1. Art and Diseño

Fabrication of Biomimetic Microporous Hydrogels Based on Chondroitin
Sulfate for Chondrocyte Culture
Xiaolong Gao1 , Chao Lin*2 , Peijun Wang*1
1
Tongji Hospital of Tongji University, Tongji University School of Medicine, Tongji University
Shanghai, 200065, PR China.
2
The Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine
Tongji University, Shanghai, 200092, PR China.
Correspondence to: Chao Lin (E-mail: [email protected]) and Pengjun Wang (E-mail:
[email protected])
ABSTRACT
The development of injectable microporous hydrogels is critical for the regeneration of injured cartilage
tissue. Herein, microporous chondroitin sulfate-tyramine (CS-TA) hydrogels crosslinked by horseradish
peroxidase (HRP)-mediated oxidation reaction is developed and their chemical and physical properties as
well as their application for cartilage tissue engineering are evaluated. CS-TA hydrogels are prepared by
mixing CS-TA solution with horseradish peroxidase (HRP) and H2 O2. The hydrogel properties such as
gelation time, gel content, swelling ratio and mechanical properties can be regulated by optimizing CSTA, HRP and H2 O2 concentrations. These CS-TA hydrogels possess a well-connected porous network
and good permeation ability for nutrients as confirmed by in vitro release of methylene blue as a model
drug from the hydrogels. Chondrocytes incorporated into the hydrogels maintain survival and can produce
collagen type II matrix after 21-day culture. The results of this study indicate that HRP-crosslinked CSTA hydrogels may serve as suitable bio-scaffolds for cartilage tissue engineering.
KEYWORDS Enzymatic crosslinking, horseradish peroxidase, injectable hydrogel, chondroitin sulfate,
cartilage regeneration
1. Introduction
Cartilage is a highly resilient tissue with limited self-repair capacity due to its avascular
nature [1, 2]. Traditional clinical strategies for cartilage repair normally encompass c hondral
shaving, total joint arthroplasty as well as osteochondral and perichondrial grafting. However,
these methods usually lead to short-term pain relief or improved mobility of patients [3].
Alternatively, tissue engineering offers an innovative method to maintain and improve cartilage
functions [4]. By this method, injured cartilage can be regenerated by making use of a temporary
macro-porous bio-scaffold for cell proliferation, differentiation and matrix production. In the
past decades, hydrogels have been widely studied as scaffolds for cartilage tissue engineering [5].
Hydrogels are three-dimensional, hydrophilic polymeric networks containing a high content of
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water, which provide a micro-environment similar to native cartilage. Moreover, hydrogels can
allow for sufficient transportation of nutrients and waste products due to their porous structure,
which is favorable for cell proliferation and differentiation. From the clinical point of view,
hydrogels that can form in-situ, also termed as injectable hydrogels, are preferred for cartilage
repair since they gain the advantages such as sufficient filling in an irregularly shaped defect,
non- invasive surgical procedure, and handy incorporation of essential cells/growth factors [6].
Different strategies have been developed for the preparation of hydrogels in cart ilage tissue
engineering [7]. One of the most commonly used methods is photocrosslinking, which affords
in-situ forming hydrogels upon UV exposure within minutes [8]. However, the drawback of the
method is that UV exposure with high intensity or long time might adversely affect cellular
metabolic activity [9]. Besides, local heat release during the crosslinking p rocess may give rise to
cellular necrosis [10]. Thus, the intensity of UV light is often limited to approximately 5-10 mW
cm-2 , in order to avoid cell damage [11]. Besides, in vivo photocrosslinking is also hampered
because of UV-light absorption by the skin and a limited penetration depth of UV light in tissues
[11]. Recently, enzymatic crosslinking method for injectable hydrogels has attracted much
attention [12]. Particularly, horseradish peroxidase (HRP)- mediating crosslinking of phenolbased polymer derivatives is of great interest. For example, Kurisawa et al. showed hyaluronic
acid–tyramine hydrogels with horseradish peroxidase (HRP) as a catalyst and hydrogen peroxide
as an oxidant [13, 14]. Park et al. prepared HRP-crosslinked hydrogels from chitosan and
polypseudorotaxane composite[15]. Similarly, Menzies et al. prepared HRP-crosslinked
poly(ethylene glycol)(PEG) hydrogels [16]. Because HRP-crosslinked hydrogels are normally
formed under mild conditions (e.g. physiological conditions), they are ideal cand idates as
scaffolds for cartilage tissue engineering [17]. A few researches showed that HRP-crosslinked
hydrogel systems based on chitosan, dextran, and PEG can maintain the proliferation of
chondrocytes in vitro and the production of collagen and GAG matrix [18-21]. However, these
systems are normally based on exogenous or synthetic materials, which lack bio- functionality for
cell adhesion, proliferation and differentiation. There is thus a fundamental need to develop
biomimetic hydrogel systems for cartilage tissue engineering.
Chondroitin sulfate (CS) is an anionic polysaccharide consisting of repeating disaccharide
units of D-glucuronic acid and N-acetyl galatosamine, sulfated at 4- or 6-positions. It can bind
with a core protein to form highly absorbent aggregan, as a major structure of cartilage [22]. CS
2
is also biodegradable in the body by the enzymes such as hyaluronidase-1 and PH-20 [23, 24].
These features make CS an ideal matrix for cartilage tissue engineering. For e xample, Anesth et
al. reported on CS and poly(vinyl alcohol) (PVA) composite hydrogels [25]. Introduction of CS
caused the composite hydrogels with increased swelling ratio when compared to PVA gels, and
meanwhile retaining good mechanical properties. Histological results indicated that the CS-based
hydrogels supported chondrogenesis of encapsulated chondrocytes. In another study, CS-based
scaffold stimulated MSCs to express cartilage specific genes and secrete cartilage specific matrix
and inhibited differentiation of MSCs into hypertrophic chondrocytes and support chondrogenic
activity [26]. Thus, CS-based hydrogels can offer a biomimetic environment for cartilageous
tissue production.
The purpose of this study is to investigate potential of porous CS-based hydrogels for cartilage
tissue engineering. To this end, injectable hydrogels based on chondroitin sulfate-tyramine (CSTA) conjugates was prepared via enzymatic crosslinking using HRP and H2 O 2 . The properties of
the hydrogels were characterized in terms of gelation time, gel content, swelling ratio and
mechanical property. In vitro release of methylene blue, as a drug model, from CS-TA hydrogels
were examined under physiological conditions. Moreover, chondrocytes were incorporated into
the hydrogels. The viability of the cells and the formation of ca rtilage tissue (collagen type II) in
the gel/cell constructs were investigated.
2. EXPERIMENTAL
2.1 Materials. Horseradish peroxidase (HRP, type VI, ~300 purpurogallin U·mg-1 ), methylene
blue (MB) and hydrogen peroxide (H2 O2 ) were purchased from Sigma-Aldrich. Chondroitin
sulfate-tyramine conjugates (CS-TA) with a degree of substitution (DS, defined as the number of
tyramine moieties per 100 repeating units of chondroitin sulfate) of 15 were synthesized using
previously reported method by coupling tyramine using carbodiimide activation chemis try [27].
2.2. Hydrogel formation and gelation time. Hydrogel samples (0.25 mL) were prepared in vials at
37 °C. In a typical procedure, to a PBS solution of CS-TA (200 μL, 12.5 wt-% stock solution),
freshly prepared stock solutions of H2 O2 and HRP in PBS were added and the mixture was
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gently mixed. The final concentration of the CS-TA conjugate was 10 wt-%. In the experiments
of preparing hydrogels, different concentrations of HRP (9.4, 18.8, 37.5 U mL-1 of stock
solution), H2 O 2 (0.25~1 wt-%) and CS-TA conjugates (2.5, 5, 10 wt-%) were applied. The time
to form a gel (denoted as gelation time) was determined using the vial tilting method.
Gel content and swelling ratio. To determine the gel content, CS-TA hydrogels (0.25 mL) were
lyophilized and weighed (W d). The dry hydrogels were then extensively extracted with 6 mL of
deionized water at room temperature for 3 days to remove uncrosslinked polymer. The water was
replaced once in each day. The non-soluble samples were freeze-dried and weighed (Wg ). The
gel content was expressed as Wg /Wd×100%.
To determine the swelling ratio, hydrogel samples (0.25 mL) were prepared in vials according
to the procedure above and accurately weighed (W i ). The samples were subsequently incubated
in 2 mL of PBS at 37°C. At regular time intervals, the buffer solution was removed from the
samples and the weight of the hydrogels was determined (W t ) to calculate the swelling ratio (S),
which is defined as Wt /Wi.
2.3 Rheological analysis. Rheological experiments were carried out with a Bohlin Gemini HR
nano rotational rheometer (Malvern) using parallel plates (25 mm diameter, 0°) configuration at
37°C. In a typical example, 75 μL of H2 O2 stock solution (0.5 wt-%, in PBS) and 75 μL of HRP
stock solution (18.8 U mL-1 , in PBS) were mixed. The HRP/ H2 O2 solution was then
immediately added to 600 μL of a solution of CS-TA (12.5 wt-%, in PBS). The mixture was
vortexed and then quickly applied to the rheometer. The upper plate was immediately lowered to
a measuring gap size of 0.5 mm, and the measurement was started. To prevent evaporation, a
layer of oil was introduced around the polymer sample. The evolution of the storage (G′) and
loss (G″) modulus was recorded as a function of time.
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2.4 Hydrogel morphology. Hydrogel samples were prepared as described in section 2.2 and
incubated in pH 7.4 PBS at 37 °C for 24 h. Next, they were freeze-dried. The morphology of the
hydrogels was studied using a Hitachi SU-1510 scanning electron microscopy (SEM).
2.5 Drug loading and in vitro release. The model drug methylene blue (MB) was loaded into the
CS-TA hydrogels by first mixing with CS-TA solution and then with HRP/H2 O 2 mixture. In a
typical example, 1 mg mL-1 of MB stock solution in PBS buffer was freshly prepared. Next, 100
μL of MB stock solution was added to CS-TA solution in PBS (100 μL, 25 wt-% stock solution),
and mixed by vortexing. Then, freshly prepared PBS solution of HRP (25 μL, 37.5 U mL-1 stock
solution) mixed with H2 O2 (25 μL, 0.5 wt-% stock solution) was added to the CS-TA/MB
solution and the mixture was gently vortexed. The hydrogels loaded with MB were kept in dark
at 37 °C for 24 h to complete the crosslinking process. Next, 2 mL of release medium (PBS) was
added on the top of the hydrogels and they were gently shaken in the incubator at 37 °C. The
release experiment was performed in triplicates. At a different time interval, the upper PBS
medium was replaced with 2 ml of fresh PBS. The concentration of release MB in the PBS was
determined at the absorption at 665 nm using U-3010 UV-Visible Spectrophotometer (Hitachi,
Japan). MB concentration was calculated using a calibration curve derived from a series of MB
solutions with different concentrations.
2.6 In situ chondrocyte incorporation into hydrogels. Chondrocytes were isolated from ear
cartilage of rabbit according to our previous reports [28, 29]. Isolated chondrocytes were
suspended in DMEM with 10% heat inactivated fetal bovine serum, 1% Penicillin/Streptomycin
(Gibco), 0.01 M MEM nonessential amino acids (Gibco), 10 mM HEPES and 0.04 mM l-proline
(Sigma) and used in Passage 1. Hydrogels containing chondrocytes were prepared under sterile
conditions by mixing a polymer/cell suspension with HRP/H2 O 2 . Polymer solutions of CS-TA
5
were made in medium and HRP and H2 O 2 stock solutions were made in PBS. All the
components were sterilized by filtration through filters with a pore size of 0.22 μm.
Chondrocytes (P1) were dispersed in the precursors of the hydrogels. The hydrogels were then
formed using the same procedure as in the absence of cells. The final polymer concentration was
10 wt-% and the cell seeding density in the gels was 5×106 cells per mL. After gelation, the
hydrogels (50 μL of each) were transferred to a culture plate and 2 mL of chondrocyte
differentiation medium was added. The samples were incubated at 37C in a humidified
atmosphere containing 5% CO 2 . The medium was replaced every 2 or 3 days. After 21 day’s of
culturing, the hydrogel constructs were rinsed in PBS and stained with Live/Dead assay Kit to
evaluate the chondrocyte viability. The morphology of chondrocytes in the hydrogels was
studied by SEM. The constructs were fixed with formalin followed by sequential dehydration
and critical point drying.
2.7 ImmunoFluorescent staining of collagen type II. The production of collagen type II was
investigated using immunofluorescent staining of type II (Pro) collagen gene (Co l2A1). After
rehydration, the samples were treated with 10 mM citric acid buffer (pH 6.0) for 10 minutes, and
then were washed with PBS/BSA 1%. Sections were incubated overnight with a Col2A1
monoclonal antibody. After washing twice for 5 min in PBS/BSA 1 %, sections were incubated
with Alexa Fluor 488 IgG1 for 1 h. After extensive washes, the sections were embedded with
DAPI mounting medium. Hydrogels containing no chondrocytes were employed as a negative
control.
2.8 Statistical analysis. Results are presented as the means ± SD values of triplicate samples
(n=3) in three independent experiments. Comparisons between two samples were performed with
the Student’s t-test. Differences were considered to be statistically significant at P<0.05.
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3. RESULTS AND DISCUSSION
3.1 Gel formation and gelation time
HRP- mediated crosslinking of chondroitin sulfate-tyramine (CS-TA) conjugates was
investigated as a function of CS-TA concentration (2.5 to 10 wt-%, HRP concentration
(0.94~3.75 U mL-1 ) and H2 O2 concentration (0.025~0.1 wt-%). The crosslinking reaction occurs
with HRP as a catalyst and H2 O2 as an oxidant. The concentration range in these studies was
chosen according to the conditions suitable for in-situ gelation as previously reported [18, 19, 30].
In general, CS-TA hydrogel was rapidly formed at room temperature by HRP- mediated
oxidative reaction. The mechanism underlying this reaction is based on coupling of phenols via a
carbon-carbon bond at the ortho positions or a carbon-oxygen bond between the carbon atom at
the ortho position and the phenoxy oxygen [31]. Table 1 shows gelation time of the hydrogels as
determined by the vial tilting method. At a low polymer concentration of 2.5 wt-%, the hydrogel
was fast formed within ~61 s. Moreover, the gelation time decreased from 61.3 to 33.0 s when
increasing the polymer concentrations from 2.5 to 10 wt-% at a constant HRP concentration
(3.75 U·mL-1 ) and H2 O2 concentration (0.05 wt-%). This faster gelation process is because more
phenol moieties involve in the crosslinking reaction with increasing polymer concentration.
Furthermore, HRP and H2 O2 concentration also has an effect on the gelation time. For example,
the hydrogel was formed within ~311 s at a HRP concentration of 0.94 U mL-1 , while a shorter
gelation time (~37.5 s) was detected at higher HRP concentration of 3.75 U mL-1 (P<0.05). This
phenomenon is likely attributed to the fact that the increased HRP concentration accelerates the
concentration of phenoxy radicals for crosslinking. On the other hand, keeping a constant HRP
(3.75 U mL-1 ) and polymer concentration (5 wt-%), the gelation time reversely increased from
approximately 26.3 to 77.1 s when increasing the H2 O2 concentrations from 0.025 to 0.1 wt-%.
This is probably due to excessive oxidation of H2 O2 , causing inactivation of HRP [32]. Similar
phenomenon was also reported towards other HRP-crosslinked hydrogel systems based on
dextran-tyramine, poly(amido amine)-tyramine and tetronic-tyramine [18, 30, 33]. Overall,
gelation time of the CS-TA hydrogels was relatively short (less than 320 s) and adjustable at a
wide range of time from seconds to minutes.
3.2 Gel content and swelling ratio
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Gel content and swelling ratio are critical parameters because they reflect the crosslinking
degree of hydrogels. The gel content of CS-TA hydrogels was investigated based on the dry gel
weight before and after extraction. As shown in Table 1, there is a significant difference in gel
content of hydrogels by varying the polymer concentrations. For example, when the po lymer
concentrations increased from 2.5 to 10 wt-%, the gel content increased from 19% to 74%. This
indicates that more polymer chains involve in the hydrogel and network formation is more
efficient at higher polymer concentrations. Besides, an increase tendency for the gel contents
could be seen when increasing the HRP concentration. For instance, at a constant polymer
concentration of 5 wt-%, the hydrogel prepared at an HRP concentration of 1.88 U mL-1
displayed higher gel content than that of 0.94 U·mL-1 (25% vs. 35%, P<0.05). Further increase of
the HRP concentration from 1.88 to 3.75 U·mL-1 gave rise to comparable gel content values
(P>0.05). However, H2 O2 had a reverse influence on the gel content. When increasing the H2 O2
concentrations from 0.025 to 0.1 wt-%, the gel content was decreased from 34% to 20%. As
mentioned above, excessive H2 O 2 may lead to inactivation of HRP and thus decreasing the
possibility of crosslinking of polymer chains.
The swelling tests of CS-TA hydrogels were performed by monitoring the wet gel weight
after adding saline on top of the gels. The swelling behavior of the gels as a function of
incubation time was illustrated in Figure 1. Keeping the same H2 O2 concentration of 0.05 wt-%,
the swelling ratio of CS-TA hydrogel with an HRP concentration of 0.94 U·mL-1 increased with
increasing incubation time and reached the plateau at day 5. The ratios of those hydrogels with
higher HRP concentrations however retained constant. Besides, the equilibrium swelling value
(at day 5) increased with decreasing HRP concentration and increasing H2 O 2 concentration
(Figure 1b). Taken the results from gel content and swelling ratio together, it can be concluded
that higher polymer and HRP concentration and lower H2 O2 concentration would lead to a higher
degree of crosslinking of the hydrogels. As was reported previously, crosslinking degree was a
critical factor in hydrogel properties that influencing the cell behavior and matrix production
when they were applied as bio-scaffolds for tissue engineering applications. For example,
Burdick et al. examined a group of photo-crosslinked hyaluronic acid hydrogels as 3D
microenvironment for supporting the chondrogenesis of mesenchymal stem cells (MSCs) and
they found that increased crosslinking degree could promote hypertrophic differentiation of
chondrogenically- induced MSCs, causing more matrix calcification in vitro [34]. Besides, an
8
overall decrease in cartilage matrix content and more restricted matrix distribution were observed
in the hydrogels with higher crosslinking degree. Another study reported by Kawakami et al. on
injectable, alginate-based hydrogels showed that increased crosslinking degree of phenol groups
resulted in a higher cellular adhesiveness due to the increased hydrophobicity. In our study,
crosslinking degree could be easily adjusted by varying the polymer, HRP and H 2 O2
concentrations, showing that the CS-TA may provide an optimal microenvironment for
chondrocytes encapsulation and would be favorable for cell proliferation and matrix production.
3.3 Rheological analysis
Previous studies elucidated that the HRP-catalyzed crosslinking reaction appeared to affect
cellular responses by varying the mechanical stiffness of the hydrogels. [16] As such, mechanical
properties of CS-TA hydrogels are important parameters for tissue engineering applications and
were investigated by oscillatory rheology experiments at 37 °C. The kinetics of the gelation was
followed by monitoring the storage modulus (G’) and loss modulus (G”) of the hydrogels in time.
The precursor solutions of CS-TA hydrogels were prepared at polymer and H2 O 2 concentrations
of 10 wt-% and 0.05 wt-%, respectively. Figure 2 shows the G’ and G” of CS-TA hydrogels at
different HRP concentrations. Generally, the G’ and G” considerably increased at the beginning
and then leveled off, indicating the completion of the crosslinking process. The G’ plateau value
was reached at about 10 min for the hydrogel prepared at an HRP concentration of 1.88 U mL -1 ,
while at about 1 h for that prepared at 0.94 U mL-1 . Moreover, G’ plateau values of CS-TA
hydrogels increased from 440 to 1900 Pa when increasing the HRP concentrations from 0.94 to
1.88 U mL-1 . This pronounced increase in the G’ may be due to increased crosslinking degree for
the hydrogel at a higher HRP concentration. For the hydrogels prepared at an HRP concentration
of 3.75 U mL-1 , it is hard to measure the modulus because the gelation was too fast and the gel
has already formed before the measuring plate came down. However, it can be deduced that the
hydrogels prepared at an HRP concentration of 3.75 U mL-1 would have a higher storage
modulus than those at 0.94 and 1.88 U mL-1 .
3.4 Morphology of hydrogels
To examine the surface and interior morphology of the hydrogels, SEM characterization of
dried hydrogels were conducted. The hydrogels with 5 and 10 wt-% polymer precursors at a
9
fixed HRP concentration of 3.75 U mL-1 and a H2 O2 concentration of 0.05 wt-% were take as
typical examples due to their comparable gelation time and different gel content. As shown in
Figure 3, all the hydrogels exhibit porous three-dimensional structures and the pores were wellinterconnected to each other. Moreover, it was found that the pore sizes were decreased with
increasing polymer concentrations. For example, the average pore size of 5 wt-% gels was
around 100 μm while the 10 wt-% gels showed a pore size of 50-70 μm and a more condensed
microstructure. The change of pore size is mainly due to increased crosslinking de nsity when the
polymer concentrations are increased, which was in accordance with the results of gel contents
(Table 1).
3.5 Model drug release from CS-TA hydrogels
Previous studies demonstrated that proper protein delivery may facilitate efficient tissue
regeneration [34, 35]. To examine the possibility of these hydrogels for protein permeation,
methylene blue (MB) as a water-soluble molecule was used as a model and loaded into the
hydrogels during the gelation process. For comparison, 5 wt-% and 10 wt-% CS-TA hydrogels
were prepared at HRP and H2 O2 concentrations of 3.75 U mL-1 and 0.05 wt-%, respectively. In
vitro release profiles of MB were studied in PBS buffer (pH 7.4) at 37 °C and are shown in
Figure 4a. For 10 wt-% gels, the release was comparatively slow and about 70% of MB was
released after 250 h. In contrast, MB was completely released from the 10 wt-% hydrogels
within the same time. This lower release rate could be due to a smaller pore size of the CS-TA
hydrogel (Figure 3). It was worth pointing out that the MB release from these hydrogels is
proportional to the square root of time (Figure 4b), indicating that the release kinetics was close
to first order. Therefore, MB release profile might be a typical diffusion-controlled release
behavior from a well- swollen hydrogel. Taken together, these data indicated that HRPcrosslinked CS-TA hydrogel can mediate sustained release under physiological cond itions,
implying good nutrient diffusion for tissue engineering applications.
3.6 Chondrocyte culture
Viability of chondrocytes encapsulated in hydrogels was visualized by Live/Dead assay. As
shown in Figure 5, most of the chondrocytes maintained survival in the CS-TA hydrogels 21-day
after encapsulation in comparison with agarose gels as a positive co ntrol. The ability of the
10
hydrogels to function as a bio-scaffold for cartilage tissue regeneration was investigated by
immunofluorescent staining of collagen type II, one of the major components in e xtracellular
matrix of cartilage. Fluorescence microscopy showed collagen type II formation by chondrocytes
incubated in CS-TA hydrogels after 21 days. Moreover, collagen type II mainly located around
the cells, which were stained blue with DAPI (Figure 6a&b). For the CS-TA hydrogels without
cells, no collagen type II was detected (Figure 6c). The morphology of the chondrocytes was
investigated by SEM. The chondrocytes retained a round shape at day 21 (Figure 6d).
Importantly, chondrocytes were surrounded by a fibrous perice llular matrix on the surface of the
cross-section of CS-TA hydrogels, which confirmed the production of cartilageous tissues.
4. CONCLUSIONS
HRP- mediated crosslinking is an effective method to prepare porous hydrogels from chondroitin
sulfate-tyramine conjugates. The gelation time of the gels may be regulated from about 26 to 310
s, depending on the concentrations of the conjugate, HRP and H2 O2 . Relatively high gel content
up to 74% can be obtained for the hydrogels at a higher polymer and HRP concentration. When
increasing HRP concentrations from 0.94 to 1.88 U·mL-1 , the storage modulus of CS-TA
hydrogels may augment from 440 to 1900 Pa. These hydrogels allow for sufficient diffusion of
nutrients and thus chondrocytes encapsulated in the hydrogels can maintain viable and produce
cartilageous tissues such as collagen type II after 21-day incubation. These results indicate that
HRP- mediated porous CS-TA hydrogels are promising for cartilage tissue engineering.
5. ACKNOWLEDGEMENTS
This work was supported by the grants of the Shanghai Municipal Natural Science Foundation
(13ZR1443600).
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Table 1. Gelation times and gel contents of chondroitin sulfate hydrogels via HRP/H 2 O2 -mediated
crosslinking
Poly mer conc.
HRP conc.
(wt-%)
(U·mL-1 )
H2 O2 conc.
(wt-%)
Gelat ion time
(s)
Gel content
(%)
2.5
3.75
0.05
61.3±6.6
19±1.4
5
3.75
0.05
37.5±1.1
35±0.8
10
3.75
0.05
33.0±1.0
74±0.9
5
0.94
0.05
310.7±5.2
24.8±1.5
5
1.88
0.05
106.4±8.4
34.2±0.8
5
3.75
0.025
26.3±0.6
26.2±2.2
5
3.75
0.1
77.1±6.1
20±1.0
13
Figure captions:
Fig. 1. Swelling ratios of 10 wt-% CS-TA hydrogels as a function of HRP (a) or H2O2 (b) concentration,
respectively, when keeping the same 0.05wt-% of H2O2 or 0.94 U·mL-1 of HRP. (*P<0.05; **P<0.005)
Fig. 2. Storage (G’) and loss (G”) modulus of 10 wt-% CS-TA hydrogels prepared at an HRP
concentration of (a) 0.94 and (b) 1.88 U mL-1 .
Fig. 3. SEM images of CS-TA hydrogels prepared at a polymer concentration of (a-c) 5 wt-% and (d-f) 10
wt-% and observed at 100×, 200× and 500× magnitude, respectively.
Fig. 4. Cumulative release of MB from 5wt % and 10 wt-% CS-TA hydrogels prepared as a function of (a)
time or (b) of the square root of time. CS-TA hydrogels prepared at an HRP concentration of 3.75 U mL -1
and a H2 O2 concentration of 0.05 wt-%.
Fig. 5. Live/Dead assay of chondrocytes incorporated into (a) agarose and (b) 10 wt-% CS-TA hydrogels
after incubation for 21 days. Scale bar: 100 μm.
Fig. 6. Immunofluorescent staining of collagen type II of 10 wt-% CS-TA hydrogels after incubation for
21 days: (a) Merged image of collagen type II (stained green) and cells (nuclei stained blue); (b)
chondrocytes inside hydrogels (nuclei stained blue); (c) hydrogels without cells; (a-c) Scale bar: 100 μm.
(d) SEM image of chondrocytes incorporated in the CS-TA hydrogels at day 21. Scale bar: 10 μm.
14
Fig. 1. Swelling ratios of 10 wt-% CS-TA hydrogels as a function of HRP (a) or H2 O2 (b) concentration ,
respectively, when keeping the same 0.05wt-% of H2O2 or 0.94 U·mL-1 of HRP. (*P<0.05; **P<0.005)
15
Fig. 2. Storage (G’) and loss (G”) modulus of 10 wt-% CS-TA hydrogels prepared at an HRP
concentration of (a) 0.94 and (b) 1.88 U mL-1 .
16
Fig. 3. SEM images of CS-TA hydrogels prepared at a polymer concentration of (a-c) 5 wt-% and (d-f) 10
wt-% and observed at 100×, 200× and 300× magnitude, respectively.
17
Fig. 4. Cumulative release of MB from 5wt % and 10 wt-% CS-TA hydrogels prepared as a function of (a)
time or (b) of the square root of time. CS-TA hydrogels prepared at an HRP concentration of 3.75 U mL -1
and a H2 O2 concentration of 0.05 wt-%.
18
Fig. 5. Live/Dead assay of chondrocytes incorporated into (a) agarose and (b) 10 wt-% CS-TA hydrogels
after incubation for 21 days. Scale bar: 100 μm.
19
Fig. 6. Immunofluorescent staining of collagen type II of 10 wt-% CS-TA hydrogels after incubation for
21 days: (a) Merged image of collagen type II (stained green) and cells (stained blue of nucleus); (b)
chondrocytes inside hydrogels (nuclei stained blue); (c) hydrogels without cells; (a-c) Scale bar: 100 μm.
(d) SEM image of chondrocytes incorporated in the CS-TA hydrogels at day 21. Scale bar: 10 μm.
20