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Research Paper

Characterization of controlled highly porous

hyaluronan/gelatin cross-linking sponges for
tissue engineering

Chia-Ling Koa, Yin-Chun Tienb, Jen-Chyan Wangc, Wen-Cheng Chend,n,1

a
School of Dentistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b
Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c
School of Dentistry, Kaohsiung Medical University, Department of Dentistry, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan
d
Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, 100, Wenhwa Road, Seatwen,
Taichung 40724, Taiwan

art i cle i nfo ab st rac t

Article history: This study examines the suitability of N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide

Received 6 May 2011 hydrochloride for use as a cross-linker in the preparation of highly porous hyaluronan (HA)/
Received in revised form gelatin sponges through the solvent casting and particulate leaching (SCPL) process. The
1 June 2012 modulus, water absorption rate, bonding, morphology, cell viability, and chondrocyte mRNA
Accepted 5 June 2012 expression of the HA/gelatin sponges were measured and compared. Water uptake by the
sponges resulted in pores with spherical diameters ranging from 100 mm to 200 mm. With

Keywords: higher porosity, the strength and modulus declined. Larger salt leaching amounts resulted in

Hyaluronic acid higher water uptake. The cross-linking bonds in the HA/gelatin sponges were mostly ester

Gelatin bonds. According to our study, the amount of salt in the SCPL process was the main factor that

Sponges influenced strength, pore interconnectivity, and water uptake ability. The results also showed

Solvent casting/particulate leaching that the scaffold had a non-viability effect on human chondrocytes, but the mRNA expression

Cross-linker level of aggrecan and type II collagen in the cartilage was significantly higher than that of the

Tissue engineering control group after 5 d of culture. The sponges developed in the present study are potential
candidates for chondrocyte proliferation and differentiation in cartilage regeneration.
& 2012 Elsevier Ltd. All rights reserved.

1. Introduction pore size and structure of scaffolds is crucial to host tissue

In tissue engineering, a porous scaffold is required to func-
tion as a template and guide in cell adhesion, extension,
proliferation, and differentiation. A target tissue would be in
growth when the scaffold is biomimetic for the physiological
need of the regenerating tissue. Therefore, controlling the
formation (Moore and Lemischka, 2006; Yaniv et al., 2006;
Tai et al., 2007). General scaffold processing techniques are
used in the development of porous materials via polymer
cross bonding and lyophilization, solvent casting, particulate
leaching, membrane lamination, melt molding, temperature-
induced phase separation, and gas foaming.

n
Corresponding author. Tel.: 886 4 2451 7250x3413; fax: 886 4 2451 4625.
E-mail addresses: wencchen@fcu.edu.tw, wincheng0925@yahoo.com.tw (W.-C. Chen).
1
Equal contribution to first author.

1751-6161/$ - see front matter & 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jmbbm.2012.06.019

Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
2 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ]
Lyophilization, more commonly known as freeze-drying, is
frequently used in producing vaccines of bulky hydrogels
through the sublimation of the medium absorbed by the
scaffold. Freeze-drying removes most of the water from the
sample. The ability of hydrogels to absorb the weight of the
medium makes freeze-dried materials become highly porous.
In tissue regeneration, pore size distribution is a major issue
related to lyophilization because it is difficult to control.
Regularity is critical in cell biology, although the addition of
water can restore the sample to nearly its original state. The
swelling process of scaffolds after medium absorption is
difficult to control with respect to the desired final pore size
and structure relative to the target tissues. Restoring the
desired sample shape is the main purpose of generating a
uniform pore size distribution at the stage of forming inter-
connective macropores.
The solvent casting and particulate leaching (SCPL) process
is another option in producing a highly porous scaffold, and
ionic salts are generally used as a particulate medium. SCPL
is one of the well-known methods for the preparation of
highly porous sponges with controlled pores (Mikos et al.,
1993). Solvent casting requires the particles to be dissolved in
an organic solvent. The particles are typically chemicals of
salts with specific desired dimensions that are then added to
the solution before the cross-linking process. Next, the
mixture of hydrogels and salts is shaped into a final three-
dimensional mold to produce a scaffold after salt leaching.
Other modified scaffold fabrication techniques include sol-
vent casting and salt leaching (Lu et al., 2000; Murphy et al.,
2002), molding and salt leaching (Hou et al., 2003; Sosnowski
et al., 2006), and gas foaming agent salt leaching (Kim et al.,
2006; Nam et al., 2000, Tai et al., 2007). However, these
conventional methods require an alternative to the use of
organic solvents, which might lead to cytotoxicity or elevated
processing temperatures that could damage the natural
properties of the material (Tai et al., 2007).
Material composition also has a critical function in provid-
ing specific biological characteristics for cells within scaf-
folds. To exhibit the desirable functional characteristics of
cell attachment or adhesion, synthetic scaffolds should have
properties similar to those of the extracellular matrix (ECM).
Hyaluronic acid (HA) is a member of the glycosaminoglycan
family, of which the ECM of liver tissue is composed (Choi
et al., 1999). HA has been used extensively in the biomedical
field and has been proven to actively influence several
cellular functions, including cell adhesion, migration, and
proliferation. HA is most commonly used as a form of
hydrogel. However, in this form, it lacks strength because of
the deficiency of ECM components, making it unable to form
structures with constant dimensions. Therefore, the applica-
tion of HA alone in tissue engineering is not the perfect
solution for tissue restoration (Liu et al., 2004). To strengthen
HA scaffolds, a soluble derivative of collagen, gelatin, was
combined as a matrix with HA. The composite of HA and
gelatin has been used in many clinical applications (Xia et al.,
2004). Gelatin is composed of a unique sequence of amino
acids (Hong et al., 2004) and numerous integrin binding sites
similar to those found in collagen. These binding sites serve
functions that are related to cell adhesion and differentiation.
Compared with other types of specific purified collagen,
Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
gelatin is more practical to use in tissue engineering applica-
tions because of its low cost and simple preparation.
Chemical cross-linkers for HA, gelatin, and HA/gelatin
composite chains include carbodiimides, hydrazides, aldehydes,
sulfides, and polyfunctional epoxides (Yannas and Burke, 1980;
Djagny et al., 2001; Ramires and Milella, 2002; Adekogbe and
Ghanem, 2005). Among these cross-linking agents, carbodiimide
is generally preferred because it prevents the initiator from being
incorporated into the cross-linked structure of the hydrogel and
allows the reaction to be easily controlled (Tomihata and Ikada,
1997; Xu et al., 2009). The resulting biomaterial is acceptable for
biomedical application.
In this study, gelatin-based HA/gelatin scaffolds that use N-
(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochlor-
ide (EDAC) as the cross-linker were produced through the
pore fabrication process. Our strategy was to use the salts
to occupy and generate interconnected pores, cross-link the
hydrogel scaffolds, remove the particulate through the salt
leaching process, and then lyophilize the scaffolds. The hypo-
thesis is that after cross-linking, the interconnected scaffolds
produced by SCPL, ion removal, and lyophilization would
have a greater ability to controllably regenerate their original
shape, and that the different ratios of salts with SCPL-
controlled pore ratios could influence their physicochemical
properties.
2. Materials and methods
2.1. Sample preparation
Gelatin powder of type B, G932500G, originally extracted
from bovine skin (Sigma Co., St. Louis, USA), and sodium
hyaluronate (HA) powder (Mw 200 kDa, Food Chemifa Co.
Ltd., Japan) were mixed at a HA/gelatin ratio of 1:100. The
procedure for fabricating the scaffolds is detailed in Table 1.
Briefly, the mixture of HA/gelatin was gelled in 1 mL
deionized-distilled water (18.2 O d.d. water) and stirred to
form a complete colloidal suspension. Pore size control was
achieved through modification of the SCPL method (Mikos
et al., 1993). Hand-ground and sieved sodium chloride salt
(Tedia Company Inc., USA) within a range of 4488 mm was
selected. Different salt weight ratios were mixed into the gels
and stirred to form a salt-based colloid. EDAC (Sigma Co., St.
Louis, USA) was used as the cross-linker. Varied volumes of
500, 600, 800, and 1000 mL of 1 vol% EDAC were added to cross
link the salt-based colloids for 5 min before the salt leach-
ing process. To eliminate ion effects, in which particulates
were leached from the colloids, a salt leaching solution of
30 mL of 50 vol% ethanol was used. The samples were
immersed in the leaching solution for 96 h. To remove
residual ions from the scaffolds, the samples were then
immersed in d.d. water at a ratio of 1 g:100 mL for another
4 h. The detailed sample fabrication processes and abbre-
viation of groups with different salt leaching are indicated
in Table 1. Before testing, the semi-finished scaffolds were
dried in a vacuum by lyophilization, immediately cut into
5 mm3 cubes, and then stored in a drier at a humidity level
below 20%. All tests were conducted on samples within 2 h
after shaping.
 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ]
Table 1 Characteristics of scaffold preparation procedures for testing in this study.
Tested sample prepared procedures
1. Gelled step: 0.35 g gelatin and 0.0035 g sodium hyaluronate
were gelled in 1 mL d.d. water to form a colloidal suspension
2. Pore fabricated step: different salt weight ratios of 1 g (S1),
2 g (S2), and 3 g (S3)with a mean particle size of 4488 mm
were mixed with colloidal suspension in Step 1 by using a
magnetic stirrer
3. Cross-linking step: the constant cross-linker concentration
(1 vol% EDAC) with different volumes of 500, 600, 800, or
1000 mL were added in Step 2 to react for 5 min to fabricate
the restricted pore sizes and volumes
4. Salt leaching step: the colloid went through the salt
leaching process with 30 mL of 50 vol% ethanol for 96 h
5. Ion-removing step: the residual ions in the preformed
scaffolds were removed again with a ratio of 1 g scaffolds/
100 mL d.d. water for 4 h
6. Lyophilized and sample preparation step: the water-
containing scaffolds were dried in a vacuum by lyophilization
for 24 h and then immediately cut into 5 mm3 cubes to avoid
the effect of humidity in the atmosphere
2.2. Mechanical testing
The loads for the compressive testing of the pore ratio-
controlled HA/gelatin dried cube-shaped scaffolds (5 mm in
length) were obtained at a crosshead speed of 1.5 mm/min
using a desktop mechanical tester (Lloyd Instrument Co., Tokyo,
Japan). The mechanical tests were performed under unconfined
compression. The modulus of elasticity was defined as
Modulus of elasticity E st=eoffset
2
where s(t) is the stress (force: N; area: m ; unit: Pa) and e is the
strain (deformation relative to the original sample length) at
time t. The modulus of the scaffolds was measured as the linear
slope of the stressstrain curve, and it fell within a strain range
of approximately 515% of the offset. Five duplicate specimens
were prepared and analyzed for each group (n 5).
2.3. Water absorption rate
The water absorption rates of the scaffolds were measured by
immersing the samples in d.d. water at 37 1C for 24 h. After
rinsing the residual solution from the surface with a water-
saturated sponge, the hydrated specimens were weighed imme-
diately (Ww). Five duplicate specimens were measured for each
group (n 5). Compared with the dried scaffold weights (Wd), the
water absorption rate was calculated using the formula
Water absorption rate Ww Wd =Wd
2.4. Microstructures, density, and porosity
The apparent/bulk density of each sample was determined by
measuring the sample mass and volume. The pore fraction of
Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
3
Abbreviation of groups with Abbreviation of constant salt
different salt leaching leaching amount in Step 2
amounts (g) in Step 2 with different cross-linker
volumes (mL) in tested
samples in Step 3
S1 500
600
800
1000
S2 500
600
800
1000
S3 500
600
800
1000
the theoretical wt% porosity was determined by comparing
the salt leaching weight to the full weight of the scaffolds
(HA/gelatin/salts). Sample masses were measured to utilize
the Archimedes method for determining the density
(ASTM, 1999). To prevent the scaffold swelling effect, the
Archimedes-principle measurement of density was con-
ducted within 30 s (n 5). To characterize the pore distribu-
tion and morphology of the foam scaffolds, the dried and
1 wetted scaffolds were observed via optical microscopy (BX51,
OLYMPUS, Tokyo, Japan) at 37 1C after 24 h of immersion. To
investigate further the pore connectivity distribution of the
scaffolds with different ratios of salt leaching (S1-3), the inner
surfaces of the samples were cut immediately after lyophili-
zation, and the perforated specimens were coated with a thin
metal film for electrical conductivity. The morphologies were
examined under a field emission scanning electron micro-
scope (S-3000 N, Hitachi, Tokyo, Japan) operated at 15 kV and
14.815.2 mm working distance. According to the method of
Barbetta et al. (2009), the mean porosity of the scaffold was
measured by analyzing the areas of the images at 100 
image contrast using an image software (ImageJ, NIH, USA).
At least five individual specimens with five images of the
different parts of the specimen from each group were
prepared and analyzed (n 5).
2.5. Fourier-transform infrared spectroscopy (FTIR)
2
The lyophilized samples were separated into portions and
then pulverized and mixed with KBr at a weight ratio of 1:100
for FTIR analysis (NICOLET 6700, Thermo, MA, USA). The
analysis was performed in the 4004000 cm1 range to esti-
mate the number of active bonds of the amide and ester
material residual in the scaffolds.
4 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ]
2.6. Isolation and culture of chondrocytes
Fresh surgical tissues were obtained from the Department
of Orthopedics, Kaohsiung Medical University Hospital,
Kaohsiung, Taiwan, after approval by its Institutional Review
Board. Articular chondrocytes were isolated following the
method described by Kuettner et al. (1982) with some mod-
ifications. Harvested cartilage was obtained from three dif-
ferent patient sources. Cells were suspended in Dulbeccos
modified Eagles medium (DMEM, Invitrogen) supplemented
with 10% fetal bovine serum (FBS, Invitrogen), 100 IU/mL
penicillin, and 100 mg/mL streptomycin (Invitrogen). The
isolated chondrocytes were then assessed for viability (via
the trypan blue exclusion test) and number. Cells (1  105)
were seeded into 10 cm diameter culture dishes and incu-
bated for 5 d in DMEM supplemented with 10% FBS, 100 IU/
mL penicillin, and 100 mg/mL streptomycin. After 5 d of
culture, the media were changed twice a week.
2.7. Cell viability
The metabolic activity effects of the sponges on chondrocytes
were determined using a commercially available tetrazolium
salt (XTT assay, Biological Industries, Israel). Sample extracts
were prepared at a ratio of 1:10 (1 g sample/10 mL medium),
in which the sponges were immersed in the culture medium
for 24 h. The cells were seeded in 96-well culture plates at a
density of 1  104 cells per well in DMEM culture medium and
then allowed to attach for 24 h. Scaffold extracts were then
added to the 96-well culture plates, and untreated culture
medium was added to the control samples. After 24 h of
culture, 50 mL of the XTT reaction solution (sodium 30 -
[1-(phenyl-aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-
nitro) benzenesulfonic acid hydrate and N-methyl dibenzo-
pyrazine methyl sulfate, 50:1) was added to the wells. After
the plates were incubated for 4 h with XTT (37 1C and 5%
CO295% air), the optical density was obtained at a wave-
length of 490 nm in an ELISA plate reader. Triplicate experi-
ments were performed throughout this study. All assays were
repeated with three different cartilage sources to ensure
reproducibility.
2.8. Quantitative real-time polymerase chain reaction
(PCR)
The 4 mm3 sample was placed in a well of a 48-well plate.
Approximately 50 mL of cell suspension was seeded into the
scaffold (density of 1  106 cells per sample). The plate was
incubated at 37 1C in a 5% CO295% air incubator. After the
primary chondrocyte cells were cultured for 1, 3, 5, 7, or 10 d,
total cell RNA was extracted for first strand synthesis. The
final volume of 20 mL was composed of 10 mL total RNA, 2 mL
reverse transcription (RT) buffer, 2 mL RT random primer,
0.8 mL 100 mM deoxyribonucleotide triphosphate, 1 mL Multi-
scribe reverse transcriptase, and 4.2 mL nuclease-free H2O
(High Capacity cDNA Rreverse Transcription Kit, Applied
Biosystems). The thermal program was executed at 25 1C for
10 min, 37 1C for 120 min, and 85 1C for 5 min. The tempera-
ture was maintained at 4 1C. After the reaction, 80 mL nuclease-
free H2O was added, and the cDNA from the first strand
Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
Table 2 Primers designed for real-time polymerase
chain reaction (real-time PCR).a
Genes Primer sequence
Type II collagen F 50 -CAACACTGCCAACGTCCAGAT-30
Type II collagen R 50 -TCTTGCAGTGGTAGGTGATGTTCT-30
GAPDH F 50 -TCTCCTCTGACTTCAACAGCGAC-30
GAPDH R 50 -CCCTGTTGCTGTAGCCAAATTC-30
Aggrecan F 50 -ACAGCTGGGGACATTAGTGG-30
Aggrecan R 50 -GTGGAATGCAGAGGTGGTTT-30
a
Mission Biotech, Taipei.
reaction was used for real-time PCR. Real-time PCR was
performed using a Maxima SYBR Green/ROX qPCR Master Mix
(2  , Fermentas). The total volume of the universal reaction
was 15 mL, which consisted of 6.25 mL of 2  SYBR Green/PCR
Master Mix, 5.75 mL of nuclease-free H2O, 0.25 mL each of the
forward and reverse primers, and 2 mL of cDNA. To correct
pipetting errors, each cDNA sample was run in triplicate. The
gene expressions of aggrecan and type II collagen were ana-
lyzed by a number of threshold cycles (Ct). The WCt values of
the genes were normalized by the housekeeping gene, GAPDH.
The primer sequences used are shown in Table 2. All real-time
PCR experiments were performed in triplicate and repeated at
least three times (n 3).
2.9. Statistical analysis
Paired t-tests were performed to compare individual sets of
data and determine their statistical significance. One-way
analysis of variance (ANOVA) and pairwise multiple compar-
ison procedures (TukeyKramer tests) were employed to
compare data groups. To clarify the combined effect of
different amounts of salt leaching and different volumes of
cross linker (1 vol% EDAC) on two variables, two-way ANOVA
statistical analysis with the JMP 8.0 software (SAS Institute,
Inc., Cary, NC, USA) was conducted. The error is reported in
the text and figures as the standard deviation. In all cases, the
results were considered statistically different when po0.05.
3. Results
3.1. Effect of salt leaching amounts and added EDAC
volumes
EDAC, a safe and effective cross-linking agent for poly-
mers with COOH, OH, or NH2 groups, was not directly
incorporated into the reaction product (Fig. 1) (Choi et al.,
1999; Barbetta et al., 2009). Typical compression curves for the
three salt leaching sponges are shown in Fig. 2a. Sponge
behavior in compression exhibited an obvious typical curve
for a closed-cell relative rigid scaffold in the lowest leaching
amount (S1) and a typical curve for an open-cell flexible
scaffold in the highest leaching amount (S3) (Fig. 2a). The
curves support the tendency and confirm the validity of
the study (Gibson and Ashby, 1982). Compared with the same
added amount of EDAC, the modulus decreased exponen-
tially with increasing salt leaching amounts (Fig. 2b). This
 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ] 5

Fig. 1 Schematic illustrations of the cross-linking structures presumed for an EDAC-catalyzed cross-link between gelatin
and hyaluronic acid and salt leaching process: (a) interpenetration polymer structure and (b) intermolecular cross-link.

Fig. 2 Typical stressstrain curves of different salt leaching amounts (a) and mechanical modulus of different EDAC and salt
leaching amounts (b).

trend indicates that higher pore content in the scaffolds
means a larger salt leaching amount, resulting in a significant
decrease in the stress at the same deformation or strain
(Fig. 2). To clarify the combined effect of varied amounts of
salt leaching and added EDAC volumes on the modulus,
two-way ANOVA was conducted. The results demonstrated
that different salt leaching amounts could significantly affect
the modulus, and the variations in the amount of EDAC were
dependent on the salt leaching amount (Table 3). All tested
scaffolds exhibited a significantly decreased modulus. This
finding may be attributed to the larger pore distribution
resulting from the salt leaching amount and not to the
variations in EDAC quantity at the same salt leaching amount
(p40.05).

Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
6 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ]

Table 3 Two-way ANOVA statistical analysis of the effects of various EDAC amounts (500, 600, 800, and 1000 lL) with a
series of salt leaching amounts ranging from 1 g to 3 g (S1S3) per scaffold specimen on the modulus of hyaluronan/
gelatin sponges (n 5).

Groups Variations DF Sum of squares Mean square F ratio p-Value

Modulus (MPa) Main effects combined 64 273.97 24.61 o0.0001

Salt amount 2 212.97 125.81 o0.0001
EDAC amount 3 6.74 2.65 0.0579
Two-way interactions 6 9.25 1.82 0.1125
Error 53 44.86 0.85

Groups are significantly different at po0.05.

Fig. 3 Water absorption abilities of sponges prepared using different cross-linker (EDAC) and salt leaching amounts after
24 h of immersion. The star symbol indicates that the scaffold was dispersed in the process of testing the water absorption
ability.

The effect of the added volume of the EDAC cross-linker on
the compressive moduli of the scaffolds was not significant.
However, the rate of water absorption was significantly
affected by the variation in the salt leaching amount and
EDAC volume. All testing groups, except for the S3 group of
scaffolds with 500 mL cross-linked EDAC, showed no obvious
degradation or dissolution phenomena after 24 h immersion.
A significant decline in the water uptake with increasing
EDAC amounts was observed when the salt leaching amount
was held constant (Fig. 3). To clarify the variable effect of the
salt leaching amount and the EDAC volume on the compres-
sive modulus and water absorption, a statistical analysis
was performed. The results are presented in Table 4. Scaf-
folds generated in the procedures with higher cross-linker
volumes in each salt leaching group all led to statistically
insignificant differences in the compressive modulus
(p40.05). However, they generally resulted in a significantly
higher water uptake, except in the S3 group. This non-
significant result occurred because the scaffold produced by
the S3/500 mL EDAC procedure was dispersed after 24 h
immersion, and the group data could not be included in the
analysis. When scaffolds from the groups with constant
EDAC volumes of 600 and 800 mL with varied salt leaching
amounts were compared, larger salt amounts generated
higher water absorption abilities.
3.2. Morphologies and porosity analysis
Scaffolds with highly interconnected pores were observed in
the S3 group with 600 mL EDAC (Fig. 4). Although the S1-2
group had porosity greater than 65%, the bulk pore distribu-
tion was much more random and non-uniform. The SEM
images imply that the group with the higher salt leaching
amount had a more uniform pore distribution and dimen-
sional properties than the other groups. As shown in the
water uptake image in Fig. 4c, all the pores were stretched
into a nearly spherical shape because of the surface ten-
sion of water, and the pore diameter fell into the range of
100200 mm.
3.3. Bond analysis
The effects of 600 mL EDAC cross linker on different salt
leaching amounts (S13) are shown in Fig. 5. The salt leaching
amounts were not strongly correlated with the absorption
bands. The ester group is assigned to the peaks at approxi-
mately 1060 and 1081 cm1. The absorption bands corre-
sponding to the CNH stretching vibration can be observed
at approximately 1633, 1538, and 1447 cm1, indicating
the reactants, gelatin, and HA, respectively. The effect of
the EDAC cross linker on the intensity of the absorbance

Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ] 7

Table 4 One-way ANOVA statistical analysis of different EDAC amounts and a series of salt leaching amounts ranging
from 1 g to 3 g (S1S3) per scaffold compared with physical properties (n 5).

Groups Variations Statistical analysis of Statistical analysis of water absorption

compressive testing modulus amount per scaffold

Salt leaching amount Added EDAC amount p-Value Group p-Value Group comparisons
(g) (mL) comparisons
S1 500/600/800/1000 0.1339 None o0.0001 5004600, 800, 1,000,600,
10004800
S2 500/600/800/1000 0.4335 None 0.0057 50041000
S3 500/600/800/1000 0.5279 None 0.0957 Nonea

Added EDAC Salt leaching p-Value Group p-Value Group

amount (lL) amount (g) comparisons comparisons

500 S1/S2/S3 o0.0001 S14S2, S3 0.1123 Nonea

600 S1/S2/S3 o0.0001 S14S2, S3 o0.0001 S34S24S1
800 S1/S2/S3 o0.0001 S14S2, S3 0.0002 S3, S24S1
1000 S1/S2/S3 o0.0001 S14S24S3 0.1082 None

Groups are significantly different at po0.05.

a
S3/500 mL group was dispersive after 24 h of immersion, and these data were not included in this statistical analysis.

Fig. 4 Typical scaffold photo image (a), morphologies of dried and wetted sponges prepared using different salt leaching
amounts with 600 lL EDAC assessed by SEM (b) and OM (c), respectively.

assigned to the amide groups in the cross-linked products 3.4. Cell viability and quantitative real-time PCR
was different from its effect on those in HA or gelatin. This
difference indicates the formation of new cross-linking bonds The viability of human chondrocytes remained high and was
(CONH) (Xu et al., 2009; Barbetta et al., 2009). not significantly affected by the salt leaching conditions used

Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
8 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ]

Fig. 6 Cell viability of sponges prepared using different salt

leaching amounts and cross-linking with 600 lL EDAC.

Fig. 5 FTIR absorption bands of the starting material and

product of cross-linking reactions with 600 lL EDAC and
various amounts of salts.

to create the scaffold variants (Fig. 6). The salt leaching and
ion removal steps led to the toleration of the scaffold extracts
by chondrocytes, at least as well as the culture medium of the
first reference. The effect of the S3 group scaffold with 600 mL
EDAC on the gene expressions of aggrecan and type II
collagen is shown in Fig. 7. Quantitation of the type II colla-
gen transcripts in the scaffold cultures by RT-PCR revealed an
approximately 20-fold increase on day 3. The expression of
type II collagen had the highest upregulation by day 5 of
scaffold culture and was significantly higher than the control
cultures within 310 d. Consistent with the profile of type II
collagen expression, aggrecan mRNA was also upregulated to
an approximately 6-fold increase after 10 d of culture. At day Fig. 7 Real-time polymerase chain reaction analysis
5, the cell/scaffold complex had greater aggrecan expres- indicating more chondrogenic markers of chondrocytes
sion than the control group. This expression progressively cultured in HA/gelatin sponges than in the control group
increased to day 10. Simultaneous measurement of cell (n 3).
viability and quantitation of the specific proteins in the
scaffold cultures by RT-PCR was incubated in the same pre-
sence after 24 h of culture. Compared with the control group, series of equations to describe the mechanical properties of
the group labeled with type II collagen and aggrecan had open- and closed-cell sponges. The results are summarized
enhanced chondrogenic marker synthesis of chondrocytes in Table 5. For the linear-elastic moduli, the salt leaching
cultured in HA/gelatin sponges instead of enhanced meta- amounts of the S1 and S2 groups coincide with the analysis of
bolic activity of chondrocytes (Fig. 7). a closed-cell model. Furthermore, using the open- and
closed-cell model equations of Gibson and Ashby, the S2
3.5. Porosities relative to moduli and densities group should behave as a closed-cell sponge as indicated in
Table 5, where the E/e3 of S2 group has a constant value with
The effect of porosity was also reflected on the stress versus the S1 group, and the E/e4 of S2 group has varied values with
strain curve with different amounts of salt leaching (Fig. 2a). the open-cell S3 group. According to the relationship curves
Based on the cubic model, Gibson and Ashby (1982) built a of the modulus of elasticity (Fig. 2b), the mechanical

Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ] 9

Table 5 Differential equations derived from an individual-based model and the related parameters.

Gibson and Ashby model equations Extended equation derived from regression lines

Closed-cell sponge E EsC1(r/rs)3 Open-cell sponge E EsC1(r/rs)2

0
Relative density (r/rs)a is
proportional to strain
e; E/e3 constant value
E Es exp(np)
Relative density (r/rs) is Youngs modulus of cell wall materials of Es and n are
proportional to e2; E/e4 constant constants, p is the pore volume fraction, and E is the
value measured modulus

Salt leaching groups S1 S2 S3 EADC amount 500 600 800 1000

(mL)
Modulus E mean 4.6634 2.4809 (0.9393) 0.4987 np 1.032 1.023 0.988 0.966
(S.D.)b (0.7116) (0.0485)
Strain e mean (S.D.) 0.1174 0.0956 (0.0147) 0.2036 Es 11.992 15.663 12.193 12.246
(0.0080) (0.0063)
E/e3 (S.D.) 2879.2 2766.2 (281.9) R2 0.997 0.998 0.999 0.944
(269.4)
E/e4 (S.D.) 29,722.3(7232.1) 294.3 (60.2) R: correlation coefficient, if R2 1, all data points fall on the
regression line

a
r is the density of sponge and rs is the density of cell wall material.
b
S.D. represents the value of standard deviation.

properties of the cross-linked structures could be summar-
ized as the equation in Table 5:
Emodulus Es exp nP
where Es and n are the experimental constants and P is the
pore volume fraction. According to the above equation and
compared with our compressive modulus results (Fig. 2b), the
lines were mostly coincident with each other, except for the
600 mL EDAC group.
The total amounts of salt present in the S1, S2, and S3
mixtures were 74, 85, and 89 wt%, respectively. The mean
image pore volumes of the S1, S2, and S3 scaffold groups were
66%, 67.6%, and 78.5 vol%, respectively. When the theore-
tical weight percentages (wt%) and the measured image pore
volumes (vol%) are substituted into Eq. (3) as the parameter P,
where Es is 12.144 [the mean value of (11.99212.19312.246)/3]
and n is 0.999 [the mean value of (1.0320.9980.966)/3], the
equation-derived moduli of the S1 group are almost identical to
the modulus values measured via compressive testing (Fig. 8a).
Obvious combined effects on the salt leaching amounts and
lyophilization were found in the S2 and S3 groups.
The density characteristics are crucial in porous scaffolds
because of their close correlation to the degree of cross-
linking and the compressive modulus. In particular, the
Archimedes-principle derived density values of the pore
scaffolds were higher than the bulk density values of the
whole scaffolds. When the density values were compared,
the Archimedes-principle derived density value decreased
exponentially as the salt leaching amount increased. Fig. 8b
shows a constant percentage decline in the bulk density
values. The tendency of the compressive modulus with
increasing salt leaching amount was more consistent with
the values of the Archimedes-principle derived density.
4. Discussion
Porous scaffolds are among the vehicles developed for cell
cultivation in tissue engineering. Interconnected porous
scaffolds enable the large-scale generation of high-yield,
anchorage-dependent cells because of their high surface area
for cell adhesion and proliferation. A variety of scaffold
3 materials, including those based on synthetic and natural
polymers such as dextran, polystyrene, cellulose, and col-
lagen- or gelatin-based sponges, have been developed
(Badylak et al., 2009). The major characteristic of three-
dimensional scaffolds is their ability to aid cells in maintain-
ing tissue-specific functions (Karageorgiou and Kaplan, 2005).
This study aims to develop a cross-linked scaffold with
highly interconnected pores. The scaffolds were composed
of HA/gelatin. The uniform pore-size distributions in the
swelling scaffolds were controlled within 100200 mm by
salt leaching. Pores of 100350 mm may be favorable for cell
colonization and vascularization, leading to the penetration of
natural tissues into the scaffolds (Simske et al., 1997). The
modified SCPL process, in which the pore sites were occupied
by salt and then cross-linked by EDAC before salt leaching and
lyophilization, resulted in a greater number of different pore
sizes. The porosity and density of the resulting scaffold can be
controlled by the amount of salt added, and the pore size is
dependent on the size of the salt. Such scaffolds can also
recover the original pore size after water uptake, and they
have a narrow pore size distribution rather than the random
sizes caused by lyophilization in the fabrication process.
The scaffolds developed in this study demonstrate that the
salt leaching amount was the primary factor determining the
compressive modulus (Fig. 2), rather than the volume of the
EDAC cross linker (Table 3). Furthermore, the cross-linking
reaction caused by EDAC was strongly related to the water
uptake ability of the scaffolds (Table 4). Larger pore volume
and lower relative density lead to reduced modulus and
strength because pores act as stress concentrators and cell
edges carry the entire load when an open-cell sponge is
deformed (Gibson and Ashby, 1982; Callister and Rethwisch,
2008). The dependence of the compressive modulus on the
Archimedes-principle derived density values and pore dis-
tribution is shown in Fig. 8. After water uptake, the samples
presented a pore diameter of 100200 mm, which was larger
than the pore size of the dried samples. The pore-size
swelling phenomenon can be attributed to the force balance

Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
10 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ]
Fig. 8 Comparisons of measured and formula-derived moduli (a) and bulk and Archimedes-principle derived densities (b) in
scaffolds after cross-linking with 600 lL EDAC.
of the matrix expansion and the surface tension effects in the
sponge after water uptake. The SCPL process employed in the
study (Mikos et al., 1994) involves the use of NaCl salt with
poly(L-lactic acid) or poly(DL-lactic-co-glycolic acid). The
results indicate that porosity can be controlled by the amount
of salt added and that the SCPL process with 70 wt% or
greater salt produces pores with high interconnectivity. The
permeability of 3-D scaffolds used for tissue engineering is
significant because it controls the rate of cell migration and
the diffusion of nutrients and waste products in and out of
the scaffold. Scaffold permeability is also related to the pore
size and the interconnectivity and distribution of the pores
(Al-Munajjed, OBrien, 2009). Pores with a diameter larger
than 100 mm should be increased in number for cell coloniza-
tion and vascularization (Simske et al., 1997). Based on our
study, the theoretical pore rates of the HA/gelatin scaffolds of
the S1, S2, and S3 groups were controlled at salt leaching
amounts of 74, 85, and 89 wt%, respectively. However, the
porosities of the HA/gelatin sponges (vol%) measured
through imaging software were less than the values predicted
by wt%, and only the S3 group (with a mean porosity of
78.5 vol%) had a porosity greater than 70 vol%. The result of
open-cell sponges with high interconnectivity was further
confirmed by the stressstrain curve (S3 group) that exhibits
three obvious regions: linear elasticity, plateau, and densifi-
cation (Fig. 2a). Thus, regions in the curve of closed-cell
sponges (S1 and S2 group) are less obvious and have a more
steeply rising stressstrain response than open-cell sponges.
The salts are dissolved in the colloidal suspension medium
before the cross-linking process, resulting in a smaller pore
volume compared with the salt leaching amount. Therefore,
highly interconnected pores were observed only in the S3
group. Larger amounts of salts should have a greater tendency
to produce uniform dimensions and homogeneous intercon-
nected pore distributions because solid salts also exhibit
aggregation phenomenon (Fig. 4). The formula-derived moduli
from the measured pores are far from the values measured by
compressive testing. The effect on the theoretical and mea-
sured moduli is strongly related to the porosity and is caused by
the fact that larger salt leaching amounts result in a higher risk
Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019
of non-uniform linking and pore size distribution in the
prepared scaffold. Thus, a larger salt leaching amount has a
greater effect on the resulting modulus (Fig. 8).
Liang et al. (2004) revealed that cross-linking bonds are
mainly ester bonds that could be damaged easily in aqueous
solutions. Some NHCO bonds were initially formed by
EDAC because of the cross-linking reaction between the
COOH and NH2 groups. By varying the amount of the EDAC
solution used (Fig. 3), the water-uptake ability of the porous
scaffolds could be altered and was the largest in the 500 mL
group. However, the scaffolds of the EDAC 500 mL group with
the S3 salt leaching condition were dispersed in the medium
after 24 h of immersion. This result could be explained by the
insufficient strength of the scaffold obtained through the
absorption of a large amount of water when a lower amount
of EDAC was used. Larger amounts of EDAC in the constant
salt leaching groups tend to decrease the water-uptake
abilities of the scaffold. This phenomenon may be attributed
to the higher amount of EDAC causing the increased forma-
tion of NHCO bonds (Fig. 1).
Furthermore, the scaffolds prepared in this study are being
developed for use in the regeneration of cartilage in humans.
The manufacturing processes used to create the material
variants tested in this study did not appear to have any
significant adverse effects on human chondrocyte viability.
Collagen type II is the main ECM protein in articular cartilage.
The mRNA expression of type II collagen in the human
chondrocytes seeded in the scaffold generally occurred ear-
lier than in the control group of human chondrocytes seeded
on a culture dish after 3 d. Aggrecan and type II collagen
are cartilage-specific markers, and their expressions were
substantially upregulated in days 10 and 3, respectively,
compared with the control group. Cartilage ECM synthesis
was characterized by greater enhancement of both type II
collagen and aggrecan in the chondrocytes cultured on deve-
loped scaffolds by days 3 and 10. Differentiating chondrocytes
undergo an initial phase of cell condensation and, subse-
quently, begin to deposit cartilage-specific matrix (Takahashi
et al., 1998). In addition, HA in HA/gelatin scaffolds could
participate in chondrogenesis. Oster et al. (1985) proposed
 journal of the mechanical behavior of biomedical materials ] (] ] ] ]) ] ] ] ] ] ]
that the osmotic changes during HA synthesis and degrada-
tion could produce sufficient swelling and deswelling to
regulate the balance between cellcell versus cellECM con-
tact. This mRNA-upregulated expression in the chondrocyte
culture shows that the scaffold could promote chondrocyte
condensation greater than the control group and could begin
to increase the expression of cartilage-specific ECM proteins
of both type II collagen and aggrecan. The early upregulated
expression of type II collagen, followed by aggrecan later than
the control group, proved their suitability as scaffolds for
tissue engineering. The present study confirms the hypoth-
esis that the controlled pore rates of these HA/gelatin scaf-
folds by the SCPL process affect their physiochemical and
gene expression properties.
A 3-D scaffold should possess morphology similar to that
of the organ it mimics. It should also possess connective
porosity, hydrophilic properties, optimal porous dimensions,
and sufficient strength to prevent the denaturing of the initial
cell cultures. Similar to the sponges prepared in this study, it
must have active amide bonds to attract proteins spontaneously.
5. Conclusion
The proper use of solvent casting/particulate leaching and
the control of the quantity of cross-linker (EDAC) in the
processing of HA/gelatin scaffolds are crucial to the produc-
tion of tissue or the organ regeneration of materials for non-
loaded defects. The designed HA/gelatin scaffolds that pos-
sess pores with high interconnectivity and size adequate to
support chondrocyte proliferation and differentiation are
suitable for cartilage regeneration supplements.
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Please cite this article as: Ko, C.-L., et al., Characterization of controlled highly porous hyaluronan/gelatin cross-linking
sponges for tissue engineering. Journal of the Mechanical Behavior of Biomedical Materials (2012), http://dx.doi.org/10.1016/
j.jmbbm.2012.06.019