Controlling the spatial organization of cells and extracellular matrix proteins in engineered tissues using ultrasound standing wave fields.
ABSTRACT Tissue engineering holds great potential for saving the lives of thousands of organ transplant patients who die each year while waiting for donor organs. However, to successfully fabricate tissues and organs in vitro, methodologies that recreate appropriate extracellular microenvironments to promote tissue regeneration are needed. In this study, we have developed an application of ultrasound standing wave field (USWF) technology to the field of tissue engineering. Acoustic radiation forces associated with USWF were used to noninvasively control the spatial distribution of mammalian cells and cell-bound extracellular matrix proteins within three-dimensional (3-D) collagen-based engineered tissues. Cells were suspended in unpolymerized collagen solutions and were exposed to a continuous wave USWF, generated using a 1 MHz source, for 15 min at room temperature. Collagen polymerization occurred during USWF exposure resulting in the formation of 3-D collagen gels with distinct bands of aggregated cells. The density of cell bands was dependent on both the initial cell concentration and the pressure amplitude of the USWF. Importantly, USWF exposure did not decrease cell viability but rather enhanced cell function. Alignment of cells into loosely clustered, planar cell bands significantly increased levels of cell-mediated collagen gel contraction and collagen fiber reorganization compared with sham-exposed samples with a homogeneous cell distribution. Additionally, the extracellular matrix protein, fibronectin, was localized to cell banded areas by binding the protein to the cell surface prior to USWF exposure. By controlling cell and extracellular organization, this application of USWF technology is a promising approach for engineering tissues in vitro.
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ABSTRACT: Accurate control over positioning of cells is a highly desirable feature in tissue engineering applications since it allows, for example, population of substrates in a controlled fashion, rather than relying on random seeding. Current methods to achieve a differential distribution of cells mostly use passive patterning methods to change chemical, mechanical or topographic properties of surfaces, making areas differentially permissive to the adhesion of cells. However, these methods have no ad hoc control over the actual deposition of cells. Direct patterning methods like bioprinting offer good control over cell position, but require sophisticated instrumentation and are often cost- and time-intensive. Here, we present a novel electronically controlled method of generating dynamic cell patterns by acoustic trapping of cells at a user-determined position, with a heptagonal acoustic tweezer device. We demonstrate the capability of the device to create complex patterns of cells using the device's ability to re-position acoustic traps by using a phase shift in the acoustic wave, and by switching the configuration of active piezoelectric transducers. Furthermore, we show that by arranging Schwann cells from neonatal rats in a linear pattern we are able to create Bands of Büngner-like structures on a non-structured surface and demonstrate that these features are able to guide neurite outgrowth from neonatal rat dorsal root ganglia.Lab on a Chip 05/2014; · 5.70 Impact Factor
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ABSTRACT: Type I collagen is the primary fibrillar component of the extracellular matrix, and functional properties of collagen arise from variations in fiber structure. This study investigated the ability of ultrasound to control collagen microstructure during hydrogel fabrication. Under appropriate conditions, ultrasound exposure of type I collagen during polymerization altered fiber microstructure. Scanning electron microscopy and second-harmonic generation microscopy revealed decreased collagen fiber diameters in response to ultrasound compared to sham-exposed samples. Results of mechanistic investigations were consistent with a thermal mechanism for the effects of ultrasound on collagen fiber structure. To control collagen microstructure site-specifically, a high frequency, 8.3-MHz, ultrasound beam was directed within the center of a large collagen sample producing dense networks of short, thin collagen fibrils within the central core of the gel and longer, thicker fibers outside the beam area. Fibroblasts seeded onto these gels migrated rapidly into small, circularly arranged aggregates only within the beam area, and clustered fibroblasts remodeled the central, ultrasound-exposed collagen fibrils into dense sheets. These investigations demonstrate the capability of ultrasound to spatially pattern various collagen microstructures within an engineered tissue noninvasively, thus enhancing the level of complexity of extracellular matrix microenvironments and cellular functions achievable within three-dimensional engineered tissues.The Journal of the Acoustical Society of America 08/2013; 134(2):1491-502. · 1.65 Impact Factor
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ABSTRACT: Histology and biochemical assays are standard techniques for estimating cell concentration in engineered tissues. However, these techniques are destructive and cannot be used for longitudinal monitoring of engineered tissues during fabrication processes. The goal of this study was to develop high-frequency quantitative ultrasound techniques to nondestructively estimate cell concentration in three-dimensional (3-D) engineered tissue constructs. High-frequency ultrasound backscatter measurements were obtained from cell-embedded, 3-D agarose hydrogels. Two broadband single-element transducers (center frequencies of 30 and 38 MHz) were employed over the frequency range of 13-47 MHz. Agarose gels with cell concentrations ranging from 1 × 10(4) to 1 × 10(6) cells mL(-1) were investigated. The integrated backscatter coefficient (IBC), a quantitative ultrasound spectral parameter, was calculated and used to estimate cell concentration. Accuracy and precision of this technique were analyzed by calculating the percent error and coefficient of variation of cell concentration estimates. The IBC increased linearly with increasing cell concentration. Axial and lateral dimensions of regions of interest that resulted in errors of less than 20% were determined. Images of cell concentration estimates were employed to visualize quantitatively regional differences in cell concentrations. This ultrasound technique provides the capability to rapidly quantify cell concentration within 3-D tissue constructs noninvasively and nondestructively.Annals of Biomedical Engineering 03/2014; · 3.23 Impact Factor
d Original Contribution
CONTROLLING THE SPATIAL ORGANIZATION OF CELLS
AND EXTRACELLULAR MATRIX PROTEINS IN ENGINEERED TISSUES
USING ULTRASOUND STANDING WAVE FIELDS
KELLEYA. GARVIN,*xDENISE C. HOCKING,*yxand DIANE DALECKI*zx
*Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA;yDepartment of Pharmacology
of Rochester, Rochester, NY, USA; andxRochester Center for Biomedical Ultrasound, University of Rochester,
Rochester, NY, USA
(Received 4 May 2010; revised 30 July 2010; in final form 13 August 2010)
Abstract—Tissue engineering holds great potential for saving the lives of thousands of organ transplant patients
who die each year while waiting for donor organs. However, to successfully fabricate tissues and organs in vitro,
methodologies that recreate appropriate extracellular microenvironments to promote tissue regeneration are
needed. In this study, we have developed an application of ultrasound standing wave field (USWF) technology
to the field of tissue engineering. Acoustic radiation forces associated with USWF were used to noninvasively
control the spatial distribution of mammalian cells and cell-bound extracellular matrix proteins within three-
dimensional (3-D) collagen-based engineered tissues. Cells were suspended in unpolymerized collagen solutions
and were exposed to a continuous wave USWF, generated using a 1 MHz source, for 15 min at room temperature.
Collagen polymerization occurred during USWF exposure resulting in the formation of 3-D collagen gels with
distinct bands of aggregated cells. The density of cell bands was dependent on both the initial cell concentration
and the pressure amplitude of the USWF. Importantly, USWF exposure did not decrease cell viability but rather
enhanced cell function. Alignment of cells into loosely clustered, planar cell bands significantly increased levels of
cell-mediated collagen gel contraction and collagen fiber reorganization compared with sham-exposed samples
with a homogeneous cell distribution. Additionally, the extracellular matrix protein, fibronectin, was localized
to cell banded areas by binding the protein to the cell surface prior to USWF exposure. By controlling cell and
extracellular organization, this application of USWF technology is a promising approach for engineering tissues
in vitro. (E-mail: email@example.com)
? 2010 World Federation for Ultrasound in Medicine & Biology.
Key Words: Ultrasound, Standing wave field, Acoustic radiation force, Tissue engineering, Extracellular matrix,
for replacing diseased or damaged tissues and organs
(Langer and Vacanti 1993). During the past decade,
several promising tissue engineering strategies have
directly into damaged tissue, implanting tissue analogs
generated in vitro from cultured cells and stimulating
tissue regeneration in situ (Griffith and Naughton 2002).
To date, these strategies have met with limited success.
With native tissue remodeling, the three-dimensional
(3-D) extracellular matrix provides cells with critical
biomechanical and biochemical signals that mediate cell
adhesion, control cell function and, in turn, guide tissue
development. As such, it has become increasingly clear
that recreating the appropriate microenvironment for en-
gineered tissues is a key step to converting basic tissue
engineering strategies into successful clinical treatments.
ogies that can specifically control cell and extracellular
matrix organization hold great potential for engineering
tissues in vitro.
Technologies currently in development to organize
cells and proteins into complex patterns can be divided
into two general categories. In the first approach, micro-
patterning of cell-adhesive contacts using extracellular
matrix proteins coated onto microfabricated stamps by
Address correspondence to: Diane Dalecki, Ph.D., Department of
Biomedical Engineering, 310 Goergen Hall, P.O. Box 270168, Univer-
sity of Rochester, Rochester, NY 14627. E-mail: dalecki@bme.
Ultrasound in Med. & Biol., Vol. 36, No. 11, pp. 1919–1932, 2010
Copyright ? 2010 World Federation for Ultrasound in Medicine & Biology
Printed in the USA. All rights reserved
0301-5629/$ - see front matter
photolithography or microcontact printing is used to
direct cell adhesion into predesigned patterns. In the
second approach, a force is applied to cells to direct
cell movement to a desired location. The applied force
can be optical, magnetic, electrokinetic or fluidic (Lin
et al. 2006). In the current study, we examine the ability
of acoustic radiation forces associated with ultrasound
standing wave fields to control the spatial distribution
of cells and the extracellular matrix protein, fibronectin,
in a collagen-based model tissue.
When an ultrasonic pressure wave is incident on an
acoustic reflector, the reflected wave interferes with the
incident wave resulting in the development of an ultra-
sound standing wave field (USWF). An USWF is charac-
terized by areas of maximum pressure, known as pressure
antinodes, and areas of zero pressure, known as pressure
nodes. Exposure of particle or cell suspensions to an
USWF can result in the alignment of particles or cells
into bands that are perpendicular to the direction ofsound
propagation and that are spaced at half-wavelength
intervals (Coakley et al. 1989; Dyson et al. 1974; Gould
and Coakley 1974; Whitworth and Coakley 1992). A
primary acoustic radiation force, (Frad), generated along
the direction of sound propagation in the USWF, is
largely responsible for this movement. Fradis defined as
where Pois the USWF peak pressure amplitude, V is the
spherical particle volume, l is the wavelength of the
sound field, z is the perpendicular distance on axis from
pressure nodal planes and f is an acoustic contrast factor
? f ? sin
where rpand bpare the density and compressibility of the
particles or cells and ro and bo are the density and
compressibility of the suspending medium, respectively
ApplicationsofUSWFinbiotechnology use the radi-
ation forces associated with USWF to aggregate cells at
defined locations within suspending media (Coakley
1997; Coakley et al. 2000). Exposure of cell suspensions
to USWF can result in cellular aggregation at areas
of minimum acoustic pressure (the pressure nodes)
(Coakley et al. 1989; Dyson et al. 1974; Gould and
Coakley 1974; Whitworth and Coakley 1992). Ultrasonic
filtration systemsuse USWF
aggregates of cells from their suspending media (Hawkes
et al. 1997; Limaye and Coakley 1998). USWF may be
used to manipulate cells within microfluidic devices for
various applications (Wiklund et al. 2006). Additionally,
half-wavelength USWF devices have been used to create
cellular aggregates in suspension to study cell behavior
following aggregation (Bazou et al. 2005; Bazou et al.
2006; Edwards et al. 2010; Kuznetsova et al. 2009).
Subsequent sedimentation and removal of cell aggregates
from similar USWF devices have been used to develop
cell culture systems (Bazou et al. 2008; Hultstro ¨m et al.
2007; Liu et al. 2007).
The acoustic radiation forces that band particles
exist only during application of the USWF. Suspending
states during USWF exposure have been used to maintain
the USWF-induced banded distribution. For example,
USWF have been used to align yeast and red blood cells
in polyacrylamide, alginate and agar (Gherardini et al.
2002; Gherardini et al. 2005). Others have localized
acrylic particles in polysiloxane resin (Saito et al. 1998,
1999). In this way, the banded pattern of particles may
be retained after removal of the sound field.
can be used to organize mammalian cells and extracel-
lular matrix proteins at defined spatial locations within
collagen-based, 3-D tissue constructs. The conversion
of soluble type-I collagen to polymerized gel during
USWF exposure was used to maintain the 3-D spatial
organization of cells after exposure. Wepresent data indi-
enhance cell function and extracellular matrix organiza-
tion and discuss applications of this technology to the
field of tissue engineering.
MATERIALS AND METHODS
The experimental set-up used for all USWF expo-
sures is depicted in Figure 1a. A plastic exposure tank
(36 3 20 3 18 cm) was filled with degassed, deionized
water at room temperature. The acoustic source consisted
of a 1 MHz unfocused transducer, fabricated from
a 2.5 cm diameter piezoceramic disk. The transducer
was mounted on the bottom of the water tank. The signal
driving the transducer was generated by a waveform
generator (Model 33120A; Hewlett Packard, Palo Alto,
CA, USA), radio-frequency (RF) power amplifier (Model
2100L; ENI, Rochester, NY, USA) and an attenuator
(Model 837; Kay Elemetrics Corp., Lincoln Park, NJ,
USA). Samples were contained within the wells of
a modified silicone elastomer-bottomed cell culture plate
(BioFlex?culture plates; FlexCellInternational Corpora-
tion, Hillsborough, NC, USA). These sample holders
were mounted to a three-axis positioner (Series B4000
Unislide; Velmex Inc., East Bloomfield, NY, USA) to
allow precise control over their location within the sound
field. The air interface above the samples was used as the
1920Ultrasound in Medicine and BiologyVolume 36, Number 11, 2010
acoustic reflector togeneratean USWFwithin the sample
Sample holder preparation
The BioFlex?culture plates used as sample holders
for our investigations are depicted in Figure 1b. They
were modified from the manufacturer’s form by reducing
the diameter of three wells per plate from 4 cm to 1 cm
using Sylgard?184 silicone elastomer (Dow Corning
Corporation, Midland, MI, USA). Through this modifica-
tion,the diameter ofthesamplewascomparable insizeto
the –6 dB beamwidth at the exposure location. The
two-part silicone elastomer was mixed in a 10:1 ratio as
recommended by the manufacturer’s instructions. The
solution was degassed at room temperature using
a vacuum chamber (Model 5830; National Appliance
Company, Portland, OR, USA) and was subsequently
poured around 1 cm diameter Teflon?mandrels (Dupont,
Wilmington, DE, USA) that were placed at the center of
elastomer at 20?C for 48 h, the mandrels were carefully
removed to leave a 1 cm diameter sample space within
three wells of each BioFlex?culture plate (Fig. 1b).
The acoustic attenuations of the silicone elastomer
well bottom of the BioFlex?plates, the Sylgard?184 sili-
cone elastomer and standard tissue culture polystyrene
(Corning/Costar, Cambridge, MA, USA) were measured
using an insertion loss technique. Using the water tank
set-up, each material was inserted into the acoustic path
and a hydrophone (either a bilaminar PVDF membrane
hydrophone [Marconi Research Center, Chelmsford,
England] or a needle hydrophone [Model HNC-0400;
Onda Corporation, Sunnyvale, CA, USA]). Peak positive
and peak negative pressure amplitudes were measured
using the hydrophone and a digital oscilloscope (Model
9310AM; LeCroy, Chestnut Ridge, NY, USA) in the pres-
ence and absence of each material for various source
amplitudes. The thickness of each material was measured
using calipers. The acoustic attenuation coefficient
(in dB/MHz/cm) was calculated for each material.
The acoustic absorption coefficient of Sylgard?184
silicone elastomer was measured using a thermocouple
Fig. 1. Schematics of the experimental set-up and sample holders used for ultrasound standing wave field (USWF) expo-
sures. (a) An unfocused, 1 MHz, 2.5 cm diameter transducer was mounted on the bottom of a plastic exposure tank filled
with degassed, deionized water. The acoustic fields were generated using a signal generator, radio-frequency (RF) power
amplifier andanattenuator tocreate anRFsignaltodrivethetransducer.Using athree-axis positioner,the sample holders
were placed with well bottoms situated at the water-air interface 12.2 cm from the transducer such that the center of the
lower, left-hand well was on the transducer axis. (b) Silicone elastomer-bottomed BioFlex?culture plates were modified
from their original form by reducing the diameter of 3 wells per plate to 1 cm using Sylgard?184 silicone elastomer
molds. The left-hand well contained the sample exposed to USWF. The right-hand wells served as sham control wells
for cell-containing and non-cell-containing collagen samples.
Application of USWF technology to tissue engineering d K. A. GARVIN et al. 1921
technique. Briefly, a 50-mm copper-constantan thermo-
couple was embedded in a sample of Sylgard?184 sili-
cone elastomer. Using the water tank set-up, the active
element of the embedded thermocouple was positioned
at the focus of a 1 MHz transducer fabricated from
a 3.8 cm diameter plane, piezoceramic disk cemented
to the back of a plano-concave lens. A laboratory ther-
mometer (Model BAT-4; Bailey Instruments Co. Inc.,
Saddle Brook, NJ, USA) and digital oscilloscope were
used to monitor the thermocouple output for various
pulsing parameters and exposure amplitudes. For each
exposure condition, the initial rate of temperature rise in
(Ispta) were measured and used to calculate the absorption
coefficient. The calculated absorption coefficients from
each exposure condition were averaged to determine the
acoustic absorption coefficient (in dB/cm) of Sylgard?
184 silicone elastomer at 1 MHz.
Temperature changes in the collagen/cell samples
were also monitored during USWF exposure using
of USWF exposures.
Acoustic field measurements
Using the water tank set-up, axial and transaxial
spatial distributions of pressure from the 1 MHz, 2.5 cm
diameter unfocused transducer were measured under
USWF exposure conditions in both the presence and
absence of the sample holder. The Onda needle hydro-
phone, connected to a three-axis positioner, and a digital
oscilloscope were used to measure the acoustic pressure.
The sample holder was placed in the far-field with the
well bottoms situated at an axial distance of 12.2 cm
were measured through a 0.5 cm distance below the air
imates the height of the collagen samples used in our
were measured at an axial distance of 12.2 cm from the
transducer in 0.1 mm intervals. A sinusoidal pulse of 50
ms duration was employed and peak positive pressures
were measured for each position.
Acoustic field calibrations
Prior to each experiment, the acoustic field was cali-
the Onda needle hydrophone under traveling wave condi-
tions. Hydrophones were calibrated regularly using the
steel sphere radiometer technique (Dunn et al. 1977).
distance of 12.2 cm from the transducer (where samples
were located during USWF exposure). Coordinates from
the exposure site to a fixedpointer were determined using
the three-axis positioner and were used to position the
center of the lower, left-hand well of the sample holder
at the exposure site (bottom of the well was 12.2 cm
from the transducer). Some water was removed from the
tank such that the sample holder was located at the expo-
sure site without full submersion.
Fibronectin-null mouse embryonic myofibroblasts
(obtained from Dr. Jane Sottile, University of Rochester)
were used for all experiments. These cells do not produce
fibronectin and have been adapted to grow under serum-
free conditions (Sottile et al. 1998). Cells were routinely
cultured in a 1:1 mixture of AimV (Invitrogen, Carlsbad,
CA, USA) and Cellgro (Mediatech, Herndon, VA, USA)
on tissue culture dishes precoated with collagen type-I.
These media do not require serum supplementation.
Thus, no source of fibronectin is present during routine
culture. On the day of USWF exposure, fibronectin-null
with 0.08% trypsin (Invitrogen) and 0.5 mM EDTA in
PBS. Trypsin activity was neutralized with 2 mg/mL
soybean trypsin inhibitor (STI; Sigma, St. Louis, MO,
USA). Cells were washed one time with 1 mg/mL STI
in PBS and were then resuspended in a 1:1 mixture of
Collagen solution preparation
A neutralized type-I collagen solution was prepared
on ice by mixing collagen type-I (isolated from rat tail
tendons [Windsor et al. 2002]) with 32 concentrated
et al. 2000). Both the 31 and 32 DMEM media were
degassed in a vacuum chamber for 30 min under sterile
conditions priortoincorporationinto thecollagenmixture.
Fibronectin-null cells were added to aliquots of
neutralized type-I collagen solutions on ice at various
final concentrations immediately prior to USWF expo-
sure. Aliquots (400 ml) of the collagen/cell solution
were then loaded into two of the 1 cm diameter Sylgard?
184 silicone elastomer molded wells of the BioFlex?
plate. For ‘‘no-cell’’ samples, an equal volume of
AimV/Cellgro was added in place of fibronectin-null
cells and aliquots were loaded into a third well. The
collagen/cell solution in the left-hand well of each plate
was exposed to a 1 MHz, continuous wave USWF for
15 min at room temperature. The two other samples in
the plate (right-hand side) served as sham control wells
1922 Ultrasound in Medicine and Biology Volume 36, Number 11, 2010
that were treated exactly as the exposed sample but were
not exposed to the USWF. The 15-min exposure duration
was sufficient to promote collagen polymerization at
room temperature. Following USWF exposure, collagen
gels were incubated for 1 h at 37?C and 8% CO2to allow
for complete collagen polymerization. An equal volume
(400 ml) of DMEM was then added to wells containing
collagen gels. In some experiments, collagen/cell and
collagen/no-cell solutions were incubated for 1 h at
polymerization before USWF exposure.
Cell viability assay
to assess cell viability (Mosmann 1983). At various time
points after USWF exposure, collagen gels were incu-
bated with 5.3 mM MTT (USB Corporation, Cleveland,
OH, USA) for 4 h at 37?C and 8% CO2. Gels were then
digested with 0.77mg/mLcollagenase (from Clostridium
histolyticum, type-I, Sigma) and formazan crystals were
dissolved using acidified isopropanol (0.04 N HCl).
Absorbance measurements at 570 nm and 700 nm (back-
ground) were determined using a spectrophotometer.
MTT absorbance was calculated by subtracting back-
ground absorbance values and nonspecific reduction of
MTT in no-cell gels from the 570 nm readings. There
was a linear relationship between cell number and MTT
absorbance. This assay is sensitive to differences of
5000 cells and greater (data not shown).
Collagen gel contraction assays
The extent of collagen gel contraction was deter-
mined using two established methods. For volumetric
gel contraction assays, collagen gels were scored around
their edges to form free-floating gels. After an additional
20 h of incubation at 37?C and 8% CO2, the gels were
removed from the wells and weighed (Model B303;
Mettler Toledo, Columbus, OH, USA). Volumetric
collagen gel contraction was calculated as a decrease in
gel weight compared with the control, no-cell gel weight
(Hocking et al. 2000). For radial gel contraction assays,
tion microscope equipped with a calibrated eyepiece
micrometer. Two measurements were recorded for each
gel and averaged to calculate gel diameter. Investigators
measuring diameters were blinded to exposure conditions.
in gel diameter compared with the original gel diameter of
1 cm (Tingstrom et al. 1992).
Soluble fibronectin binding
were incubated with 100 mg/mL of Alexa Fluor?488-
labeled human, plasma-derived fibronectin (FN-488;
labeled according to manufacturer’s instructions) in the
presence of 1 mM MnCl2for 30 min at room temperature
(Akiyama and Yamada 1985; Mastrangelo et al. 1999).
Cells were washed twice with AimV/Cellgro to remove
unbound fibronectin and were then added to neutralized
type-I collagen solutions and exposed to an USWF as
described above. In other experiments, 10 mg/mL of
FN-488wasadded toneutralized type-Icollagen solutions
in the absence of cells and exposed to an USWF as
One hour after USWF exposure, cell-embedded
collagen gels were examined using an Olympus IX70 in-
verted microscope (Center Valley, PA, USA) with a 34
phase-contrast objective and were photographed using
a digital camera (Spot RT Slider,Model 2.3.1; Diagnostic
Instruments Inc., Sterling Heights, MI, USA). FN-488
was visualized using epifluorescence microscopy. Gels
were flipped on their side to visualize cell bands through
the height of the cylindrical sample. For volumetric
collagen gel contraction experiments, gels were imaged
after obtaining weight data. Image-Pro Plus software
(Media Cybernetics, Bethesda, MD, USA) was used to
measure the linear distance between fibronectin-null cell
bands within collagen gels. Pixel distance was converted
to micron values using a micrometer calibration. A total
of 10 distances were measured on each of 20 different
images collected from three different experiments.
To visualize type-I collagen fibers, cell-embedded
collagen gels were examined using second-harmonic
generation microscopy (Freund and Deutsch 1986;
Roth and Freund 1979; Williams et al. 2005). One hour
paraformaldehyde for 1 h at room temperature. Second-
harmonic generation microscopy was performed using an
Olympus Fluoview 1000 AOM-MPM microscope equip-
Samples were illuminated with 780 nm light generated by
a Mai Tai HP Deep See Ti:Sa laser (Spectra-Physics,
Mountain View, CA, USA) and the emitted light was de-
tected with a photomultiplier tube using a bandpass filter
with a 390 nm center wavelength (Filter FF01-390/40-25;
were simultaneously visualized using a second bandpass
filter with a 519 nm center wavelength (Filter BA 495-
auto-fluorescence of cells (Monici 2005). Cell-embedded
collagen gels were photographed using a CMOS digital
camera (Moticam 1000; Motic, Xiamen, China).
were fixedin 4%
Data are presented as the mean 6SEM. Statistical
comparisonsbetweenUSWF-exposed and sham
Application of USWF technology to tissue engineering d K. A. GARVIN et al. 1923
experimental conditions were performed using either
the Student’s t- test for paired samples or one-way anal-
ysis of variance in GraphPad Prism software (La Jolla,
CA, USA). Differences were considered significant for
p values , 0.05.
Characterization of sample holders
The measured ultrasound attenuation of standard
polystyrene multi-well tissue culture plates was 4.5 6
0.7 dB/MHz/cm (n 5 3). Due to the significant attenua-
tion of the sound field by polystyrene plates, silicone
elastomer-bottomed plates were investigated as possible
samples holders for our studies. The measured acoustic
attenuation of the silicone elastomer well bottom (thick-
ness 5 1 mm) of the BioFlex?plates was only 0.7 6 0.2
dB/MHz/cm (n 5 5) indicating that there is negligible
attenuation (0.07 dB at 1 MHz) of the sound field due
to the presence of the BioFlex?sample holders.
184 silicone elastomer molding material was 2.4 6 0.04
dB/MHz/cm (n 5 3). Sound absorption at 1 MHz (1.4 6
0.03 dB/cm; n 5 3) was found to contribute ?60% of
thisattenuation. Thermocouple measurementsmonitoring
sample temperature during USWF exposure indicated
that of room temperature. Therefore, BioFlex?plates
modified with Sylgard?184 silicone elastomer molds
(Fig. 1b) were chosen as sample holders for our investiga-
tions because they did not significantly interfere with the
Characterization of acoustic fields generated in
in both the presence and absence of the BioFlex?sample
holders and are shown in Figure 2. Well-developed
USWF, characterized by pressure nodes and antinodes,
were found in both the free field (Fig. 2a) and within the
sample space (Fig. 2b). Peak pressure amplitudes of these
USWF were ?0.2 MPa and, therefore, as expected
(Blackstock 2000), were double the transducer output
distance between pressure nodes in both beam patterns
half-wavelength spacing between pressure nodes for a 1
MHz USWF generated in water (0.75 mm). Furthermore,
provide further evidence indicating that the BioFlex?
plates modified with the Sylgard?184 silicone elastomer
molds did not significantly interfere with the sound
field and, thus, are appropriate sample holders for our
USWF exposure induces the formation of cell bands in
3-D collagen gels
ulate cell organization in 3-D collagen gels, fibronectin-
null cells, suspended in unpolymerized type-I collagen
solutions, were either exposed to, or not exposed to
(sham samples), a 1 MHz, continuous wave USWF with
0.2 MPa peak pressure amplitude using the experimental
set-up depicted in Figure 1a. Fibronectin-null cells do not
produce fibronectin and are grown in serum-free condi-
tions (Sottile et al. 1998). These cells were chosen for
Fig. 2. Measurement and characterization of acoustic field.
Hydrophone measurements were used to determine the axial
spatial distributions of pressure in the free field (a) and in the
presence of the BioFlex?sample holders (b) in the water tank
set-up. The ultrasound standing wave field (USWF) was gener-
ated using a 1 MHz source with 0.1 MPa output pressure ampli-
tude. Measurements were recorded through a 0.5 cm distance
below the air interface in 0.1 mm intervals. Surface tension
effects prevented measurements directly at the air-water
interface and, therefore, measurements were started at the first
1924 Ultrasound in Medicine and BiologyVolume 36, Number 11, 2010
our initial studies to differentiate the effects of USWF on
the localization of cells and the extracellular matrix
protein, fibronectin. Collagen solutions were allowed to
polymerize during the 15-min exposure to maintain the
sound field. Cell distribution was then analyzed
copy (Fig. 3). Images shown are representative of our
results. Collagen gels polymerized in the presence of the
USWF showed a distinct banded pattern characterized
by the localization of cells to the pressure nodes of the
USWF (Fig. 3, panels b and c), while sham samples ex-
hibited a homogeneous cell distribution (Fig. 3, panel a).
The mean of the measured distance between cell bands
was 657 6 15 mm. Increasing the initial concentration
of cells in the collagen solutions led to the formation of
denser cell bands at the nodal planes (compare Fig 3b
and c). These data indicate that an USWF can spatially
To evaluate potential adverse effects of USWF
exposure on cell viability, cell number was quantified
using MTT. No differences in cell number were
observed between USWF-exposed and sham-exposed
cell-embedded collagen gels 20 h after exposure (Fig. 4).
These findings indicate that an USWF can alter the spatial
organization of cells within 3-D collagen gels without
decreasing cell viability.
To estimate the magnitude of radiation force exerted
on the cells in the applied USWF, eqn (1) was used to
calculate the maximum Frad. The acoustic exposure
parameters and the physical properties of the cells and
the suspending collagen medium used for the calculation
are listed in Table 1. Results of this calculation indicated
that the cells were subjected to a maximum radiation
force of approximately 2.2 pN.
USWF-induced formation of cell bands enhances
cell-mediated collagen gel contraction
The biomechanical properties of normal human
Fig. 3. Ultrasound standing wave field (USWF) exposure induces the formation of cells bands in 3-D collagen gels.
Fibronectin-null cells were suspended in neutralized collagen type-I solutions on ice at either 2 3 105cell/mL (a) and
(b) or 4 3 106cell/mL (c). Samples were exposed at room temperature for 15 min to a continuous wave USWF with
0.2 MPa peak pressure amplitude generated using a 1 MHz source. Sham gels were treated identically to USWF samples
but were not exposed to the sound field. Cell-embedded collagen gels were imaged as described in the Materials and
Methods section. Representative phase-contrast images, taken 1 h after USWF exposure, are shown. Scale bar, 200 mm.
Fig. 4. Ultrasound standing wave field (USWF) exposure does
not decrease cell viability. Fibronectin-null cells were sus-
pended in collagen type-I solutions (2 3 105cell/mL) and
exposed at room temperature for 15 min to a continuous wave
USWF with 0.2 MPa peak pressure amplitude generated using
a 1 MHz source. Following a 20-h incubation at 37?C and 8%
CO2, cell viability was determined using thiazolyl blue tetrazo-
lium bromide (MTT) as described in the Materials and Methods
section. Data are presented as average fold difference in absor-
bance 6SEM normalized to sham average absorbance values
(n 5 4; p . 0.05).
Application of USWF technology to tissue engineering d K. A. GARVIN et al. 1925
that tissue or tissue construct (Vogel and Sheetz 2006). In
by cell-derived forces. These forces are exerted on matrix
components through intracellular tension generation due
to cytoskeletal contractility (Hinz and Gabbiani 2003;
Hocking et al. 2000; Lee et al. 1998). Cell-mediated
lular matrix remodeling by cells (Korff and Augustin
1999; Sieminski et al. 2004; Vernon and Sage 1996).
Thus, to determine if a change in the spatial distribution
of cells affects cell-mediated extracellular matrix remod-
eling, a collagen gel contraction assay was used to
compare the extent of collagen gel contraction between
USWF cell-organized gels and sham gels.
Fibronectin-null cells, suspended in unpolymerized
collagen type-I solutions, were either exposed to, or not
exposed to (sham), an USWF (0.2 MPa peak pressure,
1 MHz source) using the experimental set-up depicted
in Figure 1a. Following an overnight incubation, sham-
exposed gels contracted 11.5 6 1.7 % (Fig. 5a). In
contrast, samples exposed to the USWF contracted
22.9 6 2.1% (Fig. 5a). Thus, a twofold increase in the
contraction of collagen gels with USWF-induced cell
organization was found compared with sham gels with
a homogeneous cell distribution (Fig. 5a and b). Addi-
tional experiments were conducted to determine whether
the increase in collagen gel contraction was due to cell
alignment or to a direct effect of USWF exposure on
cell tension generation. According to theory, increasing
the viscosity of the suspending medium inhibits USWF-
induced movement of cells to the pressure nodes
(Coakley et al. 1989). Based on this prediction, collagen
gels with a homogeneous distribution of fibronectin-null
cells were allowed to polymerize prior to USWF
exposure. Microscopic analyses confirmed the lack of
localization of cells to the nodal planes (Fig. 5d). Impor-
tantly, no differences in collagen gel contraction between
USWF-exposed and sham samples were observed
(Fig. 5c). These data indicate that aligning cells with an
USWF can enhance cell-mediated collagen matrix
The formation of cell bands depends on USWF pressure
The magnitude of the acoustic radiation force (Frad)
in an USWF has a second order dependence on pressure
amplitude (Po) and, as such, changing Powill affect the
movement of cells to the pressure nodes [eqn (1)]. Theo-
retical analysis of the forces acting on particles in an
USWF predicts a threshold pressure for banding below
which particles will not accumulate on the nodal planes
amplitude necessary to achieve cell banding within
collagen gels, fibronectin-null cells suspended in type-I
collagen solutions were exposed during the polymeriza-
tion process to an USWF of various peak pressure ampli-
tudes. As shown in Figure 6, homogeneous cell
distributions were observed within sham-exposed gels,
as well as gels fabricated using an USWF with peak pres-
sure amplitudes of 0.02 and 0.05 MPa. Exposing samples
to an USWF with peak pressure amplitude of 0.1 MPa re-
sulted in the formation of cell bands, indicating that the
threshold pressure for USWF-induced cell banding in
our system is ?0.1 MPa (Fradmax 5 0.55 pN) (Fig. 6).
When an USWF with pressure amplitude of 0.2 MPa
was used to fabricate the collagen gels, cell bands ap-
peared more dense (Fig. 6). With a pressure amplitude
of 0.3 MPa, the resulting cell bands were thicker and
more localized to the center of the gel (Fig. 6), likely
due to the influence of secondary lateral acoustic
Table 1. Parameters used for calculation of primary acoustic radiation force (Frad) using eqn (1)
Parameters (units) Numerical valueSource/reference
0.2 Hydrophone measurement
Assuming spherical particles
(Vuillard et al. 1990)
Calculation from b 5 1/c2r
900 (cells); 4.2 3 1026(FN*)
6 (cells); 0.01 (FN)
4.1310210(cells); 3.1 310210(FN)
0.13 (cells); 0.58 (FN)
1529 (cells); 1540 (FN)
Assuming collagen media has properties of water at room temperature
Chosen frequency for our studies
Using eqn (2)
Cell value (Taggart et al. 2007); FN value assumed to be sound speed in human
soft tissue (Bamber 1998)
Cell value assumed to be dominated by cytoplasm taken as low concentration
saline (Baddour et al. 2005); FN value (Fischer et al. 2004)
Axial distance between two pressure nodal planes (l/2)
Calculated using eqn (1) at z5 l/8 and 3l/8
rp(kg/m3)1050 (cells); 1350 (FN)
6 2.2 (cells); 6 4.5 3 1028(FN)
* soluble fibronectin molecules (FN).
1926 Ultrasound in Medicine and Biology Volume 36, Number 11, 2010
radiation forces acting within the pressure nodal planes
(Spengler et al. 2003). These findings indicate that
different USWF pressure amplitudes lead to variations
in the patterns of banded cells within collagen gels.
USWF pressure amplitude has a biphasic effect on
cell-mediated collagen gel contraction
Our data indicate that USWF-induced cell organiza-
tion enhances cell-mediated collagen gel contraction and
that the extent of cell banding is affected by the USWF
pressure amplitude. To determine if different cell banded
patterns affect the extent of collagen gel contraction,
radial collagen gel contraction assays were used to
compare levels of collagen gel contraction among gels
fabricated at the six different USWF pressure amplitudes
tion were observed among sham-exposed samples and
samples exposed to either 0.02 or 0.05 MPa (Fig. 7a)
Fig. 5. Cell-mediated collagen gel contraction is increased after ultrasound standing wave field (USWF)-induced forma-
tion of cell bands. Fibronectin-null cells (2 3 105cell/mL) were suspended in collagen type-I solutions and exposed to
a continuous wave USWF (0.2 MPa peak pressure, 1 MHz source) for 15 min at room temperature during the collagen
polymerization process (a) and (b). In (c) and (d), cell-containing collagen type-I solutions were allowed to polymerize at
37?C for 1 h and then exposed to the USWF. Following exposure, collagen gels were incubated for an additional 20 h at
37?C and 8% CO2and then removed from the wells and weighed. Data are presented as average percent volumetric gel
contraction 6 SEM (a, n 525; c, n 528). Representativephase-contrast images of USWF-exposed (right) and sham gels
(left) are shown in (b) (exposed to USWF during collagen polymerization) and (d)(exposed to USWFafter collagen poly-
merization). The * indicates a difference from sham group (p , 0.05). Scale bar, 200 mm.
Fig. 6. Cell banding is a function of ultrasound standing wave field (USWF) pressure amplitude. Fibronectin-null cells
wave USWF (1 MHz source) with various peak pressure amplitudes. Representative phase-contrast images, from one of
four experiments, indicate cell banding in samples exposed to 0.1 MPa and above. Scale bar, 200 mm.
Application of USWF technology to tissue engineering d K. A. GARVIN et al. 1927
where cells remained in a homogeneous distribution
(Fig. 6). In contrast, a significant 1.5-fold increase in
threshold for cell banding, where cells first become
aligned into planar bands (Fig. 6). These results, obtained
using gel diameter measurements, are similar to those re-
ported in Figure 5 using volumetric contraction assays
and, therefore, provide additional evidence that USWF-
induced cell banding enhances cell-mediated collagen
gel contraction and matrix reorganization.
As the USWF pressure amplitude was increased
above 0.1 MPa, collagen gel contraction levels decreased
(Fig. 7a). At 0.3 MPa, a 30% decrease in contraction
compared with sham levels was observed(Fig. 7a). There
were no significant differences in the number of viable
cells between sham-exposed and 0.3 MPa USWF-
exposed samples (Fig. 7b). Therefore, the decrease in
collagen gel contraction was not due to cell death but
was more likely due to effects of the cell-banded pattern
that occurred at 0.3 MPa. These findings indicate that the
effect of USWF pressure amplitude on cell-mediated
collagen gel contraction is biphasic. We attribute this
in cell-extracellular matrix contacts formed as cell bands
become more dense above the threshold pressure.
To directly assess collagen matrix organization rela-
tive to the cell-banded areas formed using USWF of
shown in Figure 8, short collagen fibrils were randomly
organized in sham-exposed cell-embedded collagen gels
and in cell-embedded collagen gels exposed to either
0.02or0.05MPawherecells remained inahomogeneous
distribution. Exposure to 0.1 MPa resulted in areas of the
gels where cells were loosely clustered and collagen
fibrils were more elongated (Fig. 8). These data clearly
show that cells aligned into planar bands at the pressure
threshold for cell banding have reorganized their
surrounding collagen matrix and, thus, provide further
evidence that USWF-induced cell banding enhances
cell-mediated collagen matrix remodeling.
Extensive areas of cell bands were clearly visible in
collagen gels exposed to 0.2 or 0.3 MPa and short
collagen fibers surrounding these areas were randomly
oriented (Fig. 8). These results indicate that as the
USWF pressure amplitude increases beyond the pressure
threshold for cell banding and cell bands become more
dense, cell-mediated collagen matrix reorganization
decreases. Therefore, the effect of USWF pressure ampli-
tude on cell-mediated collagen matrix reorganization is
biphasic and as such, these data both parallel and support
our data showing that USWF pressure amplitude has
a biphasic effect on collagen gel contraction. Taken
together, these data indicate that radiation forces associ-
ated with an USWF can indirectly influence the relative
location of extracellular proteins and thus, can be used
to control extracellular matrix-dependent functions
essential to tissue formation.
USWF localize cell-bound protein to cell bands in 3-D
Successful tissue engineering depends upon the
stimulation of key cell functions, including cell prolifer-
ation, migration and differentiation. These processes are
Fig. 7. Ultrasound standingwavefield(USWF) pressureampli-
tude leads to a biphasic effect on cell-mediated collagen gel
contraction. (a) Fibronectin-null cells (4 3 106cell/mL) sus-
pended in collagen type-I solutions were exposed at room
temperature for 15 min to a continuous wave USWF (1 MHz
source) with various peak pressure amplitudes. Following 1 h
incubation at 37?C and 8% CO2, gel diameters were measured.
Data are presented as average percent radial gel contraction 6
SEM and are normalized to the sham condition (n 5 10).
The * indicates a difference from sham group (p , 0.05). (b)
Fibronectin-null cells (2 3 105cell/mL) suspended in collagen
type-Isolutions wereexposedatroomtemperature for15minto
a continuous wave USWF (1 MHz source) with peak pressure
amplitude of 0.3 MPa. Following 1 h incubation at 37?C and
8% CO2, cell viability was assessed using thiazolyl blue
tetrazolium bromide (MTT). Data are presented as average
fold difference in absorbance 6SEM normalized to sham
average absorbance values (n 5 3; p . 0.05).
1928 Ultrasound in Medicine and BiologyVolume 36, Number 11, 2010
influenced by a variety of soluble and insoluble factors,
including growth factors, cytokines and extracellular
matrix proteins (Langer and Vacanti 1993). The extracel-
lular matrix protein, fibronectin, stimulates cell growth,
migration and contractility (Hocking et al. 2000;
Hocking and Chang 2003; Sottile et al. 1998).
Concentrating stimulatory proteins to the cell banded
areas of collagen gels may be a useful approach to
stimulate cell function.
The radiation force exerted on a spherical particle in
a standing wave field is proportional to the particle
volume [eqn (1)]. Soluble fibronectin molecules that are
20 nm in diameter (Vuillard et al. 1990) experience
a maximum primary radiation force of ?4.5 3 1028pN
when exposed to an USWF with parameters shown to
promote cell banding (Fig. 3 and Table 1). As such,
sure nodes of the USWF used in this study. This idea was
confirmed in our system by including Alexa Fluor?488-
labeled fibronectin (FN-488) in unpolymerized type-I
collagen solutions and allowing polymerization to occur
during USWF exposure. Epifluorescence microscopy
images show a homogeneous distribution of labeled
fibronectin remaining after USWF exposure (Fig. 9a).
To facilitate localization of fibronectin to the cell
bands where it may influence cell functions, soluble
FN-488 molecules were bound to fibronectin-null cells
prior to USWF exposure. Binding of FN-488 to cells
was confirmed by western blot analysis (data not shown).
FN-488-bound cells were then added to unpolymerized
type-I collagen solutions and exposed to an USWF with
a pressure amplitude of 0.2 MPa. Epifluorescent micro-
scopic analysis showed co-localization of fibronectin
molecules to USWF-induced cell bands within the poly-
merized collagen gels (Fig. 9b) indicating that USWF
radiation forces can influence the spatial organization of
cell-bound proteins within 3-D collagen gels.
We have developed the use of ultrasound standing
wave fields as a noninvasive technology for organizing
Fig. 8. Ultrasound standing wave field (USWF) pressure amplitude has a biphasic effect on cell-mediated collagen
reorganization. Fibronectin-null cells (4 3 106cell/mL) suspended in collagen type-I solutions were exposed at room
temperature for 15 min to a continuous wave USWF (1 MHz source) with various peak pressure amplitudes. Following
1 h incubation at 37?C and 8% CO2, gels were fixed with 4% paraformaldehyde and imaged using second-harmonic
generation microscopy as described in the Materials and Methods section. Representative merged images, from one of
three experiments, are shown (red - collagen type-I fibers; green - cells). Scale bar, 100 mm.
Application of USWF technology to tissue engineering d K. A. GARVIN et al. 1929
cells and cell-bound proteins within tissue engineered
biomaterials. In this study, we show that acoustic
radiation forces associated with an USWF can be used
to organize both mammalian cells and cell-associated
proteins into discrete bands within collagen hydrogels.
The density of the USWF-aligned cell bands was depen-
dent on both cell number and pressure amplitude. Expo-
sure of cells to USWF parameters utilized in the current
study did not decrease cell viability. Furthermore, the
USWF-aligned cell bands were stable for at least 20 h.
Under appropriate conditions, the organization of
cells into bands led to an increase in cell-mediated
collagen gel contraction, as measured by both volumetric
gel contraction in response to USWF exposure did not
occur if the collagen/cell samples were allowed to poly-
merize prior to USWF exposure, strongly suggesting
that the increases in cell contractility and collagen fibril
reorganization were mediated by the organization of cells
into bands and were not an indirect effect of ultrasound
exposure on individual cells. The extent of collagen
contraction was dependent upon the spatial distribution
of cells in the gel. No increase in collagen gel contraction
was observed in response to USWF exposure at pressure
amplitudes that did not produce cell banding. At the other
extreme, no increase in collagen gel contraction was
observed in response to USWF at pressure amplitudes
that produced densely packed cell bands. However, expo-
sure of samples to an USWF that led to the clustering of
cells into planar bands within the gel resulted in a signifi-
cant increase in collagen contraction above sham gels
with a homogenous cell distribution.
USWF also led to changes in collagen fibril organization
and length. Second-harmonic generation microscopic
images showed that short collagen fibers were randomly
that did not produce cell banding, as well as pressure
amplitudes that produced densely packed cell bands. In
contrast, elongated collagen fibers were observed within
loosely clustered cell banded areas indicating enhanced
cell-mediated collagen matrix remodeling in these
samples. These results are consistent with the biphasic
results of the collagen gel contraction investigations.
Hence, an important downstream effect of USWF-
mediated cell alignment is enhanced extracellular matrix
Acoustic radiation forces exerted on extracellular
in the system used in this study. However, the use of an
USWF can indirectly affect the organization of proteins
in the extracellular matrix by two avenues. First, as ex-
plained in the paragraph above, increased collagen fibril
length and organization was observed in gels exposed to
an USWF at pressure amplitudes that increased collagen
Fig. 9. Ultrasound standing wave field (USWF) exposure local-
exposed at room temperature for 15 min to a continuous wave
USWF with a peak pressure amplitude of 0.2 MPa (1 MHz
source). Fibronectin distribution was analyzed using epifluores-
cencemicroscopy.Inb,FN-488was incubated withfibronectin-
null cells as described in the Materials and Methods section.
FN-488-bound cells were then suspended in collagen type-I
above. Cell and fibronectin distribution were analyzed using
phase-contrast and epifluorescence microscopy, respectively.
Representative images, taken 1 h after USWF exposure, are
shown. Scale bar, 200 mm.
1930Ultrasound in Medicine and Biology Volume 36, Number 11, 2010
gel contraction. Thus, a downstream effect of USWF-
induced cell banding is the resultant cellular remodeling
of the surrounding extracellular matrix. Second, we
demonstrated that the extracellular matrix protein, fibro-
nectin, could be aligned into bands within the collagen
gel using an USWF if the fibronectin molecules were first
bound to the cell surface. Thus, USWF technology can be
used to spatially organize cells within engineered tissues
and to co-locate active or inactive cell-bound molecules.
The use of an USWF has numerous advantages as
a noninvasive technology to spatially organize cells and
cell-bound molecules in engineered tissues and, thereby,
influence cell function. The acoustic radiation force acts
directly on the cells and, thus, the approach does not
require any prior modification of the cell surface. Various
to this technique. Similarly, various cell types or combi-
nations could be used to engineer different tissue types.
Changing frequency of the ultrasound field will affect
spacing of cell bands and multiple transducers could be
gineered biomaterials. For example, USWF-induced
banding of human endothelial cells can give rise to
a vascular network within a 3-D construct (Garvin et al.
2010). Another potential use of this technology is the
production of dermal grafts using dermal fibroblasts or
mesenchymal stem cells. Controlling cell patterning,
cell function and extracellular matrix organization are
primary challenges to successfully engineering func-
tional tissues and organs in vitro (Khademhosseini et al.
2009). Thus, the use of USWF to specifically control
cell organization, function and extracellular matrix re-
modelingwithin3-D artificial constructshasthepotential
to address these current challenges to tissue engineering.
This study reports on the development of an applica-
tion of USWF technology to the field of tissue engi-
neering. Acoustic radiation forces associated with
USWF were used to spatially organize cells and cell-
bound proteins into distinct bands within 3-D collagen
gels. USWF-induced cell alignment increased cell
contractility and resulted in enhanced cell-mediated
extracellular matrix reorganization. By specifically
controlling cell and extracellular matrix organization,
this technology holds great potential for advancing the
fabrication of complex engineered tissues in vitro.
Acknowledgments—This work was supported in part by grants from
the NIH NIBIB (R01EB008368, R01EB008996). The authors thank
Nicholas Berry, Sally Child, Carol Raeman and Susan Wilke-Mounts
for technical assistance and Dr. Karl Kasischke (Multiphoton Core
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