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Lu, et al.
Cartilage Repair Society’s (ICRS) macroscopic grading hydroxide to obtain pale-yellow translucent hydrogel,
system [26] . Table S1 lists the grading criteria for the and the freezing/thawing process was repeated. Finally,
score, which describes cartilage wear after meniscus the white opaque hydrogel was obtained after soaking
defects. calcium chloride. SEM was performed to investigate
the microstructure of the hydrogels and the natural
2.10. Micro-magnetic resonance imaging meniscus. As illustrated in Figure 1A-C and Figure
At week 12 post-operation, the complete knee joint S3, the 40/20 PVA/dECM hydrogel showed a reticulated
structure was assessed by micro-magnetic resonance pore structure similar to the natural meniscus. The pore
imaging (micro-MRI). Micro-MRI of the knee joints size of hydrogel was about 20 μm in diameter, which well
was performed using a 9.4T Bruker Biospec 94/20 USR simulated the structure of natural meniscus. However,
Micro-MRI system (Bruker, Bremen, Germany) with a the control group showed the small number of holes
dedicated knee coil (Siemens Munich, Germany). The with different sizes. According to Figure S4, the PVA/
sagittal micro-MRI was acquired using the fat-suppressed dECM hydrogel and control group had a similar content
proton-density weighted turbo spin-echo sequences of surface element to the natural meniscus, including
(Field of view, 62 mm; echo time, 6.82 ms; repetition carbon, oxygen, chlorine, and calcium. The content of
time, 1027 ms; thickness, 1 mm). The micro-MRI sodium in the PVA/dECM hydrogel was more than that
findings were viewed using an Onis Digital Imaging and in the natural meniscus, which caused by soaking in the
sodium hydroxide.
Communications in Medicine Viewer (ver. 2.5; Digital Furthermore, the dynamic properties of the
Core Co., Ltd., Tokyo, Japan).
hydrogels were studied in a detailed manner using a
2.11. Histological evaluation rheometer at 20°C. The results of strain-sweep and the
frequency-sweep tests confirmed that this hydrogel had
The femoral condyle and tibial plateaus were decalcified obvious colloidal characteristics, which were higher
with 15% EDTA for 28 days after the collected samples according to the increased storage modulus (G’) and loss
were fixed with 10% formalin for 24 h. After that, all of modulus (G”). The results of destroy-recovery experiment
the samples were soaked in paraffin and sliced into 5-mm showed the well structural stability of hydrogel
thick slices. Hematoxylin and eosin (H&E), toluidine (Figure 1D-I, Figure S5-S7). Moreover, almost the same
blue, Safranin O, and collagen II were used to stain dynamic response was observed at 37°C, suggesting that
these sections. The tissue integrity and staining of the the performance of PVA/dECM hydrogels will not be
meniscus were compared to assess the histology results. affected by temperature in vivo.
The O’Driscoll rating system was used to evaluate
[27]
the cartilage histology data, and the scoring criteria 3.2. Compressibility of the PVA/dECM hydrogel
are listed in Table S2 . A microscope with a charge- We quantitatively measured the compressive properties
[27]
coupled device camera was used to examine all sections of the hydrogels by standard mechanical tests. The
(Olympus). typical stress-strain curves from compression-crack
2.12. Statistical analysis testing of PVA/dECM hydrogel and control hydrogel
are presented in Figure 2A and Figure S8. The 40-
Three investigators were blinded to grouping and 20 PVA/dECM hydrogel showed the best mechanical
assessed the macroscopic and histological data. SPSS properties compared with other groups. The control
19.0 software (IBM Corp., Armonk, NY, USA) and IGOR hydrogel was broken when compressed to 70%. The
Pro 6.12 software were used for statistical analysis and PVA/dECM hydrogel indicated an excellent load
exponential curve fitting (WaveMetrics Inc., Portland, bearing capacity and a distinct improvement. The
OR, USA). The results were analyzed using an unpaired Young’s modulus of 40-20 PVA/dECM hydrogel group
Student’s t-test and provided as mean standard deviation. was 0.49 MPa at 20 – 40% compression and the stress
Results with P < 0.05 was considered statistically limitation was 2.9 MPa. Meanwhile, the Young’s
significant. modulus of control group was 0.29 MPa and the stress
limitation was 1.0 MPa, which were lower than those
3. Results in the 40-20 PVA/dECM hydrogel group. The PVA/
3.1. Characterization of the PVA/dECM hydrogel dECM hydrogel exhibited significantly higher values of
Young’s modulus, fracture strain and stress compared
As shown in Figure S2, the bio-ink was a light yellow with those in the control group.
and viscous liquid after configuration. The bio-ink was The energy dissipation of hydrogels was also
irradiated by blue light to form hydrogel that was a white tested by compression-relaxation cycles. As displayed in
opaque hydrogel. The hydrogel was dipped into sodium Figure 2B and Figure S9, an obvious energy dissipation

International Journal of Bioprinting (2022)–Volume 8, Issue 4 35