Page 115 - IJB-9-2

International Journal of Bioprinting Photocurable pullulan-based bioink for 3D printing

and 40 h). The swollen weight (W ) of the samples at each per well. The culture medium in plates was removed after
t
time point and the weight reaching swollen equilibrium 8 h of incubation and 200 μL of diluted extracted solution
(W ) were recorded. The absorption rate at each time point was then added into each well. The L929s cell was co-
s
was calculated according to Equation I. The equilibrium cultured with the extracted solutions for 1, 3, and 5 days.
swelling ratio was calculated according to Equation II. At each time point, the extracted solutions were removed
W − W and 100 μL of fresh culture medium was added per well.
W = t d ×100% (I) About 10 μL of CCK-8 reagent was added to each well and
W d the plates were incubated for 1 h in the dark. The optical
W − W density (OD) values of the solution after incubation were
SR = s d ×100% (II) measured at 450 nm by a microplate reader (SpectraMax
W
d M5, Molecular Devices Corporation, USA).
2.7. Compressive mechanical property 2.10. Statistical analysis
measurement of Pul-NB hydrogels All data are presented as mean ± standard deviation.
To determine the mechanical characteristics of Pul-NB Statistical analysis between groups was performed using
hydrogels, the Pul-NB solution (5%, 6%, 7%, 8%, and hypothesis testing of OriginPro 8 software. A statistical
10% (w/v)) was prepared and DTT was added to the solution level of P < 0.05 was considered to be a significant difference
at a concentration equimolar to NB functional groups. The and a statistical level of P < 0.01 was considered to be a very
mixture was poured into a cylindrical latex mold with a significant difference.
thickness of 12 mm. After UV light treatment, the hydrogels
were placed on the sample stage of an electronic universal 3. Results and discussion
testing machine (XJ810-50N, Shanghai Xiangjie Instrument
Co., Ltd, China) for compressive testing. The test temperature 3.1. Characterization of Pul-NB
was 25°C; the preload speed was 50 mm/min; the load speed Pul-NB was synthesized through the esterification
was 50 mm/min; the posttest speed was 100 mm/min; and of hydroxyl groups of pullulan polysaccharide with
the maximum compression height was 10 mm. norbornene anhydride (Figure 1A), which was similar to
the widely used synthesis method of gelatin methacryloyl
2.8. Surface morphology observation of Pul-NB (GelMA). To prepare the photo-cured Pul-NB hydrogels,
hydrogels DTT and HDT were added for thiol-ene reaction. Thiol-
To characterize the micromorphology of Pul-NB hydrogels, ene reaction was initiated by DLP after UV irradiation, and
the UV-cured hydrogels were lyophilized and sputtered with then the Pul-NB hydrogels were formed (Figure 1B).
platinum. The cross-section and skeleton morphology were 1
observed by scanning electron microscope (Hitachi SU8010, 3.1.1. H NMR
1
Hitachi Ltd, Japan). The acceleration voltage was 1.0 kV. H NMR spectrum of Pul-NB with different DS is depicted
in Figure 2A. The resonances at 5.339, 5.305, and 4.897 ppm
2.9. In vitro cytocompatibility were assigned to the anomeric protons in glucose units via
In vitro cytocompatibility of Pul-NB hydrogels was α-1,4 and α-1,6 glycosidic linkages (represented by “b,” “c,”
1
measured with cell counting kit-8 (CCK-8) assay. and “d,” respectively). The H NMR chemical shifts between
Subcutaneous connective tissue cells of L-WRN mice (L929 3.389 and 3.972 ppm were attributed to the protons of
cells) were purchased from Shanghai Fuheng Biotechnology glucose units [33,38] . Norbornene alkene protons peak can be
Co., Ltd (Shanghai, China). Due to the inability of Pul-NB observed at ~6.178 to 6.082 ppm (denoted with the dotted
[42]
1 to form a stable hydrogel, the cytocompatibility of Pul- box), while it is absent in unmodified pullulan . The DS
NB 2, Pul-NB 3, and Pul-NB 4 hydrogels prepared with of Pul-NB was calculated according to the peak area ratio
dH O as well as Pul-NB 4 hydrogels prepared with DMEM of norbornene alkene protons to that of anomeric protons
2
culture medium (Pul-NB 4@DMEM) were studied. The of α-1,6 glycosidic linkages (Table S2). The DS of Pul-NB
extracted solutions of Pul-NB hydrogels were prepared by was increased with the increase of norbornene anhydride
immersing photocurable Pul-NB hydrogels with different concentrations. When the total norbornene anhydride
DS (after washing once with DPBS) in DMEM culture was added to get the desired -OH:NB ratio of 1:1, the DS
medium with 10% FBS and 1% penicillin-streptomycin at reached 29.93%. A higher norbornene concentration was
[43]
37°C for 24 h. The extracted solutions were sterilized by attempted to get a higher DS of Pul-NB . However, the
filtration through a 0.22 μm sterile filter (Millipore) and then DS did not increase because the amino groups on gelatin
diluted twice with fresh culture medium. The suspension- chains had a higher reactivity with norbornene than
cultured L929 cells in the exponential growth phase were hydroxyl groups on pullulan chains. As seen from the H
1
incubated into 96 well plates at a concentration of 5 × 10 NMR spectrum of unmodified pullulan (Figure 2B), no
3
Volume 9 Issue 2 (2023) 107 https://doi.org/10.18063/ijb.v9i2.657