Dohiko Terada

Fabrication of conducting electrospun nanofibers scaffold for three-dimensional cells culture

Electrospinning is a versatile method to fabricate nanofibers of a range of polymeric and composite materials suitable as scaffolds for tissue engineering applications. In this study, we report the fabrication and characterization of polyaniline-carbon nanotube/poly(N-isopropyl acrylamide-co-methacrylic acid) (PANI-CNT/PNIPAm-co-MAA) composite nanofibers and PNIPAm-co-MAA nanofibers suitable as a three-dimensional (3D) conducting smart tissue scaffold using electrospinning. The chemical structure of the resulting nanofibers was characterized with FTIR and ¹H NMR spectroscopy. The surface morphology and average diameter of the nanofibers were observed by SEM. Cellular response of the nanofibers was studied with mice L929 fibroblasts. Cell viability was checked on 7 th day of cell culture by double staining the cells with calcein-AM and PI dye. PANI-CNT/PNIPAm-co-MAA composite nanofibers were shown the highest cell growth and cell viability as compared to PNIPAm-co-MAA nanofibers. Cell viability in the composite nanofibers was obtained in order of 98% that indicates the composite nanofibers provide a better environment as a 3D scaffold for the cell proliferation and attachment suitable for tissue engineering applications.

An enzyme-free highly glucose-specific assay using self-assembled aminobenzene boronic acid upon polyelectrolytes electrospun nanofibers-mat

A highly selective enzyme-free amperometric glucose sensor based on electrostatic self-assembling of 3-aminobenzene boronic acid (ABBA) onto a poly(styrene-co-acrylamide)/polystyrene sulfonic acid (PSA/PSSA) electrospun nanofibers-mat was investigated. Emerging ability of phenylboronic acid to bind with the diols of sugars has been extended for rapid response of glucose with a pH-sensitive redox mediator, hematein natural dye. ABBA was adsorbed on the PSA/PSSA nanofibers-mat/Pt-disc electrode that resulted in an ABBA/PSA/PSSA glucose active electrode. The interaction of ABBA onto the PSA/PSSA nanofibers-mat/Pt-disc electrode was characterized with Fourier transform infrared spectroscopy (FT-IR), ζ-potential, scanning electron microscopy (SEM), contact angle and cyclic voltammetry (CV) measurements. The prepared enzyme-free sensor exhibited a fast amperometric response, i.e., about 4s and linearity ranging from 0.75 to 14mM to glucose with a sensitivity of 0.987μAmM(-1)cm(-2). Compared to other types of glucose biosensors viz. use glucose oxidase as sensing elements, present glucose sensor offers basic advantages including ease of fabrication, high affinity-selectivity to the glucose upon the electrode surface and quick response.

Influence of poly(n‐isopropylacrylamide)–CNT–polyaniline three‐dimensional electrospun microfabric scaffolds on cell growth and viability

This study investigates the effect on: (1) the bulk surface and (2) the three-dimensional non-woven microfabric scaffolds of poly(N-isopropylacrylamide)-CNT-polyaniline on growth and viability of cells. The poly(N-isopropylacrylamide)-CNT-polyaniline was prepared using coupling chemistry and electrospinning was then used for the fabrication of responsive, non-woven microfabric scaffolds. The electrospun microfabrics were assembled in regular three-dimensional scaffolds with OD: 400-500 μm; L: 6-20 cm. Mice fibroblast cells L929 were seeded on the both poly(N-isopropylacrylamide)-CNT-polyaniline bulk surface as well as non-woven microfabric scaffolds. Excellent cell proliferation and viability was observed on poly(N-isopropylacrylamide)-CNT-polyaniline non-woven microfabric matrices in compare to poly(N-isopropylacrylamide)-CNT-polyaniline bulk and commercially available Matrigel™ even with a range of cell lines up to 168 h. Temperature dependent cells detachment behavior was observed on the poly(N-isopropylacrylamide)-CNT-polyaniline scaffolds by varying incubation at below lower critical solution temperature of poly(N-isopropylacrylamide). The results suggest that poly(N-isopropylacrylamide)-CNT-polyaniline non-woven microfabrics could be used as a smart matrices for applications in tissue engineering.

An ultra sensitive saccharides detection assay using carboxyl functionalized chitosan containing Gd2O3:Eu3+ nanoparticles probe

A novel saccharides detection assay based on covalent immobilization of amino phenyl boronic acid (APBA) in thin films of carboxyl functionalized chitosan (HOOC-chitosan) containing <5 nm Gd2O3 : Eu3+ nanoparticles at a platinum disc electrode was developed. The resulting HOOC-chitosan/Gd2O3 : Eu3+ nanocomposite film exhibited excellent electrochemical response to changes in the pKa values of boronate esters yielded from different vicinal diols of sugars. The covalent interaction of APBA onto the HOOC-chitosan/Gd2O3 : Eu3+ Pt-disc electrode was characterized with FT-IR, SEM, contact angle and cyclic voltammetry, whereas Gd2O3 : Eu3+ nanoparticles and HOOC-chitosan/Gd2O3 : Eu3+ nanocomposite was identified using XRD, EDX and TEM. A wide linear response was measured to boronate esters ranging from 25 nM to 13.5 μM (r2 = 0.963) with good reproducibility. The excellent electrochemical activity of the assay might be attributed to the synergistic effects of the balanced de-/protonated HOOC-chitosan, APBA and Gd2O3 : Eu3+ nanoparticles. With APBA as a model, the HOOC-chitosan/Gd2O3 : Eu3+ nanocomposite-modified Pt-electrode was constructed through a simple drop coating method. The resulting assay exhibited a good potentiometric response to different saccharides including glucose, and could be a promising application for the precise electrochemical detection of vicinal diols of specific sugars for clinical diagnostics, medicine validation, bioscience research and food analysis.

Transient charge-masking effect of applied voltage on electrospinning of pure chitosan nanofibers from aqueous solutions

Abstract The processing of a polyelectrolyte (whose functionality is derived from its ionized functional groups) into a nanofiber may improve its functionality and yield multiple functionalities. However, the electrospinning of nanofibers from polyelectrolytes is imperfect because polyelectrolytes differ considerably from neutral polymers in their rheological properties. In our study, we attempt to solve this problem by applying a voltage of opposite polarity to charges on a polyelectrolyte. The application of this ‘countervoltage’ can temporarily mask or screen a specific rheological property of the polyelectrolyte, making it behave as a neutral polymer. This approach can significantly contribute to the development of new functional nanofiber materials.

The outermost surface properties of silk fibroin films reflect ethanol-treatment conditions used in biomaterial preparation

Silk fibroin has attracted interest as a biomaterial, given its many excellent properties. Cell attachment to silk substrates is usually weaker than to standard culture dishes, and cells cultured on silk films or hydrogels typically form spheroids and micro-aggregates. However, too little is known about the higher order structures and behavior of fibroin under different conditions to explain the features of silk fibroin as a culture substrate. For instance, different biomaterial surfaces, with distinct effects on cell culture, can be achieved by varying the conditions of crystallization by alcohol immersion. Here, we show that treatment of fibroin film with <80% ethanol results in a jelly-like, hydrated hydrogel as the outermost surface layer; fibroblasts preferably aggregate, rather than attach individually to such a hydrogel surface, and therefore aggregate into spheroids. In contrast, a fibroin film treated with >90% ethanol has a harder surface than the <80% ethanol-treated fibroin, to which individual cells prefer to attach (and then expand on the surface), rather than to aggregate. We discuss the influence of alcohol concentration on the surface properties, based on surface analysis of the films. The surface analysis involved assessment of static and dynamic contact angles, zeta potential, changes in crystallinity and microscopic morphology of electrospun fibers, and texture changes of the outermost surface at a nanometer-scale captured by a scanning probe microscope.

Efficacy of a poly glycolic acid (PGA)/collagen composite nanofibre scaffold on cell migration and neovascularisation in vivo skin defect model

Abstract Application of tissue engineering currently provides promising therapeutic options in the fields of plastic surgery and wound management. The ability of scaffold material for cell proliferation and differentiation is the key for tissue engineering. This study has developed a novel nanofibre composed of poly glycolic acid (PGA) and collagen, both of which have their own respective beneficial properties. This study aimed to estimate the in vivo efficiency of the PGA/collagen nanofibre on granulation histology and its ability to induce neovascularisation. The electrospinning technique produced the PGA/collagen nanofiber with a diameter of 500 nm and weight mixing ratio of 40%. The skin defects on the mouse model were covered with PGA/collagen or a commercially available collagen matrix (n = 9). The PGA/collagen group histologically showed significantly higher cell density and a fine microstructure with greater number of migrating cells as compared to collagen matrix. Then, both materials were applied to the microcirculatory angiogenesis model. The PGA/collagen group (n = 8) revealed significantly higher functional capillary density on days 5 and 7 after application. The findings substantiated the fact that our material had a superior ability regarding cellular migration and induction of neovascularisation compared with the elementary collagen matrix product. This better result might be attributed to the nano-size effect of fine structure and the incorporation of PGA, which has been associated with enhanced angiogenesis.

Embossed-carving processing of cytoskeletons of cultured cells by using focused ion beam technology

The focused ion beam (FIB) technology has drawn considerable attention in diverse research fields. FIB can be used to mill samples at the nanometer scale by using an ion beam derived from electrically charged liquid gallium (Ga). This powerful technology with accuracy at the nanometer scale is now being applied to life science research. In this study, we show the potential of FIB as a new tool to investigate the internal structures of cells. We sputtered Ga+ onto the surface or the cross section of animal cells to emboss the internal structures of the cell. Ga+ sputtering can erode the cell surface or the cross section and thus emboss the cytoskeletons quasi‐3 dimensionally. We also identified the embossed structures by comparing them with fluorescent images obtained via confocal laser microscopy because the secondary ion micrographs did not directly provide qualitative information directly. Furthermore, we considered artifacts during the FIB cross sectioning of cells and propose a way to prevent undesirable artifacts. We demonstrate the usefulness of FIB to observe the internal structures of cells. Microsc. Res. Tech. 76:290–295, 2013.