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Cells have a net negative outer charge which gives rise to a potential difference in a potentiometric set up. Here, Triton X-100 was used as a model toxin and the cell death was monitored by the potential change, noticed as decrease in negativity. Hence, cytotoxicity can be tested in vitro and real-time using potentiometry. However, silver ions (Ag+) and/or silver chloride ions (AgCl2)-1 lead to cell death when we used the standard Ag/AgCl reference electrode with 3 M NaCl as filling solution. Our further experimentation showed that PBS as filling solution was biocompatible and also gave stable potentiometric profiles. [ABSTRACT FROM AUTHOR]

The hypothesis was that anti-fouling diamond-like carbon polydimethylsiloxane hybrid (DLC-PDMS-h) surface impairs early and late cellular adhesion and matrix-cell interactions. The effect of hybrid surface on cellular adhesion and cytoskeletal organization, important for osteogenesis of human mesenchymal stromal cells (hMSC), where therefore compared with plain DLC and titanium (Ti). hMSCs were induced to osteogenesis and followed over time using scanning electron microscopy (SEM), time-of-flight secondary ion mass spectrometry (ToF-SIMS), immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and hydroxyapatite (HA) staining. SEM at 7.5 hours showed that initial adherence and spreading of hMSC was poor on DLC-PDMS-h. At 5 days some hMSC were undergoing condensation and apoptotic fragmentation, whereas cells on DLC and Ti grew well. DAPI-actin-vinculin triple staining disclosed dwarfed cells with poorly organized actin cytoskeleton-focal complex/adhesion- growth substrate attachments on hybrid coating, whereas spread cells, organized microfilament bundles, and focal adhesions were seen on DLC and in particular on Ti. Accordingly, at day one ToF-SIMS mass peaks showed poor protein adhesion to DLC-PDMS-h compared with DLC and Ti. COL1A1, ALP, OP mRNA levels at days 0, 7, 14, 21, and/or 28 and lack of HA deposition at day 28 demonstrated delayed or failed osteogenesis on DLC-PDMS-h. Anti-fouling DLC-PDMS-h is a poor cell adhesion substrate during the early protein adsorption-dependent phase and extracellular matrix-dependent late phase. Accordingly, some hMSCs underwent anoikis-type apoptosis and failed to complete osteogenesis, due to few focal adhesions and poor cell-to-ECM contacts. DLC-PDMS-h seems to be a suitable coating for non-integrating implants/devices designed for temporary use. [ABSTRACT FROM AUTHOR]

Background: Recurrent aphthous ulcer (RAU) is an ulcerative disease of non-keratinized oral mucosa. Colon and bronchial epithelial cells produce interleukin-17C (IL-17C) upon stimulation of Toll-like receptor 2 (TLR2), TLR3 and TLR5, which are highly expressed in epithelial cells in RAU lesions. We therefore investigated the eventual presence and function of IL-17C in cultured human oral keratinocytes (HOK) and control biopsies compared to RAU lesions.Methods: Expression of IL-17A, IL-17C, IL-17RA and IL-17RE was analysed in cultured HOK cells using quantitative real-time polymerase chain reaction (qRT-PCR). HOK cells were stimulated with IL-17C and analysed for IL-8 and tumour necrosis factor-α (TNF-α) using qRT-PCR. Control mucosa (n = 5) was immunostained for IL-17A, IL-17C, IL-8, TNF-α and mast cell tryptase and compared with RAU lesions (n = 5) using the mean grey scale value.Results: IL-17C, but no IL-17A, mRNA was found in cultured HOK cells. Components of the heterodimeric IL-17RA/IL-17RE receptor for IL-17C were also highly expressed. Stimulation of HOK with IL-17C increased TNF-α mRNA (P = 0.03; IL-8 increase was not statistically significant). HOK in RAU lesions stained intensively for IL-17C compared to controls (P = 0.006). This was associated with increased epithelial immunostaining of TNF-α (P = 0.04) and IL-8 (P = 0.02). Most of the inflammatory cells which stained for IL-17A in control mucosa and RAU lesions were also mast cell tryptase positive.Conclusion: IL-17C is highly expressed in epithelial cells in RAU lesions, where it seems to stimulate oral keratinocytes via IL-17RA/IL-17RE to produce pro-inflammatory cytokines. Human oral epithelial cells are probably important inflammatory cells in RAU. [ABSTRACT FROM AUTHOR]

Background Recurrent aphthous ulcer ( RAU) is an ulcerative disease of non-keratinized oral mucosa. Colon and bronchial epithelial cells produce interleukin-17 C ( IL-17 C) upon stimulation of Toll-like receptor 2 ( TLR2), TLR3 and TLR5, which are highly expressed in epithelial cells in RAU lesions. We therefore investigated the eventual presence and function of IL-17 C in cultured human oral keratinocytes ( HOK) and control biopsies compared to RAU lesions. Methods Expression of IL-17 A, IL-17 C, IL-17 RA and IL-17 RE was analysed in cultured HOK cells using quantitative real-time polymerase chain reaction (q RT- PCR). HOK cells were stimulated with IL-17 C and analysed for IL-8 and tumour necrosis factor-α ( TNF-α) using q RT- PCR. Control mucosa ( n = 5) was immunostained for IL-17 A, IL-17 C, IL-8, TNF-α and mast cell tryptase and compared with RAU lesions ( n = 5) using the mean grey scale value. Results IL-17 C, but no IL-17 A, m RNA was found in cultured HOK cells. Components of the heterodimeric IL-17 RA/ IL-17 RE receptor for IL-17 C were also highly expressed. Stimulation of HOK with IL-17 C increased TNF-α m RNA ( P = 0.03; IL-8 increase was not statistically significant). HOK in RAU lesions stained intensively for IL-17 C compared to controls ( P = 0.006). This was associated with increased epithelial immunostaining of TNF-α ( P = 0.04) and IL-8 ( P = 0.02). Most of the inflammatory cells which stained for IL-17 A in control mucosa and RAU lesions were also mast cell tryptase positive. Conclusion IL-17 C is highly expressed in epithelial cells in RAU lesions, where it seems to stimulate oral keratinocytes via IL-17 RA/ IL-17 RE to produce pro-inflammatory cytokines. Human oral epithelial cells are probably important inflammatory cells in RAU. [ABSTRACT FROM AUTHOR]

We hypothesized that when compared with conventional two-dimensional (2D) cultures, substrates containing 3D micropillars would allow cells to grow at levels, activating their cytoskeleton to promote osteogenesis. Fibroblasts, osteoblast-like cells, and mesenchymal stem cells (MSCs) were studied. Planar substrates were compared with 200-nm-, 5-μm-, and 20-μm-high pillars of Ormocomp®, Si, diamond-like carbon, or TiO2. Scanning electron microscopy and staining of actin cytoskeleton showed 7.5-h adhesion to pillar edges and 5-day stretching between adhesion contacts > 100-μm distances of fibroblast and MSC in 3D networks, whereas SaOS-2 cells adhered flatly and individually on horizontal and vertical surfaces. ERK and ROCK immunostaining at 14 and 21 days confirmed activation of the cytoskeleton. In contrast to expectations, success to induce osteogenesis was dominated by the cytocompatibility of the substrate over the 3D structure. This was shown using early alkaline phosphatase, intermediate osteopontin, and late mineralization markers, together with bone nodule formation, which were seen in planar substrates and low-profile TiO2 pillars, but were poor in the 20-μm landscape. The lack of intercellular contacts seems to halt the osteogenesis-promoting effects of cytoskeletal organization and tension described earlier. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 842-852, 2013. [ABSTRACT FROM AUTHOR]