Page 148 - IJB-7-3

A Dual-Sensitive Hydrogel for 3D Printing
gel state. Blunt-tipped stainless-steel needles were used as an initiator. Here, four copolymers with different
here to reduce the influence of environmental temperature. PLGA lengths, P1, P2, P3, and P4, were prepared and
During the printing process, the temperature of the syringe investigated. Their molecular weight and molecular
and print bed was controlled at 37°C and an UV light lamp weight distribution were characterized using H NMR
1
(UltraFire, 501 B) was applied to photo-crosslink the inks. and GPC. Detailed data for the four copolymers are
listed in Table 1. To obtain the dual-sensitive hydrogels,
(2) Assessment of printability all the copolymers were then reacted with acrylic acid,
To assess and compare the printability of inks, lattice while only P4 with a shorter PLGA length could result
[29]
structures (20 × 20 × 0.5 mm ) were printed onto a glass plate. in a soluble polymer due to the end-group effect . To
3
1
The printing speed was set at 10 mm/s here and the pressure verify the success of the acylation, the H NMR spectrum
was adjusted between 0.05 MPa and 0.55 MPa according to of P4DA and its precursor was showed and compared
the situation. Various needle size (0.16, 0.21, 0.31, 0.41, and (Figure 1B). Different from P4, three new peaks were
0.51 mm) and line interval (0.8, 1.0, 1.2, 1.6 mm) were used observed between 5.8 and 6.6 ppm in the spectrum of
to optimize the printing parameters. Images were captured P4DA, corresponding to the three protons of acryloyl.
by a digital microscope (Dino-lite, FC-OC1) immediately This observation indicated that the hydroxy end groups
after the printing was completed. The line width and square were replaced with acryloyl groups, and the degree of
area of grids were measured in ImageJ. Diffusion rate (D ) substitution was calculated to be 102%. This conclusion
r
was calculated according to previous research by He et al. [48] was also supported by emerging characteristic peaks
related to acryloyl in IR and UV absorption spectrums
A − A (Figure S2).
D r = t A e 100%×
t
3.2. Thermo-sensitive sol-gel transition of
copolymer solutions
where A is the theoretical area of a square and A is
t
the experimentally measured area of the square. e Initially, the vial-inverting test was used to examine the
sol-gel behavior (Figure 2A), with the results displayed
(3) Printing of large constructs in Figure 2B. The phase transition diagram showed a
gel window of “U” shape, representing a sol-gel-sol
Centimeter-scale structures cuboid (20 × 20 × 4 mm ), process. P1 showed a gelation temperature range from
3
pyramid (20 × 20 × 10 mm ), and human nose (28 × 18 × 10 33 to 46°C, and its critical gelation concentration (CGC)
3
mm ) were printed using DA40. We designed the models was around 16 wt.%. With the decrease of molecular
3
of cuboid and pyramid, and the human nose structure weight, P2 and P3 got a narrower gel window and a
was retrieved from an open-source website (https://www. higher CGC (Table 1) due to less hydrophobicity of the
thingiverse.com/thing:306031) with a slight modification. polyester block. Although P4 was quite hydrophilic and
2.11. In vitro cell viability analysis soluble even at high temperature and high concentration,
P4DA underwent sol-gel-sol transition with a gel window
Human primary fibroblasts in cell culture medium with located at 37 – 52°C. Their differences in the macroscopic
copolymers at 5 mg/mL were added to a 96-well plate at a gelation behavior are associated with the increased
cell density of 500 cells/well. Subsequently, the cells were hydrophobicity induced by new end groups. To be a
incubated for 3 days at 37°C in the presence of 5% CO . bioink, it is necessary for a hydrogel to possess a wider
2
To evaluate the cell viabilities, a cytotoxicity assay kit gel window and a lower CGC; so that P1 and P4DA were
(KeyGEN BioTECH, China) was used to stain the cells. chosen for further studies.
Briefly, the cell culture medium was removed and washed The sol-gel transition behavior was also
with PBS for 3 times. 1 mL PBS containing 0.5 μL calcein investigated with rheological measurements. Typical
AM and 0.5 μL PI was added to the samples. The samples results of temperature sweeps for P1 and P4DA are
were then incubated for 45 min at room temperature. At shown in Figure 2C. Both the storage modulus (G’)
last, a fluorescence microscope (Leica, Germany) was and loss modulus (G’’) raised rapidly from low values
used to observe live/dead fibroblasts. to a peak around body temperature and then tended to
decrease. There were two crossover points in curves of
3. Results and discussion G’ and G’’, the first intersection implied a transition of

3.1. Synthesis and structure characterization of sol-gel and the second one was believed to be a gel-sol
the triblock copolymer transition. The temperatures at the two intersection points
were remarked as T and T , respectively. T and T
gel
sol
gel
sol
The triblock copolymers were synthesized through ring- of P4DA were determined to be 38.8 and 49.2°C, which
opening polymerization of LA and GA using PEG1500 was in agreement with previous results (Figure 2B).
144 International Journal of Bioprinting (2021)–Volume 7, Issue 3