Review

Integrated Circuit-Based
Biofabrication with Common
Biomaterials for Probing
Cellular Biomechanics
Chun-Yen Sung,1 Chung-Yao Yang,1 J. Andrew Yeh,1 and
Chao-Min Cheng2,*

Recent advances in bioengineering have enabled the development of biomedi-

Trends
cal tools with modifiable surface features (small-scale architecture) to mimic
Common biomaterials for probing cel-
extracellular matrices and aid in the development of well-controlled platforms lular biomechanics are described.
that allow for the application of mechanical stimulation for studying cellular
Integrated circuit (IC)-based biofabrica-
biomechanics. An overview of recent developments in common biomaterials tion methods for constructing small-
that can be manufactured using integrated circuit-based biofabrication is pre- scale devices are described.
sented. Integrated circuit-based biofabrication possesses advantages includ-
The development of small-scale struc-
ing mass and diverse production capacities for fabricating in vitro biomedical tures for regulating cell behaviors is
devices. This review highlights the use of common biomaterials that have been presented.
most frequently used to study cellular biomechanics. In addition, the influence
Cell micropatterning technology for
of various small-scale characteristics on common biomaterial surfaces for a biological applications is presented.
range of different cell types is discussed.

Small-Scale Approaches for Cellular Biomechanics

The primary objective of biomedical engineering is to leverage engineering approaches to solve
biologically relevant problems. In particular, exploring the interaction between biomaterials and
cells is a meaningful and intriguing issue in various research communities including tissue
engineering and regenerative medicine. Notably, the past decade has brought about an
ever-increasing demand for the construction of small-scale devices that can mimic native
cellular environments and facilitate insightful bi ological research [1,2]. With such technology,
researchers can probe cellular biomechanics by observing cellular responses on these artificial
structures. The mechanisms by which cells convert mechanical input to biochemical response is
called mechanotransduction or cellular biomechanics, which can be regulated through small-
scale approaches (Box 1). The knowledge gained from research into mechanotransduction in
vitro would advance our understanding of the mechanisms of physiological and pathological
processes, including greater insight into blood vessel disease, nerve repair, drug therapy, and 1
Institute of Nanoengineering and
orthopedic implants. However, the bulk of research and the lion's share of conventional Microsystems, National Tsing Hua
knowledge regarding cell differentiation and behavior have been derived from studies using University, Hsinchu 30013, Taiwan
2
Institute of Biomedical Engineering,
planar plates or coverslips with rigidity that is orders of magnitude higher than that of mammalian National Tsing Hua University,
tissue. Despite accumulated research over recent decades, the manner by which adhesion and Hsinchu 30013, Taiwan
cell signaling is coordinated by integrin(s) clustered within the cell membrane and the precise
number of integrins that may be involved in the formation of stable adhesion remain unknown.
*Correspondence:
Such conditions considerably influence the development of cellular phenotypes, cellular differ- chaomin@mx.nthu.edu.tw
entiation potential, and other cellular behaviors. Accordingly, this produces a wide gap between (C-M. Cheng).

Trends in Biotechnology, February 2016, Vol. 34, No. 2 http://dx.doi.org/10.1016/j.tibtech.2015.11.005 171

© 2015 Elsevier Ltd. All rights reserved.
 Box 1. Cellular Biomechanics Regulation
Cells live in a milieu that is modulated via cell–ECM interactions. Microscale and nanoscale structures have been shown to
significantly affect cellular growth, differentiation, morphology, and metabolic state [69,70]. Further, cells are sensitive to
microenvironmental stimulation from biophysical components of the ECM [49,71], which provide contact guidance and
affect cellular function or phenotype. Another type of stimulation, known as applied forces, includes fluidic shear fluidics
stress, cyclic stretch, and circulatory pressure, all of which are capable of imitating biophysical stimuli on vessels, tissues,
and cells via mechanical application [24,72,73]. Cellular biomechanics (i.e., ‘mechanotransduction’) is defined as the
interaction between mechanical forces and biochemical signal responses and is related to a variety of cellular responses
including the reorientation of cell shape, actin cytoskeleton remodeling, and cell differentiation [74,75]. This mechanism is
governed by actin cytoskeleton, which acts as the critical component of intracellular force and transduces outward
mechanical stimulation through the focal adhesion complex into the cell. Notably, the cell–matrix attachment is a
preliminary, sophisticated, highly regulated process that plays a crucial role in most fundamental cellular behaviors.
Among these behaviors, focal adhesions are the primary means by which cells manage surface attachment with the ECM
[76,77]. Cells adhering to the ECM induce local accumulation of integrins and cytoplasmic proteins, and leads to the
formation of focal adhesion clusters. Ensuing actin cytoskeleton remolding generates forces to the underlying material
surface via myosin molecular motors [78]. Thus, stimulation forces transmit to cells and the nucleus regulates modula-
tions of gene expression that subsequently generate biochemical signaling cascades, resulting in a change in cell
function, including proliferation, apoptosis, migration, actin remodeling, and metabolism. We know that cell–material
interface interactions are largely and intimately determined by the interplay of adhesive molecules at the interface.
Chemical modification incorporating ECM proteins (e.g., collagen, fibronectin, laminin) or specific adhesion peptide
sequences (RGD) have been shown to enhance hydrophilicity, which in turn improves biomaterial cell attachment
[6,79,80]. These adhesive molecules affect various cellular functions, including division, proliferation, cell migration,
differentiation, and structural protein distribution [25,81]. Furthermore, physical features including microscale/nanoscale
structures are designed to resemble native tissue architecture [82–84]. The dual influences of surface topography and
adhesive molecule patterns play an important role in regulating cellular biomechanics and mimicking real ECM [85].

experimental discoveries and real biological behaviors in the field of life science. To address
these challenges, the best means for optimizing scaffold architecture, including optimization of
morphology, topography, and pore size, on suitable biomaterial interfaces for probing cellular
functions must be explored. Regulation of these structural arrangements in the cell membrane
via the manipulation of material topographies and examining consequent effects may further our
understanding of focal adhesion formation.

Recent advances in integrated circuit (IC)-based techniques have enabled the development of in
vitro cell culture models that offer greater control, efficiency, consistency, and throughput [3,4].
Further, engineered architectures incorporated into tissue scaffolds can provide functional
support to cells. Generally, the most common IC-based biofabrication processes used to
integrate small-scale devices with in vitro nanomaterial scaffolds includes lithography processes
(i.e., photolithography, soft lithography, and electron beam lithography) and etching techniques
[i.e., wet etching, reactive ion etching, and metal nanoparticle-assisted etching] (Box 2). In
addition to the effects of surface architecture, mechanical stimulation of living cells is a routine
occurrence with undeniable effects on cellular behavior; as such, its effects must be replicated
for relevant physiological research. The ability to examine the effects of mechanics at the cellular
level has recently been augmented by developments in the use of small-scale devices designed
to probe the effects of force interactions between cells and surrounding structures. Our review
presents recent novel and common techniques for creating microscale and nanoscale struc-
tures on hard or soft surfaces for studying cellular behaviors following the application of various
mechanical forces (Table 1).

Actin Cytoskeleton Remodeling

In principle, geometrical control of cytoskeletal morphology may be achieved via variations in
substrate topography (physical approaches) and/or the presence of chemical alterations
(chemical approaches) [5,6]. To understand how cells respond to specific surface morphology,
various biomaterial designs with microscale/nanoscale features have been developed to probe
cytoskeletal responses. Among these approaches, those examining cell adhesion provide the
groundwork for analyzing interactions between cells and substrate surfaces as well as the

172 Trends in Biotechnology, February 2016, Vol. 34, No. 2

 Box 2. IC-Based Biofabrication
To develop various in vitro nanomaterial scaffolds and devices to regulate cellular behaviors, the most common IC-based
microfabrication/nanofabrication processes (equivalent to ‘IC-based biofabrication’ when microfabrication/nanofabrica-
tion processes are used in biological applications) used in the semiconductor industry include lithography processes (i.e.,
photolithography, soft lithography, and electron beam lithography) and etching techniques (i.e., wet etching, reactive ion
etching, and metal nanoparticles assisted etching). Specifically, photolithography has been used to transfer geometric
designs on a mask to the surface of a substrate to facilitate patterning of biomolecules and cells [58]. This process can
generate accurate patterns with submicron resolution for a host of processes, including substrate etching, chemical
micropatterning, and replica molding [86]. More recently, soft lithography, a process developed from Professor White-
sides’ research group (Harvard University), uses a soft elastomeric stamp containing relief structures to transfer patterns
with feature sizes ranging from 30 nm to 100 mm [87]. Prior to this soft lithography process, photolithography is used to
fabricate the molds used for casting PDMS stamps, but following fabrication, the stamps can be reused infinitely. Soft
lithography can produce micropatterns of self-assembled monolayers or cells through microcontact printing (mCP),
microfluidic patterning, and stencil patterning [88]. Another process, chemical etching, is commonly used to generate
nanoscale surface architectures. Reactive ion etching, for example, is a type of dry etching that can generate desired
micropatterns via reactive plasma. The manufacturing process of reactive ion etching removes material deposited on
wafers for further cell topography examination [47]. A schematic diagram of IC-based biofabrication for constructing
small-scale devices for probing cellular biomechanics is shown in Figure I.

(A) Photolithography (B) So lithography (C) Reacve ion etching

-replica molding
IC-based Plasma
biofabricaon er
repolym Electric field
Pour p
(Common)
Peel off
PR PR

PDMS Substrate

(D) o
Nan
Biomaterial with
small-scale modificaon
Fluidic flow
ro
Contact guidance Applied forces Mic
Probe

Cellular
biomechanics

Stretch Pressure

Figure I. Probing Cellular Biomechanics through Small-Scale Devices Manufactured via Integrated Circuit
(IC)-Based Biofabrication. (A) Photolithography is the process used to transfer geometric patterns and designs on a
mask to the substrate. (B) Soft lithography uses a soft elastomeric stamp containing patterned relief structures to
generate desired patterns on peeled substrates. (C) Reactive ion etching can generate desired micropatterns via reactive
plasma to remove material deposited on wafers. (D) Biomaterials with small-scale modifications (contact guidance) can
be used with applied mechanical forces to stimulate cells on a material surface.

biocompatibility of material features. We note that nanoscale structures used were dimensionally
similar to focal adhesion clusters on cellular membranes. Accordingly, biomaterials with nano-
scale modifications not only offer biomimicking environments for cell culture but also provide a
relevant in vitro platform for modulating the cytoskeleton. A great deal of research has focused
on fabricating periodic nanostructures, including nanopillars, nanowires, nanogratings, and
ligand patterns, on hard substrates [7–10]. For instance, silicon-based sharp-tip substrates
have been fabricated via deep reactive ion etching to examine cytoskeletal morphology.
Fibroblasts have been shown to manifest a smaller cell adhesion area and lower proliferation
on needle-like nanoposts, but enhanced elongation with alignment on blade-like nanogrates
[11]. In terms of fabrication processes, electron beam lithography could be applied to construct

Trends in Biotechnology, February 2016, Vol. 34, No. 2 173

174

Table 1. Common Biomaterials for Probing Cellular Biomechanicsa

Trends in Biotechnology, February 2016, Vol. 34, No. 2

Biomaterial IC-Based Fabrication Surface Feature Biological Application Refs Characteristic

Topography Chemical Treatment Cell Type Used Response

PDMS Photolithography, DRIE, Elastomeric micropost Fibronectin patterns Stem cells, endothelial Stem cell fate, focal [28,29,32–35] The PDMS micropost
soft lithography array cells, smooth muscle adhesion organization, array can be used as a
cells, fibroblasts cell morphology, detector for traction force
cytoskeleton measurement between
contractility cells and microposts
under mechanical
stimulation

Photolithography Alternative stiffness Poly-L-lysine, Hippocampal neurons, Neurite extension and [53–56] Use of polymers with
fibronectin, laminin DRG neurons, underlying signaling alternating stiffness
neuroblasts, fibroblasts pathways, cytoskeletal characteristics to explore
and morphological cell responses
characteristics

Soft lithography Microchannels Fibronectin Fibroblasts Cytoskeletal, [42] PDMS microchannels can
morphologic changes be used to study the
coupled effects of
mechanics and
topography in cell
morphology

Photolithography, RIE, Microgrooves Poly-L-lysine, laminin Neural stem cells, cortical Neurite outgrowth [46] Microchannel width can
soft lithography neurons impact development and
differentiation of neural
stem cells

Photolithography, Angular grating patterns O2 plasma treatment, Osteoblast cells Cell migration, cell [9] Angular grating patterns
sputter deposition, treatment (hydrophilic elongation can be used to control cell
lifted-off, RIE, soft surface) motility and directionality
lithography

Soft lithography, Nanogratings Collagen Mesenchymal stem cells Cytoskeletal [10] Nanogratings can
embossing organization, cell influence stem cell
mechanical properties behaviors
 Table 1. (continued)
Biomaterial IC-Based Fabrication Surface Feature Biological Application Refs Characteristic

Topography Chemical Treatment Cell Type Used Response

Soft lithography, RIE Micropost array Poly-L-lysine Hippocampal neurons Neuronal outgrowth [7] Micropost arrays can
manipulate neuronal
outgrowth

Photolithography, Micropillars Fibronectin Fibroblasts Cell shape and [26] Micropillars can be used
Bosch process migration to affect cell shape and
migration

Photolithography, soft Geometric features Fibronectin Mesenchymal stem cells Stem cell differentiation [18,19] Geometric shape can
lithography direct MSCs to
appropriate fates

Photolithography, soft Micropatterned strips Fibronectin patterns Endothelial cells Cell apoptosis [23] Cell apoptosis can be
lithography modulated by changes in
ECM micropatterning and
mechanical forces

Silicon Photolithography, liquid Oxidized silicon APTMS-/FDTS-coated Fibroblasts, epithelial cells Cytoskeletal and [61,62] Silicon-based
vapor deposition, nanosponges morphological changes micropatterns can be
AgNPs used to influence cellular
behaviors at desired
locations at a micrometric/
nanometric level

Self-assembling Vertically silicon – Hepatic cells Cell adhesion and [15] Cell adhesion and
nanoelectrochemistry nanowire arrays spreading spreading can be
controlled by arrayed
silicon nanowires

Photolithography, DRIE Microstructures Polyornithine, laminin Neural stem cells Cell proliferation and [47] Topological parameters,
differentiation pattern, and size can
affect the differentiation of
adult neural stem cells
Trends in Biotechnology, February 2016, Vol. 34, No. 2
175
176

Table 1. (continued)
Biomaterial IC-Based Fabrication Surface Feature Biological Application Refs Characteristic
Trends in Biotechnology, February 2016, Vol. 34, No. 2

Topography Chemical Treatment Cell Type Used Response

Interference lithography, Nanoposts, nanogrates – Fibroblasts Cell proliferation and [11] Sharp-tip
DRIE morphology nanotopography can
guide filopodia extension
and formation of the
adhesion molecules
complex

Electron beam Nanodots, nanorings K-Casein, fibronectin, Endothelial cells Cell adhesion, [12] The shape of nanometer-
lithography laminin cytoskeletal scale surface patterns can
organization modulate focal adhesion
complex organization

Gold nanoparticles- Nanowires – Embryonic stem cells, Stem cell differentiation, [45] Nanowires can be used to
assisted etching embryonic kidney cells gene delivery study intracellular and
intercellular biological
processes

Glass Photolithography, RIE Nanoroughness Vitronectin, Anti- Embryonic stem cells, Cell morphology, [59,60] Nanoroughness can
EpCAM antibodies tumor cells adhesion, proliferation, regulate hESC behaviors
clonal expansion, self- and capture CTCs
renewal

Photolithography Micropatterns Neural extracellular Neural cells, skin-derived Neural differentiation [5] Contact stimulation and
matrix precursor cells signaling molecules can
induce neurons to
develop and stabilize
synaptic contacts

Hydrogel Capillary force Nanoscale cues Collagen, fibronectin Cardiac cells, fibroblasts Cell alignment, cell [13] Controlling cell–material
lithography migration, cell interactions at the
morphology nanoscale level can
manipulate cellular
structure and function
 Table 1. (continued)
Biomaterial IC-Based Fabrication Surface Feature Biological Application Refs Characteristic

Topography Chemical Treatment Cell Type Used Response

Photolithography, Microislands RGD motif Mesenchymal stem cells Cell spreading, stem cell [48] RGD nanospacing can
chemical etching, lift-off differentiation regulate cell tension and
stem cell differentiation

PLGA Soft lithography Micropatterned strips Fibronectin patterns Mesenchymal stem cells Stem cell differentiation [22] Cellular shape modulation
can be a viable strategy to
induce lineage-specific
differentiation of stem cells

Photolithography, RIE Microgroves/ – Tenocytes, osteoblasts Cell alignment [67,68] Anisotropic substrates
nanogroves with microscale/
nanoscale groves can be
used to modulate cellular
adhesion in vitro, but none
of the topographies can
promote directional cell
patterning in vivo

Chitosan Photolithography, liquid Nanotextures O2 plasma treatment Fibroblasts, cancer cells, Cellular morphology, [63–65] Physically and chemically
vapor deposition, FDTS-grafted neural cells cytoskeleton response modified chitosan can be
AgNPs used to probe the
morphology of attached
cells

a
AgNPs, silver nanoparticles; CTCs, circulating tumor cells; APTMS, (3-aminopropyl) trimethoxysilane; DRG, dorsal root ganglion; DRIE, deep reactive ion etching; ECM, extracellular matrix; FDTS, perfluor-
odecyltrichlorosilane; hESC, human embryonic stem cell; MSC, mesenchymal stem cell; PDMS, polydimethylsiloxane; PLGA, poly(lactic-co-glycolic) acid; RIE, reactive ion etching.
Trends in Biotechnology, February 2016, Vol. 34, No. 2
177
controllable periodic nanostructured surfaces to observe the organization and formation of focal
adhesions. Nanodots and nanorings coated with fibronectin have also been used to arrange the
immediate cytoskeleton into straight fibrils and diverging fibril bundles, respectively (Figure 1A)
[12]. In addition, nanoscale grooves have been employed to direct orientation and growth of
myocytes (i.e., neonatal rat ventricular myocytes). In these experiments, myocytes aligned when
cultured along poly(ethylene glycol) (PEG)-based nanoscale grooves that mimicked the in vivo
structural anisotropy of the myocardium and became manifestly longer than myocytes cultured
on an unpatterned substrate (Figure 1B) [13]. Apart from periodic nanostructures, nonperiodic
silicon nanowires were used to determine the influence of nanowire clusters on cellular behaviors
including cell attachment and spreading [14]. In this study, silicon nanowire clusters enhanced
the cell–substrate attachment force. However, nanowire clusters restricted cell spreading.
Because the distance between nanowires was consistent with the distance between two
integrin clusters on the cellular membrane, cell spreading and the formation of focal adhesions
was restricted [15].

In terms of microscale structures, adhesive regions of varying shape and size have been used to
induce cytoskeletal rearrangements, which have been shown to further affect a variety of cellular

(A) (B)

300 nm

10 µm 10 µm
FN nanodots FN nanorings

(C) (D) Paerns

Max used 102 µm

38 µm 38 µm
8 µm
64 µm
n = 80 Min
Microtubules
Centrosome
Nuclei
n = 86 3T3
Fibroblast 20 µm
(E) (F)
Non-polarized Polarized Front
Lat-front
hMSC
HUVEC

Lat-rear
Lat-front

Lat-rear

0 1 2 0 4 8 0 4 8
FA area Force per FA FA stress
(µm2) (nN) (nN µm–2)

Figure 1. Actin Cytoskeleton Modulation and Detection with Small-Scale Architectures. (A) When endothelial
cells were cultured on nanopatterns of fibronectin (FN) with a K-Casein background, cells adhered to FN nanopatterns and
formed stress fibers (red, FN; green, actin; blue, nucleus) [12]. (B) Myocytes aligned in response to the nanoscale grooves
(red, actin; blue, nucleus) (scale bar = 1 mm) [13]. (C) Fluorescent images of cells in flower and star shapes (green, F-actin;
red, vinculin; blue, nuclei; yellow, myosin IIa; right, quantitative measure of contractility, fluorescent heatmaps of cells stained
for myosin IIa) (scale bar = 20 mm) [19]. (D) Asymmetric patterns polarize and guide migrating immobilized fibroblasts. The
Golgi and the centrosome of a fibroblast are closer to the blunt end, which caused fibroblasts to extend more from the blunt
end [25]. (E) Staining images of focal adhesion (FA) area, traction force per focal adhesion, and focal adhesion stress for a
human mesenchymal stem cell (hMSC) and an endothelial cell cultured on a polydimethylsiloxane (PDMS)-based detector
(scale bars = 30 mm) [28]. (F) Atomic force microscopy deflection images of a nonpolarized and polarized 3T3 fibroblast
(yellow dots are indentation points, designated positions of the polarized fibroblast are labeled by arrows and text) [31].

178 Trends in Biotechnology, February 2016, Vol. 34, No. 2

functions [16,17]. Typically, the presence of small adhesive regions induced cells to form
spheres, while the presence of large adhesive regions induced them to flatten and to attach
to the substrate [18]. In addition to examining the impact of specifically designed adhesive areas,
substrate morphologies have been used to probe cytoskeletal response. Flower- and star-
shaped substrate patterns, for instance, have been used to explore the relationship between
environmental shape and stem cell cytoskeleton response (Figure 1C) [19]. Both physical and
chemical methods have been used to pattern substrate surfaces. From a chemical approach
perspective, microcontact printing technology has become the most common method for
transferring protein patterns onto biomaterial surfaces [20,21]. For instance, poly(lactic-co-
glycolic) acid (PLGA) biomaterials consisting of 20 mm-wide fibronectin-coated paths were
used in one study to direct human mesenchymal stem cell (hMSC) differentiation and conse-
quently rearrange the cytoskeletal network and alter nucleus shape [22]. In addition, mechanical
stimulation is often coupled with cell–substrate interactions to stimulate cytoskeletal response. In
one example, endothelial cells were cultured on micropatterned strips of fibronectin with widths
of 15, 30, and 60 mm, which allowed for the application of parallel or perpendicular shear flow.
Reacting to such stimulation, the endothelial cells cultured on 30- and 60-mm strips produced
actin stress fibers with anchoring spots of phosphorylated focal adhesion kinase. These
endothelial cells showed no significant apoptosis when fluid flow was not applied. When
endothelial cells were subjected to shear flow, parallel flow produced cell elongation with
enhanced stress fibers and phosphorylated focal adhesion kinase, and a decrease in apoptosis,
while perpendicular flow did not [23,24].

Substrate structures of varying size and morphology can induce cell polarization and further
initiate cell migration (Figure 1D) [9,25]. For instance, fibroblasts displayed more elongated and
branched shapes with fewer actin stress fibers when they were cultured on micropillar sub-
strates. It was found that modulating the spacing between micropillars affected migration paths
as a result of spatial reorganization of the actin cytoskeleton, which responded to physical
constraints and a preferential formation of focal adhesions on the micropillars [26]. Further,
geometry and dimension of substrate microstructures can affect fibroblast migration. Fibro-
blasts migrated two times faster in parallel microgrooves than they did in perpendicular micro-
grooves [27].

Cytoskeletal Contractility Detection

Recent advances in the development of a polydimethylsiloxane (PDMS) micropost array,
manufactured by a combination of photolithography, deep reactive ion etching, and soft
lithography, have allowed for the creation of an efficient detector capable of measuring dynamic
responses in cytoskeletal contractility following exposure of cells to diverse stimuli. Notably,
different post-height designs have been shown to control micropost rigidity and determine the
degree to which a micropost curves in response to traction forces between cell and substrate.
The bending of varying height microposts in response to horizontal traction forces was mea-
sured by using the finite element method. This facilitated measurement of traction forces for
individual focal adhesions but also enabled quantification of subcellular distributions (i.e., focal
adhesion area and focal adhesion stress). This experiment demonstrated that the distribution of
focal adhesion stress on focal adhesions of single hMSCs was not uniform. Instead, stresses on
interior focal adhesions were significantly higher compared with those exerted on peripheral
ones. However, endothelial cells (i.e., human umbilical vascular endothelial cells) displayed a
different behavior. They showed a relatively uniform focal adhesion stress distribution as a result
of larger traction forces and focal adhesions at the periphery (Figure 1E) [28,29]. These differ-
ences suggest that multiple approaches may exist for different cells to mechanically adjust to
their milieu. Additional research shows that micropost tips can be functionalized with extracel-
lular matrix (ECM) via microcontact printing to promote cell adhesion and control cell shape [30].
Atomic force microscopy has been used to examine a PDMS-based micropost array to measure

Trends in Biotechnology, February 2016, Vol. 34, No. 2 179

spatial distribution of force generation and filament elasticity in a migrating and polarized
fibroblast. The results indicate that cytoskeletal elasticity and force generation are types of
mechanical modulation that cells use to regulate focal adhesion complexes and migration
(Figure 1F) [31]. After integrating a PDMS micropost array onto a stretchable elastomeric
membrane to apply further stretching to adherent cells, researchers have shown that a muscle
cell can regulate cellular traction force, cell stiffness, and cell spread area under a static
equibiaxial stretch [32,33]. Additionally, this device was used to apply and examine the effects
of applied fluid shear stress, modulate substrate rigidity, and control adhesive patterning in a
microfluidic milieu. Endothelial cells examined thus modulated cytoskeletal contractile forces to
promote their morphology, specifically, their elongation process along the direction of flow [34].
In further research, ultrasound pulses were used to actuate functionalized lipid microbubbles,
which covalently attached to cells on PDMS micropost arrays designed to mediate intracellular
cytoskeleton contractility. Ultrasound-regulated intracellular cytoskeleton contractility enhance-
ment was found to be dose-dependent [35].

Cell Behavior Regulation

Within tissue, cells reside in dynamic environments that may alter their behaviors in response to a
variety of small-scale architectural and mechanical force stimuli. PDMS biomaterial is most
commonly used to probe cellular biomechanics because it can be deformed via mechanical
forces and created with small-scale architectures via IC-based biofabrication. For instance,
PDMS biomaterial has been used to probe stretch-activated mechanotransduction in sensory
neurons via deformation of an elastomeric cell–substrate material. In this study, a whole cell
patch clamp was used to record single neuron action potential response following nearby
indentation of PDMS. The merit of the device used in these experiments was that the neurites
were stretched without physical touching on deformation regions of the polymer (Figure 2A)
[36–38]. Further, mechanical stretch and shear flow could be coupled to stimulate fibroblasts
on a sheet of PDMS and produce structural responses more closely comparable to physiologi-
cal response [39,40]. Multiple modes of mechanical and chemical stimulation could affect
cellular structural response with regard to competing directions of cell alignment and orienta-
tion. One very clever use of a coupled PDMS device- and multiforce-based strategy showed
that uniaxial stretch and fluid shear stress could be recorded in a vector logic gate to
characterize cellular response as a function of cytoskeletal reorganization (Figure 2B) [41].
PDMS biomaterial has unique remodeling properties that make it highly suitable for IC-based
biofabrication aimed at creating small-scale substrate architectures. In one study, a PDMS-
microfabricated system was created by using a combination of photolithography and replica
molding to probe cellular response to compression stimulation on PDMS microchannel
surfaces [42]. This device allowed for the application of coupling effects, namely compression
and microchannel architecture, on cell cytoskeleton and morphology. Fibroblasts cultured on
this device were prone to spread along the orientation of microchannels and perpendicular to
the path of applied compression. More small-scale devices used to regulate cell behavior via
common biomaterial alterations are presented and categorized, based on purpose, in the
following paragraphs.

Notably, recent progress in the development of small-scale surfaces created via IC-based
biofabrication has allowed for the modulation of hMSC differentiation and function without
complex soluble chemical treatment. For instance, electron beam lithography has been used
as a straightforward approach to identify a nanoscale, disordered surface capable of stimulating
stem cell differentiation [43]. Poly(methyl methacrylate) (PMMA)-based nanostructures created
with varying degrees of disorder in their design have been shown to induce hMSCs to
produce bone mineral in vitro without osteogenic supplement treatment. This process
produced an outcome with similar efficiency to that of hMSCs treated with osteogenic media
[44]. In a chemically based approach to making small-scale architecture, silicon nanowires

180 Trends in Biotechnology, February 2016, Vol. 34, No. 2

(A) (B)
Stretch Vector
Recording pipee Signal logic Cell orientaon = ?
Shear gates
Indentaon pipee

5µm-5µm 10µm-10µm 20µm-20µm 10µm-60µm

(C) Ctrl

Tuj-1 Dapi

100µm

(D) (E)
Nanospacing effect?
ize

54.8% 55.6%
gs

Stem cell inducon 60.0% 60.0%

Percentage of cells
in

Percentage of cells
ad

Cell size effect? 31.0%

pre

25.0%
30.0% 30.0% 19.4%
ll s

14.3%
Ce

Integrin
RGD
0.0% 0.0%
PEG Hydrogel Au
RGD nanospacing Case I Case II Case III+IV Case V Case VI Case VII

Figure 2. Cell Behavior Regulation through Small-Scale Devices. (A) Scheme of mechanical stretching applied on a
neurite cultured on a polydimethylsiloxane (PDMS) surface. A single neurite can be stretched by substrate deformation
without contacting the neurite. A whole cell patch clamp was used to inspect the action potential of single neuron response
to nearby indentation of PDMS [36]. (B) Vector logic gates were used to classify cellular response to single and dual
mechanical inputs, including pressure-induced uniaxial stretch and fluid shear stress [41]. (C) Stem cell behaviors were
affected by topographical guidance. Fluorescence images of neural stem cells cultured on different PDMS substrate widths
after 1 week of differentiation (red, the neuronal specific marker tuj-1; blue, DAPI for nuclear staining) [46]. DAPI, 40 ,
6-diamidino-2-phenylindole. (D) RGD ligands microislands with different nanospacings and microdomain sizes for regulat-
ing stem cell differentiation [48]. (E) The percentage of different neurite responses to hard–soft–hard substrate outside and
inside of the soft channel [55].

(diameter
90 nm and length
6 mm, respectively) fabricated via chemical etching processes,
have been successfully used as an architectural substrate for the growth of cardiac myocytes
from mouse embryonic stem (mES) [45]. With regard to microstructures, microscale linear
microgrooved surfaces have been used to modulate stem cell differentiation through cell polarity
guidance. In one example, PDMS-based microgrooves with various widths were used to impact
neural stem cell differentiation. Determination of an optimal microchannel width was found to
induce higher differentiation ratio with alignment of neurites along the microchannel edges and
an enhanced neuron development rate compared with neurites grown on an unpatterned
surface (Figure 2C) [46]. Additionally, as smaller microfeature widths were achievable, better
upregulation of neuronal differentiation has been possible [47]. In addition to common linear
micropatterned surfaces, other microgeometries have been developed. Notably, microcontact
printing technique has been used to develop flower and star patterns to explore the relationship
between environmental shape and cellular differentiation of MSCs independent of soluble
factors. Geometric features promote osteogenesis by increasing actomyosin contractility.
Shape-based trends in lineage commitment play an important role in modulating focal adhesion
and myosin-generated contractility during stem cell differentiation [19]. In an elegant use of
physical feature modification to represent relevant chemical structure, specific nanoscale,
protein-based nanopatterns have been used to study stem cell biomechanics. For instance,
RGD ligand microislands, fabricated via photolithography, chemical etching, or lift-off, have been
used to regulate stem cell differentiation. MSCs displayed osteogenic or adipogenic differentia-
tion on micropatterns/nanopatterns with RGD nanoarrays when different scaled nanospacings
were regulated (Figure 2D) [48].

Trends in Biotechnology, February 2016, Vol. 34, No. 2 181

It is important to remember that the approximate stiffness for physiological tissues ranges from
hundreds of Pa (such as brain tissue) to MPa (such as cartilage tissue) [49]. As the interface
between extracellular substrates and cells, matrix stiffness plays an important role in regulating
cellular functions such as cell differentiation, phenotype, and cell orientation [50–52]. From a
neurobiological perspective, neuron adhesion, neurite outgrowth, and neural mechanotrans-
duction are all affected by substrate stiffness. In experiments modulating surface stiffness,
PDMS biomaterial is a highly suitable substrate, as varying ratios of PDMS base to curing agent
can be used to generate substrates of varying stiffness [53,54]. Substrates can even be
constructed with localized regions of alternating stiffness. For example, linear regions of a
polymer substrate can be created with specific, varying elasticities, using a combination of
photolithography and soft lithography technology, a process that had been employed to probe
neural responses [55,56]. The alternating channels in this experiment were characterized by
elastic moduli of 800 kPa and 200 kPa adjacent to one another. This experiment demonstrated
that neurites underwent outgrowth in specific paths including aligning along, turning back, or
directly crossing the elasticity interface boundary (Figure 2E).

Cell Micropatterning for Further Applications

Current promising progress in biomedical engineering has allowed for cultured cell arrangement
in desired, patterned regions that can provide an in vitro platform for biological applications, such
as artificial stents for neuron network formation, and artificial scaffolds for probing cell behaviors
during drug treatment [21,57,58]. Recent advances in the development of small-scale devices
provide an efficient strategy to arrange cells with a desired pattern on artificial scaffolds. For
instance, a microfabrication method using photolithography and reactive ion etching techniques
has been used to precisely control the micropatterning and nanoroughness of glass surfaces.
Devices manufactured in this manner can be used to regulate mechanosensory integrin-
mediated cell matrix adhesion of embryonic stem cells as well as the capture of circulating
tumor cells [59,60]. In addition, silicon wafers, a commonly used material in IC manufacturing
processes, have been easily modified with small-scale features for biological uses. For instance,
oxidized silicon nanosponges with a hydrophilic–hydrophobic boundary and pristine oxidized
silicon regions and perfluorodecyltrichlorosilane (FDTS)-grafted oxidized silicon nanosponges
have been constructed for micropatterning mammalian cells [61,62]. Biofabrication approaches
for fabricating oxidized silicon nanosponges include photolithography, Ag nanoparticle-assisted
etching, and vapor deposition (Figure 3A). In the work of Yang cited earlier, Chinese hamster
ovary (CHO) cells crossed over a nanosponge gap between two flat silicon stages, which
necessitated an adjustment in the distance between two flat silicon stages to prevent the CHO
cells from crossing (Figure 3B). Through various reexamination and redesign efforts, the ideal
nanosponge gap distance between two flat silicon stages was determined to be 80 mm. Further,
micropatterned silicon substrates could be used to verify whether membrane fusion occurred
when HIG-82 fibroblasts connected together (Figure 3C–F). The greatest advantage of silicon
material is that it is easy to fabricate because silicon is the fundamental material used in the
semiconductor industry. While it is easy to form small-scale structures on silicon surfaces, silicon
material has several disadvantages including the following: (i) nonbiocompatibility, (ii) nonbiode-
gradability, and (iii) opaqueness. By contrast, owing to its material properties, it is more
challenging to directly modify the physical features of naturally derived (i.e., ‘soft polymer’)
biomaterials using IC-based biofabrication. Further, few studies have focused on manufacturing
uniform but nonperiodic nanostructures that more closely mimic heterogeneous physiological
microenvironments.

Based on experiments using oxidized silicon nanosponges, using silicon as a parent mold to
transfer patterns onto chitosan membranes could overcome silicon material disadvantages. A
single cell chitosan microarray can be created with mixed flat and nanostructured regions
without having to induce chemical modification (Figure 3G) [63,64]. The solution casting

182 Trends in Biotechnology, February 2016, Vol. 34, No. 2

(A) Oxidized silicon nanosponge (B) (C) (E) Merged Outstanding Questions
Is in vitro scaffold with small-scale
architecture suitable for being implanted
into the tissue?
20 µm 25 µm
(D) (F) How to modify nature-driven biomaterials
through IC-based biofabrication
methods?

20 µm

(G) Nanostructured chitosan membrane (H)

(J)

Control Cyto-D Recovery 1 h Recovery 24 hrs

(I) Single-cell based microarray

Figure 3. Schematic Diagrams of Silicon and Chitosan Materials for Micropatterning Cells. (A) Biofabrication of
oxidized silicon nanosponge for micropatterning mammalian cells. The designed micropatterns were created through a
combination of photolithography and Ag nanoparticle-assisted etching. Perfluorodecyltrichlorosilane (FDTS)-grafted oxi-
dized silicon nanosponges were created via vapor phase coating deposition. (B) Scanning electron microscopy (SEM)
images of Chinese hamster ovary (CHO) cells crossed over two silicon pristine regions. (C) Fluorescence images of CHO
cells cultured on FDTS-grafted nanosponges for 3 days. (D) The distance between two silicon pristine regions that a single
CHO cell could crossover versus the suspended percentage. The maximum distance between two silicon surfaces over
which a single cell would cross was approximately 40 mm. (E) Fluorescence images of HIG-82 fibroblasts cultured on FDTS-
coated oxidized silicon nanosponges after 3 days of culture (blue, nucleus; green, membranes; red, actin filaments). (F)
Fusion ratios of CHO cells and HIG-82 fibroblasts after 3 days of culture on oxidized silicon nanosponge. (G) Biofabrication
of nanostructured chitosan membrane for drug screening. The designed flat regions and silicon nanostructures were
fabricated through a combination of photolithography and Ag nanoparticle-assisted etching. After photoresist was
removed, the chitosan solution was cast on diced silicon chips and dried in an oven at 60 8C overnight. The single cell
chitosan microarray was peeled off from silicon chips after treating with 1 M. HeLa cells were cultured on this chitosan
microarray for further drug screening applications. (H) SEM images of Madin–Darby canine kidney epithelial (MDCK) cells
(right), HIG-82 fibroblasts (middle), and HeLa cells (left) after 12 h of culture on single cell chitosan microarrays. (I)
Fluorescence images merged with phase contrast images of HeLa cells labeled with Annexin V-FITC (green) and DAPI
(40 ,6-diamidino-2-phenylindole; blue) after 1 h of incubation with cytochalasin D and clearing away cytochalasin D following
1 h and 24 h of incubation (recovery period). In the control group, HeLa cells were cultured in medium without cytochalasin
D. (J) Apoptotic cell percentage of HeLa cells cultured on a single cell chitosan microarray and pristine chitosan surfaces at
various conditions described in (I). (A–F), from [61]; (G–J), from [63,64].

technique was used in these experiments to peel off chitosan membranes from parent silicon
molds. In this case, mere surface topography can be used as a strategy to micropattern single
cells at desired positions (Figure 3H). Further, a single cell chitosan microarray could be used as a
platform for drug screening. In one study, HeLa cells were treated with cytochalasin D to inhibit
actin polymerization and facilitate drug screening using a single cell chitosan microarray
(Figure 3I,J) [63]. Furthermore, chitosan substrates have been designed with various micro-
patterns/nanopatterns to promote neural network formation [65]. This research found that
nanostructures created on chitosan membranes offered uniform but nonperiodic nanostruc-
tures that more closely mimicked the physiologically relevant matrix. Additionally, microscaled/
nanoscaled structures can be successfully integrated onto chitosan surfaces via IC-based
biofabrication

Concluding Remarks
In this review, recent developments in the use of common biomaterials with sophisticated small-
scale modifications intended for contact guidance and the probing of cellular biomechanics were
discussed. The most common IC-based microfabrication/nanofabrication processes used to

Trends in Biotechnology, February 2016, Vol. 34, No. 2 183

construct small-scale devices with in vitro nanomaterial scaffolds include lithography processes
and etching techniques. Compared with other approaches (e.g., electrospinning) for construct-
ing scaffolds composed of interwoven polymer fibers, IC-based biofabrication processes, which
primarily use etching techniques, can precisely fabricate periodic or nonperiodic substrate
topography at micrometer to nanometer resolutions by using different types of etching proce-
dures and modulating parameters for a specific process [3,66]. Further, state-of-the-art IC-
based biofabrication procedures with mass production capacity have demonstrated the poten-
tial for producing a large number of biomedical tools for constructing disease models and
advancing fundamental biology knowledge (e.g., cellular biomechanics, repair scaffolds, and
drug screening). With regard to in vitro biomedical applications, apart from their capacity to
probe the interactions between cells and surface-modified biomaterials, small-scale devices
allow for the application of different types of mechanical stimuli. In addition, because cellular
responses to contact guidance may differ depending on cell type, the influence of substrate
characteristics is presented for a variety of cell categories. Because commercial substrates with
defined microscale/nanoscale topographical features can mimic varying ECM conditions and
provide for mass examination, they offer great promise. Although in vitro cell culture platforms
using common biomaterials and IC-based biofabrication have advanced, they remain saddled
with potential limitations, namely the fact that mechanisms of interactions between materials and
cells are still not well elucidated. Further, it remains unclear whether small-scale structures can
be successfully implanted into the tissue interface [67,68]. The size, shape, and surface
properties of engineered biomaterials and properties of the material itself, such as silicon, which
is an inorganic material, may contribute to the induction of cytotoxicity in direct clinical
applications (see Outstanding Questions). In terms of tissue engineering, additional in vivo
experiments are necessary to investigate the potential for use of implantable materials for
regenerative/reparative efforts in medicine.

Acknowledgments
Support for the preparation of this review was provided by No. National Science Council (NSC) 101-2623-E-007-005-ET (to
J.A.Y.) and NSC 104-2628-E-007-001-MY3 (to C-M.C.).

References
1. Shao, Y. et al. (2014) Integrated micro/nanoengineered functional
biomaterials for cell mechanics and mechanobiology: a materials
perspective. Adv. Mater. 26, 1494–1533
2. Kim, D.H. et al. (2012) Matrix nanotopography as a regulator of cell
function. J. Cell Biol. 197, 351–360
3. Kshitiz et al. (2011) Micro- and nanoengineering for stem cell
biology: the promise with a caution. Trends Biotechnol. 29,
399–408
4. Ananthanarayanan, A. et al. (2011) Purpose-driven biomaterials
research in liver-tissue engineering. Trends Biotechnol. 29, 110–118
5. García-Parra, P. et al. (2012) Modeling neural differentiation on
micropatterned substrates coated with neural matrix components.
Front. Cell. Neurosci. 6, 10
6. Arnold, M. et al. (2008) Induction of cell polarization and migration
by a gradient of nanoscale variations in adhesive ligand spacing.
Nano Lett. 8, 2063–2069
7. Hanson, J.N. et al. (2009) Textural guidance cues for controlling
process outgrowth of mammalian neurons. Lab Chip 9, 122–131
8. Ferrari, A. et al. (2010) Neuronal polarity selection by topography-
induced focal adhesion control. Biomaterials 31, 4682–4694
9. Tang, Q.Y. et al. (2014) Influence of engineered surface on cell
directionality and motility. Biofabrication 6, 015011
10. Yim, E.K.F. et al. (2010) Nanotopography-induced changes in focal
adhesions, cytoskeletal organization, and mechanical properties of
human mesenchymal stem cells. Biomaterials 31, 1299–1306
11. Choi, C.H. et al. (2007) Cell interaction with three-dimensional
sharp-tip nanotopography. Biomaterials 28, 1672–1679
12. Pesen, D. et al. (2009) Modulation of cell adhesion complexes by
surface protein patterns. Appl. Mater. Interfaces 1, 543–548
13. Kim, D.H. et al. (2010) Nanoscale cues regulate the structure and
function of macroscopic cardiac tissue constructs. Proc. Natl.
Acad. Sci. U.S.A. 107, 565–570
14. Koufaki, N. et al. (2011) Controlling cell adhesion via replication of
laser micro/nano-textured surfaces on polymers. Biofabrication 3,
045004
15. Qi, S. et al. (2009) Cell adhesion and spreading behavior on
vertically aligned silicon nanowire arrays. Appl. Mater. Interfaces
1, 30–34
16. Gomez, E.W. et al. (2010) Tissue geometry patterns epithelial–
mesenchymal transition via intercellular mechanotransduction. J.
Cell. Biochem. 110, 44–51
17. Théry, M. et al. (2010) Micropatterning as a tool to decipher cell
morphogenesis and functions. J. Cell Sci. 123, 4201–4213
18. Gao, L. et al. (2010) Stem cell shape regulates a chondrogenic
versus myogenic fate through Rac1 and N-cadherin. Stem Cells
28, 564–572
19. Kilian, K.A. et al. (2010) Geometric cues for directing the differenti-
ation of mesenchymal stem cells. Proc. Natl. Acad. Sci. U.S.A.
107, 4872–4877
20. Poudel, I. et al. (2013) Micropatterning–retinoic acid co-control of
neuronal cell morphology and neurite outgrowth. Acta Biomater.
9, 4592–4598
21. Frimat, J.P. et al. (2010) The network formation assay: a spatially
standardized neurite outgrowth analytical display for neurotoxicity
screening. Lab Chip 10, 701–709
22. Tay, C.Y. et al. (2010) Micropatterned matrix directs differentiation
of human mesenchymal stem cells towards myocardial lineage.
Exp. Cell Res. 316, 1159–1168

184 Trends in Biotechnology, February 2016, Vol. 34, No. 2

23. Wu, C.C. et al. (2007) Directional shear flow and Rho activation
prevent the endothelial cell apoptosis induced by micropatterned
anisotropic geometry. Proc. Natl. Acad. Sci. U.S.A. 104, 1254–1259
24. Hur, S.S. et al. (2012) Roles of cell confluency and fluid shear in 3-
dimensional intracellular forces in endothelial cells. Proc. Natl.
Acad. Sci. U.S.A. 109, 11110–11115
25. Jiang, X. et al. (2005) Directing cell migration with asymmetric
micropatterns. Proc. Natl. Acad. Sci. U.S.A. 102, 975–978
26. Ghibaudo, M. et al. (2009) Substrate topography induces a cross-
over from 2D to 3D behavior in fibroblast migration. Biophys. J. 97,
357–368
27. Kaiser, J.P. et al. (2006) The effect of topographic characteristics
on cell migration velocity. Biomaterials 27, 5230–5241
28. Fu, J. et al. (2010) Mechanical regulation of cell function with
geometrically modulated elastomeric substrates. Nat. Methods
7, 733–736
29. Mann, J.M. et al. (2012) A silicone-based stretchable micropost
array membrane for monitoring live-cell subcellular cytoskeletal
response. Lab Chip 12, 731–740
30. Tee, S.Y. et al. (2011) Cell shape and substrate rigidity both
regulate cell stiffness. Biophys. J. 100, 25–27
31. Harn, H.I.C. et al. (2015) Mechanical coupling of cytoskeletal
elasticity and force generation is crucial for understanding the
migrating nature of keloid fibroblasts. Exp. Dermatol. 24, 579–584
32. Lam, R.H.W. et al. (2012) Live-cell subcellular measurement of cell
stiffness using a microengineered stretchable micropost array
membrane. Integr. Biol. 4, 1289–1298
33. Shao, Y. et al. (2014) Global architecture of the F-actin cytoskele-
ton regulates cell shape-dependent endothelial mechanotrans-
duction. Integr. Biol. 6, 300–311
34. Lam, R.H.W. et al. (2012) Elastomeric microposts integrated into
microfluidics for flow-mediated endothelial mechanotransduction
analysis. Lab Chip 12, 1865–1873
35. Fan, Z. et al. (2013) Acoustic tweezing cytometry for live-cell
subcellular modulation of intracellular cytoskeleton contractility.
Sci. Rep. 3, 2176
36. Cheng, C.C. et al. (2010) Probing localized neural mechanotrans-
duction through surface-modified elastomeric matrices and
electrophysiology. Nat. Protoc. 5, 714–724
37. Tsai, T.T. et al. (2014) Mechanotransduction in intervertebral discs.
J. Cell. Mol. Med. 18, 2351–2360
38. Lin, Y.W. et al. (2009) Understanding sensory nerve mechano-
transduction through localized elastomeric matrix control. PLoS
ONE 4, e4293
39. Steward, R.L., Jr et al. (2011) Mechanical stretch and shear flow
induced reorganization and recruitment of fibronectin in fibro-
blasts. Sci. Rep. 1, 147
40. Steward, R.L., Jr et al. (2010) Probing cell structure responses
through a shear and stretching mechanical stimulation technique.
Cell Biochem. Biophys. 56, 115–124
41. Steward, R.L., Jr et al. (2015) Cellular force signal integration
through vector logic gates. J. Biomech. 48, 613–620
42. Cheng, C.M. et al. (2009) Probing cell structure by controlling the
mechanical environment with cell–substrate interactions. J. Bio-
mech. 42, 187–192
43. McMurray, R.J. et al. (2011) Nanoscale surfaces for the long-term
maintenance of mesenchymal stem cell phenotype and multipo-
tency. Nat. Mater. 10, 637–644
44. Dalby, M.J. et al. (2007) The control of human mesenchymal cell
differentiation using nanoscale symmetry and disorder. Nat.
Mater. 6, 997–1003
45. Kim, W. et al. (2007) Interfacing silicon nanowires with mammalian
cells. J. Am. Chem. Soc. 129, 7228–7229
46. Béduer, A. et al. (2012) Engineering of adult human neural stem
cells differentiation through surface micropatterning. Biomaterials
33, 504–514
47. Qi, L. et al. (2013) The effects of topographical patterns and sizes
on neural stem cell behavior. PLoS ONE 8, e59022
48. Wang, X. et al. (2015) Fabrication of RGD micro/nanopattern and
corresponding study of stem cell differentiation. Nano Lett. 15,
1457–1467
49. Engler, A.J. et al. (2006) Matrix elasticity directs stem cell lineage
specification. Cell 126, 677–689
50. Palchesko, R.N. et al. (2015) In vitro expansion of corneal endo-
thelial cells on biomimetic substrates. Sci. Rep. 5, 7955
51. Lee, H.H. et al. (2012) Shp2 plays a crucial role in cell structural
orientation and force polarity in response to matrix rigidity. Proc.
Natl. Acad. Sci. U.S.A. 110, 2840–2845
52. Du, J. et al. (2011) Integrin activation and internalization on soft
ECM as a mechanism of induction of stem cell differentiation by
ECM elasticity. Proc. Natl. Acad. Sci. U.S.A. 108, 9466–9471
53. Chen, W.H. et al. (2013) Probing relevant molecules in modulating
the neurite outgrowth of hippocampal neurons on substrates of
different stiffness. PLoS ONE 8, e83394
54. Cheng, C.M. et al. (2011) Localized bimodal response of neurite
extensions and structural proteins in dorsal root ganglion neurons
with controlled polydimethylsiloxane substrate stiffness. J. Bio-
mech. 44, 856–862
55. Chou, S.Y. et al. (2011) Localized neurite outgrowth sensing via
substrates with alternative rigidities. Soft Matter 7, 9871–9877
56. Chou, S.Y. et al. (2009) Composite polymer systems with control
of local substrate elasticity and their effect on cytoskeletal and
morphological characteristics of adherent cells. Biomaterials 30,
3136–3142
57. Cheng, J. et al. (2013) Photopatterning of self-assembled poly
(ethylene) glycol monolayer for neuronal network fabrication. J.
Neurosci. Methods 213, 196–203
58. Lee, J.Y. et al. (2008) Use of photolithography to encode cell
adhesive domains into protein microarrays. Langmuir 24, 2232–
2239
59. Chen, W. et al. (2013) Nanoroughened surfaces for efficient cap-
ture of circulating tumor cells without using capture antibodies.
ACS Nano 7, 566–575
60. Chen, W. et al. (2012) Nanotopography influences adhesion,
spreading, and self-renewal of human embryonic stem cells.
ACS Nano 6, 4094–4103
61. Yang, C.C. et al. (2012) Micropatterning of mammalian cells on
inorganic-based nanosponges. Biomaterials 33, 4988–4997
62. Yang, C.C. et al. (2010) Cell adhesion, morphology and biochem-
istry on nano-topographic oxidized silicon surfaces. Eur. Cell
Mater. 20, 415–430
63. Shuai, H.H. et al. (2013) Using surfaces to modulate the morphol-
ogy and structure of attached cells – a case of cancer cells on
chitosan membranes. Chem. Sci. 4, 3058–3067
64. Yang, C.Y. et al. (2013) Probing cellular behaviors through nano-
patterned chitosan membranes. Sci. Technol. Adv. Mater. 14,
044406
65. Sung, C.Y. et al. Probing neural cell behaviors through micro-/
nano-patterned chitosan substrates. Biofabrication (in press)
66. Farrugia, B.L. et al. (2013) Dermal fibroblast infiltration of poly (e-
caprolactone) scaffolds fabricated by melt electrospinning in a
direct writing mode. Biofabrication 5, 025001
67. Azeem, A. et al. (2015) The influence of anisotropic nano- to micro-
topography on in vitro and in vivo osteogenesis. Nanomedicine 10,
693–711
68. English, A. et al. (2015) Substrate topography: a valuable in vitro
tool, but a clinical red herring for in vivo tenogenesis. Acta Bio-
mater. 27, 3–12
69. Fleischer, S. et al. (2013) Tissue engineering on the nanoscale:
lessons from the heart. Curr. Opin. Biotechnol. 24, 664–671
70. Dvir, T. et al. (2011) Nanotechnological strategies for engineering
complex tissues. Nat. Nanotechnol. 6, 13–22
71. Ross, A.M. et al. (2012) Physical aspects of cell culture substrates:
topography, roughness, and elasticity. Small 8, 336–355
72. Chiu, J.J. et al. (2011) Effects of disturbed flow on vascular
endothelium: pathophysiological basis and clinical perspectives.
Physiol. Rev. 91, 327–387
73. Haga, J.H. et al. (2007) Molecular basis of the effects of mechani-
cal stretch on vascular smooth muscle cells. J. Biomech. 40,
947–960
74. Goehring, N.W. et al. (2012) Cell polarity: mechanochemical pat-
terning. Trends Biotechnol. 23, 72–80
Trends in Biotechnology, February 2016, Vol. 34, No. 2 185
75. Kumar, S. et al. (2009) Dissecting the molecular basis of the
mechanics of living cells. Exp. Mech. 49, 11–23
76. Geiger, B. et al. (2009) Environmental sensing through focal
adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33
77. Zamir, E. et al. (2001) Molecular complexity and dynamics of cell–
matrix adhesions. J. Cell Sci. 114, 3583–3590
78. Balaban, N.Q. et al. (2001) Force and focal adhesion assembly: a
close relationship studied using elastic micropatterned substrates.
Nat. Cell Biol. 3, 466–472
79. Blong, C.C. et al. (2010) Differentiation and behavior of human
neural progenitors on micropatterned substrates and in the devel-
oping retina. J. Neurosci. Res. 88, 1445–1456
80. Borghi, N. et al. (2010) Regulation of cell motile behavior by
crosstalk between cadherin- and integrin-mediated adhesions.
Proc. Natl. Acad. Sci. U.S.A. 107, 13324–13329
81. Bellin, R.M. et al. (2009) Defining the role of syndecan-4 in mecha-
notransduction using surface-modification approaches. Proc.
Natl. Acad. Sci. U.S.A. 106, 22102–22107
186 Trends in Biotechnology, February 2016, Vol. 34, No. 2
82. Fozdar, D.Y. et al. (2010) Hippocampal neurons respond uniquely
to topographies of various sizes and shapes. Biofabrication 2,
035005
83. Tian, B. et al. (2012) Macroporous nanowire nanoelectronic scaf-
folds for synthetic tissues. Nat. Mater. 11, 986–994
84. Wang, Y.K. et al. (2013) Cell adhesion and mechanical stimulation
in the regulation of mesenchymal stem cell differentiation. J. Cell.
Mol. Med. 17, 823–832
85. Yang, M.T. et al. (2011) Assaying stem cell mechanobiology on
microfabricated elastomeric substrates with geometrically modu-
lated rigidity. Nat. Protoc. 6, 187–213
86. Cheng, C.C. et al. (2006) Micropatterning polyvinyl alcohol as a
biomimetic material through soft lithography with cell culture. Mol.
Biosyst. 2, 299–304
87. Xia, Y. et al. (1998) Soft lithography. Angew. Chem. Int. Ed. 37,
550–575
88. Whitesides, G.M. et al. (2001) Soft lithography in biology and
biochemistry. Annu. Rev. Biomed. Eng. 3, 335–373