Stroscio Michael A., Dutta Mitra. Biological Nanostructures and applications of Nanostructures in biology: electrical, mechanical and optical properties

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AKIL SETHURAMAN ET AL.

that would convert and couple optical signals to electrical signals on the nanoscale.

Clearly, such structures are likely to find applications in bioengineering.

10.

ADDITIONAL APPLICATIONS OF CARBON NANOTUBES

The use of CNTs as artificial muscles represent yet another possible, if not

immediate application.

These CNTs have abilities to convert electrical energy into

motion and can generate and withstand extremely high stresses. The use of CNTs as high

energy storage devices is another likely possibility that could be realized in the years to

come. CNTs have the extraordinary ability to adsorb hydrogen onto their surfaces and

adsorbed hydrogen is easier to pack densely than compressed hydrogen. Hydrogen may

also be packed inside CNTs. Potential uses would be in the field of fuel cell technology

where nanotubes are viewed as hydrogen storage devices.

The chemical sensing capabilities of CNTs are being exploited in the detection of

chemicals that are viewed as harmful pollutants. These tubes have extremely high surface

areas that help in easy adsorption of chemicals. Carbon nanothermometers employ the

use of liquid gallium inside nanotubes. The electrical properties of these nanotubes are

being researched and the use of CNTs as nanotweezers, infrared sensors and chemical

actuators have been proposed.

11.

CONCLUSION

CNTs possess extraordinary mechanical, optical and electrical properties, which

make them potentially useful for applications in fields ranging from electronics to

biotechnology. Standard manufacturing methods result in the formation of both metallic

and semiconducting nanotubes. A major concern has been the isolation of the metallic

ones from their semiconducting counterparts. The recent discovery of a means of sorting

nanotubes using a novel electrical technique has contributed to the solution of this

problem. Summarizing the biological applications of CNTs has been the main focus of

this chapter. The use of these nanotubes as sensors has drawn the attention of various

researchers and various methods to functionalize and solubilize these nanotubes have

been proposed. The remarkable mechanical properties of these nanostructures have been

exploited in the use of these tubes as tips for analytical techniques such as atomic force

microscopy and scanning probe microscopy. Moreover, these nanotubes may be used to

analyze organic samples without damaging them as they buckle if the force imparted

exceeds the critical value. Functionalization of tips with carboxyl and amine groups

would help in high precision mapping and imaging of sample surfaces. In the field of

neuroscience, they portend extensive use in the recording of electrical activity in neurons

and as substrates for neuronal growth. The recent discovery of CNTs with DNA sensing

capabilities has been a major step in the use of CNTs as biosensors. In order to enhance

the biosensing properties of CNTs, the tube surfaces have been successfully

functionalized using lipids and surfactants.

The transition from MEMS to NEMS

represents a major challenge. Just as with nanotechnology in general, the technological

revolution based on CNTs is in its infancy. If present trends continue, the ever increasing

potential for using CNTs in bioengineering will flourish in the decades to come.

CARBON NANOTUBES IN BIOENGINEERING

12.

ACKNOWLEDGEMENTS

We thank Omprakash Muppirala and Narenkumar Pandian for their help in preparing

Figure 3 and Figure 4.

13.

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NANOPHYSICAL PROPERTIES OF LIVING CELLS

The Cytoskeleton

Gregory Yourek, Adel Al-Hadlaq, Rupal Patel, Susan McCormick, Gwen-

dolen C. Reilly, Jeremy J. Mao*

INTRODUCTION

This chapter concerns the nanostructural and nanomechanical characteristics of the

cell membrane of bone marrow derived mesenchymal stem cells (MSCs) and how cell

membrane nanocharacteristics can affect cytoskeletal changes in response to flow-

induced shear stresses. The present approaches are motivated by recent advances in both

the fields of cellular micromechanics and cell-based tissue engineering. For example,

computational models and experimental evidence have converged to support the notion

that tissue-borne mechanical stresses outside the cell can be transmitted via the cytoskele-

ton to the cell nucleus.

1, 2

We have recently characterized the nanostructural and

nanomechanical properties of marrow-derived mesenchymal stem cells.

Further evi-

dence has suggested that exogenous mechanical stresses can upregulate and downregulate

an increasing amount of mechanosensitive genes.

4-8

On the tissue-engineering front,

adult MSCs have been differentiated into chondrogenic and osteogenic lineages and have

been encapsulated into two stratified layers in hydrogel polymers, leading to tissue-

engineered neogenesis of human-shaped articular condyle.

Despite these advances, a

fundamental aspect of mesenchymal stem cells has been understudied – their biophysical

characteristics in response to mechanical stresses. Because MSCs give rise to all skeletal

tissues such as bone, cartilage, skeletal muscle, tendons, and ligaments, mechanical

Gregory Yourek, Tissue Engineering Laboratory, Depts of Bioengineering and Orthodontics, Univ of IL at

Chicago, Chicago, IL 60612. Adel Al-Hadlaq, Tissue Engineering Laboratory, Depts of Anatomy and

Physiology and Orthodontics, Univ of IL at Chicago, Chicago, IL 60612. Rupal Patel, Tissue Engineering

Laboratory, Depts of Dentistry and Orthodontics, Univ of IL at Chicago, Chicago, IL 60612. Susan M.

McCormick, Depts of Bioengineering, Univ of IL at Chicago, Chicago, IL 60612. Gwendolen C. Reilly,

Tissue Engineering Laboratory, Depts of Bioengineering and Orthodontics, Univ of IL at Chicago, Chicago,

IL 60612. Jeremy J. Mao, Tissue Engineering Laboratory, Depts of Bioengineering and Orthodontics, Univ

of IL at Chicago, Chicago, IL 60612.

G.YOUREK

ET AL

modulation of MSCs is of paramount importance to their differentiation behavior.

Hence, there is much interest in investigating the effects of the nanostructural and

nanomechanical characteristics of MSCs on their differentiation and metabolism, using

combined biological and engineering approaches. In this chapter we describe our current

studies using stem cell culture and differentiation, atomic force microscopy, and applica-

tion of shear stresses. We hope that the present combined biological and bioengineering

approaches, will provide much needed insight into how adult stem cells are regulated by

micromechanical stresses toward purposeful differentiation pathways.

PHYSIOLOGICAL ASPECTS

2.1.

Human Bone Marrow Stromal Cells

Mesenchymal stem cells (MSCs), also known as bone marrow stromal cells

(BMSCs), or mesenchymal progenitor cells, are defined as self-renewing multipotential

cells with the capacity to differentiate into several distinct mesenchymal cell types.

MSCs represent a group of mesenchymal precursors that contribute to the regeneration of

bone, cartilage, adipose, tendon, muscle, neural, and other connective tissues.

11-18

Sus-

pensions of MSCs form a fibrous osteogenic tissue when incubated within diffusion

chambers implanted in vivo.

This implies that the differentiating capacity of these cells

is not dependent on the structural relationship of the cells in situ and that cells capable of

differentiating in an osteogenic direction are present in marrow stroma.

When ex-

panded ex vivo, MSCs have been shown to generate a bone-like tissue.

20-22

MSCs can be

directed towards osteogenic differentiation in vitro when cultured in the presence of dex-

amethasone, and ascorbic acid.

There is much interest in the po-

tential of using stem cells populations in the tissue engineering of bone and cartilage for

the treatment of musculoskeletal trauma and disease. Adult mesenchymal stem cells offer

certain advantages over embryonic stem cells for tissue engineering including their readi-

ness and availability as they can be obtained from the same individual.

The studies

described here were designed to investigate the biophysical and nanostructural character-

istics of MSCs, in parallel with recent meritorious effort on biochemical characteriza-

tion.

25-28

2.2.

Cytoskeleton

The cytoskeleton plays an important role in cell morphology, adhesion, growth, and

signaling. The actin cytoskeleton consists of 3 components, actin filaments, intermediate

filaments and microtubules. The “backbone” of the cytoskeleton is the actin filaments

composed of F-actin. Actin filaments consist of repeating subunits of monomers arranged

in a right-handed double helical structure. The widths of these filaments have a diameter

of 5-9 nm.

Actin filaments are dynamic structures and can be elongated or shortened by

polymerization or dissociation of monomers at the filaments ends

, this creates a

“treadmilling” effect, in which one end is polymerized while the other is depolymerized.

Filaments can be bundled or cross-linked to each other by several actin binding proteins

to create a network. Actin and intermediate filament systems have been described as

THE CYTOSKELETON

“molecular guy wires” to mechanically stiffen the nucleus and hold it in place.

There is

a direct link between integrins, the actin cytoskeleton, and the nucleus.

The actin network plays a major role in the determination of the mechanical proper-

ties of living cells.

31, 32

Changes within the cytoskeleton of the cell allow the cell to mi-

grate, divide, or maintain its shape.

33, 34

Actin polymerization and depolymerization are

important actions in cell signaling and metabolism, as has been shown by subjecting cells

to actin disrupting drugs such as cytochalasin D (cytD), latrunculin A (latA), or Jasplaki-

nolide.

35-37

The effects of cytochalasins on cell elasticity has been studied by both a de-

vice designed to poke individual cells

and more quantitatively by AFM.

Rotsch and

Radmacher

demonstrated the importance of the actin network for mechanical stability

of living cells by showing that disaggregation of actin filaments always resulted in a de-

crease in the cell’s average elastic modulus.

2.3. Mechanobiology

An increase in bone mass and strength across multiple species occurs as a result of

mechanical loading, especially associated with cyclic mechanical stresses.

6, 8, 39-41

Con-

versely, bone loss results from bedrest, immobilization, and weightlessness.

42-46

It seems

clear that both bone resorption and bone formation are mediated by mechanical signals.

Among the multiple theories regarding which is the specific physical signal to which

bone cells respond, strain-induced fluid flow has received the most abundant experimen-

tal support. Fluid flow occurs in the interstitial spaces around bone cells due to circula-

tion and repetitive loading and unloading of bones during activity.

It is hypothesized

that the sensing mechanism in mechanical load-induced bone remodeling is that the load

induced fluid flow produces shear stress at bone cell membranes and these shear stresses

induce cell signaling events which translate the mechanical signal to a cellular response.

Changes in interstitial fluid flow due to internal bone pressure changes influence bone

remodeling whereas normal pressures serve to maintain a baseline level of flow and bone

maintenance.

2.4. Fluid Flow

Fluid flow is a mechanical stimulus likely experienced by all cells. A fluid flow sig-

nal experienced at a cell membrane partially transduced into a response by the cytoskele-

ton. Either transient, or more likely sustained, signals may lead to upregulation and/or

downregulation of certain genes. At least two mechanisms are likely to be involved in

the transmission of these mechanical signals from the cell membrane to the cell. These

processes are termed “mechanotransduction”. The first depends on second messengers

released close to the membrane that then activate a chemical reaction cascade, leading to

transcription factor activation and translocation into the nucleus. The second is that di-

rect cytoskeletal displacement is transmitted to the nucleus and other cell communication

points through physical coupling rather than chemical reaction cascades. It has been hy-

pothesized that in mature osteoblasts, fluid flow creates a drag on the glycocalyx (a pro-

teoglycan rich cell coat) and this drag causes the cytoskeleton to be stimulated and trans-

mit a signal to the cell nucleus.

Experimental data show that a proteoglycan rich gly-

cocalyx is necessary for biological responses to fluid flow

and that the actin cytoskele-

ton is necessary for other biological responses to fluid flow.

However, although MSCs

G.YOUREK

ET AL

have been shown to also respond to a fluid flow stimulus,

51-54

it is not known how the

MSC cytoskeleton differs from the mature osteoblast cytoskeleton or if the cytoskeleton

can contribute to a fluid flow induced differentiation.

BASICS FOR STUDYING CONTRIBUTION OF CYTOSKELETON TO

DIFFERENTIATION

3.1. Differentiating Factors

The pluripotency of MSCs has been demonstrated by exposing the cells to their nor-

mal growth medium, typically Essential Medium Eagle) or

DMEM (Dulbecco’s Modified Eagle Medium) plus 10-20% fetal bovine serum (FBS)

and 1% antibiotics with supplementation by growth factors and other pharmacological

agents. The virtually infinite expansion and differentiation of these cells into other cell

types not as easily cultured indicate that MSCs will eventually play a major role in treat-

ment for trauma, disease, or aging. MSCs have been successfully differentiated into adi-

pocytes (fat cells), chondrocytes (cartilage cells), and osteoblasts (bone cells) by a num-

ber of laboratories under a wide variation of culture conditions, demonstrating the rela-

tive ease with which these cells can be utilized.

Stem cells have been differentiated towards an osteogenic lineage by supplementa-

tion with (i) dexamethasone, a member of the glucocorticoid family of steroids which

may modulate Bone Morphogenetic Proteins (BMPs),

which are major inducers of os-

teogenesis, (ii) to promote calcium phosphate deposition

9, 56-59

and

(iii) ascorbic acid which plays an important role in the production of the collagenous

bone extracellular matrix. Bone marrow stromal cells have been differentiated towards

the chondrogenic lineage by supplementation with a number of different factors, the most

potent of these being members of the transforming growth family.

9, 23,

60,61

For example, is found in great supply in embryonic cartilage

. Finally,

MSCs have been differentiated towards a adipogenic lineage by supplementation with

dexamethasone, 1-methyl-3-isobutylxanthine (MIX), and indomethacin (both inhibitors

of cAMP and reducers of the synthesis of lipogenic enzymes).

23, 60, 63

3.2. Pharmacological Cytoskeleton Disrupting Agents

Pharmacological agents have recently become a tool for clinicians and researchers

for disrupting the cytoskeleton of cells for both therapeutic and research reasons. These

agents are derived from plant sources where they are used as defense mechanisms for

otherwise defenseless organisms.

When consumed by an organism, these poisons at-

tack one of the three cytoskeletal components (actin filaments, intermediate filaments, or

microtubules) and cause great damage to the infected organism. When used in the labo-

ratory for studies on the importance of cytoskeletal components in cellular events or in a

clinic to combat certain types of cancer (for reviews see: 64, 65), they can be a useful

mechanism for cell modification.

Cytochalasins are isolated from fungi and have an abundance of cytotoxic activites,

including the disruption of actin filaments.

These compounds “cap” actin filaments by

THE CYTOSKELETON

binding to the growing end of actin

67-72

and cleave actin filaments.

71, 73-75

There are

agents which are specific to each of the three parts of the cytoskeleton. For example,

phalloidin, cytochalasin, swinholide, and latrunculin have all been found to disrupt actin

filaments, each acting through a specific mechanism.

Taxol (a chemotherapeutic), col-

chine, vinblastine, and nocodazole disrupt microtubules.

3.3.

Mechanical Stimulation of Cells

Parallel-plate flow chambers have been used to examine the effects of flow-induced

shear stresses on the biochemical and morphological response of many types of cells.

They provide a well-controlled mechanical stimulus of shear stress to a relatively homo-

geneous population of the cells of interest.

48-50, 76-82

Low level shear stress may have

beneficial effects on cellular metabolism.

Fluid flow through parallel-plate flow cham-

bers is laminar and has been well characterized

and since most of the apparatus is as-

sembled of glass, polycarbonate, or rubber, there is no loss of medium due to permeation

through the tubing or evaporation.

Cone-and-plate viscometers have also been used to study the effects of shear stress

on a monolayer of cells;

84-87

however, these systems have a smaller cell-to-volume ratio,

do not permit continuous sampling of the cell culture medium and have significant cul-

ture medium evaporation, requiring continuous infusion of fresh medium.

Recently, an apparatus known as Flexercell has been developed by Flexcell (Hills-

borough, NC) that consists of six-well culture dishes with flexible silicone rubber bot-

toms which uses positive pressure to compress samples with a piston. Simmons et al.

used this system to study the effects of equibiaxial cyclic strain on hMSCs cultured in

osteogenic media. They found a decrease in proliferation and an increase in matrix min-

eralization over unstrained cells. Flexcell has also been used by a group in the Nether-

lands

to show that the stage of differentiation that a osteogenic cell is at affects its re-

sponse to stretch in vitro by noting differences in proliferation and apoptosis of a differ-

entiating human fetal osteoblast cell line.

ATOMIC FORCE MICROCOPY (AFM)

4.1. Operation Principles

The fundamental principle of AFM imaging is to “sense” the sample surface with a

probe. This “sensing” or probing of the sample surface is accomplished in a precisely

controlled manner by the movement of a piezoelectric scanner, which has the sample

mounted on top, against a micro-fabricated sharpened tip mounted at the end of a cantile-

ver. The directional movement of the cantilever and the scanning tip is a function of the

sample’s surface topographic features including peaks and valleys, causing a deflection

of the scanning tip as it moves across the sample surface. A laser diode tracks the canti-

lever movement by a laser beam focused on the cantilever surface that is opposite, but

preferably over, the scanning tip. Therefore, each vertical and lateral deflection of the

cantilever from the sample surface is coupled with a directional reflection of the laser

beam. Most modern AFMs are equipped with a four-segment photodiode detector that

registers the vertical and lateral reflections of the laser beam off the cantilever surface as

G.YOUREK

ET AL

a function of the laser intensity difference between different segments. These input photo

signals are then amplified and transformed into AFM surface images through a digital

control system.

Generally, AFM scanning results in two types of images: height image and force

image. Whereas height images represent the sub-micron digital replication of the sample

surface topographic characteristics, force images are a function of the elastic responses of

the sample surface to nanoindentational forces applied on the surface by the scanning tip.

Height and force images, individually or combined, provide fundamental characteristics

of biological structures including cells and their surrounding matrices, thus offering great

promise for elucidation of rich structural and functional details at unparalleled levels of

resolution.

Another distinctive feature of the AFM is substantial minimization of sample prepa-

ration in comparison with other imaging modalities. Compared with electron micros-

copy, three dimensional AFM images are obtainable without expensive and time-

consuming sample preparation, and yet yield more detailed representation than the two

dimensional profiles available from cross-sectioned samples. AFM imaging eliminates

the need for vacuum that is required by most electron microscopes to image samples, and

yet has the potential to image samples in ambient air or liquid. The AFM’s ability to

view and scan samples in their quasi-native environment has resulted in the unsurpassed

advantage of real-time imaging of live biological samples. In addition, compared to SEM

that requires sample coating, AFM provides extraordinary topographic contrast with di-

rect height measurements and unobscured view of surface features. Moreover, the ex-

tended ability of the AFM to measure the viscoelastic behavior of samples, where appli-

cable, has broadened the versatility and implications of the AFM in various scientific

fields.

Despite its advantages, AFM has several practical limitations. For example, because

the depth of field in AFM is limited by the travel distance of the scanning tube (about 5.3

in the current model), AFM is best suited for imaging relatively soft samples. Depth

of field is also limited by the relationship between the scanning tip geometry and the

sample’s surface topography. If the tip is too thick to reach into a groove or recess on the

surface, a true image cannot be produced and thus the depth of field is reduced. The tip

geometry relative to the surface features presents another challenge. Sheer walls and

undercuts of the sample’s surface may represent difficulties during AFM scanning due to

the nature of the surface topography and tip geometry. Thus, it is critical to select appro-

priate AFM scanning tips for various biological samples.

4.2.

Preparation of Cells for AFM Imaging

In general, the AFM can be used to scan and image any cell type provided that the

cells can adhere or attach to a substrate to be mounted on the AFM scanner. Cells float-

ing in suspension or lacking substrate support to withstand the interactions with the scan-

ning tip are not suitable for AFM imaging.

The amount of the glycocalyx halo sur-

rounding the cells

can present another limiting factor in obtaining a high-resolution im-

age of the cell surface. Similarly, rapid cellular events that exceed the time required to

obtain an AFM image, such as rapid cell growth, division, or movement, can result in low

quality imaging. Typically, obtaining an AFM topographic image with acceptable re-

THE CYTOSKELETON

solving power for an area of about requires at least 2-3 minutes, and thus only

slower cellular events are considered suitable candidates for the real-time AFM imaging.

A widely used method to prepare living cells for AFM imaging is to culture them on

glass coverslips, usually treated with a material that facilitates cellular attachment such as

the positively charged poly-D-lysine.

Also, trapping cells in millipore filters with pore

size comparable to the dimensions of the cell can be used to image rounded living cells

and to overcome the attachment requirement.

Another established methodology in-

volves the use of AFM in conjunction with micropipette aspiration for cellular immobili-

zation.

4.3. Imaging Conditions

The driving motive to image samples in fluid is to study biological samples under

normal physiologic conditions by minimizing surface interactive forces on soft samples.

AFM imaging procedures using fluid are similar to those under air, but with several nota-

ble differences. Fluid imaging with the AFM minimizes the attractive surface tension

forces between the scanning tip and the sample surface in the contact mode imaging, and

thus also minimizes the scanning forces exerted by the cantilever and the tip deflection on

the sample surface. Fluid imaging has shown its applicability especially for larger-scaled

biological structures such as cells and extracellular matrices.

The imaging under fluid generally requires an additional piece of hardware such as a

fluid cell to contain the sample and the imaging medium. The fluid cell is a small glass

assembly that houses the liquid phase. The glass surface provides a flat beveled interface

to avoid distortion of the AFM laser beam from fluid movement. The sample is mounted

on a metal disk that fits magnetically on top of the AFM piezoscanner in a similar fashion

to imaging in ambient air. The AFM fluid cell fits over the sample/disk assembly and

holds the cantilever in a position above the sample. Fluid imaging can be performed by

means of fluid cell with or without O-ring seal. Certain criteria are worth considering

when choosing a liquid medium to image living cells with the AFM. For example, the

liquid should not be too viscous, so to avoid potential interferences with tip/sample inter-

actions and consequent image distortion. The liquid should also be clear of particles that

can build up on the sample surface or the tip and affect their proper interaction. When

imaging living cells, chemical and physical characteristics of the imaging liquid should

resemble, as close as possible, physiologic conditions of the native extracellular fluids.

Phosphate saline buffer (PBS) is commonly available and relatively inexpensive, and thus

has broad acceptance among AFM users as a suitable imaging liquid-medium. However,

when PBS dries, salt crystals form, making accurate scanning difficult. Regular serum-

free culture medium, Dulbecco’s Modified Eagle’s culture Medium (DMEM) for exam-

ple, or a mixture of DMEM and PBS can also suffice the imaging requirement. Which-

ever liquid medium is used, the pH of the solution should be maintained constant and

within the normal physiologic range by frequent exchange or continuous flow of medium

in case of extended imaging sessions.

4.4. AFM Probe Selection