Cellular responses to a nanofibrous environment

Cells are supported in vivo by a three-dimensional extracellular

matrix (ECM) of nanofibers with different chemical ligands that

interact with cell surface receptors1-4. The interplay between cells

and the ECM is a dynamic and complex process where the physical

and chemical properties of the ECM elicit different cellular

responses1,5. An engineered substrate seeking to emulate the

functions of the native ECM should, therefore, recapitulate its

three dimensionality and nanofibrous topography, as well as its

plethora of chemical motifs6,7. Advances in nanotechnology in

recent years have enabled us to engineer novel biomaterials with

these levels of complexities7-9. The eventual use of such complex

biomaterials for specific applications will require an iterative

process of understanding the mechanisms guiding cell-matrix

interactions so that we can precisely control biomaterial

properties to elicit desirable cellular responses (Fig. 1). Here, we

emphasize the importance of the three-dimensional nanofibrous

features of extracellular environments in modulating cellular

responses with local or subcellular resolutions in space- and time-

dependent manners. We also highlight state-of-the-art

technologies to fabricate and characterize nanofibrous

environments for relevant applications.

Influence of the nanofibrous environmenton cell phenotype and signalingCells interact with their nanofibrous extracellular environment via cell

surface proteins, such as integrins, to activate various intracellular

signaling pathways that regulate cellular processes, e.g. cell shape,

mobility, and proliferation (Fig. 2). Understanding the specific biological

Cells respond profoundly to the mechanical rigidity and three-dimensionalnanotopology of substrates, as well as the spatial and temporalarrangements of extracellular cues. We summarize the latestdevelopments in probing and engineering biocompatible nanofibrousextracellular environments at the cell and molecular level for applicationsin tissue engineering and biological research. This will, in turn, guidefurther development of three-dimensional nanofibrous scaffolds in orderto elicit specific cellular responses for relevant applications.

Yi-Chin Toh1,2, Susanne Ng1,2, Yuet Mei Khong1,2, Xin Zhang2, Yajuan Zhu2, Pao-Chun Lin3, Chee-Min Te3, Wanxin Sun1, and Hanry

Yu1,2,3*1Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 1386992Graduate Program in Bioengineering, NUS Graduate School of Integrative Sciences and Engineering, National University of Singapore,

Singapore 1175973Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597

*E-mail: [email protected]

ISSN:1748 0132 © Elsevier Ltd 2006AUGUST 2006 | VOLUME 1 | NUMBER 3 34

Cellular responses to ananofibrous environment

NT103p34_43.qxd 07/06/2006 14:44 Page 34

mailto:[email protected]

response triggered by various aspects of the nanofibrous environment

is important in guiding the design and engineering of novel substrates

that mimic the native cell-matrix interactions in vivo.

Influence of three-dimensional topographyThree-dimensional nanofibers allow cells to interact with a pliable,

three-dimensional mesh in contrast to two-dimensional flat and rigid

substrates. The three-dimensional nanofibrous environments promote

in vivo-like cellular phenotypes and promote tissue morphogenesis10-13.

Molecular events associated with cells interacting with three-

dimensional nanofibers are different to those on two-dimensional

substrates such as petri dishes. The three dimensionality of the

nanofibers alone plays a role in the activation of the Rac-mediated

signal transduction pathway within NIH 3T3 fibroblasts and normal rat

kidney cells, when cells are cultured on electrospun nanofibers in the

absence of ECM macromolecules14. Fibroblast motility in three-

dimensional collagen matrices (important in collagen remodeling

during wound healing) depends heavily on nonmuscle myosin heavy

chain II (NMHC II-B), whereas cell motility on two-dimensional rigid

surfaces does not15. Paxillin (in focal adhesion) and α5 integrin (in

fibrillar adhesion) co-localize in three dimensions rather than localizing

separately into classical focal and fibrillar adhesions on two-

dimensional substrates16. Artificially flattening three-dimensional

nanofibrous matrices to form virtually flat two-dimensional mats by

mechanical compression results in the loss of the characteristic triple

co-localization of α5 integrin, paxillin, and fibronectin that is found in

three-dimensional matrix adhesions16. Thus, three-dimensional

nanofibrous topography plays a very important role in influencing cell-

ECM interactions at the molecular level.

Influence of mechanical propertiesCells respond to a variety of chemical ligands1 and also to mechanical

cues17,18 in the extracellular environment. Stretching cells on rigid

substrates induces cell proliferation and alters the differentiation

lineage of stem cells19. Cells are sensitive to shear stress and

compression forces, which in turn influence the remodeling and

mechanical properties of tissue constructs20-23. In three dimensions,

cells are also sensitive to nanofiber rigidity16,24,25. External forces affect

the cell via ECM-integrin-cytoskeleton connections, inducing the

Tools in biologicalresearch

Engineered artificial nanofibrous extracellular environment for biological research or

clinical applications

Naturalnanofibrous

basedsystems

Understanding cellular responses to nanofibrous extracellular environment

Engineering capabilities to createand probe nanofibrous

extracellular environmentGuiding principles forbiomaterials design

Tools in biologicalresearch

AUGUST 2006 | VOLUME 1 | NUMBER 3 35

Cellular responses to a nanofibrous environment REVIEW FEATURE

Fig. 1 Paradigm for engineering three-dimensional nanofibrous extracellular environments with desirable cellular responses for cell biology research and tissueengineering applications. Precision engineering of the nanofibers enables systematic and quantitative studies of cell behaviors that, in turn, enable the constructionof models to guide the future design of nanofibrous environments for specific applications.

Fig. 2 Simplified schematic of cell signaling mediated by cell-matrixinteractions.

NT103p34_43.qxd 07/06/2006 14:44 Page 35

assembly of focal adhesions that trigger tyrosine kinase/phosphatases

and Src family kinases (SFKs) with subsequent activation of small

G proteins and MAP kinases for the regulation of downstream cellular

events26. It will be important to quantify these events systematically

and establish models of cellular response to mechanical and chemical

properties of the nanofibers to guide the development of three-

dimensional nanofibrous environments for applications.

Influence of spatial and temporal arrangements ofextracellular cuesCellular responses to the mechanical and chemical properties of three-

dimensional nanofibers are often space- and time-dependent27-29.

Neural stem cells and smooth muscle cells, when cultured on aligned

electrospun nanofibers, elongate and orient themselves along the

fibers30,31. This sensitivity to the spatial arrangement of the nanofibers


REVIEW FEATURE Cellular responses to a nanofibrous environment

Glossary α5 integrin One of the integrin family of transmembrane proteins that are involved in the adhesion of

cells to the ECM.

angiogenesis The generation of new blood vessels.

collagen Fibrous ECM protein rich in glycine and proline. At least 12 types of collagen have been

identified. Collagen I is commonly found in tendon and bone, while collagen II is commonly

found in cartilage.

complex coacervation The separation of two liquid phases in a colloidal system caused by the interaction of two

oppositely charged colloids.

DsRed2-calreticulin Calreticulin, a highly conserved Ca-binding protein within nonmuscle smooth muscle cell

ERs, conjugated to the fluorescent probe DsRed.

endoplasmic reticulum (ER) Membrane-bounded compartment in the cytoplasm of eukaryotic cells, responsible for lipid

synthesis, as well as the synthesis and sorting of membrane-bound and secretory proteins.

fluorescence resonance energy transfer (FRET) Describes the phenomenon of energy transfer between two fluorescent molecules. A

fluorescent donor is excited at one wavelength and its emission excites a neighboring

fluorescent acceptor, whose emission wavelength is detected.

mitogen-activated protein (MAP) kinases Protein kinases that perform a crucial step in relaying signals from the plasma membrane to

the nucleus. They regulate various cellular activities, such as gene expression, mitosis,

differentiation, and cell survival/apoptosis.

morphogenesis The process of forming a tissue or organ via regulated growth and differentiation of cells.

messenger ribonucleic acid (mRNA) An RNA molecule that specifies the amino acid sequence of a protein during protein synthesis.

NIH 3T3 fibroblasts A cell line derived from fibroblasts of disaggregated Swiss mouse embryos.

paxillin Focal adhesion protein involved in the binding of the actin cytoskeleton at sites of cell

attachment to the ECM.

phenotype The observable characteristics or traits of a cell or an organism, e.g. size, eye color.

Rac-mediated signal transduction pathway Signaling pathway regulated by Rac guanosine triphosphate (GTP)-binding protein, which is

involved in controlling the organization of cytoskeletal filaments.

RGD sites Peptide sequence consisting of three amino acids (arginine-glycine-aspartic acid) commonly

associated with cell attachment.

small G proteins A large family of monomeric GTP-binding proteins that activate target proteins on binding

GTP.

solid free-form fabrication (SFF) Three-dimensional, computer-aided designs are converted into stereolithography data and a

format recognizable by software for printing layer by layer. Reverse SFF fabricates the desired

construct by casting over a negative mould created via SFF.

Src family kinases A tyrosine kinase family of proteins, which interact with a variety of cell-surface receptors and

participate in intracellular signal transduction pathways.

tyrosine kinases/phosphatases Enzymes that catalyze the phosphorylation/dephosphorylation, respectively, of tyrosine

residues in proteins. Tyrosine kinase/phosphatase pairs regulate cellular signal transduction and

may play a role in cell growth control and carcinogenesis or cancer formation.

NT103p34_43.qxd 07/06/2006 14:44 Page 36

possibly results from localized interactions of cells with the

extracellular cues at subcellular resolutions. Most of the structural or

signaling proteins involved in rigidity sensing, ECM anchorage, and cell

migration are found in specific locations in a cell, such as the leading

tip of migrating fibroblasts or focal adhesion sites, but not in other

locations even 0.5 µm away28. A single ECM-integrin-cytoskeleton

linkage at one specific location can be dynamically modulated in

response to an extracellular force without affecting a nearby

linkage32,33. Clustering of integrin molecules to form focal adhesions

and stress fibers only occurs when sufficient RGD sites are clustered

within a discrete spot of less than 70 nm27-29.

Molecules associated with the integrin-cytoskeleton linkages

involved in cell-ECM adhesion also change in a time-dependent manner

throughout the life cycle of the adhesion sites34 and are sensitive to the

stiffness of the ECM nanofibers32,35-38. The formation of cell contacts

with the ECM is not a continuous process, but involves cycles of

contraction and relaxation. We have recently shown that presenting

extracellular nanofibers to hepatocytes at different times or sequences

in sandwich culture elicits different cellular responses39. Thus, cells are

sensitive to temporal as well as spatial arrangements of extracellular

cues in the environment. It seems that the sum of local responses to

extracellular cues over time helps determine cell morphology and gene

expression26,40. It is important therefore to understand and engineer the

local cellular responses to spatially and temporally distributed

extracellular cues in further development of three-dimensional

nanofibrous environments.

Extracellular cues elicit local cellular responsesUpon stimulation by a discrete and sufficiently strong extracellular cue,

cells first respond locally followed by an eventual global response. The

endoplasmic reticulum (ER) and many of the ER-associated complexes

are normally distributed uniformly in cells as a network-like structure

throughout the cytoplasm in a two-dimensional culture (Fig. 3a). They

can dynamically rearrange their distributions upon extracellular

stimulation or when engaged in cell attachment, proliferation, or

migration (Fig. 3b). When a fibronectin-coated bead interacts with the

upper surface of a cell, some ER membrane proteins such as kinectin,

receptor-associated protein (RAP), calreticulin41, and eukaryotic

elongation factor-1δ (EEF-1δ)42 are rapidly translocated to integrin-

associated complexes (IACs) formed transiently around the fibronectin-

coated bead. They do not respond to diffuse fibronectin in solution or

fibronectin distributed evenly on large, two-dimensional surfaces

(Fig. 4). EEF-1δ has been shown to anchor the entire EEF complex onto

the ER membrane via kinectin, and regulates the synthesis of

membrane/secretory and cytosolic proteins43. In addition, ribosomes

and mRNA are observed to target IACs around fibronectin-coated

beads44 and rapid protein synthesis occurs before changes in

transcription are detectable41,45. We hypothesize, therefore, that local

synthesis of proteins on-site might be a general cellular process that

responds to strong and discrete localized extracellular cues in a space-

and possibly time-dependent manner (Fig. 5). This local translation

hypothesis (LTH) suggests that a cell mobilizes its protein synthesis

factory, including ribosomes, mRNA, and translation regulators such as

micro RNA, to a location near the extracellular cue, which might be a

fibronectin-coated bead41, force exertion by optical tweezers46, or

other rigid, substrate attachment site47. Local translation is likely to

supplement the movement of ready-made proteins from intracellular

pools to the site of local cellular response to extracellular stimulation

when the extracellular cue is discrete and above a certain threshold

value. It implies that the local intensity and distribution of extracellular

cues in three-dimensional nanofibrous environments need to be

precisely engineered at subcellular (i.e. micron- or nanoscale)

resolutions, especially when more detailed and quantitative

information that tests the LTH becomes available in coming years. For



(b)(a)

Fig. 3 The endoplasmic reticulum (ER) is a dynamic intracellular network-like structure that changes its intracellular distribution. (a) ER in a HeLa cell transfectedwith an ER-retention peptide KDEL coupled to yellow fluorescence protein (YFP). (b) ER dynamics in a dividing HeLa cell stably transfected with a DsRed2-calreticulin fusion construct was imaged in three dimensions over time under the microscope. Image stacks were collected every 4 min, beginning at metaphase.

NT103p34_43.qxd 07/06/2006 14:44 Page 37

example, nanoscale features on engineered substrates, e.g. ligands

patterned in a discrete manner instead of a uniform layer, could elicit

stronger cellular responses at the stimulated sites.

Fabricating three-dimensionalnanofibrous environmentsCurrently, nanofiber fabrication is dominated by electrospinning

techniques and self-assembling peptides (SAPs), but we also summarize

technologies based on polyelectrolyte complex coacervation and phase

separation. Most approaches aim to emulate the native ECM for tissue

engineering applications. The ability to control the mechanical and

chemical properties of the nanofibers precisely will also allow us to

study cellular responses to three-dimensional nanofibrous

environments systematically and quantitatively at subcellular

resolutions, which can in turn inform the design of biomaterials.

ElectrospinningElectrospinning of polymeric materials has emerged as a convenient

tool to construct nanofibrous scaffolds because of its versatility48,49.

Electrospinning uses high voltages (5-20 kV) to charge up polymer

solutions that emerge from a nozzle, which are then accelerated

toward a grounded collecting substrate. Solvent evaporates from the

jet of polymeric solution during this process, forming a random,

nonwoven fabric of polymeric nanofibers on the collecting substrate50.

The properties of electrospun nanofibers, such as fiber diameter, can be

controlled readily via manipulation of the polymer solution and



Fig. 5 Side-view schematic of a cell with a single fibronectin-coated bead attached on its upper surface illustrating the local translation hypothesis (LTH). Weak anddistributed extracellular cues such as cell attachment on a two-dimensional flat surface yield evenly distributed ER networks and associated protein synthesisapparatus throughout cytoplasm. Anything required to support cellular responses at a specific location can be transported from other intracellular locations. Whena strong and discrete extracellular cue, such as the single fibronectin-coated bead, is presented to the upper surface of the cell, there is a transient mobilization andassembly of protein synthesis factories around the bead.

Fig. 4 Translocation of ER membrane proteins to integrin-associated complexes (IACs) formed transiently around fibronectin (FN)-coated beads. (a) ER redistribution and accumulation around fibronectin-coated beads attached to a cell surface (left, indicated by white arrow). Beads were incubated withDsRed2-calreticulin transfected HeLa cells for 30 min at 37°C for IAC formation. Uncoated beads were used as a control (right). (b) Quantitative analysis of ERredistribution and accumulation around fibronectin-coated beads versus control beads. p-value < 0.0005 and n = 19.

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electrospinning operational parameters48,50,51. Three-dimensional ECM

models can be constructed for quantitative correlations of cellular

responses to defined three-dimensional nanofibrous environments, as

well as tissue engineering applications. Nanofibers can be electrospun

from synthetic polymers or natural biomaterials (Table 1), and

functionalized with chemical motifs49 to improve biocompatibility52

and cellular functions53. Electrospun nanofibrous scaffolds have been

used successfully to culture different cell types for tissue engineering of

cartilage, bone, arterial blood vessels, heart tissue, and ligaments

(Table 1). Electrospun nanofibers can be aligned to provide contact

guidance for cell alignment along the direction of the fibers54,55.

Generally, electrospun nanofibers yield a flat mat that has limited three

dimensionality48,56 and suffers from cell infiltration problems because

of the small pore size of the mats. These problems can be addressed by

electrospinning nanofibers onto microfibrous scaffolds56,57, using

multiple nozzles for sequential deposition of nanofiber layers58, or

co-spinning the nanofibers with cells59.

Self-assembling peptidesAnother strategy to engineer nanofibers relies on the molecular

assembly of synthetic oligopeptides to generate fibrous networks

capable of supporting cells in three dimensions. Synthetic peptides are

an ideal choice as building blocks for fabricating three-dimensional

nanofibrous scaffolds as they are biocompatible and can be designed to

incorporate motifs, such as RGD peptides, that are reminiscent of those

found in natural ECMs to modulate cellular phenotypes60,61. Moreover,

a great bank of knowledge on protein folding and protein-protein

interactions has been accrued over the years, facilitating the design of

oligopeptides that will self-assemble62. These SAPs can be triggered to

spontaneously assemble into a three-dimensional nanofibrous network

around cells under physiological conditions, eliminating cell-seeding

problems associated with using prefabricated nanofibrous scaffolds.

SAPs can be synthesized from naturally occurring amino acids8,9,60,62

or synthetic amino acids63, although the former is more commonly

employed to emulate natural protein motifs that interact with cells. The

nanofibrous extracellular environment formed by SAPs can be based on

either α-helix or β-sheet conformations at the secondary structural

level. Lacking mechanical strength, they are often administered directly

into patients as cell- or drug-delivery vehicles64, or further incorporated

into microfibrous scaffolds65 for tissue engineering and cell-based

applications (Table 1). Researchers have envisioned ‘designer scaffolds’

with fine-tuned microenvironments to support specific cell types66,

which would be especially attractive in determining cellular responses to

three-dimensional nanofibrous environments. For instance, SAP scaffolds

could simulate angiogenic microenvironments for microvascular

endothelial cells better than collagen can, thereby providing a good

model for understanding angiogenesis in tissue-engineered constructs67.

Angiogenesis is vital to coax production of blood vessels for nutrient

delivery into large tissue-engineered constructs and to facilitate

integration of the construct with the host’s tissue upon implantation.

Polyelectrolyte complex coacervationA nanofibrous environment may also be fabricated by complex

coacervation of oppositely charged polyelectrolytes, a method



Biomaterials Cell culture compatibility

EElleeccttrroossppiinnnniinnggRandom two-dimensional Synthetic: poly(ethylene oxide), poly-ε-caprolactone, Bone-marrow-derived mesenchymal stem cells (MSCs), aortic mats50,78-87 poly(glycolic acid), poly(lactic-co-glycolic acid), smooth muscle cells, NIH 3T3 fibroblasts, normal rat kidney cells,

polyamide chondrocytes, osteoblasts, cardiomyocytesNatural: collagen, silk fibrion, chitosan, hyaluronic acid

Aligned nanofibers54,55 Poly(L-lactic-co-ε-caprolactone), polyurethane Smooth muscle cells, ligament fibroblastsThree-dimensional Poly(lactic-co-glycolic acid), starch/polycaprolactone Bone-marrow-derived MSCs, human osteoblast-like osteosarcomas nanofibrous scaffolds56-58 (SPCL), collagen, gelatin, polyurethane, poly(ethylene (SaOs-2)

oxide)

SSeellff--aasssseemmbblliinngg ppeeppttiiddeessβ-sheet-forming SAPs Synthesized ionic amphiphilic HIT-T15, chicken embryo fibroblasts, human fibroblasts, HepG2,

oligopeptides8,60,88-94 MG63, HeLa, HEK293, NIH 3T3, CHO, PC12, hippocampal neural cells, Lig-8, chondrocytes

Oligopeptides with fatty acid chains9,61,95,96 MC3T3-E1, neural progenitor cellsRecombinant DNA expressed polypeptides89,90,97 Not evaluated

α-helix-forming SAPs Synthesized polypeptides98-100 Not evaluated

CCoommpplleexx ccooaacceerrvvaattiioonn71-75 Water soluble chitin/alginate, Human dermal fibroblasts, bovine pulmonary artery endothelial Chitosan/alginate, methylated collagen/ cells, MSCs, HepG2, hepatocytesHEMA-MMA-MAA terpolymer

PPhhaassee sseeppaarraattiioonn76,101-104 Poly(L-lactic acid), hydroxyapatite/poly(L-lactic acid) MC3T3-E1 osteoblasts, neonatal mouse cerebellum C17-2 stem cells

Table 1 Technologies for engineering extracellular three-dimensional nanofibrous environments.

NT103p34_43.qxd 07/06/2006 14:44 Page 39

originally developed for the macro- or microencapsulation of cells68-70.

Polyelectrolyte complex coacervation can take place under mild

aqueous conditions, and is therefore favorable for in situ formation of

nanofibers in the presence of cells. A plethora of polyelectrolytes have

been developed and evaluated for cell encapsulation68,69. Complex

coacervated nanofibers generally lack mechanical strength; therefore, it

is difficult to cast them into three-dimensional scaffolds for tissue

engineering applications. The nanofibers can be readily incorporated

into cell culture constructs such as three-dimensional microfibrous

scaffolds and microfluidic channels to immobilize and support sensitive,

anchorage-dependent cells71,72. Alternatively, nanofibers may be drawn

directly into microfibers, with nanofibrous features to support cultures

of various mammalian cells73-75.

Phase separationPhase separation, an approach commonly used to fabricate

microfibrous scaffolds, can also be used to fabricate nanocomposite

scaffolds. Polymer- and solvent-rich domains of a polymer solution are

separated either by cooling the solution or exchanging a

nonsolvent for the solvent. A nanofibrous (fibers with diameters of

50-500 nm) three-dimensional scaffold has been constructed with

poly(L-lactic acid) via thermally induced phase separation76. The

scaffold has controllable high porosity (up to 98.5%) and surface-to-

volume ratios, as well as defined mechanical properties (Table 1).

A reverse solid freeform fabrication (SFF) technique to control

scaffold architecture and dimensions by integrating paraffin spheres for

pore connectivity has recently been developed77, enabling the

fabrication of scaffolds of any desired shape or size. Nanofiber

distribution and uniformity is subject to the controllability of the

processing. Scaffold fabrication also involves a five-step process and

has, so far, only been applied to a few polymers such as

poly(L-lactic acid) and its blends49. This technique can be readily

applied in tissue engineering applications.

Characterizing the three-dimensionalnanofibrous environmentNew developments in microscopy techniques and fluorescence probes

have enabled dynamic imaging of three-dimensional nanofibrous

matrices and their interactions with cells.

Introduction of fluorescence probes normally interferes with the

biological system under scrutiny to a certain extent by changing the

matrix properties or interacting with the cells in the system. Intensive

efforts are being made to develop less invasive fluorescence dyes

and proteins with high specificity to characterize the chemical

properties of the cellular environment. Several imaging techniques can

be used to quantify structural ECM dynamics without fluorescence

probes, and force-sensing methods such as atomic force microscopy and

optical tweezers have great potential for mechanical property

measurements.

Structural characterization using imaging techniquesCellular interactions with three-dimensional nanofibrous networks have

been monitored using differential interference contrast (DIC)

microscopy, which is a contrast-enhancing imaging technique105. Two-

dimensional images are obtained at different depths, though it is

intrinsically difficult to construct three-dimensional images from DIC

images even after image deconvolution processing.

Reflection or backscattering confocal microscopy exploits the light

scattering properties of nanofibrous ECM molecules to construct images.

It has been used to image collagen fibers and cell-ECM interactions5,106.

Fig. 6 is a projection of a three-dimensional collagen matrix obtained by

backscattering confocal microscopy with an illumination wavelength of

488 nm. In three-dimensional imaging, the resolution and image quality

deteriorate greatly because of light scattering when the focus depth

exceeds 30 µm. Multiwavelength excitation has been proposed to

achieve both resolution and penetration depth107. To enhance contrast,

metallic decoration108, e.g. Au nanoparticles, can be applied to fixed

specimens.

Second harmonic generation (SHG) is a second-order nonlinear

optical process in which two photons are scattered coherently into one

with twice the energy. Unlike fluorescence, SHG is a nonabsorption

process and, thus, the specimen sustains no photochemical damage.

SHG was first integrated into an optical microscope to visualize

microscopic crystal structure109. Collagen structures in rat tail

tendon110, skin111-113, cornea114,115, brain116, tumors117 and fish

embryos118 have been imaged using SHG microscopy. With

improvement in sensitivity and resolution, SHG microscopy is now used

to image three-dimensional ECM nanofibers even in tissues with



Fig. 6 Backscattered confocal image of collagen nanofibers in a microcapsuleformed by complex coacervation of methylated collagen with a HEMA-MMA-MAA terpolymer.

NT103p34_43.qxd 07/06/2006 14:44 Page 40

significant light scattering such as the liver (Fig. 7). Here, the SHG signal

is generated by collagen nanofibers without any exogenous labeling.

Since the deleterious effect of light scattering is greatly relieved with

the use of infrared light and reduced effective point-spread function119,

the imaging depth is increased to at least hundreds of microns or even

more than 1 mm for translucent specimens, and the image resolution

and quality is improved.

Three-dimensional nanofibers in the extracellular environment,

including collagen fibers, are often optically anisotropic and can be

detected by birefringence in a polarization microscope. The specimen is

illuminated by linear polarized light and the transmitted light is analyzed

by a perpendicular polarizer. The birefringence of the specimen is

converted to light intensity and registered by a detector. As shown in

Fig. 8, the birefringent nanofibers appear as bright objects in a dark

background. Polarization microscopy has been extensively used in

quantitative characterization of collagen in unstained tissues120. To

enhance birefringence, collagen nanofibers in fixed tissues are normally

labeled with Sirius red, where the dye molecules align along the collagen

fibers121.

Chemical characterization using fluorescence confocalmicroscopyDynamic imaging of three-dimensional nanofiber scaffolds can also be

achieved by labeling using fluorescent proteins, for example122. In

general, fluorescence labeling conveys high biological specificity and

good contrast at the cost of interfering with the structural integrity of

the nanofibrous environment as well as the cellular responses. To

relieve the adverse interference effects of the large fluorescent

proteins, promising small molecule labeling techniques have been

developed123. Fluorescence resonance energy transfer (FRET) is an

optical ruler that can measure the distance between donor and

receptor fluorophores by characterizing the near-field coupling by

either fluorescence intensity or lifetime. This technique enables

discrimination between protein conformations to give insight into how

cells alter protein structure in the extracellular environment, and how

the structural changes correlate with changes in cell function124,125.

Fibronectin has been tagged with donor and receptor fluorophores at

different sites, and intramolecular FRET shows fibronectin unfolding

and stretching in response to cytoskeletal tension exerted by NIH 3T3

fibroblasts. Two-photon-excited fluorescence has also extended the

imaging depth to hundreds of microns126 for deep tissue construct

imaging.

Mechanical characterization using force-sensingmethodsDevelopments in force-sensing methods have allowed measurements

of weak forces at piconewton and even lower levels. Besides geometry

measurements, the mechanical properties of individual nanofibers can

also be derived, including the spring constant and Young’s modulus.

Atomic force microscopy (AFM) was invented about 20 years ago

as a high-resolution imaging technique127. In addition to topographic

measurements, AFM can sense piconewton forces and subnanometer

displacements, allowing studies of inter- and intramolecular forces

through force-distance curves128. Measuring the binding force

between integrins and ECM molecules129-131, and correlating this

with the stiffness of natural132 and engineered nanofibers133,

researchers can effectively employ AFM to study cell-ECM

interactions. AFM is also compatible with optical microscopy, such

that an AFM scan head can be mounted onto an inverted optical

microscope to record both mechanical properties and structural or



Fig. 8 Polarization microscopy image of electrospun collagen nanofibers.(Courtesy of Cathy Boutin, Cambridge Research and Instrumentation.)

Fig. 7 SHG image of the ECM in a 100 µm rat liver slice. The image stack wasrecorded by transmission SHG excited by a Ti:sapphire femtosecond laser at900 nm and visualized with a maximum intensity projection algorithm.

NT103p34_43.qxd 07/06/2006 14:44 Page 41



REFERENCES

1. Rosso, F., et al., J. Cell. Physiology (2004) 119999, 174

2. Cukierman, E., et al., Curr. Opin. Cell Biol. (2002) 1144, 633

3. Flemming, R. G., et al., Biomaterials (1999) 2200, 573

4. Lutolf, M. P., and Hubbell, J. A., Nat. Biotechnol. (2005) 2233, 47

5. Ng, S., et al., Biomaterials (2005) 2266, 3153

6. Stevens, M. M., and George, J. H., Science (2005) 331100, 1135

7. Ma, Z., et al., Tissue Eng. (2005) 1111, 101

8. Zhang, S., Nat. Biotechnol. (2003) 2211, 1171

9. Hartgerink, J. D., et al., Science (2001) 229944, 1684

10. Schindler, M., et al., Biomaterials (2005) 2266, 5624

11. Fassett, J. T., et al., J. Biol. Chem. (2003) 227788, 31691

12. Chua, K. N., et al., Biomaterials (2005) 2266, 2537

13. Xu, C., et al., Tissue Eng. (2004) 1100, 1160

14. Nur-E-Kamal, A., et al., Biochem. Biophys. Res. Commun. (2005) 333311, 428

15. Meshel, A. S., et al., Nat. Cell Biol. (2005) 77, 157

16. Cukierman, E., et al., Science (2001) 229944, 1708

17. Li, S., et al., Annu. Rev. Biomed. Eng. (2005) 77, 105

18. Ingber, D. E., Circ. Res. (2002) 9911, 877

19. McBeath, R., et al., Developmental Cell (2004) 66, 483

20. Courtney, T., et al., Biomaterials (2006) 2277, 3631

21. Gemmiti, C. V., and Guldberg, R. E., Tissue Eng. (2006) 1122, 469

22. Grodzinsky, A. J., et al., Annu. Rev. Biomed. Eng. (2000) 22, 691

23. Neidert, M. R., and Tranquillo, R. T., Tissue Eng. (2006) 1122, 891

24. Paszek, M. J., et al., Cancer Cell (2005) 88, 241

25. Fringer, J., and Grinnell, F., J. Biol. Chem. (2001) 227766, 31047

26. Giannone, G., and Sheetz, M. P., Trends Cell Biol. (2006), in press

27. Wang, H.-B., et al., Proc. Natl. Acad. Sci. USA (2001) 9988, 11295

28. Nishizaka, T., et al., Proc. Natl. Acad. Sci. USA (2000) 9977, 692

29. Koo, L. Y., et al., J. Cell Sci. (2002) 111155, 1423

30. Yang, F., et al., Biomaterials (2005) 2266, 2603

functional information simultaneously. The correlation between the

two sets of information provides quantitative insight into cellular

responses to three-dimensional nanofibrous environments with spatial

and temporal arrangements of the extracellular mechanical and

chemical cues.

Optical tweezers use light to manipulate microscopic objects, even

single atoms at low temperatures134,135. The fundamental principle

relies on the radiation pressure exerted by a light beam on dielectric

objects. The trapping force on a single atom or molecule is tiny. To

manipulate a single molecule at room temperature, ‘handles’, for

example polystyrene beads, are usually tethered to it. Besides

manipulation of tiny objects, optical tweezers can be used to measure

intermolecular forces in the range of piconewtons136 and the elasticity

of fibers137. By tethering polystyrene beads to nanofibers, optical

tweezers are able to bend fibers and measure their stiffness. Multipoint

measurements in three dimensions can be more readily implemented

here than in AFM. Besides single fibers, optical tweezers can characterize

the rheological properties of defined volumes through localized

viscoelastic measurements138. The rheological properties are dependent

on the macro- or supramolecular structure of the ECM or three-

dimensional nanofibrous environment.

Future outlookSystematic studies at a molecular level on cellular responses to three-

dimensional nanofibrous matrices have so far been conducted mainly

with natural ECMs, despite the existence of fabrication technologies

capable of engineering nanofibers with complex features. There is a

great need to use these engineered nanofibrous substrates for the

systematic understanding of cell-matrix interactions and the iterative

process of designing novel biomaterials for specific applications. Small,

three-dimensional nanofiber inserts have recently been marketed as

three-dimensional surfaces for cell culture (Ultra-Web™ Synthetic ECM

by Donaldson). Commercial production of this and other, larger

nanofiber substrates will accelerate the use of such three-dimensional

environments in cell biology research as well as tissue engineering

applications. Three-dimensional nanofibers and scaffolds with greater

uniformity and controllable mechanical/chemical properties will

become possible as new instruments and processes are developed.

These advances, together with imaging and force-sensing techniques

for probing nanofibers in three-dimensions over time, will support

research on how different molecular and cellular processes respond to

these scaffolds at subcellular resolutions. LTH will be tested and

refined. Systematic and quantitative measurements of molecular and

cellular parameters at discrete sites of cell-ECM interaction, as well as

at the whole-cell level, will enable the construction of computational

and systems biology models of cellular responses to nanofibers of

various mechanical and chemical properties, distributed spatially and

temporally. Such models will guide the further development of new

generations of three-dimensional nanofibrous environments for

applications.

AcknowledgmentsWe would like to thank P. C. Cheng (Buffalo, New York) and Cathy Boutin(Cambridge Research and Instrumentation) for their kind assistance in imageacquisition. We acknowledge technical support and stimulating discussionswith other members of the Cell and Tissue Engineering Program at the Instituteof Bioengineering and Nanotechnology (IBN), A*STAR, Singapore. This work issupported in part by intramural funding of the Biomedical Research Councilthrough IBN. YCT, SN, and PCL are A*STAR graduate scholars. YMK, XZ, andYJZ are National University of Singapore graduate research scholars.

NT103p34_43.qxd 07/06/2006 14:44 Page 42

31. Venugopal, J., et al., Cell Biol. Int. (2005) 2299, 861

32. Jiang, G., et al., Nature (2003) 442244, 334

33. Jiang, G., et al., Biophys. J. (2006) 9900, 1804

34. Webb, D. J., et al., Nat. Cell. Biol. (2004) 66, 154

35. Zaidel-Bar, R., et al., J. Cell Sci. (2003) 111166, 4605

36. Giannone, G., et al., J. Cell Biol. (2003) 116633, 409

37. Laukaitis, C. M., et al., J. Cell Biol. (2001) 115533, 1427

38. Zamir, E., et al., Nat. Cell Biol. (2000) 22, 191

39. Ng, S., et al., Tissue Eng. (2006), in press

40. Vogel, V., and Sheetz, M. P., Nat. Rev. Cell Mol. Biol. (2006), in press

41. Tran, H., et al., J. Cell Sci. (2002) 111155, 2031

42. Ong, L.-L., et al., J. Biol. Chem. (2003) 227788, 32115

43. Ong, L. L., et al., J. Biol. Chem. (2006), in press

44. Chicurel, M. E., et al., Nature (1998) 339922, 730

45. Benecke, B. J., et al., Cell (1978) 1144, 931

46. Choquet, D., et al., Cell (1997) 8888, 39

47. Lo, C.-M., et al., Biophys. J. (2000) 7799, 144


49. Zhang, Y., et al., J. Mater.Sci.: Mater. Med. (2005) 1166, 933

50. Dieitzel, J. M., et al., Polymer (2001) 4422, 8163

51. Shin, Y. M., et al., Polymer (2001) 4422, 9955

52. Kenawy, E.-R., et al., J. Controlled Release (2002) 8811, 57


54. Xu, C. Y., et al., Biomaterials (2004) 2255, 877

55. Lee, C. H., et al., Biomaterials (2005) 2266, 1261

56. Sahoo, S., et al., Tissue Eng. (2006) 1122, 91

57. Tuzlakoglu, K., et al., J. Mater. Sci.: Mater. Med. (2005) 1166, 1099

58. Kidoaki, S., et al., Biomaterials (2005) 2266, 37

59. Stankus, J. J., et al., Biomaterials (2006) 2277, 735

60. Holmes, T. C., Trends Biotechnol. (2002) 2200, 16

61. Beniash, E., et al., Acta Biomater. (2005) 11, 387

62. Rajagopal, K., and Schneider, J. P., Curr. Opin. Struct. Biol. (2004) 1144, 480

63. Lashuel, H. A., et al., J. Am. Chem. Soc. (2000) 112222, 5262

64. Hsieh, P. C. H., et al., J. Clinical Investigation (2006) 1166, 237

65. Harrington, D. A., et al., J. Biomed. Mater. Res. Part A (2006), in press

66. Zhang, S., et al., Seminars Cancer Biol. (2005) 1155, 413

67. Narmoneva, D. A., et al., Biomaterials (2005) 2266, 4837

68. Uludag, H., et al., Adv. Drug Delivery Rev. (2000) 4422, 29

69. Bhatia, S. R., et al., Curr. Opin. Colloid Interface Sci. (2005) 1100, 45

70. Chia, S.-M., et al., Tissue Eng. (2000) 66, 481

71. Toh, Y. C., et al., Biomaterials (2005) 2266, 4149

72. Toh, Y.-C., et al., Assay Drug Development Technol. (2005) 33, 169

73. Wan, A. C. A., et al., Macromolecules (2004) 3377, 7019

74. Wan, A. C. A., et al., Adv. Mater. (2006) 1188, 641

75. Wan, A. C. A., et al., J. Biomed. Mater. Res. Part A (2004) 7711AA, 586

76. Ma, P. X., and Zhang, R., J. Biomed. Mater. Res. (1999) 4466, 59

77. Chen, V. J., et al., Biomaterials (2006) 2277, 3973

78. Bognitzki, M., et al., Polymer Eng. Sci. (2001) 4411, 982

79. Boland, E. D., et al., J. Macromol. Sci.-Pure Appl. Chem. (2001) 3388, 1231

80. Matthews, J. A., et al., Biomacromolecules (2002) 33, 232

81. Min, B.-M., et al., Biomaterials (2004) 2255, 1289

82. Bhattarai, N., et al., Biomaterials (2005) 2266, 6176

83. Um, I. C., et al., Biomacromolecules (2004) 55, 1428

84. Schindler, M., et al., Biomaterials (2005) 2266, 5624

85. Li, W.-J., et al., Biomaterials (2005) 2266, 599

86. Zong, X., et al., Biomaterials (2005) 2266, 5330

87. Yoshimoto, H., et al., Biomaterials (2003) 2244, 2077

88. Zhang, S., et al., Biomaterials (1995) 1166, 1385

89. Petka, W. A., et al., Science (1998) 228811, 389

90. Nowak, A. P., et al., Nature (2002) 441177, 424

91. Holmes, T. C., et al., Proc. Natl. Acad. Sci USA (2000) 9977, 6728

92. Semino, C. E., et al., Tissue Eng. (2004) 1100, 643

93. Semino, C. E., et al., Differentiation (2003) 7711, 262

94. Kisiday, J., et al., Proc. Natl. Acad. Sci USA (2002) 9999, 9996

95. Hartgerink, J. D., et al., Proc. Natl. Acad. Sci USA (2002) 9999, 5133

96. Silva, G. A., et al., Science (2004) 330033, 1352

97. Schneider, J. P., et al., J. Am. Chem. Soc. (2002) 112244, 15030

98. Potekhin, S. A., et al., Chem. Biol. (2001) 88, 1025

99. Ryadnov, M. G., and Woolfson, D. N., Nat. Mater. (2003) 22, 329

100. Zhou, M., et al., J. Am. Chem. Soc. (2004) 112266, 734

101. Wei, G. B., and Ma, P. X., Biomaterials (2004) 2255, 4749

102. Liu, X., et al., Biomaterials (2006) 2277, 3980

103. Woo, K. M., et al., J. Biomed. Mater. Res. Part A (2003) 6677AA, 531

104. Yang, F., et al., Biomaterials (2004) 2255, 1891

105. Petroll, W. M., and Ma, L., Cell Motil. Cytoskeleton (2003) 5555, 254

106. Brightman, A. O., et al., Biopolymers (2000) 5544, 222

107. Friedl, P., Histochem. Cell Biol. (2004) 112222, 183

108. Cheng, P. C., and Kriete, A., In Handbook of Biological Confocal Microscopy, Pawley, J. B., (ed.), Plenum Press, New York, (1995), 281

109. Hellwarth, R., and Christensen, P., Optics Commun. (1974) 1122, 318

110. Freund, I., et al., Biophys. J. (1986) 5500, 693

111. König, K., and Riemann, I., J. Biomed. Opt. (2003) 88, 432

112. Yasui, T., et al., J. Biomed. Opt. (2004) 99, 259

113. Lin, X. S., et al., Prog. Biochem. Biophys. (2004) 3311, 83

114. Yeh, A. T., et al., Opt. Lett. (2002) 2277, 2082

115. Han, M., et al., J. Biomed. Opt. (2004) 99, 760

116. Dombeck, D. A., et al., Proc. Natl. Acad. Sci. USA (2003) 110000, 7081

117. Brown, E., et al., Nat. Med. (2003) 99, 796

118. Sun, C. K., et al., J. Struct. Biol. (2004) 114477, 19

119. Gan, X., and Gu, M., Opt. Lett. (1999) 2244, 741

120. Király, K., et al., Histochem. J. (1997) 2299, 317

121. Kocsis, K., et al., Microsc. Res. Tech. (1998) 4433, 511

122. Heim, R., and Tsien, R. Y., Curr. Biol. (1996) 66, 178

123. Griffin, B. A., et al., Science (1998) 228811, 269

124. Baneyx, G., et al., Proc. Natl. Acad. Sci. USA (2001) 9988, 14464

125. Baneyx, G., et al., Proc. Natl. Acad. Sci. USA (2002) 9999, 5139

126. Denk, W., et al., Science (1990) 224488, 73

127. Binnig, G., et al., Phys. Rev. Lett. (1986) 5566, 930

128. Cappella, B., et al., IEEE Eng. Med. Biol. (1997) 1166, 58

129. Lehenkari, P. P., and Horton, M. A., Biochem. Biophys. Res. Commun. (1999) 225599, 645

130. Moy, V. T., et al., Science (1994) 226666, 257

131. Sun, Z., et al., Am. J. Physiol. Heart Circ. Physiol. (2005) 228899, H2526

132. Graham, J. S., et al., Exp. Cell Res. (2004) 229999, 335

133. Lee, S. H., et al., Mater. Sci. Eng. A (2005) 339988, 77

134. Ashkin, A., Phys. Rev. Lett. (1970) 2244, 156

135. Chu, S., Science (1991) 225533, 861

136. Kuo, S. C., and Sheetz, M. P., Science (1993) 226600, 232

137. Smith, S. B., et al., Science (1996) 227711, 795

138. Velegol, D., and Frederick, L., Biophys. J. (2001) 8811, 1786.



NT103p34_43.qxd 07/06/2006 14:44 Page 43

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Cellular responses to a nanofibrous environment