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

(collagen, gelatin, and silk) [20-23] are preferable for 3D display true 3D configurations (e.g., cylinder, triangle,
bioprinting due to their excellent biocompatibility and pentagram, and superimposed rectangles) and satisfactory
cell-binding affinity. However, the natural material-based shape fidelity through digital light processing.
hydrogels exhibit weak mechanical performance and
brittleness [24-26] . Therefore, the natural polymers were 2. Material and methods
modified with photo-functional groups to realize 3D
printing by light-induced technique . 2.1. Materials and chemicals
[27]
Sodium carbonate anhydrous (AR), sodium biocarbonate
To obtain photo-curable hydrogels made from (AR), and methacrylic anhydride (94%) were purchased
natural polymers, the most commonly used groups from Shanghai Aladdin Chemistry Company Limited
for light-triggered crosslinking are acrylic and methyl (Co., Ltd). Pullulan was provided by Feiyu Biotechnology
acrylic ester functional groups . When performing Co., Ltd. Norbornene anhydride and 1, 6-hexanediol
[28]
optical functionalization, attention should be paid to the (HDT) were supplied by Shanghai Macklin Biochemical
manufacturability, key properties, reaction conditions, Co., Ltd. Dithiothreitol (DTT) was obtained from Beijing
sterilization conditions, and biocompatibility of final solarbio Science & Technology Co., Ltd. Sodium hydroxide
product, as these photo-crosslinkable derivatives are (NaOH) was obtained from Sinopharm Chemical Reagent
required for successful clinical applications. Pullulan is Co., Ltd. Photoinitiator Lithium phenyl-2,4,6-trimethyl
a microbial extracellular polysaccharide produced by benzoylphosphinate (LAP) was provided by Jiangyin
Aureobasidium pullulans. Due to its biodegradability, non- StemEasy Biotechnology Company. All the reagents
toxicity, non-immunogenicity, non-mutagenicity, and non- were kept as received condition before use. Deionized
carcinogenicity, the U.S. Food and Drug Administration water (dH O) used in all the experiments was self-
(FDA) approved pullulan as a GRAS (generally recognized prepared. Dulbecco’s modified eagle medium (DMEM)
2
as safe) substance in 2002. Due to the existence of a large and Dulbecco’s phosphate-buffered saline (DPBS) were
number of hydroxyl groups on the molecular chains, purchased from Thermo Fisher Scientific Ltd (Beijing,
new chemical functional groups can be introduced into China). Fetal bovine serum (FBS) was purchased
pullulan through esterification, oxidation, etherification, from NEWZERUM Ltd (Beijing, China). About 0.25%
copolymerization, sulfonation, and chlorination, which Trypsin-EDTA solution (without phenol red), CCK-8 cell
will modify its chemical and/or physical properties, proliferation and cytotoxicity assay kit, and penicillin-
thus widely extending its applications [29-31] . At present, streptomycin liquid were purchased from Beijing Solarbio
the preparation of pullulan derivatives by different Science & Technology Co., Ltd (Beijing, China).
modification methods has become a key research area in
the biomedical field [32-36] . Pullulan was also functionalized 2.2. Synthesis and characterization of
with methacrylic groups to realize 3D printing through norbornene-functionalized pullulan
radical polymerization reactions [37,38] . Massimo et al. Four gram of pullulan was dissolved in 40 mL of 0.1 M
[38]
synthetized photopolymerizable pullulan by introducing carbonate-bicabonate buffer (pH 9.0). Then, to get the

methacrylic groups on the polysaccharide chains and then desired -OH:NB ratio (1:5; 2:5; 4:5; 1:1), 1/8 (w/w) of
performing 3D printing by ultraviolet (UV)-initiated free the total norbornene anhydride (NB) was added to the
radical polymerization. solution. The reaction was left to proceed for 30 min
before adjustment of the pH to 9 with 1 M NaOH. Under
The thiol-ene hydrogels exhibited mechanical
properties closer to those of native fatty tissue compared continuous stirring, pH adjustment and sequential
addition of NB were repeated seven times with an interval
to acrylate and methacrylate functionalized hydrogels [39-41] . of 30 min. The solution was stirred at 50°C for another 5 h.
In addition, thiol-ene chemistry is simple, efficient, and Unreacted NB was separated by filtration. The mixture was
insensitive to environmental oxygen, and consumes then dialyzed against dH O using dialysis membrane with
reactive oxygen species (ROS) during its progressive a molecular weight (MW) cutoff of 8 kDa with frequent
2
growth polymerization, which has no negative effects change of water for 48 h at 40°C. The solution was freeze-
on cells. Inspired by this, we introduced norbornene dried to obtain a white product Pul-NB. The Pul-NB
photo-functional groups onto pullulan molecular chains, samples prepared with different desired ratio (1:5; 2:5; 4:5;
and a series of photo-curable hydrogels were prepared 1:1) of -OH:NB were abbreviated as Pul-NB 1, Pul-NB 2,
by thiol-ene click reaction. The composition of photo- Pul-NB 3, and Pul-NB 4, respectively.
functionalized pullulan (Pul-NB) ink for 3D printing was
optimized by adjusting the substitution degree (DS), Pul- To determine the degree of modification of the
NB concentration, and thiol crosslinking agent. The above pullulan backbone with norbornene functional groups,
1
ink material allows the printed objects to successfully proton nuclear magnetic resonance spectra (H NMR)

Volume 9 Issue 2 (2023) 105 https://doi.org/10.18063/ijb.v9i2.657