Tissues and organs consist of a complex organization of cells, extracellular matrix (ECM), and signaling molecules. In particular, blood vessels and skin are a highly organized hierarchical layer composed of various types of cells and ECM layers. The construction in vitro of three-dimensional (3D) cell-polymeric material composites has created notable advances in tissue regeneration.[1] However, an effective methodology to fabricate a 3D multilayer composed of cells and an ECM layer with the appropriate components and thickness has not yet been achieved. Recently, new technologies such as a cell sheet, [2a] magnetic liposomes, [2b] and a chitosan membrane [2c] have been reported to fabricate layered tissues. Although these methods are intriguing, complicated manipulation is required and the thickness of the ECM layer is not controllable.We focused on a layer-by-layer (LbL) technique, which is an appropriate method to prepare nanometer-sized films on a substrate through the alternate immersion into interactive polymer solutions.[3] The preparation of nanometer-sized multilayer films composed of ECM components on the surface of the first layer of cells provides a cell-adhesive surface for the second layer of cells. Rajagopalan et al. demonstrated a bilayer structure composed of hepatocytes and other cells by preparing a polyelectrolyte multilayer consisting of chitosan and DNA on the hepatocyte surface.[4a]However, chitosan cannot dissolve in neutral buffer solutions and fabrication of polyelectrolyte multilayers onto the cell surface is limited owing to the cytotoxicity of polycations. [4b,c] Furthermore, a highly organized cellular multilayer with more than three layers will be required for the creation of functional artificial tissues that are similar to natural tissues. The use of natural ECM components for nanofilms is significant because the typical ECM presents with celladhesive moieties such as RGD (arginine-glycine-aspartic acid) and other amino acid sequences for cellular functions. [5] In the present study, fibronectin (FN) and gelatin were selected to prepare nanometer-sized ECM films (nano-ECM film) on the cell surface. FN is a flexible multifunctional glycoprotein that plays an important role in cell attachment, migration, differentiation, etc. [6a,b] FN is well known to interact not only with a variety of ECM proteins, such as collagens (gelatins) and glycosaminoglycans, but also with the a 5 b 1 integrin receptor on the cell surface.[6c] Recently, we reported FN-based protein multilayers composed of FN and ECM components, such as gelatin, heparin, and elastin, constructed by LbL assembly.[7] Although FN and gelatin have a negative charge under physiological conditions, they interact with each other because FN has a collagen binding domain.[6b] The preparation of FN-gelatin nanofilms on the surface of the first layer of cells will provide a suitable celladhesive surface that is similar to the natural ECM for the second layer of cells. Herein, we report well-organized, fourlayered architectures...
Various nanometer-sized multilayers were directly prepared onto the surface of mouse L929 fibroblast cells by a layer-by-layer (LbL) assembly technique to control the cell surface microenvironment and cell functions, such as viability, morphology, and proliferation. The species of LbL nanofilms strongly affected the cell morphology and growth. Polyelectrolyte (PE) multilayers induced a round-shaped morphology of the adhered cells, although each component of the multilayers had high cytocompatibility, whereas fibronectin (FN)-gelatin (G) and -dextran sulfate (DS) multilayers with FN-binding domain interactions (FN films) showed extended morphologies of the cells similar to that of control cells (without films). A clear difference in cell proliferation was observed for PE and FN films. The cells with FN films on their surfaces showed good proliferation profiles independent of the film thickness, but cell proliferation was not observed using the PE films although the cells survived during the culture period. Fluorescence microscopic and scanning electron microscopic observations clearly suggested a nanometer-sized meshwork morphology of the FN films on the cell surface after 24 h of incubation, whereas the PE films showed homogeneous film morphologies on the cell surface. These nanomeshwork morphologies seemed to be similar to the fibrous structure of the natural extracellular matrix. The results of this study demonstrated that the components, charge, and morphology of LbL nanofilms prepared directly on the cell surface strongly affected cell functions, and the effects of these LbL nanofilms on cell functions differed vastly as compared to PE films prepared on a substrate. The preparation of LbL nanofilms onto a cell surface might be a novel and interesting technique to control cell functions.
Chronic salt loading up-regulated the expression of neuronal nitric oxide synthase (NOS) mRNA in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus with a concomitant increase in NOS activity in the posterior pituitary. Once daily ip injection of N-omega-nitroarginine (N-Arg), a NOS inhibitor, significantly inhibited NOS activity in the posterior pituitary in a dose-dependent manner, but did not influence NOS mRNA levels. Two percent salt loading for 3 or 4 days significantly depleted the contents of both arginine vasopressin (AVP) and oxytocin (OT) in the posterior pituitary, and simultaneous treatment with daily injections of N-Arg at a dose of 10 mg/kg significantly enhanced the depletion of both AVP and OT. This effect was dose dependent and paralleled the inhibition of NOS activity in the posterior pituitary. N-Arg treatment had no effect on the levels of both AVP and OT transcripts in PVN or SON. These results suggest that NOS gene expression in the SON and PVN of the rat hypothalamus is increased during hyperosmotic stimulation and suggest a neuromodulatory role for NO in the rat hypothalamo-hypophysial system as an inhibitory regulator of AVP and OT secretion.
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