Efficient electroporation of peptides into adherent cells: investigation of the role of mechano-growth factor in chondrocyte culture

Peptide therapeutics are of increasing interest due to their biological specificity. We used a simple technique to study the efficacy of inducing peptides into adherent chondrocytes by transiently permeabilizing the membrane with electric pulses (in situ electroporation). Mechano-growth factor (MGF) was selected as a model peptide. FITC-labeled MGF was added to cultures of adherent primary chondrocytes grown on ITO coated glass slides. Cells were subjected to 3-9pulses of 175-275V and evaluated by flow cytometry. Under optimal conditions, an electroporation efficiency of close to 50% could be achieved. This technique can be used to study the functional domains of intracellular peptides, peptide inhibition of signal transduction and intracrine-mediated effects of peptides in adherent cell

Equine epidermis : a source of epithelial-like stem/progenitor cells with in vitro and in vivo regenerative capacities

Besides the presence of somatic stem cells in hair follicles and dermis, the epidermis also contains a subpopulation of stem cells, reflecting its high regenerative capacity. However, only limited information concerning epidermis-derived epithelial-like stem/progenitor cells (EpSCs) is available to date. Nonetheless, this stem cell type could prove itself useful in skin reconstitution after injury. After harvesting from equine epidermis, the purified cells were characterized as EpSCs by means of positive expression for CD29, CD44, CD49f, CD90, Casein Kinase 2, p63, and Ki67, low expression for cytokeratin (CK)14 and negative expression for CD105, CK18, Wide CK, and Pan CK. Furthermore, their self-renewal capacity was assessed in adhesion as well as in suspension. Moreover, the isolated cells were differentiated toward keratinocytes and adipocytes. To assess the regenerative capacities of EpSCs, six full-thickness skin wounds were made: three were treated with EpSCs and platelet-rich-plasma (EpSC/PRP-treated), while the remaining three were administered carrier fluid alone (PRP-treated). The dermis of EpSC/PRP-treated wounds was significantly thinner and exhibited more restricted granulation tissue than did the PRP-treated wounds. The EpSC/PRP-treated wounds also exhibited increases in EpSCs, vascularization, elastin content, and follicle-like structures. In addition, combining EpSCs with a PRP treatment enhanced tissue repair after clinical application

Screening the Beach – Probing the Past: The Baltic Sea in Contemporary German Cinema

Since the fall of the Wall, unification and the subsequent re-invention of the nation, filmmakers have revisited the German Heimat film / homeland film cinematic tradition with a view to placing themselves creatively in the context of its intellectual and artistic heritage. However, German directors like Ute Badura, Wolfgang Koepp, Andreas Dresen, Peter Welz and Andreas Kleinert, who, in their work and autobiographies, bring East and West together, choose an Eastern setting for their films – rather than alpine or heath-landscapes – as they ascribe symbolic value to the Baltic Region and former German territories in the East. In many instances their films culminate at the sea which stands for the rough elements of nature as experienced in numerous maritime disasters in the untamed tidal waters of Germany’s limited coastline. The ocean drives home the message that the only certainty in life is change. But why did they choose the contested Eastern German territories and the Baltic Sea? Is this re-orientation and paradigm shift in the Heimat genre from the west to the east a rapprochement or, rather, a territorial claim? Are the shores of the Baltic Sea perhaps expressing a yearning for former German territories further east that were lost after 1945? This article will probe several interpretations of the Baltic shore as a cinematic motif

Quantitative Topographical Analysis of Nuclear Pore Complex Function Using Scanning Force Microscopy

The only avenue for macromolecular communication between nucleoplasmic and cytoplasmic compartments of the cell is through the nuclear pore complexes (NPCs), which are thus situated at a key location for the control of downstream transcriptional processes. The translocation of cargo through the NPC is mediated by transport receptors, which have the difficult task of making specific, yet transitory, interactions with the NPC in order to support efficient transport. In this report we have examined two stages in the translocation process using scanning force microscopy. We show that the initial docking of importin β 45-462 is rapid and occurs at nanomolar concentrations. Later stages of transport involve translocation through the central lumen of the NPC and are thought to involve hydrophobic interactions between transport receptors and the NPC. Using calcium-depleted nuclear envelopes we argue that luminal regions of the NPC exhibit hydrophobic characteristics that are not observed for other regions of the NPC

Graphene-Induced Osteogenic Differentiation Is Mediated by the Integrin/FAK Axis

Graphene is capable of promoting osteogenesis without chemical induction. Nevertheless, the underlying mechanism(s) remain largely unknown. The objectives here were: (i) to assess whether graphene scaffolds are capable of supporting osteogenesis in vivo and; (ii) to ascertain the participation of the integrin/FAK mechanotransduction axis during the osteogenic differentiation induced by graphene. MSC-impregnated graphene scaffolds (n = 6) were implanted into immunocompromised mice (28 days). Alternatively, MSCs were seeded onto PDMS substrates (modulus of elasticity = 130, 830 and 1300 kPa) coated with a single monomolecular layer of graphene and cultured in basal medium (10 days). The ensuing expressions of FAK-p397, integrin, ROCK1, F-actin, Smad p1/5, RUNX2, OCN and OPN were evaluated by Western blot (n = 3). As controls, MSCs were plated onto uncoated PDMS in the presence of mechanotransduction inhibitors (echistatin, Y27632 and DMH1). MSC-impregnated graphene scaffolds exhibited positive immunoexpression of bone-related markers (RUNX2 and OPN) without the assistance of osteogenic inducers. In vitro, regardless of the stiffness of the underlying PDMS substrate, MSCs seeded onto graphene-coated PDMS substrates demonstrated higher expressions of all tested osteogenic and integrin/FAK proteins tested compared to MSCs seeded onto PDMS alone. Hence, graphene promotes osteogenesis via the activation of the mechanosensitive integrin/FAK axis

Real Time Analysis of Myogenesis in Behaving Myoblasts

Effective Tissue engineering ultimately depends on understanding the interplay between the proliferation and differentiation stages of development. One of the best systems to examine these processes is myogenesis. Activated myoblasts first undergo many rounds of cell division. Depending on the needs of the cells, myoblasts may next withdraw from the cell cycle to fuse to form myotubes. Division and fusion are differentially modulated by mechanical stimuli transmitted to the cell’s biosynthetic mechanisms via the actin-based cytoskeleton; Specifically cyclic stretch promotes proliferation and inhibits fusion. By inference, the nature of cell attachment to the substrate should also influence transition through myogenesis. We therefore investigated the effect of the extracellular matrix on behaving cells undergoing either cytokinesis or fusion using atomic force microscopy (AFM) as well as confocal laser scanning microscopy (CLSM) in fluorescent mode

Real Time Analyses of Myogenesis in Behaving Myoblasts

Effective Tissue engineering ultimately depends on understanding the interplay between the proliferation and differentiation stages of development. One of the best systems to examine these processes is myogenesis. Activated myoblasts first undergo many rounds of cell division. Depending on the needs of the cells, myoblasts may next withdraw from the cell cycle to fuse to form myotubes.Division and fusion are differentially modulated by mechanical stimuli transmitted to the cell’s biosynthetic mechanisms via the actin-based cytoskeleton; Specifically cyclic stretch promotes proliferation and inhibits fusion. By inference, the nature of cell attachment to the substrate should also influence transition through myogenesis. We therefore investigated the effect of the extracellular matrix on behaving cells undergoing either cytokinesis or fusion using atomic force microscopy (AFM) as well as confocal laser scanning microscopy (CLSM) in fluorescent mode