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Dong, et al.
3.4. Shear-thinning, thermosensitivity, and 3.5. Orientation structure alignment induces
printability of CN+HAMA hydrogel precursors directional cell alignment
The CN+HAMA hydrogel precursors before UV Ideal biomaterial inks should possess acceptable
cross-linking exhibited different properties under cytocompatibility. To investigate the cell safety of
different temperature treatments. Through rheological CN+HAMA hydrogels, we assessed the cytotoxicity
measurements and differential scanning calorimetry, with L929 cells. Based on the above evaluation of the
CN+HAMA hydrogel precursors demonstrated shear- printing effect, grid scaffolds printed with CN+1%HAMA
thinning and thermal response properties, which hydrogels were used to further investigate the
corresponded with the results shown in Figure 3. As biocompatibility of printed hydrogels. Therefore, we
further demonstrated in Figure 4a, the shear-thinning began testing the cell safety of the hydrogels after
behavior of hydrogel precursors at high shear rates, obtaining the printed scaffolds. The CN+1%HAMA
with the curves indicating that the precursors could be hydrogel-printed scaffold stabilized with UV irradiation
used for printing at equivalent shear rates. In addition, and then seeded with L929 cells. Specifically, to measure
Figure 4b shows the oscillatory temperature sweeps the viability of the cells, scaffolds were fluorescence
of the four different hydrogel precursors. For all imaged after 1, 3, and 7 days in culture by staining cells
samples, the G’ and G″ of the CN+HAMA hydrogels with calcein-AM (green) and ethidium homodimer (red)
precursors increased with the temperature, which can (Figures 5b-d and S5). The results demonstrated that the
be ascribed to the temperature-dependent property 3D-printed scaffolds supported cell adhesion by allowing
of the CN hydrogel precursors [42,43] . The CN and cells to extend projections. The cells adhered to the
CN+1%HAMA hydrogel precursors were in liquid scaffold and multiplied with increasing culture time.
states below 30°C. The data suggested that CN and The cytotoxicity results revealed that the difference
CN+1%HAMA hydrogel precursors gelled due to self- between the CN+1%HAMA hydrogels and the no pattern
association interactions between cellulose aggregates group was not significant, and compared with the other
above 37°C. Furthermore, at elevated temperatures, the types of hydrogels, the CN+1%HAMA hydrogels had
combination of LiOH and urea hydrate on the cellulose the highest cell viability (Figure 5e). To check the
chain was disturbed. Due to the self-binding force of cytoskeleton of the cells, scaffolds were also imaged
cellulose, cellulose molecules connected, forming a after 3 days (Figure S6) in culture by staining cells with
network structure. In addition, the CN+3%HAMA phalloidin (green) and DAPI (blue). As expected, the
and CN+5%HAMA hydrogel precursors were both morphology of L929 cells did not change significantly
in the gelation state due to their high concentrations. in the culture with 3D-printed scaffolds. These data
Collectively, CN+1%HAMA hydrogel precursors suggested that the printed CN+1%HAMA hydrogel
are appealing, because they can transit from a liquid scaffold had low in vitro cytotoxicity and no effect on cell
state at low temperatures to a gel state at elevated proliferation.
temperatures (Figure 4c). Compared with the other As shown in Figure S3, the prepared CN+1%HAMA
hydrogels, the temperature change results revealed that hydrogel scaffold had ridge and groove nanosurface.
the CN+1%HAMA precursors were clearly thermally When cells recognize the surface characteristics of
responsive. the hydrogel, they can respond to the micro-nano-
Due to their thermal responsiveness, CN+HAMA scale surface of the topological structure and produce a
hydrogel precursors could adjust and physically cross- contact guidance effect . The grooves of the material
[44]
linked during 3D printing by thermal gelation, which surface can affect the arrangement balance between cells
helped to maintain the shapes of the printed structures. and force cells to rearrange to adapt to the contacted
According to the rheological curves and thermal analysis material. Cells can adjust their size and orientation along
results, the thermosensitive CN+1%HAMA precursors the groove direction. The microstrips on the surface
exhibited the optimal printability at approximately of the hydrogel can regulate the signal transduction
30°C. To prove this, we printed hydrogels at various of cells and matrix, affecting the cell’s adhesion, the
concentrations and different temperatures. From development of cytoskeleton and the movement of the
Figure 4d, it was clear that the printed grid structure of cell, thereby forming highly-oriented cells patterns [45,46] .
CN+1%HAMA precursors had the most uniform lines and As shown in Figure 5g, the L929 cells were seeded on
most stable structure at 30°C. The printed structure of the the CN+1%HAMA hydrogel scaffold for 7 days and
other groups exhibited unclear lines and forms, because rearranged to adapt to the contacted hydrogel. Cells could
the concentration was so high that the ink coalesced and adjust their size and orientation along the groove direction,
was extruded unequally. The results again proved that eventually forming a cell arrangement layer parallel to the
CN+1%HAMA precursors were the most appropriate ink CN direction in the hydrogel (Figure 5a). The L929 cells
for 3D printing. grew and arranged in one direction, forming oriented

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