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Cui, et al.
The cytocompatibility of hydrogels was also the calculation of integral area in H NMR spectra
1
investigated. 20 wt% PCA +4.4 wt% hydrogel was (Figure 2A), the chain length of each polyester block
2
chosen for the investigation while cells were seeded was around 2000 Da. That means the block ratio of
on its surface. The following process was identical to polyester decreased with the increase of PEG chains.
the previous procedure. The fluorescent images were Meanwhile, the molar ratio of the constitutional
captured after 12-h co-incubation. repeating unit between CL and LA was calculated,
(c) The cell viability was also carried out using Cell respectively, as 0.98, 0.9, and 1.08, which were close
Counting Kit-8 (CCK-8) cell viability assay. The CCK-8 to the equal distribution of 1:1, ensuring the amorphous
stock solution (Dojindo, Kumamoto, Japan) was diluted state of the hydrophobic block. In addition, the GPC
(1:10) in culture medium to form a working solution. results illustrated that the molecular weight of all
The human primary fibroblasts and copolymer at a given copolymers was narrowly distributed (Figure 2B).
concentration were submerged for 2 h. 100 μL of the
submerged solution was extracted and transferred into 3.2. Gel-sol transition of the CNC-enhanced
a plate of 96 wells. The measurements were conducted copolymer gels
using a microplate reader (BioTek Instrument, USA)
with the absorbance at 450 nm. The samples were The hydrogels formed by these copolymers exhibited
washed with PBS until the working solution was totally thermal-sensitive properties with gel-sol transition, that
removed; and fresh medium was added subsequently for is, these gels have a lower critical gelation temperature
incubation. (LCGT). As the temperature is lower than LCGT, the
system is in a gel state. When the temperature is above
3. Results and discussion LCGT, lower concentration gels transform into a flowable
sol state and the gels of higher concentration will dehydrate
3.1. Synthesis and properties of the copolymers and precipitate. Therefore, LCGT can effectively reflect the
According to our previous study, dihydroxy terminated thermal stability of the hydrogels. Since the polyester block
PEGs with different molecular weight (6, 8, and 10 kDa) is an amorphous block formed by the copolymerization of
were chosen as the macroinitiator. By regulating the two monomers, the formed hydrogel often has a low LCGT
feed ratio, the molecular weight of polyester was and is not as stable as a gel formed by a crystalline copolymer
controlled around 2 kDa. Thus, three PCLA-PEG- with the same molecular weight. Thus, CNCs with different
PCLA copolymers with different chain lengths, named, concentrations were introduced to the hydrogels (20 wt%)
respectively, as PCA , PCA and PCA , were prepared to evaluate the enhancement effect (Figure 3A). It can be
3
2
1
via ring-opening polymerization using Sn(Oct) as the seen that the overall LCGT was at a relatively low level in
2
catalyst (Figure 1). To ensure the elimination of the the absence of CNC, and as the molecular weight of PEG
intrinsic crystallinity of polyester, the constitutional increases, the LCGT gradually decreases. The reduction of
repeating unit between caprolactone (CL) and lactide hydrophobic block ratio will be detrimental to the thermal
(LA) were adjusted to the same molar number. The stability of gels, which is consistent with our previous
obtained samples were characterized through H study . The thermal stability of the hydrogels is improved
1
[16]
NMR and GPC, which is shown in Table 1. Through to varying degrees when different concentrations of CNC

Figure 1. The synthesis strategy of PCLA-PEG-PCLA triblock copolymers.

Table 1. Properties of PCLA-PEG-PCLA triblock copolymers
Samples M n,NMR a,c M n,GPC b M /M n b Polyester/PEG (M /M ) CL/LA (mol/mol)
a
a
n
w
n
PCA 1 2 170-6 000-2 170 10 500 1.01 0.72 0.98
PCA 2 1 820-8 000-1 820 13 400 1.11 0.46 0.9
PCA 1 880-10 000-1 880 16 100 1.25 0.38 1.08
b
a M n,NMR 3 , polyester/PEG ratios and CL/LA ratios of the copolymers were calculated from 1H NMR spectra. M n,GPC and M /M of copolymers were calculated
w
n
by GPC. The difference among the PCA , PCA and PCA was the molecular weight of PEG, which was 6, 8, and 10 kDa, respectively, while the hydrophobic
c
1
2
3
block was kept at around 2 kDa for the convenience of subsequent comparison
International Journal of Bioprinting (2021)–Volume 7, Issue 4 115