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2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Healthcare Mater. 2012, 1, 112116
Aurlie Jean and George C. Engelmayr Jr.*
Anisotropic Collagen Fibrillogenesis Within Microfabricated
Scaffolds: Implications For Biomimetic Tissue Engineering
Dr. A. Jean
Department of Bioengineering
The Pennsylvania State University
223 Hallowell Building, University Park
PA 16802, USA
Dr. G. C. Engelmayr Jr.
Department of Bioengineering
The Pennsylvania State University
223 Hallowell Building
University Park
PA 16802, USA
E-mail: gce101@psu.edu
DOI: 10.1002/adhm.201100017
Collagen gels nd widespread application as three-dimensional
substrates in cell culture assays,
[

1

]
drug delivery,
[

2

]
and tissue
engineering.
[

3

]
At the macroscale, boundary constraints inu-
ence cell-laden collagen gel anisotropy;
[

4

]
at shorter length
scales, composites of collagen gels with microfabricated mate-
rials
[

57

]
raise questions concerning how brillogenesis itself
may be inuenced by the geometry of such microstructures. In
particular, collagen gel morphology imparted by compartmen-
talization within microfabricated materials could impact func-
tional performance parameters (e.g., cell mobility, shape, or
alignment;
[

814

]
drug diffusion;
[

2

]
hierarchical engineered tissue
mechanics
[

1517

]
) of such composite devices.
In biomimetic tissue engineering,
[

18

]
collagen gels have been
used for generating functional myocardium from heart cells.
[

3

]

Collagen gels are capable of promoting cell alignment under
boundary constraint
[

4

,

10

]
or cyclic loading,
[

19

]
however they tend
to be mechanically inferior to myocardium.
[

8

]
We generated
tissue engineered myocardium by cultivating heart cells on an
accordion-like honeycomb (ALH) scaffold rendered by laser
microablation of poly(glycerol sebacate) (PGS).
[

20

]
The ALH
scaffold provided cardio-mimetic anisotropic elastic proper-
ties and a capacity to guide preferential cell alignment. Toward
enhancing heart cell-mediated contractility, we developed peri-
odic nite element simulations for investigating changes in
ALH scaffold geometry
[

21

]
and investigated improving heart cell
seeding efciency via Matrigel.
[

22

]
Matrigel, however, did not
promote cardiomyocyte elongation.
Based on observations of directional collagen brillogenesis
in collagen-doped microuidic devices
[

23

]
and that elongated
scaffold pores can promote cell-secreted collagen alignment,
[

9

]

we speculated that directional collagen brillogenesis might
manifest within ALH pores.
[

18

]
Anisotropic collagen brillogen-
esis could potentially overcome the limited heart cell elongation
observed in Matrigel-ALH composites.
[

22

]

To elucidate if ALH scaffolds can induce anisotropic col-
lagen brillogenesis, the three-dimensional bril organizations
within ALH ( Figure 1 A) and square diamond (Figure 1 B) pores
were imaged by confocal reectance microscopy (Figure 1 C,D)
and compared to collagen gelled unconstrained on glass
slides (Figure S1, Supporting Information). ALH scaffolds
preferentially oriented brils along the long axis of the pore
(Figure 1 C,E), with an orientation index OI
ALH
= 17.75 6.55%
signicantly higher than that measured for glass slides ( OI
glass
=
1.45 0.40%; p < 0.05). Indeed, neither glass slides nor square
diamond scaffolds (Figure 1 D,E; OI
square
= 0.26 0.16%)
induced preferential bril orientation along the pore long axis.
Providing a measure of both the density and homogeneity of
the collagen gel, the inter-bril distance distributions were
quantied (Figure 1 F). The mean interbril distance was 5.06
0.05 m for collagen gelled on glass slides; values for the ALH
(4.075 0.5 m) and square diamond (4.08 0.3 m) scaffolds
were lower ( p < 0.05). Hence, the organization of brils tended
to be denser upon compartmentalization within the pores of
ALH and square diamond scaffolds than on glass slides. The
entropy value calculated for ALH pores (
ALH
= 3.8 0.23) was
signicantly lower than that calculated for glass slides (
glass
=
4.6 0.10; p < 0.05), demonstrating that the organization of
brils was more ordered in ALH pores. Square diamond pores
exhibited an intermediate value
square
= 4.18 0.12.
To predict the mechanical stiffnesses and anisotropy of
ALH scaffold-collagen composites, compare these with native
heart muscle, and to investigate the ramications of aniso-
tropic collagen brillogenesis in the ALH scaffold, periodic
FE simulations
[

21

]
were conducted. FE predicted effective stiff-
nesses E
PD
and E
XD
, and anisotropy ratio r = E
PD
/ E
XD
, and Voigt
( Equation 4 ) and Reuss ( Equation 5 ) elastic bounds of the com-
posite initially assumed the collagen was isotropic using upper
(24.3 kPa
[

15

]
) and lower (5 kPa
[

24

]
) bounds of collagen stiffness.
The Reuss bound (range 7.335.1 kPa) was dictated by the most
compliant component of the composite (i.e., the collagen); the
Voigt bound (aka the rule-of-mixtures; range 265278 kPa)
was dictated by the stiffer component, and therefore varied
only slightly with the stiffness of the collagen. By contrast, FE
predicted effective stiffnesses E
PD
(range 108215 kPa) and
E
XD
(range 62.2173 kPa) depended strongly on the stiffness
of the collagen and were comparable to values measured by
uniaxial tensile testing (Figure S3, Supporting Information).
As expected, FE predicted and measured values of E
PD
and E
XD

with 3 mg mL
1
collagen gelled within the pores were higher
than those reported for the ALH scaffold itself ( E
PD
= 83 kPa
and E
XD
= 31 kPa
[

20

]
). The FE predicted anisotropy ratios r =
1.2 and r = 1.7 associated with the upper and lower collagen
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2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Healthcare Mater. 2012, 1, 112116
stiffness bounds, respectively, were signicantly lower than that
predicted for the ALH scaffold without collagen (2.5)
[

21

]
. Rec-
ognizing that the collagen within the ALH scaffold was itself
anisotropic (Figure 1 ), we simulated collagen anisotropy via an
orthotropic material model targeting left ventricular stiffnesses
in the circumferential (157 kPa) and longitudinal (84 kPa)
directions ( r = 1.87)
[

20

]
and solved for the requisite collagen gel
stiffnesses in the PD and XD directions. FE simulations pre-
dicted collagen gel stiffnesses E
coll
PD
= 47.0 kPa and E
coll
PD
= 26.5
kPa ( r
coll
= 1.77). Finally, comparing the spatial distribution
of equivalent von Mises strain within the collagen matrix for
isotropic ( Figure 2 A) and orthotropic (Figure 2 B) assumptions,
orthotropic yielded a more homogeneous strain distribution
(range 0.040.05) along the central PD axis versus isotropic
collagen gel (range 0.030.07). Hence, FE simulations can be
used to predict collagen gel properties required to match ALH
scaffold-collagen composite stiffnesses and anisotropy to native
heart muscle.
Composite devices comprised of microfabricated materials
and collagen gels offer the prospect of controlled bridging
Figure 2 . Predicted spatial distribution of strain (equivalent von Mises) within the ALH pore simulating (A) isotropic versus (B) orthotropic collagen gel
lling the pore. For each simulation a macroscopic strain of 0.1 was prescribed along the PD direction. (C) The orthotropic collagen gel was predicted
to yield a more homogeneous strain distribution (range 0.040.05) along the central PD axis of the ALH pore compared with an isotropic collagen gel
(range 0.030.07). Of note, in the isotropic case ( E
coll
= 10 kPa) the spatial strain distribution pattern was dictated solely by the shape of the ALH pore;
by contrast, in the orthotropic case ( E
coll
PD
/ E
coll
XD
= 47. 0 kPa / 26.5 kPa ) the pattern was further inuenced by simulation of PD-aligned collagen. Color
bar and associated numeric values indicate equivalent von Mises strain and apply to all gure panels AC.
Figure 1 . Representative scanning electron microscopy images of ALH (A) and square diamond (B) scaffolds (100 original magnication; scale bars =
1 mm). Confocal reectance micrographs of collagen brils within a representative pore of an ALH (C) and square diamond (D) scaffold (400 original
magnication; scale bars = 100 m). Collagen bril angular orientation distributions (E) and inter-bril distance distributions (F) measured by image
analysis of collagen-lled ALH (top; red) and square diamond pores (bottom; red) compared with collagen gelled unconstrained on a glass slide
(blue).
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2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Healthcare Mater. 2012, 1, 112116
between the micro-to-nanometer length scales, potentially
yielding novel in vitro cell culture assays,
[

1

]
drug delivery
systems,
[

2

]
and engineered tissues.
[

3

]
Hierarchically, engineered
tissues formed by seeding cells onto microfabricated scaffolds
evolve through the structuralmechanical interplay between
scaffold, cells, and extracellular matrix. Without accounting
for extracellular matrix, we demonstrated that heart cell-seeded
ALH scaffolds could mimic aspects of cardiac anisotropy.
[

20

]

Extracellular collagen structures, however, play important
roles in myocardium.
[

18

]
We demonstrated three key ndings
regarding collagen gelled within the ALH pore versus uncon-
strained on a glass slide: 1) increased order of the bril distri-
bution (i.e., decreased entropy;
ALH
= 3.8 0.23 versus
glass
=
4.6 0.10; p < 0.05), 2) increased bril density (i.e., decreased
mean inter-bril distance; d
ALH
= 4.075 0.5 m versus
d
glass
= 5.06 0.05 m; p < 0.05), and 3) increased bril alignment
along the reference angle dened by the ALH pore long axis
( OI
ALH
= 17.75 6.55% versus OI
glass
= 1.45 0.40%; p < 0.05).
For comparison, Bayan et al. reported entropy values ranging
from 6.376.5 and OI values ranging from 9.4513.46% in
similar acellular collagen gels.
[

25

]
Of note, Bayan et al. did not
detect signicant differences in OI when comparing 1, 2, and
3 mg mL
1
collagen gels. Further, when gelled on a glass slide
and compared with the 3 mg mL
1
gel, we did not detect any dif-
ference in the collagen orientation distribution in a 6 mg mL
1

collagen gel (Figure S4, Supporting Information). Of note,
the degree of collagen bril alignment mediated by the ALH
pore geometry alone ( OI = 17.75 6.55%) was less than that
observed by Bayan et al. in a 3 mg mL
1
cell laden gel cultivated
for 12 days ( OI = 30.86 14.76%).
[

25

]

A combination of mechanisms may have contributed to
the anisotropic collagen brillogenesis observed herein. For
example, when 3 mg mL
1
collagen solution was owed into
and gelled within the channels of a collagen-doped alginate
microuidic device, Gillette et al. observed that a number of
collagen brils appeared to bridge contiguously, in straight
lines, from the collagen-doped alginate (i.e., the channel walls)
into the collagen gelled within the channel.
[

23

]
Coupled with the
preference for collagen bril tip growth predicted by diffusion
limited aggregation models by Parkinson et al.,
[

26

]
the results
from Gillette et al. suggest that collagen brils can grow in a
straight line from the tips of collagen brils exposed at a sur-
face into the bulk of a collagen solution. In the present study
collagen solution was gelled in direct contact with the PGS
structural elements of the ALH scaffold. In a previous study,
Sales et al. demonstrated that type I collagen can adsorb to a
PGS foam scaffold from dilute solutions, reaching a maximum
surface concentration from solutions as dilute 20 L mL
1
col-
lagen.
[

27

]
We thus expect that the surfaces of the PGS struts
were saturated with adsorbed collagen under the conditions
tested herein, and that upon exhausting the available PGS strut
surface area, the growing tips of the collagen brils would tend
to progress outward from the struts into the bulk collagen solu-
tion lling the pore. Indeed, proximal to the collagen gel-PGS
strut interfaces, confocal reectance micrographs qualitatively
revealed that collagen brils were arranged not in parallel, but
rather at nite angles or roughly perpendicular to the PGS
struts (Figure 1 C,D). In the case of the square diamond pore, in
which the PGS struts were oriented at opposing angles of 45
and at equal distances from each other, essentially equal frac-
tions of the collagen brils were oriented at 45 , yielding no
single preferential angle of alignment (Figure 1 E). By contrast,
while the PGS struts were likewise oriented at 45 in the ALH
scaffold, the distances between opposing struts were longer
along the PD versus XD direction, thereby offering a longer
path for extension of collagen brils along the PD direction of
the ALH pore.
We undertook FE simulations to predict what stiffnesses
the collagen matrix would need to manifest in order for the
effective stiffnesses and anisotropy of the ALH-collagen com-
posite to match those of native left ventricular myocardium.
FE simulations predicted the collagen would need to exhibit
E
coll
PD
= 47.0 kPa and E
coll
XD
= 26.5 kPa ( r
coll
= 1.77) in order
for the composite to reach 157 kPa and 84 kPa ( r = 1.87).
[

20

]

Simulations suggested two potential routes toward matching
ALH-based constructs to left ventricular mechanical proper-
ties. In the context of heart cell-seeding,
[

20

,

22

]
the stiffness of
the collagen gel would be expected to increase as the gel is
contracted by the seeded cells; a potential limitation, however,
could be debonding of the collagen from the PGS scaffold
upon cell-mediated gel contraction. Toward such approaches,
we have demonstrated that cells and collagen can be retained
within the ALH pore upon stretching the ALH scaffold
(Figure S5, Supporting Information). A broad range of cell-
seeded collagen gel stiffnesses have been reported ranging
from 37 kPa (estimated from Figure 4 of Feng et al.
[

8

]
) to
5.33 1.33 MPa.
[

28

]
These studies suggest simulation predicted
collagen stiffnesses of 26.547.0 kPa could be achieved by an
appropriate combination of collagen gel concentration, cell
seeding density, and cultivation time. An alternative approach
could involve co-varying the ALH scaffold structure (e.g., strut
width) and PGS curing conditions (i.e., PGS modulus).
[

21

]

We demonstrated by FE simulations that two distinct values
of strut width ( w ) are capable of yielding an anisotropy ratio
equal to that of left ventricular myocardium (i.e., r = 1.87): w =
20 m or w = 140 m.
[

21

]
The 20 m strut width would be both
feasible to microfabricate and provide allowance for increased
collagen matrix stiffnesses associated with heart cell-mediated
contraction. As collagen ber alignment alone is not suf-
cient to explain the high degree of anisotropy observed in
broblast-seeded collagen gels,
[

29

]
we speculate that the ani-
sotropic collagen brillogenesis demonstrated herein, while
signicant, represents only a starting point in understanding
the interplay between pore geometry, collagen morphology,
and cell morphology. In future studies, Voronoi tessellation-
based models could be useful in coupling collagen gel mor-
phology to mechanical behavior.
[

30

]
Further, the evolution of
collagen anisotropy demonstrated in the present study may
be extendable to other hydrogels, such as brin and Matrigel.
Of particular note, Bian et al. demonstrated that muscle cell-
laden brin-based hydrogels can be spatially patterned into
anisotropic tissue bundles by casting within microfabricated
poly(dimethysiloxane) molds.
[

31

]
More broadly, collagen gel-
based cell culture assays and drug delivery systems may mani-
fest and potentially exploit anisotropic collagen brillogenesis,
in particular in miniaturized composites of collagen gel and
microfabricated or microscale materials. For example, in a
miniaturized aortic ring assay introduced by Reed et al., 30 L
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2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Healthcare Mater. 2012, 1, 112116
of collagen solution was gelled within and supported by a
nylon mesh ring (3 mm inside diameter) comprised of 50 m
diameter bers arranged in a square lattice ( 125 125 m
inside pore dimensions).
[

1

]
As such systems are further mini-
aturized for high throughput screening, collagen morphology
induced by the system boundaries could potentially impact
the directionality of capillary sprouting; similar phenomena
could potentially be exploited in microuidic collagen gels
mimicking human microvascular networks.
[

32

]
In the context
of drug delivery, De Paoli et al. have reported on the effects of
oscillating magnetic elds on drug release from magnetic col-
lagen gels (i.e., collagen gels containing iron oxide particles of
up to 3 m diameter).
[

33

]
In magnetic collagen gels, structural
changes within the gel associated with oscillating magnetic
elds were demonstrated to impact drug release kinetics; sim-
ilar effects could potentially be mediated by collagen gelation
within the compartments of microfabricated drug delivery
devices.
[

34

]
In concert with auxiliary biophysical and bio-
chemical regulators, compartmentalizing collagen gels within
microfabricated materials represents a promising strategy for
controlling collagen bril anisotropy and associated functional
performance parameters in advanced cell culture assay, drug
delivery, and tissue engineering applications.
Experimental Section
PGS ScaffoldCollagen Gel Composite Preparation : The ALH and
square diamond scaffolds (5 mm 5 mm) were fabricated by 213 nm
laser microablation (LSX-213; CETAC Technologies, Inc., Omaha, NE)
of 250 m thick PGS cured under vacuum ( < 50 mTorr) for 7.5 h at
160 C.
[

22

,

35

]
The ALH and square diamond scaffolds each comprised
a periodic tessellation of unit cells with a strut width of 50 m and a
strut length (inside the pore) of 200 m (Figure 1 A,B). The long axis of
the ALH pore was dened as the preferred direction (PD) and the short
axis the cross-preferred direction (XD). Collagen gel was prepared on ice
by adding 100 L of 10X phosphate buffered saline (PBS) to 800 L of
3 mg mL
1
bovine dermal collagen solution (Sigma, St. Louis, MO). The
solution was neutralized to pH 7.27.6 by 75 L of 0.1 M NaOH and
300 L was pipetted onto a glass microscope slide (VWR, West Chester,
PA) or the scaffold, coverslipped, and then incubated for 1 h in a CO
2

incubator at 37 C. Specimens were then imaged immediately without
xation.
Confocal Reectance Imaging : The collagen brils were imaged using
a confocal microscope (FluoView 1000; Olympus America, Center Valley,
PA) in reectance mode (488 nm) (Figure 1 C,D).
[

36

]
The interval between
z -stack images corresponded to the ( x , y ) plane resolution (i.e, 0.621 m
per pixel for 40X objective and 512 512 pixel image). Images were rst
segmented batch-wise via a custom automated algorithm implemented
in Matlab
[

37

]
(Figure S1, Supporting Information). The algorithm
comprised: RGB to grayscale conversion, a median lter, local adaptive
thresholding to yield binary images, morphological opening with a
1 pixel sized structural element.
Image Analysis of Collagen Fibril Organization : The morphology of
the collagen brils was analyzed from confocal micrographs based
on set theory for image analysis using custom code written in Matlab
and Python.
[

38

]
Measures were averaged over the entire z -stack (i.e.,
5070 images) and over 10 pores (or locations for the glass slide).
The size (i.e., interbril distance) distribution of the inter-bril spaces
was computed by opening granulometry with a disk D of diameter d
on complementary binary images. Opening granulometry consisted of
morphologically opening the set of pixels in each image corresponding
to the inter-bril spaces using a sequence of increasing larger structural
elements (i.e., disks D of diameter d ). At each value of d, the probability
P of one point (i.e., one pixel in the discretized image) belonging to the
opened set was calculated ( O ( d )) ( Equation 1 ) (Figure S2, Supporting
Information).
[

39

]
The opening operation included an erosion ( ) with
D ( d ) following by a dilation () with an identical D ( d ), removing all
disconnected groups of pixels with a size less than d .

O(d ) = P(x ((A
brils
)
C
(d )) D(d ))

(1)
The bril angular orientation distribution was measured using the
fast Fourier transform (FFT),
[

40

]
which yields the intensity angular
distribution I ( ). The angle
m
for which the intensity was maximum
(m) was identied on I ( ) and dened the predominant bril direction.
The FFT-based image analysis used a Matlab program previously used
to quantify cellular F-actin lament orientation.
[

20

]
An orientation index
OI (
m
) was calculated from I ( ) at
m
to yield the percentage of brils
strictly oriented in the reference angle direction (herein designated as 0
to correspond with the PD direction of the ALH pore) ( Equation 2 )
[

25

]
.

OI (
m
) = 100
_
2
_
_
180
0
I(

)
_
cos
2
(

m
)
_
d

_
180
0
I(

)d

_
1
_

(2)

Of note, the orientation index used herein
[

25

]
was distinct from that
used in our previous study.
[

20

]
The entropy (i.e., a measure of disorder)
was measured by processing the Hough transform
[

41

]
using a custom
Matlab program. The Shannon entropy of the probability distribution
p ( ) of predominant directions was calculated ( Equation 3 ).
[

25

]


=

p()
_
log [ p()]
_

(3)
Statistical differences were determined by two-tailed student t-tests
(Matlab) with a p -value < 0.05 considered signicant. Values reported
represent mean standard deviation for n = 6 samples.
Periodic Finite Element Simulations : A multiscale periodic FE method
previously applied to the ALH scaffold itself
[

21

]
was used herein to predict
the elastic stiffnesses of the ALH scaffold-collagen gel composite ( Z-set
software
[

42

]
). The Youngs modulus and Poisson ratio of the PGS forming
the scaffold struts were specied as 825 kPa and 0.45, respectively.
[

21

]

Upper and lower bounds on Youngs modulus (linear elastic isotropic
assumptions) of a 3 mg mL
1
collagen were specied as 24.3 kPa
[

15

]
and
5 kPa
[

24

]
and a Poisson ratio of 0.45 was assumed. The elastic stiffnesses
of the ALH scaffold-collagen gel composite in the PD ( E
PD
) and XD
( E
XD
) directions were predicted. An anisotropy ratio r = E
PD
/ E
XD
was
calculated.
[

21

]
In addition, the Voigt ( Equation 4 ) and Reuss ( Equation 5 )
bounds known as the upper (i.e., the rule-of-mixtures) and lower elastic
bounds were determined:

E
Voigt
= E
PGS
Vv
PGS
+ E
coll
Vv
coll

(4)

E
Reuss
=
_
Vv
PGS
E
PGS
+
Vv
coll
E
coll
_
1

(5)

Simulations were conducted allowing for anisotropic elastic behavior
of the collagen gelled within the ALH pores via an orthotropic material
model in which elastic constants were optimized (augmented Lagrangian
method) based on the criteria that E
PD
and E
XD
equaled values measured
previously for adult rat left ventricular myocardium in the respective
circumferential and longitudinal directions.
[

20

]
In the orthotropic material
model the collagen gel stiffness in the z (i.e., thickness) direction was
assumed to take an intermediate value (i.e., 10 kPa) between upper
[

15

]

and lower
[

24

]
bounds.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
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Acknowledgements
Funding for this work was provided by National Institutes of Health
(NIH) Grant 1-R01-HL086521-01A2 (PI Dr. Lisa E. Freed, Subaward
PI GCE). This funding was made possible by the American Recovery and
Reinvestment Act. The authors gratefully acknowledge Dr. Lisa E. Freed
(The Charles Stark Draper Laboratories and Massachusetts Institute of
Technology, Cambridge, MA) for helpful discussions in preparing the
manuscript and Sarah R. Bass, John L. Dzikiy, and Harshal Sawant for
their assistance conducting experiments.
Received: November 4, 2011
Published online: December 15, 2011