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A strong bio-ink for Meniscus Regeneration
compression, respectively. The approximate linear fitting 10 min after 48 h. The cells were stained with phalloidine
results at a strain deformation of 20 – 40% were used to and 4’,6-diamidino-2-phenylindole (DAPI) after being
calculate the Young’s modulus. treated with 0.1% Triton X-100, and the morphology
of the cells was studied using a laser scanning confocal
2.5. 3D printing test microscope.
A Bio-Architect® WS 3D printer was used for the 2.8. In vivo study
3D printing test (Regenovo, Hangzhou, China). The
cylindrical scaffolds with angles of 0° and 90°, as well as We used the 40-20 PVA/dECM hydrogels with excellent
the filaments, were extruded using a nozzle with a diameter mechanical properties and well cytocompatibility as
of 310 m and a 0.25 MPa air pressure. The printing speed experimental group. All animal researches were carried
was set at 6 mm/s, and the filament separation distance out in accordance with the Ethics Committee of Drum
was set to 600 m. The model’s thickness was set at 280 m. Tower Hospital Affiliated to Nanjing University’s Medical
A BX-53 microscope (Olympus, Tokyo, Japan) was used School (Nanjing, China) legislation and guidelines, as
to inspect the printed samples for printing correctness. well as the Institutional Animal Care and Use Committee
recommendations.
2.6. Cytotoxicity assay A total of 24 female New Zealand white rabbits
with a bodyweight of 2.5 kg were used in the present
The biocompatibility test was performed using human study and randomly divided into four groups (PVA/
umbilical vein endothelial cells (HUVECs; Warner, dECM hydrogel group, control group, blank group, and
Wuhan, China). The cells were kept in a 5% CO sham group; n = 6 rabbits per each group). The surgery
2
environment at 37°C in Dulbecco’s modified Eagle’s was carried out under general anesthesia with a 0.2 mL/
medium (Gibco, New York, NY, USA) supplemented kg intramuscular injection of xylazine HCl (Jilin Huamu
with 10% fetal bovine serum (Gibco) and 100 IU/mL Animal Health Products Co., Ltd., Jilin, China). Knee
penicillin/streptomycin. The materials were cut into surgery was carried out under sterile conditions. Briefly,
cylinders of 8 mm diameter and 2 mm height and sterilized a lateral parapatellar incision was made and the lateral
by UV irradiation overnight before in vitro testing. The meniscus was disclosed. A 3-mm full-thickness circular
samples were then transferred into 24-well plates, and the defect was created in the central part of lateral meniscus
cells suspension (3000 cells/cm ) was dropped onto the using sharp blades (Figure S1). The defects in the
−2
surface of samples. The blank group received the same hydrogel group were implanted with pre-fabricated PVA/
amount of HUVECs in the blank dishes. After culturing dECM hydrogel and control hydrogel, and the defects
for 48 h, the cell counting kit-8 solution was added to in the blank group remained blank. Only the articular
each group (10 μL per each group). After incubation at cavity was opened in the sham group. All the hydrogels
37°C for 2 h, the optical density (OD) was measured at used for implantation were from the same batch. The
450 nm, and the cell viability was calculated. The cells lateral collateral ligament was securely sutured after
were then dyed with live/dead assay. Briefly, the cells the lateral meniscus was reduced, the joint capsule
and samples were dyed with calcein AM and propidium was repaired with 3-0 nylon sutures, and the skin was
iodide (PI) for 45 min in the dark and were then fixed closed with 1-0 nylon sutures. After surgery, the rabbits
with paraformaldehyde for 30 min. The cells were were returned to their cages and permitted to resume
subsequently observed under a laser scanning confocal full weight-bearing activities. To prevent infection,
microscope (141 FV3000; Olympus). penicillin was administered intramuscularly for 3 days
Cell proliferation was calculated using the following following surgery. At week 12 after surgery, all animals
equation: Cell proliferation (%) = (OD hydrogel −OD baseline )/ were slaughtered to assess meniscus regeneration and
OD baseline × 100%. The absorbance of cells cultured on cartilage preservation.
the surface of hydrogels and dishes for 4 h was taken as
OD baseline , and the absorbance of cells cultured for 48 h 2.9. Macroscopic evaluation
was taken as OD .
hydrogel The meniscus, femoral condyle, and tibial plateaus
2.7. Cell adhesion were photographed and the evaluation of gross
morphology was performed. The meniscus defect
The phalloidine/DAPI staining was performed to repair was compared using ImageJ software (National
investigate cell adhesion. The HUVECs were passaged, Institutes of Health, Bethesda, MD, USA). Color,
and the cell suspension was dropped onto the surface of integrity, shape, and smoothness were assessed on the
samples, while the blank group was passaged onto the surface of the cartilage worn zone. The quality of the
glass bottom dishes with no hydrogel. The cells were repair was noted. The macroscopic appearance of the
rinsed in PBS and fixed in 4% paraformaldehyde for restored tissue was evaluated using the International

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