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Cells Tissues Organs 2011;194:13–24
Engineered Vascular Tissue Fabricated
from Aggregated Smooth Muscle Cells
Tracy A. Gwyther Jason Z. Hu Alexander G. Christakis Jeremy K. Skorinko
Sharon M. Shaw Kristen L. Billiar Marsha W. Rolle
Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, Mass. , USA
fusion between ring subunits. This unique system provides
a versatile new tool for optimization and functional assess-
ment of cell-derived tissue, and a new approach to creating
tissue-engineered vascular grafts.
Copyright © 2011 S. Karger AG, Basel
Over the past three decades, tissue engineering has
emerged as a promising approach to create blood vessel
substitutes for clinical transplantation, as well as model
systems to study vascular tissue function in vitro. To date,
the majority of strategies for tissue-engineered blood ves-
sel (TEBV) synthesis have involved seeding cells within
scaffolds made from synthetic [Shinoka et al., 1998; Nik-
Biomechanics ? Cell-derived matrix ? Smooth muscle cell ?
Vascular tissue engineering
The goal of this study was to develop a system to rapidly
generate engineered tissue constructs from aggregated
cells and cell-derived extracellular matrix (ECM) to enable
evaluation of cell-derived tissue structure and function. Rat
aortic smooth muscle cells seeded into annular agarose
wells (2, 4 or 6 mm inside diameter) aggregated and formed
thick tissue rings within 2 weeks of static culture (0.76 mm at
8 days; 0.94 mm at 14 days). Overall, cells appeared healthy
and surrounded by ECM comprised of glycosoaminoglycans
and collagen, although signs of necrosis were observed near
the centers of the thickest rings. Tissue ring strength and
stiffness values were superior to those reported for engi-
neered tissue constructs cultured for comparable times. The
strength (100–500 kPa) and modulus (0.5–2 MPa) of tissue
rings increased with ring size and decreased with culture du-
ration. Finally, tissue rings cultured for 7 days on silicone
mandrels fused to form tubular constructs. Ring margins
were visible after 7 days, but tubes were cohesive and me-
chanically stable, and histological examination confirmed
Accepted after revision: November 1, 2010
Published online: January 19, 2011
Dr. Marsha W. Rolle
Department of Biomedical Engineering
Worcester Polytechnic Institute
100 Institute Road, Worcester, MA 01609 (USA)
Tel. +1 508 831 4145, Fax +1 508 831 4121, E-Mail mrolle @ wpi.edu
© 2011 S. Karger AG, Basel
Accessible online at:
Abbreviations used in this paper
Dulbecco’s modified Eagle’s medium
fetal bovine serum
maximum tangent modulus
smooth muscle cell
tissue-engineered blood vessel
ultimate tensile strength
T.A.G. and J.Z.H. contributed equally to this work.
Cells Tissues Organs 2011;194:13–24
lason et al., 1999; Opitz et al., 2004; Hashi et al., 2007;
Nieponice et al., 2008] or natural polymers [Weinberg
and Bell, 1986; Seliktar et al., 2000; Grassl et al., 2003;
Swartz et al., 2005; Stegemann et al., 2007; Zavan et al.,
2008]. Alternatively, ‘scaffold-free’ tissue engineering ap-
proaches have been explored in which TEBV are fabri-
cated entirely from self-assembled cells and cell-derived
extracellular matrix (ECM), such as rolling cultured cell
sheets [L’Heureux et al., 1998; Gauvin et al., 2010], organ
printing [Mironov et al., 2009; Norotte et al., 2009] or as-
sembly and fusion of clustered cells [Kelm et al., 2010].
Autologous vascular grafts produced by the cell sheet-
based engineering method exhibit comparable tensile
strength to human saphenous veins [Konig et al., 2009]
although graft fabrication and maturation requires 2–3
months [L’Heureux et al., 2006]. However, vascular grafts
created with this method have already shown clinical
promise as arteriovenous fistulas [McAllister et al., 2009].
Despite the promise and increasing number of reports
using cell-based approaches to tissue engineering, few
studies to date have examined the mechanical strength or
other functional properties of engineered tissue con-
structs created entirely from cells and cell-derived ECM.
Safe and successful in vivo application of TEBV made
entirely from cells will depend on achieving adequate
strength and mechanical stability. The aim of this study
was therefore to develop a simple system to generate
strong three-dimensional tissue constructs from aggre-
gated cells within an experimentally useful time frame
(1–2 weeks) in a format that is conducive to mechanical
and physiological testing. To achieve this aim, we chose
to create ring-shaped constructs due to their simple ge-
ometry and the precedent for using vascular tissue rings
for mechanical and physiological analysis of blood vessel
function. We predict that this model system will enable
systematic assessment of the roles of cell source and cul-
ture parameters on cell-derived tissue structure and
To create ring-shaped tissue constructs, rat aortic
smooth muscle cells (SMCs) were seeded into custom
round-bottomed, annular wells cast in agarose, with post
sizes of 2, 4 or 6 mm (to produce rings with 2, 4 or 6 mm
inner diameters). Tissue rings were cultured for 8 or 14
days prior to thickness measurements and analysis of
handling and mechanical properties. Uniaxial tensile
testing was performed to measure ultimate tensile
strength, stiffness and failure strain, and tissue structure
and ECM composition were examined by histology. Fi-
nally, we assessed the feasibility of using tissue rings as
subunits to generate larger, tube-shaped constructs.
Materials and Methods
Custom Cell Culture Well Fabrication
A custom polycarbonate mold was created by machining an-
nular wells with inner post diameters of 2, 4 and 6 mm (Small
Parts Inc., Miramar, Fla., USA). The wells were machined with
round bottoms to facilitate cell settling and self-aggregation to
form rings. Polydimethylsiloxane (PDMS, Sylgard 184; Dow
Corning, Midland, Mich., USA) was mixed at a 10:
of base to curing agent, degassed for 2 h, and poured onto the
polycarbonate mold. After curing at 60 ° C for 4 h, the PDMS was
peeled from the mold and used as a template. Two percent agarose
(w/v; Lonza, Rockland, Me., USA) was dissolved in Dulbecco’s
modified Eagle medium (DMEM; Mediatech, Herndon, Va.,
USA), autoclaved and poured onto the PDMS template to form the
wells for cell seeding. Individual agarose wells were cut away from
the PDMS template and placed into 6-well plates. The agarose
wells were incubated in DMEM supplemented with 10% fetal bo-
vine serum (FBS; PAA, Etobicoke, Ont., Canada) and 1% penicil-
lin/streptomycin (Mediatech) and equilibrated in an incubator
for 1 h prior to cell seeding at 37 ° C and 5% CO 2 . A schematic of
this process is shown in figure 1 .
1 ratio (w/w)
SMC Culture and Seeding
Rat aortic SMCs (WKY 3M-22; a cell line derived from SMCs
isolated from 3-month-old adult male Wistar-Kyoto rat aortas by
enzymatic digestion [Lemire et al., 1994; Lemire et al., 1996]; gen-
erously provided by Dr. Thomas Wight) were cultured in DMEM
(Mediatech) supplemented with 10% FBS (PAA) and 1% penicil-
lin/streptomycin (Mediatech). At 90% confluence, SMCs were
trypsinized and re-suspended in culture medium. The number of
SMCs seeded into each well was scaled to the size of the channel
(0.66, 1.3 and 2.0 ! 10 6 cells per well seeded into 2-, 4- and 6-mm
inner diameter wells, respectively). Plates were left undisturbed in
the incubator for the first 48 h after seeding, after which the cul-
ture medium was changed every 48 h for the duration of the 8- or
14-day culture period. Four batches of 2-, 4- and 6-mm rings were
produced for mechanical testing studies (two batches harvested
at 8 days and 2 batches harvested at 14 days) as described below.
An additional two batches (one at each time point) of 4-mm rings
were created for histological evaluation of tissue rings not sub-
jected to mechanical testing (3 rings per time point).
Tissue Ring Thickness Measurements
On the final day of each study, the tissue rings were removed
from the agarose wells and transferred to 60-mm Petri dishes
filled with phosphate-buffered saline (PBS) at room temperature.
The rings were centered under a machine vision system (model
630; DVT Corporation, Atlanta, Ga., USA) and thickness mea-
surements were acquired in three separate positions along the cir-
cumference of the ring using edge detection software (Framework
2.4.6; DVT). Three measurements were averaged to yield a mean
thickness value for each sample.
Mechanical properties of tissue rings were measured using a
uniaxial testing machine (ElectroPuls E1000; Instron, Norwood,
Mass., USA). The tissue rings were mounted between two small
stainless steel pins (referred to as ‘grips’) and submerged in PBS.
One grip was connected to an electromagnetic actuator and the
Cell-Derived Tissue Rings
Cells Tissues Organs 2011;194:13–24
strength or stiffness. Interestingly, preliminary studies
suggest that culturing tissue rings in culture medium
supplemented with sodium ascorbate and amino caproic
acid, conditions that have been shown to increase colla-
gen synthesis and cross-linking, also improved tissue
ring strength and stiffness (data not shown). It may be
possible to optimize culture conditions (by decreasing or
eliminating serum, and adding growth factors or me-
chanical stimulation, as described above) to make tissue
rings stronger without increasing thickness.
Given the large number of cells needed to generate 4
batches of tissue rings in 3 different sizes to establish the
basic parameters (for example, initial cell seeding number
per well, culture duration and mechanical testing proto-
col) for creating and analyzing cell-derived tissue rings,
we chose to use the WKY 3M-22 rat SMC line for the ex-
periments reported in this study. However, we recently
applied the same techniques to successfully assemble pri-
mary human coronary artery SMCs into cell-derived tis-
sue rings, which were then cultured for 14 days. Despite
their slower doubling time, in preliminary experiments
the human SMC rings exhibited greater mechanical
strength than the rat SMCs reported here (data not shown),
thereby demonstrating that this cell aggregation system
can be applied to create tissue rings from primary cells.
Ongoing studies are focused on histological and biochem-
ical analysis of the human SMC tissue constructs.
An important difference between the tissue ring con-
structs and vascular ring segments from native arteries is
the lack of an endothelium or adventitia. Like many in
vitro reports of TEBV construction, our study focused on
a single cell type, SMCs, to mimic the vascular media.
Recent studies have shown that cell sheet-based vascular
grafts comprised of both SMCs and fibroblasts exhibit
greater ECM synthesis and higher burst pressures com-
pared to constructs made from SMCs alone [Gauvin et
al., 2010]. Furthermore, microtissue aggregation studies
have shown that endothelial cells can co-aggregate with
fibroblasts to form spheroids [Napolitano et al., 2007a, b;
Kelm et al., 2010]. It may therefore be possible to add fi-
broblasts and endothelial cells to SMCs to increase
strength and more closely mimic blood vessel structure
and function in cell-derived tissue rings.
Upon successful fabrication and handling of cell-
based ring constructs, it became evident that cell-derived
tissue rings could be used as building blocks to form tis-
sue tubes. Here, we report proof of concept that tissue
rings cultured in close proximity fuse to form a cohesive
tissue tube within 14 days (7 days for ring fabrication and
7 days for fusion). Culturing the tubes for an extended
period may result in further fusion and elimination of
ring boundaries. A recent study by Livoti and Morgan
 showed that toroid microtissues (600 ? m inner di-
ameter) self-assembled from H35 hepatocytes cultured
for 48 h could be stacked and cultured, with fusion of ad-
jacent toroids within 72 h. The ease with which 2-mm
SMC rings could be handled after 7 days in our study sug-
gests that it may be possible to harvest our rings even ear-
lier to accelerate the process of graft fabrication. Finally,
histological evaluation demonstrated that individual
rings had fused to form a contiguous tissue mass within
7 days. However, burst pressure analysis will be a critical
benchmark to determine the feasibility of transplanting
vascular grafts created with this method.
In conclusion, we have shown that tissue constructs
that are suitable for manipulation and functional testing
can be created from aggregated SMCs within a few days.
Although these rings are not as strong as ring segments of
native blood vessels or TEBV generated from cultured cell
sheets for 2–3 months, their strength compares favorably
to other engineered tissue constructs reported to date.
Given the short time frame and simplicity of this system
(which relies on commercially available materials and
methods), it may enable systematic assessment of a variety
of parameters on tissue structure and function (for ex-
ample, cell source, culture medium composition and dy-
namic culture regimens). The ring-shaped geometry of
these constructs is useful for mechanical testing, and
based on the ease with which they could be mounted onto
wire grips, may also be used in a myograph system to mea-
sure tissue responses to pharmacologic agents. This sys-
tem has potential as a new three-dimensional in vitro
model of vascular tissue function, and a versatile tool to
advance development of cell-derived vascular grafts.
We gratefully acknowledge Dr. Elizabeth Ryder for her guid-
ance with statistical analysis of the data and Dr. Raymond Page
for training and use of equipment for polarized light microscopy
as well as helpful comments on the manuscript. The authors also
thank Neil Whitehouse for his assistance with CNC machining,
Adriana Hera for her assistance with MATLAB programming,
and the Histology Department of the University of Massachusetts
Medical School for assistance with sample processing. This work
was funded by Worcester Polytechnic Institute (Summer Under-
graduate Research Fellowship to J.Z.H. and institutional start-up
funds to M.W.R.), the UMass Medical School-WPI Pilot Research
Initiative, the American Heart Association (undergraduate re-
search fellowship to J.Z.H.) and the National Institutes of Health
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