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Lu, et al.
2. Materials and methods tube and exposed to blue light for 20 min to complete the
photocrosslink reaction. Hydrogel samples were stored at
2.1. Preparation of PVA/dECM hydrogel room temperature.
The meniscal dECM was prepared as previously
reported . Rabbit menisci were sliced into thin slices 2.2. Surface characterization of the PVA/dECM
[16]
(1 mm thick), frozen at −80°C, and then pulverized into hydrogel
coarse powders. For 72 h, the powders were mixed in A scanning electron microscope (SEM) was used to
a solution of 1% SDS/phosphate-buffered saline (SDS/ examine the surface topography (SU8020; Hitachi,
PBS) (w/v) (Sigma-Aldrich, St. Louis, MO, USA). Tokyo, Japan). All samples (n = 3 for each group) were
Every 24 h, the solution was updated. The sample was dehydrated and dried at room temperature using a series
then submerged overnight in a considerable volume of of graded alcohols. The dried samples were sputter-
deionized water to remove the leftover compounds after coated with gold-palladium and viewed with a SEM at
being treated with 0.1% w/v ethylenediaminetetraacetic 1 kV. A diffractometer was used to measure the X-ray
acid (EDTA)/PBS solution (Sigma-Aldrich) for 24 h. diffraction patterns of materials at 40 kV and 40 Ma using
Finally, the resulting dECM was frozen at −80°C for Cu radiation. Diffractograms were set to 6 – 70° (2θ),
3 days before being lyophilized. The dECM was finely with a step size of 0.020° (2θ), at a rate of 1.20°/min (2θ).
powdered before being added to a pepsin/0.01 M
hydrochloride (HCl) solution (Sigma-Aldrich) and stirred 2.3. Rheological test
at 15 mg/mL for 48 h at room temperature. 0.1 M sodium
hydroxide (NaOH; Sigma-Aldrich) and PBS were used to A Haake Mars 40 Rotational Rheometer was used for
neutralize the viscous solution (Sigma-Aldrich). the rheological test (Thermo Fisher Scientific, Waltham,
PVA (Sigma-Aldrich) was dissolved in deionized MA, USA). Thin slices of the samples were cut (1 cm in
water with a 10% mass-to-volume ratio. The solution diameter and 1 mm in thickness). The frequency range
was stirred in 55℃ water bath for 4 h until PVA crystals for the frequency sweep experiment was adjusted at 0.1
were completely dissolved. Then, the 10% w/v PVA – 100 Hz with a strain of 0.1%. The strain range for the
and 10% w/v dECM were added to deionized water and strain sweep experiment was adjusted at 0.1 – 100% with
stirred evenly. The 10% w/v PEGDA (Sigma-Aldrich) a frequency of 1 Hz. G’ and G” were measured at 0.1%
and 0.05% w/v 2-hydroxy-4’-(2-hydroxyethoxy)-2- strain and 6.28 rad/s rotational velocity for the destroy-
methylpropiophenone (Sigma-Aldrich) were added to recovery experiment. The strain was then increased to
the solution as crosslinking agent and photoinitiator, 300% for 60 s to destroy the hydrogels, and then reduced
respectively. After that, 6% w/v alginic acid sodium salt to 0.1% for 600 s to monitor mechanical property recovery
(Sigma-Aldrich, St. Louis, MO, USA) was added and while keeping the angular velocity at 6.28 rad/s. All of the
kept in a 4℃ refrigerator overnight. trials were carried out 3 times.
The prepared solution was placed into a 2 mL 2.4. Compression test
centrifuge tube and exposed to blue light for 20 min
to complete the photocrosslink reaction. The hydrogel The samples with a height of 10 mm and a diameter
was cut into small pieces and soaked into the 6 mol/L of 8 mm were used for the compressive test. The
NaOH at 3 time points (20/40/120 min). After washing compressive stress-strain test was carried out using
with deionized water, the prepared hydrogel was frozen an Instron-5944 universal instrument (Thermo Fisher
at −80℃ for 1 h and thawed for 24 h. The freezing/ Scientific) equipped with a 2 kN sensor in air at room
thawing process was repeated for 10/20 times. According temperature. In the compression-relaxation cycle test and
to differences in alkaline treatment time and freezing/ the compression-crack test, the rate of compression was
thawing times, samples were divided into 6 groups of maintained at 2 mm/min. In the stress relaxation test, the
20-10, 20-20, 40-10, 40-20, 120-10, and 120-20. Finally, strain in each compression process was set to 10% and
the PVA/dECM hydrogel was immersed into 80 mmol/L repeated 5 times. Afterward, the hydrogel was quickly
calcium chloride (CaCl ; Sigma-Aldrich) for 10 min to preloaded to different strains at a rate of 60 mm/min and
2
achieve the ionic crosslinking. Hydrogel samples were kept for 5 min to achieve the stress-relaxation curves. All
stored at room temperature. the experiments were repeated thrice.
The control group was treated with deionized Ef=x0 xf(x)dx, where x0 and xf denote the starting
water by adding 10% w/v dECM, 10% w/v PEGDA, distance and fracture distance of the compression,
and 0.05% w/v 2-hydroxy-4′-(2-hydroxyethoxy)-2- respectively, was calculated from the area below the
methylpropiophenone. The 6% w/v alginic acid sodium compressive stress-strain curve until fracture using the
salt was added and kept in a 4℃ refrigerator overnight. following equation: Ef=x0 xf(x)dx, where x0 and xf
The prepared solution was put into a 2-mL centrifuge denote the starting distance and fracture distance of the

International Journal of Bioprinting (2022)–Volume 8, Issue 4 33