ARTICLE IN PRESS

Biomaterials 26 (2005) 7628–7635

www.elsevier.com/locate/biomaterials

Fabrication of tubular tissue constructs by centrifugal casting of cells

suspended in an in situ crosslinkable hyaluronan-gelatin hydrogel
Vladimir Mironova,, Vladimir Kasyanova,b, Xiao Zheng Shuc,
Carol Eisenberga, Leonard Eisenberga, Steve Gondad, Thomas Truska,
Roger R. Markwalda, Glenn D. Prestwichc
a
Medical University of South Carolina, Charleston, SC 29426, USA
b
Riga Stradins University, Riga, LV-1007, Latvia
c
Center for Therapeutic Biomaterials and Department of Medicinal Chemistry,
The University of Utah, Salt Lake City, UT 84108-1257, USA
d
NASA Johnson Space Center, Houston, TX 77058, USA

Received 17 March 2005; accepted 16 May 2005

Available online 14 July 2005

Abstract

Achieving the optimal cell density and desired cell distribution in scaffolds is a major goal of cell seeding technologies in tissue
engineering. In order to reach this goal, a novel centrifugal casting technology was developed using in situ crosslinkable hyaluronan-
based (HA) synthetic extracellular matrix (sECM). Living cells were suspended in a viscous solution of thiol-modified HA and thiol-
modified gelatin, a polyethyleneglycol diacrylate crosslinker was added, and a hydrogel was formed during rotation. The tubular
tissue constructs consisting of a densely packed cell layer were fabricated with the rotation device operating at 2000 rpm for 10 min.
The majority of cells suspended in the HA mixture before rotation were located inside the layer after centrifugal casting. Cells
survived the effect of the centrifugal forces experienced under the rotational regime employed. The volume cell density (65.6%)
approached the maximal possible volume density based on theoretical sphere packing models. Thus, centrifugal casting allows the
fabrication of tubular constructs with the desired redistribution, composition and thickness of cell layers that makes the maximum
efficient use of available cells. Centrifugal casting in this sECM would enable rapid fabrication of tissue-engineered vascular grafts,
as well as other tubular and planar tissue-engineered constructs.
r 2005 Elsevier Ltd. All rights reserved.

Keywords: Centrifugal casting; Thiol-modified gelatin; Glycosaminoglycan; Tubular construct; Synthetic extracellular matrix

1. Introduction using self-assembling ‘‘bio-ink’’ and ‘‘bio-paper’’ [7],

Tissue engineering has the potential to create living
and growing tissues and organs suitable for transplanta-
tion [1]. During the last decades, several tissue engineer-
ing technologies have been developed: (i) embedding
cells in 3-D hydrogels [2,3], (ii) seeding cells on mesh-
work 3-D biodegradable scaffolds [4], (iii) cell sheet
technology [5,6], (iv) tissue and organ printing with
Corresponding author. Tel.: +843 792 7630; fax: +843 792 0664.
E-mail address: mironovv@musc.edu (V. Mironov).
and (v) injectable tissue engineering [8]. For directed
tissue self-assembly, the density of cells and their precise
positioning and distribution within the tissue constructs
is crucial for biological function. Moreover, the
manufacture of engineered constructs of desired geome-
trical form is also a fundamental goal for any new tissue
engineering technology. Efficiency has emerged as a
further criterion for the development and optimization
tissue assembly technologies destined for commerciali-
zation. Time-consuming, costly, and physiologically
stressful cell seeding and tissue assembly techniques

0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2005.05.061
 ARTICLE IN PRESS
V. Mironov et al. / Biomaterials 26 (2005) 7628–7635
would become limiting factors in the development of
clinically useful tissue-engineered products.
Recently, it was suggested that using centrifugal
forces could be used to assemble a scaffold tube from
a synthetic polymer [9], and that cells could be seeded
onto biodegradable synthetic scaffolds using centrifugal
forces [10–12]. Although centrifugal casting has been
used for centuries in traditional manufacturing, to the
best of our knowledge this approach has not yet been
applied to the field of tissue engineering. Briefly,
centrifugal casting combines cell embedding or molding
technologies with a rotational device generating cen-
trifugal force. To employ centrifugal casting for cell-
seeded materials for tissue engineering, an in situ
crosslinkable biopolymer that forms a biocompatible
hydrogel in presence of living cells was a prerequisite.
That is, our protocols required that living cells could be
suspended in a viscous medium that would solidify by
gelation as rotation provided the centrifugal force to
cast a tubular hydrogel.
Recently, such hydrogels have been developed and
described as synthetic extracellular matrices (sECMs)
for in vitro and in vivo tissue engineering. In particular,
in situ crosslinkable hydrogels based on thiol-modified
hyaluronan (HA) [13–16] satisfy many of the design
criteria for centrifugal casting of a cell-seeded viscous
liquid. It was shown that anchorage-dependent cell types
can survive polymerization of hydrogel and maintain
their viability in 3-D HA hydrogels with addition of
thiol-modified gelatin or in thiol-modified HA hydrogels
containing covalently linked RGD peptides [17].
The goal of this research was to investigate the
possibility of using this in situ crosslinkable sECM to
develop a novel centrifugal casting technology for tissue
engineering. We demonstrate herein the feasibility of
using centrifugal casting with viable living cells sus-
pended in an in situ crosslinkable sECM as a fast and
effective method to assemble a tubular tissue.
2. Materials and methods
2.1. Rotation device
A stirrer, model RW 20.n, (IKA Labortechnik, Germany)
with constant power drive was used for spinning glass tube
with hydrogel and cells. This model is equipped with an
integrated speed control display and two speed ranges allow
usage over entire 60–2000 rpm range. The stirrer was fastened
to a plate stand and its spindle was oriented horizontally. This
model allows a smooth increase in spindle rotation speed.
2.2. Spin tube preparation
Pyrex glass tubes B-YF-WG6 (Small Part Inc.) with inner
diameter 770.1 mm were used for spinning. Tubes were
7629
4570.2 mm in length, and the ends were closed using
translucent Fisherbrand solid silicon stoppers.
2.3. Cell culture
Quail QCE-6 cells with proven cardiogenic potential [18]
were transfected with green fluorescent gene (GFP) and then
grown in a medium consisting of media M199 (Fisher)
supplemented with 10% chicken serum (Fisher) 100 U/ml
penicillin, and 100 mg/ml streptomycin sulfate. All cells were
incubated in a humidified 37 1C 5% CO2 environment with
growth media being replaced every 3–4 days. The cells were
expanded to 80–90% confluence prior to trypsinization for
passage or centrifugal casting.
2.4. Scaffold preparation
Custom-made silicon tubes and woven hybrid vascular
grafts [19] with outer diameter 7.070.1 mm were used as
scaffolds. Silicon tubes were prepared by three sequential
dippings of appropriate glass rods in the solution, followed by
drying in an oven (70 1C) after each dipping. Woven hybrid
vascular grafts [19], which were prepared from a combination
of two different type of yarns (polyurethane and polyester) and
had a wall thickness 0.4770.01 mm. Prior to placing the
mixture of hydrogel and cells, hybrid scaffolds were pre-wet
in spin tubes using media M199 and incubated overnight
at 37 1C.
2.5. Preparation and cell seeding of the sECM
Thiolated HA and thiolated gelatin [13,14] were dissolved in
media 199 to give 1.25% (w/v) and 3.0% (w/v) solutions,
respectively, and the solution pH was adjusted to 7.4 by adding
0.1 M NaOH solution. Polyethylene glycol diacrylate (PEGDA
Mw 3400, Nektar) was also dissolved in media 199 to give a
4.5% (w/v) solution. All the above solutions were then
sterilized by filtering through 0.45 mm filter. Next, a solution
of thiolated HA and thiolated gelatin was prepared by mixing
2.8 ml thiolated HA solution and 1.2 ml thiolated gelatin
solution. The pellet of centrifuged cells was then very gently
mixed to suspend the cells in the with the HA/gelatin solution
(volume of cells: volume of solution ¼ 1:3); immediately
after adding cells, the PEGDA solution was added to cell
suspension in a volume ratio of 1:4 to initiate the crosslinking
and gelation [15,17]. Thus cells occupied approximately 20%
of volume of gel/cell mixture. After careful mixing, the final
cell suspension in the gelling solution was placed in glass tubes
for centrifugal casting.
2.6. Centrifugal casting procedure
Before fabrication, the volume of hydrogel for the first
hydrogel layer was calculated on the basis of given thickness of
the layer (1 mm) and length of the tube (less the length of
stoppers in the tube). This volume of the hydrogel was placed
in the tube. The tube was tightly closed with silicon stoppers
and then fixed in the spinner. The tube was spun with a speed
of 2000 rpm for 10 min, during this period the solution became
crosslinked to form a tubular hydrogel. The next required
 ARTICLE IN PRESS
7630 V. Mironov et al. / Biomaterials 26 (2005) 7628–7635
volume of hydrogel and cells was calculated starting from the
thickness of the second layer. The viscous cell suspension was
placed into the tube and spun for an additional 10 min. After
crosslinking of the second layer, the scaffold was removed
from the tube and placed either in 4% paraformaldehyde or in
media M199 with 10% fetal bovine serum (FBS).
2.7. Histology and microscopic analysis
After completion of the centrifugal casting procedure, the
tubular tissue constructs were carefully removed from the glass
tube, and then cut in 1 mm segments. One group of specimens
was analyzed in non-fixed conditions under phase/fluorescent
microscope (Nikon TE2000S, Japan). The frozen sections were
prepared on a cryomicrotome. A second group of specimens
was fixed and embedded in epoxy resin (Polyscience Inc,
USA). Semi-thin sections were made on LKB ultramicrotome
(Sweden). Finally, a third group of specimens was placed in
4% neutral buffered paraformaldehyde solution overnight,
rinsed in PBS, and then dehydrated by passing through series
of ethanol solutions increasing concentration prior to embed-
ding in paraffin. Histological sections were analyzed by light
microscopy (Olympus, Japan).
2.8. Morphometric analysis and statistics
The percentage of dead cells was estimated before and
after centrifugation in HA hydrogel at magnification  20.
Cell density was estimated on semi-thin and histological
sections of tubular construct at magnification  40 using a
standard stereologic grid method and with computational
image analysis software (Caligari trueSpace V6.6). Volume
cell density was defined and estimated stereologically as the
percent of all analyzed surface area versus surface area
occupied by cell profiles. Groups of control and experimental
data (n ¼ 10) were analyzed by single-factor ANOVA. For
pair-wise comparisons, heteroscedastic t tests were used to
determine significance in differences between population
means. Statistically different pairs were defined as having
po0:05.
3. Results
3.1. Optimization of rotational speed
The rotational speed was determined to be optimal
when sufficient force was generated to overcome surface
tension and gravity and thereby allow formation of a
uniform tubular-like redistribution of the gelling hydro-
gel during rotation. We found that reproducible tubular-
like redistribution of the HA hydrogel in these glass
tubes was achieved at 2000 rpm, whereas a lower speed
(e.g., 1000 rpm) failed to permit the formation of a
tubular-like structure (Fig. 1a, b). Thus, the rotation
speed was set constant at 2000 rpm for all subsequent
experiments. This equates to a relative centrifugal force
(RCF) of 11.2g at the cell layer, using a formula [12]:
Fig. 1. Fabrication of a tubular construct using in situ crosslinking of
a hydrogel sECM in rotating glass tube. Panel (a) shows that tubular
hydrogel films do not form in glass tube at low (1000 rpm) speed of
rotation. Panel (b) shows a concentric tubular redistribution of the
hydrogel in glass tube at 2000 rpm. Panel (c) shows a cross-section of
the crosslinked tubular sECM hydrogel construct from in glass tube
after 10 min of rotation. Panel (d) shows the crosslinked tubular
hydrogel in a silicon tube after removal from the glass tube.
RCF ¼ 1.119  105 r rpm2, where r ¼ radius the layer
of cells in cm, or 0.25 cm.
3.2. Fabrication of tubular scaffold
The crosslinked sECM tube was fabricated by a
horizontal rotation at 2000 rpm for 10 min. After
removal from the glass casting support tube, fabricated
construct retained its tubular shape and round cross
section (Fig. 1c, d). There was no significant difference
in rate of crosslinking between the rotated and static,
non-rotated sECM mixtures (data not shown). Impor-
tantly, in preliminary experiments rotated collagen
hydrogels were not able to maintain a tubular shape
(data not shown). Moreover, tubular constructs could
not be fabricated using alginate hydrogel due to the
rapid crosslinking of this gel (data not shown). While
agarose can be fabricated into a tubular construct, the
temperature required for this process damaged living
cells (data not shown). Thus, we focused our efforts on
using in situ crosslinkable HA-based sECM hydrogel as
the preferred biomaterial for the development of the
centrifugal casting technology. The fabrication proce-
dure to obtain a tubular tissue construct by concentric
centrifugal casting includes several sequential steps.
After fabrication of first layer of cell-free sECM, a
suspension of cells in the sECM mixture was placed
inside the glass tube and a second concentric tubular
layer was formed by centrifugal casting. As a result of
the centrifugal forces during rotation, the cells moved to
the border area between crosslinked first layer of
hydrogel and in situ crosslinked second hydrogel layer.
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V. Mironov et al. / Biomaterials 26 (2005) 7628–7635
After completion of the second rotation and cross-
linking cycle, a second concentric tube had formed. The
cells form a densely packed cellular layer sandwiched
between two crosslinked sECM layers. Thus, cells are
embedded in an sECM, which serves as a scaffold, and
support their tubular spatial arrangement and redis-
tribution.
3.3. Cell survival during centrifugal casting
In order to estimate the effect of centrifugal forces on
cell survival (versus cell damage), we used green
fluorescent protein (GFP)-labeled quail QCE-6 cells.
These cells express GFP, and living cells with an intact
cell membrane fluoresce green. If cell damage occurs
and the cell membrane is compromised, the GFP leaks
out of the cell cytoplasm through the damaged cell
membrane, and cells lose their green fluorescence.
Mixing of GFP-labeled QCE cells with the ungelled
sECM induces limited cell damage, as observed by a
small number of non-fluorescent cells. Living fluorescent
QCE cells in hydrogel incubated for10 min without
centrifugation and living fluorescent QCE-6 cells in the
sECM experiencing 10 min centrifugation at 2000 rpm
were similar in appearance and numbers. Quantitative
cytometry of dead (green negative) QCE cells in control
(10 min incubation in sECM without centrifugation)
and in experiments (10 min centrifugation in sECM)
confirmed the absence of a statistically significant effect
(p40.05) of centrifugal forces of cell damage (Fig. 2).
Using live/dead and other cell viability assays provided
similar results (data not shown). We concluded that
centrifugal forces (11.2g) employed during centrifugal
casting technology do not significantly damage cells.
Fig. 2. Centrifugal forces do not affect cell survival in the sECM. Key:
(1) Percentage of dead QCE cells in in situ crosslinked sECM without
centrifugation; (2) Percent of dead cells in sECM after 10 min
centrifugation. Cell survival is not statistically different (p40:05).
7631
Thus, centrifugal casting using an in situ crosslinkable
HA-gelatin sECM provides satisfactory cell survival to
proceed experimentally.
3.4. Cell distribution and cell density
Centrifugal casting allows the redistribution of cells
into the form of tubular construct with a circular cross-
sectional cell layer. This layer consists of densely packed
cells (Fig. 3). The fluorescence of the labeled cells
indicates that most of the cells in this layer were viable
and undamaged (Fig. 3a). The packing density is very
high as shown both on frozen and histological sections
(Fig. 3b, c). Morphometric stereological estimation of
surface cell density based on histological sections
demonstrated that packing volume density (65.6%)
(Fig 4.) approaches the packing density calculated
as average theoretically maximum packing density
(62.3%). This estimate was made as the average between
two theoretical sphere-packing models when spheres are
touching each other: face-centered cubic lattice packing
(FCLP) (75.3%) and 3-D simple cubic lattice packing
(SCLP) (53.2%). Moreover, the experimentally obtained
density is very close to volume density of natural
myocardium in adult animal (75%) (see Section 4 for
details).
3.5. Centrifugal casting of woven hybrid vascular graft
In order to estimate suitability of centrifugal casting
technology for placing of cells on tissue-engineered
scaffolds for vascular tissue engineering, a wetted woven
hybrid vascular graft [19] was employed. It was placed in
glass tube and then centrifugal casting procedure was
performed. In the second experiment, two steps cen-
trifugal casting procedure was employed. After prepar-
ing the first acellular layer with the sECM hydrogel, a
second layer was formed with 10 min at 2000 rpm with
Fig. 3. Cell redistribution in a tubular tissue construct fabricated by
centrifugal casting of an sECM hydrogel. Panel (a) shows the
fluorescent microscopy of the wall segment of a tubular tissue
construct formed by living (green) GFP-labeled QCE-6 cells; panel
(b) shows a frozen, unstained section of tubular construct with dense-
packed QCE-6 cells; and panel (c) provides a higher magnification
image of a toluidine blue semi-thin section of tubular constructs with
densely packed QCE-6 cells.
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7632 V. Mironov et al. / Biomaterials 26 (2005) 7628–7635
Fig. 4. Cell packing (volume density) analysis after centrifugal casting.
Panel (a) face-centered cubic lattice packing (FCLP) of homogenous
spheres (three-dimensional view); panel (b) FCLP of homogenous
spheres (cross-sectional view); panel (c) three-dimensional (3-D) simple
cubic lattice packing (SCLP) of homogenous spheres (3-D view); panel
(d) 3-D SCLP of homogenous spheres (cross-sectional view); panel (e)
automatic image analysis of volume density of QCE-6 cells fabricated
by centrifugal casting using automatic cell contouring using image
analysis software. Panel (f) shows the volume density of cell in
centrifugally casting tubular construct as compare with natural volume
density of myocardium and volume density based on two theoretical
spheres packing models. Key for panel (f): (1) maximal volume density
of closely packing spheres of equal size according to FCLP (75.3%);
(2) maximal volume density of closely packing spheres of equal size
according to 3-D SCLP (53.2% ); (3) packing density (63.2%)
calculated as an average between two theoretical sphere packing
models; (4) volume density (85%) of myocardium in perinatal rat (1
day); (5) volume density (75%) of myocardium in adult rats; and (6)
volume density ( 65.6%) of QCE-6 cells after centrifugal casting in in
situ crosslinkable sECM hydrogel.
the cells suspended in the HA-gelatin sECM solution.
This second crosslinked concentric layer of cells in
sECM cell thus gave a vascular construct containing
living cells on lumenal surface. Fluorescent microscopy
demonstrates a high density of redistributed cells on the
lumenal surface of vascular construct. Strong fluores-
cence indicates that virtually all cells survived the
centrifugal casting procedure. The fabricated tissue-
engineered construct contains HA-based sECM hydro-
gel and circular layer consisted of several rows densely
packed living cells.
In another experiment designed to place a cellularized
sECM into wetted woven hybrid vascular graft, a one-
step centrifugal casting method was used. This technique
produced a thin, densely packed layer of fluorescent cells
Fig. 5. Centrifugal casting of woven hybrid compliant vascular graft.
Panel (a) shows a cross section of the woven hybrid vascular graft with
concentric centrifugally cast mixture of sECM hydrogel with
fluorescently labeled QCE-6 progenitor cells. Cells form a thin layer
on the lumenal surface of the vascular graft. Panel (b) shows a similar
woven graft in which the cells are redistributed homogeneously
through the graft wall.
on the lumenal surface of woven hybrid vascular graft
after concentric centrifugal casting (Fig. 5a) and
remarkably homogeneous redistribution of fluorescent
cells throughout whole wall thickness of vascular graft
(Fig. 5b). Thus, the centrifugal casting technique in
combination with the in situ crosslinkable sECM could
be used to rapidly (10 min) fabricate a bioreactor-free
cellularized composite hybrid compliant biodegradable
vascular graft. In principle, such a construct would be
and ‘‘off-the-shelf’’ tissue-engineered vascular graft for
in vivo implantation.
4. Discussion
The primary outcome of the studies described herein
is the development of a novel centrifugal casting
technology that is suitable for rapid tissue assembly
and cell packing in tubular, layered tissue-engineered
constructs. Centrifugal casting is an ancient manufac-
turing technology, and centrifugal forces have been
previously employed for making engineering tissue-
engineered synthetic scaffolds [9] and for cell seeding
into preformed porous scaffolds [10–12] We have
described herein the first example in which an in situ
crosslinkable hydrogel can be used for centrifugal
casting of living cells in a layered sECM.
4.1. Optimal rotation speed
The optimal speed of rotation for the centrifugal
casting technology was defined as a speed at which (i) a
hydrogel tube would be formed by crosslinking, (ii) the
desired redistribution of cells was achieved, and (iii) cell
viability was not compromised on. We have shown
experimentally that tubular-like redistribution of ro-
tated solutions could be achieved at low rotation speed.
However, reducing the diameter with each sequentially
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V. Mironov et al. / Biomaterials 26 (2005) 7628–7635
placed hydrogel layer during concentric centrifugal
casting, as well as the increasing of viscosity arising as
the sECM crosslinking proceeds, demands a higher
rotation speed. We have found that 2000 rpm (a
maximal rotation speed achievable on employed rota-
tion device) is an optimal speed for centrifugal casting.
The force generated was 11.2g for the diameter of
tubes used herein. This speed is significantly lower
by an order of magnitude than centrifugal forces
(50–500g) that were employed to study the effect of
centrifugal forces on cell seeding in porous scaffolds
[10–12]. However, we clearly show that the reduced
force used herein is sufficient enough to reproduce
fabricated tubular constructs containing viable, unda-
maged cells.
4.2. Centrifugal casting provides layer-like cell
redistribution
One important advantage of centrifugal casting is that
it provides a high density of redistributed cells, thus
placing cells in a closely packed layer of desired
thickness. Maximizing highly close packing is a crucial
and essential requirement for fast and effective tissue
self-assembly. Moreover, we have demonstrated that
fabrication of sequential layers of sECM hydrogels is
technically achievable. Thus, by using the so-called
concentric centrifugal casting or repeatable multiple
centrifugal casting it would be possible eventually to
create desired spacing between concentric cell layers.
Sequential centrifugation of hydrogels with or without
cells could create concentric multilayered constructs
with hydrogel spacing between sequential cell layers.
Finally, concentric centrifugal casting also allows one to
create tubular tissue-engineered construct consisting
from concentric heterogeneous cell layers from different
cell types.
4.3. Effect of centrifugal forces during centrifugal casting
on cell viability
An important question for the feasibility the proposed
casting technology was the ability of cells to survive
potential damaging forces experienced during rotation
as the sECM crosslinked. Although cell centrifugation is
a routine procedure in cell culture research, and during
routine centrifugation the centrifugal forces were much
higher then centrifugal forces used in this study, we must
realize that mixing and centrifugation of cells in HA
hydrogel can create different mechanical conditions and
the potentially damaging effects of centrifugal forces
could be higher in a highly viscous medium. Thus,
survival of cell during centrifugal casting under these
conditions was not a trivial issue, and could not be
readily extrapolated from data on cell survival of the
effect of centrifugal forces in solution.
7633
Previous studies have shown that the components and
crosslinkers employed in making sECMs were not
cytotoxic [13–17]. In order to investigate if any
damaging effect of centrifugal forces on cells occurred
during centrifugation in forming the sECM hydrogels,
we used GFP-transfected quail QCE-6 cells. The
qualitative and quantitative data demonstrated that
centrifugal forces at 2000 rpm were equal to 11.2g and
did not damage the cells. That is, the percentage of
viable cells placed in a static hydrogel for 10 min without
centrifugation was not statistically different from the
percentage of viable cells after 10 min centrifugation
at 2000 rpm in the sECM hydrogel. These data
support previously reported data that centrifugal forces
are not induce significant cell damage [10–12]. It is
not surprising that in our experiments we observe
excellent cell survival. In previously published experi-
ments, the centrifugal forces were much higher
(35–52.5g) [11] or 50–500g [12]. However, it was
discovered (data not shown) that the most damaging
step for cells appears to be the pipetting during
transfer of cell pellets to the viscous pre-gelled sECM
mixture. Thus, the most dangerous step for cell viability
in the proposed centrifugal casting technology is not
centrifugation per se, but rather the shear forces
generated when cells are mixed with viscous solutions.
This mandates careful cell manipulation and gentle
mixing during this step to maintain cell viability.
Subsequently, 10 min centrifugation at 2000 rpm al-
lowed both complete hydrogel formation and preserva-
tion of cell viability.
4.4. Centrifugal casting optimizes cell seeding efficiency
and cell seeding density
If we assume that cells represent ideal spheres of
standard size and that cells are touching each other, then
the maximum possible packing could be calculated
based on two theoretical models of space packing
theory: a SCLP and a FCLP [20]. First, the space
packing model (SCPL model) predicts that volume
occupied by spheres (volume density) will be 52.3%,
whereas according to more effective packing in second
theoretical model (FCLP model) the same parameter
will be equal to 75.4%. Our stereological measurement
indicates that volume density of cells (packing density)
in fabricate tubular construct is equal to 65.6%. This
represents more effective packing as compared with
SCLP model and even 87% as compared with theore-
tically maximal effective FCPL sphere packing model. It
is interesting to mention that packing density achieved
by centrifugal casting is close to the level of packing
calculated as average between two theoretical model
(63.6%) [20] or packing density occurring in case of so-
called random close packing of spheres (64%) [21]. The
initially calculated volume cell density employed for the
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7634 V. Mironov et al. / Biomaterials 26 (2005) 7628–7635
sECM hydrogels was around 20% or 1.5  104 cells/ml.
Thus, centrifugal casting technology provides cell
packing or cell density is also pretty close to volume
density of myocardium in newborn (85%) and adult
animals (75%) [22] (Fig. 4).
4.5. Further applications of centrifugal casting in tissue
engineering
Although the most immediate application of centri-
fugal casting technology would be in the area of vascular
tissue engineering, it has not escaped our notice that
centrifugal casting has a broader potential utility. For
example, centrifugal casting could be used to engineer
complex multilayer tubular organs such as the esopha-
gus, the bronchi, intestine, uterus, urethra, or vagina.
Each of these tissues would require sequential concentric
tissue layers composed of heterogeneous cell popula-
tions. Centrifugal casting with large diameter tube with
sequential longitudinal cutting will also allow the use of
this technology for fast assembly of flat tissue-engi-
neered organs such as skin, cornea, and myocardial
patches. Thus, using centrifugal casting technology and
employing centrifugal forces is a very promising and
perspective approach in the development of fast tissue
assembling and effective cell seeding and cell packing
technologies. It would be interesting to evaluate the
suitability for centrifugal casting with so-called ‘‘smart’’
hydrogels, i.e., photo-sensitive, pH-sensitive, or thermo-
sensitive biomimetic hydrogels. The development of
concentric centrifugal casting as well as centrifugal
packing of heterogeneous cell populations would be the
next logical step in the further development and
advancing of proposed centrifugal casting technology.
In vivo testing of tissue-engineered constructs fabricated
by centrifugal casting technique and implanted in
experimental animals is a final step in preclinical
validation of proposed bioreactor-free tissue engineering
technology.
5. Conclusion
The feasibility of using novel centrifugal casting
tissue-engineering technology in conjunction with a
cytocompatible in situ cross-linkable HA-based sECM
has been demonstrated. The centrifugal casting technol-
ogy described herein could be used to fabricate
tissue-engineered constructs of tubular and flat geome-
trical form with desired cell composition, layer
thickness and concentric order with highly controlled
and precise distribution of living cells in 3-D hydrogels.
Centrifugal forces employed in this technology bring
cells close together in 3-D hydrogels, but they do
not induce significant cell damage and thus allow to
achieve a level of cell density which is critically
important for sequential fast and effective tissue self-
assembly. Centrifugal casting could serve as variant of
highly desired bioreactor-free ‘‘off-the-shelf’’ tissue
engineering technology, For example, manufacturing
of tissue-engineered tubular or flat tissue constructs
ready for implantation could be accomplished just
during several minutes, not even during days, weeks or
months.
Acknowledgements
We thank Mrs. Carol Moscovic and Ms. Aimee
Phelps for assistance with histological processing and
sectioning. We also thank Ms. Lisa Sooy for helping
with cell culturing. We appreciate the kindly provided
professional consultation by Prof. Piero Anversa
and Prof. Robert Tomanek about volume density of
myocardium in natural tissue. This research was funded
by NASA/EPSCR grant ‘‘Cardiovascular Tissue En-
gineering’’. Work at Utah was supported by the Utah
Centers of Excellence Program and the NIH (DC
04336).
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