antígenos CD40/genética , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antígenos CD40/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Progressão da Doença , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To evaluate the effects of different titanium particle concentrations on viability of human calvarial osteoblasts and human gingival fibroblasts. MATERIALS AND METHODS: Primary human calvarial osteoblasts (HCO, 3H Biomedical) and human gingival fibroblasts (HGF-1, ATCC) were cultivated and allowed to adhere for 24 hours. Titanium powder concentrations (0.01 to 1.0 mg/mL) were added, and samples were analyzed at three time points (24 hours, 7 days, 21 days). Cell viability was analyzed using living cell count, proliferation (MTT) assay, and a live/dead staining. Cytotoxic effects were evaluated using lactated dehydrogenase assay. Qualitative analysis of cell viability was performed. In addition, scanning electron microscopy (SEM) analysis was performed. Release of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-±) was estimated with Human IL-6 / Human TNF-± ELISA. RESULTS: Titanium concentrations of 0.1 mg/mL and 1.0 mg/mL showed medium- and long-term effects on cell growth and proliferation rates. Cytotoxic effects by release of lactate dehydrogenase were observable during the first 24 hours. Human gingival fibroblast cells showed a release factor between 2.6 to 3.4. Titanium powder seemed to be more cytotoxic to human gingival fibroblast cells than to human calvarial osteoblast cells. For human calvarial osteoblasts, only the highest concentration showed cytotoxic effects with a release factor of 2.7. Human calvarial osteoblasts secreted IL-6 only during the first 24 hours and only in the highest titanium concentration, whereas human gingival fibroblasts secreted IL-6 during the entire period. The lowest titanium concentration showed stronger secretion of IL-6 compared to control. Incorporation of smaller and single titanium particles by cells was identified under SEM analysis. CONCLUSION: Cell viability is negatively correlated with titanium concentration. Further, titanium debris might lead to an inflammatory biologic response of dental peri-implant tissue. Also, cells interact with the debris, eg, with incorporation of particles.
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Implantes Dentários , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Titânio/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Humanos , Interleucina-6/metabolismo , Microscopia Eletrônica de Varredura , Osteoblastos/metabolismo , Crânio/citologia , Fator de Necrose Tumoral alfa/análiseRESUMO
Background: Ectodermal dysplasia describes a heterogeneous group of hereditary, congenital malformations involving developmental dystrophies of ectodermal structures. The aim of this study was to analyse the oral health-related quality of life (OHRQoL) in people with ectodermal dysplasia and to evaluate the influence of different variables. Methods: The study was designed as an anonymous epidemiological survey study among people with ectodermal dysplasia to evaluate oral symptoms, satisfaction with the health system and their respective OHRQoL using the validated German version of the OHIP-14 (Oral Health Impact Profile) questionnaire. Results: When asked about oral symptoms, 110 of the participants provided responses, of which 109 (99.09%) described oral symptoms. The average age of the female participants at the time of diagnosis was 17.02 years (range: 0 to 48 years), the average age of men was 5.19 years (range: 0 to 43 years). The average OHIP-14 overall score for female participants was 12.23 points (SD: 12.39), for male participants an average OHIP score of 11.79 points was recorded (SD: 11.08 points). Difficulty in finding a dentist (p = 0.001), and the dissatisfaction with the health system (p = 0.007) showed a negative impact on the OHRQoL. Conclusion: People with ectodermal dysplasia rate their OHRQoL worse than is usually prevalent in the normal German population (4.09 points); women are diagnosed with "ectodermal dysplasia" later than men. Participants who reported difficulties in finding a dentist for treatment exhibited higher OHIP values. Likewise, dissatisfaction with the health system demonstrated a negative impact on the oral health-related quality of life.
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Displasia Ectodérmica/epidemiologia , Displasia Ectodérmica/psicologia , Saúde Bucal/estatística & dados numéricos , Aceitação pelo Paciente de Cuidados de Saúde/psicologia , Qualidade de Vida , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Alemanha/epidemiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Autoavaliação (Psicologia) , Adulto JovemRESUMO
PURPOSE: To evaluate the effects of different titanium particle concentrations on viability of human calvarial osteoblasts and human gingival fibroblasts. MATERIALS AND METHODS: Primary human calvarial osteoblasts (HCO, 3H Biomedical) and human gingival fibroblasts (HGF-1, ATCC) were cultivated and allowed to adhere for 24 hours. Titanium powder concentrations (0.01 to 1.0 mg/mL) were added, and samples were analyzed at three time points (24 hours, 7 days, 21 days). Cell viability was analyzed using living cell count, proliferation (MTT) assay, and a live/dead staining. Cytotoxic effects were evaluated using lactated dehydrogenase assay. Qualitative analysis of cell viability was performed. In addition, scanning electron microscopy (SEM) analysis was performed. Release of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-±) was estimated with Human IL-6 / Human TNF-± ELISA. RESULTS: Titanium concentrations of 0.1 mg/mL and 1.0 mg/mL showed medium- and long-term effects on cell growth and proliferation rates. Cytotoxic effects by release of lactate dehydrogenase were observable during the first 24 hours. Human gingival fibroblast cells showed a release factor between 2.6 to 3.4. Titanium powder seemed to be more cytotoxic to human gingival fibroblast cells than to human calvarial osteoblast cells. For human calvarial osteoblasts, only the highest concentration showed cytotoxic effects with a release factor of 2.7. Human calvarial osteoblasts secreted IL-6 only during the first 24 hours and only in the highest titanium concentration, whereas human gingival fibroblasts secreted IL-6 during the entire period. The lowest titanium concentration showed stronger secretion of IL-6 compared to control. Incorporation of smaller and single titanium particles by cells was identified under SEM analysis. CONCLUSION: Cell viability is negatively correlated with titanium concentration. Further, titanium debris might lead to an inflammatory biologic response of dental peri-implant tissue. Also, cells interact with the debris, eg, with incorporation of particles.
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BACKGROUND: Adult stem cells appear to be a promising subject for tissue engineering, representing an individual material for regeneration of aged and damaged cells. Especially adipose derived stromal cells (ADSC), which are easily to achieve, allow an encouraging perspective due to their capability of differentiating into miscellaneous cell types. Here we describe the in vitro formation of human subcutaneous, visceral and omental ADSC micromasses and compare their histological attributes while being cultivated on collagen membranes. METHODS: Subcutaneous, visceral and omental fat tissue derived cells were isolated and processed according to standard protocols. Positively stained cells for CD13, CD44 and CD90 were cultivated on agarose in order to study micromass formation using a special method of cell tracking. Stained paraffin-embedded micromasses were analysed morphologically before and after being plated on collagen membranes. RESULTS: The micromass formation process was similar in all three tissue types. Subcutaneous fat tissue derived micromasses turned out to develop a more homogeneous and compact shape than visceral and omental tissue. Nevertheless all micromasses adhered to collagen membranes with visible spreading of cells. The immune histochemical (IHC) staining of subcutaneous, visceral and omental ADSC micromasses shows a constant expression of CD13 and a decrease of CD44 and CD 90 expression within 28 days. After that period, omental fat cells don't show any expression of CD44. CONCLUSION: In conclusion micromass formation and cultivation of all analysed fat tissues can be achieved, subcutaneous cells appearing to be the best material for regenerative concepts.
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Células-Tronco/citologia , Células Estromais/citologia , Gordura Subcutânea/citologia , Engenharia Tecidual/métodos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Omento/citologia , Medicina Regenerativa/métodos , Sensibilidade e Especificidade , Células-Tronco/metabolismo , Células Estromais/metabolismo , Gordura Subcutânea/metabolismo , Vísceras/citologiaRESUMO
The aim of this study was to evaluate the effect of an epoxy resin-based (AH-Plus), a zinc oxide eugenol containing (Pulp-Canal-Sealer) and two calcium silicate containing (MTA-Fillapex and BioRoot-RCS) sealers on primary human osteoblasts (hOB) in freshly mixed and set state. All sealers were mixed strictly according to the manufacturers´ instructions and identically samples were produced. In a pretest cytotoxic sealer concentrations were determined. Thus, for the main cell culture study, dilutions of sealer extract 1:1, 1:2, and 1:10 were used. To simulate a clinical scenario, extracts from freshly mixed sealer were added to the cells on day one. Extracts form set sealers were used for subsequent culturing for 24h, 7d, 14d, and 21d. Cell viability was analyzed by living-cell-count, MTT-assay, and living/dead-staining, cytotoxicity by LDH-assay, and changes by Richardson-staining. All data were statistically evaluated by one way ANOVA and a posthoc analysis with Bonferroni-Holm testing (p<0.05). AH-Plus was cytotoxic in a freshly mixed state, but not when the sealer was set. MTA-Fillapex and Pulp-Canal-Sealer were cytotoxic in a fresh as well as in a set state. BioRoot-RCS showed the lowest toxicity in both states; where as a regeneration of the cells could be observed over time (p<0.05). Contact of freshly mixed AH-Plus to osteoblasts should be avoided. Pulp Canal Sealer and MTA-Fillapex showed no biocompatibility in contact with osteoblasts at all. BioRoot-RCS had a positive influence on the cell metabolism (bioactivity) and is biocompatible.
Assuntos
Teste de Materiais , Osteoblastos/metabolismo , Materiais Restauradores do Canal Radicular/farmacologia , Células Cultivadas , Feminino , Humanos , Masculino , Osteoblastos/citologia , Materiais Restauradores do Canal Radicular/efeitos adversosRESUMO
BACKGROUND Rifampin-soaked synthetic prosthetic grafts have been widely used for prevention or treatment of vascular graft infections (VGIs). This in vitro study investigated the effect of the antibiotics daptomycin and vancomycin and the new recombinant bacteriophage endolysin HY-133 on vascular cells, as potential alternatives compared to rifampin. MATERIAL AND METHODS Primary human ECs, vascular smooth muscle cells (vSMC), and fibroblasts were cultivated in 96-well plates and incubated with rifampin, daptomycin, vancomycin, and endolysin HY-133 for 24 h. Subsequently, after washing, cell viability was determined by measuring mitochondrial ATP concentration. Antibiotics were used in their corresponding minimum and maximum serum concentrations, in decimal multiples and in maximum soaking concentration. The experiments were performed in triplicate. RESULTS The 10-fold max serum concentrations of rifampin, daptomycin, and vancomycin did not influence viability of EC and vSMC (100 µg/ml, p>0.170). Higher concentrations of rifampin (>1 mg/ml) significantly (p<0.001) reduced cell viability of all cell types. For the other antibiotics, high concentrations (close to maximum soaking concentration) were most cytotoxic for EC and vSMC and fibroblasts (p<0.001). Endolysin did not display any cytotoxicity towards vascular cells. CONCLUSIONS Results of this in vitro study show the high cytotoxicity of rifampin against vascular cells, and may re-initiate the discussion about the benefit of prophylactic pre-soaking in high concentrations of rifampin. Further studies are necessary to determine the influence of rifampin on the restoration of vessel functionality versus its prophylactic effect against VGIs. Future use of recombinant phage endolysins for alternative prophylactic strategies needs further investigations.
Assuntos
Antibacterianos/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Enxerto Vascular/métodos , Antibacterianos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Daptomicina/farmacologia , Endopeptidases/farmacologia , Células Endoteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Rifampina/farmacologia , Vancomicina/farmacologiaRESUMO
PURPOSE: Therapeutic strategies attacking oral squamous cell carcinoma have not essentially succeeded to improve long-term prognosis and overall survival over the last decades. Therefore, in this study, we aimed to illuminate the molecular regulation of angiogenesis in this tumour entity in order to demask novel markers of prognosis or therapeutic approach. MATERIALS AND METHODS: A panel of significant transcriptional alterations in angiogenic genes of 83 cancer samples was established by comparison to 30 samples of healthy oral mucosa with microarray technique. Immunohistochemistry (IHC) was performed to trace the signalling cascade from gene to protein level. RESULTS: A distinctive expression profile of VEGFA, EFNB2, PECAM1/CD31, ANGPT1 and ANGPT2 was revealed: VEGFA, EFNB2, and ANGPT2 were found overexpressed in 84 % to 95 % of tumour samples. In contrast, the expression of CD31 and ANGPT1 was downregulated in 80 % to 95 % of tumour samples. IHC confirmed results of the microarray analysis. Tumours with lymphatic spread showed higher gene expression rates of VEGFA, EFNB2 and ANGPT2 in moderately differentiated tumours and of VEGFA and EFNB2 in small tumours, respectively. The ANGPT1/ ANGPT2 transcription ratio was found decreased in larger tumours and especially in tumours without lymphatic spread. CONCLUSIONS: A characteristic expression profile of angiogenic markers was established. The specific overexpression of EFNB2 in small tumours with lymphatic spread and the typical decrease of the ANGPT1/ ANGPT2 ratio in larger tumours give weight to EFNB2 and angiopoietins as prognostic factors and potential therapeutic targets.
Assuntos
Angiopoietina-1/genética , Angiopoietina-2/genética , Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Neovascularização Patológica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Mucosa Bucal/patologia , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologia , Prognóstico , Estudos Retrospectivos , Medição de Risco , Análise de SobrevidaRESUMO
The gene queD encoding quercetinase of Streptomyces sp. FLA, a soil isolate related to S. eurythermus (T), was identified. Quercetinases catalyze the 2,4-dioxygenolytic cleavage of 3,5,7,3',4'-pentahydroxyflavone to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The queD gene was expressed in S. lividans and E. coli, and the recombinant hexahistidine-tagged protein (QueDHis(6)) was purified. Several flavonols were converted by QueDHis(6), whereas CO formation from the 2,3-dihydroflavonol taxifolin and the flavone luteolin were not observed. In contrast to bicupin quercetinases from Aspergillus japonicus and Bacillus subtilis, and bicupin pirins showing quercetinase activity, QueD of strain FLA is a monocupin exhibiting 35.9% sequence identity to the C-terminal domain of B. subtilis quercetinase. Its native molecular mass of 63 kDa suggests a multimeric protein. A queD-specific probe hybridized with fragments of genomic DNA of four other quercetin degrading Streptomyces strains, but not with DNA of B. subtilis. Potential ORFs upstream of queD probably code for a serine protease and an endoribonuclease; two ORFs downstream of queD may encode an amidohydrolase and a carboxylesterase. This arrangement suggests that queD is not part of a catabolic gene cluster. Quercetinases might play a major role as detoxifying rather than catabolic enzymes.
Assuntos
Dioxigenases , Flavonóis/metabolismo , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Monóxido de Carbono/metabolismo , Dioxigenases/química , Dioxigenases/genética , Dioxigenases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Quercetina/metabolismo , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces lividans/enzimologia , Streptomyces lividans/genéticaRESUMO
Quinaldine 4-oxidase (Qox), which catalyzes the hydroxylation of quinaldine to 1H-4-oxoquinaldine, is a heterotrimeric (LMS)2 molybdo-iron/sulfur flavoprotein belonging to the xanthine oxidase family. Variants of Qox were generated by site-directed mutagenesis. Replacement in the large subunit at E736, which is presumed to be located close to the molybdenum, by aspartate (QoxLE736D) resulted in a marked decrease in kcat app for quinaldine, while Km app was largely unaffected. Although a minor reduction of the glutamine substituted variant QoxLE736Q by quinaldine occurred, its activity was below detection, indicating that the carboxylate group of E736 is crucial for catalysis. Replacement of cysteine ligands C40, C45, or C60 (FeSII) and of the C120 or C154 ligands to FeSI in the small subunit of Qox by serine led to decreased iron contents of the protein preparations. Substitutions C40S and C45S (Fe1 of FeSII) suppressed the characteristic FeSII EPR signals and significantly reduced catalytic activity. In QoxSC154S (Fe1 of FeSI), the g-factor components of FeSI were drastically changed. In contrast, Qox proteins with substitutions of C48 and C60 (Fe2 of FeSII), and of the C120 ligand at Fe2 of FeSI, retained considerable activity and showed less pronounced changes in their EPR parameters. Taken together, the properties of the Qox variants suggest that Fe1 of both FeSI and FeSII are the reducible iron sites, whereas the Fe2 ions remain in the ferric state. The location of the reducible iron sites of FeSI and FeSII appears to be conserved in enzymes of the xanthine oxidase family.