Ex vivo expansion of limbal stem cells is affected by substrate properties

Limbal epithelial stem cells play a key role in the maintenance and regulation of the corneal surface. Damage or destruction of these cells results in vascularisation and corneal opacity. Subsequent limbal stem cell transplantation requires an ex vivo expansion step and preserving cells in an undifferentiated state remains vital. In this report we seek to control the phenotype of limbal epithelial stem cells by the novel application of compressed collagen substrates. We have characterised the mechanical and surface properties of conventional collagen gels using shear rheology and scanning electron microscopy. In doing so, we provide evidence to show that compressive load can improve the stiffness of collagen substrates. In addition Western blotting and immunohistochemistry display increased cytokeratin 3 (CK3) protein expression relating to limbal epithelial cell differentiation on stiff collagen substrates. Such gels with an elastic modulus of 2900 Pa supported a significantly higher number of cells than less stiff collagen gels (3 Pa). These findings have substantial influence in the development of ocular surface constructs or experimental models particularly in the fields of stem cell research, tissue engineering and regenerative medicine.

Development of an ex vivo human skin model for intradermal vaccination : tissue viability and Langerhans cell behaviour

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

Greater impact of dietary fat manipulation than apolipoprotein E genotype on ex-vivo cytokine production – insights from the SATgenε study

Apolipoprotein E (APOE) genotype is believed to play an important role in cardiovascular risk. APOE4 carriers have been associated with higher blood lipid levels and a more pro-inflammatory state compared with APOE3/E3 individuals. Although dietary fat composition has been considered to modulate the inflammatory state in humans, very little is known about how APOE genotype can impact on this response. In a follow-up to the main SATgene study, we aimed to explore the effects of APOE genotype, as well as, dietary fat manipulation on ex vivo cytokine production. Blood samples were collected from a subset of SATgene participants (n = 52/88), prospectively recruited according to APOE genotype (n = 26 E3/E3 and n = 26 E3/E4) after low-fat (LF), high saturated fat (HSF) and HSF with 3.45 g docosahexaenoic acid (DHA) dietary periods (each diet eight weeks in duration assigned in the same order) for the measurement of ex vivo cytokine production using whole blood culture (WBC). Concentrations of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha were measured in WBC supernatant samples after stimulation for 24 h with either 0.05 or 1 lg/ml of bacterial lipopolysaccharide (LPS). Cytokine levels were not influenced by genotype, whereas, dietary fat manipulation had a significant impact on TNF-a and IL-10 production; TNF-a concentration was higher after consumption of the HSF diet compared with baseline and the LF diet (P < 0.05), whereas, IL-10 concentration was higher after the LF diet compared with baseline (P < 0.05). In conclusion, our study has revealed the amount and type of dietary fat can significantly modulate the production of TNF-a and IL-10 by ex vivo LPS-stimulated WBC samples obtained from normolipidaemic subjects.

1,8-cineol potentiates IRF3-mediated antiviral response in human stem cells and an ex vivo model of rhinosinusitis

Common cold is one of the most frequent human inflammatory diseases caused by viruses and can facilitate bacterial super-infections resulting in sinusitis or pneumonia. The active ingredient of the drug Soledum, 1,8-cineole, is commonly applied for treating inflammatory diseases of the respiratory tract. However, the potential of 1,8-cineole for treating primary viral infections of the respiratory tract remains unclear. In the present study, we demonstrate for the first time that 1,8-cineole potentiates Poly(I:C)-induced activity of the anti-viral transcription factor Interferon Regulatory Factor 3, while simultaneously reducing pro-inflammatory NF-κB-activity in human cell lines, inferior turbinate stem cells (ITSCs) and ex vivo cultivated human nasal mucosa. Co-treatment of cell lines with Poly(I:C) and 1,8-cineole resulted in significantly increased IRF3 reporter gene activity compared to Poly(I:C) alone, whereas NF-κB-activity was reduced. Accordingly, 1,8-cineole- and Poly(I:C)-treatment led to increased nuclear translocation of IRF3 in ITSCs and a human ex vivo model of rhinosinusitis compared to the Poly(I:C)-treated approach. Nuclear translocation of IRF3 was significantly increased in ITSCs and slice cultures treated with LPS and 1,8-cineole compared to the LPS-treated cells mimicking bacterial infection. Our findings strongly suggest that 1,8-cineole potentiates the antiviral activity of IRF3 in addition to its inhibitory effect on pro-inflammatory NF-κB-signalling and may thus broaden its field of application.

Ex Vivo Accuracy of an Apex Locator Using Digital Signal Processing in Primary Teeth

The purpose of this study was to evaluate ex vivo the accuracy an electronic apex locator during root canal length determination in primary molars. Methods: One calibrated examiner determined the root canal length in 15 primary molars (total=34 root canals) with different stages of root resorption. Root canal length was measured both visually, with the placement of a K-file 1 mm short of the apical foramen or the apical resorption bevel, and electronically using an electronic apex locator (Digital Signal Processing). Data were analyzed statistically using the intraclass correlation (ICC) test. Results: Comparing the actual and electronic root canal length measurements in the primary teeth showed a high correlation (ICC=0.95) Conclusions: The Digital Signal Processing apex locator is useful and accurate for apex foramen location during root canal length measurement in primary molars. (Pediatr Dent 200937:320-2) Received April 75, 2008 vertical bar Lost Revision August 21, 2008 vertical bar Revision Accepted August 22, 2008

Ex vivo evaluation of the accuracy of two electronic apex locators during root canal length determination in primary teeth

Aim To evaluate ex vivo the accuracy of two electronic apex locators during root canal length determination in primary incisor and molar teeth with different stages of physiological root resorption. Methodology One calibrated examiner determined the root canal length in 17 primary incisors and 16 primary molars (total of 57 root canals) with different stages of root resorption based on the actual canal length and using two electronic apex locators. Root canal length was measured both visually, with the placement of a K-file 1 mm short of the apical foramen or the apical resorption bevel, and electronically using two electronic apex locators (Root ZX II - J. Morita Corp. and Mini Apex Locator - SybronEndo) according to the manufacturers` instructions. Data were analysed statistically using the intraclass correlation (ICC) test. Results Comparison of the actual root canal length and the electronic root canal length measurements revealed high correlation (ICC = 0.99), regardless of the tooth type (single-rooted and multi-rooted teeth) or the presence/absence of physiological root resorption. Conclusions Root ZX II and Mini Apex Locator proved useful and accurate for apex foramen location during root canal length measurement in primary incisors and molars.

Accuracy of the iPex multi-frequency electronic apex locator in primary molars: an ex vivo study

P>Aim To evaluate ex vivo the accuracy of the iPex multi-frequency electronic apex locator (NSK Ltd, Tokyo, Japan) for working length determination in primary molar teeth. Methodology One calibrated examiner determined the working length in 20 primary molar teeth (total of 33 root canals). Working length was measured both visually, with the placement of a K-file 1 mm short of the apical foramen or the most coronal limit of root resorption, and electronically using the electronic apex locator iPex, according to the manufacturers` instructions. Data were analysed statistically using the intraclass correlation (ICC) test. Results Comparison of the actual and the electronic measurements revealed high correlation (ICC = 0.99) between the methods, regardless of the presence or absence of physiological root resorption. Conclusions In this laboratory study, the iPex accurately identified the apical foramen or the apical opening location for working length measurement in primary molar teeth.

Micropartículas contendo pantoprazol sódico : preparação, caracterização físico-química e avaliação anti-ulcerativa in vivo e da absorção intestinal ex vivo

O pantoprazol (PAN) é um inibidor da bomba de prótons clinicamente empregado para o tratamento de úlcera gástrica e refluxo gastro-esofágico. Estudos relacionados à estabilidade físico-química mostraram que a degradação do PAN está diretamente relacionada com a acidez do meio, determinando a necessidade de administrá-lo em uma forma gastrorresistente. Desse modo, este trabalho propôs-se a desenvolver micropartículas à base de polímero gastrorresistente (Eudragit S100®), polímero de baixa permeabilidade (Eudragit RS30D®) ou de blenda polimérica (Eudragit S100®/ Eudragit RS30D®), contendo PAN pela técnica de spraydrying . O estudo de dissolução in vitro utilizando célula de fluxo demonstrou que o PAN foi liberado das micropartículas em 120 minutos, seguindo cinética de primeira ordem, de acordo com o modelo monoexponencial. A avaliação da gastrorresistência in vitro em célula de fluxo e em dissolutor evidenciou que as formulações de micropartículas à base de Eudragit S100® e da blenda (Eudragit S100®/ Eudragit RS30D®), garantiram adequada proteção ao fármaco em ambiente ácido. Estudos in vivo confirmaram esses resultados, pois possibilitaram a constatação da proteção do fármaco pelas micropartículas durante a passagem pelo estômago, o que possibilitou absorção entérica do PAN em quantidade adequada para exercer atividade farmacológica. Por fim, a investigação ex vivo da permeação do PAN carreado por micropartículas no epitélio intestinal mostrou que estes sistemas foram capazes de garantir a absorção da totalidade do fármaco carreado, constatando-se ainda que este processo ocorreu segundo o modelo monoexponencial.

Altered Ex Vivo Expression of Caspase 8, Caspase 9, and Bcl-2 Is Associated with T-Cell Hyporeactivity in Patients with Paracoccidioidomycosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synthesis, ex Vivo and in Vitro Hydrolysis Study of an Indoline Derivative Designed as an Anti-Inflammatory with Reduced Gastric Ulceration Properties

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mechanical resistance of the modified stabilization method for the tibial tuberosity advancement technique: ex vivo experimental study in dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of salivary stimulation on erosion of human and bovine enamel subjected or not to subsequent abrasion: An in situ/ex vivo study

This in situ/ex vivo study evaluated whether saliva stimulated by chewing gum could prevent or reduce the wear and the percent change in microhardness (%SMH) of bovine and human enamel submitted to erosion followed by brushing abrasion immediately or after 1 h. During 2 experimental 7-day crossover phases, 9 previously selected volunteers wore intraoral palatal devices, with 12 enamel specimens (6 human and 6 bovine). In the first phase, the volunteers immersed the device for 5 min in 150 ml of cola drink, 4 times per day (at 8, 12, 16 and 20 h). Immediately after the immersions, no treatment was performed in 4 specimens, 4 other specimens were immediately brushed (0 min) using a fluoride dentifrice, and the device was replaced into the mouth. After 60 min, the remaining 4 specimens were brushed. In the second phase, the procedures were repeated, but after the immersions, the volunteers stimulated the salivary flow rate by chewing a sugar-free gum for 30 min. Changes in wear and %SMH were measured. ANOVA and Tukey's test showed statistical differences (p < 0.05) for the following comparisons. The chewing gum promoted less wear and %SMH. A decreasing %SMH and an increasing enamel wear were observed in the following conditions: erosion only, 60 min and 0 min. The human enamel presented greater %SMH and less wear compared to bovine enamel. The data suggest that the salivary stimulation after an erosive or erosive/abrasive attack can reduce the dental wear and the %SMH.

Short-term in situ/ex vivo study of the anticariogenic potential of a resin-modified glass-ionomer cement associated with adhesive systems

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of different concentrations of fluoride in dentifrices on dentin erosion subjected or not to abrasion in situ/ex vivo

This in situ/ex vivo study assessed the effect of different concentrations of fluoride in dentifrices on dentin subjected to erosion or to erosion plus abrasion. Ten volunteers took part in this crossover and double-blind study performed in 3 phases (7 days). They wore acrylic palatal appliances containing 4 bovine dentin blocks divided in two rows: erosion and erosion plus abrasion. The blocks were subjected to erosion by immersion ex vivo in a cola drink (60 s, pH 2.6) 4 times daily. During this step, the volunteers brushed their teeth with one of three dentifrices D (5,000 ppm F, NaF, silica); C (1,100 ppm F, NaF, silica) and placebo (22 ppm F, silica). Then, the respective dentifrice slurry (1: 3) was dripped on dentin surfaces. While no further treatment was performed in one row, the other row was brushed using an electric toothbrush for 30 s ex vivo. The appliances were replaced in the mouth and the volunteers rinsed with water. Dentin loss was determined by profilometry and analyzed by 2-way ANOVA/Bonferroni test (alpha = 0.05). Dentin loss after erosive-abrasive wear was significantly greater than after erosion alone. Wear was significantly higher for the placebo than for the D and C dentifrices, which were not significantly different from each other. It can be concluded that the presence of fluoride concentrations around 1,100 ppm in dentifrices is important to reduce dentin wear by erosion and erosion + abrasion, but the protective effect does not increase with fluoride concentration. Copyright (C) 2008 S. Karger AG, Basel.