RESUMO
The effects of compressive strain during orthodontic treatment on gene expression profiles of periodontal ligament fibroblasts (PDLFs) have mostly been studied in 2D cell culture. However, cells behave differently in many aspects in 3D culture. Therefore, the effect of pressure application on PDLFs in different 3D structures was investigated. PDLFs were either conventionally seeded or embedded into different 3D structures (spheroids, Mebiol® gel, 3D scaffolds) and exposed to compressive force or incubated without pressure. For one 3D scaffold (POR), we also tested the effect of different compressive forces and application times. Expression of an angiogenic gene (VEGF), a gene involved in extracellular matrix synthesis (COL1A2), inflammatory genes (IL6, PTGS2), and genes involved in bone remodelling (OPG, RANKL) were investigated by RT-qPCR. Depending on the used 3D cell culture model, we detected different effects of compressive strain on expression profiles of PDLFs. COL1A2 was downregulated in all investigated 3D culture models. Angiogenetic and proinflammatory genes were regulated differentially between models. In 3D scaffolds, regulation of bone-remodelling genes upon compressive force was contrary to that observed in 3D gels. 3D cell culture models provide better approximations to in vivo physiology, compared with conventional 2D models. However, it is crucial which 3D structures are used, as these showed diverse effects on the expression profiles of PDLFs during mechanical strain.
Assuntos
Fibroblastos/metabolismo , Ligamento Periodontal/citologia , Engenharia Tecidual/métodos , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Fibroblastos/citologia , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Pressão , Cultura Primária de Células/métodos , Ligante RANK/genética , Ligante RANK/metabolismo , Alicerces Teciduais/química , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The maxilla is formed by the medial nasal and maxillary processes fusion. The dental lamina develops from 2 origins connecting in the lateral incisor. The maxillary lateral incisor region is often affected by dental anomalies and clefting. It is possible that genes involved in oral cleft could also be associated with a variety of phenotypic variations in the maxillary lateral incisor. In this phenotype-genotype study, we explored the association between polymorphisms in the oral-cleft-related genes BMP2 and BMP4 and root curvature of maxillary lateral incisors.Cross-sectional study.Universities and private clinics.Panoramic radiographs and DNA from 231 patients were analyzed.Schneider method (1971) was applied to estimate the degree of root curvature of the maxillary lateral incisors and to classify the root as straight (5° or less) or curved (higher than 5°). Genetic polymorphisms in BMP2 (rs235768 and rs1005464) and BMP4 (rs17563) were genotyped. Statistical analysis was performed.A total of 401 teeth (199 left and 202 right) were evaluated. Genetic analysis demonstrated trends toward association for the rs1005464 in BMP2 (P = .025) in co-dominant model and in dominant model (P = .026) for left incisors. The rs235768 in BMP2 showed trends toward association with the degree of root curvature in left incisors in the recessive model (P = .031). rs17563 in BMP4 also showed trends toward association with the degree of the root curvature in left incisors (P = .019).BMP2 (rs235768 and rs1005464) and BMP4 (rs17563) might be involved in maxillary lateral incisor root curvature.
RESUMO
BACKGROUND: In orthodontic tooth movement (OTM), pseudo-inflammatory processes occur that are similar to those of nicotine-induced periodontitis. Previous studies have shown that nicotine accelerates OTM, but induces periodontal bone loss and dental root resorption via synergistically increased osteoclastogenesis. This study aimed to investigate the role of hypoxia-inducible factor 1 alpha (HIF-1α) in nicotine-induced osteoclastogenesis during OTM. MATERIALS/METHODS: Male Fischer-344 rats were treated with l-Nicotine (1.89 mg/kg/day s.c., N = 10) or NaCl solution (N = 10). After a week of premedication, a NiTi spring was inserted to mesialize the first upper left molar. The extent of dental root resorption, osteoclastogenesis, and HIF-1α protein expression was determined by (immuno)histology, as well as bone volume (BV/TV) and trabecular thickness (TbTh) using µCT. Receptor activator of nuclear factor of activated B-cells ligand (RANK-L), osteoprotegerin (OPG), and HIF-1α expression were examined at the protein level in periodontal ligament fibroblasts (PDLF) exposed to pressure, nicotine and/or hypoxia, as well as PDLF-induced osteoclastogenesis in co-culture experiments with osteoclast progenitor cells. RESULTS: Nicotine favoured dental root resorptions and osteoclastogenesis during OTM, while BV/TV and TbTh were only influenced by force. This nicotine-induced increase does not appear to be mediated by HIF-1α, since HIF-1α was stabilized by force application and hypoxia, but not by nicotine. The in vitro data showed that the hypoxia-induced increase in RANK-L/OPG expression ratio and PDLF-mediated osteoclastogenesis was less pronounced than the nicotine-induced increase. CONCLUSIONS: Study results indicate that the nicotine-induced increase in osteoclastogenesis and periodontal bone resorption during OTM may not be mediated by hypoxic effects or HIF-1α stabilization in the context of nicotine-induced vasoconstriction, but rather by an alternative mechanism.
Assuntos
Perda do Osso Alveolar , Reabsorção Óssea , Reabsorção da Raiz , Animais , Reabsorção Óssea/induzido quimicamente , Masculino , Nicotina/toxicidade , Osteoclastos , Ligamento Periodontal , Ligante RANK , Ratos , Reabsorção da Raiz/etiologia , Técnicas de Movimentação Dentária/efeitos adversosRESUMO
For accurate interpretation of quantitative real-time PCR (qPCR) data, stable reference genes are essential for normalization of target genes. To date, there is no information on reliable housekeeping genes in CD4+ T cells in a three-dimensional (3D) matrix under pressure stimulation. This in vitro study describes for the first time a method for pressure stimulation of CD4+ T cells in a 3D matrix in the context of orthodontic tooth movement (OTM) and identifies a set of reliable reference genes. CD4+ T cells were isolated from murine spleen and activated with anti-CD3/-CD28 Dynabeads (Thermo Fisher, Langenselbold, Germany) on standard cell culture plates or in 3D scaffolds with or without compressive strain. Expression stability of nine potential reference genes was examined using four mathematical algorithms. Gene expression of Il2 was normalized to all potential reference genes to highlight the importance of correct normalization. Cell proliferation and the expression of the surface markers CD25 and CD69 were also determined. The 3D matrix did not inhibit proliferation after immunological activation of T cells and embedded the cells sufficiently to expose them to pressure load. Expression of ubiquitin C (Ubc) and hypoxanthine phosphoribosyltransferase (Hprt) was the most stable under all conditions tested. A combination of these two genes was suitable for normalization of qPCR data. Normalization of Il2 gene expression showed highly variable results depending on the reference gene used. Pressure reduced cell proliferation and the number of CD69-positive T cells. This study provides a basis for performing valid and reliable qPCR experiments with CD4+ T cells cultured in 3D scaffolds and exposed to compressive forces simulating OTM.
RESUMO
We investigated the physical properties of head and neck cancer cells to develop new cell-selective treatment strategies for squamous cell carcinoma. For better characterization and subsequent differentiation of cancerous and healthy cells, we developed MATLAB-based software to analyze image stacks obtained using a confocal laser-scanning microscope. A confocal laser-scanning microscope was used for three-dimensional (3-D) imaging of a cell line from the head and neck area. The volume of cell organelles of interest was calculated using our newly developed software. Our software enables 3-D visualization and volume calculation as well as data analysis associated with cell morphology. Using filter and semi-automatic segmentation algorithms, our software recognizes individual cell organelles in each slice of an image stack. It matches the corresponding cell cross section areas to produce a 3-D image and to determine the volume of the imaged organelles. We calculated the volume of the nucleus, actin filaments and microtubules in relation to total cell volume. Our software enables 3-D visualization and calculation of organelle volume, which improves cell characterization and comparison of healthy and cancerous cell lines. Differences between cell lines can be observed in detail and used to develop new cancer treatment strategies.
Assuntos
Imageamento Tridimensional , Software , Algoritmos , Tamanho Celular , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/métodos , Microscopia Confocal/métodosRESUMO
Background: A considerable amount of evidence has shown that acute cardiovascular diseases exhibit specific temporal patterns in their onset. Aim: This study was performed to determine if takotsubo syndrome (TTS) shows chronobiological variations with short and long-term impacts on adverse events. Design: Our institutional database constituted a collective of 114 consecutive TTS patients between 2003 and 2015. Methods: Patients were divided into groups defined by the onset of TTS as per time of the day, day of the week, month and quarter of year. Results: TTS events were most common afternoon and least common in the night, indicating a wave-like pattern (p = 0.001) of manifestation. The occurrence of TTS events was similar among days of the week and weeks of the month. TTS patients diagnosed in the month of November and subsequently in the fourth quarter showed a significantly longer QTc interval. These patients also revealed a significantly lower event-free-survival over a 1-year follow-up. In a multivariate Cox regression analysis, TTS events occurring in the fourth quarter of year (HR 6.8, 95%CI: 1.3-35.9; p = 0.02) proved to be an independent predictor of lower event-free-survival. Conclusions: TTS seems to exhibit temporal preference in its onset, but nevertheless this possibly coincidental result needs to be analyzed in a large multicenter registry.
RESUMO
During orthodontic tooth movement (OTM) mechanical forces trigger pseudo-inflammatory, osteoclastogenic and remodelling processes in the periodontal ligament (PDL) that are mediated by PDL fibroblasts via the expression of various signalling molecules. Thus far, it is unknown whether these processes are mainly induced by mechanical cellular deformation (mechanotransduction) or by concomitant hypoxic conditions via the compression of periodontal blood vessels. Human primary PDL fibroblasts were randomly seeded in conventional six-well cell culture plates with O2-impermeable polystyrene membranes and in special plates with gas-permeable membranes (Lumox®, Sarstedt), enabling the experimental separation of mechanotransducive and hypoxic effects that occur concomitantly during OTM. To simulate physiological orthodontic compressive forces, PDL fibroblasts were stimulated mechanically at 2 g·cm-2 for 48 h after 24 h of pre-incubation. We quantified the cell viability by MTT assay, gene expression by quantitative real-time polymerase chain reaction (RT-qPCR) and protein expression by western blot/enzyme-linked immunosorbent assays (ELISA). In addition, PDL-fibroblast-mediated osteoclastogenesis (TRAP+ cells) was measured in a 72-h coculture with RAW264.7 cells. The expression of HIF-1α, COX-2, PGE2, VEGF, COL1A2, collagen and ALPL, and the RANKL/OPG ratios at the mRNA/protein levels during PDL-fibroblast-mediated osteoclastogenesis were significantly elevated by mechanical loading irrespective of the oxygen supply, whereas hypoxic conditions had no significant additional effects. The cellular-molecular mediation of OTM by PDL fibroblasts via the expression of various signalling molecules is expected to be predominantly controlled by the application of force (mechanotransduction), whereas hypoxic effects seem to play only a minor role. In the context of OTM, the hypoxic marker HIF-1α does not appear to be primarily stabilized by a reduced O2 supply but is rather stabilised mechanically.