Medical Engineering & Physics 27 (2005) 754–762

Magnetic micro- and nanoparticle mediated activationof mechanosensitive ion channels

Steven Hughes, Alicia J. El Haj, Jon Dobson∗

Institute of Science and Technology in Medicine, Keele University, Thornburrow Drive, Hartshill, Stoke-on-Trent, ST4 7QB, UK

Received 7 February 2005; accepted 11 April 2005

Abstract

Most cells are known to respond to mechanical cues, which initiate biochemical signalling pathways and play a role in cell membraneelectrodynamics. These cues can be transduced either via direct activation of mechanosensitive (MS) ion channels or through deformation ofthe cell membrane and cytoskeleton. Investigation of the function and role of these ion channels is a fertile area of research and studies aimedat characterizing and understanding the mechanoactive regions of these channels and how they interact with the cytoskeleton are fundamentalto discovering the specific role that mechanical cues play in cells. In this review, we will focus on novel techniques, which use magnetic micro-and nanoparticles coupled to external applied magnetic fields for activating and investigating MS ion channels and cytoskeletal mechanics.©

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eywords:Biomagnetism; Mechanotransduction; Bone cells; Nanoparticle; Ion channels

. Introduction

The activation of mechanosensitive (MS) ion channels inivo is the first step in the initiation of biochemical reac-ion pathways that leads to the fundamental developmentnd normal functioning of many types of tissue. Despite

he importance of mechanotransduction, the mechanisms andathways by which it occurs are not fully understood. Forxample, mechanotransduction in bone is a complex event,equiring the selective involvement of many different sig-alling pathways, however it is clear from a number of studies

hat changes in intracellular calcium and membrane potentialre one of the earliest, and possibly most influential responsesf bone cells to mechanical stimulation[1–4].

Mechanosensitive, or stretch-activated ion channels areound within the cell membranes of almost all cell types.he activity of mechanosensitive channels is directly influ-nced by changes in membrane tension and they are known toe important for osmoregulation and cell protection, but arelso though to be involved in the transduction of mechanicaltimuli by a variety of cell types, including endothelial cells

[5], smooth muscle cells[6–8], heart cells[9,10], neuronacells[11–13], fibroblasts[14,15], chondrocytes[16–18], andosteoblast-like cells[19–24].

Many stretch-activated ion channels are permeabcations in general, with high permeability for divalent catisuch as calcium. Activation of these channels typically rein the influx of extracellular calcium across the cell memband into the intracellular regions of the cell. Other classemechanosensitive ion channels are selectively permeasodium ions, potassium ions, chloride ions or to aniongeneral.

Mechanosensitive ion channels often show bursts of aity followed by periods of inactivity, where membrane strereduces the length of the inactive periods, although the prgating mechanisms involved in the actions of these chaare poorly understood. There is mounting evidence to sua kinetic scheme for many mechanosensitive ion channewhich there are a number of closed states in a linear faswhere potentially only the transition from the state furthfrom the open state is affected by stretch and that the chamay also be dependent on other factors such as the st

∗ Corresponding author. Tel.: +44 1782 555261; fax: +44 1782 747319.E-mail address:bea22@keele.ac.uk (J. Dobson).

phosphorylation[25,26]or the presence of a specific ligand[27]. For the majority of examples of mechanosensitive ionchannels, it is not clear what aspect of ion channel structure

350-4533/$ – see front matter © 2005 IPEM. Published by Elsevier Ltd. All rights reserved.

oi:10.1016/j.medengphy.2005.04.006

S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762 755

is responsible for stretch sensitivity, or how indeed externalmechanical stimuli interact with this component of the ionchannel in order to influence the open probabilities of thepore. The majority of structural studies have been carriedout on bacterial and archaeal mechanically gated channels,which provide evidence that these channels can sense mem-brane tension directly. The tension is created in the lipidbilayer and the mechanism is described by the bilayer model[28] (Markin and Martinac) but little parallel work has beenconducted in mammalian systems. Xenopus stretch activatedcation channels do provide evidence of similar bilayer the-ory mechanisms to those described for bacterial systems[29].However, it has been proposed that a number of external fac-tors may regulate the activity of these channels.

Forces transmitted to the cytoskeleton may lead toincreased tension or curvature of the cell membrane, andthus the cytoskeleton has the potential to regulate ion channelactivity[30–33]. It is plausible that the cytoskeleton may indi-rectly regulate the activity of mechanosensitive ion channelsby controlling the biomechanical properties of the lipid mem-brane. For example, stiffening of the membrane (mediated bychanges in the cytoskeleton that result in increasing levels ofpre-stress or tension) will leave it less prone to deformationand thus the likelihood of a channel opening in response to afixed stimulus may be reduced.

A direct link between the cytoskeleton and mechanosen-s anismb ctlyl sultsh form oulds ay bedN srup-t abil-i ioncP ther nnelp esulto rachi-d letonrT ctiv-i byi xes[

an-n pro-v thet ud-i iadae d notw

Bone cells have been shown to express a range ofmechanosensitive ion channels. Early work on rat calvar-ial cultures described potentially mechanosensitive L- andT-type voltage operated calcium channels[47]. This workhas been confirmed in other bone derived cell types (bonemarrow stromal cells and femoral osteoblasts[19,48] andbone cell lines[20,49]. Duncan and Misler[20] demonstratedand characterised a 20pS calcium stretch sensitive permeablechannel. Further evidence that potassium and non-selectivemechanosensitive channels are present in osteoblasts showsthe potential complexity of ion signalling events across themembrane[22].

It has been suggested that the strain induced activationof mechanosensitive calcium channels can cause an influxof Ca2+ great enough to induce the activation of T-type(LVA) and possibly L-type (HVA) voltage operated calciumchannels (VOCCS)[19], and thus this is one mechanism bywhich mechanical forces may be transduced into a meaning-ful biochemical signal. In support of this, the responses ofosteoblasts to fluid flow in the presence of PTH have beenshown to be dependent on the activation of both mechanosen-sitive ion channels and VOCCs[24].

2. Magnetic particles as a tool for applying forces tocells in vitro

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itive ion channels has been proposed as another mechy which deformations of the cytoskeleton may dire

ead to increased ion channel activity. Experimental reave demonstrated the affinity of cytoskeletal proteinsechanosensitive ion channel proteins and thus it w

eem plausible that membrane bound ion channels mirectly linked to the cytoskeleton of cells in vivo[25,34].umerous studies have shown that pharmacological di

ion of the cytoskeleton leads to changes in the open probty of a number of different classes of mechanosensitivehannel (for example see Schwiebert et al.[35], Xu et al.[36],iao et al.[37]. However, whether or not this response is

esult of a change in the direct interactions of the ion charoteins with the cytoskeleton, or whether they are the rf increased release of signalling molecules, such as aonic acid or phospholipases, from the disrupted cytoskeemains largely unanswered (for review, see Janmey[38]).here is however, limited evidence to suggest that the a

ty of specific ion channels may be directly influencednteractions with integrins and focal adhesion comple39,40].

Pharmacological inhibition of mechanosensitive ion chels (with agents such as gadolinium for example) hasided evidence for the activity of these channels inransduction of various forms of mechanical stimuli inclng static fluid flow[41,24], cyclical mechanical strain veformable membranes or four point bending models[42–46]nd mechanical manipulation with optical tweezers[1]. How-ver, the specific classes of ion channels that are involveell understood.

Recently a number of techniques have been describellow mechanical forces to be applied to specific recepn the surfaces of cultured cells through the use of microanoscale magnetic particles coated in ligands for cell

ace receptors (e.g. Wang et al.[33], Glogauer et al.[14,50]).hese particles typically are composed of a magneticith a polymer coating, which can be functionalizedross-linked to biological molecules of interest[51,52]. Bothhe type and amount of magnetic material can be varieroduce particles of differing magnetic properties (pargnetic, superparamagnetic and ferromagnetic, for examnd the polymer used can be varied in order vary the mial properties of the particle as a whole (i.e. surface charotein binding capacity, surface topography and biocom

bility).The magnetic techniques reported can be broadly spli

wo types; those utilising magnetic drag to apply translatiorces, and those relying on magnetic rotation or twistinmpart torque on magnetic particles attached to the cell mrane. Magnetic drag techniques, such as those reportlogauer et al.[14,50]are technically simpler than the pale rotation techniques such as magnetic twisting cytomMTC) developed by Wang et al.[33].

. Magnetic drag

Glogauer and others have developed a simple techsing a permanent ceramic magnet to apply magnetic

756 S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762

Fig. 1. Left – Schematic representation of magnetic particles attached tothe dorsal surface of a substrate attached cell. Right – Application of a high-gradient magnetic field causes translational displacement of the particles andresults in membrane deformation. Image adapted from Glogauer and Ferrier[50].

to particles attached to integrin receptors on the dorsal sur-face of substrate attached cells in order to cause a verticaldisplacement of the cells (Fig. 1). Similar approaches havealso been developed where the magnetic field is supplied byan electromagnet that produces a highly localised magneticfield [53]. In both cases the underlying principles are largelythe same, the high gradient magnetic fields are used to dragand pull the particles in a given direction and thus imparta translational force causing tension and deformation of thecell membrane. Systems exerting both vertical[14,50] andhorizontal shear forces[54] have been reported.

The direction and size of the membrane deformationinduced is dependent on both the magnetic field applied andthe number and location of the particles attached to the cellsurface. Thus, by regulating these factors it is possible tocontrol the forces applied to the cells.

During binding to the cell surface the particles becomefirmly attached to the cell membranes through the accumu-lative actions of many simultaneous interactions over theparticle surface (demonstrated by SEM images, see Fabryet al.[55]). Thus, forces up to and in excess of normal physi-ological stresses can be applied without either detachment ofthe particle or damage of the cell membrane while allow-ing the cells to be monitored visually, biochemically andelectrophysiologically in real time (e.g. Glogauer et al.[14],Pommerenke et al.[55], Ingber[56], Browe and Baumgarten[

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mechanical properties of the cytoskeleton[33,61,62]. Thistechnique is based on the application of highly localisedtorque to specific membrane receptors without deforming thecell membrane as a whole. Ferro(ferri)magnetic micropar-ticles are attached to the cell surface through the use ofspecific antibody or ligand coatings in much the same wayas described above. In this case, however, magneticallyblocked particles (particles which retain a magnetization inthe absence of a field) are used and the torque is exerted viathe application of an external magnetic field that is orientedat an angle to the particle’s magnetization vector. This hasthe effect of “twisting” the particle.

Following attachment the particles are first magnetised ina given direction with a short pulse of a strong magnetizingfield that aligns the magnetization vector of all the particles.As the particles used are ferromagnetic they retain this direc-tion of magnetisation after the initial magnetising pulse haspassed. Following magnetisation a weaker second ‘twisting’field is applied to the particles in a direction perpendicular tothat of the magnetising field. This ‘twisting’ field is not strongenough to remagnetise the particles in the new direction, butinstead causes the particles to rotate, as they attempt to aligntheir magnetic poles in the direction of the new weaker mag-netic field (Fig. 2). The rotation of the particles is resisted bytheir attachments to the cell membrane surface and thus withlow magnitude twisting fields, little rotation is seen and littlet hicht ld isi andt tors.B uslym chedt raget

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57]).Using magnetic drag in this way it is possible to apply c

rolled physical forces to single cells or to populations of cpotentially in 3D) with relative ease, and as such this tique is becoming increasing popular as a tool for stud

he biomechanical properties of living cells[58–62]. How-ver, the main advantage of this approach compared toore traditional methods of applying forces to cultured c

uch as fluid flow and substrate bending, is that the aporces can be ‘localised’ to the vicinity of specific celluomponents or surface bound receptors, such as integroating the particles with specific ligands or antibodies.

. Magnetic twisting cytometry (MTC)

A more complex technique, termed magnetic twisytometry (MTC), has been developed by Wang et al., wakes use of magnetic particle rotation to investigate

orque is applied to the cell membrane receptors to whe particles are bound. As the size of the twisting fiencreased higher magnitudes of rotation are observedhus higher torque forces are transmitted to the recepy using a highly sensitive magnetometer to simultaneoeasuring the rotation of around 50,000 particles atta

o 20–40,000 cells, MTC provides a measure of the aveorque applied per particle.

The main advantage of MTC over other simpler metic drag techniques is that, although the applied to

orces remain highly localised to the specific cell sureceptor of interest, the forces used do not cause a ge

ig. 2. MTC – Magnetisation field is applied briefly in theYplane.H rep-esents the twisting field used to rotate the particle, whereΦ represents thngle of particle rotation. Rotation of the particle is computed from cha

n the magnetic field measured by a magnetometer. Diagram re-prorom Puig-De-Morales et al.[109].

S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762 757

deformation of the cell membrane as a whole (for finite ele-ment model see Mijailovich et al.[63]). Consequently MTCrepresents an ideal tool for investigating the role of specificmembrane receptors and adhesion proteins in mechanotrans-duction. Furthermore, by comparing the levels of particlerotation seen at different levels of twisting field, or by com-paring the rate at which the ‘twisted’ particles return to theiroriginal positions following the removal of the magnetic field,it is possible to deduce information concerning the materialproperties of the cytoskeleton and the cell as a whole (i.e. stiff-ness, creep response and viscoelasticity[64,33,61,62,59,65].

4.1. Cellular responses to magnetic loading

Combined, magnetic particle based techniques have beenused to apply forces to a wide range of cell types includingfibroblasts[14,15,50,66,67], endothelial cells[33,56,68–71],mesenchymal stem cells[72], osteoblast cells[54,73–75],myoblast cells[76], alveolar epithelial cells 60, vascularsmooth muscle cells[77], bladder smooth muscle cells[78],hepatocytes[79] (Nebe et al.), neurons[80], macrophages[59], astrocytes, glioma cells and a range of kidney derivedcells[69].

The majority of work using this technique has focused onthe direct stimulation of candidate mechanotransducers, suchas integrins[33,56,73], E-cadherins[15,81], E-selectins[82]a es e tot

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different cell types) that ‘non’ responsive cells that fail toelicit intracellular calcium transients in response to this typeof magnetic loading are often spindle shaped or well flat-tened cells with long processes, whereas responsive cells areusually characterised by a well-rounded morphology.

Niggel et al.[69] have investigated the calcium responsesof a variety of cell types (over 10) including fibroblasts,bovine aortic endothelial cells, UMR-106 osteocyte cellline, HEK-293 human kidney cells, primary chick cardiacmyocytes, rat primary astrocytes and glioma cells. Theyfound that although all cells responded to magnetic stim-ulation of integrin receptors with increases in intracellularcalcium levels, the responses varied in a number of respectsbetween the different cell types. The magnitude, duration andpattern of response varied markedly between cells. There wasalso a difference in the source of calcium, with some cellsrequiring the activity of membrane bound mechanosensitiveion channels, while other cell types relied entirely on releasefrom intracellular stores.

5. Quantification of applied forces and uniformity ofparticle binding

In many published papers authors have attempted to defineor calculate the magnitude of force that is being applied tot mpled eachp oper-t l perp thisc a-d e tog eringt cellm rcea raneb eent d lit-e /cmf sedt

rquea rota-t ana sim-p ey don ach-m ctronm torc ly tov th oft nicals emt callya ly be

nd the urokinase receptor[83], in an attempt to elude thpecific signalling pathways that are initiated in responshe mechanical activation of these receptors.

Stimulation of integrin receptors on osteoblast cells uagnetic drag has demonstrated a range of responses i

ng; adaptive changes to the cytoskeleton (including accumulation and formation of focal adhesion complex

ncreased tyrosine phosphorylation and MAP kinase ac74], changes in intracellular pH[75] and generation of intraellular calcium transients[54,73]. These responses weypically absent when other ‘control’ receptors such asransferrin receptor (CD71) or LDL receptor were stimula54,73–75]. In addition, the applied magnetic fields usedhese examples have been shown to have no effect on che absence of magnetic particles.

Responses observed in other cell types include mrane stiffening and changes in cytoskeletal architecture[67],hanges in intracellular calcium levels, recruitment of mRnd ribosomes to focal adhesion complexes[68], elevation o

ntracellular cAMP[56], increased CREB phosphorylatiop-regulation of endothelin-1 gene expression[70] and theownstream activation of a range of ion channels via Finase and src based mechanisms[57].

Wu et al.[66] have shown that the level of cellular acrganisation is a key factor in determining the naturealcium transients in human fibroblast cells. Freshly plbroblasts showed a three-fold increase in the magnitualcium transients compared to cells that have been allo attach and spread for up to 16 h. These formal findingupported by the observations of a number of authors (u

-

he cells on which magnetic particles are attached. For sirag techniques approximations of the force applied toarticle can be obtained by considering the magnetic pr

ies of the particle (type and mass of magnetic materiaarticle, etc. – for review and mathematical treatment ofoncept, see Pankhurst et al.[84]) and the magnitude and grient of the magnetic field applied. From this it is possiblet some idea of the forces acting on each cell by consid

he number and location of the particles attached to theembrane. However, it is important to note that the fopplied is not evenly distributed across the cell membut is instead localised to the points of attachment betwhe cell membrane and the magnetic particle(s). Staterature values typically range between 0 and 165 dynes2

or MTC, or 4–150 pN per particle for magnetic drag baechniques.

MTC provides an indirect measure of the average tocting on each particle by simultaneously measuring the

ion of ∼50,000 particles and subsequently calculatingverage. The problem with MTC based calculations andler approximations based on drag techniques is that thot take into account the heterogeneity of particle attent that has been demonstrated with scanning eleicroscopy[55]. The number of physical ligand/recep

ontacts between a cell and a particular particle is likeary, as is the surface area of particle to cell contact. Bohese factors will affect the exact nature of the mechatimulus that is applied to the cell. In addition, it would sehat not all particles present on a cell surface are specifittached to the receptor of interest but some may on

758 S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762

loosely attached to the cell through extracellular matrix orFCS protein-mediated interactions.

Fabry et al.[55] have investigated the influence of hetero-geneous particle behaviour on calculations made using MTCand have shown that the values of cell stiffness can be hugelyunder estimated even when only 1% of particles are indi-rectly attached (loosely attached particles are free to rotateeven at low twisting fields and therefore introduce error intocalculations of average particle rotation). The authors haveconcluded that the apparent shear modulus obtained fromMTC calculations may underestimate the real shear modulusexperienced by the cell membrane, but that these differencescan be minimised when the magnetic torque applied is greater(when even tightly bound particles are rotated maximally).Apparent changes in the calculated value of cell stiffness withincreasing twisting fields have previously been attributed toa cell stiffening response and interpreted as evidence for thetensegrity model of cellular biomechanics[85].

6. Particle internalisation

Magnetic nanoparticles are highly biocompatible(dextran–magnetite has no measurable toxicity[86]),however the one factor that potentially limits the usefulnessof magnetic particle techniques for long-term stimulationo tiono eriorr

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of internalisation employed. It is clear that particles maybe internalised by cells by a number of complex and dis-tinctly different cellular mechanisms. Endocytotic pathwayscan be broadly split into phagocytosis and pinocytosis typeevents. Clatherin-dependent pinocytosis, caveolin-dependentpinocytosis and macropinocytosis are all mechanisms com-monly employed by cells of various lineages to ingest smallparticles (typically <200 nM), however this and other formsof endocytosis are thought to be limited to ingesting parti-cles smaller than one to two microns in diameter (for review,see Liu and Shapiro[96]). A number of different cell types,including macrophages and osteoblasts, are capable of inter-nalising relatively large particles (>10�m) via ‘true’ phago-cytotic mechanisms[97–99]. Phagocytosis is distinguishedfrom pinocytosis by the involvement of actin polymerisationand the size of particles internalised (for review, see Aderemand Underhill[100]).

Phagocytosis is not only important for immune mediateddefence, but is also an essential process for tissue remodellingand repair and thus many other cell types other than the tra-ditional phagocytotic leukocytes (granulocytes, monocytesand macrophages) demonstrate some degree of phagocytoticactivity (for review, see Rabinovitch[101]). Phagocytosis isinitiated by the stimulation of specific receptors on the phago-cytotic cell by a ligand on the surface of a particle or invadingorganism. Stimulation of phagocytotic receptors (comple-m icallyl ctinp tosiso aga-s mentt them d it isc thano somec esiona rhill[

aysh ple,K ter-n raneb l sur-f alisa-ti

thei sm ofi iclest ents[ ee ghoutt war[ ed ino ures,i clear

f membrane receptors is the relatively rapid internalisaf the attached particles from the cell surface to the integions of the cells.

For example, Bierbaum and Notbohm[75] have shownthat both RGD and fibronectin-coated particles with 4�mdiameter are internalised into osteoblast and fibroblastwithin hours, where the time scale for internalisationrelatively similar for both cell types and both coatingsO’Connor et al.[87] have shown that Ti particles rangifrom 1.5 to 4.0�m in diameter are internalised and moto the peri-nuclear space in <8 h in rat osteoblasts.

Almost all cell types studied have shown the abilitynternalise very small particles (around 10–50 nm), aighlighted by the growing popularity of using magneanoparticles for transfection studies (for examples, seeasetwar et al.[88], Prabha et al.[89]). However there arlear cell type specific differences concerning the size oficles that can be internalised. This effect is evident inange of 50–500 nm sized particles[89], but becomes mucore apparent as particle sizes are increased to around�mnd above[90,91].

Overall, the rate of particle internalisation is dependn a wide range of factors including cell type[92], particleize[87,92–94], the hydrophobicity and surface charge ofarticle polymer[93,94], the nature of the surface ‘proteoating on the particle[95] and the proliferate rate of the ce92].

However, one factor that is key to governing thef particle internalisation for a given cell is the meth

ent receptors, Fc receptors or scavenger receptors) typeads to the internalisation of the ‘foreign’ particle via an aolymerisation based mechanism resulting in the endocyf the particle into a phagocytotic compartment (a phome), which then merges with the lysosomal comparto facilitate degradation of the foreign material. Althoughechanism of phagocytosis is not completely understoo

lear that ‘foreign’ particles may be recognised by morene type of receptor and also that a particular receptor (lasses of integrins for example) may mediate both adhnd internalisation (for review, see Aderem and Unde

100]).In addition a number of specialised endocytotic pathw

ave been described in particular cell types. For examruth et al.[102] have shown that macrophages can inalise hydrophobic polystyrene micoparticles into membound compartments that remain connected to the cel

ace, a process termed patocytosis. This type of internion is limited to hydrophobic particles of less than 0.5�mn diameter.

The ‘route of entry’ has implications for the fate ofnternalised particles, where depending on the mechaninternalisation and to a large extent the size of the parthese may become ‘trapped’ in phagocytotic compartm97] remain connected to the cytoskeleton[102] or escapnolysomal compartments and become dispersed throu

he cytoplasm[103](for review, see Panyam and Labhaset104]). It is possible that these differences can be exploitrder to apply mechanical forces to numerous cell struct

ncluding the cell membrane, the cytoskeleton, the nu

S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762 759

membrane, the endoplasmic reticulem membrane or intra-cellular regions of membrane bound receptors.

The phenomenon of internalisation does not however pre-vent long-term stimulation of surface bound receptors. Theinsertion of a cleavable linker sequence between the ‘lig-and’ coating and the physical surface of the particle will, inprinciple, allow the particles to be separated from the cell sur-face bound receptor at a time point before internalisation hasoccurred. It is conceivable that in an in vitro bioreactor envi-ronment (see below) magnetic particles could be added to themedia perfusing a cell seeded construct. The flow of mediacould then be temporarily suspended to allow the particlesto attach before being resumed to remove unbound particlesand continue nutrient supply. Following a period of mag-netic loading (possibly only 20 min at a time for example)the enzyme required to cleave the particles from the ligandcoating (that effectively anchors them to the cell membrane)could be added to the cell culture media to induce detachmentof the membrane bound particles. Detached particles wouldthen be washed away by media perfusion and could be easilycollected and removed from the circulating culture media viathe use of a ‘magnetic trap’.

7. Use of internalised particles for magnetic loading

edp kele-t L ora genee nter-n spiteb lace-m in thec ionc lisedp areao

ter-n ons.I ingh ells)w chedfi tra-c ticm sulti “A”c cal-c eticd berso

um-b lism( G3Pd haven ells.

Fig. 3. The model system used by Koh et al.[15]. Cells containing inter-nalised particles (S cells) are allowed to form intracellular adheren junctionswith substrate attached cells (A cells). Application of magnetic field to S cellsis capable of inducing mechanical load responses in A cells via stimulationof adheren junctions. Image reproduced from Koh et al.[15].

Only two studies to date have involved long-term stimu-lation by magnetic particles. Yuge and Kataoka[76] showedthat the alignment and differentiation of the L6 rat myoblastcell line is accelerated when cultured in a continuous staticmagnetic field (0.05 T) for 3 weeks following the electropora-tion of nanoscale magnetic particles into the cytoplasm of thecells. Cell viability was reportedly unaffected. More recently,Cartmell et al.[105] cultured human derived osteoblastsunder exposure to a 1 Hz magnetic field for 1 h/day over aperiod of 3 weeks to investigate mechanical condition ofbone cells for tissue engineering (see below). Though rel-atively large particles were used (Sphereotech,d= 4.5�m),there were no reported effects on cell viability.

7.1. Magnetic activation of ion channels for tissueengineering and stem cell conditioning

Recently, magnetic activation of MS ion channels hasfound practical application within the field of tissue engineer-ing and stem cell research, in the development of magneticforce bioreactors (MFB)[105,106]. In order to grow func-tional bone in a bioreactor the cells must experience mechan-ical loading stimuli similar to those experienced in vivo[19]– in other words, the in vivo stress environment must be mim-icked inside the bioreactor. This has not been achieved withp esigno toc eredt

eliv-e centm Thisi d/orn s area ordert peri-e ion-i ting

Chen et al.[70] have shown that twisting of internalisarticles (internalised and associated with the cytos

on) coated with RGD peptide sequence, but not AcLDnti-HLA antibody, caused an increase in endothelin-1xpression in HUVEC cells. The authors suggest that ialised particles are still associated with integrins deeing stored in endocytic compartments, and that dispent of these particles causes conformational changes

ytoskeleton that lead to activation of stretch activatedhannels. The authors also suggest that twisting internaarticles resulted in a torque being applied to a greaterf the cell compared to cells twisted on the cell surface.

Koh et al.[15] have shown that it is possible to use inalised particles to stimulate intracellular cadherin juncti

n this model system populations of fibroblasts containigh concentrations of anti-cadherin coated particles (S cere incubated with a second population of substrate attabroblasts (designated A cells) and allowed to form inellular cadherin junctions (Fig. 3). Subsequent magneanipulation of the particle population was shown to re

n a rise in intracellular calcium in the substrate attachedells but not the suspension “S” cells. Interestingly noium response was seen following application of magnrag to substrate attached “S” cells containing large numf internalised particles.

It is also interesting to note that the presence of large ners of internalised particles did not affect cell metaboas measured by cytochrome oxidase (COX-1) assay andehydrogenase activity) and these cells were found to beormally and proliferate at a comparable rate to normal c

resent technology and presents a challenge for the df bioreactors[107,108]. This challenge is not restrictedonnective tissue and is also relevant to other bioengineissue constructs.

In the case of the MFB, the mechanical cues are dred directly to the cell membrane (which activates adjaechanosensitive ion channels), or to the channel itself.

s done by attaching biocompatible magnetic micro- ananoparticles to membrane receptors. Intermittent forcepplied via a permanent magnet array on a drive sled in

o simulate time-varying stress cycles similar to those exnced in vivo. This has the effect of mechanically condit

ng the cells by activating MS ion channels as well as initia

760 S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762

cytoskeletal deformation. The technique is also being inves-tigated for active compound screening in a dynamic environ-ment.

8. Discussion

Magnetic micro- and nanoparticle-mediated activation ofmechanosensitive ion channels represents a powerful toolfor investigating the interaction between ion channels andthe cytoskeleton as well as for the development of biomedi-cal applications. Recent results have demonstrated its use-fulness for a variety of applications and investigations inwhich magnetic particles have been used to manipulate allof the major candidates for mechanosensors, including inte-grins, the cytoskeleton, kinase-type enzymes, mechanosen-sitive enzymes (bound to the membrane and cytoskeleton)and mechanosensitive ion channels (via membrane deforma-tion). Potentially we can use different particles to selectivelymanipulate these pathways simultaneously or in isolation, asand when required for particular experimental conditions. Itis clear that these techniques have an important role to playin developing our understanding of how MS ion channelsoperate and elucidating the complex biochemical processesinvolved in their function.

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[11] Oh Y. Ion channels in neuroglial cells. Kaohsiung J Med Sci1997;13(1):1–9.

[12] Quasthoff S. A mechanosensitive K+ channel with fast-gating kinet-ics on human axons blocked by gadolinium ions. Neurosci Lett1994;169(1–2):39–42.

[13] Oliet SH, Bourque CW. Mechanosensitive channels transduceosmosensitivity in supraoptic neurons. Nature 1993;364(6435):341–3.

[14] Glogauer M, Ferrier J, McCulloch CAG. Magnetic fields applied tocollagen coated beads induce stretch-activated Ca2+ flux in fibrob-lasts. Am J Physiol 1995;38:C1093–104.

[15] Koh SK, Arora PD, McCulloch CAG. Cadherins mediate intra-cellular mechanical signalling in fibroblasts by activation ofstretch-sensitive calcium permeable channels. J Biol Chem2001;276(38):35967–77.

[16] Guilak F, Zell RA, Erickson GR, Grande DA, Rubin CT, McLeodKJ, et al. Mechanically induced calcium waves in articular chon-drocytes are inhibited by Gadolinium and Amiloride. J OrthopaedRes 1999;17:421–9.

[17] Lee HS, Millward-Sadler SJ, Wright MO, Nuki G, Salter DM. Inte-grin and mechanosensitive ion channel-dependent tyrosine phos-phorylation of focal adhesion proteins and�-catenin in humanarticular chondrocytes after mechanical stimulation. J Bone MinerRes 2000;15(8):1501–9.

[18] Mobasheri A, Carter SD, Martin-Vasallo P, Shakibaei M. Integrinsand stretch activated ion channels; putative components of func-tional cell surface mechanoreceptors in articular chondrocytes. CellBiol Int 2002;26(1):1–18.

[19] El Haj A, Walker LM, Preston MR, Publicover SJ. Mechanotrans-duction pathways in bone: calcium fluxes and the role of voltage-operated calcium channels. Med Biol Eng Comp 1999;37:403–9.

Ba06).

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er-ses to

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eferences

[1] Walker LM, Holm A, Cooling L, Maxwell L, Oberg A, Sundqviset al. Mechanical manipulation of bone and cartilage cells‘optical tweezers’. FEBS Lett 1999;459:39–42.

[2] Jorgenson NR, Henriksen Z, Brot C, Eriksen EF, SorensonCivitelli R, et al. Human osteoblastic cells propagate intracelcalcium signals by two different mechanisms. J Bone Miner2000;15(6):1024–32.

[3] Salter DM, Wallace WHB, Robb JE, Caldwell H, Wright MHuman bone cell hyperpolarisation response to cyclical mechastrain is mediated by an interleukin-1� autocrine/paracrine loop.Bone Miner Res 2000;15(9):1746–55.

[4] Charras GT, Lehenkari PP, Horton MA. Atomic force microsccan be used to mechanically stimulate osteoblasts and evcellular strain distributions. Ultramicroscopy 2001;86:85–95.

[5] Naruse K, Yamada T, Sakabe M. Involvement of SA ion chanin orienting response of cultured endothelial cells to cyclic streAm J Physiol 1998;274:1532–8.

[6] Davis MJ, Donovitz JA, Hood JD. Stretch-activated single-chaand whole cell currents in vascular smooth muscle cells. APhysiol 1992;262(4.1):1083–8.

[7] Farrugia G, Holm AN, Rich A, Sarr MG, Szurszewski JH, RaeA mechanosensitive calcium channel in human intestinal smmuscle cells. Gastroenterology 1999;117(4):900–5.

[8] Wu X, Davis MJ. Characterization of stretch-activated cationrent in coronary smooth muscle cells. Am J Physiol HeartPhysiol 2001;280(4):1751–61.

[9] Hu H, Sachs F. Stretch-activated ion channels in the heart. JCell Cardiol 1997;29(6):1511–23.

[10] Kamkin A, Kiseleva I, Wagner KD, Bohm J, Theres H, Gther J, et al. Characterization of stretch-activated ion currenisolated atrial myocytes from human hearts. Pflugers Arch 2446(3):339–46.

[20] Duncan RL, Misler S. Voltage-activated and stretch-activated2+

conducting channels in an osteoblast-like cell line (UMR1FEBS 1989;251(1):17–21.

[21] Duncan RL, Hruska KA, Misler S. Parathyroid hormone activaof stretch-activated cation channels in osteosarcoma cells (U106.01). FEBS Lett 1992;307(2):219–23.

[22] Davidson RM, Tatakis DW, Auerbach AL. Multiple formsmechanosensitive ion channels in osteoblast-like cells. PflArch 1990;416:646–51.

[23] Rawlinson SCF, Pitsillides AA, Lanyon LE. Involvement of diffent ion channels in osteoblasts’ and osteocytes’ early responmechanical strain. Bone 1996;19:609–14.

[24] Ryder KD, Duncan RL. Parathyroid hormone enhances fluid sinduced [Ca2+]I signalling in osteoblastic cells through activatof mechanosensitive and voltage sensitive Ca2+ channels. J BonMiner Res 2001;16:240–8.

[25] Park SM, Liu G, Kubal A, Fury M, Cao L, Marx SO. Direct interation between BKCa potassium channel and microtubule-assoprotein 1A. FEBS Lett 2004;570(1–3):143–8.

[26] Bockenhauer D, Zilberberg N, Goldstein SAN. KCNK2: reversconversion of a hippocampal potassium leak into a voltdependent channel. Nat Neurosci 2001;4:487–91.

[27] Paoletti P, Ascher P. Mechanosensitivity of NMDA receptorcultured mouse central neurons. Neuron 1994;13:645–55.

[28] Markin VS, Martinac B. Mechanosensitive ion channelsreporters of bilayer expansion. A theoretical model. Biophy1991;60(5):1120–7.

[29] Zhang Y, Hamill OP. Calcium voltage and osmotic stress sencurrents in Xenopus oocytes and their possible relationship to smechanically gated channels. J Physiol (London) 2000;523:8

[30] Davies E. Intercellular and intracellular signals and their transtion via the plasma membrane-cytoskeleton interface. SeminBiol 1993;4(2):139–47.

[31] Diamond SL, Sachs F, Sigurdson WJ. Mechanically inducalcium mobilisation is dependent on actin and phospholipArtheroscler Thromb 1994;14:2000–6.

S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762 761

[32] Sachs F. Mechanical transduction in biological systems. CRC CritRev Biomed Eng 1988;16:141–69.

[33] Wang N, Butler JP, Ingber DE. Mechanotransduction across thecell surface and through the cytoskeleton. Science 1993;260(5111):1124–7.

[34] Suzuki M, Hirao A, Mizuno A. Microfilament-associated protein 7increases the membrane expression of transient receptor potentialvanilloid 4 (TRPV4). J Biol Chem 2003;278(51):51448–53.

[35] Schwiebert EM, Mills JW, Stanton BA. Actin-based cytoskeletonregulates a chloride channel and cell volume in a renal corticalcollecting duct cell line. J Biol Chem 1994;269(10):7081–9.

[36] Xu WX, Kim SJ, So I, Kim KW. Role of actin microfilamentin osmotic stretch-induced increase of voltage-operated calciumchannel current in guinea-pig gastric myocytes. Pflugers Arch1997;434(4):502–4.

[37] Piao L, Ho WK, Earm YE. Actin filaments regulate the stretch sen-sitivity of large-conductance Ca2+-activated K+ channels in coro-nary artery smooth muscle cells. Pflugers Arch 2003;446(5):523–8.

[38] Janmey PA. The cytoskeleton and cell signalling: component local-isation and mechanical coupling. Biol Rev 1998;78(3):763–81.

[39] Schwartz MA, Brown EJ, Fazeli B. A 50 kDa integrin-associatedprotein is required for integrin-regulated calcium entry in endothe-lial cell. J Cell Biol 1993;268:19931–4.

[40] Rezzonico R, Schmid-Alliana A, Romey G, Bourget-Ponzio I,Breuil V, Breittmayer V, et al. Prostaglandin E2 induces interac-tion between hSlo potassium channel and Syk tyrosine kinase inosteosarcoma cells. J Bone Miner Res 2003;17(5):869–78.

[41] Hung CT, Allen FD, Pollack SR, Brighton CR. IntracellularCa2+ stores and extracellular Ca2+ are required in the real timeCa2+ response of bone cells experiencing fluid flow. J Biomech1996;29(11):1411–7.

ch-. Am

pon-

H,ovelytes.

J.ani-ysiol

Hajtionhem

cal-:291–

per-s in

renton)

to

, Tan.anor-nts.

al anded byinyl

[53] Goldschmidt PL, Devillechabrolle A, Ait-Arkoub Z, Aubin JT.Comparison of an amplified enzyme-linked immunosorbent assaywith procedures based on molecular biology for assessing humanimmunodeficiency virus type 1 viral load. Clin Diagn Lab Immunol1998;5(4):513–8.

[54] Pommerenke H, Schreiber E, Durr F, Nebe B, Hahnel C, MollerW, et al. Stimulation of integrin receptors using a magnetic dragforce device induces intracellular free calcium response. Eur J CellBiol 1996;70:157–64.

[55] Fabry B, Maksym GN, Hubmayr DR, Butler JP, Fredburg JJ.Implications for heterogeneous bead behaviour on cell mechani-cal properties measured via magnetic twisting cytometry. J MagnMagn Mater 1999;194:120–5.

[56] Ingber DE. Mechanical control of cyclic cAMP signalling and genetranscription through integrins. Nat Cell Biol 2000;2:666–8.

[57] Browe DM, Baumgarten CM. Stretch of beta 1 integrin activatesan outwardly rectifying chloride current via FAK and Src in rabbitventricular myocytes. J Gen Physiol 2003;122(6):689–702.

[58] Alenghat FJ, Fabry JB, Tsai KY, Goldmann WH, Ingber DE. Anal-ysis of cell mechanics in single vinculin-deficient cells using a mag-netic tweezer. Biochem Biophys Res Commun 2000;277(1):93–9.

[59] Bausch AR, Moller W, Sackmann E. Measurement of local vis-coelasticity and forces in living cells by magnetic tweezers. Bio-phys J 1999;76:573–9.

[60] Berrios JC, Schroeder MA, Hubmayr RD. Mechanical properties ofalveolar epithelial cells in culture. J Appl Physiol 2001;91:65–73.

[61] Wang N. Mechanical interactions among cytoskeletal filaments.Hypertension 1998;32(1):162–5.

[62] Wang N, Stamenovic D. Contribution of intermediatary fila-ments to cell stiffness, stiffening, and growth. Am J Physiol2000;279(1):188–94.

J.ead

hite-obeds.anndher-ys J

u-ctin

hmoughem

ingomes

ciumd C6

torsAm J

annthe-ogy

es-cal997.

r P,ntra-

[42] Duncan RL, Hruska KA. Chronic intermittent loading alters menosensitive ion channels characteristics in osteoblast like cellsJ Physiol 1994;267:F909–16.

[43] Thomas GP, El Haj AJ. Bone marrow stromal cells are load ressive in vitro. Calcif Tissue Int 1996;58(2):101–8.

[44] Millward-Sadler SJ, Wright MO, Lee HS, Nishida K, CaldwellNuki G, et al. Integrin-regulated secretion of interleukin-4: a npathway of mechanotransduction in human articular chondrocJ Cell Biol 1999;145:183–9.

[45] Peake MA, Cooling LMM, Agnay JL, Tomas PB, El Haj ASelected contribution: regulatory pathways involved in mechcal induction of cfos gene expression in bone cells. J Appl Ph2000;89(6):2498–507.

[46] Walker LM, Publicover SJ, Preston MR, Said Ahmed MA, ElAJ. Celcium channel activation and matrix protein upregulain bone cells in response to mechnical strain. J Cell Bioc2000;79(4):648–61.

[47] Chesnoy-Marchais D, Fritsch J. Voltage gated sodium andcium channels in rat osteoblasts. J Physiol (London) 1988;398311.

[48] Preston MR, El Haj AJ, Publicover SJ. Expression of voltage oated Ca2+ channel currents in rat bone marrow stromal cellvitro. Bone 1996;19:101–6.

[49] Amagai Y, Kasai S. A voltage dependent calcium curin mouse MC3T3-E1 osteogenic cells. J Physiol (Lond1989;398:291–311.

[50] Glogauer M, Ferrier J. A new method for application of forcecells via ferric oxide beads. Eur J Physiol 1998;435:320–7.

[51] Santra SR, Tapec N, Theodoropoulou J, Dobson J, Hebard ASynthesis and characterization of silica-coated iron oxide nparticles in microemulsion: the effect of non-ionic surfactaLangmuir 2001;17:2900–6.

[52] Pardoe HW, Chua-anusorn T, St. Pierre G, Dobson J. Structurmagnetic properties of nanoscale magnetic particles synthesiscoprecipitation of iron oxide in the presence of dextran or polyvalcohol. J Magn Magn Mater 2001;225:41–6.

[63] Mijailovich SM, Kojic M, Zivokovic M, Fabry B, Fredburg JA finite leement model of cell deformation using magnetic btwisting. J Appl Physiol 2002;93:1429–36.

[64] Hu S, Eberhard L, Chen J, Love JC, Butler JP, Fredburg JJ, Wsides GM, Wang N, Mechanical anisotropy of adherent cells prby a 3D magnetic twisting device. Am J Cell Physiol, in pres

[65] Bausch AR, Ziemann F, Boulbitch AA, Jacobson K, SackmE. Local measurements of viscoelastic parameters of aent cell surfaces by magnetic bead microrheometry. Bioph1998;75:2038–49.

[66] Wu Z, Wong K, Glogauer M, Ellen RP, McCulloch CAG. Reglation of stretch activated intracellular calcium transients by afilaments. Biochem Biophys Res Commun 1999;261:419–25.

[67] D’Addario M, Arora PD, Fan J, Ganss B, Ellenm RP, McCullocCAG. Cytoprotection against mechanical forces delivered thr�1 integrins requires the induction of filamin A. J Biol Ch2001;276(34):31969–77.

[68] Chicurel ME, Singer RH, Meyer CJ, Ingber DE. Integrin bindand mechanical tension induce movement of mRNA and ribosto focal adhesions. Nature 1998;392:730–3.

[69] Niggel J, Sigurdson W, Sachs F. Mechanically induced calmovements in astrocytes, bovine aortic endothelial cells, anglioma cells. J Membr Biol 2000;174:121–34.

[70] Chen J, Fabry B, Schiffrin EL, Wang N. Twisting integrin recepincreases endothelin-1 gene expression in endothelial cells.Physiol Cell Physiol 2001;280:C1475–84.

[71] Bausch AR, Hellerer U, Essler M, Aepfelbacher M, SackmE. Rapid stiffening of integrin receptor-actin linkages in endolial cells stimulated with thromb. A magnetic bead microrheolstudy. Biophys J 2001;80:2649–57.

[72] Bierbaum S, Notbohm H. Magnetomechanical stimulation of menchymal cells. In: Hafeli EA, editor. In Scientific and CliniApplications of Magnetic Carriers. New York: Plenum Press; 1p. 311–22.

[73] Pommerenke H, Schmidt C, Durr F, Nebe B, Luthen F, Mulleet al. The mode of mechanical integrin stressing controls i

762 S. Hughes et al. / Medical Engineering & Physics 27 (2005) 754–762

cellular signalling in osteoblasts. J Bone Miner Res 2002;17:603–11.

[74] Schmidt C, Pommerenke H, Durr F, Nebe N, Rychly J. Mechan-ical stressing of integrin receptors induces enhanced tyrosinephosphorylation of cytoskeletally anchored proteins. J Biol Chem1998;273(9):5081–5.

[75] Bierbaum S, Notbohm H. Tyrosine phosphorylation of 40 kDaproteins in Osteoblastic cells after mechanical stimulation of�1-integrins. Eur J Cell Biol 1998;77:60–7.

[76] Yuge L, Kataoka K. Differentiation of myoblasts is acceleratedin culture in a magnetic field. In vitro Cell Dev Biol Anim2000;36:383–6.

[77] Goldschmidt ME, Kenneth J, McLeod W, Taylor R. Integrin medi-ated mechanotransduction in vascular smooth muscle cells. CircRes 2001;88:674–80.

[78] Kushida N, Kabuyama Y, Yamaguchi O, Homma Y. Essentialrole for extracellular Ca(2+) in JNK activation by mechanicalstretch in bladder smooth muscle cells. Am J Physiol Cell Physiol2001;281(4):1165–72.

[79] Nebe B, Rychly J, Knopp A, Bohn W. Mechanical induction of�1 integrin-mediated signalling in a Hepatocyte cell line. Exp CellRes 1995;218:479–84.

[80] Fass JN, Odde DJ. Tensile force-dependent neurite elicitationvia anti-beta1 integrin antibody-coated magnetic beads. BiophysJ 2003;85(1):623–36.

[81] Potard US, Butler JP, Wang N. Cytoskeletal mechanics in confluentepithelial cells probed through integrins and E-cadherins. Am JPhysiol 1997;272(5.1):1654–63.

[82] Yoshida M, Westlin WF, Wang N, Ingber DE, RosenzweigA. Leukocyte adhesion induces E-selectin association with thecytoskeleton. J Cell Biol 1996;133:445–55.

imoncross

ag-1.plinghem

loodpoth

wffectRes

rans-ulture

ncy ofnated

Theco-2

73.ke

ithe-

[92] Zauner W, Farrow N, Haines MR. In vitro uptake of polystyrenemicrospheres: effect of particle size, cell line and cell density. JContr Rel 2001;71:39–51.

[93] Pisken E, Tuncel A, Denzili A, Ayhan H. Monosize microbeadsbased on polystyrene and their modified forms for some selectedmedical and biological applications. J Biomater Sci 1994;5:451.

[94] Yamamato N, Fukai F, Ohshima H, Terada H, Makino K. Depen-dence of the phagocytotic uptake of polystyrene microspheres bydifferentiated HL60 upon the size and surface properties of themicrospheres. Colloids Surf 2002;25:157–62.

[95] Wihelm C, Billotey C, Roger J, Pons JN, Bacri JC, Gazeau F.Intracelluar uptake of anionic superparamagnetic nanoparticles asa function of their surface coating. Biomaterials 2003;24:1001–11.

[96] Liu J, Shapiro JI. Endocytosis and signal transduction: basic scienceupdate. Biol Res Nursing 2003;5(2):117–28.

[97] Moller W, Kreyling WG, Kohlhaufl M, Haussinger K, Heyder J.Macrophage functions measure by magnetic microparticles in vivoand in vitro. J Magn Mater 2001;225:218–25.

[98] Heinmann DE, Lohman C, Siggelkow H, Alves F, Engel I, KosterG. Human osteoblast-like cells phagocytose metal particles andexpress the macrophage marker CD68 in vitro. J Bone Joint SurgBr Vol 2000;82(2):283–9.

[99] Ruiz C, Perez E, Vallecillo-Capilla M, Reyes-Botella C. Phagocyto-sis and allogeneic T cell stimulation by cultured human osteoblast-like cells. Cell Physiol Biochem 2003;13(5):309–14.

[100] Aderem A, Underhill DM. Mechanisms of phagocytosis inmacrophages. Annu Rev Immunol 1999;17:593–623.

[101] Rabinovitch M. Professional and non-professional phagocytes: anintroduction. Trends Cell Biol 1995;5:85–7.

[102] Kruth HS, Chang J, Zhang WY. Characterisation of patocytosis:ts. Eur

etwar)B J

drugRev

entcellsosci

of a0–1.ngi-for, in

terr in295–

N,neticppl

[83] Wang N, Planus E, Pouchelet M, Fredberg JJ, Barlovatz-MeG. Urokinase receptor mediates mechanical force transfer athe cell surface. Am J Physiol 1995;268(4.1):1062–6.

[84] Pankhurst QA, Connoly, Jones SK, Dobson J. Applications of mnetic nanoparticles in biomedicine. J Phys D 2003;36:R167–8

[85] Wang N, Ingber DE. Probing transmembrane mechanical couand cytomechanics using magnetic twisting cytometry. BiocCell Biol 1995;73(7–8):327–35.

[86] Babincova M, Sourivong P, Leszczynska D, Babinec P. Bspecific whole body electromagnetic hyperthermia. Med Hy2000;55:459–60.

[87] O’Connor DT, Choi MG, Kwon SY, Paul, Sung KL. Neinsight into the mechanism of hip prosthesis loosening: eof titanium debris size on osteoblast function. J Orthopaed2004;22(2):229–36.

[88] Labhaesetwar V, Bonadion J, Goldstein SA, Levy RJ. Gene tfection using biodegradable nanospheres: results in tissue cand a rat osteotomy model. Colloids Surf 1999;16:281–90.

[89] Prahba S, Zhou W, Panyam J, Labhaesetwar V. Size-dependenanoparticle-mediated gene transfection: studies with fractionanoparticles. Int J Pharmaceut 2002;244(1–2):105–15.

[90] Desai MP, Labhasetwar V, Walter E, Levy RJ, Amidon GI.mechanism of uptake of biodegradable microparticles in Cacells is size dependent. Pharmaceut Res 1997;14(11):1568–

[91] Foster KA, Yazdanian M, Audus KL. Microparticulate uptamechanisms if in-vitro cell culture models of the respiratory eplium. J Pharm Pharmacol 2001;53(1):57–66.

endocytosis into macrophage surface-connected compartmenJ Cell Biol 1999;78(2):91–9.

[103] Panyam J, Zhou WZ, Prabha S, Sahoo SK, LabhasV. Rapid endolysomal escape of poly(d,l-lactide-co-glycolidenanoparticles: implications for drug and gene delivery. FASE2002;10:1217–26.

[104] Panyam J, Labhasetwar V. Biodegradable nanoparticles forand gene delivery to cells and tissue. Adv Drug Deliv2003;55(3):329–47.

[105] Cartmell SH, Dobson J, Verschueren S, El Haj A. Developmof magnetic particle techniques for long-term culture of bonewith intermittent mechanical activation. IEEE Trans Nanobi2002;1:92–7.

[106] Dobson J, Keramane A, El Haj AJ. Theory and applicationsmagnetic force bioreactor. Eur Cells Mater 2002;4(Suppl. 2):13

[107] El Haj AJ, Cartmell SH. Mechanical bioreactors for tissue eneering. In: Chaudhuri JB, Al Rubeai M., editors. BioreactorsTissue Engineering. Dordrecht: Kluwer Academic Publisherspress.

[108] Guldberg RE, Coldwell NJ, Guo XE, Goulet RW, HollisSJ, Goldstein SA. Mechanical stimulation of tissue repaithe hydraulic bone chamber. J Bone Miner Res 1997;12:1302.

[109] Puig-De-Morales M, Grabulosa M, Alcaraz J, Mullol J, MaksymFredburg JJ, et al. Measurement of cell microrheology by magtwisting cytometry with frequency domain demodulation. J APhysiol 2001;91:1152–9.