Kazuya Furusawa

Reagent-free crosslinking of aqueous gelatin: manufacture and characteristics of gelatin gels irradiated with gamma-ray and electron beam.

In order to obtain a gelatin hydrogel crosslinked by a reagent-free method, gamma-ray and electron beam radiation was applied to porcine, bovine and fish gelatin gels and the products were characterized by measuring the gel fraction, the swelling ratio and the enzymatic degradability. On increasing the radiation dose, the gel fraction increased and both the swelling ratio and the enzymatic degradability decreased. The transition temperature from gel to sol of the hydrogel containing more than 5% mammal gelatins increased up to more than 90°C when gamma-ray or electron beam were irradiated by more than 10 kGy. The results show that the degree of crosslinking of irradiated gelatin hydrogels increases with increasing irradiation dose and with decreasing concentration. It is suggested that the radiation crosslinking occurs around the physical crosslinking point or multiple helix structure of gelatin gel.

Anisotropic structure of calcium-induced alginate gels by optical and small-angle X-ray scattering measurements

It was more than 50 years ago that an appearance of birefringence in alginate gels prepared under cation flow was reported for the first time, however, the anisotropic structure of the alginate gel has not been studied in detail. In the present study, anisotropic Ca-alginate gels were prepared within dialysis tubing in a high Ca(2+)-concentration external bath, and optical and small-angle X-ray scattering (SAXS) measurements were performed to characterize the structure of the gel. The observations of the gel with crossed polarizers and with circular polarizers revealed the molecular orientation perpendicular to the direction of Ca(2+) flow. Analyses of the SAXS intensity profiles indicated the formation of rod-like fibrils consisting of a few tens of alginate molecules and that the anisotropy of the gel was caused by the circumferential orientation of the large fibrils. From the observed asymmetric SAXS pattern, it was found that the axis of rotational symmetry of the anisotropic structure was parallel to the direction of Ca(2+) flow. The alignment factor (A(f)) calculated from the SAXS intensity data confirmed that the orientation of the fibrils was perpendicular to the direction of Ca(2+) flow.

Studies on the Formation Mechanism and the Structure of the Anisotropic Collagen Gel Prepared by Dialysis-Induced Anisotropic Gelation

We have found that dialysis of 5 mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 151 mM [corrected] at 25 °C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that the collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

Universality and specificity in molecular orientation in anisotropic gels prepared by diffusion method

Molecular orientation in anisotropic gels of chitosan, Curdlan and DNA obtained by dialysis of those aqueous solutions in gelation-inducing solutions was investigated. In this diffusion method (or dialysis method), the gel formation was induced by letting small molecules diffuse in or out of the polymer solutions through the surface. For the gels of DNA and chitosan, the polymer chains aligned perpendicular to the diffusion direction. The same direction of molecular orientation was observed for the Curdlan gel prepared in the dialysis cell. On the other hand, a peculiar nature was observed for the Curdlan gel prepared in the dialysis tube: the molecular orientation was perpendicular to the diffusion direction in the outermost layer of the gel, while the orientation was parallel to the diffusion direction in the inner translucent layer. The orientation parallel to the diffusion direction is attributed to a small deformation of the inner translucent layer caused by a slight shrinkage of the central region after the gel formation. At least near the surface of the gel, the molecular orientation perpendicular to the diffusion direction is a universal characteristic for the gels prepared by the diffusion method.

Effect of pH on Anisotropic Gelation of DNA Induced by Aluminum Cations

To investigate the effects of pH on the structure and the properties of DNA anisotropic gels and their growth process, we have observed the morphology of DNA anisotropic gel films prepared from DNA solutions with various initial pH, and measured the pH dependences of the shrinking ratio, the birefringence, and the relaxation modulus of the gel as well as the time courses of the gel front and pH-change front lines. The gel films prepared from DNA solutions with high pH have inhomogeneous macroscopic structure, large shrinking ratio, and high optical anisotropy whereas those prepared from DNA solutions with low pH have homogeneous macroscopic structure, small shrinking ratio, and low optical anisotropy. The difference observed at different pH is attributed to the difference in the interaction between DNA molecules and aluminum cations. The time courses of the gel front and pH-change front lines were analyzed with theories based on assumptions for each condition. Both two-stage dynamics observed under high initial pH and one-stage dynamics under low initial pH were explained consistently with the theories.

Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate.

Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis.

Multiscale analysis of changes in an anisotropic collagen gel structure by culturing osteoblasts.

Mimicking the complicated anisotropic structures of a native tissue is extremely important in tissue engineering. In a previous study, we developed an anisotropic collagen gel scaffold (ACGS) having a hierarchical structure and a properties gradient. In this study, our objective was to see how cells remodel the scaffolds through the cells-ACGS interaction. For this purpose, we cultured osteoblastic cells on ACGS, which we regarded as a model system for the cells-extracellular matrix (cell-ECM) interaction. Changes in the ACGS-cell composites structure by cell-ECM interactions was investigated from a macroscopic level to a microscopic level. Osteoblastic cells were also cultured on an isotropic collagen gel (ICGS) as a control. During the cultivation, mechanical stimuli were applied to collagen-cell composites for adequate matrix remodeling. Confocal laser scanning microscope (CLSM) was used to observe macroscopic changes in the ACGS-cell composite structure by osteoblastic cells. Small-angle X-ray scattering (SAXS) measurements were performed to characterize microscopic structural changes in the composites. Macroscopic observations using CLSM revealed that osteoblastic cells remained only in the diluted phase in ACGS and they collected collagen fibrils or formed a toroidal structure, depending on the depth from the ACGS surface in the tubular diluted phase. The cells were uniformly distributed in ICGS. SAXS analysis suggests that collagen fibrils were remodeled by osteoblastic cells, and this remodeling process would be affected by the structure difference between ACGS and ICGS. These results suggest that we directly regulate cell-ECM interaction by the unique anisotropic and hierarchical structure of ACGS. The cell-gel composite presented in this study would promise an efficient scaffold material in tissue engineering.

Small-angle X-ray and light scattering analysis of multi-layered Curdlan gels prepared by a diffusion method

Curdlan, a microbial polysaccharide, forms a multi-layered gel consisting of four layers with different turbidity when its alkaline solution is dialyzed against aqueous solutions containing Ca2+ (diffusion-set gel). The present study clarified the microstructure of each layer of the diffusion-set Curdlan gel by small-angle X-ray scattering (SAXS) and small-angle light scattering (SALS). The SAXS data showed that Curdlan chains assume a helical ordered conformation in the gel and that the gel consists of the fibrils formed by the association of Curdlan chains and the aggregates of fibrils. The SAXS results also indicated that the gelation is induced by the formation of a network of Ca2+-cross-linked fibrils in the outer region of the gel, whereas by the network formation of the aggregation of fibrils in the neutralization process in the inner region of the gel. A structural anisotropy of the gel was investigated by analysis of two-dimensional SAXS images, showing that the fibril is oriented circumferentially in the outer region of the cylindrical gel, whereas it is oriented randomly in the inner region of the gel. The SALS data showed that a characteristic length of an inhomogeneous structure in the turbid layers is of the order of micrometers. The observed spatial variation of the microscopic structure is caused by the difference in the paths of pH and [Ca2+] traced in the gelation process.

Expression of xSDF-1α, xCXCR4, and xCXCR7 during gastrulation in Xenopus laevis.

Chemokines play a crucial role in developmental processes and recent studies have revealed that they also control gastrulation movements. In this paper, we report the expression patterns of xSDF-1α, xCXCR4 and xCXCR7 and regulation of the expression of xSDF-1α and xCXCR4 during gastrulation. We performed whole mount in situ hybridization (WISH) and quantitative real-time RT-PCR (qRT-PCR) analyses to examine the distribution of transcripts. The effect of activin/nodal signaling on the expression of xSDF-1α and its receptors was examined by animal cap assay and microinjection of cer-s mRNA. We have demonstrated that the xSDF-1αtranscript is increased in the blastocoel roof during gastrulation, but not in the involuted mesoderm. xCXCR4 was expressed in the mesendoderm at late blastula and was retained throughout gastrulation. xCXCR7 was found in the dorsal lip around the blastopore in the early gastrula stage and became localized in the presumptive notochord later. We also show that the expression of xCXCR4 and xSDF-1transcript is increased in the blastocoel roof during gastrulation, but not in the involuted mesoderm. xCXCR4 was expressed in the mesendoderm at late blastula and was retained throughout gastrulation. xCXCR7 was found in the dorsal lip around the blastopore in the early gastrula stage and became localized in the presumptive notochord later. We also show that the expression of xCXCR4 and xSDF-1α were reciprocally regulated by activin/nodal signaling. These results suggest that xSDF-1α and its receptors contribute to the cell arrangement of mesoderm cells and their expression patterns are partially regulated by activin/nodal signaling.

ALUMINIUM ION‐INDUCED ANISOTROPIC GELATION OF DNA

Water‐insoluble anisotropic gel has been prepared from salmon milt DNA by immersing concentrated DNA aqueous borate solutions sandwiched between two circular glass plates in concentrated aluminium chloride solutions. To clear the dynamics of anisotropic gelation in immersion process, the time course of gel formation process has been measured with varying concentrations of DNA and aluminium chloride as well as diameter of glass plates. Scaled plots predicted by the theory based on “moving boundary picture” shows two stages; early stage and late stage, characterized by different values of a parameter K related to the diffusion coefficient of aluminium cations in the gel layer, the critical concentration of aluminium cations for forming gel, and the concentration of aluminium cations in immersing solution. The results are explained by the change in the interaction between DNA molecules and aluminium ions associated with pH change during gel formation. The entire process is expressed with a master curve by reducing the time and the gel layer thickness with the values at gel completion.