High-throughput physically based approach for mammalian cell

encapsulation
Jiashing Yu, Po-Chen Wu, Chi-Hui Huang, Chung-Yao Yang, and Chao-Min Cheng

Citation: Appl. Phys. Lett. 103, 153704 (2013); doi: 10.1063/1.4824851

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 APPLIED PHYSICS LETTERS 103, 153704 (2013)

High-throughput physically based approach for mammalian cell

encapsulation
Jiashing Yu,1 Po-Chen Wu,1 Chi-Hui Huang,1 Chung-Yao Yang,2 and Chao-Min Cheng2,a)
1
Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan
2
Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan
(Received 4 May 2013; accepted 27 September 2013; published online 10 October 2013)
Herein, we wish to tear down the traditional boundaries between physics and life sciences by
demonstrating a physically based, flow-focusing method to encapsulate mammalian cells into
alginate-based microspheres in a very short period of time. We paid particular attention to the
physical properties of the alginate solution as it was critical to create a physiologically relevant
environment within the alginate microspheres. The cells we cultured when re-culturing them on
Petri dishes could still be maintained for at least 4 days after microsphere encapsulation. We
believe that this study would provide interesting insight in biophysics, polymer physics, and
C 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4824851]
applied physics. V

This paper describes the development of a robust and
easy-to-handle, physically based method to encapsulate
mammalian cells (i.e., NIH-3T3 fibroblasts, human adipose-
derived stem cells, and HeLa cells) into alginate-based
microspheres of various sizes, with careful attention paid to
the precise measurement of physical alginate solution prop-
erties in the preparation of these microspheres. The process
for preparing biocompatible polymeric spheres at the nano-
/micro-meter level using either physically or chemically
based methods with high-throughput capacity to allow for
the practical development of clinically relevant applications
has become a longstanding but interesting objective within
various research communities including the development of
both naturally derived and synthesized biomaterials, polymer
physics, tissue engineering, and regenerative medicine.1–3
Cell therapy, for instance, is one of the more promising and
practical directions in regenerative medicine and has been
extensively developed to deal with clinically relevant issues
such as myocardial repair.4 This approach, however, suffers
from a physically based issue that we wish to address–how
to effectively deliver specific types of cells (e.g., stem or pro-
genitor cells) to desired regions (e.g., damaged tissues) with-
out either destroying or losing a significant amount of the
delivered cells. To overcome this inherent cell therapy issue,
multiple engineering-based approaches have been demon-
strated including the use of injectable hydrogels, nanofibers,
and micropatterned porous structures and spheres.5–8 In this
study, we have chosen to focus on the development of a
physically based method that allows us to stably encapsulate
mammalian cells into microspheres, and do so with high-
throughput capacity. Our aim was to develop a practical and
efficient means of encapsulating a fair amount of mammalian
cells into microspheres in a short period of time, i.e., a few
seconds. This would increase manufacturing efficiency,
reduce the cost of the manufacturing and materials prepara-
tion, and enhance the delivery of viable, living cells to tar-
geted regions for therapeutic intent.
a)
Author to whom correspondence should be addressed. E-mail:
chaomin@mx.nthu.edu.tw
The microspheres (in this study, alginate-based micro-
spheres), which were prepared using an air-pressure-driven
method that we developed for this study, are notable for a va-
riety of exciting characteristics as follows: (1) tunable sphere
diameter and surface pore size; (2) stable diffusion (mass
transfer) while releasing either nutrients (for culturing specific
types of cells) or drugs (chemical compounds or biomole-
cules) from single microspheres; (3) the ability to modify the
sphere surface using either chemically or physically based
approaches including the conjugation of Arg-Gly-Asp (RGD)
peptides (chemical-based modification) or the incorporation
of extracellular matrix molecules such as fibronectin through
physical adsorption (physically based modification); and, (4) a
great combination of three tissue-engineering-based ele-
ments—cells, matrices, and signaling molecules (mammalian
cells, alginate-based microspheres, and RGD peptides in this
study).9,10 In addition to preparing alginate-based micro-
spheres using our physically based method (once again, with
appropriate procedures), we have also measured the physical
properties of the alginate solution (i.e., viscosity) for two main
reasons: (1) to better understand how said physical properties
affect the development of an appropriate procedure for opti-
mizing our physically based method; and (2) to create an
in-vitro three-dimensional matrix designed to maintain the
physiologically relevant functionality of mammalian cells,
thus mimicking the in-vivo physiologically relevant environ-
ment. Note, the alginate solution that we used as the matrix
material for cell encapsulation is a naturally derived and US
FDA-approved biomaterial, though it has not previously been
used as an encapsulation material as in this study. Together,
we have demonstrated a physically based method that builds
off of automated system groundwork and has characteristics
of robustness, ease of use, high throughput, and potential
expandability to mass production for biotechnologically rele-
vant applications (e.g., improving/leveraging interactions
between targeted cells and cell-specific drugs) using a flow-
focusing approach (i.e., a nitrogen pressure gradient)11,12 to
effectively and stably encapsulate specific types of cells into
alginate-based microspheres (i.e., our cell-based carrier of
choice). We also have developed an appropriate procedure to
encapsulate three types of mammalian cells with different

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V
153704-2 Yu et al.
physiological or pathological functionality into uniform
alginate-based microspheres (with and without the conjuga-
tion of RGD peptides) by physically controlling flow rate,
nitrogen pressure, and nozzle diameter. Once again, we
have attempted to build the undeniable link between two
longstanding distinct research communities—physics and life
sciences—through this interdisciplinary study. We believe
that this study, which has significant physics analysis and
basis, would provide deeper understanding of alginate-based
microsphere preparation and use (not limited to the use of
alginate as an encapsulation material) for mammalian cell
encapsulation using a physically based method (i.e., flow-
focused) with characteristics of high-throughput capacity, and
application versatility, especially for such applications as the
development of synthesized biomaterials and tissue engineer-
ing in either biophysics or bioengineering, the development of
soft materials in polymer physics, and the development of
fluid mechanics and instrumentations in applied physics.
Alginic acid sodium salts (MVG and LVM; MVG:
viscosity > 200 mPa s sodium alginate with minimum of 60%
guluronate monomer; LVM: viscosity 20–200 mPa s sodium
alginate with minimum of 50% mannuronate monomer) were
purchased from NovaMatrix (BP 1103-01 and BP 0711-02,
Norway). Arg-Gly-Asp (RGD) peptides (GGGGRGDY) that
we used in this study were prepared by Yao-Hong
Biotechnology Inc. (Taiwan) with purity ranging from 97% to
99%. To achieve the objective of this study, sodium alginate
powders (both MVG and LVM, but mainly LVM)13 were first
mixed with distilled deionized water under stirring for two
hours in order to form an alginate aqueous solution with a con-
centration of 2% (w/v). This aqueous solution was placed into
a MWCO 3500 dialysis cast and the assembly underwent dial-
ysis in distilled deionized water for another 3 days (in order to
perform purification). Dialyzed solution was then freeze-dried
and then re-dissolved in distilled deionized water to achieve a
concentration of 10% (w/v). We subsequently diluted this
FIG. 1. (a) The cell-alginate solution-based mixture was loaded into the Nisco Var J30 Encapsulation Unit using a syringe pump, and a stable spray of the cell-
contained alginate solution was formed within a few seconds based on the mechanism of flow focusing. (b) The cells that we attempted to encapsulate were
trypsinized from the cell culture plate and then mixed in an alginate solution after re-suspension. Next, the cell-alginate solution in the syringe was injected
into the Nisco Var J30 Encapsulation Unit for cell encapsulation.
Appl. Phys. Lett. 103, 153704 (2013)
aqueous solution with an equal volume of 0.5 M sodium meta-
periodate solution under stirring for 19 h to oxidize the algi-
nate. This reaction was terminated by placing 1/20 volume
ratio of ethylene glycol into this alginate aqueous solution
(total volume), which was then loaded into a MWCO 3500
dialysis cast (to perform an additional purification), and the
assembly underwent dialysis in distilled deionized water for
3 days (in order to remove both surplus sodium metaperiodate
and ethylene glycol). Dialyzed solution was then freeze-dried,
and then reformulated to be a 1.2% (w/v) alginate solution
with 0.9% (w/w) sodium chloride and 0.1 M 2-(N-morpholino)
ethanesulfonic acid buffer. Regarding the incorporation of
RGD peptides while preparing the alginate solution that we
used to perform these experiments, the procedure that we
developed was described as follows: (1) both 0.2 mmol and
0.1 mmol of solutions of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide and N-hydroxysuccinimide were pipetted into
10 ml of an alginate solution that we prepared; (2) after it
was completely dissolved, RGD peptides (GGGGRGDY) with
a concentration of 22 lmol were placed into this alginate
solution under stirring for 1 day. The resulting solution was
then dialyzed with a MWCO 3500 dialysis cast; and, (3) the
final solution was freeze-dried and stored for future experi-
ments, and the RGD peptide grafting ratio was measured and
confirmed with an O.D. value of 280 nm in this study.
Both viscosity and surface tension of the alginate solu-
tion with different ratios that we prepared were measured by
a DV-III Rheometer and the pendant drop method, respec-
tively. The alginate solution with a ratio ranging from 1% to
4% (w/v) was loaded into the rheometer spindle and its vis-
cosity was measured under different shear rates. In addition,
the same alginate solution (as used to measure the viscosity)
was injected to form complete droplets in air. The time-lapse
droplet images were then recorded and analyzed using a pub-
lic software, ImageJ (from National Institutes of Health), in
order to obtain the surface tension of the alginate solution.
153704-3 Yu et al. Appl. Phys. Lett. 103, 153704 (2013)

The nozzle of Nisco Var J30 Encapsulation Device (with
other device components) was sterilized using an autoclave
before we encapsulated different types of mammalian cells
into alginate-based microspheres (with and without the
incorporation of RGD peptides), and the nitrogen inlet
was connected with a filter disk of 0.22 lm in diameter.
Mammalian cells (with a cell number of about 2.5  106
cells/ml) for encapsulation were trypsinized from the cell
culture plate and re-suspended into an alginate solution as
well. The cell-alginate solution-based mixture was then
loaded into a Nisco Var J30 Encapsulation Unit via a syringe
pump. Note the nitrogen inlet pressure was also set to control
sphere diameter (i.e., as higher pressure was used, smaller
cell-encapsulated alginate microspheres were obtained in
this study). A stable spray of the alginate solution containing
different types of mammalian cells was formed within a few
seconds (i.e., high throughput), coherently reducing the
opportunity to generate fluidic shear stress (while carrying
out flow focusing), which could influence the cellular behav-
iors of mammalian cells while performing mammalian cell
encapsulation (Fig. 1(a)).10 This spray, after entering the
isotonic calcium chloride solution, was then immediately
crosslinked, and the resulting alginate microspheres were
collected through centrifugation. The isotonic calcium
chloride solution was replaced by cell culture medium. Note
the schematic of our physically based method for our
mammalian cell encapsulation process is shown in Fig. 1(b).
Once we obtained alginate-based microspheres via our
method, we performed diameter analysis of resultant micro-
spheres using a static light scattering method (LS230 Particle
Size Analyzer) in order to better understand microsphere
size distribution and determine whether our procedure was
appropriate with and without the incorporation of RGD pep-
tides. Our physically based method, based on the mechanism
of flow focusing, provides a robust and easy-to-handle (i.e.,
automatically based) approach to generate a fair amount of
uniform cell-encapsulated alginate microspheres, once again,
with and without the incorporation of RGD peptides, in a
very short period of time, i.e., with high throughput.
The surface tension of the 2% (w/v) alginate solution was
about 53 mN/m (N ¼ 4). We also have performed the viscosity
measurement of the alginate solution with different ratios, as
shown in Fig. 2(a). We found that the viscosity of the alginate
solution with a ratio of 1% (w/v) was about the same as blood

FIG. 2. (a) The viscosity of alginate solution used was measured by DV-III Rheometer (N ¼ 3) at various concentrations of 1 to 4 (w/v). (b) The shear thinning
property of 2% alginate solution was observed by changing the shear rate in 12 days of preservation (N ¼ 3); the fluid property remained the same over twelve
days of storage in a 4  C environment. The average diameter of alginate microspheres that we prepared using this physical-based approach versus the air pres-
sure of this automatic device while using a nozzle with different diameters was (c) 0.35 mm and (d) 0.25 mm at different flow rates. Data are mean 6 standard
deviation (N ¼ 100) for all detections. Histogram of the measured diameter (e) with and (f) without the RGD-peptide modification were determined using the
LS230 Particle Size Analyzer. The average diameters of modified and unmodified alginate microspheres were 118 and 109 lm, respectively.
153704-4 Yu et al. Appl. Phys. Lett. 103, 153704 (2013)

viscosity,14 reinforcing the use of alginate of this particular
viscosity as an appropriate matrix material for physiologically
relevant mammalian cell encapsulation. This allowed for
physiologically relevant functionality and enhanced opportu-
nity for delivery of these cells to desired locations within the
human body without diminishing their viability. It is impor-
tant to note that the mechanical strength of these alginate
microspheres was relatively low, and it was very challenging
to maintain the shape of alginate microspheres and perform
further experiments such as mammalian cell encapsulation
while using 1% alginate (w/v) solution spheres because they
were so soft microspheres. For this reason, we elected to use
2% (w/v) alginate solution spheres to perform the mammalian
cell encapsulation. This allowed us to do the following: (1) de-
velop an appropriate procedure (once again, for mammalian
cell encapsulation, using our physically based method); (2)
provide cell-encapsulated alginate microspheres with a better
mechanical strength; and, (3) provide a physiologically mim-
icking environment, once again, in order to maintain the phys-
iologically relevant functionality of mammalian cells that we
attempted to deliver (to the targeted regions of the body) for
cell therapy. We found that increasing the shear rate resulted
in a decrease in the viscosity of the 2% (w/v) alginate solution
(Fig. 2(b)). This is the shear thinning of the alginate solution.
Additionally, in order to verify the storage viability of our 2%
(w/v) alginate solution (i.e., how long the alginate solution
could still be used after the storage)—a critical issue in the
translational shift from lab to commercial use)—we examined
alginate solution viscosity after storage in a 4  C environment
for 12 days. We found that both viscosity and shear thinning
of the alginate solution were about the same as the alginate
solution that we kept under the same condition for 24 h, as
shown in Fig. 2(b).
One of the advantages to using the Nisco Var J30
Encapsulation Device was that the liquid-phase synthesized
biomaterials (e.g., alginate, before crosslinking) could be
easily loaded into this automatic device, in order to form
sprays (spheres) with various diameters (i.e., larger sprays
produced larger spheres). Figs. 2(c) and 2(d) show the aver-
age diameter of alginate microspheres that we prepared using
our physically based method (with appropriate procedures),
versus nitrogen pressure (used to form alginate-based micro-
spheres using this automatic device), while using nozzles
with different diameters (0.35 mm versus 0.25 mm) and a 2%
(w/v) alginate solution. With an increase in flow rate using
this automatic device and nitrogen pressure gradient (an
approach based on the mechanism of flow focusing), larger
alginate microspheres could be obtained; however, while
increasing nitrogen pressure, the diameter of spheres (i.e.,
the aero-cutting efficiency),15 decreased, and the minimum
sphere diameter that we could obtain was approximately
10 lm when nitrogen pressure was increased to (500 mbar
amount) and nozzle diameter was adjusted to 0.25 mm. In
this study, which takes into consideration cell density, injec-
tion feasibility, and mass transfer efficiency,6,16 the average
diameter (i.e., optimal diameter) of alginate microspheres
that we could obtain was approximately 100 lm. The diame-
ter distributions of the alginate microspheres with and with-
out RGD-peptide modification were determined using an
LS230 Particle Size Analyzer (Figs. 2(e) and 2(f)), and the
average diameters of both modified and unmodified alginate
microspheres were 118 and 109 lm, respectively.

FIG. 3. (a) Human adipose-derived stem cells were re-cultured on Petri dishes after dissolving cell-encapsulated alginate microspheres. The figure shows that
released cells would both spread and proliferate normally on the dishes. (b) Human adipose-derived stem cell viability was examined using live versus dead
staining after a few days of culture using a LIVE/DEADV R Cell Imaging Kit that incorporated FITC (green fluorescence; live cells) and Texas Red (red fluores-

cence; dead cells). (c) The cell encapsulation number (i.e., the number of cells encapsulated in the microsphere) of modified and unmodified with the RGD-
peptide was both approximately 3.3  105 (N ¼ 3). (d)The viability of cells cultured in modified and unmodified microspheres was observed using a fluorescent
microscope (N ¼ 3). The cell viability while culturing mammalian cells in the RGD-peptide-modified alginate microspheres was relatively higher than that for
the unmodified alginate microspheres. More than 90% of the mammalian cells maintained their viability after 4 days of culture in both alginate microspheres.
153704-5 Yu et al.
Because oxidation that would occur in the preparation
of RDG-peptide-modified alginate-based microspheres
would change the conformation of L-guluronic acid,17 gela-
tion would not be achieved by simply mixing with a cal-
cium ion solution. A mixture of two types of alginates (both
MVG and LVM, but greater MVG for the incorporation of
RGD peptides) was used in this study while thoroughly
considering the gelation characteristics, biodegradability,
and compatibility of alginates.18 The alginate conjugation
ratio was measured via the absorbance of aromatic groups
with an O.D. value at 280 nm. In order to further verify
whether both cell adhesion and proliferation would be
enhanced via RGD-peptide modification, human adipose-
derived stem cells were cultured in both modified and
unmodified alginate microspheres after mammalian cell
encapsulation and use of a 2% (w/v) alginate solution. The
stem cells cultured in unmodified alginate microspheres
showed a spherical shape and did not adhere onto the
sphere surface; however, the stem cells spread out and
adhered onto the RGD-peptide-modified sphere surface
(data not shown), indicating that the alginate-based micro-
spheres with an RGD-peptide modification did improve
cell-matrix interactions, i.e., stem-cell-alginate interactions
(RGD-based specificity versus non-specificity in this
study),19,20 in order to enhance the capacity for both carry-
ing and delivering more mammalian cells to the desired
regions of the body, ultimately, for cell therapy. Our results
also indicated that it would be very challenging to use algi-
nate microspheres without the incorporation of signaling
molecules such as RGD peptides (i.e., pure alginate micro-
spheres) for carrying and delivering mammalian cells, in
particular, human adipose-derived stem cells, even though
we attempted to make “blood-mimicking microspheres.”
To promote a more comprehensive understanding of
cell-matrix interactions between mammalian cells and RGD-
peptide-modified alginate microspheres, three types of mam-
malian cells—HeLa cells, NIH-3T3 fibroblasts, and human
adipose-derived stem cells—with different physiological or
pathological functionality were encapsulated in RGD-peptide-
modified alginate microspheres (based on the appropriate pro-
cedure that we developed in this study). We first dissolved
cell-encapsulated alginate microspheres and re-cultured these
mammalian cells (releasing from RGD-peptide-modified algi-
nate microspheres) on Petri dishes, as shown in Fig. 3(a),
showing that these released cells would both spread and pro-
liferate normally on the dishes. In this study, we provided the
results from culturing human adipose-derived stem cells on
Petri dishes, and we obtained the similar results when cultur-
ing both NIH-3T3 fibroblasts and HeLa cells (data not
shown). We then examined mammalian cell viability via live
versus dead staining after a few days of culture, as shown in
Fig. 3(b). Up to 95% of the mammalian cells were observed
right after mammalian cell encapsulation (i.e., our physically
based method), and more than 90% of the mammalian cells
maintained their viability after 4 days of culture. The cell via-
bility while culturing mammalian cells in the RGD-peptide-
modified alginate microspheres was relatively higher than that
for the unmodified alginate microspheres, as we expected
(Figs. 3(c) and 3(d)).
Appl. Phys. Lett. 103, 153704 (2013)
In summary, we have demonstrated a robust and easy-to-
handle physically based method (with appropriate proce-
dures), based on the mechanism of flow focusing, which can
be used to generate a fair amount of uniform cell-
encapsulated alginate microspheres (with and without the con-
jugation of RGD peptides), in a very short period of time, i.e.,
with high-throughput capacity. We also measured the physical
properties of the alginate solution and took such characteris-
tics into account when designing the experiments, in order to
create a physiologically mimicking environment using algi-
nate microspheres themselves to maintain the physiologically
relevant functionality of mammalian cells (once culturing
them inside the microspheres). Cell viability of mammalian
cells, in particular, human adipose-derived stem cells, when
re-cultured on Petri dishes, indicated that they can be main-
tained for at least 4 days after mammalian cell encapsulation.
We believe that this study, which is supported by significant
physics analysis, would provide researchers with interesting
insights into different divisions of physics including biophy-
sics, polymer physics, and applied physics.
We would like to thank the National Science Council of
Taiwan for financially supporting this research under
Contract Nos. NSC 99-2314-B-002-145 (to J. Yu) and NSC
101-2628-E-007-011-MY3 (to C.-M. Cheng), and Dr. Nai-
Chen Cheng in the Department of Surgery, National Taiwan
University for the kind assistance of the adipose tissues
obtained from multiple patients. The procedure in this study
has been approved by the Internal Ethical Committee of
National Taiwan University Hospital.
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