oaches to studying

ineering, National University of Singapore, 9 Engineering Drive 1,

576, SingaporeDrive 1, Singapore 117576, Singapore

is the availability of biophysical and nanotechnological

Review TRENDS in Biotechnology Vol.25 No.3

Glossaryandmechanical properties of cells as well as from abnormalmechanotransduction (see Glossary) [1,2]. This not onlygives rise to the breakdown of physiological functions indiseasedstates, it alsodisrupts orderegulates themolecularmechanisms by which cells sense mechanical signals andconvert them into biochemical responses. Some examples ofsuch diseases are arthritis, asthma, cancer, elliptocytosis,malaria, sickle cell anemia and spherocytosis (Table 1).

Nanobiomechanics an emerging topic of researchinterest involves the study of the mechanics of livingcells and biomolecules and their connections to humandiseases [3]. One reason for the emergence of this research

Biorheological: relating to the deformation and flow of cells and biological

fluids in a physiological environment.

Creep behavior: a time-dependent deformation behavior of a body as it

attempts to relieve itself of an applied stress.

Displacement resolution: smallest division of distance measurable.

Mechanotransduction: mechanism by which cells convert mechanical stimuli

into biochemical responses.

Merozoites: daughter protozoan cells that result from the asexual division of

parasitic sporozoan cells during their life cycle.

Rheoscope: instrument using fluid sheer stress to measure the deformability of

RBCs.

Shear elastic modulus: parameter that provides a measure of the resistance of

a body to shearing or twisting.

Schizonts: late-stage malaria-infected RBCs.

Trophozoites: mid-stage malaria-infected RBCs.

Youngs modulus: also known as elastic modulus, it is a parameter that

provides a measure of the resistance of a material to elongation or

compression.Corresponding author: Lim, C.T. (ctlim@nus.edu.sg).Available online 25 January 2007.

www.sciencedirect.com 0167-7799/$ see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2007.01.005of awide range of humandiseases have suggested that theiretiologymighthaveresulted fromdeviation in thestructuralsiological outcomes. Recent studies on the pathophysiologyBiomechanics apprhuman diseasesGabriel Y.H. Lee2 and Chwee T. Lim1,2,3

1Division of Bioengineering and Department of Mechanical EngSingapore 117576, Singapore2SingaporeMIT Alliance, 4 Engineering Drive 3, Singapore 1173NUS Nanoscience & Nanotechnology Initiative, 9 Engineering

Nanobiomechanics has recently been identified as anemerging field that can potentially make significantcontributions in the study of human diseases. Researchinto biomechanics at the cellular and molecular levels ofsome human diseases has not only led to a betterelucidation of the mechanisms behind disease pro-gression, because diseased cells differ physically fromhealthy ones, but has also provided important knowl-edge in the fight against these diseases. This articlehighlights some of the cell and molecular biomechanicsresearch carried out on human diseases such as malaria,sickle cell anemia and cancer and aims to provide furtherimportant insights into the pathophysiology of suchdiseases. It is hoped that this can lead to new methodsof early detection, diagnosis and treatment.

Nanobiomechanics and its connections to humandiseasesHumandiseasecanbedefinedasacondition, stateorprocessoccurring in our body that not only impairs our bodilystructures and functions but also threatens our healthand well-being. Every disease is unique and can vary insymptoms, signs and outcomes. Disease not only causesbiological and functional alterations but also results inabnormalities in the physical and structural characteristicsof cells. Current research on diseases mainly focuses on themolecular, microbiological, immunological and pathologicalaspects, rather than on the mechanical basis, which mightmake direct contributions to the symptoms and pathophy-tools that can mechanically probe cells and biomoleculesin their physiological states at forces and displacementresolutions at the piconewton and nanometer scales,respectively. Such measurements were previously notachievable. Some of the recently developed biophysicaland nanotechnological tools and experimental techniquesinclude atomic force microscopy (AFM), molecular forcespectroscopy, cytoindenter, flow cytometry, magnetictwisting cytometry, microfluidics, magnetic tweezers,microplate manipulation, optical tweezers or laser traps,and optical stretcher (see [4,5] for reviews). This hasfacilitated quantitative experimental and computationalstudies at the nano- and microscale on how the mechanicalproperties of a cell can be altered by the organizational ormolecular changes occurring within the cell that can leadto or can arise from human diseases [1] (Table 1).

Studying human diseases from a biomechanicsperspective can lead to a better understanding of thepathophysiology and pathogenesis of a variety of humandiseases because changes occurring at the molecular andcellular levels will affect, and can be correlated to, changesoccurring at the macroscopic level. This will provide analternative and better approach to assess the onset orprogression of diseases as well as to identify targets fortherapeutic interventions. This review article will high-light some of the research in cell and molecular biomecha-nics being performed to study three types of diseases that

manifest structural and mechanical property changes:malaria, sickle cell anemia and cancer.

Role of mechanics in the pathophysiology of humandiseasesMalariaHealthy red blood cells (RBCs) are highly deformable theytransport oxygen to various parts of the body by squeezingtheir way through narrow capillaries. From a mechanisticperspective, two important outcomes in thepathophysiologyof malaria (Box 1) are the increased rigidity and cytoadher-ence (stickiness) of infected RBCs. These not only causeserious impairment of blood flow but also result in severeanemia, coma or even death. Thus, investigating how aninfectedRBCundergoes extensivemolecular and structural

infected RBCs [12]. Two proteins the knob-associatedhistidine rich protein (KAHRP) and the Plasmodiumfalciparum erythrocyte membrane protein 3 (PfEMP3) are known to have been exported from the parasite to theRBC membrane during infection. In 2002, Glenister et al.[13] used micropipette aspiration to study the change inthe shear elastic modulus of RBCmembranes infectedwithtransgenic parasites (with either of the KAHRP or PfEMP3genes deleted) to determine the contribution of these

Box 1. Malaria

Malaria is one of the most severe parasitic diseases on earth. Itdwarfs all other infectious diseases in that it infects 350500 million and results in 1.33 million deaths each year [54].Malaria arises from the protozoan vertebrate blood parasites of thegenus Plasmodium and is transmitted by the female Anophelesmosquito. Of the four Plasmodium strains of the malarial parasite( falciparum, vivax, ovalae and malariae), the Plasmodium falcipar-um strain accounts for 80% of all known cases and 95% of themalaria-associated mortalities [55].

In the event of an infection by the malarial parasite, the followingchanges take place in a RBC: decreased deformability and mechan-ical and rheological property changes; export of parasite proteins tothe surface membrane; development of cytoadherent and rosettingproperties, resulting in the sequestration of RBCs containing late-stage trophozoites and schizonts in deep vascular beds; anddigestion of hemoglobin. When a malarial parasite invades and

further export of parasite proteins to the cell surface membrane. Asa result, distortion of the cell cytoskeleton and membrane occurs,and the infected RBC becomes more spherical than biconcave. Atthe end of the schizont stage, which is 48 h after invasion, themerozoites break out of the cell and begin to invade other healthyRBCs. Owing to the extensive cell modification caused by theparasite, as well as the direct specific interaction of the exportedparasite proteins with the membrane and spectrin network of theRBC, the cell becomes stiff and sticky [56]. This contributes to thepathophysiology of malaria, which can result in blood clogging,anemia, coma or even death [58].

ical techniques and tools for detecting them

hysical techniques and tools Refs

opipette aspiration [65]

cytometry [66]

cal magnetic twisting cytometry, traction microscopy [67]

ic force microscopy, optical stretcher, scanning acousticoscopy

[3537,47,68]

cytometry [66]

amic flow adhesion assay [69]

ometer, rheoscope, laminar flow assay, micropipetteration, optical or laser tweezers, microfluidics

[4,715,18,19]

cal or laser tweezers, micropipette aspiration [2730]

r diffraction viscometer [70]

112 Review TRENDS in Biotechnology Vol.25 No.3changes and how this eventually contributes towards theincreased stiffness and cytoadherence will be important inthe understanding of the pathophysiology of malaria [6].

Research into the biomechanics of malaria was firstperformed in 1971 byMiller et al., who studied suspensionsof monkey RBCs infected with two different strains of themalarial parasites, using a viscometer and cell-filtrationtechnique [7,8]. Their results were the first to indicate thatinfection by the Plasmodium parasites could impair thedeformability of RBCs and, hence, the biorheological prop-erties of blood. Progressing from looking at a suspension ofcells to observing the deformability of an individual cell,Cranston et al. [9], in 1984, used a rheoscope to study thedeformability of individual malaria-infected RBCs atdifferent stages of infection. Similarly, Suwanarusket al. [10] used a laminarshear flow system to evaluatethe deformability of RBCs at different stages of infectioncaused by the Plasmodium falciparum and Plasmodiumvivax malarial parasites.

The use of the micropipette aspiration technique [4](Figure 1) and optical tweezers [4] (Figure 2) enables moreaccurate probing of the mechanical response of singleinfected cells down to piconewton and micrometer resol-utions. Nash et al. [11], in 1989, were the first to usemicropipette aspiration to study the abnormalities of RBCsinfected withPlasmodium falciparum at different stages ofinfection. Subsequently, the micropipette aspiration tech-nique was used in 1993 to evaluate the effect of differentstrains of the malarial parasite on the deformability of

Table 1. Some human diseases and the accompanying biophys

Human disease Pathophysiological outcomes Biop

Arthritis Chondrocyte stiffening andincreased viscosity

Micr

Asian ovalocytosis Erythrocyte stiffening Flow

Asthma Airway smooth-muscle cellstiffening and contracting

Opti

Cancer Epithelial and fibroblast cells softening andmetastasis

Atommicr

Cryohydrocytosis Erythrocyte stiffening Flow

Elliptocytosis Erythrocyte stiffening andincreased adherence

Dyn

Malaria Erythrocyte stiffening and cytoadherence Viscaspi

Sickle cell anemia Erythrocyte stiffening andincreased viscosity

Opti

Erythrocyte stiffening and LaseSpherocytosisincreased adherence

www.sciencedirect.commatures within a RBC, the cell becomes stiff and cytoadherent(sticky). The erythrocytic stage of malaria affects human RBCs and isassociated with the pathophysiology and pathogenesis of thedisease. The erythrocytic developmental stage of the parasitebegins with the ring stage at 30 min after invasion of a parasiteinto a RBC [5658]. At 20 h after invasion, it develops into atrophozoite, where the parasite continues to grow inside the RBCwhile exporting parasite proteins to the surface membrane of thecell. At 25 to 40 h after invasion, it finally develops into a late-stageschizont, where the nuclear division of the parasite results in theproliferation of between 12 to 20 (or more) merozoites as well as

proteins to the increased rigidity of infected RBCs. Resultsshowed that the removal of either protein reduced therigidity of the membrane of the parasitized cell. Thisexperiment demonstrates how the contribution of specificparasite-exported proteins to the stiffening of Plasmodium

falciparum-harbored RBCs can be determined quantitat-ively. Certainly, the effects of other parasite-exportedproteins, as well as the mechanisms by which theseproteins stiffen the cell membrane, still need to be furtherstudied. In addition, optical tweezers were used, in 2004, to

Figure 1. (a) Schematic diagram of the micropipette aspiration of a cell. This technique uses a suction pressure, DP, to aspirate a single cell, wholly or partially, into a

micropipette of inner radius, RP, and measures the cell elongation, L, as a function of suction pressure to determine the elastic shear modulus, G, of the cell membrane

using the relation.

L

RP kDP

G

where k is a constant whose value depends on the mechanical model used in analyzing the experimental results. (b) An optical image showing the micropipette aspiration

of a RBC. Figures reprinted from [4], Copyright 2006, with permission from Elsevier.

Review TRENDS in Biotechnology Vol.25 No.3 113Figure 2. (a) Schematic diagram showing an example of the stretching of a single RB

attached diametrically across the RBC, as shown in the optical image, with the left bead

radiation pressure from a focused beam of laser gives rise to a force that could physically

force is due to gradients in light intensity. The RBC is subsequently stretched by moving

to large deformation. The optical tweezers technique is capable of applying forces in the

with permission from Elsevier.

www.sciencedirect.comC using an optical tweezers setup. (b) Two silica microbeads are non-specifically

anchored to the glass slide. (c) A laser beam is used to trap the right bead as the

trap, control and manipulate small particles such as microbeads. The origin of this

the glass slide to the left. The optical image shows the stretching of a healthy RBC

piconewton range. Figures in (b) and (c) were reprinted from [17], Copyright 2004,

perspective, we can provide useful information to clinicianson how they can better reduce parasite virulence. This willalso assist in developing testing strategies that can quan-titatively evaluate the effectiveness of drugs being devel-oped to prevent or inhibit stiffening and cytoadherence ofinfected RBCs.

Sickle cell anemiaSickle cell anemia is a hereditary blood disorder that givesrise to blood circulatory problems due to an alteration in themolecular structure of hemoglobin (Box 2). Affected RBCstake the shape of a curved sickle and becomemore rigid andsticky. The mechanical properties of sickle-shaped RBCshave been probed using optical tweezers and micropipetteaspiration. RBCs obtained from patients with sickle cellanemia are found to be stiffer and more viscous comparedwith healthy RBCs [2730]: instead of moving through thebloodstream with great ease, these stiffer sickle-shapedRBCs end up clogging blood vessels and hence deprivetissues and organs of oxygen. According to Brandao et al.[27] and Itoh et al. [31], because sickle cell anemia affectsindividual RBCs, the key advantage of using the micropip-ette aspiration and optical tweezers techniques is that theyenable the study of individual diseased cells as comparedwith the average cell mechanical properties obtained from a

114 Review TRENDS in Biotechnology Vol.25 No.3measure the mechanical property differences betweenhealthy and infected RBCs at different stages of infection[14,15]. The stiffness of the infected RBCs was found toincrease as the disease progresses. In all of these works,the researchers have focused on the changes in the shearelastic modulus of the membrane of infected RBCs, basedon a RBC membrane model [16,17].

More recently, Shelby et al. [18] and Cheng et al. [19]were among the first to use microfluidics to investigatewhether the rigidified infected RBCs can block capillariesthat have diameters smaller than those of the infectedRBCs (Figure 3). Microfluidics enables the observation ofthe rheological behavior of individual cells flowing throughmicrofluidic devices with micrometer-scale channels andchambers [20]. In 2003, Shelby et al. qualitatively showedthat late-stage schizonts can cause blockages in narrowelastomeric channels, which were used to mimic thenarrow capillaries found in the human body.

Although much research has been focused oninvestigating the stiffening of malaria-infected RBCs,relatively less research has been done to quantify the

Figure 3. (a) Schematic diagram showing the geometry of a elastomeric

microchannel used to study the flow behaviour of different stage malaria-

infected RBCs with the width (w) of the microchannel sized at 2, 4, 6 and 8 mm,

and height of the microchannel fixed at 2 mm. The arrow points to the entrance of

the microchannel and indicates the flow direction in general. (b) An optical image

of a healthy RBC squeezing through the entrance of the elastomeric microchannel

depicted in (a), with w = 2 mm. (c) The optical image shows the stiffer late-stage

schizonts clogging the entrance of the elastomeric microchannel depicted in (a)

with w = 2 mm.physical adhesion involved in cytoadherence (stickiness).Studies have revealed that knob-like structures mani-fested on the infected cell membrane contain parasite-exported proteins that form the focal adherent points,onto which the infected cell sticks to the endothelialcells lining the blood vessels and capillaries. Someligandreceptor pairs involved in cytoadherence havebeen identified [21], but the contributions of each specificcytoadherent binding-protein has not been well quanti-fied. Almost all cytoadherence is mediated by the inter-action of the parasite protein PfEMP1 [22] with variousendothelial receptor molecules, such as CD36 [23,24],intercellular-adhesion molecule 1 (ICAM-1) [25], E-selec-tin, and vascular cell-adhesion molecule 1 (VCAM-1) [26].Thus, more quantitative measurements of the interactionforce between the cytoadherent binding-proteins and thereceptor molecules of the endothelial cells need to beperformed.

These studies demonstrate the relevance ofbiomechanics in studying malaria. With a better under-standing of malaria pathophysiology from a biomechanics

www.sciencedirect.comcell population. This enables direct comparisons between ahealthy and a diseased cell.

In addition, Ballas et al. [32] and Brandao et al. [27]studied the effects of hydroxyurea, an anti-tumor drugcommonly used in the treatment of sickle cell disease. Theyfound that the deformability of RBCs in sickle cell anemiapatients undergoing hydroxyurea treatment for at least sixmonths is almost identical to that of healthy RBCs. Thissuggests that cellular elasticity might also be used as a

Box 2. Sickle cell anemia

Sickle cell anemia, an inherited blood disorder, is a result of agenetic error occurring in the hemoglobin molecules. An autosomalrecessive gene causes the formation of an abnormal hemoglobin(hemoglobin S [HbS]) that results in deformed RBCs. Major changesin cellular mechanics occur upon deoxygenation, when the HbSpolymerizes. The deformed RBCs are rigid and come in the form of acurved sickle or crescent shape. Because these rigid sickle-shapedRBCs have difficulty flowing through the small blood vessels andcapillaries in the body, this gives rise to blood circulatory problems,which deprive tissues and organs of oxygenated blood. The RBCsalso rupture more easily and are removed from circulation by thespleen.

Millions of people throughout the world are affected by sicklecell anemia. Each year, an estimated 120 000 infants are born withthe disease in Africa (http://www.cwru.edu/med/epidbio/mphp439/Sickle_Cell_Disease.htm), whereas in the USA 72,000 people areaffected (http://www.nhlbi.nih.gov/health/dci/Diseases/Sca/SCA_Summary.html). Some serious consequences of sickle cell anemiainclude stroke and infection. Pneumonia, however, remains theleading cause of death in children born with this disease; hence,early screening and diagnosis in infancy is vital for preventingdeath in children born with this type of hereditary blood disorder.

Nevertheless, a person with the sickle cell trait (carrier) is knownto demonstrate an increased resistance to malaria. First, in such acarrier, the malarial parasite can easily rupture the RBC and is,therefore, unable to reproduce. Second, owing to the polymeriza-tion of hemoglobin the parasite is unable to digest it. Therefore, inmalaria-prone areas, the chance of surviving is actually higher for

people carrying the sickle cell trait.

Review TRENDS in Biotechnology Vol.25 No.3 115potential drug-response indicator to monitor the effective-ness of drugs used in the treatment of diseases. Never-theless, for carriers and patients, early diagnosis isstill important in providing preventive care or propertreatment for some of the devastating complications aris-ing from this disease.

CancerCancer can be characterized by the uncontrolled division ofabnormal cells, which spread to various parts of the bodyand infiltrate and destroy healthy body tissue (Box 3). In thefight against cancer, complications in surgical treatment,and the inefficient and non-specific action, as well as theunwanted side effects, of chemotherapeutic drugs have beenmajor hurdles for progress [33]. One pathophysiologicaloutcome of cancer is that the affected cells aremore deform-able than non-malignant cells [3436]; therefore, the differ-ence in cell deformability can be exploited to distinguishcancer cells from healthy cells. Another outcome is thecapacity of malignant cancer cells and tumors to infiltrate,invade or metastasize to distant sites. Currently, there islittle understanding of how changes in the biomechanical

Box 3. Cancer

Cancer is currently one of the leading causes of death worldwide,with >11 million cases and 7 million deaths attributed to it each year(http://www.who.int/cancer/en/). It is estimated that by 2020 therewill be 15 million new cancer cases expected anually [59]. Canceris a disease that results from a rapid, unrestricted and uncontrolledproliferation of abnormal cells, which possess increased deform-ability and adhesion. Cancer occurs when the genes responsible forcellular growth and repair undergo changes. To effectively treat orcure for cancer, there is a need to be able to detect the presence ofcancer cells at the earliest stage possible. This will help to preventthem from spreading to other parts of the body in a process calledmetastasis (Box 4). When cancer is detected at an early stage, theodds of curing it are normally high.

Although current diagnostic approaches have enabled the detec-tion of cancer, they are still not sensitive enough to quickly, reliablyand accurately detect the presence of cancer at an early stage [60]. Inmost cases, cancer is not diagnosed or detected until the cancercells have begun to metastasize or migrate; and metastasis is themajor cause of death due to cancer.properties of cancer cells and tumors can contribute tocancer metastasis.

The use of AFM in cell andmolecular biology is emergingas apowerful nanobiomechanical probebecause of its abilityto function as a high-resolution topographical imaging tooland a force sensor with piconewton resolution [4]. One keyadvantageof theAFMis its inherentability to carry out real-time measurements on biological samples in their physio-logical conditions. Lekka et al. [37] used theAFM, in1999, todetermine the elasticity of normal and cancerous humanbladder epithelial cells; they found the Youngs modulus ofcancer cells to be about one-tenth that of healthy cells.

The optical stretcher is a recently developed tool tostudy the deformation of single suspended cells[36,38,39] (Figure 4). One advantage of this technique isthat the entire cell surface is acted upon by a distribution ofthe laser-induced forces, thus revealing the overall mech-anical property of the cell [36]. This is in direct contrast tosome of the other techniques, such as the micropipetteaspiration [40,41], optical tweezers [4] and AFM [4245],

www.sciencedirect.comwhich involve the application of forces at localized areas ofthe cell surface. However, because the optical stretcherenables the testing of cells in suspension, adherent celltypes are not tested in their physiological state.

In 2005, Wottawah et al. [36] studied the creep behaviorof individual suspended fibroblasts using the opticalstretcher, and found that cancerous mouse fibroblast cellswere 50% more deformable than healthy cells. Thisincrease in deformability is consistent with the results ofearlier works carried out on cancer cells usingmicropipetteaspiration and AFM techniques [37,46]. In a separatestudy, Lincoln et al. [47] used the optical stretcher toinvestigate the malignant transformation of human breastepithelial cells. They found that cancer cells stretchapproximately five times more than healthy cells, andmetastatic cancer cells stretch about twice as much asnon-metastatic cancer cells. Moreover, the opticalstretcher technique can be easily incorporated into amicro-fluidic device to produce a high-throughput flow-cytometricsystem that is capable of sorting and trapping cells withoutintruding into their underlying biological functions so thatfurther analysis and therapeutic measurements can bemade. The optical stretcher is an example of a biomecha-

Figure 4. Schematic diagram showing the axial stretching of a cell using an optical

stretcher. The cell is trapped by the optical forces from two divergent laser beams,

and is subsequently stretched along the laser beam axis when a higher laser power

is applied. The optical stretcher makes use of the fact that the total force acting on a

dielectric object is zero, with surface forces being additive when it is placed

between two divergent laser beams. This results in a force pulling on either side of

the cell, thus stretching it. Figure adapted from [38], Copyright 2001, with

permission from the Biophysical Society.nical characterization tool that might be useful in thediagnosis of cancer because it can potentially detectthe disease by measuring how deformable cells are, giventhe fact that cancer cells are, in general, more deformablethan their non-cancerous counterparts.

Metastasis (Box 4) is the process whereby cancer cellsspread from their primary site to other parts of the body. Itinvolves the breaking away of cancer cells from a primarytumor, penetration into blood vessels and then settlingand growing in a normal tissue at a distant site (metas-tasize) in the body (Figure I). Because metastasis is thepredominant cause of death due to cancer, it is importantto understand the detailed mechanisms involved so thatstrategies can be developed to keep the disease undercontrol. Although some work on the mechanics of tumorcell migration has been done by Dong et al. [48,49], morework is still needed to better elucidate the detailed mech-anisms involved in the metastasis of cancer cells. Theseinclude the abnormal receptor-mediated adhesion ofcancer cells to the extracellular matrix, breaching of

116 Review TRENDS in Biotechnology Vol.25 No.3Box 4. Invasion and metastasis in cancer

During metastasis, cancer cells penetrate into blood vessels, lympha-basement membranes, intravasation of cancer cells intothe circulation, interaction with lymphoid cells and plate-lets to form tumor-cell emboli, rheology of the tumor-cellemboli in the circulation, arrest and adhesion of tumor

tics and body cavities, thus enabling the cancer to spread (Figure I).Metastasis is initiated by the down-regulation of E-cadherin expres-sion: E-cadherins are adhesion molecules that keep epithelial cellstogether. The cancer cells then lose their ability to adhere to eachother, become detached from the primary tumor, and begin to invadethe surrounding tissues. Hence, at the site of the primary tumor, acarcinoma will first breach the underlying basement membrane,traverse the interstitial connective tissue and penetrate the vascularbasement membrane, before gaining access to circulation.

To penetrate into the surrounding extracellular matrix (ECM), tumorcells must be able to adhere to it. Some studies indicate that receptor-mediated attachment of tumor cells to laminin and fibronectin isnecessary for invasion and metastasis. Normal (polarized) epithelialcells are known to express receptors with high-affinity for thebasement membrane laminin on their basal surface. However, somecancer cells express many more receptors and integrins, which aredistributed throughout the cell membrane and serve as receptors forthe various components of the ECM. In fact, there seems to be acorrelation between the density of these receptors and the invasive-ness of cancer [61]. After attaching to components of the basement

Figure I. Cancer metastasis. Schematic diagram showing the different stages whereby

modified from [61], Copyright 2005, with permission from Elsevier.

www.sciencedirect.commembrane or interstitial ECM, tumor cells then create passagewayscells in capillary beds at distant sites, and extravasationthrough the vascular basement membrane. Specific andeffective therapies can then be developed to target thesemigratory cancer cells or disrupt the metastatic process.

and migrate towards the vascular basement membrane. Once thevascular basement membrane is breached, and cancer cells intrava-sate into the circulation, some tumor cells will aggregate and travel inclumps as emboli.

For extravasation of tumor cell emboli (egress of cancer cellsthrough the vascular basement membrane) to occur at distant sites,tumor cells must first be arrested at and adhere to the capillary beds.During the extravasation process, this will, again, involve adhesionmolecules such as integrins and laminin receptors and proteolyticenzymes. Normally, these tumor cells might contain adhesionmolecules, the ligands of which are expressed preferentially on theendothelial cells of the distant target organs [62]. Alternatively,chemokines can also have an important role in determining thetarget sites. For example, cancer cells express the chemokinereceptors CXCR4 and CCR7, which have affinity for the chemokinesthat are highly expressed in the tissues and sites where breast cancercommonly metastasizes [63]. Finally, chemoattractants might also bereleased by some target organs; these subsequently recruit cancercells to their sites. At the new site, tumor cells will begin to proliferateand develop a vascular supply while evading the host defences [64].

cancer cells spread from a primary tumor site to a distant site in the body. Figure

red blood-cells caused by Plasmodium falciparum. Blood 74, 855861

mechanical properties of malaria-infected red blood cells. Blood 99,

Review TRENDS in Biotechnology Vol.25 No.3 117Potential role in the detection and diagnosis of humandiseasesThe research work reviewed here suggests thatbiomechanics can, indeed, play an important role inbetter understanding the pathophysiology of a variety ofdiseases. The knowledge obtained can also be useful in thedevelopment of new and improved assays and diagnosticdevices and techniques that are not only sensitive enoughin the early detection of diseases but are also highlyaccurate, even when the symptoms or signs of the diseasesare hardly discernable. This is particularly needed fordiseases where early diagnosis and detection are crucialfor their prevention and control. For example, cell deform-ability, which can be determined using appropriate mech-anical probes, could be used as a potential biologicalmarker in the detection and diagnosis of human diseasesbecause the change in the cellular mechanical propertiescan quantitatively reflect their diseased states. This emer-ging field of research can also assist in the development ofsuitable testing strategies that can evaluate the efficacy ofcertain agents and drugs being developed to prevent ortreat some of the diseases.

Future directionsThe use of biomechanics approaches to studying humandiseases is still in its infancy, and there are many out-standing questions that need to be addressed. For example,human diseases can be caused by hereditary factors,changes in the internal physiological condition or invasionby foreign organisms such as viruses and parasites; there-fore, how do these factors induce the cellular andmolecularchanges that eventually lead to structural and biomecha-nical property changes in the cells, such as cell adhesion,cell elasticity, motility and rheology? Furthermore, how dochanges in the structural and biomechanical properties incells eventually lead to diseases?

To address the above questions, newer and noveltechniques need to be developed. Although powerfulstate-of-the-art tools exist to probe human diseases atthe cellular and molecular levels, as described in thisarticle, they are, nevertheless, tedious and difficult touse. There is a need to develop techniques and devicesthat can mechanically characterize not only individualcells but also populations of cells more rapidly and withenhanced sensitivity and accuracy. Also, because cells arefrequently subjected to multiple cues in addition to bio-chemical and mechanical stimuli in their physiologicalenvironment, devices and systems must be designed sothat they can also present cells with these cues and stimuliin a controlled and reproducible way. Thus, an integratedhigh-throughput approach will be needed that must beable to perform the following tasks: accurately manipulateand handle small sample volumes of bodily fluids and cells;provide the necessary controlled stimuli; and incorporatehigh-resolution techniques that can analyze the functionsand gene expression in these cells. Emerging technologies,such as laboratory-on-chip or micro total analysis system(mTAS) [5052], could be used to integrate and automateall the necessary processes, to provide for the rapid, sensi-

tive and effective analysis of diseased cells in their phys-iological environment. These will not only lead to the

www.sciencedirect.com1060106314 Mills, J.P. et al. (2004) Nonlinear elastic and viscoelastic deformation of

the human red blood cell with optical tweezers.Mech. Chem. Biosyst. 1,169180

15 Suresh, S. et al. (2005) Connections between single-cell biomechanicsand human disease states: gastrointestinal cancer and malaria. ActaBiomater. 1, 1530

16 Dao, M. et al. (2003) Mechanics of the human red blood cell deformedusing optical tweezers. J. Mech. & Phy. Solids 51, 22592280 (Also seeErrata at J. Mech. & Phy. Solids 53, 493494 (2005).)

17 Lim, C.T. et al. (2004) Large deformation of living cells using lasertraps. Acta Mater. 52 (7), 18371845 (Also see Corrigendum at ActaMater. 52, 40654066 (2004).)

18 Shelby, J.P. et al. (2003) A microfluidic model for single-cell capillaryobstruction by Plasmodium falciparum-infected erythrocytes. Proc.Natl. Acad. Sci. U. S. A. 100, 1461814622

19 Cheng, T.M. et al. (2005) Microrheology study of malaria infected12 Paulitschke, M. and Nash, G.B. (1993) Membrane rigidity of red bloodcells parasitized by different strains ofPlasmodium falciparum. J. Lab.Clin. Med. 122, 581589

13 Glenister, F.K. et al. (2002) Contribution of parasite proteins to altereddevelopment of new, novel and inexpensive medical diag-nostic devices and techniques but also enable the minia-turization and integration of sophisticated toolsand complex processes on a chip; hence, making themaccessible to the developing world, where some of thesediseases are rampant [53].

Finally, for such research to succeed, we will need todevelop close multi-disciplinary collaborations not onlyamong biologists, biochemists, life scientists and cliniciansbut also with engineers and physicists. Ultimately, it ishoped that this effort will lead to a better understanding ofdiseases, provide alternativemeans to assess their onset orprogression and assist in developing better treatment, oreven aid in their prevention.

AcknowledgementsThe support provided by the Singapore-MIT Alliance is gratefullyacknowledged.

References1 Lim, C.T. et al. (2006) Mechanical models for living cells a review.J. Biomech. 39, 195216

2 Ingber, D.E. (2003) Mechanobiology and diseases ofmechanotransduction. Ann. Med. 35, 564577

3 Fitzgerald, M. (2006) Ten emerging technologies: nanobiomechanics.Technol. Rev. 109, 67

4 Lim, C.T. et al. (2006) Experimental techniques for single cell andsingle molecule biomechanics. Mat. Sci. Eng. C 26, 12781288

5 Van Vliet, K.J. et al. (2003) The biomechanics toolbox: experimentalapproaches for living cells and biomolecules.ActaMater. 51, 58815905

6 Lim, C.T. (2006) Single cell mechanics study of the human diseasemalaria. J. Biomech. Sci. & Engrg. 1, 8292

7 Miller, L.H. et al. (1971) Alteration in the rheologic properties ofPlasmodium knowlesi-infected red cells. A possible mechanism forcapillary obstruction. J. Clin. Invest. 50, 14511455

8 Miller, L.H. et al. (1972) Decreased deformability of Plasmodiumcoatneyi-infected red cells and its possible relation to cerebralmalaria. Am. J. Trop. Med. Hyg. 21, 133137

9 Cranston, H.A. et al. (1984) Plasmodium falciparum maturationabolishes physiologic red cell deformability. Science 223, 400403

10 Suwanarusk, R. et al. (2004) The deformability of red blood cellsparasitized by Plasmodium falciparum and P. vivax. J. Infect. Dis.189, 190194

11 Nash, G.B. et al. (1989) Abnormalities in the mechanical properties oferythrocytes flowing through microfluidic channel. Proceedings ofthe 12th International Conference on Biomedical Engineering, p. 184

20 Huang, H. et al. (2004) Cell mechanics and mechanotransduction:pathways, probes, and physiology. Am. J. Physiol. Cell Physiol. 287,111

21 Cooke, B. et al. (2000) Falciparum malaria: sticking up, standing outand out-standing. Parasitol. Today 16, 416420

22 Magowan, C. et al. (1988) Cytoadherence by Plasmodium falciparum-infected erythrocytes is correlated with the expression of a family of

43 Mahaffy, R.E. et al. (2004) Quantitative analysis of the viscoelasticproperties of thin regions of fibroblasts using atomic force microscopy.Biophys. J. 86, 17771793

44 Rotsch, C. et al. (1999) Dimensional andmechanical dynamics of activeand stable edges in motile fibroblasts investigated by using atomicforce microscopy. Proc. Natl. Acad. Sci. U. S. A. 96, 921926

45 Rotsch, C. et al. (2000) Drug-induced changes of cytoskeletal structure

118 Review TRENDS in Biotechnology Vol.25 No.3variable proteins on infected erythrocytes. J. Exp. Med. 168, 13071320

23 Barnwell, J.W. et al. (1989) A human 88 kD membrane glycoprotein(CD36) functions in vitro as a receptor for a cytoadherence ligand onPlasmodium falciparum-infected erythrocytes. J. Clin. Invest. 84, 765772

24 Ockenhouse, C.F. et al. (1989) Identification of a platelet membraneglycoprotein as a falciparum malaria sequestration receptor. Science243, 14691471

25 Berendt, A.R. (1989) Intercellular-adhesion molecule-1 is anendothelial cell adhesion receptor for Plasmodium falciparum.Nature 341, 5759

26 Ockenhouse, C.F. et al. (1992)Human vascular endothelial cell adhesionreceptors for Plasmodium falciparum-infected erythrocytes: roles forendothelial leukocyte adhesion molecule 1 and vascular cell adhesionmolecule 1. J. Exp. Med. 176, 11831189

27 Brandao, M.M. et al. (2003) Optical tweezers for measuring red bloodcell elasticity: application to the study of drug response in sickle celldisease. Eur. J. Hematol. 70, 207211

28 Evans, E.A. and La Celle, P.L. (1975) Intrinsic material properties ofthe erythrocyte membrane indicated by mechanical analysis ofdeformation. Blood 45, 2943

29 Hochmuth, R.M. et al. (1979) Red cell extensional recovery and thedetermination of membrane viscosity. Biophys. J. 26, 101114

30 Nash, G.B. et al. (1984) Mechanical properties of oxygenated red bloodcells in sickle cell disease. Blood 63, 7382

31 Itoh, T. et al. (1995) Effects of hemoglobin concentration ondeformability of individual sickle cells after deoxygenation. Blood85, 22452253

32 Ballas, S.K. et al. (1989) Effect of hydroxyurea on the rheologicalproperties of sickle erythrocytes in vivo. Am. J. Hematol. 32, 104111

33 Lundstrom, K. and Boulikas, T. (2002) Breakthrough in cancertherapy: encapsulation of drugs and viruses. Curr. Drug Discov. 1923 November

34 Gast, F.U. et al. (2006) The microscopy cell (MicCell), a versatilemodular flowthrough system for cell biology, biomaterial research,and nanotechnology. Microfluid. Nanofluid. 2, 2136

35 Guck, J. et al. (2005) Optical deformability as inherent cell marker fortesting malignant transformation and metastatic competence.Biophys. J. 88, 36893698

36 Wottawah, F. et al. (2005) Characterizing single suspended cells byoptorheology. Acta Biomater. 1, 263271

37 Lekka, M. et al. (1999) Elasticity of normal and cancerous humanbladder cells studied by scanning force microscopy.Eur. Biophys. J. 28,312316

38 Guck, J. et al. (2001) The optical stretcher: a novel laser tool tomicromanipulate cells. Biophys. J. 81, 767784

39 Guck, J. et al. (2000) Optical deformability of soft biological dielectrics.Phys. Rev. Lett. 84, 54515454

40 Discher, D.E. et al. (1994) Molecular maps of red cell deformation:hidden elasticity and in situ connectivity. Science 266, 10321035

41 Hochmuth, R.M. (2000) Micropipette aspiration of living cells.J. Biomech. 33, 1522

42 Mahaffy, R.E. et al. (2000) Scanning probe-based frequency-dependentmicrorheology of polymer gels and biological cells. Phys. Rev. Lett. 85,880883www.sciencedirect.comand mechanics in fibroblasts: an atomic force microscopy study.Biophys. J. 78, 520535

46 Ward, K.A. et al. (1991) Viscoelastic properties of transformed cells:role in tumor cell progression and metastasis formation. Biorheology28, 301313

47 Lincoln, B. et al. (2004) Deformability-based flow cytometry.CytometryA 59A, 203209

48 Dong, C. et al. (2002) In vitro characterization and micromechanics oftumor cell chemotactic protrusion, locomotion, and extravasation.Ann.Biomed. Eng. 30, 344355

49 Dong, C. et al. (2005) Micromechanics of tumor cell adhesion andmigration under dynamic flow conditions. Front. Biosci. 10, 379384

50 Daw, R. et al. (2006) Lab on a chip. Nature 442, 36751 Whitesides, G.M. (2006) The origins and the future of microfluidics.

Nature 442, 36837352 El-Ali, J. et al. (2006) Cell on chips. Nature 442, 40341153 Yager, P. et al. (2006) Microfluidic diagnostic technologies for global

public health. Nature 442, 41241854 Campbell, N.A. and Reece, J.B. (2005) Biology, Addison Wesley

Longman, Inc.55 Engwerda, C.R. et al. (2005) The importance of the spleen in malaria.

Trends Parasitol. 21, 758056 Bannister, L.H. et al. (2000) A brief illustrated guide to the

ultrastructure of Plasmodium falciparum asexual blood stages.Parasitol. Today 16, 427433

57 Miller, L.H. et al. (2002) The pathogenic basis of malaria. Nature 415,673679

58 Cooke, B.M. et al. (2001) The malaria-infected red blood cell: structuraland functional changes. Adv. Parasitol. 50, 186

59 International Agency for Research on Cancer (2003) World CancerReport (B.W. Stewart and P. Kleihues, eds), World HealthOrganization.

60 Wulfkuhle, J.D. et al. (2003) Proteomic applications for the earlydetection of cancer. Nat. Rev. Cancer 3, 267275

61 Kumar, V. et al. (2005)Robbins and Cotran Pathologic Basis of Disease,(7th edn), Elsevier Saunders

62 Ruoslahti, E. (2002) Specialization of tumor vasculature. Nat. Rev.Cancer 2, 8390

63 Muller, A. et al. (2001) Involvement of chemokine receptors in breastcancer metastasis. Nature 410, 5056

64 Fidler, I.J. (2003) The pathogenesis of cancer metastasis: the seed andsoil hypothesis revisited. Nat. Rev. Cancer 3, 453458

65 Trickey, W.R. et al. (2000) Viscoelastic properties of chondrocytes fromnormal and osteoarthritic human cartilage. J. Orthop. Res. 18, 891898

66 King, M.J. et al. (2000) Rapid flow-cytometric test for the diagnosis ofmembrane cytoskeleton-associated haemolytic anaemia. Br. J.Haematol. 111, 924933

67 An, S.S. et al. (2006) Do biophysical properties of the airway smoothmuscle in culture predict airway hyperresponsiveness? Am. J. Respir.Cell Mol. Biol. 35, 5564

68 Lemons, R.A. and Quate, C.F. (1975) Acoustic microscopy: biomedicalapplications. Science 188, 905911

69 Wandersee, N.J. et al. (2004) Increased erythrocyte adhesion in miceand humans with hereditary spherocytosis and hereditaryelliptocytosis. Blood 103, 710716

70 Clark, M.R. et al. (1983) Osmotic-gradient ektocytometry:comprehensive characterization of red cell volume and surfacemaintenance. Blood 61, 899910

Biomechanics approaches to studying human diseasesNanobiomechanics and its connections to human diseasesRole of mechanics in the pathophysiology of human diseasesMalariaSickle cell anemiaCancer

Potential role in the detection and diagnosis of human diseasesFuture directionsAcknowledgementsReferences