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Dong, et al.
wide-angle X-ray diffraction (2D-WAXD) was performed. After coculture for 1, 3, and 7 days, cell counting kit-8
An in-house micro X-ray source (Incoatec, GmbH) with (CCK-8) solution was added to each well and incubated
Cu Kα radiation (λ = 0.154 nm) was used. The scattering for 2 h. Subsequently, the reaction solution transferred
signal was collected by a multiwire proportional chamber to another 96-well plate, the optical density (OD) of
detector (Bruker, Vantec-500) with a resolution of the solution was measured by a spectrophotometer at
2048*2048 pixels (pixel size of 68 μm). The distance 450 nm .
[38]
between the sample and detector was fixed at 1038 mm.
2.12. Animal experiment in vivo
2.8. 3D printing of the hydrogels All procedures were performed with the approval of
The hydrogel precursors were placed in a 5 mL syringe that the Animal Ethics Committee of Tsinghua Shenzhen
was fixed to the extrusion printhead. Biomaker software International Graduate School, Tsinghua University.
was used to design the printing model, and a 3D printer In vivo biosafety experiments were tested through
(SunP CPD1/Biomaker) was used to print the designed subcutaneous implantation of Kunming mice (KM).
hydrogel. A 25 G needle was used during the process. The CN+HAMA hydrogels were cut into slices 10 mm
The materials were printed at different temperatures, with in diameter and 1 mm in height. A small incision was
a printing speed of 5 mm/s, and an extrusion speed of made in the back of each mouse to insert a slice of the
0.7 mm³/s. The hydrogel underwent shear thinning inside hydrogel. The wound was stitched up after the hydrogel
the extrusion needle and regained its viscosity outside the was inserted. After 1 week, 3 weeks, and 10 weeks of
extrusion needle. Therefore, the hydrogel could be printed implantation, skin and mucous tissue on the wound
as one-dimensional (1D) filaments or 3D structures. After side, including the inserted hydrogel slices or not, were
printing, the printed structures were exposed to UV light removed to perform hematoxylin-eosin (H&E) staining
to enhance their stability. and immunofluorescence staining. H&E staining was
performed according to the manufacturer’s instructions.
2.9. Cell seeding and cell cultures on scaffolds Immunofluorescence staining was performed with CD3

Mouse fibroblasts (L929 cells) were cultured in Roswell (goat anti-rabbit, Servicebio, and GB13014) and CD68
Park Memorial Institute (RPMI 1640) medium with (goat anti-rabbit, Servicebio, and GB113109) as primary
10% fetal bovine serum at 37°C and 5% CO . Before antibodies, anti-goat as secondary antibodies, and DAPI
2
cell seeding, the CN+HAMA hydrogels scaffolds were (Servicebio, G1012). A fluorescence microscope (Leica,
immersed in 75% ethanol overnight and then exposed to DMi8) was used to image the immunofluorescence of the
UV light for 1 h. After UV irradiation, the scaffolds were tissue sections.
washed 3 times with phosphate-buffered saline (PBS). An 2.13. Statistical analysis
L929 cell suspension with a density of 3×10 cells/mL
6
was seeded onto the scaffold. The scaffolds were cultured All data were expressed as at least three independent
at 37°C with 5% CO , and the medium was changed experiments. Except that data analyzed in Figure 2o was
2
3 times per week. using one-way analysis of variance (ANOVA), all the
data elsewhere were analyzed using two-way ANOVA.
2.10. Live/dead staining and cytoskeletal staining Significant differences were indicated at *P < 0.05 and
of cell-seeded scaffolds **P < 0.01.

The culture medium was removed from the L929 3. Results and discussion
cell-seeded scaffolds, and then, the scaffolds were
washed with 1 × PBS 3 times (5 min each time). 3.1. The design of the CN+HAMA hydrogels
Calcein-AM/ethidium homodimer dye was added for As previously reported, the solvent alkali and urea can
15 min to stain the live/dead cells on the scaffolds. dissolve nanocellulose at low temperatures . Active
[39]
Phalloidin and DAPI dye were added for 1 h to stain the alkali hydrates were combined with cellulose and added
cytoskeleton of the cells on the scaffolds. The results were to the solvent. Urea hydrates served as hydrogen-bonding
observed with a fluorescence microscope (Leica, DMi8). donors and receptors, preventing cellulose molecules from

2.11. Cytotoxicity test coming into contact with one another. The nanocellulose
molecular chain was untangled, and the solvent had a good
L929 cells with a cell density of 4 × 10 /mL were seeded dispersion of nanocellulose (Figure 1a). While dissolving
4
into a 48-well plate and incubated for 24 h. Various cellulose, a thermosensitive phenomenon occurred, and
concentrations of the cross-linked CN+HAMA hydrogels the nanocellulose solution was in the liquid stage at low
scaffold sheets with a diameter of 8 mm and a thickness temperatures (≈ 4°C). When the temperature increased
of 2 mm were added to the cell medium in different wells. to body temperature (>30°C), the cellulose solution

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