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The clinical benefits of electromagnetic field (EMF) therapy in enhancing osteogenesis have been acknowledged for decades, but agreement regarding the underlying mechanisms continues to be sought. Studies have shown EMFs to promote osteoblast-like cell proliferation, or contrarily, to induce differentiation and enhance mineralization. Typically these disparities have been attributed to methodological differences. The present paper argues the possibility that the chosen osteoblast model impacts stimulation outcome. Phenotypically immature cells, particularly at low seeding densities, appear to be prone to EMF-amplified proliferation. Conversely, mature cells at higher densities seem to be predisposed to earlier onset differentiation and mineralization. This suggests that EMFs augment ongoing processes in cell populations. To test this hypothesis, mature SaOS-2 cells and immature MC3T3-E1 cells at various densities, with or without osteo-induction, were exposed to sinusoidal 50 Hz EMF. The exposure stimulated the proliferation of MC3T3-E1 and inhibited the proliferation of SaOS-2 cells. Baseline alkaline phosphatase (ALP) expression of SaOS-2 cells was high and rapidly further increased with EMF exposure, whereas ALP effects in MC3T3-E1 cells were not seen until the second week. Thus both cell types responded differently to EMF stimulation, corroborating the hypothesis that the phenotypic maturity and culture stage of cells influence stimulation outcome. [ABSTRACT FROM AUTHOR]

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]

Osteogenic responses of human mesenchymal stromal cells (hMSCs) were compared on square-patterned, inverse square-patterned, and planar titanium, chromium, diamond-like carbon (DLC), and tantalum; hypothesis was that both the materials and patterns affect osteogenesis. Samples were produced using photolithography and physical vapor deposition. Early-marker alkaline phosphatase (ALP) and mid-markers, small body size and mothers against decapentaplegic-related protein-1 (SMAD1), runt-related transcription factor-2 (RUNX2), and osteopontin were studied using quantitative real-time polymerase chain reaction. ALP and hydroxyapatite, were colorimetrically studied. ALP reached highest values on both patterned titanium samples, but mid-markers disclosed that it was already lagging behind planar and inverse patterned tantalum. Hydroxyapatite formation disclosed that osteo-induced hMSCs passed all the differentiation stages (except on planar chromium). Presence of hydroxyapatite disclosed that both types of patterning promoted (p < 0.001) osteogenesis compared to planar samples. Results suggest that the osseocompatibility/integration of implants could be improved by changing the monotonous and featureless implant–host interface into micro-patterned interface to provide physical differentiation cues. [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]

Introduction: Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA. Methods: Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-α. Results: Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1β after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions. Conclusion: We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients. [ABSTRACT FROM AUTHOR]

Regenerative medicine is a multidisciplinary field utilizing the potential of stem cells and the regenerative capability of the body to restore, maintain, or enhance tissue and organ functions. Stem cells are unspecialized cells that can self-renew but also differentiate into several somatic cells when subjected the appropriate environmental cues. The ability to reliably direct stem cell fate would provide tremendous potential for basic research and clinical therapies. Proper tissue function and regeneration rely on the spatial and temporal control of biophysical and biochemical cues, including soluble molecules, cell-cell contacts, cell-extracellular matrix contacts, and physical forces. The mechanisms involved remain poorly understood. This review focuses on the stem cell-extracellular matrix interactions by summarizing the observations of the effects of material variables (such as overall architecture, surface topography, charge, ζ-potential, surface energy, and elastic modulus) on the stem cell fate. It also deals with the mechanisms underlying the effects of these extrinsic, material variables. Insight in the environmental interactions of the stem cells is crucial for the development of new material-based approaches for cell culture experiments and future experimental and clinical regenerative medicine applications. [ABSTRACT FROM AUTHOR]

It was hypothesized that human mesenchymal stromal cell (hMSC) can be guided by patterned and plain amorphous diamond (AD), titanium (Ti), tantalum (Ta) and chromium (Cr) coatings, produced on silicon wafer using physical vapour deposition and photolithography. At 7.5 h hMSCs density was 3.0–3.5× higher ( P < 0.0003, except Ti) and cells were smaller (68 vs. 102 μm, P 0.000006–0.02) on patterns than on silicon background. HMSC-covered surface of the background silicon was lower on Ti than AD patterns ( P = 0.015), but at 5 days this had reversed ( P = 0.006). At 7.5 h focal vinculin adhesions and actin cytoskeleton were outgoing from pattern edges so cells assumed geometric square shapes. Patterns allowed induced osteogenesis, but less effectively than plain surfaces, except for AD, which could be used to avoid osseointegration. All these biomaterial patterns exert direct early, intermediate and late guidance on hMSCs and osteogenic differentiation, but indirect interactions exist with cells on silicon background. [ABSTRACT FROM AUTHOR]