Shigeo Wada

Honeycomb-patterned thin films of amphiphilic polymers as cell culture substrates

Abstract Recently, we have found honeycomb patterns with sub-micron line width that form during the non-equilibrium process of cast film formation. The honeycomb-patterned films were fabricated using four macromolecular compounds (amphiphilic copolymers containing lactose units or carboxyl groups as side-chains and polyion complexes composed of anionic polysaccharides). The specific binding of lactose by lectin confirmed that the lactose moieties contained in the honeycomb films work as biologically active ligands. Bovine serum albumin (BSA) labeled with fluorescein was covalently attached to the honeycomb films using water-soluble carbodiimide (WSC) as an activator. Using fluorescence imaging of the modified film, we could show that the proteins are immobilized on the honeycomb patterns. Adhesion of bovine aorta endothelial cells (ECs) to the honeycomb films indicates that the honeycomb structure works as an adhesive site for the cells.

Mesoscopic patterning of cell adhesive substrates as novel biofunctional interfaces

In this report, cell adhesion to honeycomb-patterned films is described with respect to the dimensions of the honeycomb structure. The honeycomb-patterned films can be fabricated by casting a dilute solution of amphiphilic polymers on solid substrates. The honeycomb structure is not homogeneous in all dimensions. Analysis of distribution of the honeycomb hole sizes demonstrates a gradual decrease in honeycomb hole diameter along the radius of the cast area. The largest holes were located near geometric center of the cast area. The diameter of the largest honeycomb holes in the cast area could be controlled by varying the cast volume of the polymer solution. Cell cultures on the honeycomb films demonstrated that cell adhesion could be inhibited at the outer region of the cast area. The extent of the inhibition of cell adhesion was influenced by the chemical properties of the polymers constituting the honeycomb films.

Theoretical prediction of low-density lipoproteins concentration at the luminal surface of an artery with a multiple bend.

AbstractTo elucidate the mechanisms of localization of atherosclerotic lesions in man, the effects of various physical and hemodynamic factors on transport of atherogenic low-density lipoproteins (LDL) from flowing blood to the wall of an artery with a multiple bend were studied theoretically by means of a computer simulation under the conditions of a steady flow. It was found that due to a semipermeable nature of an arterial wall to plasma, flow-dependent concentration polarization of LDL occurred at the luminal surface of the vessel, creating a region of high LDL concentration distal to the apex of the inner wall of each bend where the flow was locally disturbed by the formation of secondary and recirculation flows and where wall shear stresses were low. The highest surface concentration of LDL occurred distal to the acute second bend where atherosclerotic intimal thickening developed. At a Re0=500, the values calculated using estimated diffusivities of LDL in whole blood and plasma were respectively 35.1% and 15.6% higher than that in the bulk flow. The results are consistent with our hypothesis that the localization of atherosclerotic lesions results from the flow-dependent concentration polarization of LDL which creates locally a hypercholesterolemic environment even in normocholesterolemic subjects, thus augmenting the uptake of LDL by vascular endothelial cells existing at such sites.

Theoretical study of the effect of local flow disturbances on the concentration of low-density lipoproteins at the luminal surface of end-to-end anastomosed vessels.

To elucidate the mechanisms of localisation of intimal hyperplasia in anastomosed arteries, the effects of flow disturbances on the transport of lowdensity lipoproteins (LDLs) from the flowing blood to the wall of end-to-end anastomosed arteries, with and without a moderate stenosis, were studied theoretically by means of a computer simulation under the condition of steady flow. In an artery with moderate stenosis at the anastomotic junction and intimal thickening distal to it, we found that, owing to the water-permeable nature of the arterial wall, the surface concentration of LDL was elevated up to 20% higher than that of the bulk flow distal to the stenosis, where a recirculation zone was formed and wall shear stresses were low. In contrast to this, no significant elevation of surface concentration of LDLs occurred in another anastomosed vessel in which no stenosis was formed and no intimal thickening was observed. These results suggest that flowdependent concentration polarisation of LDLs plays a causative role in the localisation of anastomotic intimal hyperplasia in the human arterial system by locally elevating the surface concentration of LDLs, thus augmenting their uptake by endothelial cells.

Computational Study on LDL Transfer from Flowing Blood to Arterial Walls

It is suspected that flow-dependent concentration polarization of low density lipoproteins (LDL) occurs at a blood/endothelium boundary due to an water-permeable nature of an arterial wall, creating favourable conditions for the genesis and development of several vascular diseases such as atherosclerosis, intimal hyperplasia and aneurysmus in the arterial system. Hence the effect of flow patterns and flow-induced wall shear stress (shear rate) on LDL concentration at a blood/endothelium boundary was investigated theoretically by means of a computer simulation of LDL transport from flowing blood to water-permeable walls of arteries having various geometry under conditions of a steady flow. It was found that in a straight artery, accumulation of LDL occurs near the vessel wall depending on the magnitude of wall shear rates, filtration velocity of water at the vessel wall, and diffusivity of LDL in blood under physiological conditions. Furthermore, through extension of the study to the cases of an axisymmetric stenosis, a T-junction, and curved segments, it was found that surface concentration of LDL is locally elevated in regions where wall shear stresses are low and where blood reaches after traveling a long distance along the vessel wall. These results strongly suggest that flow-dependent concentration polarization of LDL is playing an important role in the localization of various vascular diseases.

Simultaneous viscometry and particle sizing on the basis of dynamic light scattering

The ultimate purpose of our research is to demonstrate experimentally the relationship of the deformation and aggregation of red blood cells to the viscosity of blood. We need to measure simultaneously the viscosity of blood and the aggregation of red blood cells. Therefore, we propose a new method to measure simultaneously the viscosity of fluid and the aggregation of particles. The method is developed by combining a cone-plate viscometer with the technique of the particle sizing based on the dynamic light scattering. We show theoretically that a temporal autocorrelation function on the intensity basis is a square of a sum of the autocorrelation functions on the amplitude basis of light scattered from particles and the dynamic speckle produced from the rough surface of a rotating cone in the viscometer. The theoretical prediction is confirmed by experiments for using the solutions of polystyrene latex particles.

Optical monitoring of the concentration profile of submicron latex particles in flow through a translucent water-permeable tube: demonstration of flow-dependent concentration polarization of plasma proteins at a blood/endothelium boundary

It is well accepted that hemodynamics plays an important role in atherogenesis in man. However, the precise mechanisms have not been elucidated yet. Recently, Karino and his coworkers hypothesized that flow-dependent concentration polarization of low-density lipoproteins (LDL: a carrier of cholesterol) may occur at a blood/endothelium boundary, leading to a high risk of atherogenesis in regions of slow flow and low wall shear rate where the concentration of LDL builds up. In this study, we attempted to confirm experimentally their predictions by measuring optically the concentration profile of polystyrene microspheres (used as a model of LDL) flowing in steady flow through a dialyses tube (used as a model of an artery) by transversing a laser beam across the tube and detecting the intensity of the transmitted light. It was found that surface concentration of the microsphere certainly increases with decreasing the flow rate (hence wall shear rate) and it occurs even under the conditions of a very low water filtration velocity encountered in normal arteries in vivo, thus giving a strong support to the hypothesis proposed by Karino et al.

Mechanical Model and Simulation of Respiratory System

A simulation approach based on the detailed mechanical model is applied to understand the complex system of lung respiration in terms of computational biomechanics. Mechanical models were established for the various elements of the respiratory processes such as air flow, tissue deformation, blood flow, and gas diffusion, and they are unified into a system model in a coupled manner. The simulation using the mechanical model represents the overall behavior of lung respiration as well as the behavior in detail of the elements. The mechanical model for each element is verified through system behavior, which is observed by experiments, and the respiratory system is analyzed by synthesis. Taking into account the hierarchical interaction of the mechanical phenomena in lung respiration, we attempted to seek an appropriate pressure condition in artificial ventilation.