Page 118 - IJB-7-4

CNC-enhanced Hydrogels for 3D Bioprinting
(Shanghai, China) and used without further purification, 2.6. Scanning electron microscopy (SEM)
unless otherwise specified.
The copolymers-formed hydrogels (20 wt%) were
2.2. Synthesis of the triblock copolymer lyophilized at -50°C under 10 Pa (Vacuum freeze dryer,
FD-1A-50, BiLon, Shanghai, China) overnight, which
The PCLA-PEG-PCLAs were all synthesized through was followed by metal-spraying using an MSP-2S, an
ring-opening polymerization. Taking PCA as an example, ion sputtering instrument (EIKO Corporation, Japan) for
1
3 g dihydroxyl PEG (M = 6 000 Da, 0.5 mmol), 1.14 g 1 min. The SEM images were obtained through a Hitachi
n
ε-CL (1.11 mL, 10 mmol), 1.44 g LLA (10 mmol), and S-4800 SEM instrument.
3% Sn (Oct) were added into a blank round-bottom flask.
2
The mixture was melted at 60°C and then purified with 2.7. Degradation properties
argon under stirring for 3 times. The polymerization was 20 wt% PCA hydrogel was chosen to study the
conducted under vacuum at 160°C for 8 h. The reaction degradation properties. 0.5 g of PCA was added to a
2
was terminated at –20°C. The obtained crude product was vial and 1 mL phosphate buffer (PB) containing 0.1 mg
2
dissolved in around 5 mL CH Cl and precipitated in over lipase (Aladdin, Shanghai, China) was subsequently
2
2
100 mL diethyl ether for 3 times. The solid product was added. The experiments were conducted in triplicates at
then dried in a vacuum oven overnight. 37°C and the buffer was refreshed every day. The residual
2.3. Characterization of copolymer structure samples were taken out and lyophilized at specific time
intervals. The weight of each sample was recorded and
1 H NMR spectra of triblock copolymers were recorded the average value of weight ratio of weight loss and the
using a Brucker Avance-400 nuclear magnetic resonance original weight before incubation was calculated as the
instrument. The solvent was CDCl . The gel permeation degradation rate for the analysis.
3
chromatography (GPC) was determined through Waters
PL-GPC-50 instrument. The eluent was THF with a flow 2.8. 3D printability of hydrogels
rate of 1.0 mL/min. The extrusion-based 3D printing was carried out with

2.4. Vial-inverting test an instrument of Bio-Architect® PRO (Regenovo). The
hydrogels were filled into the barrels and then placed into
The enhancement effect of CNC on phase transition an oven at 37°C overnight to eliminate the bubbles. The
of hydrogels was evaluated by vial-inverting test. The corresponding GCodes and software were supported by
hydrogels of the copolymers were prepared at the Regenovo. The filament collapse experiments and micro-
concentration of 20 wt% while 0, 2.2, 4.4, and 8.8 wt% extrusion were performed by adjusting the nozzle size, the
CNC were used. The vials were incubated in a water bath. pneumatic pressure value, and translational speed. Taking
The temperature was regulated from 25 to 70°C with an the 20 wt% PCA +4.4 wt% CNC gel with the 0.41 mm
2
increment of 1°C per step. Each sample was equilibrated of nozzle as an example, the thickness was set as 0.4 mm
for 5 min at each temperature and the state was evaluated. and the 3D constructs were printed at 37°C under 0.3
Once a flowable or dehydrated state was observed after MPa of air pressure with 10 mm/s of transitional speed.
inverting the vial, the transition temperature would be
recorded. 2.9. In vitro cytotoxicity assay

2.5. Rheological experiments Primary human fibroblasts (HDFs) were used for
evaluation. HDFs were obtained from discarded human
The rheological properties of hydrogels were foreskin and used until 4–6 passage. The cell seeding
th
characterized through a TA ARES-G2 instrument using a density was 500/well. The Dulbecco’s modified Eagle’s
25 mm parallel plate geometry. The samples were loaded medium, Sigma, USA was used as culture medium.
on the Peltier platform. Temperature sweep experiments The cytocompatibility of the copolymers with
were conducted at 1 Hz from 25 to 75°C with a ramp rate different concentrations was evaluated through staining
of 3°C/min. Frequency sweep experiments were carried with cytotoxicity assay kit (KeyGEN BioTECH,
out at 25°C from 0.1 to 100 rad/s with a strain value of China). The culture medium was removed and washed
0.5%. Strain sweep experiments (25°C) were performed with PB saline (PBS) for 3 times. The work solution
at a strain ranging from 0.01% to 1000% with a frequency (0.5 μL propidium iodide (PI) + 0.5 μL calcein
value of 1 Hz. Shear thinning tests was carried out at acetoxymethyl ester (AM) in 1 mL PBS) was added into the
37°C with a frequency value of 1 Hz. The shear rates samples and then incubated for 45 min at room temperature.
were from 0.01 to 100 rad/s. The thixotropy experiments The fluorescent images were observed through fluorescence
(25°C) were performed at an alternating strain of 0.01% microscope (Leica, Germany); the living and dead cells
and 100% for 100 s, respectively, per cycle. showed as green and red regions, respectively.

114 International Journal of Bioprinting (2021)–Volume 7, Issue 4