Yusuke Arima

Effects of surface functional groups on protein adsorption and subsequent cell adhesion using self-assembled monolayers

We investigated initial cell adhesion on self-assembled monolayers (SAMs) of alkanethiols carrying different functional groups including methyl (CH3), hydroxyl (OH), carboxylic acid (COOH), and amine (NH2). The combination of a surface plasmon resonance (SPR) instrument and a total internal reflection fluorescence microscope (TIRFM) allowed us to examine the kinetics of protein adsorption and correlating cell adhesion. Upon exposure of the SAM surface to a serum-containing medium, serum proteins rapidly adsorbed, and cells subsequently approached the surface. Adhesion of human umbilical vein endothelial cells (HUVECs) was greatly affected by surface functional groups; HUVECs adhered well to COOH– and NH2–SAMs, whereas poorly to CH3– and OH–SAMs. The amount of adsorbed protein from the serum-containing medium varied slightly with the terminal groups of the SAMs. On COOH– and NH2–SAMs, HUVECs adhered to bovine serum albumin (BSA)-preadsorbed surfaces with a few minutes delay, suggesting that displacement of preadsorbed BSA with cell-adhesive proteins, such as fibronectin or vitronectin, supports cell adhesion to these surfaces. Since the concentration of cell-adhesive proteins is much less than that of non-adhesive proteins such as BSA, displacement of adsorbed proteins with cell-adhesive proteins plays an important role in initial cell adhesion.

Complement Activation by Polymers Carrying Hydroxyl Groups

Hydrogels of polymers carrying surface hydroxyl groups strongly activate the complement system through the alternative pathway, although it has also been reported that solutions of polymers do not. To address these curious, inconsistent results, we examined the effect of polymer states, either immobilized on a surface or soluble in serum, on the complement activation using a surface plasmon resonance apparatus and enzyme-linked immunosorbent assay. We clearly showed that dextran- and poly(vinyl alcohol)-immobilized surfaces strongly activated the complement system but that soluble polymers could not, even when the amounts of the soluble polymers added to serum were 4-2000 times higher than those on the polymer-immobilized surfaces.

Surface plasmon resonance in monitoring of complement activation on biomaterials

When artificial materials come into contact with blood, various biological responses are induced. For successful development of biomaterials used in biomedical devices that will be exposed to blood, understanding and control of these interactions are essential. Surface plasmon resonance (SPR) spectroscopy is one of the surface-sensitive optical methods to monitor biological interactions. SPR enables real-time and in situ analysis of interfacial events associated with biomaterials research. In this review, we describe an SPR biosensor and its application to monitor complement activation onto biomaterials surface. We also discuss the effect of surface properties of the material on complement activation.

Preferential adsorption of cell adhesive proteins from complex media on self-assembled monolayers and its effect on subsequent cell adhesion

UNLABELLED We examined the effect of surface chemistry on adsorption of fibronectin (Fn) and vitronectin (Vn) and subsequent cell adhesion, employing self-assembled monolayers (SAMs) of alkanethiols carrying terminal methyl (CH3), hydroxyl groups (OH), carboxylic acid (COOH), and amine (NH2). More Fn and Vn adsorbed to COOH- and NH2-SAMs than to CH3- and OH-SAMs from a mixture with bovine serum albumin (BSA) and from 2% fetal bovine serum. Adhesion of human umbilical vein endothelial cells (HUVECs) on CH3- and OH-SAMs preadsorbed with Fn and BSA decreased with decreasing adsorbed Fn; however, HUVECs adhered to COOH- and NH2-SAMs even in the presence of BSA at 1000-fold more than Fn in a mixture because of the preferential adsorption of Fn and/or displacement of preadsorbed BSA with Fn and Vn in a serum-containing medium. SAMs coated with a mixture of Vn and BSA exhibited adhesion of HUVECs regardless of surface functional groups. A well-organized focal adhesion complex and actin stress fibers were observed only for COOH- and NH2-SAMs when SAMs were preadsorbed with Vn and BSA. These results suggest that COOH- and NH2-SAMs allow for both cell adhesion and cell spreading because of the high density of cell-binding domains derived from adsorbed Vn. STATEMENT OF SIGNIFICANCE Adsorption of cell adhesive proteins including fibronectin (Fn) and vitronectin (Vn) plays an important role in cell adhesion to artificial materials. However, for the development of biomaterials that contact with biological fluids, it is important to understand adsorption of Fn and Vn in complex media containing many kinds of proteins. Here, we focused on adsorption of Fn and Vn from complex media including mixed solution with albumin and fetal bovine serum, and its role on cell adhesion using self-assembled monolayers (SAMs). Our result demonstrates that SAMs carrying carboxylic acid or amine allow for both cell adhesion and cell spreading because of preferentially adsorbed Vn. The result provides insights into surface design of cell culture substrates and tissue engineering scaffolds.

Array-based functional screening of growth factors toward optimizing neural stem cell microenvironments

To gain insights into the effect of various growth factors on the behaviors of neural stem cells, cell culture assays were performed on the array that displayed five different growth factors including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, brain-derived neurotrophic factor, and ciliary neurotrophic factor. These factors were expressed in Escherichia coli as fusion proteins with a hexahistidine sequence and arrayed on a nickel ion-functionalized chip as single factors or the combination of two factors. Neural stem cells obtained from the fetal rat brain were cultured on the array to investigate their proliferation and differentiation. It was shown that the five growth factors displayed as a single component had significant impacts on cell behaviors. These effects are overall in accordance with those reported previously. On the other hand, in the case that two different growth factors were co-displayed on a single spot, the behaviors of neural stem cells could not be simply predicted from their individual effects. We performed a multivariate cluster analysis for the quantitative data on cell proliferation and differentiation. It was shown that the effect of two growth factors co-displayed was competitive, synergistic, or destructive depending on the combinations. In other peculiar cases, the effect of growth factors was totally different from those of individual factors.

Complement activation on degraded polyethylene glycol-covered surface

Surface modification with polyethylene glycol (PEG) has been employed in the development of biomaterials to reduce unfavorable reactions. However, unanticipated body reactions have been reported, with activation of the complement system being suggested as having involvement in these responses. In this study, we prepared a PEG-modified surface on a gold surface using a monolayer of alpha-mercaptoethyl-omega-methoxy-polyoxyethylene. We observed neither protein adsorption nor activation of the complement system on the PEG-modified surface just after preparation. Storage of the PEG-modified surface in a desiccator under ambient light for several days or following ultraviolet irradiation, reflection-adsorption (FTIR-RAS) and X-ray photo spectrometry revealed deterioration of the PEG layer, which became a strong activator of the complement system through the alternative pathway.

Surface Plasmon Resonance and Surface Plasmon Field-Enhanced Fluorescence Spectroscopy for Sensitive Detection of Tumor Markers

Surface plasmon resonance (SPR), which provides real-time, in situ analysis of dynamic surface events, is a valuable tool for studying interactions between biomolecules. In the clinical diagnosis of tumor markers in human blood, SPR is applied to detect the formation of a sandwich-type immune complex composed of a primary antibody immobilized on a sensor surface, the tumor marker, and a secondary antibody. However, the SPR signal is quite low due to the minute amounts (ng-pg/mL) of most tumor markers in blood. We have shown that the SPR signal can be amplified by applying an antibody against the secondary antibody or streptavidin-conjugated nanobeads that specifically accumulate on the secondary antibody. Another method employed for highly sensitive detection is the surface plasmon field-enhanced fluorescence spectroscopy-based immunoassay, which utilizes the enhanced electric field intensity at a metal/water interface to excite a fluorophore. Fluorescence intensity attributed to binding of a fluorophore-labeled secondary antibody is increased due to the enhanced field in the SPR condition and can be monitored in real time.

Effect of swelling of poly(vinyl alcohol) layers on complement activation

Polymers carrying hydroxyl groups have the potential ability to activate the complement system when in contact with blood. However, the effects of their surface structure on complement activation are still not fully understood. In this study, we examined complement activation by poly(vinyl alcohol) (PVA) layers formed on a gold surface modified with aldehyde groups. The complement system was strongly activated by a PVA surface with a dry thickness of 2.9 nm, while it was poorly activated by a PVA surface with a dry thickness of 7.4 nm. Annealing of the latter for 2 h at 150 degrees C converted the surface into a complement activating surface. The difference in complement activation between PVA layers was associated with the water content of PVA layers. These results suggest that complement activation by hydrated polymers highly depends on the water content of the polymer layers.

Multifunctional chimeric proteins for the sequential regulation of neural stem cell differentiation.

Controlling the dynamics of growth factor signaling is a challenge in regenerative medicine for various tissues including the central nervous system. Here, we report on the development of the biomolecular system that facilitates sequential regulation of growth factor signals acting on neural stem/progenitor cells. Recombinant technology was employed to synthesize the multifunctional chimeric protein that contained multiple domains, including epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), globular capping domain, thrombin-cleavable sequence, and substrate-binding domain with affinity for Ni(II) ions. The chimeric protein is expected to expose CNTF upon elimination of the capping domain by digestion with endogenous thrombin in vivo. When the multifunctional chimeric protein was immobilized onto a substrate through the coordination of the substrate-binding domain with surface-immobilized Ni(II) ions, the substrate served to proliferate neural stem cells, maintaining the population of undifferentiated cells at 85%. This effect is primarily due to the activity of EGF, while CNTF activity is temporally veiled with the capping domain. Upon digesting the thrombin-cleavable sequence to remove the capping domain, the activity of CNTF emerged to induce differentiation of astrocytes in situ from the proliferated neural stem cells. The fraction of differentiated astrocytes reached 68% of total cells. These results demonstrate the feasibility of the system for controlling the dynamics of growth factor signals.

Effect of dielectric spacer thickness on signal intensity of surface plasmon field-enhanced fluorescence spectroscopy.

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) combines enhanced field platform and fluorescence detection. Its advantages are the strong intensity of the electromagnetic field and the high signal/noise (S/N) ratio due to the localized evanescent field at the water/metal interface. However, the energy transfer from the fluorophore to the metal surface diminishes the fluorescence intensity, and this reduces the sensitivity. In this study, we tested whether polystyrene (PSt) could act as a dielectric layer to suppress the energy transfer from the fluorophore to the metal surface. We hypothesized that this would improve the sensitivity of SPFS-based immunoassays. We used α-fetoprotein (AFP) as a model tumor biomarker in the sandwich-type immunoassay. We determined the relationship between fluorescent signal intensity and PSt layer thickness and compared this to theoretical predictions. We found that the fluorescence signal increased by optimally controlling the thickness of the PSt layer. Our results indicated that the SPFS-based immunoassay is a promising clinical diagnostic tool for quantitatively determining the concentrations of low-level biomarkers in blood samples.