The State of the Art of Nanobioscience in Japan

54 IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 5, NO. 1, MARCH 2006

The State of the Art of Nanobioscience in JapanShoogo Ueno , Fellow, IEEE, Joji Ando, Hiroyuki Fujita, Member, IEEE, Tadashi Sugawara,

Yasuhiko Jimbo, Member, IEEE, Keiji Itaka, Kazunori Kataoka, and Takashi Ushida

Abstract—This paper reviews a part of the state of the art ofnanobioscience in Japan. The importance of combination andintegration of interdisciplinary principles is emphasized for thedevelopment of nanobioscience. Biomagnetics, biomechanics,nanomachining, self-replicating cell model, neuronal network,drug delivery system, and tissue engineering are discussed.

Index Terms—Biomagnetics, biomechanics, drug deliverysystem, nanomachining, neuronal network, self-replicating cellmodel, tissue engineering.

I. INTRODUCTION

NANOBIOSCIENCE is an interdisciplinary field whereengineering, bioengineering, nanotechnology, biology,

and medicine overlap and integrate for medicine today andtomorrow. This paper introduces a part of the state of theart of nanobioscience in Japan. This review paper includesbiomagnetics, biomechanics, physiome, nano- and micoroma-chining, self-replicating biological membrane system, neuronalnetworks on electrode arrays, drug delivery systems, and tissueengineering. The interaction, combination, and integration ofdifferent disciplines in different fields are essential for thedevelopment of a new field, and, in particular, nanobioscienceis a good example of the integrated science with heterogeneousprinciples and ideas.

Due to space limitations, only part of nanobioscience is dis-cussed in this paper. Other important topics, such as bioimagingand regenerative medicine, will be introduced in the near future.

Manuscript received April 1, 2005; revised June 9, 2005. This work wassupported in part by the Japan Society for Promotion of Science (JSPS) underGrant-in-Aid Scientific Research (S) 17100006. The work of S. Ueno was sup-ported by the Ministry of Education, Culture, Sports, Science and Technologyof Japan under Grant-in-Aid for Specially Promoted Research 12002002. As-terisk indicates corresponding author.

*S. Ueno is with the Department of Biomedical Engineering, GraduateSchool of Medicine, University of Tokyo, Tokyo 113-0033, Japan (e-mail:[email protected]).

J. Ando is with the Department of Biomedical Engineering, Graduate Schoolof Medicine, University of Tokyo, Tokyo 113-0033, Japan (e-mail: [email protected]).

H. Fujita is with the Institute of Industrial Science, University of Tokyo,Tokyo 153-8505, Japan (e-mail: [email protected]).

T. Sugawara is with the Department of Basic Science, Graduate Schoolof Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan (e-mail:[email protected]).

Y. Jimbo is with the Department of Precision Engineering, GraduateSchool of Engineering, University of Tokyo, Tokyo 113-8656, Japan (e-mail:[email protected]).

K. Itaka and K. Kataoka are with the Department of Materials Science andEngineering, Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan (e-mail: [email protected]).

T. Ushida is with the Center for Disease Biology and Medicine, GraduateSchool of Medicine, University of Tokyo, Tokyo 113-0033, Japan (e-mail:[email protected]).

Digital Object Identifier 10.1109/TNB.2005.864022

The sections and contributors are as follows:I. Introduction by S. Ueno;

II. Biomagnetics and Magnetic Control of Cell Growthand Cell Destruction by S. Ueno;

III. Biomechanics and the Physiome by J. Ando;IV. Nano- and Micromachining for Biomedicine by

H. Fujita;V. Minimal Cell Model as a Programmable Nanomachine

by T. Sugawara;VI. Neuronal Networks on Electrode Arrays by Y. Jimbo;

VII. Drug Delivery Systems by K. Itaka and K. Kataoka;VIII. Tissue Engineering by T. Ushida.

II. BIOMAGNETICS AND MAGNETIC CONTROL OF CELL

GROWTH AND CELL DESTRUCTION

Biomagnetics is an interdisciplinary field for investigation ofthe relations between living organisms and magnetism. Biomag-netic stimulation and magnetic control of cell orientation usinghigh pulsed magnetic fields and strong static magnetic fieldshave promising medical applications [1]–[5]. Building bridgesbetween biomagnetics and nanoscience opens a new horizon innanobioscience.

Recent developments in high-power pulsed magnetic fieldtechnology and superconducting magnets have enabled us tostudy the magnetic stimulation of nanoparticles and the mag-netically controlled alignment and growth of biological cells.

Magnetic force acting on magnetic materials moves the ma-terials along magnetic field gradients. A new method to de-struct targeted cells was developed using magnetizable beadsand pulsed magnetic force [6], [7]. TCC-S leukemic cells werecombined with magnetizable beads (diameter 4.5 0.2 m,magnetic mass susceptibility 16.3 3 10 m kg). Aftercombination, the cell/bead/antibody complexes were placed ona magnet for enough aggregation. The aggregated beads werethen stimulated 10 times at 5-s intervals by a circular shapedcoil (inner diameter 15 mm, outer diameter 75 mm),which produced monophasic pulses of 150 s in duration anda maximum of 2.4 T at the center of the coil. After stimula-tion, the viability of the cells was measured, and the cells wereobserved under a scanning electron microscope. The viabilityof the aggregated and stimulated cell/bead/antibody complexeswas significantly decreased, and the cells were destructed by thepenetration of the beads into the cells or rupturing of the cellsby the beads, as shown in Fig. 1. We hypothesize that the in-stantaneous pulsed magnetic forces cause the aggregated beadsto forcefully penetrate or rupture the targeted cells. In principle,the magnetic force acting on any particular material is propor-tional to the magnetic field, magnetic field gradient, and themagnetic susceptibility of the material; however, the magnetic

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UENO et al.: THE STATE OF THE ART OF NANOBIOSCIENCE IN JAPAN 55

Fig. 1. Scanning electron micrographs of the cell/bead/antibody complexeswith and without pulsed magnetic stimulation [6]. (a) The nonstimulated cell/bead/antibody complex was not damaged. (b) The stimulated cell/bead/antibodycomplexes were damaged by penetration of the beads. (c) and (d) The stimu-lated cell/bead/antibody complexes were damaged by rupturing by the beads.Scale bars = 4.5 �m.

force acting on the individual nanoscale magnetic particles in-side the beads are too weak to affect them, because the magneticsusceptibility of each individual particle is very low. In contrast,when the nanoscale particles inside the beads are closely as-sembled, the magnetic mass susceptibility is sufficiently high toforce the attachment of the beads to the cells by the magneticforce. Thus, the magnetic force acting on the aggregated beadswas strong enough to shift the beads and damage the cells. Theabsence of heat and microbubbles makes this a potentially vi-able treatment modality for solid tumor type cancers as well asleukemia in which cells are distributed throughout the wholebody.

When diamagnetic materials such as fibrin, collagen,osteoblasts, and smooth muscle cells are exposed to static mag-netic fields of tesla order, these materials align either parallel orperpendicular to the direction of the magnetic field dependingon the magnetic anisotropy of the materials [8]–[15]. Themagnetic torque acting on the diamagnetic components in bio-logical cells causes this phenomenon of magnetic orientation.Fig. 2 shows magnetically oriented biological macromoleculesand cells. The magnetic orientation of fibrin fibers and collagenhas been observed under strong magnetic fields [8]–[11]. Redblood cells and platelets are also oriented in magnetic fields[12], [13]. In a recent experiment, Schwann cells with andwithout magnetically oriented collagen were oriented in thedirection of the magnetic field [14]. The magnetic control ofbiological cells may translate into potentially viable tissue andmedical engineering applications including nerve regeneration.

The introduction of bone formation to an intentional orienta-tion is a potentially viable clinical treatment for bone disorders.The effects of static magnetic fields of 8 T on bone formation

Fig. 2. Magnetic orientation of biological macromolecules and cells. Fibrin,osteoblasts, endothelial cells, smooth muscle cells, and Schwann cells were ori-ented in the direction parallel to the magnetic field. Collagen was oriented in thedirection perpendicular to the magnetic field.

were investigated in both in vivo and in vitro systems [10], [11].After 60 h of exposure to the magnetic field, cultured mouse os-teoblastic MC3T3-E1 cells were transformed to rod-like shapesand were oriented in the direction parallel to the magnetic field.Although the magnetic field exposure did not affect cell prolif-eration, it up-regulated cell differentiation and matrix synthesisas determined by alkaline phosphatase and Alizarin red stain-ings, respectively. The magnetic fields also stimulated ectopicbone formation in and around subcutaneously implanted bonemorphogenetic protein-2 (BMP-2) containing pellets in mice,in which the orientation of bone formation was parallel to themagnetic field. Strong magnetic fields have the potency to stim-ulate bone formation as well as to regulate its orientation in bothin vitro and in vivo models. We propose that the combinationof strong magnetic fields and a potent osteogenic agent such asBMP may possibly lead to an effective treatment of bone frac-tures and defects.

Schwann cells aid in neuronal regeneration in the periph-eral nervous system by guiding the regrowth of axons. Schwanncells provide a supportive role in the peripheral nervous system,forming a layer or myelin sheath along single segments of an


Fig. 3. P2X4 purinoceptors mediate shear-stress-induced Ca influx in endothelial cells. Upper panel shows pseudocolor images of Ca responses in HPAECsloaded with a calcium indicator Indo-1. Shear stress increased intracellular Ca concentrations (F405/F480) in a dose-dependent manner in human pulmonaryartery endothelial cells (HPAECs) (Control). When the expression of P2X4 receptors was suppressed by the treatment of cells with antisense-oligonucleotides, theshear stress-dependent Ca influx was markedly inhibited (P2X4-Antisense oligos). Each graph represents 12 Ca responses by a group of 8–10 cells. Thesefindings suggest that P2X4 receptors play a crucial role in the shear-stress-dependent Ca influx in HPAECs.

axon. Schwann cells were exposed to 8 T magnetic fields, orthe cells were cultivated in magnetically oriented collagen gels[14]. After 60 h of exposure, Schwann cells oriented parallel tothe magnetic fields. In contrast, the Schwann cells and collagenmixture aligned along the magnetically oriented collagen fibersor perpendicular to the magnetic field after 2 h of exposure. Al-though further experiments are necessary to determine whethermagnetic field exposure promotes Schwann cell proliferationand to clarify the mechanisms for the apparent orientation, themagnetic control of Schwann cell alignment is potentially usefulfor nerve regeneration techniques in tissue engineering and re-generative medicine.

III. BIOMECHANICS AND THE PHYSIOME

Research on human biomechanics has made rapid progress.Bioengineering experiments on cardiovascular biomechanicsinvolving the exposure of cultured cells to controlled levels ofmechanical stimuli in flow- and/or stretch-loading devices havebeen widely used, and the molecular mechanisms for vascularcell responses to hemodynamic forces, such as shear stress fromflowing blood and cyclic stretch, from pulsatile blood pressurehave been identified. The data on gene responses to mechanicalstimuli, in particular, has been rapidly accumulated. We havedemonstrated that shear stress decreases the transcription of thevascular cell adhesion molecule-1 (VCAM-1) gene in murinelymph node vein endothelial cells [16]. The transcription factor

c-jun and its binding sites on the promoter (shear stress responseelement) of VCAM-1 play an essential role in this responseto shear stress [17]. We also observed that the transcriptionfactor SP1 binding site functions as a shear stress responseelement in the shear stress-induced downregulation of P2X4purinoceptor gene transcription in endothelial cells, indicatingthat the shear stress response element is not universal and dif-fers among different genes [18]. On the other hand, shear stressregulates the expression of the hematopoietic cytokine granu-locyte/macrophage-colony stimulating factor (GM-CSF) genevia mRNA stabilization, not transcription [19]. mRNA stabi-lization is also involved in the shear stress-mediated regulationof cyclin-dependent kinase inhibitor p21 and cyclooxygenase-2(COX2) gene expression [20], [21]. A DNA microarray analysisshowed that when human umbilical vein endothelial cells wereexposed to a laminar shear stress of 15 dynes/cm for 24 h,approximately 3% of the total genes increased their expressionby more than twofold or decreased their expression by morethan half. This means that if approximately 20 000 genes areexpressed in endothelial cells, approximately 600 genes areshear stress-responsive. A global analysis of the genes thatrespond to turbulent shear stress is now underway in the hopeof identifying anti- or pro-atherogenetic genes.

Shear stress-sensing mechanisms have also been activelyinvestigated, and it has become apparent that shear stress istransmitted as a signal into the cell interior via ion channels,


adhesion molecules such as integrin and PECAM-1, and thecytoskeleton. We observed that Ca signaling plays an im-portant role in shear stress signal transduction [22]. Shearstress induces both Ca release and influx in endothelialcells. Ca release from intracellular stores, such as the endo-plasmic reticulum, occurs at caveolae, and the Ca increaseis propagates throughout the entire cell in the form of a Cawave [23]. Ca influx is mediated by an ATP-gated cationchannel, P2X4, that is predominantly expressed in endothelialcells (Fig. 3) [24], [25]. Recently, endogenously released ATPhas been shown to be involved in shear stress-dependent Cainflux via P2X4 [26]. Mechano-ATP coupling has also beenshown to occur in other types of cells, such as mammary gland,airway, and intestinal epithelial cells. An attempt is now beingmade to identify the initial sensor that recognizes shear stress.Integrin and platelet endothelial cell adhesion molecule-1(PECAM-1) have been reported to be potential candidatesfor this shear stress sensor [27], [28]. The elucidation of themechanotransduction mechanism would be beneficial notonly in clarifying physiological significance of hemodynamicforces in circulatory system homeostasis, but for improvingour understanding of the pathophysiology of cardiovasculardiseases, such as atherosclerosis, aneurysms, and hypertension,and possibly leading to the development of new therapies.

IV. NANO- AND MICROMACHINING FOR BIOMEDICINE

Nano- and micro-machining based on semiconductorprocesses have been applied to biomicrosystems and mi-cromachined tools for nanobiotechnology [29]. There arenumerous opportunities in diagnosis, pharmacology, andtherapy. Biomicrosystems range from DNA array chips, mi-crofluidic microelectromechanical systems (MEMS) devices[30] for DNA electrophoresis on-a-chip or chemical synthesisand screening, chips for cell handling and culture, and MEMSintegrated with biomolecules.

The typical dimensions of biological objects are around1–10 m for biological cells, while macromolecules arenanometers in thickness and micrometers in length. The elec-tric field distribution obtained by microfabricated electrodescan be controlled on the same order of these object sizes and issuitable for manipulating them. Washizu et al. [31] developed acell fusion system using both a microfluidic system and manip-ulation with an electric field. They also succeeded in orientingDNA molecules along the field [32] and modifying them usingan enzyme attached to a laser manipulated bead [29].

MEMS for DNA multiplication and detection are underdevelopment [33]. Arrays of small vessels for cell fusionand culture were fabricated. Precise delivery of fluid in smallamount is required in such vessels. In medicine, endoscopes,microblood-analysis systems, implantable artificial organs, andtools for minimally invasive surgery have high potential forMEMS application. An active catheter composed of multipleactive links for shape control has also been fabricated [34].

The cell handling and culture in MEMS has been investigated.Biological cells can be transported by dielectric force betweenmicroelectrodes [35]. Holes and chambers of micrometers indimensions were fabricated on a silicon wafer for cell fusionand culture [36]. Neuron cells were cultured in microfluidic

channels made by polydimethylsiloxane (PDMS); cells formedwell-defined networks guided by the channel [37]. The guid-ance structures are transparent to allow observation of cells. Thefluid handling system was improved so that cells can surviveover a week. The microchannels were aligned with commercialbio electrodes for multichannel neural signal detection. We havesuccessfully recorded spontaneous neural signals from culturedneural networks [38].

We have integrated liner motor molecules on a chip and uti-lized them as a nano actuator. We attached long chain molecules,named microtuble, that work as rails to direct the motion. Smallstructures such as polymer beads and silicon microstructureswere coated with kinesine molecules that generate force anddisplacement on the microtuble. We have success fully carriedthose objects on the immobilized rail molecule [39]. We havealso fabricated microfluidic devices in which we can observe therotation of a single molecule named ATP-ase of 10 nm in diam-eter over wide rage of temperature and chemical concentration.

A typical example of micromachined tools for nanobiotech-nology is the family of scanning probe microscopes (SPMs) in-cluding the atomic force microscope (AFM). SPM is well suitedto image nano-objects such as DNA or other biomolecules butis limited in handling of these objects because it has only onesharp tip. We have developed multiprobe devices. Microma-chined tweezers with a pair of sharp tips were coated with Al anddipped in an aquaous solution of 16- m-long DNA moleculeswhich were fluorescent-dyed. The high-frequency electrostaticfield between tips attracted molecules, some of which were cap-tured between them. As shown in Fig. 4, we have retrieved DNAmolecules in the air and observed fluorescence from molecules[40]. Current investigation directs toward four terminal charac-terization of the electrical conduction through DNA.

V. MINIMAL CELL MODEL AS A

PROGRAMMABLE NANOMACHINE

In order to obtain a solution to the origin of life problem,the constructive approach is promising: namely, prepare a min-imal cell artificially using well-defined fundamental moleculesand watch how it works. Such a minimal cell, at the same time,would be an ultimate nanomachine made of soft materials. Theminimal cell consists of a compartment which separates theinner reaction system from the outer world, a catalyst which cat-alyzes important metabolic reactions and an informational sub-stance which delivers the information to the descendant [41].Here we consider two replicating processes to realize a minimalcell model. One is a self-replication of the cell membrane, rec-ognized as a lipid world [42]. The other is a self-replication ofthe informational substance, recognized as an RNA world. Ifthese two self-replicating cycles are synchronizing, a self-repli-cating minimal cell will emerge.

A schematic illustration of our self-replication membranesystem is shown in Fig. 5 [43]. The original vesicle is composedof amphiphile V which is composed of two parts, A and B [44].If one supplies amphiphile A and lipophilic molecule B, whichare the precursors of amphiphile V, A reacts with B inside thevesicle to form the membrane molecule V. Incidentally, thereactive A should be supplied as an inactive form A , which canbe unlocked to the active form A in the presence of catalyst C.


Fig. 4. (a) Micrograph of nanotweezers. (b) DNA molecules captured between two tips [40].

Fig. 5. A schematic illustration of the self-replicating membrane system.

In the real system shown in Fig. 2, lipophilic molecule Bexists as oil droplets, in advance, in a water pool inside theoriginal vesicle [Fig. 6(a)]. After about 20 min, all the dropletswere consumed. In compensation for the disappearance of oildroplets, daughter vesicles were generated inside the water pool[Fig. 6(b)], and they were extruded from the original vesicle bythe increased osmotic pressure inside [Fig. 6(c)]. Since thesevesicles of the second generation are composed of the samecomponents as the original ones, such a process can be regardedas a self-replication of giant vesicles.

The information is a vital element for life because it charac-terizes life. The program of life is written by the sequence ofnucleosides of DNA, and it must be passed down to descen-dants. A key molecule that we invented was a DNA–cholesterolconjugate molecule shown in Fig. 7(a) [45]. DNA (or RNA) cancarry the information, and the cholesterol part which has thelipophilic nature can firmly anchor into the membrane of thevesicle. DNA (or RNA), which is hydrophilic, never mixes withthe lipid which has the hydrophobic (i.e., lipophilic) long alkyltails. Therefore, this ambiguous conjugate becomes a bridgemolecule between the lipid and the RNA worlds.

Fig. 6. Microscopic images and corresponding diagrams of the self-replicatingmembrane dynamics.

Fig. 7(b) and 7(c) shows DNA hybridization occurred onthe outer membrane of a giant vesicle. The conjugate moleculecomposed of a DNA 15 mer (rectangle), a polyethyleneglycol


Fig. 7. (a) Structure of DNA-cholesterol conjugate molecule. (b) Fluorescencemicroscopic image of the giant vesicle growing from the platinum electrode.A band of fluorescence near the giant vesicle was the fluorescein-labeled DNAprobe that hybridized with cholesterol-tagged DNA in the nonswelling lipidspreaded over the electrode. (c) Diagram of the hybridization event occuringon the surface of the above giant vesicle.

(PEG)-spacer (rumpled string) [46], and cholesterol (oval)locates in and on the membrane. When a probing molecule,which is composed of a green fluorescent dye (ball) and a com-plementary DNA 15 mer, was added to the vesicle containingthe conjugate molecule, we found that only the surface of thevesicle shone green. This observation proves that the high po-tenciality of the conjugate to hybridize with the complementaryDNA 15 mer on the surface of the membrane. Furthermore,since DNA is anchored onto the membrane of the vesicle, theinformation on the DNA part must be surely delivered frommother to daughter vesicles on the division of self-replicatingmother vesicle, regardless of the types of the division (forexample separation, birthing, and so on) [47].

The replication of information on the DNA part of the con-jugate on the “inner” membrane of the vesicle with a proteincatalyst is progressing [48]. Since we are obtaining the tworeplicating systems, the synchronized coupling of these systemsshould generate a prototype of the minimal cell system whichoperates as a programmable nanomachine.

VI. NEURONAL NETWORKS ON ELECTRODE ARRAYS

A. Microelectrode Arrays for Cultured Neurons

Various techniques have been developed to visualize spa-tiotemporal activity patterns in neuronal networks. Amongthose, the microelectrode array (MEA), which was first in-troduced by Gross [1] and by Pine [2], is one of the mostpromising tools. An MEA is a dish for cell culture, on thesurface of which multiple microelectrodes are embedded. Itsunique advantages are noninvasiveness, quite high temporalresolution allowing observation of spike propagation in a singleneuron, and the capability of multisite stimulation. Cells can

Fig. 8. Cultured neuronal networks on an electrode-array substrate. Clear neu-ronal spikes could be recorded at 64 sites. Recent progress in surface modifica-tion techniques allowed cell positioning as well as growth guidance of neurites.

Fig. 9. Tetanus-induced modification of evoked responses. Both potentiationand depression were observed.

be kept on the array for a long time, up to several months[3]. To see developmental changes during neuronal networkformation, we recorded spontaneous activity from cultured ratcortical neurons continuously for two months [4]. An example


Fig. 10. Features of polymeric micelles that are relevant for drug delivery.

of cultured cortical neurons on an MEA and their spontaneousactivity observed at 64 recording sites is shown in Fig. 1(a)–(c).The first spontaneous activity was detected on the third day invitro. The activity then became gradually synchronized amongcells, and its frequency increased. The spontaneous activityreached a steady state after about one month. Spatio-tem-poral properties of both spontaneous and evoked responseswere characterized [5], [6]. Recently, cell-positioning andgrowth-guidance techniques have been combined with MEAs[7]. As shown in Fig. 1(d), we can visualize individual neuronsin the network. The relationship between network structuresand electrical activity has been extensively studied.

B. Multisite Electrical Stimulation

MEA-based multisite stimulation is simple in principle.Practically, however, several aspects should be carefully con-sidered. The effective stimulation intensity is around 1 V,whereas the typical amplitude of extracellular spikes is 0.1 mV.This large discrepancy causes high stimulus-related artifact,which often hides early components of the evoked responses.Switching individual electrodes between recording and stim-ulation is a useful strategy. The inputs of the preamplifiersare detached from the electrodes and are kept at properlydetermined dc levels during stimulation. To achieve long-termmeasurements, biologically inactive materials must be used forelectrodes. Those materials, such as gold or platinum, formpolarized electrode/electrolyte interface, so that drifts of theelectrode potentials are inevitable. Therefore, dc offsets mustbe tracked for individual electrodes and the inputs should bemaintained at each tracked level during stimulation. Otherwiseswitching between recording and stimulation causes anothertransient. These tracked levels are also useful to produce con-stant stimulation intensity. Stimulation pulses are added to thestored levels, ensuring constant stimulation intensity. Finally,to reduce stimulus-related transients, the net injection chargesshould be minimized. Biphasic pulses are effective for thispurpose. Because of the nonlinear properties of the interface,complete compensation of charge injection is difficult. The

injected charges are at least partly compensated by biphasicpulses. Based on the considerations above, we designed inte-grated modules containing preamplifiers, switching devices,and sample/hold circuits. All the switching timings as well asstimulation waveforms could be controlled by a PC. Using thisstimulation system, we achieved spike detection within 2 msafter the stimulus at the stimulation site, and confirmed repro-ducibility of the evoked responses at a constant stimulationintensity [8]. Such PC-controlled multisite stimulation systemswill be useful tools to study network physiology.

C. Plasticity in Cultured Neuronal Networks

Though long-term potentiation and depression (LTP/LTD)have been extensively studied, little was known about howthese synaptic modifications were integrated and reflected inthe network activity. Using the multisite stimulation systemdescribed above, we tried to see tetanus-induced modificationof evoked responses [9]. First, a test stimulus pulse was appliedfrom each of the 64 sites and the stimulation site was scannedsequentially. The corresponding 64 evoked network activitieswere recorded. Then, tetanic stimulation was applied to asingle site. Both enhancement and depression of activity wereproduced by the same tetanic stimulus in different pathways.Representative examples are shown in Fig. 2. In the case ofpotentiation, more intense, more long-lasting activity wasobserved after tetanus. In contrast, some pathways were clearlyweakened and the evoked activity was terminated earlier.To explore the factors governing these opposite changes, weexamined the influence of spike timing, by calculating thecross-correlation function of spike trains during stimulationthrough a particular pathway with the spike trains evoked bysingle pulses in the tetanized pathway. In the case of potentia-tion, the spikes initially showed a relatively tight correlation totetanic pathway spikes. After tetanus, the peak became still nar-rower. In depressed pathways, spikes before tetanus were muchmore loosely correlated to tetanic pathway spikes, but the effectof tetanus was qualitatively the same: a pronounced contrac-tion of the cross-correlation function around its central peak,


enhancing the frequency of spikes in the tetanized pathway anddepressing the frequency of those not contained in the pathway.Tetanic stimulation caused a relative strengthening of the partsof each stimulus pathway that are closest in correlation to thetetanus-activated pathway and depression of the rest.

VII. DRUG DELIVERY SYSTEMS

Drug targeting for efficient accumulation in the body isoften hampered by the rapid recognition of carrier system bythe reticuloendothelial system (RES) and by the subsequentkidney and/or hepatic elimination. Moreover, for modulateddrug delivery to solid tumors, which locate outside the bloodcompartment, the carrier is required to exhibit not just a suffi-cient half-life in the blood compartment, but also the capabilityof extravasation at the tumor site. Recent developments led tothe design of drug carriers with prolonged circulation in thevascular system. Cancer chemotherapy may cause severe sideeffects, leaving patients under extreme distress. To overcomethis problem, an interest has been raised in the application ofblock copolymer micelles as novel carrier systems for anti-cancer agents because of the high drug-loading capacity of theinner core as well as of the unique disposition characteristics inthe body [59]–[61]. Compared to surfactant micelles, polymericmicelles are generally more stable, with a remarkably loweredcritical micelle concentration (CMC), and have a slower rateof dissociation, allowing retention of loaded drugs for a longerperiod of time and, eventually, achieving higher accumulationof a drug at the target site. Polymeric micelles have a size of30–50 nm in diameter, ranging closely to that of viruses, andapparently, this size range is favorable for extravasation toachieve so-called enhanced permeation retention effect (EPReffect) [62].

Special focus is focused here to polymeric micelles formedfrom heterobifunctional block copolymers. A challenge in thedevelopment of novel micellar carrier systems is to designtargetable polymeric micelles in which pilot molecules areinstalled on their surface to achieve a specific-binding prop-erty to target cells. Of particular importance in this regardis the establishment of a novel and effective synthetic routefor end-functionalized amphiphilicblock copolymers withappreciable biocompatibility and biodegradability, allowingconjugation of the pilot molecules at the tethered end of thehydrophilic segment.

The concept of polymeric micelle stabilization through theformation of a hydrophilic palisade surrounding a water-in-compatible core can be extended to include the case ofmacromolecular association through electrostatic interaction[63], [64]. It was shown that a pair of block copolymers withan oppositely charged polyelectrolyte segment, poly (ethyleneglycol)-block-poly(L-lysine) (PEG-b-PLL) and poly(ethyleneglycol)-block-poly(a,b-aspartic acid) [PEG-b-P(Asp)], spon-taneously associates to form micelles with a core composedof polyion complex of poly(L-lysine) and poly(a,b-asparticacid) segments [65]. The polyion complex (PIC) micellesopened the way to incorporate charged macromolecules ofsynthetic and biological origins including proteins and nucleicacids into the micelles and have been developed as nonviralDNA delivery systems. The strategy to coat the polyion

complex core of a polycation and DNA with a hydrophilicsegment of the cationic block copolymer was applied to pre-pare a variety of nanoassociates. These includes PEG-b-PLL[66], [67], poly(ethylene glycol)-block-poly(ethyleneimine)(PEG-b-PEI) [68], and poly(ethylene glycol)-block-poly(dimethylaminoethyl methacrylate) (PEG-b-PAMA) [69], andrecently the PEG-poly-(3-[(3-aminopropyl)amino] propylas-partamide (PEG-DPT), carrying a diamine side chain withdistinctive pKa, which was especially applied for siRNA de-livery [70].

The tremendous progress has been attained in the character-ization and application and of nanostructured materials usingblock copolymers. The properties of polymeric micelles formedthrough the multimolecular assembly of block copolymers ishighly useful as novel core-shell typed colloidal carriers for drugand gene delivery.

VIII. TISSUE ENGINEERING

Tissue engineering is one of the typical interdisciplinarydisciplines, involving material science, cell biology, molecularbiology, and mechanical engineering. Among them, stem cellbiology has especially achieved remarkable progress also inJapan. However, cells are not sufficient for regenerating livingtissues, although they are still one of the major elements forthat purpose. One of the major elements other than cells isrecognized as scaffolds, which enable cells to adhere andreconstruct in three dimensional manner. It is known for cellsto be regulated by intracellular signals evoked by other thanbiochemical stimulation. Cell adhesion assemblies involvingintegrins are well known to regulate cell adhesion, spreading,and migration by inside-out signals, and regulate at the sametime cell differentiation and tissue regeneration by outside-insignals, which are caused by integrin–matrix interaction.Another interaction regulating cell differentiation and tissue re-generation is cell–cell interaction, which is realized for examplein cell aggregation at developmental steps. Thus, an approachis presumed probable to regulate cell differentiation and tissueregeneration by means of realizing cell–matrix interaction andcell–cell interaction from the engineering point of view.

Cartilage is one of the promising candidates for tissue re-generation in vitro by using tissue engineering, because chon-drocytes could endure low oxygen concentration and nutritionsupply. It is necessary for regenerated cartilage to be biggerthan one taken with biopsy in order to exceed mosaic plasty.For that purpose, it is inevitable to make chondrocytes prolifer-ated in vitro. Articular cartilage is composed of type II collagen,although fibrous cartilage is composed of type I collagen. How-ever, it is well recognized that chondrocytes are dedifferentiatedaccording to proliferation in vitro, producing not type II but typeI collagen. Therefore, tissue engineering is requested to estab-lish a new methodology how to make dedifferentiated chondro-cytes redifferentiated in vitro.

Biomaterials derived from tissues such as collagen,hyaluronan have been used in tissue engineering. Thosematerials have sites to be able to interact with integrins orreceptors on the plasma membrane and evoke intracellularsignals, although they are relatively weak in mechanical prop-erties. On the other hand, biodegradable polymers such as


Fig. 11. SEM micrograph of: (a) PLGA–collagen hybrid mesh. (b) Phase contrast micrograph of two passaged cells immediately after seeding in the hybrid mesh.(c)–(d) SEM micrographs of chondrocytes cultured in the hybrid mesh for: (c) 1 week and (d) 4 weeks. (left) Northern blot analysis of the genes encoding type Icollagen, type II collagen, and aggrecan of two passaged chondrocytes cultured in the hybrid mesh for 0, 2, 4, and 12 weeks.

poly glycolic acid (PGA), poly lactic acid (PLLA), and theircopolymer poly DL-lactic-co-glycolic acid (PLGA) have alsobeen frequently used in tissue engineering. Those polymershave enough mechanical strength, although they are relativelyhydrophobic and have no sites to interact with cells.

In order to compensate mutually demerits of biomaterialsand biodegradable polymers, their hybridization has been tried.For that context, we have made the hybridization of collagenmicrosponges with PLGA sponges [71]–[73], the hybridiza-tion of collagen microsponges with PLGA meshes [74], thehybridization of collagen microsponges with hydroxyapatitemicrobeads and PLGA sponges [75], and the hybridization ofcollagen gels including chondrocytes with PLLA nonwovenfiber scaffolds [76]. Thus, those hybridization could promotenot only efficiencies of cell seeding and cell adhesion but alsoregeneration ability of cartilage-like tissues in vitro. Fig. 11shows: (a) SEM micrograph of PLGA-collagen hybrid mesh;(b) phase contrast micrograph of two passaged cells immedi-ately after seeding in the hybrid mesh; and SEM micrographsof chondrocytes cultured in the hybrid mesh for: (c) 1 weekand (d) 4 weeks. The hybrid mesh was prepared by formingcobweb-like collagen microsponges in the openings of a knittedmesh made of PLGA [Fig. 11(a)]. Observation by a phase con-trast microscope indicates that the cells were entrapped bythe collagen microsponges in the hybrid mesh immediatelyafter cell seeding [Fig. 11(b)]. Chondrocytes adhered to thecobweb-like hybrid mesh and showed uniform distributionon the mesh. They proliferated and regenerated cartilaginousmatrices, filling the void spaces in the hybrid mesh [Fig. 11(c),(d)]. Fig. 11 also shows Northern blot analysis of the genesencoding type I collagen, type II collagen, and aggrecan oftwo passaged chondrocytes cultured in the hybrid mesh for 0,2, 4, and 12 weeks. When the dedifferentiated chondrocyteswere seeded and cultured in the hybrid mesh, the expressionof mRNAs for type II collagen and aggrecan was up-regulatedand that of type I collagen mRNA was down-regulated. After

culture in the hybrid mesh for 12 weeks, the gene expression oftype I collagen was very weakly detectable, but those of type IIcollagen and aggrecan reached their highest levels. Those re-sults show a possibility for such hybridized scaffolds to possessthe function enabling dedifferentiated chondrocytes rediffer-entiated in coincidence with cartilage-like tissue regeneration[77]. It could be also thought possible to regulate cell differen-tiation and tissue regeneration in more complicated tissue suchas blood vessel and bone, using signals from matrices and threedimensional structures of those hybridized scaffolds [78], [79].

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Shoogo Ueno (M’72–SM’96–F’01) was born inKumamoto, Japan, on October 1, 1943. He receivedthe B.S., M.Sc., and Ph.D. (Dr.Eng.) degrees fromKyushu University, Fukuoka, Japan, in 1966, 1968,and 1972, respectively.

From 1976 to 1986, he was an Associate Professorwith the Department of Electronics, Kyusyu Uni-versity. From 1979 to 1981, he spent his sabbaticalwith the Department of Biomedical Engineering,Linkoping University, Linkoping, Sweden, as aGuest Scientist. From 1986 to 1994, he was a

Professor with the Department of Electronics, Kyushu University. Since 1994,he has been a Professor with the Department of Biomedical Engineering,Graduate School of Medicine, the University of Tokyo, Tokyo, Japan. Hedeveloped a method for localized magnetic stimulation of the human brainusing a figure-eight coil, a computed topographic electroencephalography(EEG) mapping system, and impedance magnetic resonance imaging (MRI).His primary research interests include biomagnetics, transcranial magneticstimulation (TMS), magnetoencephalography (MEG), neuronal current MRI,magnetic orientation and control of biological cells for tissue engineering, andthe biological effects of magnetic and electromagnetic fields.

Dr. Ueno was elected as a Fellow of the American Institute for Medicaland Biological Engineering (AIMBE) in 2001. Dr. Ueno was president ofthe Bioelectromagnetics Society (BEMS) (2003–2004), and president of theJapanese Society for Medical and Biological Engineering (2002–2004). He ispast chairman of the International Scientific Radio Union (URSI) CommissionK on Electromagnetics in Biology and Medicine (2000–2003), past presidentof the Magnetics Society of Japan (2001–2003), and past president of the JapanBiomagnetism and Bioelectromagnetics Society (1999–2001). He is also activein the IEEE Magnetics Society as an Administrative Committee (AdCom)member and the chair of the Biomagnetism Committee. He is a member ofthe Steering Committee and Editorial Board for the IEEE TRANSACTIONS

ON NANOBIOSCIENCE. He was the recipient of the Doctores Honoris Causa(honorary doctor) presented by Linkoping University in 1998.

Joji Ando was born in Hokkaido, Japan, in 1948.He received the M.D. degree and the Ph.D degree ininternal medicine from Hokkaido University in 1973and 1981, respectively.

He was on the medical staff in the Departmentof Internal Medicine from 1973 to 1975 and inthe Department of Cardiovascular Medicine from1975 to 1983 in the Hokkaido University Hospital.From 1983 to 1991, he worked for the Instituteof Electronic Science at Hokkaido University asan Assistant Professor. In 1991, he moved to the

Department of Cardiovascular Biomechanics as Visiting Associate Professorat the University of Tokyo, Tokyo, Japan. From 1997 to 1999, he was anAssociate Professor in the Department of Biomedical Engineering, and iscurrently a Professor of the Department of Biomedical Engineering at theUniversity of Tokyo. His research has focused on the role of hemodynamicforces such as shear stress generated by blood flow in the regulation of vascularfunctions and in the pathogenesis of vascular diseases including atherosclerosis.He has published more than 70 peer-reviewed papers in the fields of vascularphysiology and cellular biomechanics.

Dr. Ando is a member of several professional societies, including theJapanese Society for Medical and Biological Engineering, the Japanese Societyof Biorheology, the Japanese College of Angiology, and the Japanese Societyfor Microcirculation.


Hiroyuki Fujita (S’76–M’80) received the B.S.,M.S., and Ph.D. degrees in electrical engineeringfrom the University of Tokyo, Tokyo, Japan, in 1975,1977, and 1980, respectively.

Since 1980, he has been with the Institute ofIndustrial Science, University of Tokyo, as a Lecturer(1980–1981), Associate Professor (1981–1993),Professor (1993–present). Since 2000, he has alsobeen Director of the Center for International Re-search on MicroMechatronics (CIRMM), Tokyo.He has been engaged in the investigation of micro-

electromechanical systems fabricated by integrated circuit-based processes andapplications to optics, hard-disk drives, and bio- and nanotechology. He is alsointerested in autonomous distributed microsystems.

Tadashi Sugawara received the B.S., M.S., andPh.D. degrees in science from the Department ofScience, University of Tokyo, Tokyo, Japan in 1969,1971, and 1974, respectively.

After being a postdoctoral fellow in Minnesota andMaryland from 1975 to 1978, he joined the Institutefor Molecular Science, Okazaki, Japan, as a ResearchAssociate in 1978 and engaged in the research of highspin molecules and molecular assemblies. He movedto the Department of Pure and Applied Sciences, Uni-versity of Tokyo, in 1986, and became professor in

1991. His major research interests are design and synthesis of organic con-ducting and/or magnetic materials, novel organic dielectrics based on protondynamics in hydrogen-bonded crystals, molecular dynamics in reactive organicsolids, and self-replicating giant vesicles.

Dr. Sugawara is a member of the Chemical Society of Japan, the PhysicalSociety of Japan, the Crystallographic Society of Japan, the Society of PolymerScience Japan, and the American Chemical Society. He chaired the 9th Interna-tional Conference on Molecule-based Magnets in 2004, Tsukuba, Japan.

Yasuhiko Jimbo (M’05) received the B.E., M.E., andPh.D. degrees in electronic engineering from the Uni-versity of Tokyo, Tokyo, Japan, in 1983, 1985, and1988, respectively.

He joined Nippon Telegraph and Telephone(NTT) Basic Research Laboratories in 1988. From1992 to 1993, he was a Visiting Researcher atCNRS, Marseilles, France. Since 2003, he has beenan Associate Professor at the University of Tokyo,where he has been a member of Department ofPrecision Engineering. His research interests are

neuroengineering and neuroscience based on microfabrication technology.Dr. Jimbo is a Member of IEE Japan, IEICE Japan, and Japanese Society for

Medical and Biological Engineering.

Keiji Itaka received the M.D. and Ph.D. degreesfrom the University of Tokyo, Tokyo, Japan, in 1991and 2003.

He worked as an orthopedic surgeon at the HaibaraGeneral Hospital, the Kanto Central Hospital of theMutual Aid Association of Public School Teachers,and the Mitsui Memorial Hospital from 1991 to 1996.He was an Assistant Professor of the Department ofOrthopaedic Surgery at the University of Tokyo Hos-pital in 1997–1998. Then he began research in thefield of bioengineering at both Graduate School of

Medicine and Engineering. He is currently a Research Associate Professor of theClinical Biotechnology at Center of Disease Biology and Integrative Medicine,University of Tokyo, and a core member of the Nano Bioengineering EducationProgram (NBEP). His current major research interest includes the developmentof new polymeric carrier systems, especially block copolymer micelles, for drugand gene targeting, and their application to the clinical medicine.

Dr. Itaka received the Society Award from the Controlled Release Societyin 2001 and the Outstanding Presentation Award from The Japanese Society ofGene Design and Delivery in 2002.

Kazunori Kataoka received the B.S. and Ph.D. de-grees from the University of Tokyo, Tokyo, Japan, in1974 and 1979, respectively.

He served as a Research Associate and an As-sociate Professor at the Institute of BiomedicalEngineering, Tokyo Women’s Medical College,from 1979 to 1989. In 1989 he assumed an AssociateProfessor position at the Science University ofTokyo. He became full Professor there in 1994. InApril 1998, he moved to the University of Tokyo as aProfessor of Biomaterials. He is currently a Professor

of Biomaterials in the Graduate School of Engineering, University of Tokyo,having been appointed to a joint position since 2004 from Graduate Schoolof Medicine, University of Tokyo, as a Professor of Clinical Biotechnologyat the Center of Disease Biology and Integrative Medicine. He held visitingprofessorship at the University of Paris XIII in 1992, 1996, and also in 2000.He is Editor of the Journal of Biomaterials Science, Polymer Edition, AssociateEditor of Journal of Controlled Release (Controlled Release Society) andBiomacromolecules (American Chemical Society), and an Executive Editorof Advanced Drug Delivery Reviews, and is on the Editorial Board of Journalof Drug Targeting, Journal of Materials Science: Materials in Medicine(European Society of Biomaterials), and Materials and Engineering: C.He is the author of more than 300 scientific papers. His current major researchinterest includes the molecular design of cellular specific polymers and thedevelopment of new polymeric carrier systems, especially block copolymermicelles, for drug and gene targeting.

Dr. Kataoka is a member of the Controlled Release Society, the American As-sociation for the Advancement of Science, the New York Academy of Sciences,the Japanese Chemical Society, the American Chemical Society, the Society ofPolymer Science, Japan, the Japanese Society for Biomaterials, DDS Society,Japan, and the Japanese Society of Artificial Organs. He has been a Fellow ofthe American Institute of Medical and Biological Engineering since 1999 anda Fellow of Biomaterials Science and Engineering since 2004. He received theSociety Award from the Japanese Society for Biomaterials in 1993. In 1994,he received the Journal of Controlled Release Jeorge Heller Outstanding PaperAward from the Controlled Release Society. Also from the Controlled ReleaseSociety, he received the 1996 Outstanding Pharmaceutical Paper Award. He re-ceived the Society Award from the Society of Polymer Science, Japan, in 2000.In April 2005, he received the Clemson Award in Basic Research from the So-ciety for Biomaterials.

Takashi Ushida was born in Aichi, Japan, onNovember 5, 1955. He received the B.S., M.Sc.and Ph.D. (Dr.Eng.) degrees from the Universityof Tokyo, Tokyo, Japan, in 1979, 1982, and 1985,respectively.

From 1985 to 1992, he was a Researcher at Biome-chanics Division, Mechanical Engineering Labora-tory, Agency of Industrial Science and Technologies.From 1992 to 2000, he was an Associate Professorat Institutes of Medical Science, Tsukuba University.From 1993 to 2000, he was an Senior Researcher at

Bionic Design Research Group, Institute for Advanced Interdisciplinary Scienceand Technologies. From 2000 to 2003, he was an Associate Professor at Depart-ment of Mechanical Engineering, Graduate School of Engineering, Universityof Tokyo. Since 2003, he is a Professor in the Division of Biomedical Mate-rials and Systems, Center for Disease Biology and Integrative Medicine, Schoolof Medicine, University of Tokyo. He investigates regenerated cartilage, bone,and blood vessels in combination with 3-D scaffold technologies and tissue en-gineering. He also focuses on the elucidation of cellular mechanism of signaltransduction evoked by physical stimulations such as stretching, shear, and hy-drostatic pressure.

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The State of the Art of Nanobioscience in Japan