Fibronectin Films Research Articles

Poly-L-Lysine and Human Plasmatic Fibronectin Films as Proactive Coatings to Improve Implant Biointegration.

The success of stable and long-term implant integration implies the promotion, control, and respect of the cell microenvironment at the site of implantation. The key is to enhance the implant–host tissue cross talk by developing interfacial strategies that guarantee an optimal and stable seal of soft tissue onto the implant, while preventing potential early and late infection. Indeed, implant rejection is often jeopardized by lack of stable tissue surrounding the biomaterial combined with infections which reduce the lifespan and increase the failure rate of implants and morbidity and account for high medical costs. Thin films formed by the layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes are particularly versatile and attractive for applications involving cell–material contact. With the combination of the extracellular matrix protein fibronectin (Fn, purified from human plasma) and poly-L-lysine (PLL, exhibiting specific chain lengths), we proposed proactive and biomimetic coatings able to guarantee enhanced cell attachment and exhibiting antimicrobial properties. Fn, able to create a biomimetic interface that could enhance cell attachment and promote extracellular cell matrix remodeling, is incorporated as the anionic polymer during film construction by the LbL technic whereas PLL is used as the cationic polymer for its capacity to confer remarkable antibacterial properties.

Single-Cell Probe Force Studies to Identify Sox2 Overexpression-Promoted Cell Adhesion in MCF7 Breast Cancer Cells.

The replacement of the cantilever tip by a living cell in Atomic Force Microscopy (AFM) experiments permits the direct quantification of cell–substrate and cell–cell adhesion forces. This single-cell probe force measurement technique, when complemented by microscopy, allows controlled manipulation of the cell with defined location at the area of interest. In this work, a setup based on two glass half-slides, a non-fouling one with bacterial S-layer protein SbpA from L. sphaericus CMM 2177 and the second with a fibronectin layer, has been employed to measure the adhesion of MCF7 breast cancer cells to fibronectin films (using SbpA as control) and to other cells (symmetric vs. asymmetric systems). The measurements aimed to characterize and compare the adhesion capacities of parental cells and cells overexpressing the embryonic transcription factor Sox2, which have a higher capacity for invasion and are more resistant to endocrine therapy in vivo. Together with the use of fluorescence techniques (epifluorescence, Total Internal Fluorescence Microscopy (TIRF)), the visualization of vinculin and actin distribution in cells in contact with fibronectin surfaces is enabled, facilitating the monitoring and quantification of the formation of adhesion complexes. These findings demonstrate the strength of this combined approach to assess and compare the adhesion properties of cell lines and to illustrate the heterogeneity of adhesive strength found in breast cancer cells.

Self-stabilized fibronectin films at the air/water interface

ABSTRACTFibronectin (FN) is a mediator molecule, which can connect cell receptors to the extracellular matrix (ECM) in tissues. This function is highly desirable for biomaterial surfaces in order to support cell adhesion. Controlling the fibronectin adsorption profile on substrates is challenging because of possible conformational changes after deposition, or due to displacement by secondary proteins from the culture medium. Here, we aim to develop a method to realize self-stabilized ECM glycoprotein layers with preserved native secondary structure on substrates. Our concept is the assembly of FN layers at the air-water (A-W) interface by spreading FN solution as droplets on the interface and transfer of the layer by the Langmuir-Schäfer (LS) method onto a substrate. It is hypothesized that 2D confinement and high local concentration at A-W interface supports FN self-interlinking to form cohesive films. Rising surface pressure with time, plateauing at 10.5 mN·m-1 (after 10 hrs), indicated that FN was self-assembling at the A-W interface. In situ polarization-modulation infrared reflection absorption spectroscopy of the layer revealed that FN maintained its native anti-parallel β-sheet structure after adsorption at the A-W interface. FN self-interlinking and elasticity was shown by the increase in elastic modulus and loss modulus with time using interfacial rheology. A network-like structure of FN films formed at the A-W interface was confirmed by atomic force microscopy after LS transfer onto Si-wafer. FN films consisted of native, globular FN molecules self-stabilized by intermolecular interactions at the A-W interface. Therefore, the facile FN self-stabilized network-like films with native anti-parallel β-sheet structure produced here, could serve as stable ECM protein coatings to enhance cell attachment on in vitro cell culture substrates and planar implant materials.

Quartz Crystal Microbalance as Cell-Based Biosensor to Detect and Study Cytoskeletal Alterations and Dynamics.

Several techniques can be used to monitor cell dynamism after a perturbation. Among these, Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) offers the great advantage to study the mechanical properties of cells in real-time and with a great sensitivity. Here, we used QCM-D to investigate the effects of two cytoskeleton-targeting agents, cytochalasin D (CytoD) and Y27632, on human MCF-7 cells. Cell adhesion on the sensor surface, crucial for in-flow experiments, was obtained by covalent adsorption of a fibronectin (FN) film, an extracellular matrix (ECM) protein. Direct analysis of MCF-7 cells on FN-coated sensor, shows a specific cellular response that was revealed and quantified by QCM-D after drugs exposure. Notably, upon treatment with Y27632, we observed a two-regime dissipation behavior that we associated with specific modifications of actin filaments and signaling proteins providing a link between biophysical and molecular mechanisms. Overall, this approach opens new opportunities for studying cellular response to mechanical cues in different biological conditions.

Surface Features of Recombinant Spider Silk Protein eADF4(κ16)‐Made Materials are Well‐Suited for Cardiac Tissue Engineering

Cardiovascular diseases causing high morbidity and mortality represent a major socioeconomic burden. The primary cause of impaired heart function is often the loss of cardiomyocytes. Thus, novel therapies aim at restoring the lost myocardial tissue. One promising approach is cardiac tissue engineering. Previously, it is shown that Antheraea mylitta silk protein fibroin is a suitable material for cardiac tissue engineering, however, its quality is difficult to control. To overcome this limitation, the interaction of primary rat heart cells with engineered Araneus diadematus fibroin 4 (κ16) (eADF4(κ16)) is investigated here, which is engineered based on the sequence of ADF4 by replacing the glutamic acid residue in the repetitive unit of its core domain with lysine. The data demonstrate that cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells attach well to eADF4(κ16) films on glass coverslips which provide an engineered surface with a polycationic character. Moreover, eADF4(κ16) films have, in contrast to fibronectin films, no hypertrophic effect but allow the induction of cardiomyocyte hypertrophy. Finally, cardiomyocytes grown on eADF4(κ16) films respond to pro‐proliferative factors and exhibit proper cell‐to‐cell communication and electric coupling. Collectively, these data demonstrate that designed recombinant eADF4(κ16)‐based materials are promising materials for cardiac tissue engineering.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS.

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In vitro construction of vascularized iPSC-derived 3D-cardiac myoblast tissues for drug assessment

Event Abstract Back to Event In vitro construction of vascularized iPSC-derived 3D-cardiac myoblast tissues for drug assessment Michiya Matsusaki1, Yuto Amano1, Manabu Seo2*, Takashi Yamaguchi2, Shigeru Miyagawa3, Yoshiki Sawa3* and Mitsuru Akashi1 1 Osaka University, Department of Applied Chemistry, Graduate School of Engineering, Japan 2 Ricoh Company, Ltd., Advanced Technology Research & Development Center, Ricoh Institute of Technology, Japan 3 Osaka University Graduate School of Medicine, Department of Cardiovascular Surgery, Japan Introduction: Recent drug discovery systems are time-consuming and costly because the pharmaceutical assays using cell monolayer and animal models have many issues due to different drug responses as compared to human body. Therefore, the use of induced pluripotent stem cell (iPSC), which can provide many types of normal and diseased human cell sources, has enormous potential for pharmaceutical assays. However, since nearly all tissues are integrated three-dimensional (3D) structures of multiple types of cells and extracellular matrices (ECMs), and since intercellular signaling is important for tissue functions, it is difficult to evaluate actual tissue functions by 2D-culture method. In this study, we aim to develop 3D-cardiac myoblast tissues (3D-iPSC-CM) composed of iPS-CMs, cardiac fibroblast, and cardiac endothelial cells by the cell-accumulation technique (Figure 1). We reported a bottom-up approach, termed “accumulation technique”[1] which was improved method of our previous technique, hierarchical cell manipulation[2], to develop multilayered thick tissues (>100 µm) by cell surface coating with nanometer-sized ECM-films[3]. Less than 10 nm sized ECM-films, such as fibronectin (FN)-gelatin (G), induced cell-cell interaction in three dimensions. By this method, iPSC-derived 3D-cardiac muscle tissues were successfully fabricated. The obtained tissues showed the synchronized contractions, and the tolerance to the cytotoxicity of anticancer drugs[4]. The iPSC-derived 3D-cardiac muscle tissues have a great potential for pharmaceutical applications. Figure 1. Schematic illustration of the vascularized 3D-iPSC-CM for drug assessments. Methods: iPSC-CM with 50~60% differentiation was used in this study. After repeating the nine steps of layer-by-layer (LbL) deposition, the (FN/G)4 FN films with about 10 nm thickness were prepared on the cell surface by cell accumulation technique. Normal human cardiac fibroblasts (NHCF) and normal human cardiac endothelial cells (NHCMEC) were also coated as the same way. The coated cells were seeded into a 24 micro well cell culture insert (1 x 105 cells/layers) and cultured to construct multilayered tissues. The ratio of NHCF was varied to optimize for the highest beating number, synchronization, and vascularization. 10% NHCMEC was added for blood capillary formation. Results: The ratio of NHCF was varied to 0, 25, 50, 75, and 100% to optimize beating number and synchronization of the tissues. The 3D-iPSC-CM tissues with 25~50% of NHCF clearly showed the highest beating number and synchronization. In the case of blood capillary formation, the network clearly found at over 25% NHCF condition and the network area increased with increasing NHCF ratio. Accordingly, 50% NHCF was used for the drug assessment. Interestingly, the vascularized 3D-tissues showed higher tolerance to the drug as compared to the monolayer culture (2D). We also found that the capillary was damaged by the treatment of doxorubicin in dose dependent manner. These results indicated that the vascularized 3D-iPSC-CM tissues might be useful for drug assessment. Figure 2. (a) Beating number of 3D-iPSC-CM tissues (NHCF50) and 2D monolayr of iPSC-CMs (NHCF50) after 3 days of incubation with 1, 50, or 1000 nM doxorubicin. *indicates 0. (b) CLSM images of the 3D-tissues and 2D-monolayer after incubation with doxorubicin by immunostaining with troponin T antibody. (c) CLSM images of blood capillary networks in 3D-iPSC-CM tissues after treatment with doxorubicin. The networks were immunostained with anti CD31 antibody. (d) Quantified capillary area in images of (c). ††p < 0.01 statistically significant difference as compared to 2D. Conclusions: We successfully constructed vascularized iPSC-CM tissues by coating of FN-G nanofilms. Drug responses and tolerance mechanism of the 3D-tissues will report in the conference. NEXT Program (LR026); Grant-in-Aid for Scientific Research (S) (A232250040); Grant-in-Aid for Scientific Research (B) (26282138); Grant-in-Aid for Scientific Research on Innovative Areas (26106717); SENTAN-JST Program (13A1204); Health Labour Sciences Research Grant from the Japanese Ministry of Health, Labour and Welfare, and Grand-in-Aid for JSPS Fellows (24・622)

Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand–Receptor Bonds

Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the bacterial bond with a substratum and play important roles in bacterial adhesion, detachment and susceptibility to antimicrobials.

Micro Patterning, Tension Force and the Growth of Osteoblasts

The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying mechanism remains largely unclear. It has been reported that tension force is a key issue for osteogenesis of human mesenchymal stem cells (MSCs) and osteoblasts. In the present study, fibronectin film covered on glass substrate was made into square-shaped micro patterns. Osteoblasts cultured on this geometric substrate first located at the edge of the square. Next day the cells extended to the whole square. The results showed that geometric cues can control the growth of osteoblasts. Possible explanation is that the edge of the square provides higher tension force to the cell. These findings demonstrate that tension force from geometric shape is a positive signal for osteogenesis of osteoblasts and bone remodeling.

Adhesive Bond Stiffness of Staphylococcus aureus with and without Proteins That Bind to an Adsorbed Fibronectin Film

Staphylococcus aureus is known to cause biomaterial-associated infections of implants and devices once it has breached the skin and mucosal barriers. Adhesion is the initial step in the development of a biomaterial-associated infection, and strategies to prevent staphylococcal adhesion and thus biomaterial-associated infections require understanding of the adhesive bond. The aim of this study was to compare the adhesive bond stiffnesses of two S. aureus strains with and without fibronectin-binding proteins (FnBPs) adhering to a fibronectin-coated quartz crystal microbalance (QCM) sensor surface on the basis of a coupled- resonance model. Both fibronectin adsorption and staphylococcal adhesion were accompanied by negative frequency shifts, regardless of the absence or presence of FnBPs on the staphylococcal cell surfaces. This is the opposite of the positive frequency shifts often observed for other bacterial strains adhering to bare sensor surfaces. Most likely, adhering staphylococci sink into and deform the adsorbed protein layer, creating stiff binding with the sensor surface due to an increased bacterium-substratum contact area. S. aureus 8325-4 possesses FnBPs and yields less negative frequency shifts (Δf) that are further away from the zero-crossing frequency than S. aureus DU5883. This suggests that FnBPs on S. aureus 8325-4 create a stiffer bond to the fibronectin coating than has been observed for S. aureus DU5883. Due to a limited window of observation, as defined by the available resonance frequencies in QCM, we could not determine exact stiffness values.

Optimization of protein patterns for neuronal cell culture applications

In the present study, we fabricated two-component extracellular matrix protein patterned substrates with fibronectin (FN) and laminin (LN) because of our interest in the mechanism of axonal regeneration and injury in the central and peripheral nervous systems. The authors investigated how the patterning order and method of attachment affected the spatial distribution and biological activity of the immobilized proteins. Micro-contact printing (μCP) techniques in concert with reactive surface chemistry were used to modify glass substrates with one- and two-component films of FN and LN, including micrometer-scale patterns of FN and LN. The composition and spatial distributions of both proteins on the patterned surfaces were characterized by x ray photoelectron spectroscopy, epi-fluorescence microscopy, atomic force microscopy, and time-of-flight secondary-ion mass spectrometry. The authors also characterized the biological activity of the top-most protein layer in a two-layer protein system as well as the ability of the top-most protein layer to mask the biological activity of an underlying protein layer using a fluorescence-based enzyme-linked immunosorbent assay. The order of protein deposition significantly affected the relative biological activity of the upper-most and underlying immobilized proteins. As a result of these optimization studies, maximum biological activity per surface protein was achieved by first immobilizing FN from solution, followed by μCP of LN on the FN. Addition of μCP LN films was able to mask ∼84% of the underlying FN activity, whereas μCP FN films were only able to mask ∼27% of the underlying LN activity.

The effect of coimmobilizing heparin and fibronectin on titanium on hemocompatibility and endothelialization

Currently available cardiovascular implants, such as heart valves and stents, exhibit suboptimal biocompatibility because of the incomplete endothelialization and sequential thrombosis formation especially after a long-term implantation. To improve the blood compatibility and endothelialization simultaneously and ensure the long-term effect of the cardiovascular implants, a technique of combining electrostatic interaction and coimmobilization was developed to form heparin and fibronectin (Hep/Fn) films on aminosilanized titanium (Ti) surfaces. The Hep/Fn coimmobilized films were stable after immersion in PBS for five days, probed by wettability studies and by the release kinetics of heparin and fibronectin. Blood compatibility tests showed that the coimmobilized Hep/Fn films displayed lower hemolysis rate, prolonged blood coagulation time, higher AT III binding density, less platelets activation and aggregation, and less fibrinogen conformational change compared with Ti surface. Endothelial cells (ECs) seeding and fibronectin bioactivity results showed more attached and proliferated ECs and exposed cell-binding sites on the Hep/Fn immobilized samples than that on Ti surfaces. Thus, the Hep/Fn coimmobilized films kept excellent bioactivity even after immersion in PBS for five days. Systemic evaluation suggests that the coimmobilization of Hep/Fn complex improves the blood compatibility and promotes the endothelialization simultaneously. We envisage that this method will provide a potential and effective selection for biomaterials surface modification of cardiovascular implants.

Protein patterning by atmospheric-pressure plasmas

Low-frequency (LF) atmospheric-pressure plasma (APP) jets have been used for patterning of thin fibronectin films deposited on a silicon (Si) wafer with the use of a metal stencil mask. Since fibronectin is an adhesion protein that can be found in the extracellular matrix (ECM), a micro-patterned fibronectin film may be used for arranging living cells in a desired pattern on a surface. Removal of fibronectin from the surface by plasma application was observed by Fourier transform infrared spectroscopy (FT-IR) and atomic force microscopy (AFM). It has been found that removal of fibronectin takes place even in the location away from the direct plasma jet application spot, which indicates that desorption of fibronectin is likely to be caused by chemically reactive charge-neutral species that can diffuse away from the plasma without emitting visible light.

Control of Cell Surface and Functions by Layer-by-Layer Nanofilms

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.

Localized and Sustained SDF-1 Gene Release Mediated by Fibronectin Films: A Potential Method for Recruiting Stem Cells

Gene-activated matrix has wide potential utilization in tissue engineering. It may genetically modify cells with plasmid DNA encoding therapeutic genes and allow sustained expression and release of the proteins to surrounding tissues. In this study, we assessed the feasibility of the local gene release from human fibronectin (HFN) substrate and the efficacy of local release of stromal cell-derived factor-1 (SDF-1) gene on c-kit+ cell homing. Cationic polymer polyethylenimine (25kDa PEI) was used as non-viral DNA vector. Gene-activated HFN (GAH) was prepared by mixing PEI/DNA complexes with HFN substrate. The DNA retardation, the complex size, and the DNA release speed from the GAH were studied. The in vitro transfection was optimized by luciferase expression and cell viability assay in the COS7 cell line. Localized gene expression in COS7 cells cultured on the GAH was assessed by LacZ and GFP-N3-SDF-1 marker genes. Ckit+ cell homing was investigated in response to the local in vitro SDF-1 expression from rat mesenchymal stem cells (RMSCs) cultured on GAH. Results showed GAH allows long time-sustained DNA release, localized gene delivery, and high transfection efficiency. Local SDF-1 expression with GAH is a promising method to induce targetable stem cell homing.

Streptococcus mutans and Streptococcus intermedius Adhesion to Fibronectin Films Are Oppositely Influenced by Ionic Strength

Bacterial adhesion to protein-coated surfaces is mediated by an interplay of specific and nonspecific interactions. Although nonspecific interactions are ubiquitously present, little is known about the physicochemical mechanisms of specific interactions. The aim of this paper is to determine the influence of ionic strength on the adhesion of two streptococcal strains to fibronectin films. Streptococcus mutans LT11 and Streptococcus intermedius NCTC11324 both possess antigen I/II with the ability to bind fibronectin from solution, but S. intermedius binds approximately 20x less fibronectin than does the S. mutans strain under identical conditions. Both strains as well as fibronectin films are negatively charged in low ionic strength phosphate buffered saline (PBS, 10x diluted), but bacteria appear uncharged in high ionic strength PBS. Physicochemical modeling on the basis of overall cell surface properties (cell surface hydrophobicity and zeta potentials) demonstrates that both strains should favor adhesion to fibronectin films in a high ionic strength environment as compared to in a low ionic strength environment, where electrostatic repulsion between equally charged surfaces is dominant. Adhesion of S. intermedius to fibronectin films in a parallel plate flow chamber was completely in line with this modeling, while in addition atomic force microscopy (AFM) indicated stronger adhesion forces upon retraction between fibronectin-coated tips and the cell surfaces in high ionic strength PBS than in low ionic strength PBS. Thus, the dependence of the interaction on ionic strength is dominated by the overall negative charge on the interacting surfaces. Adhesion of S. mutans to fibronectin films, however, was completely at odds with theoretical modeling, and the strain adhered best in low ionic strength PBS. Moreover, AFM indicated weaker repulsive forces upon approach between fibronectin-coated tips and the cell surfaces in low ionic strength PBS than in high ionic strength PBS. This indicated that the dependence of the interaction on ionic strength is dominated by electrostatic attraction between oppositely charged, localized domains on the interacting surfaces, despite their overall negative charge. In summary, this study shows that physicochemical modeling of bacterial adhesion to protein-coated surfaces is only valid provided the number of specific interaction sites on the cell surfaces is low, such as on S. intermedius NCTC11324. Nonspecific interactions are dominated by specific interactions if the number of specific interaction sites is large, such as on S. mutans LT11. Its ionic strength dependence indicates that the specific interaction is electrostatic in nature and operative between oppositely charged domains on the interacting surfaces, despite the generally overall negatively charged character of the surfaces.

Can matrix metalloproteinases be targeted in hepatic ischemia/reperfusion injury?

Can matrix metalloproteinases be targeted in hepatic ischemia/reperfusion injury?

Structure and Reactivity of Adsorbed Fibronectin Films on Mica

Understanding the interactions of adsorbed fibronectin (Fn) with other biomolecules is important for many biomedical applications. Fn is found in almost all body fluids, in the extracellular matrix, and plays a fundamental role in many biological processes. This study found that the structure (conformation, orientation) and reactivity of Fn adsorbed onto mica is dependent on the Fn surface concentration. Atomic force microscopy and x-ray photoelectron spectroscopy were used to determine the surface coverage of adsorbed Fn from isolated molecules at low surface coverage to full monolayers at high surface coverage. Both methods showed that the thickness of Fn film continued to increase after the mica surface was completely covered, consistent with Fn adsorbed in a more upright conformation at the highest surface-Fn concentrations. Time-of-flight secondary ion mass spectrometry showed that relative intensities of both sulfur-containing (cystine, methionine) and hydrophobic (glycine, leucine/isoleucine) amino acids varied with changing Fn surface coverage, indicating that the conformation of adsorbed Fn depended on surface coverage. Single-molecule force spectroscopy with collagen-related peptides immobilized onto the atomic force microscope tip showed that the specific interaction force between the peptide and Fn increases with increasing Fn surface coverage.

Static time‐of‐flight secondary ion mass spectrometry and x‐ray photoelectron spectroscopy characterization of adsorbed albumin and fibronectin films

AbstractStatic time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), monochromatized x‐ray photoelectron spectroscopy (XPS) and 125I radiolabeling have been used to characterize albumin films adsorbed onto titanium, gold, polytetrafluoroethylene and r.f. glow discharge‐deposited tetrafluoroethylene (TFE) substrates. A comparison between albumin and fibronectin films also was made. The intensities of characteristic amino acid mass fragments (immonium ions) detected in the static ToF‐SIMS experiments depended on the protein type, the substrate type and the adsorption conditions, demonstrating the sensitivity of static ToF‐SIMS for probing the structure of adsorbed protein films. Based on the results from albumin and fibronectin, static ToF‐SIMS can provide information about the identity of adsorbed proteins and their conformation, orientation, denaturation, etc. X‐ray photoelectron spectroscopy can distinguish pure protein films, but the higher molecular specificity of static ToF‐SIMS is more useful than XPS for examining complex protein films. The 125I radiolabeling experiments and the XPS atomic percentage of nitrogen were used to quantify the amount of adsorbed protein. Copyright © 2001 John Wiley &amp; Sons, Ltd.

Static time‐of‐flight secondary ion mass spectrometry and x‐ray photoelectron spectroscopy characterization of adsorbed albumin and fibronectin films

Static time‐of‐flight secondary ion mass spectrometry and x‐ray photoelectron spectroscopy characterization of adsorbed albumin and fibronectin films