Page 398 - IJB-9-5

International Journal of Bioprinting A sturgeon cartilage extracellular matrix-derived bioactive bioink

5 mg, 10 mg, and 15 mg/mLdSC-ECMMA, respectively, and SerMA hydrogel (Q) can be calculated as the following
which were correspondingly defined as dSC-ECM-5, formulation:
dSC-ECM-10, and dSC-ECM-15. A bioink precursor Q = (W – W )/ W × 100(%) (I)
solution containing 150 mg/mLSerMA and 5mg/mL LAP swollen dry dry
only was prepared as the control bioink sample. Finally, 2.6. Isolation and cultivation of rabbit chondrocytes
those samples were solidified by exposure to blue light All animal experiments in this research were approved
(wavelength: 405 nm) to test their ability to photocuring by the Animal Care and Ethics Committee of College
and operability for further cell culture. Hydrogel samples of Biology, Hunan University. The isolation and culture
prepared from the dSC-ECMMA containing bioink of rabbit chondrocytes followed the protocol described
precursor solutions are simply described as dSC-ECM-5, in the publication without any modification .
[22]
dSC-ECM-10, and dSC-ECM-15 hydrogels, respectively, Chondrocytes in the second passage were harvested for
in later statements. further experiments.
2.5. Characterizations of hydrogels prepared with 2.7. Cytocompatibility of dSC-ECM hydrogels
dSC-ECM-derived and the control bioinks AlamarBlue assays and fluorescein diacetate (FDA)
2.5.1. H NMR tests staining were carried out to evaluate the cytocompatibility
1
The key component of bioinks, dSC-ECMMA and SerMA, of prepared hydrogel samples, which encapsulated living
were characterized and compared with proton nuclear chondrocytes. SerMA hydrogels, which were prepared
magnetic resonance ( H NMR) measurements. For the by photocrosslinking SerMA bioink precursor solution
1
1 H NMR analysis,10 mg of SerMa were completely carrying chondrocytes (10×10 cells/mL), were used as
5
dissolved in 1 mL of deuterium oxide (D O; Sigma-Aldrich, the control samples. The dSC-ECM hydrogel samples
2
St. Louis, USA). The dSC-ECM and dSC-ECMMA samples were produced by irradiating dSC-ECM-derived bioinks
were dissolved as much as possible in D O. All samples in (10×10 chondrocytes/mL), which contained 5, 10, and
5
2
D O were analyzed by H NMR (Bruker 400 MHz Advance, 15 mg/mL dSC-ECMMA, respectively (described at 2.4),
1
2
Switzerland) to obtain a corresponding NMR spectrum. with blue light. Then, these samples with encapsulated
chondrocytes were transferred to cell culture plates
2.5.2. Rheological properties with high glucose DMEM containing 10% FBS. The
Rheological properties of dSC-ECM hydrogels and SerMA cell culture plates with samples were incubated in a
hydrogel were evaluatedusing a rheometer (MCR 92, humidified 37°C, 5% CO incubator. In brief, the cell-
Anton Paar GmbH) with oscillation frequency from 1 to loaded hydrogel samples incubated for 1 and 7 days
2
10 Hz. The cylinder samples, which were 0.3 mm in height were taken out, stained with FDA, and observed with
and 15 mm in diameter cylinder shape, were prepared for an inverted fluorescent microscope (IX-73, Olympus,
the rheological properties test. Japan). The cellular proliferation was analyzed following
TM
2.5.3. Mechanical properties tests 1 and 5 days of incubation using the AlamarBlue Assay
The mechanical properties of hydrogel samples were kit (Thermo Fisher Scientific). Briefly, all samples were
measured using the electronic universal testing machine washed three times with PBS and incubated in a fresh
TM
(AGS-V universal testing machine with a 20 N sensor, medium containing 10% v/v AlamarBlue dye solution
Shimadzu Corporation). As-prepared cylindrical hydrogel at 37°C. After 6 h incubation, 200μL of the medium was
samples (d = 10 mm, h = 3 mm) were placed at the center collected and measured the fluorescence with excitation at
of the horizontal measuring platform. All tests were done 570 nm and emission at 630 nm with a spectrophotometer
on 3 samples (n=3). (Thermo Multiskan MK3, USA).

2.5.4. Equilibrium swelling ratio 2.8. Scanning electron microscopy and porosity
The swelling behavior of dSC-ECMMA hydrogels and measurement of lyophilized dSC-ECM-5 and
SerMA hydrogels was measured via the gravimetric SerMA hydrogels
method. Briefly, samples were immersed for 0.125, 0.5, 1, The microscopic morphology of samples, which were
and 1.5 h in 1× PBS (pH 7.4) at 37°C. The hydrogels were lyophilized dSC-ECM-5 and SerMA hydrogels, were
washed three times with ddH O, and the filter paper was observed by scanning electron microscope (SEM) at
2
used to blot the surface liquid of the hydrogel as much as an accelerating voltage of 30 kV (FEI Quanta 200, FEI
possible. The hydrogels were weighed at the different time Company, Czech Republic). The average pore sizes of the
points described above to obtain W swollen . Then the gels were lyophilized hydrogels were analyzed from the SEM images
lyophilized and measured the dried weight (W ). The by using ImageJ (50 random pores for each sample were
dry
ratio of water uptake from dried dSC-ECMMA hydrogels calculated).

Volume 9 Issue 5 (2023) 390 https://doi.org/10.18063/ijb.768