Matrix-dependent adhesion of vascular and valvular endothelial cells in microfluidic channels

The interactions between endothelial cells and the underlying extracellular matrix regulate adhesion and cellular responses to microenvironmental stimuli, including flow-induced shear stress. In this study, we investigated the adhesion properties of primary porcine aortic endothelial cells (PAECs) and valve endothelial cells (PAVECs) in a microfluidic network. Taking advantage of the parallel arrangement of the microchannels, we compared adhesion of PAECs and PAVECs to fibronectin and type I collagen, two prominent extracellular matrix proteins, over a broad range of concentrations. Cell spreading was measured morphologically, based on cytoplasmic staining with a vital dye, while adhesion strength was characterized by the number of cells attached after application of shear stresses of 11, 110, and 220 dyn cm(-2). Results showed that PAVECs were more well spread on fibronectin than on type I collagen (P < 0.0001), particularly for coating concentrations of 100, 200, and 500 microg mL(-1). PAVECs also withstood shear significantly better on fibronectin than on collagen for 500 microg mL(-1). PAECs were more well spread on collagen compared to PAVECs (P < 0.0001), but did not have significantly better adhesion strength. These results demonstrate that cell adhesion is both cell-type and matrix dependent. Furthermore, they reveal important phenotypic differences between vascular and valvular endothelium, with implications for endothelial mechanobiology and the design of microdevices and engineered tissues.     

Direct functionalization of cell-adhesion promoters to hydrogel microparticles synthesized by stop-flow lithography

Polyethylene glycol (PEG) hydrogel microparticles generated via stop-flow lithography can be utilized for efficient microparticle-based cell culture processes because of their high biocompatibility, the molecular diffusion capability in the gel structure, and the tunability of their shape and size. However, the typical functionalization process of PEG microparticles with cell-adhesion promoters has inevitable limitations, requiring additional linker molecules and the preconjugation of linkers to cell-adhesion promoters and microparticles. In this study, a simple and direct cell-adhesion promoter functionalization process of the PEG microparticles is presented by use of aza-Michael reaction between remnant unreacted acrylate groups in particles and amine groups in cell-adhesion promoters. On the basis of proposed process, particles are directly conjugated with poly-l -lysine (PLL), a typical cell-adhesion promoter that can electrostatically interact with cellular membranes, in a controllable manner. We demonstrate enhanced cell-adhesion capabilities of the particles along with the increased amount of conjugated PLL in the particles. Furthermore, to validate extended applicability, the particles are directly conjugated with Gly-Arg-Gly-Asp-Ser (GRGDS) peptides, in which RGD sequence is involved in the cell-adhesion behavior of extracellular matrix proteins, including fibronectin. The introduced GRGDS peptides increase the cell-adhesion capacity of the microparticles binding to integrin proteins in cellular membranes.     

Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with alpha3beta1 integrin

Betaig-h3 (betaig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of betaig-h3 is responsive to TGF-Beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating betaig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of betaig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of betaig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed betaig-h3 and the expression was dramatically induced by exogenous TGF-Beta1 treatment. betaig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known alpha3beta1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in betaig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that alpha3beta1 integrin mediates the adhesion and migration of RPTECs on betaig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that betaig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with alpha3beta1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.     

The surface energy of various biomaterials coated with adhesion molecules used in cell culture

This study calculates the surface energy of polystyrene tissue culture plastic, silicon, silicon dioxide and indium tin oxide, all of which have applications in tissue culture. The adhesion molecules: collagen, fibronectin, poly-L-ornithine and poly-D-lysine, were coated onto these various surfaces, and the surface energy of the coated substrates calculated. Coating with fibronectin was found to produce a monopolar acidic surface while poly-D-lysine, poly-L-ornithine and collagen coatings were found to produce monopolar basic surfaces. The calculated surface energy components of the coated materials were then used to give a quantitative determination of the magnitude of their hydrophobicity. It was concluded that collagen, polylysine and polyornithine could provide a hydrophobic or hydrophilic surface depending on the underlying substrates they were coated on. The measurement obtained for fibronectin, unlike the other adhesion molecules, was independent of the underlying surface and remained hydrophobic on all substrates tested. Wetting experiments were carried out on the coated substrates, using the tissue culture medium Dulbeccos modified eagles medium, both containing and not containing serum proteins, and saline solution. These liquids that are commonly used in tissue culture, were then used to provide information how these liquids behave on various substrates coated with the adhesion molecules. Results show that fibronectin coated surfaces represent the most phobic surface for all three liquids. The findings of this study can be used in cell manipulation studies and provide a valuable data set for the biomedical and research industries.     

Cross talk between cell-cell and cell-matrix adhesion signaling pathways during heart organogenesis: implications for cardiac birth defects.

The anterior-posterior and dorsal-ventral progression of heart organogenesis is well illustrated by the patterning and activity of two members of different families of cell adhesion molecules: the calcium-dependent cadherins, specifically N-cadherin, and the extracellular matrix glycoproteins, fibronectin. N-cadherin by its binding to the intracellular molecule beta-catenin and fibronectin by its binding to integrins at focal adhesion sites, are involved in regulation of gene expression by their association with the cytoskeleton and through signal transduction pathways. The ventral precardiac mesoderm cells epithelialize and become stably committed by the activation of these cell-matrix and intracellular signaling transduction pathways. Cross talk between the adhesion signaling pathways initiates the characteristic phenotypic changes associated with cardiomyocyte differentiation: electrical activity and organization of myofibrils. The development of both organ form and function occurs within a short interval thereafter. Mutations in any of the interacting molecules, or environmental insults affecting either of these signaling pathways, can result in embryonic lethality or fetuses born with severe heart defects. As an example, we have defined that exposure of the embryo temporally to lithium during an early sensitive developmental period affects a canonical Wnt pathway leading to beta-catenin stabilization. Lithium exposure results in an anterior-posterior progression of severe cardiac defects.     

Inhibition of blood coagulation factor XIIIa-mediated cross-linking between fibronectin and collagen by polyamines

Soluble fibronectin is found in body fluids and media of cultured adherent cells. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for factor XIIIa (plasma transglutaminase) and can be cross-linked to collagen and to the alpha chain of fibrin. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate the possibility that factor XIIIa-mediated cross-linking is influenced by polyamines. Spermidine inhibited cross-linking between fibronectin and type I collagen, isolated alpha 1 (I) collagen chains, or iodinated cyanogen bromide fragment 7 of alpha 1 (I) chains (125I-alpha 1 (I)-CB7). Half-maximal inhibition of cross-linking between 125I-alpha (I)-CB7 and fibronectin was observed when 0.1 mM spermine or spermidine was present. Spermidine, 0.7 mM, partially inhibited cross-linking between fibronectin and the alpha chain of fibrin but failed to inhibit cross-linking between the fibrin monomers of a fibrin clot. Spermidine also failed to inhibit cross-linking between fibronectin molecules when aggregation of fibronectin was induced with dithiothreitol. In contrast, 0.7 mM monodanyslcadaverine inhibited fibronectin-collagen, fibronectin-fibrin, fibronectin-fibronectin, and fibrin-fibrin cross-linking. Spermidine or spermine, 0.7 mM, enhanced the cross-linking between molecules of partially amidinated fibronectin, suggesting that N1,8-(di-gamma-glutamyl)-polyamine cross-linkages were formed. Spermidine and spermine failed to enhance cross-linking between monomers of amidinated fibrin. These results indicate that physiologic concentrations of polyamines specifically disturb transglutaminase-catalyzed cross-linking between fibronectin and collagen.     

Micropatterning gradients and controlling surface densities of photoactivatable biomolecules on self-assembled monolayers of oligo(ethylene glycol) alkanethiolates

Background: Bioactive molecules that are covalently immobilized in patterns on surfaces have previously been used to control or study cell behavior such as adhesion, spreading, movement or differentiation. Photoimmobilization techniques can be used, however, to control not only the spatial pattern of molecular immobilization, termed the micropattern, but also the surface density of the molecules — a characteristic that has not been previously exploited.Results: Oligopeptides containing the bioactive Arg-Gly-Asp cell-adhesion sequence were immobilized upon self-assembled monolayers of an oligo(ethylene glycol) alkanethiolate in patterns that were visualized and quantified by autoradiography. The amount and pattern of immobilized peptide were controlled by manipulating the exposure of the sample to a LIV lamp or a laser beam. Patterns of peptides, including a density gradient, were used to control the location and number of adherent cells and also the cell shape.Conclusions: A photo immobilization technique for decorating surfaces with micropatterns that consist of variable densities of bioactive molecules is described. The efficacy of the patterns for controlling cell adhesion and shape has been demonstrated. This technique is useful for the study of cell behavior on micropatterns.     

Probing fibronectin-surface interactions: a multitechnique approach

The development of adhesive as well as antiadhesive surfaces is essential in various biomaterial applications. In this study, we have used a multidisciplinary approach that combines biological and physicochemical methods to progress in our understanding of cell-surface interactions. Four model surfaces have been used to investigate fibronectin (Fn) adsorption and the subsequent morphology and adhesion of preosteoblasts. Such experimental conditions lead us to distinguish between anti- and proadhesive substrata. Our results indicate that Fn is not able to induce cell adhesion on antiadhesive materials. On adhesive substrata, Fn did not increase the number of adherent cells but favored their spreading. This work also examined Fn-surface interactions using ELISA immunoassays, fluorescent labeling of Fn, and force spectroscopy with Fn-modified tips. The results provided clear evidence of the advantages and limitations of each technique. All of the techniques confirmed the important adsorption of Fn on proadhesive surfaces for cells. By contrast, antiadhesive substrata for cells avoided Fn adsorption. Furthermore, ELISA experiments enabled us to verify the accessibility of cell binding sites to adsorbed Fn molecules.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollagen type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Endothelial cell adhesion on polyelectrolyte multilayer films functionalised with fibronectin and collagen

The seeding of endothelial cells on biomaterial surfaces has become a major challenge to achieve better haemocompatibility of these surfaces. Multilayers of polyelectrolytes formed by the layerby-layer method are promising in this respect. In this study, the interactions of endothelial cells with multilayered polyelectrolytes films were investigated. The build-ups were prepared by selfassembled alternatively adsorbed polyanions and polycations functionalised with fibronectin and collagen. Anionic poly(sodium 4-styrenesulfonate) and cationic poly(allylamine hydrochloride) polyelectrolytes were chosen as a model system. Elaborated surfaces were characterised by electrochemical impedance spectroscopy and cyclic voltammetry. The modified electrode showed good reversible electrochemical properties and high stability in an electrolyte solution. The film ohmic resistance was highest when the film was coated with fibronectin; the parameters so determined were correlated with atomic force microscopy images. Cell colorimetric assay (WST-1) and immunofluorescence were used to quantify the cell viability and evaluate the adhesion properties. When cultured on a surface where proteins were deposited, cells adhered and proliferated better with fibronectin than with collagen. In addition, a high surface free energy was favourable to adhesion and proliferation (48.8 mJ m⁻² for fibronectin and 39.7 mJ m⁻² for collagen, respectively). Endothelial cells seeded on functionalised-polyelectrolyte multilayer films showed a good morphology and adhesion necessary for the development of a new endothelium.     

Issues of ligand accessibility and mobility in initial cell attachment

The influence of lateral ligand mobility on cell attachment and receptor clustering has previously been explored for membrane-anchored molecules involved in cell-cell adhesion. In this study, we considered instead a cell binding motif from the extracellular matrix. Even though the lateral mobility of extracellular matrix ligands in membranes does not occur in vivo, we believe it is of interest for cell engineering in vitro. As is the case for cell-cell adhesion molecules, lateral mobility of extracellular matrix ligands could influence cell attachment and, subsequently, cell behavior in cell culture. In this paper, the accessibility and functionality of extracellular matrix ligands presented at surfaces were evaluated for the conditions of laterally mobile versus non-mobile ligands by studying ligand-antibody binding events and early cell attachment as a function of ligand concentration. We compare the initial attachment of rat-derived adult hippocampal progenitor (AHP) cells on laterally mobile, supported phospholipid bilayer membranes to non-mobile, poly-L-lysine-grafted-poly(ethylene glycol) (PLL-g-PEG) polymer films functionalized with a range of laminin-derived IKVAV-containing peptide densities. To this end, synthesis of a new PLL-g-PEG/PEG-IKVAV polymer is described. The characterization of available IKVAV peptides on both surface presentations schemes was explored by studying the mass uptake of anti-IKVAV antibodies using a combination of the surface-sensitive techniques quartz crystal microbalance with dissipation monitoring, surface plasmon resonance spectroscopy, and optical waveguide lightmode spectroscopy. IKVAV-containing peptides presented on laterally mobile, supported phospholipid bilayers and non-mobile PLL-g-PEG were recognized by the anti-IKVAV antibody in a dose-dependent manner, indicating that the amount of available IKVAV ligands increases proportionally with ligand density over the concentrations tested. Attachment of AHP cells to IKVAV-functionalized PLL-g-PEG and supported phospholipid bilayers followed a sigmoidal dependence on peptide concentration, with a critical concentration of approximately 3 pmol/cm2 IKVAV ligands required to support initial AHP cell attachment for both surface modifications. There appeared to be little influence of IKVAV peptide mobility on the initial attachment of AHP cells. Although the spread in the cell attachment data was larger for the PLL-g-PEG surface modification, this was reduced when observed after 24 h, indicating that the cells might need longer times to establish attachment strengths equivalent to those observed on peptide-functionalized supported lipid bilayers. The present study is a step toward understanding the influence of extracellular-matrix-derived ligand mobility on cell fate. Further analysis should focus on the systematic tuning of lateral ligand diffusion, as well as a comparison between the response of non-spreading cells (i.e., AHPs), versus spreading cells (i.e., fibroblasts).     

Fibronectin and proteoglycans as determinants of cell-substratum adhesion.

When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.     

Short peptides enhance single cell adhesion and viability on microarrays

Single cell patterning holds important implications for biology, biochemistry, biotechnology, medicine, and bioinformatics. The challenge for single cell patterning is to produce small islands hosting only single cells and retaining their viability for a prolonged period of time. This study demonstrated a surface engineering approach that uses a covalently bound short peptide as a mediator to pattern cells with improved single cell adhesion and prolonged cellular viability on gold patterned SiO2 substrates. The underlying hypothesis is that cell adhesion is regulated by the type, availability, and stability of effective cell adhesion peptides, and thus covalently bound short peptides would promote cell spreading and, thus, single cell adhesion and viability. The effectiveness of this approach and the underlying mechanism for the increased probability of single cell adhesion and prolonged cell viability by short peptides were studied by comparing cellular behavior of human umbilical cord vein endothelial cells on three model surfaces whose gold electrodes were immobilized with fibronectin, physically adsorbed Arg-Glu-Asp-Val-Tyr, and covalently bound Lys-Arg-Glu-Asp-Val-Tyr, respectively. The surface chemistry and binding properties were characterized by reflectance Fourier transform infrared spectroscopy. Both short peptides were superior to fibronectin in producing adhesion of only single cells, whereas the covalently bound peptide also reduced apoptosis and necrosis of adhered cells. Controlling cell spreading by peptide binding domains to regulate apoptosis and viability represents a fundamental mechanism in cell-materials interaction and provides an effective strategy in engineering arrays of single cells.     

Enhancement of biocompatibility on aligned electrospun poly(3‐hydroxybutyrate) scaffold immobilized with laminin towards murine neuroblastoma Neuro2a cell line and rat brain‐derived neural stem cells (mNSCs)

Electrospinning has been extensively used to construct tissue‐engineered scaffolds because of its ability to provide the fibrous scaffold with structurally analogous to the naturally occurring protein in the extracellular matrix of native tissues. In addition, the modification of scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation, and differentiation. In the present contribution, we prepared and investigated the potential used of aligned electrospun poly(3‐hydroxybutyrate) (PHB) scaffold immobilized with bioactive molecule to serve as nervous scaffold. Laminin was successfully immobilized on the surface using covalent binding between functional groups of modified scaffolds and protein. The ability to use for neural regeneration was evaluated in vitro towards murine neuroblastoma Neuro2a cell line and mouse brain‐derived neural stem cells. The surface modification with laminin immobilized on the PHB fibrous scaffolds supported the attachment and promoted the proliferation of Neuro2a very wells. Despite the good attachment and proliferation of Neuro2a and mouse brain‐derived neural stem cells were not able to proliferate on the neat PHB, hydrolyzed PHB and laminin immobilized on hydrolyzed PHB fibrous scaffold.     

能够促进细胞黏附的生物活性表面的制备

报道了一种能够促进细胞黏附的生物活性表面的制备方法.首先通过表面引发原子转移自由基聚合方法在硅表面接枝了聚(N-甲基丙烯酰氧基琥珀酰亚胺)(PNMASI)聚合物刷.随着反应时间的增加,接枝层厚度基本呈线性增长,表明聚合反应具有一定的可控性.蛋白质吸附测试表明PNMASI改性后的表面具有高密度固定生物分子的能力.同时,通...     

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronectin coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4-fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties.     

Cell shape and spreading of stromal (mesenchymal) stem cells cultured on fibronectin coated gold and hydroxyapatite surfaces

In order to identify the cellular mechanisms leading to the biocompatibility of hydroxyapatite implants, we studied the interaction of human bone marrow derived stromal (mesenchymal) stem cells (hMSCs) with fibronectin-coated gold (Au) and hydroxyapatite (HA) surfaces. The adsorption of fibronectin was monitored by Quartz Crystal Microbalance with Dissipation (QCM-D) at two different concentrations, 20 μg/ml and 200 μg/ml, and the fibronectin adsorption experiments were complemented with antibody measurements. The QCM-D results show that the surface mass uptake is largest on the Au surfaces, while the number of polyclonal and monoclonal antibodies directed against the cell-binding domain (CB-domain) on the fibronectin (Fn) is significantly larger on the (HA) surfaces. Moreover, a higher number of antibodies bound to the fibronectin coatings formed from the highest bulk fibronection concentration. In subsequent cell studies with hMSC's we studied the cell spreading, cytoskeletal organization and cell morphology on the respective surfaces. When the cells were adsorbed on the uncoated substrates, a diffuse cell actin cytoskeleton was revealed, and the cells had a highly elongated shape. On the fibronectin coated surfaces the cells adapted to a more polygonal shape with a well-defined actin cytoskeleton, while a larger cell area and roundness values were observed for cells cultured on the coated surfaces. Among the coated surfaces a slightly larger cell area and roundness values was observed on HA as compared to Au. Moreover, the results revealed that the morphology of cells cultured on fibronectin coated HA surfaces were less irregular. In summary we find that fibronectin adsorbs in a more activated state on the HA surfaces, resulting in a slightly different cellular response as compared to the fibronectin coated Au surfaces.     

Effects of a serum spreading factor on growth and morphology of cells in serum-free medium

A heat-sensitive, trypsin-sensitive factor that promoted growth and spreading of cells in serum-free, hormone-supplemented medium was partially purified from human serum. The major portion of the proteins in these preparations migrated upon SDS-polyacrylamide gel electrophoresis with a mobility consistent with molecular weights between 60,000 and 90,000. The spreading activity, which we have termed serum spreading factor, stimulated growth and spreading of a wide variety of cell types. The serum spreading factor was similar to fibronectin in that it showed an affinity for the plastic cell culture substrate but was shown to be distinct from fibronectin by several criteria. This factor may prove useful in studies of cell attachment and spreading and in studies of the relationship of cell shape and cell proliferation.     

Multifunctional mixed SAMs that promote both cell adhesion and noncovalent DNA immobilization

The ability of DNA strands to influence cellular gene expression directly and to bind with high affinity and specificity to other biological molecules (e.g., proteins and target DNA strands) makes them a potentially attractive component of cell culture substrates. On the basis of the potential importance of immobilized DNA in cell culture and the well-defined characteristics of alkanethiol self-assembled monolayers (SAMs), the current study was designed to create multifunctional SAMs upon which cell adhesion and DNA immobilization can be independently modulated. The approach immobilizes the fibronectin-derived cell adhesion ligand Arg-Gly-Asp-Ser-Pro (RGDSP) using carbodiimide activation chemistry and immobilizes DNA strands on the same surface via cDNA-DNA interactions. The surface density of hexanethiol-terminated DNA strands on alkanethiol monolayers (30.2-69.2 pmol/cm2) was controlled using a backfill method, and specific target DNA binding on cDNA-containing SAMs was regulated by varying the soluble target DNA concentration and buffer characteristics. The fibronectin-derived cell adhesion ligand GGRGDSP was covalently linked to carboxylate groups on DNA-containing SAM substrates, and peptide density was proportional to the amount of carboxylate present during SAM preparation. C166-GFP endothelial cells attached and spread on mixed SAM substrates and cell adhesion and spreading were specifically mediated by the immobilized GGRGDSP peptide. The ability to control the characteristics of noncovalent DNA immobilization and cell adhesion on a cell culture substrate suggests that these mixed SAMs could be a useful platform for studying the interaction between cells and DNA.     

Effects of Danggui Buxue Tang, a traditional Chinese herbal decoction, on high glucose-induced proliferation and expression of extracellular matrix proteins in glomerular mesangial cells

Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidney, but the efficacy of currently available strategies for the prevention of DN remains unsatisfactory. In this study, we investigated the effects of Danggui Buxue Tang (DBT), a Chinese herbal decoction prepared from Radix Astragali (RA) and Radix Angelicae sinensis (RAS), on high glucose-induced proliferation and expression of laminin, type IV collagen (collagen IV) and fibronectin in glomerular mesangial cells (GMCs). The cell proliferation was determined by MTT assay, and the expression of collagen IV, laminin and fibronectin in GMCs was detected by ELISA assay. It was shown that high glucose clearly induced the proliferation of GMCs and increased the release of collagen IV, laminin and fibronectin. Treatment with RA, RAS and DBT inhibited cell proliferation and the expression of collagen IV, laminin and fibronectin induced by high glucose, with DBT, especially at the highest concentration (DBT20), exhibiting a stronger effect than RA and RAS alone. Thus, it is concluded that DBT inhibits increased cell proliferation and the expression of major extracellular matrix proteins that are induced by high glucose, indicating its value for prophylaxis and therapy of DN at the early stages.