Published April 30, 2012 JCB: Review

Matrix nanotopography as a regulator of cell function

Deok-Ho Kim,1 Paolo P. Provenzano,2 Chris L. Smith,3 and Andre Levchenko3
1
Department of Bioengineering, University of Washington, Seattle, WA 98195
2
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
3
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21218

The architecture of the extracellular matrix (ECM) directs pathophysiologic and physiological events ranging from glioma
cell behavior by providing spatial and mechanical cues to progression (Fig. 1 C) to development and remodeling of car-
diovascular tissue (Fig. 1 D). As cells contain nanoscale fea-
which cells respond. In addition to soluble chemical factors,
tures whose sizes are compatible with these ECM structures,
physical interactions between the cell and ECM regulate such as focal contacts/adhesions, and fine processes (e.g., cilia
primary cell processes, including differentiation, migra- and filopodia), one can reasonably conclude that nanoscale
tion, and proliferation. Advances in microtechnology and, features of ECM influence cell function in vivo.
THE JOURNAL OF CELL BIOLOGY

more recently, nanotechnology provide a powerful means Despite the complex topographical features of 3D cell

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to study the influence of the ECM on cell behavior. By re- microenvironments in various tissues in vivo (Fig. 1), most
in vitro studies examine cells cultured on flat 2D rigid sub-
capitulating local architectures that cells encounter in vivo,
strates. Although these studies have been instrumental in elu-
we can elucidate and dissect the fundamental signal cidating fundamental principles in cell biology, they do not
transduction pathways that control cell behavior in critical recapitulate the complexity found in 3D microenvironments.
developmental, physiological, and pathological processes. This difference is important, as extensive experimental evi-
dence suggests that cell behavior is often profoundly differ-
ent in deformable 3D matrices versus flat, rigid 2D culture
substrates made of glass or plastic (Cukierman et al., 2001;
Grinnell, 2003; Yamada and Cukierman, 2007). Arguably, the
Introduction primary reason for consistent use of standard 2D culture is its
Living tissues are intricate ensembles of multiple cell types ease and simplicity. Indeed, it is considerably more difficult
embedded in a complex, well-defined ECM. The ECM pos- to measure and control the details of 3D microenvironments.
sesses topographical and adhesive features ranging from nano- Thus, a useful compromise allows one to mimic aspects of the
meters to micrometers. Many ECM proteins form large-scale natural 3D cell environment while retaining the convenience of
structures up to several hundred micrometers in size that inter- working in 2D. Recent technological advances have made this
act with multiple individual cells to coordinate complex multi- possibility a reality.
cellular behavior. For instance, collagen fibrils, with diameters The utilization of microtechnology has heralded attractive
ranging from 20–200 nm, can form hierarchically structured innovations to classical cell biology experimentation (Bhadriraju
microscale collagen fibers (Birk et al., 1989, 1995; Canty et al., and Chen, 2002; Paguirigan and Beebe, 2008), including the
2004). Interestingly, the nano- and microscale architecture of use of sophisticated microfluidic devices that allow detailed
these fibrils/fibers influence cell polarity and promote migra- analysis and control of live cells (Li et al., 2003; Gupta et al.,
tion along collagen fibrils by providing contact guidance cues 2010). Microengineered or micropatterned cell adhesion sub-
(Dickinson et al., 1994; Wang et al., 2002; Meshel et al., 2005; strates have enabled selective cell attachment to study cell pheno­
Provenzano et al., 2008; Perentes et al., 2009). Basement mem- type and signaling (Chen et al., 1997; Tan et al., 2003; Xia
brane complexes are another class of ECM superstructures. et al., 2008). As a result, the impact of microscale ECM fea-
Recent ultrastructural analysis using EM reveals that the base- tures has been well documented and is viewed as being central
ment membrane of epithelia and endothelia exhibits a complex to many cellular functions (Lim and Donahue, 2007; Ruiz and
3D texture in the nanometer range (Fig. 1 A; Abrams et al., Chen, 2008). More recent advances in nanoscale fabri­cation
2002, 2003). Moreover, during several pathological conditions, techniques have allowed rapid accumulation of evidence sup-
such as cancer cell invasion, the ECM is commonly remodeled porting the significance of ECM nanotopography on cellular
(Fig. 1 B), whereas ECM architecture influences numerous
© 2012 Kim et al.  This article is distributed under the terms of an Attribution–Noncommercial–
Correspondence to Deok-Ho Kim: [email protected]; or Andre Levchenko: Share Alike–No Mirror Sites license for the first six months after the publication date (see
http://www.rupress.org/terms). After six months it is available under a Creative Commons
[email protected]
License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at
Abbreviation used in this paper: hMSC, human mesenchymal stem cell. http://creativecommons.org/licenses/by-nc-sa/3.0/).

The Rockefeller University Press

J. Cell Biol. Vol. 197 No. 3  351–360
www.jcb.org/cgi/doi/10.1083/jcb.201108062 JCB 351
 Published April 30, 2012

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Figure 1.  Anatomical features of highly oriented ECM in various tissues. (A) SEM images showing interaction of an aortic endothelial cell with the base-
ment membranes in an intact vessel. The left image shows the edges of the cell membrane (arrowheads) interacting with the rough ECM structures forming
the basement membrane (asterisks). The boxed area marks the area that is magnified in the right image and displays an end process of the cell membrane
adhering to the basement membrane. The higher magnification allows better visualization of the bumps, ridges, pits, and grooves forming the complex
topography of the basement membrane. The arrow marks the area magnified in the inset (top right), which highlights the specific interaction between an
end foot of the cell membrane and the nanotopography of the ECM. The images are adapted from Liliensiek et al. (2009), reprinted with permission from
Mary Ann Liebert, Inc. (B) Remodeling of ECM structures by motile HT1080 fibrosarcoma cells. Transition from individual to collective invasion is displayed
in 3D spheroids cultured within a 3D collagen lattice. Single cells (white arrowheads) generate small proteolytic tracks (black arrowheads in inset; detected
by cleavage site–specific COL2 3/4C antibody) that become further remodeled and widened by solid strands of multiple cells (Str). The box marks the area
magnified in the inset (bottom right). Images are from Friedl and Wolf (2008), reprinted with permission from American Association for Cancer Research.
(C, left) Invading brain tumor cells move along the corpus callosum of the human brain. An MRI of brain tumor (bright mass) and cells migrating along the
corpus callosum (arrow) is shown. (right) Individual brain cancer cells (arrow) can be seen migrating along myelinated fibers (blue) of white matter tracts.
The images are adapted from Bellail et al. (2004), reprinted with permission from Elsevier. (D) SEM images of ex vivo myocardium of adult rat heart. The
side view (top) and top view (middle) show well-aligned myocardium. The inset in the middle image and the magnified view (bottom) demonstrate that
the structural organization of the myocardium correlates with matrix fibers (arrows) aligned in parallel. Bars, 10 µm. The images are adapted from Kim
et al. (2010b), reprinted with permission from Proceedings of the National Academy of Sciences USA.

behavior (Curtis, 2004; Bettinger et al., 2009; Kim et al., 2010a). Nanotopographic cues as regulators
A variety of nanoscale topographic features, <1 µm in size, can of cell behavior
now be incorporated into in vitro experimental platforms to Nanotopography-induced changes in cell shape and
model the structural and mechanical intricacies of 3D in vivo polarity. Characteristic functions of complex tissues depend
ECM environments. Experiments using engineered substrates heavily on cell shape and polarity (Chen et al., 1997; McBeath
with nanoscale features (Fig. 2) demonstrate that nanotopog- et al., 2004; Kim et al., 2010b). For instance, establishment and
raphy strongly influences cellular form and function. However, maintenance of epithelial cell polarity is critical for organ de-
detailed understanding of the mechanisms by which nanotopo- velopment (Bryant and Mostov, 2008; Miller and McCrea, 2010),
graphic cues are integrated to regulate cell behaviors remains and loss of cell polarity is involved in many human disease pro-
largely unknown, but, with growing advances in nanotechnology cesses, including cancer metastasis (Bryant and Mostov, 2008;
tools, we are poised to answer fundamental questions at the Petrie et al., 2009). Cell–cell- and cell–matrix-associated signaling
single-cell level. Here, we survey current efforts using nanoscale complexes, particularly the Par (partitioning defective) complex
technologies to investigate the influences of ECM nanotopogra- (composed of Par3, Par6, and aPKC) and associated Rho GTPase
phy on cell behaviors, such as polarity, migration, proliferation, signaling, have emerged as regulators of polarity during numerous
and differentiation. cellular processes, including basal–apical polarity, asymmetric cell

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division, and directional migration (Etienne-Manneville and Hall,

2003; Aranda et al., 2008; Petrie et al., 2009). However, the role of
local ECM architecture on cell polarity is not well understood.
Substrate topography can strongly influence the polarity
of many different cell types through a process known as contact
guidance (Dunn and Ebendal, 1978; Clark et al., 1991; Dickinson
et al., 1994). Arrays of parallel nanogrooves, or nanoridges
(Fig. 2), have been used to provide an in vitro experimental
model of the nanotopography of the in vivo ECM. Interestingly,
contact guidance cues from these substrates compel multiple
cell types to preferentially elongate and align parallel to these
grooved nanopatterns (Fig. 3 A, left; Rajnicek et al., 1997;
Rajnicek and McCaig, 1997; Karuri et al., 2004; Teixeira et al.,
2004; Diehl et al., 2005; Yim et al., 2005; Biela et al., 2009;
Kim et al., 2009a, 2010b). Neurons in the peripheral nervous
system polarize along nanogrooves fabricated specifically to imi-
tate polarization along neurite bundles (Nagata and Nakatsuji,
1991; Nagata et al., 1993), suggesting that nanotopographic
features of neurite bundles may regulate polarity of neuronal

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cells. Furthermore, quantitative analysis with corneal epithelial
cells showed that the extent of alignment varied with the specific
dimensions (e.g., depth and pitch) of the nanogrooves and culture Figure 2.  Nanotopographically defined in vitro cell culture tools. Sche-
matics of three representative nanotopography geometries commonly used
media conditions, suggesting a synergy between substrate archi-
as cell culture substrates, including nanogroove/ridge arrays, nanopost
tecture and soluble chemical factors (Teixeira et al., 2003, 2006). arrays, and nanopit arrays. Anisotropic topographies are directionally de-
The geometry of the underlying nanotopography also affected pendent, in this case, providing cues along a single axis. Isotropic topog-
raphies are uniform in all directions, providing cues along multiple axes.
cell shape and polarity (Fig. 3, B and C). Filopodia and lamel-
Topography gradients provide cues through gradual changes in physical
lipodia generally extended parallel to the underlying nanoridge features (e.g., groove spacing) along a particular direction. Schematics
features with aligned actin stress fibers and vinculin-positive are not drawn to scale. The ranges of relevant feature sizes could vary
between 100 nm and 1 µm, depending on the design dimensions of the
focal adhesions that scaled with the width of the underlying
substrates’ nanofeatures.
substrate (Teixeira et al., 2003, 2006). The nanoscale width of
the grooves can also limit cell penetration into them, thus limiting
the cell–substratum adhesion (Fig. 3 D). Yet, the role of these and metastasis (Sahai, 2007; Petrie et al., 2009; Friedl and
features in regulating cell polarity in response to nanotopography Wolf, 2010). Although a great deal is known about the fun-
remains poorly understood. However, these studies do demon- damental mechanisms of cell migration, the molecular mecha-
strate that even extremely small (nanoscale) differences in architec- nisms by which cells integrate signals resulting from local 3D
ture can profoundly influence cell polarity and provide a unique matrix architectures to promote directional migration are not
forum to dissect the molecular mechanisms driving cell polarity. well understood (Petrie et al., 2009). In conjunction with its ef-
The influence of nanoarchitecture on cell morphology is fects on cell polarity, nanotopography influences cell migration.
further exemplified by the cells’ ability to acutely sense vari- Numerous human cell types exhibit enhanced motility when
ability in topographic cues. For instance, nanoscale differences grown on nanostructured surfaces (Ranucci and Moghe, 2001;
in ligand spacing significantly influenced polarity of osteoblasts Mello et al., 2003; Brammer et al., 2008). In contrast to flat sub-
and fibroblasts (Arnold et al., 2008; Kim et al., 2009a). Interest- strates where the trajectory of migration is essentially random,
ingly, growing NIH 3T3 cells on a gradient of nanoridges with corneal epithelial cells migrate parallel to nanoridges (Diehl
varying groove widths led to increased cell polarity on denser et al., 2005), consistent with in vivo findings with carcinoma
patterns, i.e., those with narrower groove widths (Fig. 3 A, right; cells migrating along aligned ECM (Provenzano et al., 2006;
Kim et al., 2009a). Organization of the actin cytoskeleton was Provenzano et al., 2008). Furthermore, neutrophils migrate more
also sensitive to the local topographic pattern density. Polarized rapidly on grooved surfaces than on flat surfaces (Tan and
fibroblasts aligned with dense groove patterns showed aligned Saltzman, 2002), which may have important implications for
actin stress fibers and robust focal adhesions at the cell’s leading immune surveillance as well as wound repair in which ECM
edge. In contrast, on locally sparser patterns (i.e., those with architecture is disrupted and reestablished. Hence, these studies
greater groove widths), vinculin-positive focal adhesions were bring to light the influence of nanoscale architectures as regula-
more randomly distributed, demonstrating that varying topogra- tors of directional migration, a behavior that is important during
phy alters adhesion organization, which may play a role in dic- both normal and pathological conditions such as proper tissue
tating cell polarity. organization during development, wound repair, and focal inva-
Nanotopographic guidance during cell migra- sion leading to metastasis.
tion. Cell migration is essential for numerous physiological and Cell migration is also sensitive to variation in the density
pathological processes, including embryogenesis, wound repair, of nanotopographic cues. For example, the migration speed of

Matrix nanotopography regulates cell function • Kim et al. 353

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Figure 3.  Mechanosensitivity of cells to changes in substrate nanotopography. (A) SEM images of NIH 3T3 fibroblasts cultured on nanoridge arrays with
regular spacing (left) and graded spacing (right). The yellow rectangle represents regularity in spacing between nanoridges, whereas the triangle repre-
sents a gradient of spacing between the nanoridges. The white arrow indicates membrane protrusion extending toward more closely spaced ridges. The

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images are adapted from Kim et al. (2009a), reprinted with permission from Elsevier. (B) Representative SEM image of the morphology of cells on square
lattice pattern arrays of different local densities. The gray triangles describe the changing dimensions of the rectangular gaps in the lattice. A magnified
image at the motile edge is shown in the inset. The images are adapted from Kim et al. (2009b), reprinted with permission from Wiley. (C) Directed cell
migration along grooves of different curvature. Bar, 50 µm. (D) Differential degree of primary cardiac cell protrusion into a 400-nm-wide (left) and an
800-nm-wide (right) groove. Mf, myofilament. Bars, 200 nm. The images are adapted from Kim et al. (2010b), reprinted with permission from Proceedings
of the National Academy of Sciences USA.

NIH 3T3 fibroblasts is dependent on the width of nanopatterned modulated have an increased potential to provide additional in-
ridges. With this biphasic dependence, nanoridges that are either sights into the physical mechanisms of cell motility. With nano­
too close or too far apart do not promote directed migration to scale tools, quantitative models can be tested with additional
the degree of nanoridges 200–300 µm apart (Kim et al., 2009a), rigor, and more complex models can be formulated to gain
suggesting a complex interplay between cell sensing of micro­ valuable insights into the mechanisms of cell migration. For
scale and nanoscale features. In these cases, cell movement could example, seminal work by Lauffenburger and Horwitz (1996)
be guided by gradients in patterned substrates, which can be provided an important insight into the physical mechanisms
termed topotaxis (i.e., directional migration toward particular of cell migration on planar surfaces. Later work has confirmed
topography density gradients), as seen by the trajectories of early findings and produced additional mechanistic data to des­
migrating fibroblasts toward denser regions of patterns (Fig. 3 A; cribe mechanical aspects of cell migration (Gardel et al., 2010).
Kim et al., 2009a). Interestingly, this behavior was also observed As many of the fundamental processes that regulate cell
for both individual and collective cell migration, suggesting a role migration identified to date operate on the microscale and,
for matrix nanotopography in coordinated cellular responses such particularly, the nanoscale (Arnold et al., 2008; Gardel et al.,
as wound repair. Aggregation and orientation of fibroblasts in 2010; Kanchanawong et al., 2010), tools specific to these scales
areas of optimal ECM content and organization might facilitate should be instrumental in obtaining new findings that can shape
very important functions of these cells in the reconstruction of more complete quantitative models to accurately describe cell
damaged ECM. As ECM is repaired and the density of orga- migration in complex microenvironments.
nized matrix is increased, fibroblasts can naturally migrate to Nanotopographic control of cell proliferation.
areas of less-organized matrix, thus ensuring self-organized repair Cell proliferation is heavily influenced by the structure of the
propagation (Fig. 4). By analyzing fibroblasts grown on a rect- surrounding microenvironment. Seminal work by Chen et al.
angular lattice of variable local density and anisotropy (Fig. 3 B), (1997) defined a role for cell shape as a determinant of cell pro-
which mimics the complexity of randomly organized ECM com­ liferation and apoptosis using microscale-patterned substrates.
po­nents, preferential migration and planar congregation toward More recent work demonstrated that proliferation in human cell
areas of optimal topographic density were observed (Kim et al., lines is sensitive to nanoarchitecture by culturing cells on sub-
2009b). The direction of cell movement was also guided by gro­ strates consisting of randomized nanoscale bumps or nanois-
oves of different curvature (Fig. 3 C). These results support the lands of varied heights <100 nm (Lim et al., 2005; Milner and
conclusion that nanotopography may influence cell localization Siedlecki, 2007). Cells maintained significantly lower densities
and migration during tissue repair. Thus, cell motility can be en- and lower rates of proliferation on patterns with larger nanoscale
hanced and directed by both the local density and anisotropy of features. Continued reduction of feature sizes eliminated the dis­
the nanoscale features of their immediate matrix environment. crepancy between nanotextured and flat substrates with respect
Studies in which surface architecture, stiffness, ligand to cell proliferation (Milner and Siedlecki, 2007). Indeed, cell
type and density, and chemical stimuli can be simultaneously proliferation in multiple cell types is reduced on nanopatterned

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surfaces when compared with flat substrates (Yim et al., 2005;

Liliensiek et al., 2006; Bettinger et al., 2008; Dulgar-Tulloch
et al., 2009), which may explain, in part, why cells proliferate at
much greater rates in vitro than in native in vivo microenviron-
ment. In fact, corneal epithelial cells showed reduced prolifera-
tion when cultured on nanogrooves, mimicking the effective
sizes of collagen fibrils in the corneal stroma (Liliensiek et al.,
2006). Furthermore, culturing cells on surfaces with nanoscale
roughness showed that even altering roughness by a few nano-
meters influenced cell proliferation (Washburn et al., 2004;
Brunetti et al., 2010), highlighting the exquisite sensitivity to
even minute nanoscale cues.
Further support for the extreme sensitivity of cell prolif-
eration to the specific size of nanoscale features was observed
through experiments with rat neural stem cells cultured on meshes
composed of nanofibers (Christopherson et al., 2009). The num-
ber of proliferative cells in a population depended heavily on the
diameters of the nanofibers, wherein proliferation increased with
decreasing fiber size. In contrast, Oh et al. (2006) reported en-

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hanced proliferation of mouse osteoblasts on hollow nanotubes
of diameters near 100 nm versus the flat control. Hence, nanotop-
ography may regulate cell proliferation in a cell-specific manner, Figure 4.  Model of the role of fibroblasts in ECM remodeling during wound
consistent with differences in cell proliferation found for cells healing. During the wound healing process, fibroblasts are functionally
essential cells that migrate (arrows) toward and into the open wound, deposit
residing in diverse ECM microenvironment in vivo. collagen, and restructure the ECM. Fibroblasts can aggregate in zones
Nanotopographic control of cell differentia- of intermediate ECM density that may become the zones of active ECM
tion. During development and tissue homeostasis, pluripotent repair, which can change as cells progressively create denser matrix and
clear these now dense zones for the less-dense adjacent zone. This, along
cells integrate numerous signals that determine their ultimate with apoptosis, can be an efficient mechanism of cell clearance from the
fates. Among the numerous cues affecting differentiation (soluble restructured wound zones before reepithelialization. The schematics are
factors, ECM composition, etc.), the nanotopography of the cells’ adapted from Kim et al. (2009a), reprinted with permission from Elsevier.
surroundings also plays an important role (Kshitiz et al., 2011).
For instance, either nanopits or nanotubes stimulated osteogenic differentiate in the presence of NGF, the authors found that the
differentiation of human mesenchymal stem cells (hMSCs) in NGF threshold for induction of neuritogenesis was lowered
the absence of osteogenic induction media (Dalby et al., 2007; when combined with specific surface nanotopography (Foley
Oh et al., 2009), suggesting that nanotopography may be suffi- et al., 2005). Hence, these studies suggest that nanotopographic
cient to guide differentiation but not optimal, as osteogenesis cues of precise dimensions can be used to bias precursor, pluripo-
of hMSCs was synergistically enhanced by culture on nano- tent, and adult stem cells toward particular fates; yet, the molecu-
structured surfaces with osteogenic induction media (You et al., lar mechanisms driving these processes are not currently known.
2010). Likewise, Sjöström et al. (2009) reported skeletal differ-
entiation by exposing hMSCs to nanopillar structures of different Synergistic effects of nanotopography
heights (0–100 nm), finding maximal differentiation on pillars and mechanical stimuli on cell motility
of 15 nm, again highlighting the influence of even subtle differ- In the complex microenvironment, cells encounter a multitude of
ences in nanoarchitecture on cell behavior. distinct, simultaneously active stimuli that are biochemical, struc-
In addition to hMSC osteogenic differentiation, the role tural, and mechanical in nature. For example, directed migration
of nanotopography as a factor promoting differentiation toward can be controlled by many different mechanisms, which may in-
other lineages has been investigated. Dang and Leong (2007) clude chemotaxis, galvanotaxis (i.e., directional movement of cells
found that aligned nanofibers promote cytoskeletal reorganiza- in response to an electric field), haptotaxis (i.e., the directional
tion as well as cellular and nuclear elongation of hMSCs, which motility guided by gradients in adhesion), and durotaxis (i.e., cell
biases hMSCs toward myogenic differentiation. Nanostructures migration guided by gradients in substrate rigidity) as well as con-
have also been used to bias MSCs and mouse embryonic stem tact guidance. Cells exposed to combinatorial stimuli decipher
cells toward neuronal lineages (Christopherson et al., 2009; their crosstalk to determine cell behavior in any given situation.
Tsuji et al., 2009; Xie et al., 2009; Migliorini et al., 2011). Further- Although a limited body of work has been presented on synergistic
more, the diameter of nanofibers influenced the differentiation effects between nanoarchitecture and growth factor stimulation
of rat neural progenitor cells, showing a 40% increase in oligo- (Teixeira et al., 2003, 2006; Foley et al., 2005; Patel et al., 2007;
dendrocyte differentiation on 283-nm fibers and a 20% increase You et al., 2010) as well as nanoarchitecture and electric field
in neuronal differentiation on 749-nm fibers, relative to standard stimuli (Au et al., 2007; Rajnicek et al., 2007; Heidi Au et al.,
tissue culture surfaces (Christopherson et al., 2009). In a sepa- 2009), here, we will discuss recent exciting data demonstrating
rate study using rat pheochromocytoma cells, which terminally crosstalk between nanoarchitecture and mechanical signals.

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The rigidity of the surrounding ECM plays a role in regulat- Integrins transduce extracellular forces into biochemical
ing cell behavior (Pelham and Wang, 1997; Lo et al., 2000; Paszek signals through focal adhesions, a cluster of integrin-associated
et al., 2005; Provenzano et al., 2009; Provenzano and Keely, 2011) proteins localized at the interface between integrin receptors and
and distinctly influences cell migration (Pelham and Wang, 1997; the actin cytoskeleton. Integrins play a major role in adhesion-
Lo et al., 2000). Interestingly, the intricate nano­topographic fea- and force-dependent signal transduction, serving as key regula-
tures of the cell environment com­prise a critical element of me- tors of cell motility, growth, and differentiation (Giancotti and
chanical stimulation (Tzvetkova-Chevolleau et al., 2008; Park Ruoslahti, 1999; Katsumi et al., 2004). Interestingly, work using
et al., 2012). When using nanostructured polymeric materials to substrates with defined microscale features has provided novel
simulate the cell environment, it is possible to vary the stiffness of insight into focal adhesion signaling (Balaban et al., 2001; Tan
the substrate by altering its chemical composition. For example, et al., 2003; Goffin et al., 2006). Gallant et al. (2005) used micro­
CHO cells are more polarized along stiffer nanogrooved sub- patterned substrates to measure focal adhesion strength and
strates (Park et al., 2012), whereas NIH 3T3 and cancerous SaI/N reported a novel role for FAK, a primary regulator of focal ad-
mouse fibroblasts cultured on nanoposts and nanogrooves (Fig. 2) hesion signaling, in integrin activation and adhesion strengthening
display changes in morphology and motility in response to modu- (Michael et al., 2009). Extending these studies using nanopat-
lated nanosubstrate rigidity (Tzvetkova-Chevolleau et al., 2008). terning techniques presents cell-adhesive signals on scales com-
NIH 3T3 fibroblasts obtained more polarized morphologies on parable with individual focal adhesion complexes (Arnold et al.,
softer patterned nanosubstrates, whereas SaI/N fibroblasts demon- 2004; Zhu et al., 2005) and subsequently provides tools to faci­
strate enhanced migration speed on patterned surfaces, which was litate novel insight into focal adhesion signaling. For example,
further amplified on more rigid nanosubstrates. Hence, changing CHO cell polarization along nanogrooves is 1-integrin depen-

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the stiffness of nanostructures has a profound influence on cell dent (Park et al., 2012), whereas culturing fibroblasts on nano-
motility. As cells sense the stiffness of their local environment, column substrates increases filopodia (Dalby et al., 2004). This
which in turn influences cellular traction force, and many of the fun- increased surface contact guided cell migration by regulating
damental processes that regulate cell migration are in the nanoscale the strength of focal adhesions through FAK and myosin II
(Arnold et al., 2008; Gardel et al., 2010; Kanchanawong et al., (Frey et al., 2006), whereas aligned fibers or grooves promote
2010), nanotechnology tools should be instrumental in obtaining actin polymerization and protrusion in the direction parallel
new findings regarding the physical and molecular mechanisms to the fibers/grooves (Li et al., 2005). This process can result
of cell migration. in aligned focal adhesions and traction force through the actin
In addition to force transmitted to and from the ECM, cytoskeleton in the same direction, which likely plays a role in
many cells types (e.g., vascular endothelial cells) encounter me- cell polarity and directed migration. Furthermore, the addition
chanical stimulation from fluid flow in vivo. Accordingly, many of actin-disrupting agents attenuates alignment of human em-
in vitro experimental platforms intentionally and unintentionally bryonic stem cells in response to nanotopography (Gerecht
expose cells to this phenomenon. Interestingly, bovine aortic endo- et al., 2007). These findings illustrate the importance of inter-
thelial cells respond to flow-induced shear stress differentially play between adhesion signaling, the actin cytoskeleton, and
when cultured on parallel grooves versus flat control conditions substrate interactions in mediating contact guidance.
(Uttayarat et al., 2008). Under static conditions, these cells align Other contact guidance studies have focused more heavily
their focal adhesions and migrate parallel to the grooves. High on intracellular signaling cascades. RACK1 (receptor of acti-
levels of shear stress alter this response. The number of aligned, vated protein kinase C) inhibits the response to contact guidance
parallel migrating cells are increased by parallel flow and de- by nanogrooves while positively promoting adhesion (Dalby
creased by orthogonal flow (Uttayarat et al., 2008). Hence, these et al., 2008). In hippocampal neuritis, calcium influx and pro-
studies suggest complex mechanisms for integrating multiple tein kinase C activity regulate alignment on nanoarchitectured
mechanical signals arising at different scales and modalities substrates (Rajnicek and McCaig, 1997), whereas the PI3 kinase
as well as a combinatorial influence from ECM structure that is pathway was similarly necessary for contact guidance in fibro-
yet to be well described. blasts and cardiomyocytes (Au et al., 2007). Likewise, STRO-1+
skeletal stem cells cultured on nanotopographies displayed
Mechanisms of cellular responses significant FAK-dependent down-regulation of extracellular
to nanotopography signal–regulated kinase (ERK)/MAPK signaling molecules
Significant research has now been conducted to observe the (Biggs et al., 2009). In addition, ERK/MAPK pathways exhibited
behaviors of cells in the presence of nanotopographic cues. marked changes in transcription factor expression as a result of
However, the underlying molecular mechanisms governing variation in nanoarchitecture, whereas nanotopography-induced
these processes remain elusive. One of the most challeng- changes in many MAPK pathway component proteins (e.g., heat
ing questions is: how do cells sense matrix nanotopography? shock protein 70 [Hsp70] and galectin-8 [Gal-8]) have also been
In efforts to describe these complex systems, scientists have reported (Kantawong et al., 2009).
recently used nanofabricated substrates that mimic the orga- Examination of the temporal sequence of dynamic events
nized matrix in vivo. Although, to date, few distinct answers involved in contact guidance has provided interesting insight
have arisen, initial clues come from defined micro- and na- into the mechanisms of the guidance response. Immuno­cy­to­
noscale substrates and the integrin family of transmembrane che­mical analysis of microtubules, focal contacts, and actin
adhesion molecules. filaments in fibroblasts aligned along grooved substrates

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Figure 5.  Engineering nanoarchitectures to study cell behavior in 3D environments. (A) Combined multiphoton excitation (red; endogenous cellular
fluorescence) and second harmonic generation (green; collagen) microscopy capturing 3D cell migration into magnetically aligned collagen matrices.
MDA-MB-231 breast carcinoma cells (asterisks; dashed lines highlight the leading edge of each cell) preferentially migrate through aligned collagen (top)
versus randomly oriented collagen (bottom; highlighted by #). The image was adapted from Provenzano et al. (2008), reprinted with permission from
Elsevier. (B) Bovine aortic endothelial cell expressing GFP-tubulin (green) on collagen aligned in microchannels (white) shows cell alignment (asterisks)
along the collagen matrix versus random alignment on a flat surface (#; bottom left of the micrograph). The image was adapted from Lee et al. (2006),
reprinted with permission from Springer. (C) Consistent generation of highly aligned collagen fibrils using flow-through microchannels. The image was
adapted from Lanfer et al. (2008), reprinted with permission from Elsevier. (D) SEM micrograph of polyamide nanofibers coating a glass slide. The image
was adapted from Schindler et al. (2005), reprinted with permission from Elsevier. If technologies such as these can be extended to provide highly defined
3D microenvironments with nanoscale and microscale features for embedded cells, they may be of great value to unlocking fundamental questions in cell
biology. Bars: (A, B, and C) 10 µm; (D) 5 µm.

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(Oakley and Brunette, 1993) showed that microtubules are the recapitulated on 1D microtracks of fibrillar fibronectin (Doyle
first element to orient parallel to the grooves followed by et al., 2009). Interestingly, 1D guidance more closely mimics
actin microfilaments and then focal contacts. Based on the motility in 3D matrices than planar 2D surfaces (Doyle et al.,
assumption that the initially aligned cytoskeletal component is 2009), consistent with observations of carcinoma cells moving
the major determinant of cell orientation, these findings impli- along collagen fibers in vivo (Wang et al., 2002), suggesting
cate microtubules as a key regulator of contact guidance. Combin- that fundamental aspects of 3D cell migration in vivo may be
ing live-cell time-lapse imaging with fluorescent proteins could successfully studied in more reductionist 1D and 2D culture
further enhance our knowledge of the molecular mechanisms systems that provide relevant architecture. To date, efforts to
under­lying topographic guidance. engineer 3D microenvironments with defined architecture have
focused on microscale technologies, particularly microfluidics
Applications and outlook (Lee et al., 2006; Lanfer et al., 2008; Sung et al., 2009). Using
It is clear from the numerous investigations described herein microscale channels, collagen alignment is achieved in a flow-
and elsewhere that the nanotopography of the ECM plays a crit- mediated fashion (Fig. 5, B and C; Lee et al., 2006; Lanfer et al.,
ical role in regulating cell behavior. These interactions, which 2008) and can be prepared to contain embedded cells (Fig. 5 B;
occur at an extremely small-length scale, are often overlooked Sung et al., 2009). Whereas the matrices are formed in micro-
when experiments are conducted in the context of larger-length fluidic channels, nanoscale features arise from the dimensions
scales. However, specific tools to study the influence of intri- of the comprised collagen fibrils (Fig. 5 C). As many synthetic
cate nanoscale features of the ECM are available, and scientists matrices can be integrated with natural ECM components
should now be able to take full advantage to gain additional and/or reconstituted ECMs (Fig. 5 D; Schindler et al., 2005) and
insight into fundamental mechanisms driving cell behavior. have tunable mechanical properties, such nanoscale constructs
Although no in vitro system can perfectly emulate the character- may provide valuable resources to study fundamental cell biol-
istics of the in vivo environment, these systems hold advantages ogy in 3D microenvironments.
in their reduced, but relevant, complexity. Nanotopographically Precise nanotopographic control of cell behavior will likely
defined cell culture models described in this review provide a allow better understanding of signaling and cellular functions
unique middle ground, maintaining the simplicity of traditional and inspire novel strategies to manipulate cell motility, prolif-
in vitro systems while mimicking small-scale 3D features of the eration, and differentiation. By predefining nanoarchitectures
ECM down to the molecular level. found in vivo with the capability to manipulate surface architec-
The numerous studies with micro- and nanoengineered tures as well as stiffness, ligand type and density, and chemical
substrates described in this review demonstrate influences on stimuli to essentially provide single cells with choices, a wealth
cell morphology, migration, proliferation, and differentiation. of new information becomes available. Although such levels of
In 3D, ECM composition, density, and architecture have a control are not yet available, they may play a fundamental role in
profound influence on cell behavior (Amatangelo et al., 2005; the future of nanotechnology in cell biology. For instance, it will
Alcaraz et al., 2008; Provenzano et al., 2008). For instance, be of interest to investigate whether the presentation of nanoto-
3D collagen matrices provide contact guidance, wherein cells pographic cues that mimic the anisotropic, filament-like proper-
preferentially orient parallel to aligned collagen fibrils/fibers ties of ECM would lead to results distinct from cell exposure
and migrate in the direction of collagen alignment (Fig. 5 A; to disorganized, rough nanoscale surfaces, whose features lack
Guido and Tranquillo, 1993; Dickinson et al., 1994; Dallon particular geometric definition (Washburn et al., 2004; Brunetti
et al., 1999; Provenzano et al., 2008). This behavior is often et al., 2010). By virtue of being able to vary the cues regulating

Matrix nanotopography regulates cell function • Kim et al. 357

 Published April 30, 2012

cell behavior in a very precise manner down to nanometer scale, Biggs, M.J., R.G. Richards, N. Gadegaard, C.D. Wilkinson, R.O. Oreffo, and M.J.
Dalby. 2009. The use of nanoscale topography to modulate the dynamics
quantitative models can be devised and refined by comparing of adhesion formation in primary osteoblasts and ERK/MAPK signalling
differing experimental and theoretical outcomes in response to in STRO-1+ enriched skeletal stem cells. Biomaterials. 30:5094–5103.
http://dx.doi.org/10.1016/j.biomaterials.2009.05.049
even minute variations in cellular environment. As such, one can
Birk, D.E., E.I. Zycband, D.A. Winkelmann, and R.L. Trelstad. 1989. Collagen
now revisit many of the dogmas about regulation of fundamental fibrillogenesis in situ: Fibril segments are intermediates in matrix
cellular processes with a more quantitative rigor. assembly. Proc. Natl. Acad. Sci. USA. 86:4549–4553. http://dx.doi.org/
10.1073/pnas.86.12.4549
D.-H. Kim thanks the Department of Bioengineering at the University of Washington Birk, D.E., M.V. Nurminskaya, and E.I. Zycband. 1995. Collagen fibrillogenesis
for the new faculty startup fund. D.-H. Kim is also supported by a Perkins Coie in situ: Fibril segments undergo post-depositional modifications result-
Award for Discovery. C.L. Smith acknowledges the Ford Foundation Predoc- ing in linear and lateral growth during matrix development. Dev. Dyn.
toral Fellowship. 202:229–243. http://dx.doi.org/10.1002/aja.1002020303
This work was also supported by the National Institutes of Health Brammer, K.S., S. Oh, J.O. Gallagher, and S. Jin. 2008. Enhanced cellular mo-
(CA152249 to P.P. Provenzano and EB008562 to A. Levchenko). P.P. Provenzano bility guided by TiO2 nanotube surfaces. Nano Lett. 8:786–793. http://
is also supported by a Jaconette L. Teitze Young Scientist Award. dx.doi.org/10.1021/nl072572o
Brunetti, V., G. Maiorano, L. Rizzello, B. Sorce, S. Sabella, R. Cingolani, and
P.P. Pompa. 2010. Neurons sense nanoscale roughness with nanometer
Submitted: 10 August 2011 sensitivity. Proc. Natl. Acad. Sci. USA. 107:6264–6269. http://dx.doi.org/
Accepted: 6 April 2012 10.1073/pnas.0914456107
Bryant, D.M., and K.E. Mostov. 2008. From cells to organs: Building polar-
ized tissue. Nat. Rev. Mol. Cell Biol. 9:887–901. http://dx.doi.org/10
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