Scaffolds for Engineering Smooth

Muscle Under Cyclic Mechanical

Byung-Soo Kim
Graduate Student,
Department of Chemical Engineering,
University of Michigan,
Ann Arbor, MI 48109
e-mail: kim – b@hub.tch.harvard.edu
David J. Mooney
Associate Professor,
Departments of Chemical Engineering and
Biologic and Materials Sciences,
University of Michigan,
Ann Arbor, MI 48109-2136
e-mail: mooneyd@umich.edu
Strain Conditions
Cyclic mechanical strain has been demonstrated to enhance the development and function
of engineered smooth muscle (SM) tissues, but appropriate scaffolds for engineering
tissues under conditions of cyclic strain are currently lacking. These scaffolds must dis-
play elastic behavior, and be capable of inducing an appropriate smooth muscle cell
(SMC) phenotype in response to mechanical signals. In this study, we have characterized
several scaffold types commonly utilized in tissue engineering applications in order to
select scaffolds that exhibit elastic properties under appropriate cyclic strain conditions.
The ability of the scaffolds to promote an appropriate SMC phenotype in engineered SM
tissues under cyclic strain conditions was subsequently analyzed. Poly(L-lactic acid)-
bonded polyglycolide fiber-based scaffolds and type I collagen sponges exhibited partially
elastic mechanical properties under cyclic strain conditions, although the synthetic poly-
mer scaffolds demonstrated significant permanent deformation after extended times of
cyclic strain application. SM tissues engineered with type I collagen sponges subjected to
cyclic strain were found to contain more elastin than control tissues, and the SMCs in
these tissues exhibited a contractile phenotype. In contrast, SMCs in control tissues ex-
hibited a structure more consistent with the nondifferentiated, synthetic phenotype. These
studies indicate the appropriate choice of a scaffold for engineering tissues in a mechani-
cally dynamic environment is dependent on the time frame of the mechanical stimulation,
and elastic scaffolds allow for mechanically directed control of cell phenotype in engi-
neered tissues. 关S0148-0731共00兲00103-5兴

Introduction effects of cyclic strain on the phenotype of SMCs in tissues engi-

To engineer functional tissues, the interactions of the cells in
the tissue with their microenvironment must be appropriately con-
trolled during the process of tissue development 关1兴. The signals
that cells receive from their microenvironments include cell bind-
ing molecules 关2兴, cell–cell adhesive interactions 关3兴, soluble
growth factors 关4兴, and mechanical stimuli imposed on the cells
关5兴. Smooth muscle cells 共SMCs兲, a functional element of a num-
ber of cardiovascular, gastrointestinal, and urinary tissues, typi-
cally reside in a mechanically dynamic environment in vivo 关6兴.
Many studies have shown that mechanical strain significantly
regulates the phenotype of SMCs in two-dimensional culture sys-
tems 关7–11兴. Recently, we and others have demonstrated that cy-
clic mechanical strain enhanced the development and function of
engineered smooth muscle 共SM兲 tissues 关12,13兴. Specifically, cy-
clic strain increased elastin and collagen production and tissue
organization, resulting in over an order of magnitude increase in
the mechanical properties of the engineered SM, but only if cells
are adherent to scaffolds via specific cell adhesion ligands 关12兴.
The development of scaffolds that can maintain their mechani-
cal integrity and transduce the mechanical signals to adherent
cells during long-term mechanical strain application is likely to be
necessary if one wishes to engineer SM under cyclic mechanical
strain conditions. The scaffolds must be elastic and capable of
withstanding cyclic mechanical strain without cracking or signifi-
cant permanent deformation for time periods ranging from days to
months. In addition, the scaffolds must promote the appropriate
cellular phenotype in response to mechanical signals. Therefore,
we have designed the present study both to characterize the me-
chanical properties of tissue engineering scaffolds during varying
time periods of cyclic strain application, and to investigate the
Contributed by the Bioengineering Division for publication in the JOURNAL OF
BIOMECHANICAL ENGINEERING. Manuscript received by the Bioengineering Divi-
sion October 21, 1999; revised manuscript received February 6, 2000. Associate
Technical Editor: R. Vanderby, Jr.
neered with the selected scaffolds. Solid scaffolds formed from
type I collagen and fibers of polyglycolic acid 共PGA兲 physically
bonded with poly共L-lactic acid兲 共PLLA兲 were utilized in these
studies, as these scaffolds have been frequently utilized to engi-
neer SM containing tissues 关12,14–16兴, and the phenotype of en-
gineered SM tissues has been previously demonstrated to be con-
trolled by these two types of scaffold 关17兴.
Materials and Methods
Scaffolds. PGA fiber-based scaffolds 共fibers approximately
12 ␮m in diameter兲 assembled into nonwoven arrays were pur-
chased from Albany International, Inc. 共Taunton, MA兲. The po-
rosity of the PGA matrix was approximately 97 percent. The crys-
tallinity of PGA fibers was 57 percent, as measured by differential
scanning calorimetry. The inherent viscosity of PGA was 1.23
dl/g, and the residual monomer content was less than 0.3 percent.
To stabilize scaffolds, a solution of PLLA 共Boehringer Ingelheim,
Winchester, VA兲 dissolved in chloroform 共5 percent w/v兲 was
placed in an atomizer 共Devilbus Corp.兲 and sprayed over both
sides of PGA matrix from a distance of 3 in. using a nitrogen
stream 共18 psi兲 关18兴. The solvent was subsequently allowed to
evaporate, and scaffolds were lyophilized to remove residual sol-
vent. Type I collagen sponges were purchased from Integra Life
Science, Inc. 共Plainsboro, NJ兲. These sponges were fabricated by
lyophilizing bovine type I collagen solution and cross-linking with
glutaraldehyde.
Scaffold Characterization. For scanning electron micro-
scopic examination, samples were coated with gold using a Sput-
ter Coater 共Desk II, Denton Vacuum, Cherry Hill, NJ兲, and a
scanning electron microscope 共Hitachi, model S-800兲 was oper-
ated at a 5 kV voltage to image samples. The tensile properties of
the scaffolds (n⫽3) were tested using a mechanical tester 共model
810, MTS Systems Corp., Eden Prairie, MN兲. A 10-Newton-
maximum load cell was used, and the cross-head speed was 1

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 Fig. 1 Apparatus utilized to subject scaffolds to cyclic strain. Scaffolds were immersed
in PBS or medium and clamped in the tissue culture chamber. The scaffolds were sub-
jected to cyclic strain by periodical movement of a crank back and forth as an eccentric
disk that was driven by a motor and connected to the crank rotated. The frequency and
amplitude of cyclic strain were regulated by controlling the speed of motor rotation with
a controller and the position of the crank connection to the eccentric disk, respectively.

mm/min. Before the testing, samples were pre-wet by placing the
samples in a vial containing water for 24 hours. Young’s modulus
was obtained from the slope of the initial linear section of the
stress–strain curve. Elastic limit of the scaffolds was determined
by the strain at the end point of the initial linear section of the
stress–strain curve. The mass of PLLA bonded to PGA scaffolds
was determined by weighing PGA devices before and after spray-
ing. Dynamic mechanical tests of the scaffolds were performed
using a custom-made mechanical strain apparatus 共Fig. 1兲. The
scaffolds were subjected to cyclic strain at various amplitude be-
tween 2.5 and 9.0 percent and at a frequency of 0.5 Hz 共30 cycles
per minute兲 in PBS at 37°C over various time periods. Elastic
properties of the scaffolds were determined by measuring perma-
nent deformation of the scaffolds following cyclic strain applica-
tion for various time periods.
SMC Isolation. SMCs were isolated from rat aortas using an
adaptation of a previously published technique 关19兴. In brief, the
descending aortas of 300–350 g adult male Lewis rats 共Charles
River Laboratories, Wilmington, MA兲, after the removal of endo-
thelium, adventitia, fat, and connective tissue, were cut into mul-
tiple small pieces and incubated for 90 minutes at 37°C in a sterile
spinner flask 共100 ml, Bellco Glass, Inc., Vineland, NJ兲 containing
an enzymatic dissociation buffer. This buffer contains 0.125
mg/ml elastase 共Sigma Chemical Co., St. Louis, MO兲, 1.0 mg/ml
collagenase 共CLS type I, 204 units/mg, Worthington Biochemical
Corp., Freehold, NJ兲, 0.25 mg/ml soybean trypsin inhibitor 共type
1-S, Sigma兲, and 2.0 mg/ml crystallized bovine serum albumin
共BSA, Gibco/Life Technologies, Gaithersburg, MD兲. The result-
ant tissue suspension was filtered through a 100 ␮m Nitex filter
共Tetko, Inc., Briarcliff Manor, NY兲 and centrifuged at 200 g for 5
minutes. The pellet was resuspended in growth medium 共Medium
199兲 共Gibco兲 supplemented with 20 percent fetal bovine serum
共FBS兲共Gibco兲, 2 mM L-glutamine 共Gibco兲, 100 units/ml penicillin
共Gibco兲, and 0.1 mg/ml streptomycin 共Gibco兲. SMCs were main-
tained in growth media containing 20 percent FBS until the first
passage, while all subsequent cultures were grown in the presence
of 10 percent FBS. SMCs between passage 3 and 5 were used in
this study. Serum starvation, which is a common method to syn-
chronize cells in a segment of the cell cycle, was not utilized in
this study.
Engineering SM Tissues Under Cyclic Strain Condition. A
cell suspension 共3.5⫻107 cells/ml, 3 ml per sponge兲 was injected
onto type I collagen sponges (50⫻35⫻0.25 mm) in petri dishes.
Cells were not seeded at the edges of the collagen sponges where
the collagen sponges were to be clamped in an apparatus utilized
to subject the collagen sponges to cyclic strain. The dishes con-
taining the seeded collagen sponges were agitated at 100 rpm with
an orbital shaker for 24 hours in a humidified 5 percent CO2
atmosphere. This dynamic seeding method enhanced the cell
seeding efficiency dramatically compared to static seeding meth-
ods 关16兴. The seeded scaffolds were subsequently washed with
PBS 共Gibco兲, transferred to new petri dishes, and maintained in a
humidified 5 percent CO2 atmosphere for 2 days with the medium
changed every day. SMCs were subjected to cyclic strain in vitro
using custom-made strain units 共Fig. 1兲. The seeded scaffolds
were clamped in the tissue culture chamber and subjected to cy-
clic strain by movement of the crank back and forth as the eccen-
tric disk connected to the crank rotates. The strain units are placed
in a humidified incubator with 5 percent CO2 at 37°C. The seeded
scaffolds were subjected to cyclic strain at a frequency of 1 Hz 共1
cycle per second兲 and an amplitude of 7 percent of initial length
共unless otherwise specified兲, which are similar to those of SM
tissues in vivo 关20,22,23兴, for up to 20 weeks. As a control,
seeded scaffolds were fixed at only one end of the clamps and
moved back and forth at the same frequency and amplitude as the
mechanical strain conditions.
Engineered Tissue Characterization. For histological
analysis, both clamped edges of the collagen sponges were cut off,
and the rest of tissue samples were fixed in 10 percent 共v/v兲 buf-
fered formalin, dehydrated with a series of ethanol, paraffin em-
bedded, sectioned 共5 ␮m thick兲, and stained with Verhoeff’s stain-
ing. For transmission electron microscopic analysis, tissue
samples were fixed in 2 percent glutaraldehyde for 2 hours at
4°C, postfixed in 1 percent osmium tetroxide for 30 minute at
4°C, dehydrated with acetone, and embedded in polybed 812
resin 共Analytical Standards, Kungsbacka, Sweden兲. Thin sections
共70 nm thick兲 were cut by a Reichert Ultracut ultramicrotome and
stained with lead citrate and uranyl acetate. The specimens were
examined in a Jeol 100CX electron microscope 共Jeol, Tokyo,
Japan兲.
Results
The response of PGA scaffolds 共Fig. 2共a兲兲, PLLA-bonded PGA
scaffolds 共Fig. 2共b兲兲, and type I collagen sponges 共Fig. 2共c兲兲 to
cyclic mechanical strain was assessed. Nonwoven PGA fiber-
based scaffolds lack mechanical strength, and deformed perma-

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 nently even when subjected to low levels of stress. Tensile tests of
PLLA-bonded PGA scaffolds indicated the physical bonding dra-
matically increased the Young’s modulus and ultimate tensile
strength of the PGA scaffolds 共Fig. 3共a兲兲.. The Young’s modulus
of the bonded PGA scaffolds linearly increased with the mass of
PLLA utilized to bond the PGA scaffolds, and reached a value
378-fold over unbonded PGA scaffolds at the highest bonding
level 共Fig. 3共a兲兲. However, the elastic limit of the bonded PGA
scaffolds was constant in the range of 3–6 percent of initial
length, regardless of the bonding PLLA mass 共Fig. 3共c兲兲. The
tensile tests of type I collagen sponges exhibited typical elastic
mechanical behaviors, as the stress continuously increases with
the strain until the failure point 共Fig. 4兲, which is a typical stress–
strain relation for elastic rubbers 关21兴.
To determine whether PLLA-bonded PGA scaffolds and type I
collagen sponges would exhibit elastic properties under cyclic me-
chanical strain conditions, dynamic mechanical tests of these scaf-

Fig. 3 Mechanical properties of tissue-engineering scaffolds:

Fig. 2 Scanning electron microscopic photomicrographs of:
„a… nonwoven PGA fiber-based scaffold, „b… PLLA-bonded PGA
scaffold, and „c… type I collagen sponge. The size bars in „a…,
„b…, and „c… indicate 100 ␮m, 100 ␮m, and 200 ␮m, respectively.
„a… Typical tensile stress–strain curve of nonwoven PGA and
bonded PGA scaffolds. „b… Young’s moduli of PGA scaffolds
bonded with various amounts of PLLA. The moduli were ob-
tained from the slopes of the initial linear sections of tensile
strain–stress curves. „c… Elastic limits of PGA scaffolds
bonded with various amounts of PLLA. The elastic limits were
determined as the strain at the end points of the initial linear
sections of tensile strain–stress curves.

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 Fig. 4 Typical stress–strain curve of type I collagen sponges
subjected to tensile loading
folds were performed. The scaffolds were subjected to cyclic
strain at various amplitudes between 2.5 and 10 percent and at a
frequency of 0.5 Hz in PBS at 37°C for 24 hours. Unbonded
共nonwoven兲 PGA scaffolds deformed completely under these cy-
clic strain conditions 共not shown兲. Bonded PGA scaffolds exhib-
ited partially elastic properties under the cyclic strain conditions,
as the permanent deformation increased with the magnitude of the
applied strain following 24 hours of strain application 共Fig. 5兲.
The extent of permanent deformation of the bonded PGA scaf-
folds under cyclic strain conditions did not depend on the mass of
PLLA utilized to bond the PGA scaffolds 共data not shown兲. The
type I collagen sponges were almost completely elastic under cy-
clic strain for 24 hours, independent of the magnitude of applied
strain, as the permanent deformation was less than 9 percent of the
applied strain magnitude for all strain amplitudes tested 共Fig. 5兲.
Long-term dynamic mechanical tests of these scaffolds were
next performed to determine their response to strain application
over time periods of weeks. The scaffolds were subjected to a
Fig. 5 Permanent deformation of bonded PGA scaffolds and
type I collagen sponges subjected to cyclic tensile loads with
various amplitude and a frequency of 0.5 Hz for 24 hours „ n
Ä3…
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Fig. 6 Permanent deformation of bonded PGA scaffolds and
type I collagen sponges subjected to a cyclic tensile load with
an amplitude of 7 percent of initial length and a frequency of 0.5
Hz for various time periods. The mass of bonding PLLA in the
bonded PGA scaffolds was 82 percent of initial PGA mass „ n
Ä3….
cyclic strain at an amplitude of 7 percent and a frequency of 0.5
Hz in PBS at 37°C for up to 20 weeks. The bonded PGA scaffolds
exhibited an increasing permanent deformation with time of strain
application, as expected from the shorter term tests, and the
bonded PGA scaffolds failed 共cracked兲 at week 2.5 共Fig. 6兲. Type
I collagen sponges remained almost completely elastic for up to
20 weeks, and the permanent deformation was less than 2 percent
of the applied strain magnitude during the entire time course 共Fig.
6兲. The longterm tests were repeated at a higher frequency 共1 Hz兲,
and no significant differences in scaffold permanent deformation
were observed at the two different frequencies 共0.5 and 1 Hz, data
not shown兲.
The effects of cyclic mechanical strain on the phenotype of
vascular SMCs in tissues engineered with type I collagen scaf-
folds subjected to mechanical strain were next assessed. Our pre-
vious study has demonstrated that cyclic strain can significantly
alter SMC phenotype on both PGA and type I collagen scaffolds
关12兴. Type I collagen sponges seeded with SMCs 共Fig. 7兲 were
subjected to cyclic strain 共1 Hz, 7 percent兲 for 10 weeks. Cyclic
Fig. 7 Scanning electron microscopic photomicrograph of rat
aortic SMCs following seeding onto type I collagen sponge.
The size bar indicates 20 ␮m.
JUNE 2000, Vol. 122 Õ 213

Fig. 8 Photomicrographs of Verhoeff’s stained cross sections
of SM tissues engineered with type I collagen sponges for 10
weeks. The tissue constructs were subjected to „a… cyclic strain
or „b… no strain. The dark color represents positive staining for
elastin. The original magnification of the photographs was
Ã1000.
Fig. 9 Transmission electron microscopic photomicrographs of SM tissues engineered with
type I collagen sponges for 10 weeks. The tissue constructs were subjected to „a… no strain or
„b… cyclic strain. The arrows and arrow heads indicate rough endoplasmic reticulum and dense
body, respectively. N: nucleus. Size barsÄ2 ␮ m.
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strain stimulation significantly promoted elastin production com-
pared to the control condition 共no strain兲, as demonstrated by
histological analysis 共Fig. 8兲. SMCs in tissues engineered under
cyclic strain exhibited the properties of contractile phenotype, as
analyzed by transmission electron microscopy, while cells in con-
trol tissues exhibited the properties of the synthetic, nondifferen-
tiated phenotype 共Fig. 9兲. Bundles of myofilaments coalescing in
dense bodies, a contractile apparatus, were evident in SMCs under
cyclic strain, whereas the cytoplasm of cells in control tissues was
dominated by significant amounts of rough endoplasmic reticu-
lum, a synthetic organelle.
Discussion
It will be necessary to utilize elastic scaffolds if one wishes to
engineer any tissue under conditions of cyclic mechanical strain
for extended time periods. The specific PLLA-bonded PGA fiber-
based scaffolds and type I collagen sponges utilized in this study
exhibited elastic properties without a significant permanent defor-
mation under cyclic strain condition for short time periods, but
only the collagen scaffolds demonstrated elastic properties under
extended 共multiple weeks兲 application of cyclic strain. The impor-
tance of these properties was confirmed by the finding that SM
tissues engineered with type I collagen sponges under cyclic strain
condition contained more elastin, and the phenotype of SMCs in
these tissues was more consistent with a contractile, differentiated
phenotype than control tissues.
Both PLLA-bonded PGA fiber-based scaffolds and type I col-
lagen sponges possessed appropriate mechanical properties for cy-
clic strain application for time periods under 2 weeks. Physical
bonds between adjacent PGA fibers likely are responsible for the
partially elastic mechanical properties of the nonwoven PGA scaf-
folds. The physical bonds dramatically increased the mechanical
properties of the PGA scaffolds 共e.g., Young’s modulus and ulti-
mate tensile strength兲, depending on the mass of PLLA utilized to
bond the PGA fibers. However, the elastic limit of the bonded
PGA scaffolds remained constant, regardless of the mass of
PLLA. The bonded PGA scaffolds exhibited appreciable elastic
properties under cyclic strain conditions with various amplitude
Transactions of the ASME
 between 2.5 and 9.0 percent of initial length that is similar to
strain amplitudes of SM tissues in vivo 关20,22,23兴. The failure of
these scaffolds on week 2.5 is likely due to the degradation of the
scaffolds. The bonded PGA scaffolds began to degrade in PBS at
37°C on week 2, and the mechanical properties of scaffolds at this
time were dramatically decreased 关15兴. It may be possible to alter
the elastic mechanical properties of PGA scaffolds by changing
assembly of PGA fibers. Type I collagen sponges also exhibited
elastic properties under cyclic strain condition for up to 20 weeks.
Permanent deformation of the sponges was only 2–9 percent of
initial length under cyclic strain conditions with amplitude in the
range of 5–10 percent.
Utilization of appropriate scaffolds when engineering SM with
mechanical stimulation may result in tissue engineering of func-
tional SM. The functions of the SM element in many tissues 共e.g.,
blood vessels, intestines, and bladder兲 include providing appropri-
ate mechanical properties to withstand fluid pressures, maintain-
ing tissue structures as a fluid conduit or reservoir, and contracting
to regulate blood pressures or transfer fluids. Our previous study
has demonstrated that cyclic strain dramatically enhances the me-
chanical properties of SM tissues engineered with collagen
sponges by stimulating collagen and elastin synthesis and tissue
organization 关12兴. In addition, another study has shown that elas-
tin stabilizes arterial structure by regulating proliferation and or-
ganization of vascular SMCs 关24兴. In that study, arteries in mice
that lack elastin were obliterated by subendothelial proliferation of
SMCs. There have been several attempts to engineer blood vessels
关13,25,26兴, but these engineered tissues commonly exhibited low
elastin contents. The stimulation of elastin synthesis by mechani-
cal stimulation 共Fig. 8兲 may aid in maintaining the luminal struc-
tures of engineered, SM-containing tissues. In addition, induction
of contractile phenotype 共Fig. 9兲 and alignment 关12兴 of SMCs by
mechanical stimulation may allow the engineered SM tissues to
exhibit contractile functions.
Development of adequate scaffolds is critical in engineering
functional tissues for the replacement of lost or malfunctioning
tissues. If mechanical stimulation is necessary for engineering
functional SM tissues, appropriate scaffolds should have elastic
mechanical properties and surface characteristics capable of trans-
ferring mechanical signals to the cells through specific cellular
adhesion 关12兴. The scaffolds selected in this study could be ap-
propriate scaffolds for SM tissue engineering, because these scaf-
folds possess elastic mechanical properties and promote func-
tional SM tissue development under mechanical strain conditions.
These scaffolds could be utilized in further investigations of the
role of mechanical signals in SM tissue development. These stud-
ies may lead to clinically relevant engineered SM tissues, and in
turn, SM-containing tissues such as blood vessels, intestines, and
bladder. These scaffolds could also be useful in tissue engineering
of skeletal muscle and tendon, in which the cellular phenotype is
also strongly influenced by cyclic mechanical strain 关27,28兴.
Acknowledgment
Funding was provided by the NSF 共BES-9501376兲.
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