Materials Science & Engineering C 115 (2020) 111096

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Materials Science & Engineering C

journal homepage: www.elsevier.com/locate/msec

Injectable anti-inflammatory hyaluronic acid hydrogel for osteoarthritic T

cartilage repair
Yinji Jina,1, Rachel H. Koha,1, Su-Hwan Kimb,c, Kyung Min Kimb, G. Kate Parkc,
⁎
Nathaniel S. Hwanga,b,c,
a
School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea
b
Interdisciplinary Program in Bioengineering, Seoul National University, Seoul 08826, Republic of Korea
c
Institute of Engineering Research, Seoul National University, Seoul 08826, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Osteoarthritis (OA) is one of the most common cartilage disorder that results from breakdown of joint cartilage
Osteoarthritis and underlying bone tissues. Once OA is induced in the joint, subsequent immune reaction leads to chronic
Cartilage regeneration inflammation that results in the progression of OA and deconstruction of the cartilage. In this study, an injectable
Epigallocatechin-3-gallate hydrogel with epigallocatechin-3-gallate (EGCG) is introduced to control inflammation and enhance cartilage
Anti-inflammation
regeneration. EGCG has intrinsic properties that can modulate inflammation and scavenge radical species.
Injectable hydrogel
Hyaluronic acid (HA), as a major component of the cartilage ECM, is commonly used for cartilage tissue en-
gineering. In this study, EGCG was combined with tyramine-conjugated HA and gelatin to create a composite
hydrogel at an optimized concentration of 50 μM EGCG and 5% w/v HA. The composite hydrogel provided
protection to chondrocytes against the pro-inflammatory factor, IL-1β. Additionally, the composite hydrogel led
to chondrogenic regeneration in vitro. Histological analysis in vivo showed that EGCG-HA/Gelatin hybrid hy-
drogel minimized cartilage loss in surgically induced OA model. This study demonstrates that inflammation-
modulating HA-based hydrogel may provide a therapeutic option for OA treatment.

1. Introduction to have therapeutic effects such as anti-cancer, anti-oxidant, and anti-

Osteoarthritis (OA) is known as the most common chronic disease of
the cartilage. Risk factors include age, obesity and traumatic injury,
which lead to excessive or altered joint loading. As the cartilage breaks
down due to wear-and-tear and/or mechanical stress, it causes swelling,
pain and inflammation, leading to extracellular matrix (ECM) loss [1].
The imbalance between anabolic and catabolic processes destroys
normal cartilage ECM turnover and homeostasis [2,3]. The main cata-
bolic cytokine involved in OA pathogenesis is interleukin-1 beta (IL-
1β). OA patients have elevated levels of IL-1β in the synovium and
cartilage [4]. IL-1β significantly affects the metabolism of the chon-
drocytes and its ECM [5]. A previous study by Stove et al. demonstrated
that IL-1β blocks the synthesis of ECM components such as type II
collagen (COL2) and aggrecan (ACAN) [6]. Thus, anti-inflammatory
medications deserve attention in therapeutic strategies to delay OA
progression.
Nowadays, many researchers have focused their attention on poly-
phenol that is a pharmaceutical-grade nutrient. Polyphenols are known
inflammation for many diseases [7–10]. Found in green tea, epigallo-
catechin-3-gallate (EGCG) is one of the polyphenols that can scavenge
radical oxygen species and prevent inflammation-induced oxidative
damage. Furthermore, EGCG has been shown to directly suppress in-
flammation in various cell types, including vascular endothelial cells,
fibroblasts, and chondrocytes [11,12]. EGCG has been shown to directly
inhibit upstream targets of pro-inflammatory signaling cascades by in-
hibiting phosphorylation of p38 and JNK, and limiting nuclear trans-
location of NF-kB [8,13,14]. Over the last decade, many studies have
demonstrated inflammation-regulating and chondroprotective effects of
EGCG on monolayer-cultured chondrocytes [12,15,16]. EGCG has been
shown to inhibit metalloproteinases (MMP13) cyclooxygenase-2 (COX-
2), and nuclear factor-kappa B (NF-kB) in IL-1-stimulated chondrocytes
[17–19].
Current treatments are limited to mitigation of OA symptoms by
taking some analgesics and nonsteroidal anti-inflammatory drugs
(NSAIDs), or intra-articular injection of steroid drugs [20]. These
treatments, when carried out long-term, have detrimental side effects

⁎
Corresponding author at: School of Chemical and Biological Engineering, Seoul National University, Republic of Korea.
E-mail address: nshwang@snu.ac.kr (N.S. Hwang).
1
These two authors contributed equally to this paper.

https://doi.org/10.1016/j.msec.2020.111096
Received 8 January 2020; Received in revised form 23 April 2020; Accepted 12 May 2020
Available online 15 May 2020
0928-4931/ © 2020 Elsevier B.V. All rights reserved.
Y. Jin, et al.
on gastrointestinal and cardiovascular systems. Therefore, OA therapy
still needs development. The on-site delivery of drugs via controlled
releasing systems is a new breakthrough. For the delivery system,
hyaluronic acid (HA)-based hydrogel has been established as a useful
vehicle for drug delivery [21]. HA is a substance that occurs naturally in
the joints and is known as a major component of GAG. HA is widely
used in many tissue engineering approaches because of its biocompat-
ibility, biodegradability and low immunogenicity. Since the elimination
of injected HA occurs rapidly, HA is usually crosslinked to last longer
[22,23]. So, HA-based hydrogels are widely studied for OA treatment.
However, HA-based hydrogels have limitation cell adhesion [24]. Ge-
latin, the production of collagen hydrolysis, contains many Arg-Gly-Asp
(RGD) residues that can provide a good cell adhesive and proliferative
environment [25,26]. In this study, we fabricated hybrid hydrogels that
consist of HA and gelatin to mimic the cartilage ECM. For HA and ge-
latin hydrogel formation, HA was conjugated with tyramine (HA_t) to
crosslink via oxidative reaction of tyramine residues [27]. Recently, we
have reported enzyme-based hydrogel crosslinking systems [27,28].
Enzyme-mediated HA_t hydrogel has advantages, including rapid
crosslinking, non-cytotoxicity, and injectability.
In this study, we investigated EGCG-loaded HA/gelatin (HTG-E)
hybrid hydrogel for OA therapy. We first studied EGCG and HA/gelatin
(HTG) hybrid hydrogel, independently. The evaluation of the anti-in-
flammatory effect of EGCG was performed by OA-like chondrocyte
laden hydrogel. The chondrogenic potential of HTG hydrogel was in-
vestigated by cartilage formation for 3 weeks in vitro. Finally, we
combined the composite hydrogel with EGCG to provide a suitable 3-
dimensional (3D) environment for the growth of inflammation-induced
chondrocyte in vitro. The in vivo evaluation of EGCG-loaded composite
hydrogel demonstrated a synergetic anti-inflammatory and chon-
droprotective effects on OA therapy. We have established a novel
method for OA therapy with inflammation-modulating HA and gelatin-
based hybrid hydrogels.
2. Materials and methods
2.1. Porcine articular chondrocyte isolation
Articular cartilage was harvested from femoral condyle of domestic
pigs, which were purchased from a local slaughterhouse. Cartilage
tissue was minced into 1mm3 pieces and digested with 0.2% (w/v) type
II collagenase for 18 h on an orbital shaker at 37 °C. Subsequently, the
digested cell suspension was strained by 70 μm cell strainer and cen-
trifuged. The cell pellet was washed 2–3 times with PBS and cultured in
high glucose Dulbecco's modified Eagles' medium (Hyclone), supple-
mented with 10% (v/v) fetal bovine serum (Corning), 1% (v/v) peni-
cillin/streptomycin (Gibco), 10 mM HEPES (Gibco), 0.1 mM NEAA
(Gibco), 8 μg mL−1 proline (Sigma-Aldrich), and 50 μg mL−1 ascorbic
acid (Sigma-Aldrich).
2.2. Tyramine-conjugated HA synthesis
HA_tyramine (HA_t) synthesis was performed by EDC/NHS coupling
protocol which was demonstrated by previous work [27]. Briefly, so-
dium hyaluronate (Lifecore Biomedical) was dissolved into 0.2 M 2-(N-
morpholino) ethanesulfonic acid (MES) buffered saline (Thermo Fisher
Scientific) to make 1% (w/v) HA solution. While stirring, N-hydro-
xysulfosuccinimide (sulfo-NHS; Sigma-Aldrich) and N-(3-dimethylami-
nopropyl)-N-ethylcarbodiimide (EDC; Sigma-Aldrich) were added to
the HA solution. Tyramine hydrochloride (Sigma-Aldrich) was added at
1:1 mole ratio to HA. The reaction was performed overnight, and the
product was dialyzed against distilled water for 3 days. Synthesized
HA_t was lyophilized for later use.
HA_t was characterized via 1H NMR. Peaks at 7.3 and 7.5 ppm are
due to aromatic protons of tyramine. The tyramine content in HA_t was
determined to be 13.38%.
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Materials Science & Engineering C 115 (2020) 111096
2.3. Hydrogel preparation
Synthesized HA_t was dissolved in PBS at 4% or 10% (w/v). In ad-
dition, gelatin was dissolved in PBS at 10% (w/v). Then HA_t and ge-
latin solutions were sterilized by 0.22 μm syringe filter and mixed at 1:1
ratio to make 2% HTG (2% HA_t and 5% gelatin) and 5% HTG (5% HA_t
and 5% gelatin) hydrogel precursors. 0% HTG hydrogel precursor was
composed of 5% (w/v) gelatin solution only (without HA_t). Upon
precursor solution preparation, 6 μM of tyrosinase (SA_Ty) was added
into the precursor solution to induce enzymatic cross-linking as pre-
viously described [28]. HTG hydrogel containing EGCG (HTG-E) were
fabricated by physical mixing of EGCG (0–100 μM) into HTG hydrogel
precursor solution prior to addition of tyrosinase.
2.4. Rheological test
For rheological measurements, hydrogels were fabricated in 8 mm
diameter and 2 mm thickness cylindrical PDMS mold. The test was
carried out in Anton Paar Korea's demo lab using a rheometer (MCR
302, Anton-Paar). Amplitude sweep was performed with increasing
shear strain (%) from 0.1 to 10%. Frequency sweep was carried out at a
constant strain of 1% from 0.1 to 10 Hz. To determine the gelation time,
time-dependent storage (G′) and loss (G″) moduli at 1% shear strain and
10 Hz measured upon adding SA_Ty. The time at which G′ crosses over
G″ indicates sol-to-gel transition, which corresponds to gelation.
2.5. FT-IR analysis and swelling ratio analysis
For FT-IR analysis, prepared HTG hydrogels and HTG-E hydrogels
were lyophilized and analyzed via FT-IR Spectrometer (PerkinElmer).
For measuring the swelling ratio (Q), hydrogels (n = 3) were immersed
in PBS for 24 h. The swollen (Wwet) and dry (Wdry) weights of the hy-
drogels were measured. The swelling ratio was calculated by the fol-
lowing Eq. (1).
Wwet
Q=
Wdry (1)
2.6. Enzymatic degradation test
Hydrogels were prepared at the same volume (50 μL/construct;
n = 3/time point). 10 units of hyaluronidase (Worthington) and col-
lagenase II (Worthington) were used independently for enzymatic de-
gradation test. Enzyme solution was changed daily. Each time point
samples were collected and lyophilized, and the dry weight was mea-
sured. The weight loss of the dry weight of hydrogel was calculated.
2.7. Cell encapsulation
Articular chondrocytes (Passage 1–2) were prepared for 3D cell
encapsulation. Cells were initially suspended with HTG hydrogel pre-
cursor solution containing EGCG (HTG-E). Then, SA_Ty was added for
hydrogel formation and cell encapsulation. The final cell concentration
in the hydrogel was 2 × 107 cells mL−1. After gelation, cell-laden
hydrogels were transferred to 24-well plate and supplied with DMEM,
supplemented with 1% penicillin/streptomycin, 1% ITS + Premix (BD
Biosciences), 100 nM dexamethasone (Sigma-Aldrich), 50 mg mL−1
ascorbic acid-2-phosphate (Sigma-Aldrich), and 10 ng mL−1 TGF-β3
(Life technologies). The media was changed every 2 days. Encapsulated
chondrocytes were treated with 10 ng mL−1 IL-1β to induce OA-like in
vitro condition.
2.8. Cytotoxicity evaluation
After cell encapsulation, cell cytotoxicity was measured by live/
dead assay kit (Invitrogen), according to the manufacturer's instruction.
Y. Jin, et al. Materials Science & Engineering C 115 (2020) 111096

Table 1 2.12. Histological analysis

Primer list.
Primer sequences (5′-3′) The in vitro hydrogel samples were collected after 3 weeks of cul-
ture, and mice with intra-articular injection were sacrificed 4 weeks
GAPDH Forward TCA GCT GCT GGG GAG TCA CA post-injection. All samples were fixed with 4% PFA at 4 °C for 24 h.
Reverse CCT AAG CCC CTC CCC TTC TT
Mice joint samples were decalcified in 0.5 M
IL-1β Forward TCT CTG GAC CCA AAG GAG GG
Reverse ACG GCT GCT TTC ACG GGT GA
Ethylenediaminetetraacetic acid (Sigma) for a week. Fixed cell-laden
TNF-a Forward AGC TGG TGG TGC CGA CAG AT hydrogels and mice joint samples were then embedded in paraffin
Reverse TGC GAT GCG GCT GAT GGT GT blocks and sectioned into 3-μm thickness slides. For histological
MMP13 Forward CCT GTG CTC CTG CCA TTT GG staining analysis, the sections were deparaffinized and rehydrated. For
Reverse GAA TGG GCA GCT CCA TGG CT
H&E staining, sections were stained with hematoxylin (Ricca Chemical
ADAMTS5 Forward CGC TCC CTG GCT GTC TTT GA
Reverse TCA AAG TGT AGC GCG CGT GC Company) for 5 min and Eosin-Y (Richard-Allan Scientific) for 1 min,
COL2 Forward TAG AGA GGT TTC CTG GGC CG followed by dehydration in graded series of ethanol. For Safranin-O
Reverse AGG AGG ACG CTG GAA CAG AG staining, the sections were dipped in 0.1% (w/v) Safranin-O solution
SOX9 Forward CAG TCC CAG CGA ACG CAC AT
(ScholAR Chemistry) for 5 min, followed by dehydration in ethanol.
Reverse TGC TGC TGC TGC TCG CTG TA
ACAN Forward CTG CCC CGA AAC ATC ACC GA
Types I, II and X collagen immunofluorescence staining was per-
Reverse GTA AAG GGC TCC TCA GGC TC formed on sectioned slices of in vivo samples. Deparaffinized sections
were permeabilized in 0.1% (v/v) Triton-X (Sigma), and blocked with
10% (v/v) normal goat serum for 45 min. Sections were incubated with
Cell-laden hydrogels were stained for 20 min and washed with PBS. anti-collagen II (ab34712, Abcam; 1:100 dilution), anti-collagen X
Live/dead images were captured via LSM 720 confocal microscope (ab58632, Abcam; 1:100 dilution) or anti-collagen I (ab3471, Abcam;
(Zeiss) or EVOS FL Imaging System AMF4300 (Life Technologies). Cell 1:100 dilution) at 4 °C for overnight. After washing thrice with 0.1%
viability was quantified via ImageJ software. BSA/PBS, sections were incubated with secondary antibodies (Goat
anti-rabbit Alexa-Fluor 546 or 488; Life Technologies). The nuclei were
stained with DAPI for 5 min at room temperature. Imaging was carried
2.9. Real-time PCR analysis out using EVOS FL Imaging System AMF4300 (Life Technologies).
ImageJ was used to quantify the average cartilage thickness from
Cell-laden hydrogels (n = 3) were collected on week 3 for gene Safranin-O staining and the mean fluorescence intensity of immuno-
expression analysis. Samples were ground within TRIzol reagent (Life fluorescence stainings.
Technologies) and total mRNA was extracted. The RNA concentration
was measured using Infinite 200 PRO NanoQuant Microplate Readers 2.13. Statistical analysis
(TECAN). 1 μg of RNA was used for cDNA synthesis by M-MLV cDNA
Synthesis Kit (Enzynomics), according to the manufacturer's instruc- All data are presented as mean ± standard deviation (SD).
tion. Real-time PCR was performed using SYBR Green PCR Master Mix Statistical significance between groups was determined by one-way
(Enzynomics) and ABI StepOnePlus™ Real-Time RCR system (Applied ANOVA with *p < 0.05, **p < 0.01, ***p < 0.005.
Biosystems). Primer sequence of glyceraldehyde 3-phosphate dehy-
drogenase (GAPDH, housekeeping gene), type II collagen (COL2), Sex 3. Results
determining region Y box 9 (SOX 9), aggrecan (ACAN), Tumor necrosis
factor-alpha (TNF-α), interleukin 1 beta (IL-1β), Matrix metalloproteinase 3.1. Effect of EGCG on 3D-encapsulated chondrocytes
13 (MMP13) and A disintegrin and metalloproteinase with thrombospondin
motifs 5 (ADAMTS5) are listed in Table 1. The relative gene expression EGCG is a polyphenolic bioactive compound isolated from green tea
was calculated by −2ΔΔCt method. extracts [30]. Many studies have demonstrated the therapeutic effects
of EGCG on inflammatory diseases [31,32]. However, previous studies
that claim the therapeutic potential of EGCG were limited to 2D cell
2.10. Biochemical assay culture. In this study, we aimed to fabricate immune-modulating hy-
drogel composed of EGCG and apply this hydrogel to OA disease model
For biochemical analysis, cell-laden hydrogels (n = 3) were di- for functional recovery. EGCG-incorporated hydrogels were fabricated
gested using papain solution (Worthington) for 16 h at 60 °C. DNA by introducing various amounts of EGCG into the mixture of HA_t and
content was quantified by Quant-iT™ Pico-Green®dsDNA assay kit gelatin. We have previously reported the enzyme kinetics of SA_Ty for
(Invitrogen) according to the manufacturer's instruction. sGAG accu- facilitated enzyme-based crosslinking of phenolic compounds [27].
mulation was measured by dimethylmethylene blue (DMMB) spectro- Therefore, a combination of HA_t, gelatin and EGCG can be crosslinked
photometric assay at A525. For total collagen content, the hydro- to form a hydrogel in a rapid manner by SA_Ty. EGCG-loaded HA_t/
xyproline amount was measured by chloramine-T colorimetric assay. gelatin (HTG-E) hydrogels were examined for their in vitro anti-in-
flammatory responses and subsequently, applied to murine OA model
to evaluate therapeutic efficacy (Fig. 1).
2.11. Intra-articular injection In order to optimize EGCG concentration, we examined cytotoxicity
and anti-inflammatory effects of EGCG on OA chondrocyte in a 3D
Twenty male Balb/c mice (10-week-old) were used for the in vivo environment. Chondrocytes (2 × 107 cells/construct, 50 μL/construct)
experiment following the protocols approved by the Institutional were encapsulated in 5% (w/v) gelatin hydrogel crosslinked with
Animal Care and Use Committee (IACUC) of Seoul National University. SA_Ty, containing various concentrations of EGCG (0–100 μM).
The mice were randomly divided into 5 groups (n = 4/group): normal, Encapsulated chondrocytes were then treated with 10 ng mL−1 IL-1β
PBS, EGCG 50 μM, 5% HTG and 5% HTG-E. OA induction surgery was throughout the entire culture period to re-create an OA-like condition in
performed by anterior cruciate ligament transection (ACLT) [29]. A vitro. After 1 day of encapsulation, live/dead staining showed that
week after surgery, 5 μL of PBS, 50 μM EGCG, 5% HTG or 5% HTG-E EGCG did not cause significant cytotoxicity in 3D culture systems
were injected bilaterally into the knee. After 4 weeks post-injection, the (Fig. 2A, B). OA chondrocytes maintained above 90% viability even at
knee joint samples were collected for histological analysis. 100 μM EGCG concentration. Interestingly, in the monolayer culture

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Y. Jin, et al. Materials Science & Engineering C 115 (2020) 111096

Fig. 1. Schematic illustration of evaluating inflammation-modulatory function of HA-based hydrogel.

system, EGCG concentration above 20 μM showed cytotoxicity, which
may be attributed to its low stability in free-standing form (Supple-
mentary Fig. 3A). The polyphenol can undergo fast degradation in
aqueous solution via auto-oxidation or epimerization, and the de-
gradation products may have damaging effects on cells [33]. These
results suggest that SA_Ty successfully incorporated EGCG into the
hydrogel as crosslinkers and allowed limited free-standing EGCG in the
medium. We observed that EGCG did not cause any cytotoxicity to
chondrocyte when incorporated into the hydrogel. Therefore, hydrogels
incorporated with EGCG can support cell survival for long-term culti-
vation.
The anti-inflammatory effect of EGCG on monolayer-expanded
chondrocytes was reported in several studies [12,16,34]. These studies
demonstrated that EGCG suppressed the expressions of pro-in-
flammatory genes such as COX2, MMP1, MMP13, and ADMTS5 [35,36].
We evaluated whether incorporation of EGCG into the hydrogel would
exhibit similar effects on OA chondrocytes. IL-1β is the main pro-
inflammatory cytokine in synovial fluid that causes inflammatory
symptoms in OA. To mimic OA inflammation, chondrocytes were en-
capsulated in the gelatin-based hydrogel and maintained with culture
media containing IL-1β (10 ng mL−1). After 2 days, cell-laden con-
structs were collected for real-time PCR analysis (Fig. 2C). The in-
flammatory and catabolic genes were all increased after IL-1β stimu-
lation. However, EGCG-dependent downregulation of inflammatory
response genes was observed. In particular, gelatin hydrogel with >
30 μM EGCG concentration resulted in significant downregulation of
IL-1β, TNF-α, MMP13, and ADAMTS5. Gene expression of IL-1β and
TNF-α showed similar levels as that of the control group with the 30 μM
EGCG incorporation. In addition, MMP13 and ADAMTS5 gene expres-
sion levels were downregulated to that of the control group with 50 μM
EGCG incorporation. It demonstrated that EGCG has the ability to
regulate inflammation-induced by IL-1β in the hydrogels. Based on real-
time PCR analysis, we selected 50 μM EGCG for further in vitro and in
vivo applications.

Fig. 2. EGCG effect on 3D encapsulated OA chondrocytes. (A) Live/dead assay for dose-dependent EGCG treatment; Live cell was stained with by Calcein AM (green)
and dead cell was stained by Ethidium homodimer-1 (red). Scale bar = 100 μm. (B) Cell viability assay was measured by AlamarBlue™ reagent. (C) Real-time PCR
analysis of IL-1β, TNF-α, MMP13 and ADAMTS5 genes (n = 3), RFI = relative fold induction. *p < 0.05, **p < 0.01, ***p < 0.005. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)

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Y. Jin, et al. Materials Science & Engineering C 115 (2020) 111096

Fig. 3. Characterization of HTG hydrogel. (A) Gross images of 0%, 2% and 5% HTG hydrogels. The final concentration of each hydrogel was composed of 0%, 2%, 5%
(w/v) HA_t and 5% (w/v) gelatin. (B) SEM images of internal pore structure at 100× magnification. (C) Rheological analysis of HTG hydrogels to determine gelation
time. G′ = storage modulus and G″ = loss modulus. (D) Rheological analysis of HTG to determine viscoelastic properties of pre-formed hydrogels. (E) Swelling ratio
(n = 3). (F) Enzymatic degradation of HTG hydrogels using 10 U/mL of each enzyme (n = 3). *p < 0.05, **p < 0.01, ***p < 0.005. (For interpretation of the
references to color in this figure, the reader is referred to the web version of this article.)

3.2. Characterization of HTG hydrogel and its chondrogenic effects
It is well known that HA promotes chondrogenesis because HA is a
major component of the cartilage ECM [37]. As a natural polymer, non-
crosslinked, unmodified HA has limitations in use for cartilage tissue
engineering, such as rapid clearance and low mechanical properties.
Several approaches to crosslink HA have been studied [38]. In a pre-
vious study, our lab reported the fabrication of HA-based injectable
hydrogel [27]. Briefly, tyramine hydrochloride was conjugated onto HA
backbone via EDC/NHS chemistry. Then, tyramine-conjugated
HA(HA_t) was mixed with gelatin and crosslinked via tyrosinase-
mediated oxidative reaction. This enzyme-based crosslinking is rela-
tively rapid and can be applied as an injectable system. 2% or 5% (w/v)
HA_t was mixed with 5% (w/v) gelatin to produce 2% or 5% HTG hy-
drogels. We used gelatin without HA_t as a control hydrogel (0% HTG).
Since gelatin itself has tyrosinase-reactive functional groups (i.e. amine
and tyrosine), HA/Gelatin hydrogel without HA_t showed brown color
(0% HTG), as a result of SA_Ty crosslinking. Additionally, HA/Gelatin
(HTG) hydrogels showed gradually darker brown color as HA_t con-
centration was increased (Fig. 3A). The rheological properties of HTG
hydrogels were evaluated by the rheometer (Fig. 3C & D). Gelation
time, which can be determined from the time point at which G′ exceeds
G″, was 420 s, 108 s and 132 s for 0%, 2% and 5% HTG, respectively. As
expected, gelation time was much faster for HA_t-containing groups
than 0% HTG hydrogel. However, 5% HTG hydrogel exhibited slightly
slower gelation time than 2% HTG hydrogel, presumably due to high
viscosity of 5% HTG precursor solution. Both amplitude and frequency
sweep exhibited HA_t concentration-dependent manner. 5% HTG hy-
drogel showed the highest storage modulus (G′). The swelling ratio
analysis showed reciprocal trends as the storage modulus G′ (Fig. 3E).
5% HTG hydrogel showed the lowest degree of water uptake. Both
rheology and swelling test results confirmed the highest crosslinking
degree and strength of 5% HTG hydrogel.
We also measured enzymatic degradation of hydrogels (Fig. 3F).
The hydrogels were exposed to hyaluronidase or collagenase
(10 U mL−1). After 2 weeks of hyaluronidase degradation, all groups
were degraded completely. However, in the collagenase degradation
test, all groups except 0% HTG hydrogel remained after 2 weeks. This
demonstrated that gelatin-related crosslinking undergoes rapid enzy-
matic degradation and HA_t-related crosslinking is strong so that the
degradation is not easy.
3.3. Evaluation of chondrogenic effects of HTG hydrogel
To evaluate whether HTG hydrogel can be applied to cartilage tissue
regeneration, porcine articular chondrocytes (2 × 107 cells/construct)
were encapsulated in HTG hydrogels and evaluated the tissue con-
structs after 3 weeks of culture. After 3 weeks of culture, constructs
were harvested and biochemical analysis was performed. We quantified
sulfated GAG amount by DMMB assay and collagen content by hydro-
xyproline content analysis. All the measurements were normalized to
the respective DNA amount. 0% HTG hydrogel on day 1 served as
control. As HA_t content increased, overall collagen and GAG contents
within the hydrogel also increased (Fig. 4A). Additionally, 5% HTG

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Y. Jin, et al. Materials Science & Engineering C 115 (2020) 111096

Fig. 4. Comparison of the chondrogenic properties of 2% and 5% HTG hydrogel through in vitro cell encapsulation. (A) Biochemical analysis of sGAG, collagen and
DNA content (n = 3). 0% HTG hydrogel collected at day 1 was served as control. (B) Histological analysis of H&E, Safranin-O and type II collagen im-
munohistochemistry. Scale bar = 200 μm (C) Real-time PCR results of COL2, SOX9, and ACAN genes (n = 3). 0% HTG hydrogel collected on day 1 served as control.
RFI = Relative fold induction. *p < 0.05.

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Y. Jin, et al.
exhibited the highest level of collagen compared to 0% and 2% HTG
groups. Histological analysis confirmed with biochemical analysis
(Fig. 4B). H&E staining showed reduced cellularity in 5% HTG com-
pared to the 2% HTG, confirming chondrocytes within 5% HTG pro-
duced more ECM. Additionally, Safranin-O staining showed intense red-
stained area in 5% HTG hydrogel compared to 2% HTG hydrogel as a
result of an increased in GAG accumulation within the hydrogel. Fi-
nally, type II collagen immunostaining showed that the cells in 5% HTG
hydrogel retained circular morphology and type II collagen was re-
tained within the cellular periphery. Cells in 2% HTG hydrogels were
spindle-shaped and elongated. Furthermore, counterstaining with DAPI
confirmed increased cellularity within 2% HTG hydrogel. Interestingly,
0% HTG (HA/Gelatin hydrogel without HA_t), resulted in complete
degradation within 5 days of culture. This may be due to the reduced
crosslinking of the hydrogel as a result of 0% HA_t along with active
cellular reorganization and cell-mediated degradation.
We also measured cartilage-specific gene expression levels of the
cell-laden HTG hydrogels. Real-time PCR was used to compare the level
of chondrogenic genes such as COL2, SOX9 and ACAN at week 3. The
data is shown in Fig. 4C. All gene expressions were normalized to 0%
HTG hydrogel collected on day 1. Real-time PCR data showed similar
trends as biochemical analysis. Chondrogenic gene levels of 5% HTG
hydrogel group were higher than those of other groups. We confirmed
that HTG composite hydrogel can promote chondrogenesis, with higher
chondrogenic property as HA content increases.
3.4. Synergetic effect of HTG hydrogel combined with EGCG (HTG-E)
To confirm the synergetic effect of EGCG and HTG hydrogel, we
encapsulated chondrocytes in 5% HTG hydrogels or 5% HTG-E hydro-
gels (5% HTG hydrogel with 50 μM EGCG) and treated with
10 ng mL−1 of IL-1β for 3 weeks to mimic inflammatory OA environ-
ment. EGCG was mixed with hydrogel precursors to evaluate its anti-
inflammatory effect. Fig. 5A showed histological analysis 5% HTG and
5% HTG-E cultured for 3 weeks. Safranin-O staining indicated more
GAG accumulation in 5% HTG-E group than 5% HTG group. Type II
collagen immunostaining results showed collagen production in the
pericellular area in both groups. However, the expression of type II
collagen protein was more prominent in 5% HTG-E hydrogel compared
to the EGCG-absent group. The presence of IL-1β leads to cartilage ECM
degradation in OA knee joint. This may lead to reduced syntheses of
ECM like GAG and type II collagen by chondrocyte in 5% HTG, which
were preserved in EGCG-containing hydrogel. EGCG not only has an
anti-inflammatory effect as shown in Fig. 2C but also a chon-
droprotective effect in long-term inflammation.
The chondrogenic and inflammatory gene expressions were eval-
uated by real-time PCR (Fig. 5B and C). As anticipated, inflammatory
gene expressions were down-regulated in the EGCG-containing group,
confirming EGCG's inflammatory effects in the HTG hydrogel in vitro.
The chondrogenic genes were upregulated in 5% HTG-E group, which is
consistent with the histological analysis.
3.5. In vivo OA treatment with HTG-E hydrogel in mouse OA model
EGCG with HTG hydrogel was intra-articularly administered to OA-
induced mice to determine the effect on OA progression. A week after
OA induction surgery, PBS, 50 μM EGCG, 5% HTG, and 5% HTG-E were
each injected into the knee. The knee joints were collected for histo-
logical analysis after 4 weeks (Fig. 6A). Safranin-O staining and type II
collagen immunostaining demonstrated that the PBS injection group
had very severely eroded and thinned cartilage and notable GAG and
type II collagen loss. Although less severe than the PBS group, 50 μM
EGCG and 5% HTG hydrogel groups showed some degree of cartilage
ECM loss and surface erosion compared to normal cartilage. When
EGCG and the hydrogel were injected together as in 5% HTG-E group,
GAG and collagen content were up to par with that of native cartilage.
7
Materials Science & Engineering C 115 (2020) 111096
Furthermore, in 5% HTG-E, cartilage surface and thickness (Fig. 6B)
were completely intact, showing no sign of wear and tear.
Types I and X collagen, which are absent in healthy articular car-
tilage, have been additionally examined to observe chondrocyte ded-
ifferentiation and hypertrophy in OA mouse knee joints. Elevated ex-
pression levels of collagen I and X were observed in PBS-injected OA
joints, while OA joints that received any form of treatment indicated
lower expression levels of these markers. In particular, the mean
fluorescence intensity of 5% HTG-E group had no significant difference
with that of normal group (Fig. 6C). These results indicate that the
combination of EGCG and HTG hydrogel retarded the progression of
OA, while each of the components alone also had mild beneficial ef-
fects.
4. Discussion
OA is a type of cartilage degenerative disease that results from the
breakdown of joint cartilage and underlying bone [39]. With OA pro-
gression, ECM components in cartilage degrade gradually as pro-in-
flammatory environment promotes ECM deconstruction. IL-1β is a main
pro-inflammatory cytokines in osteoarthritic joint synovium. Therefore,
the sequestration of pro-inflammatory cytokine like IL-1β may have
significant therapeutic benefits in preventing OA progression and re-
storing tissue homeostasis.
In recent years, several biomaterials or cell secretome-based ap-
proaches have attempted to modulate the immune response in a way
that can more effectively promote cartilage regeneration at the site of
injury [26,40–43]. These include the modulation in biomaterial surface
chemistry to influence protein adsorption. The selectivity of adsorbed
proteins on biomaterials may mediate the interactions with immune
cells, which may ultimately control immune cell activation. In addition,
a strategy of chemical crosslinking of natural ECM has been employed
to increase the stability of naturally derived biomaterials. Interestingly,
recent evidence supports the notion that types of crosslinking strategies
may impact the immune response. Recently, Brown et al. showed that
acellular ECM without chemical modification often results in M2
macrophage response while carbodiimide (CDI) cross-linked scaffolds
induce M1 response [44]. In our study, we employed tyrosinase-in-
duced crosslinking of tyramine-conjugated HA and gelatin. Tyrosinase
activity is involved in our body as a natural crosslinking process that
induces the accumulation of melanin. Our previous report has sug-
gested that SA_Ty based crosslinking of natural ECM (such as gelatin)
allowed a minimal immune response. Furthermore, SA_Ty-based
crosslinking allowed a very stable hydrogel system that can withstand
collagenase or hyaluronidase-induced degradation (Fig. 3F). In addi-
tion, SA_Ty-based crosslinking system not only allowed in situ hydrogel
formation but also induced stable adhesion of the HTG hydrogel onto
the cartilage surface. In a previous study, Liu et al. fabricated a free
radical scavenging HA-EGCG conjugate hydrogel that is crosslinked via
horseradish peroxide and hydrogen peroxide, which is injectable and
anti-inflammatory like our HTG-E hydrogel [45]. However, under our
hydrogel system, tyramine residues conjugated on HA backbone and
amine functional groups of gelatin enable a high degree of adhesion
onto cartilage surface, a huge advantage for intra-articular injection.
We and others have demonstrated injectability of a tyrosinase-based
crosslinking system and adhesiveness of gelatin-based hydrogels on
various tissue surface [27,28,46]. As the hydrogel can imbibe a large
amount of water, our HTG hydrogel may have contributed to the se-
questration of inflammatory cytokines upon injection into the OA an-
imal model, creating a local anti-inflammatory microenvironment that
influenced the regeneration.
In this study, we also incorporated EGCG and hyaluronic acid into
our hydrogel platform to the added benefit of controlling the OA in-
flammatory microenvironment. EGCG is a polyphenolic compound that
has been shown to possess anti-inflammatory activity with a potential
for anti-arthritic effects [15]. Due to its phenolic functional groups, it
Y. Jin, et al. Materials Science & Engineering C 115 (2020) 111096

Fig. 5. In vitro evaluation of 5% HTG and 5% HTG-E hydrogel in OA mimetic environment. (A) Histological analysis of H&E, Safranin-O and type II collagen
immunohistochemistry. Scale bar = 200 μm (B) Real-time PCR results of IL-1β, MMP13, and ADAMTS5 (n = 3). (C) Real-time PCR results of COL2, SOX9, and ACAN
(n = 3). RFI = Relative fold induction. *p < 0.05, **p < 0.01.

can scavenge radical oxygen species (ROS), which are overproduced in
inflammatory osteoarthritic joints. Elevated ROS levels put oxidative
stress on chondrocytes, leading to cell senescence, apoptosis as well as
lower levels of ECM synthesis and elevated levels of ECM degradation.
By scavenging ROS, EGCG can rescue chondrocytes from inflammation-
induced cell damage. Moreover, as mentioned previously, EGCG can
suppress pro-inflammatory signaling cascades by inhibiting phosphor-
ylation of p38, JNK and nuclear translocation of NF-kB. Indeed, we
have confirmed that chondrocytes in pathophysiological conditions
exhibited significant downregulation of inflammatory gene expression
levels when treated with EGCG (Fig. 2C). However, we noticed that
EGCG over 20 μM resulted in cellular cytotoxicity in monolayer (Sup-
plementary Fig. 3A). However, when we observed cell viability in the
HTG-E hydrogel, we were able to load up to 100 μM of EGCG onto the
HTG-E hydrogel with minimal cytotoxicity. This could be the result of
covalent conjugation of EGCG (with its phenolic functional groups)

8
Y. Jin, et al.
Fig. 6. Intra-articular injection of 5% HTG-E hydrogel in OA mouse joint. (A) Histological analysis by Safranin-O and immunofluorescence staining of types I, II, X
collagen. Scale bar = 200 μm. (B) Quantification of cartilage thickness. (C) Quantification of mean fluorescence intensity (MFI) of types I, II and X collagen
immunofluorescence staining. *p < 0.05, **p < 0.01, ***p < 0.005.
onto HA_t or gelatin, resulting in covalent incorporation of EGCG onto
the hydrogel. Indeed, physical and mechanical properties comparing
5% HTG vs 5% HTG-E showed slightly decreased swelling ratio along
with increased mechanical properties as a result of EGCG incorporation
(Supplementary Fig. 2). As a significant amount of added EGCG was
covalently incorporated onto the hydrogel, we hypothesize that EGCG-
induced cytotoxicity was minimized in the 3D hydrogel environment
compared to the 2D culture platform. In addition, our study demon-
strates the added benefit of incorporating EGCG onto the tyrosinase-
induced crosslinking hydrogel for effective anti-inflammatory control
via EGCG.
HA is one of the most abundant glycosaminoglycans (GAGs) in the
cartilage ECM. In addition, HA in synovial fluid provides lubrication,
along with lubricin [47]. Even though the therapeutic effect of intra-
articular injection of HA to OA joint has been reported the HA injection
provide only a transient treatment option for OA patient as it is not
directly involved in the cartilage regeneration and has relatively rapid
clearance [48]. As a way to provide a cell-friendly environment for the
cartilage and provide a lubricating component to the injected hydrogel,
we introduced tyramine-conjugated HA.
When chondrocytes were encapsulated in HTG hydrogel of various
HA_t concentration, we observed a HA_t concentration-dependent ECM
accumulation. Previously, crosslinked HA-based hydrogel has been
extensively studied for cartilage tissue engineering for past decades.
Levett et al. and Moulisova et al. have reported crosslinked HA-based
9
Materials Science & Engineering C 115 (2020) 111096
hydrogels promoted the GAG and collagen II accumulation [49,50]. In
accordance with the previous study, we also demonstrated that higher
the HA content more ECM was accumulated within the hydrogel [51].
Histological analysis and immunostaining confirmed the increased ECM
accumulation in 5% HTG when compared to the 2% HTG constructs.
Interestingly, cellular phenotype and organization of type II collagen
were drastically different in 5% HTG versus 2% HTG (Fig. 4B). In 5%
HTG hydrogel, chondrocytes maintained circular phenotype and type II
collagen were typically located around the cells. However, within 2%
HTG hydrogel, cells appeared to be stretched and collagen orientation
was distinct. Relatively lower HA content in 2% HTG hydrogel may
have allowed increased cell adhesion to gelatin within the hydrogel.
Although cellular adhesion to biomaterials is beneficial for cell survival
and proliferation, cytoskeletal tension within chondrocytes is re-
sponsible for dedifferentiation and phenotypical change to fibroblast-
like morphology. This is one of the primary reasons why chondrocytes
cultured in 2D platform are not able to synthesize GAGs and down-
regulate chondrocyte-specific genes such as COL2, SOX9, and ACAN
[52]. In contrast, 3D cultivation of chondrocytes can support the re-
differentiation by keeping the cell in spherical shape [53]. In addition
to cytoskeletal tension due to cell adhesion, different physical proper-
ties of the HTG hydrogels may have influenced chondrocyte phenotype
within the hydrogel. Compared to 5% HTG hydrogel, 2% HTG hydrogel
has a higher swelling ratio and larger internal pores as seen in SEM
images (Fig. 3B and E). This implies that a larger living space is
Y. Jin, et al.
available in 2% HTG hydrogel for encapsulated chondrocytes, leading
to a higher degree of cell spreading and dedifferentiation. Toh et al. and
many others have reported that chondrocytic phenotype is more well-
maintained on lower stiffness hydrogels [54,55]. Although 2% HTG has
lower stiffness than 5% HTG hydrogel, the relative availability of cell
adhesion sites and size of cell living space seemingly have more influ-
ence on chondrocyte phenotype and genotype.
Based on EGCG-induced anti-inflammation effects and optimized
HA_t concentration to induce optimal ECM production, we incorporated
EGCG into 5% HTG hydrogel for the treatment of OA. Prior to appli-
cation in OA animal model, cell-laden hydrogels with or without EGCG
were cultured in IL-1β-containing media for 3 weeks to investigate
whether the HTG-E hydrogel could resist to OA environment. Our
findings suggested that 5% HTG-E hydrogel provides a protective effect
from the OA-like inflammatory environment. The accumulation of ECM
was detected in the 5% HTG-E group rather than in the 5% HTG group,
and the inflammatory gene was down-regulated through the 3-week
culture. On the other hand, the cartilage forming gene was upregulated
in the 5% HTG-E group. In vivo evaluation of intra-articular injection of
5% HTG-E showed progressive results in OA treatment. OA was in-
itiated by destabilization of the medial meniscus, and 5 μL of hydrogels
(5% HTG with or without EGCG) were injected. All the tissues were
evaluated after 4 weeks. In the PBS injection control group, the carti-
lage was completely damaged. In 50 μM EGCG and 5% HTG hydrogel
injection groups, the cartilage was thinned out, but not completely
eroded. These results indicated that injection of 50 μM EGCG or 5%
HTG hydrogel alone provided therapeutic benefit to OA. Our study is in
accordance with previous studies that reported the beneficial effects of
EGCG on the OA model. Leong et al., have reported that the daily in-
traperitoneal injection of EGCG to OA induced mice exhibited relief of
OA-related pain and reduction of proinflammatory cytokine levels [56].
In another study by Natarajan et al., intra-articular injections of poly-
phenols such as EGCG and tannic acid showed resistant to degradation
despite ongoing inflammation [57]. This study was performed with
EGCG and Tannic acid injection every 3 days for the significant out-
come. Although intra-articular injection of drugs provides one option
for drug delivery in a joint, the rapid clearance of the injected drug
limits the therapeutic efficacy of the intra-articular injection.
In our study, we have allowed crosslinking of EGCG into our hy-
drogel. Our HTG-E hydrogel can be retained in the joint for a long time.
Furthermore, due to the adhesive property of HTG-E hydrogel, anti-
inflammatory EGCG components can be retained within the surface of
articular cartilage. A recent study by Emoto et al. demonstrated the in
situ cross-linkable HA hydrogels. This HA-based hydrogel was able to
sustain the release of anti-cancer drugs within several days [58]. For the
combination treatment of EGCG and HA-based hydrogel, 5% HTG hy-
drogel injection group showed hyaline cartilage similar to that of the
normal control group. We did not detect any wear and ECM loss. The
injected HTG-E hydrogel may have formed a thin layer on the cartilage
surface to protect cartilage from physical abrasion. Furthermore, the
EGCG component within HTG-E hydrogel may provide an anti-in-
flammatory microenvironment that allowed the maintenance of ar-
ticular cartilage. Based on our initial finding, we believe that this in-
flammatory modulating hydrogel can be applied in various tissue
augment applications.
5. Conclusion
In summary, we demonstrated that HTG hydrogel promotes ECM
accumulation and EGCG-loaded composite hydrogel has anti-in-
flammatory and chondroprotective capacities in IL-1β-induced OA-like
environment. Injection of the EGCG-loaded composite hydrogel to
surgically induced OA knee joint can protect the cartilage from in-
flammation and “wear-and-tear.” Therefore, we believe that this EGCG-
loaded injectable HTG hydrogel is a promising tissue engineering
strategy and has great potential for clinical recovery of OA.
10
Materials Science & Engineering C 115 (2020) 111096
CRediT authorship contribution statement
Yin Ji Jin: Writing - original draft, Methodology, Formal analysis,
Investigation. Rachel H. Koh: Writing - review & editing,
Methodology. Su-Hwan Kim: Conceptualization. Kyung Min Kim:
Resources. G. Kate Park: Resources. Nathaniel S. Hwang:
Conceptualization, Funding acquisition, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The Institute of Engineering Research at Seoul National University
provided research facilities for this work. This research was supported
by National Research Foundation of Korea Grant funded by the Korean
Government (NRF-2016R1E1A1A01943393). This research was a part
of the project titled ‘Biochemical modification of marine based bio-
materials for therapeutic ischemic cardiovascular disease treatment’,
funded by the Ministry of Oceans and Fisheries, Korea.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.msec.2020.111096.
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