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Dong, et al.
tissues during regeneration to prevent any treatment was purchased from Zhengzhou Alfa Chemical Co., Ltd
invalidation caused by the detachment of the two parts . (Zhengzhou, China). TA and 1,1-diphenyl-2-picrylhydrazyl
[16]
Under osteoarthritis conditions, hydrogels with antioxidant (DPPH) were purchased from Aladdin Co., Ltd (Shanghai,
properties are indispensable . These diseased conditions China). 2,2-azinobis (3-ethylbenzothiazoline -6-sulfonic
[17]
could produce excess reactive oxygen species and generate acid mmonium salt) (ABTS), potassium persulfate
oxidative stress that damages the redox balance, which (K S O ), 2-Phenyl-4,4,5,5- tetramethylimidazoline-3-
2 2
8
suppresses osteogenic differentiation [18,19] . Hence, for oxide-1-oxyl (PTIO), and borax were purchased from
hydrogel applications in osteochondral repair, it is essential to Beijing Innochem Science and Technology., Ltd (China).
obtain a synergistic effect involving good biocompatibility,
high mechanical performance, excellent oxidation 2.2. GelMA synthesis
resistance, and adhesion to realize multifunctionality. GelMA was synthesized following previous methods [26,27] .
Tannic acid (TA) is a plant polyphenol with good Briefly, 10 g gelatin was dissolved in 100 ml carbonate-
anti-inflammatory, antibacterial, and antioxidant properties bicarbonate buffer at 50°C under constant stirring. MA
that have attracted widespread interest in biomedical (2 ml) was slowly added to the above-mentioned solution.
applications . Recent studies have demonstrated that TA After reacting for 3 h, the pH of the solution was adjusted
[20]
combines with polyacrylamide or PVA to form a composite to neutral using hydrochloric acid or sodium hydroxide.
hydrogel with solid adhesion, toughness, and oxidation GelMA was obtained after the resulting solution was
resistance [21-23] . However, because of the polymerization dialyzed, filtered, and freeze-dried.
inhibition properties of TA, it is challenging to introduce
TA directly into photoinitiated polymers. In addition, a 2.3. Preparation of composite hydrogel
hydrogel synthesized in one pot has submicrometer- or A series of NAGA/GelMA/Laponite (denoted as NGL,
nano-sized networks, which obstruct the exchange of where N, G, and L represent NAGA, GelMA, and
nutrients and oxygen. It drastically inhibits the proliferation LPN, respectively) composite hydrogels (Table 1) were
and differentiation of cells . 3D printing technology can prepared. First, deionized water (5 g), NAGA (3 g), and
[24]
solve this problem by precisely controlling the macropore GelMA (1 g) were mixed and added to a 20 mL beaker
structure within the hydrogel scaffold. [24,25] under constant stirring for 30 min. Then, the photoinitiator
Therefore, it was hypothesized that TA incubation Irgacure 1173 (100 µL) was added to the above solution.
could provide a simple and effective strategy for preparing LPN (0.3 g) was then dispersed in 5 mL of deionized water.
multifunctional hydrogels. The TA, which contained many Subsequently, the NAGA/GelMA solution was mixed
pyrogallol groups, was inserted into a covalent polymer with the LPN suspension. After complete dissolution, the
consisting of gelatin methacrylate (GelMA) and NAGA. resulting solution was added into plastic molds (diameter
The copolymerization of GelMA and NAGA formed 10 mm and thickness 5 mm) and exposed to an ultraviolet
a leading network. By adding polyphenol compounds, (UV) crosslinker (Chitang Co., Ltd., Shanghai, China,
multiple hydrogen bonds occurred between the phenolic 16 W) for 40 min. The obtained NGL3 hydrogel was
hydroxyl group of TA, the carbonyl group, and the amino immersed in an aqueous solution with various amounts
group of NAGA/GelMA. The two-step method prevented of TA (5 and 10 w/v %) for 24 h to obtain T5 and T10
the polymerization inhibition effect of polyphenols. The hydrogels, respectively (the 1 w/v % borax was added at
hydrogel’s morphology, chemical structure, swelling a specific time).
behavior, mechanical properties, and cell compatibility were
characterized. The adhesion and oxidation resistance of the 2.4. Physicochemical characterization of
hydrogels were tested. Subsequently, the influence of clay hydrogel
and GelMA on the printability of the hydrogel was examined.
In addition, the biocompatibility of the hydrogel and the The microstructures of freeze-dried samples were
potential of osteogenic and chondrogenic were evaluated. analyzed by a field emission scanning electron microscope,
Zeiss, Germany. Fourier transform infrared spectroscopy
2. Materials and methods (FTIR, Nicolet iN10, Thermo Scientific, USA) was used
to identify the chemical structure of the samples.
2.1. Materials
(1) Mechanical property
Gelatin (from porcine skin, Type A, powder, gel strength
240~270 Bloom) was purchased from Yeasen (Shanghai, Compression testing of the hydrogel was performed
China), methacrylic anhydride (MA), and 2-hydroxy-2- using a Zwick Z050 in the press mode. In brief, a
methyl-1-phenyl-1-propanone (IRGACURE 1173, 98%) photocrosslinked hydrogel cylinder (6 mm in height
were purchased from Sigma Aldrich (USA), Laponite and 10 mm in diameter; n = 3) was placed on the
XLG (LPN) was purchased from BYK (UK), NAGA lower plate, and the speed was set at 1 mm/min. The

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