Plasma treatment is a method to modify the physicochemical properties of biomaterials, which consequently may affect interactions with cells. Based on the rationale that physical cues on the surface of culture substrates and implants, such as surface roughness, have proven to alter cell behaviour, we used electrospinning to fabricate fibrous three-dimensional scaffolds made of a poly (ethylene oxide terephthalate)/poly (butylene terephthalate) copolymer to mimic the physical microenvironment of extracellular matrix and applied radio-frequency oxygen plasma treatment to create nanoscale roughness. Scanning electron microscopy (SEM) analysis revealed a fibre diameter of 5.49 ± 0.96 µm for as-spun meshes. Atomic force microscopy (AFM) measurements determined an exponential increase of surface roughness with plasma treatment time. An increase in hydrophilicity after plasma treatment was observed, which was associated with higher oxygen content in plasma treated scaffolds compared to untreated ones. A more pronounced adsorption of bovine serum albumin occurred on scaffolds treated with plasma for 15 and 30 min compared to untreated fibres. Clinically relevant human mesenchymal stromal cells (hMSCs) were cultured on untreated, 15 and 30 min treated scaffolds. SEM analysis confirmed cell attachment and a pronounced spindle-like morphology on all scaffolds. No significant differences were observed between different scaffolds regarding the amount of DNA, metabolic activity and alkaline phosphatase (ALP) activity after 7 days of culture. The amount of ALP positive cells increased between 7 and 21 days of culture on both untreated and 30 min treated meshes. In addition, ALP staining of cells on plasma treated meshes appeared more pronounced than on untreated meshes after 21 days of culture. Quantitative polymerase chain reaction showed significant upregulation of bone sialoprotein and osteonectin expression on oxygen plasma treated fibres compared to untreated fibres in basic culture medium after 7 days of culture, while no differences were observed in the expression of other osteogenic markers. At 21 days, no osteocalcin protein could be detected by ELISA at any of the substrates. In conclusion, this study shows that oxygen plasma treatment can successfully be applied to modify the nanoscale surface properties of polymeric electrospun fibre meshes, which in turn may positively affect osteogenic differentiation of hMSCs.
Electrospun fiber meshes are patterned at length scales comparable to or lower than their fiber diameter. Simple nano- and microgrooves and closed geometric shapes are imprinted in different tones using a fast imprint process at physiological temperatures. Human mesenchymal stromal cells cultured on patterned scaffolds show differences in cellular morphology and cytoskeleton organization. Microgrooved electrospun fibers support upregulation of alkaline phosphatase and bone morphogenetic protein-2 gene expression when cells are cultured in osteogenic medium.
A murine line haploinsufficient in the cardiac sodium channel has been used to model human Brugada syndrome: a disease causing sudden cardiac death due to lethal ventricular arrhythmias. We explored the effects of cholinergic tone on electrophysiological parameters in wild-type and genetically modified, heterozygous, Scn5a+/− knockout mice. Scn5a+/− ventricular slices showed longer refractory periods than wild-type both at baseline and during isoprenaline challenge. Scn5a+/− hearts also showed lower conduction velocities and increased mean increase in delay than did littermate controls at baseline and blunted responses to isoprenaline challenge. Carbachol exerted limited effects but reversed the effects of isoprenaline with coapplication. Scn5a+/− mice showed a reduction in conduction reserve in that isoprenaline no longer increased conduction velocity, and this was not antagonized by muscarinic agonists.
Many studies have shown the influence of soluble factors and material properties on the differentiation capacity of mesenchymal stromal cells (MSCs) cultured as monolayers. These types of two-dimensional (2D) studies can be used as simplified models to understand cell processes related to stem cell sensing and mechano-transduction in a three-dimensional (3D) context. For several other mechanisms such as cell-cell signaling, cell proliferation and cell morphology, it is well-known that cells behave differently on a planar surface compared to cells in 3D environments. In classical tissue engineering approaches, a combination of cells, 3D scaffolds and soluble factors are considered as the key ingredients for the generation of mechanically stable 3D tissue constructs. However, when MSCs are used for tissue engineering strategies, little is known about the maintenance of their differentiation potential in 3D scaffolds after the removal of differentiation soluble factors. In this study, the differentiation potential of human MSCs (hMSCs) into the chondrogenic and osteogenic lineages on two distinct 3D scaffolds, additive manufactured electrospun scaffolds, was assessed and compared to conventional 2D culture. Human MSCs cultured in the presence of soluble factors in 3D showed to differentiate to the same extent as hMSCs cultured as 2D monolayers or as scaffold-free pellets, indicating that the two scaffolds do not play a consistent role in the differentiation process. In the case of phenotypic changes, the achieved differentiated phenotype was not maintained after the removal of soluble factors, suggesting that the plasticity of hMSCs is retained in 3D cell culture systems. This finding can have implications for future tissue engineering approaches in which the validation of hMSC differentiation on 3D scaffolds will not be sufficient to ensure the maintenance of the functionality of the cells in the absence of appropriate differentiation signals.
Background We explored the hypothesis that increased cholinergic tone exerts its pro-arrhythmic effects in Brugada Syndrome (BrS) through increasing dispersion of transmural repolarisation in patients with spontaneous and drug induced Brugada Syndrome. Methods BrS and Supraventricular tachycardia (SVT) patients were studied after deploying an Ensite Array in the right ventricular outflow tract and a Cardima catheter in the great cardiac vein to record endo & epicardial signals respectively. S1-S2 restitution curves from the RV apex were conducted at baseline and after edrophonium challenge to promote increased cholinergic tone. The local unipolar electrograms were then analyzed to study transmural conduction and repolarisation dynamics. Results The study included 8 BrS patients: (5M: 3F; mean age 56y) and 8 controls patients with SVT (5M: 3F; mean age 48y). Electrophysiological studies in controls demonstrated shorter endocardial than epicardial right ventricular (RV) activation times (AT's) (mean difference: 26 ms, p<0.001). In contrast, BrS patients showed longer endocardial than epicardial AT (mean difference:-15 ms, p=0.001). BrS hearts, compared to controls, showed significantly larger transmural gradients (TMG) in their activation recovery intervals (ARIs) (mean intervals 20.5 vs 3.5 ms; p<0.01), with longer endocardial than epicardial ARIs. Edrophonium challenge increased such gradients in both controls (to a mean of 16 msecs (p<0.001) and BrS (to 29.7 ms; p<0.001). However, these were attributable to epicardial and endocardial ARI prolongations in control and BrS hearts respectively. Dynamic changes in repolarisation gradients were also observed across the BrS RV wall in BrS. Conclusions: Differential contributions of conduction and repolarisation were identified in BrS which critically modulated transmural dispersion of repolarisation with significant cholinergic effects only identified in the BrS patients. This has important implications for explaining the pro-arrhythmic effects of increased vagal tone in BrS aswell as evaluating autonomic modulation & epicardial ablation as therapeutic strategies.
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