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Dong, et al.
As shown in Figure 1b, CN+HAMA hydrogels CN+HAMA hydrogels were neater and more directionally
consisting of CNs and HAMA were designed to aligned after extrusion (π = 68%, f = 0.28) (Figure 2g)
achieve temperature/UV dual-responsiveness. The dual- from the nozzle than before extrusion (f = 0.06, Figure 2f),
responsiveness of the CN+HAMA hydrogels could induce which was consistent with the previous results . The
[25]
gelation in situ (body temperature). These properties pave results proved that CNs in the hydrogels can be aligned
the way for potential applications in soft-tissue repair. after extrusion from the nozzle.
In Figure 1c, the CN+HAMA mixture was in a liquid To further evaluate the orientation effects of the
state at a low temperature of 4°C and solidified as the CN+HAMA hydrogels, we used a Mueller matrix imaging
temperature increased to 37°C. When the nanocellulose microscope to analyze the inner fiber arrangement. The
inside the hydrogel was extruded through a 3D printing depolarization of the whole sample area was high before
nozzle, the internal structure was rearranged from a hydrogel extrusion (Figure 2i). In addition, the birefringent
random order to directional alignment. To optimize the samples had different directions and arrangements, and
degree of nanocellulose alignment, the shear stress of the internal structure was homogeneously arranged
the nozzle (τ max ) during extrusion was calculated with (Figure 2j). After hydrogel extrusion, the depolarization
the equation . τ= ΔP⋅r/2L, where ΔP is the maximum of the sample center area was reduced (Figure 2k)
[41]
pressure, applied at the nozzle (ΔP = 1.132 × 10 Pa). r is and the angle of the hydrogel aligned along the axial
6
the radius of the nozzle (r = 1.25 × 10 −4 m ), and L is the direction was reduced, with a fiber alignment angle of
nozzle length (L = 1.277 × 10 −2 m ). After the above formula approximately 20° (Figure 2l). The Mueller matrix data
calculation, we got the T max = 6460 Pa. As shown in Figure show that the fibers were arranged in the same direction
S2, the maximum pressure applied at the nozzle exceeded after extrusion, indicating that the alignment of the
the yield stress of each of the three concentrations (0%, CNs in the hydrogel was more regular than that before
1%, and 3%) of CN+HAMA hydrogels, resulting in extrusion. In addition, the H NMR spectrum of HAMA
1
differential flow (τ < τ max ). The nanocellulose in these shows the existence of peaks at 5.4 and 5.6 ppm, which
three hydrogel concentrations could be oriented after corresponded to the double bonds of methacrylamides
extrusion in 3D printing. The hydrogels were exposed to (Figure 2m). In addition, the mechanical properties of
secondary UV curing after extrusion from nozzle. the synthesized hydrogels were determined through the
compressive stress−strain curves and elastic moduli. As
3.2. Printed-induced quantified CN+HAMA demonstrated from the results (Figure 2n and o), the
alignment and characterization mechanical properties of the CN+1%HAMA hydrogels

To confirm our hypothesis, we first improved the were 50% higher than those of the CN hydrogels. As the
synthesis method of cellulose hydrogel and synthesized concentration of HAMA increased, the elastic moduli of
HAMA according to previously published reports . the CN+HAMA hydrogels increased.
[37]
Furthermore, we synthesized temperature-sensitive CNs 3.3. Temperature and UV dual-responsiveness of
by utilizing the temperature change during the synthesis CN+HAMA hydrogels
process. SEM images show that the synthesized cellulose
fibers were neatly oriented after extrusion and that HAMA In addition to the directional arrangement mode,
had a honeycomb porous structure (Figure 2a-d). The the CN+HAMA hydrogels demonstrated reversible
inner structure of CN+HAMA composite hydrogels after temperature sensitivity. The CN+HAMA hydrogels were
extrusion had neat and directional alignment. Furthermore, treated with different temperatures and UV conditions,
as shown in Figure S3, the prepared CN+1%HAMA and the hydrogels showed different gel states, which
hydrogel structure had a grooved and ridged nanosurface, indirectly proved their dual-responsiveness (UV response
the inner structures are nanofibers directional alignment. and thermal response). Figure 3a shows that the CN-
To explore the flow-induced orientation of related hydrogel precursors underwent reversible solution-
CN+HAMA hydrogels, we carried out two-dimensional gelation conversion with as the temperature changed. When
(2D)-WAXD measurements and Mueller matrix imaging the temperature was low, the hydrogel precursors were in
of the CN+HAMA hydrogels before and after extrusion. a solution state similar to a liquid; when the temperature
The CNs disordered inside the container and gradually was high, at approximately 30°C, they formed solid gels.
aligned during the extrusion process (Figure 2e). As Next, we tested the UV (intensity=15 mW/cm ) response
2
shown in Figure 2h, the degree of orientation (π) performance of the hydrogels at 4°C (Figure 3b). The
was calculated based on the azimuthal integration. In CN-related hydrogels maintained their state regardless of
addition, the Herman’s order parameter (f) value reflects how long they were exposed to UV light. In contrast, the
the orientation degree: the larger the f value is, the greater HAMA-related hydrogels solidified under UV irradiation
the orientation of the nanocellulose alignment. According for various times, after which the hydrogels remained in
to the calculations shown in the Supplementary File, the a solid gel state. Figure 3b indicates that the curing time

International Journal of Bioprinting (2022)–Volume 8, Issue 3 131