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Dong, et al.
sweeps of the samples (0.1 – 100 Hz) were performed (1000 cm ), the characteristic N-H peak at 1558 cm −1
−1
at room temperature in the obtained linear viscoelastic arose from NAGA, the characteristic peaks at 1658 cm
−1
region. The storage modulus (G’) and loss modulus (G”) corresponding to C=O, -OH, and N-H stretching bands
of the samples were measured in a frequency range of at 3300 cm originated from GelMA and NAGA. The
−1
0.1 – 100 Hz. The recovery properties were quantitatively polymerization reaction caused the 1622 cm band for
−1
characterized at room temperature using a rheometer. NAGA (representing the characteristic peak of C=C) to
An alternate shear strain sweep test was performed at disappear, indicating that the composite hydrogel was
a frequency of 1 Hz to test the recovery ability of the successfully formed after UV irradiation . Compared with
[32]
hydrogel. The shear strain was switched successively NGL, the TA-mediated hydrogel exhibited overlapping
from a minor strain of 1% (100 s) to a large strain of TA signals, indicating that the TA was incorporated into
100%, 200%, or 300% (100 s). After each phase of the the NGL hydrogel network. The formation of broad bands
test, the next stage began immediately without retention. at 3290 (-OH stretching vibration), and the movement of
the -OH stretching vibration to a lower wavenumber in the
2.6. Biocompatibility and osteochondral activity T5 spectrum, indicated that a large number of hydrogen
of hydrogel bonds were constructed between the TA and NGL .
[4]
Bone marrow mesenchymal stem cells (BMSCs) were The high swelling rate of the hydrogel could
chosen to evaluate the biocompatibility of the hydrogel by squeeze the surrounding tissues of the affected area,
cell counting kit-8 assays. The osteogenic differentiation thereby affecting the repair of a defect. Figure 2B shows
on the hydrogels was evaluated by alizarin red staining the swelling characteristics of four different hydrogels.
(ARS), alkaline phosphatase (ALP) staining, and Because the hydrophilic groups of the GelMA backbones
quantitative real-time polymerase chain reaction (qRT- were easily hydrated, the equilibrium swelling rates of
PCR) assays. The chondrogenic differentiation on the the prepared hydrogels ranged from 600 to 900%. The
hydrogels was evaluated by qRT-PCR assays. The details TA-mediated hydrogel reached swelling equilibrium
are available in Supplementary File. within 12 h. It has been previously shown that TA has a
positive impact on the swelling properties of TA-mediated
2.7. Statistical analysis composite hydrogel, and the swelling mainly occurred

Data were analyzed by one-way analysis of variance with within the first 4 h, which also confirmed the formation
Turkey’s post hoc test (SPSS, version 17.0, USA) and of hydrogen bonds in the TA-mediated hydrogel. As
expressed as the mean ± standard deviation. P < 0.05 was previously mentioned, the addition of TA produced a
reported as significant for all statistical tests (*P < 0.05), hydrogel with a higher crosslink density and smaller pore
and P < 0.01 was reported as highly significant for all size, obstructing the diffusion of water into the hydrogel,
statistical tests (**P < 0.01). which reduced the swelling ratio.

3. Results and discussion 3.2. Mechanical properties of hydrogels
Ideal mechanical properties are essential for biomaterials
3.1. Morphology, FTIR, and in vitro swelling of because hydrogels need sufficient mechanical strength
the hydrogel to withstand the squeezing of surrounding tissues. As
Microstructural characterization revealed that the freeze- shown in Figure 3A, the compressive modulus doubled.
dried NAGA hydrogel exhibited a microfibrous structure The compressive modulus reached 0.65 MPa for T5
(Figure 1A). This might have resulted from the strong (Figure 3B and Table S1), close to the compression
intermolecular interactions of the hydrogen bonds. modulus and compressive strength of human articular
[33]
Figure 1B and C showed that the clay was intercalated cartilage (0.4 – 0.8 MPa) . There was no significant
into the NAGA/GelMA hydrogel . As the clay content difference in the compressive modulus of NGL3 and
[14]
and tannin content increased, the gel network became NGL5 (Figure 3B and C). LPN by forming physical
denser (Figure 1B-E). In addition, as the TA concentration entanglement with NAGA/GelMA chains as well as
increased, the pore size of the TA-mediated hydrogel themselves can disrupt the covalent network (established
was obviously reduced, and the T10 sample possessed during NAGA/GelMA polymerization) and weaken the
morphology similar to that of TA (Figure 1E and F). This compressive modulus. At the same time, an increase
might have been because the higher TA concentration in the concentration of nanoclay used as the “filler”
improved the crosslink density by forming hydrogen increases the compression modulus. Therefore, balancing
bonds . The macroscopic observations of NGL3, T5, these two effects would result in no significant change
[30]
and T10 hydrogel were presented in Figure S1. in the compressive modulus . However, when the TA
[34]
As shown in Figure 2A, in the NGL3 spectrum, it content reached 10%, the compressive modulus of the
was easy to observe the characteristic Si-O peak of LPN material was significantly reduced, which might be
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