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Huang, et al.
and cell live/dead staining kit were obtained from 2.5. Proliferation assay
Dojindo Company (Japan). Other instruments include NIH-3T3 cells were cultured in DMEM containing 10%
fluorescence microscope from Leica (Germany), scanning FBS and 1% penicillin/streptomycin at 37°C under the
electron microscope (SEM) (TESCAN Company, Czech ambience of 5% CO . Cells were seeded on the scaffold
Republic), microplate reader (Thermo Company, USA), at a density of 1.0 × 10 per scaffold. In the control group,
2
5
and CPD1 3D bioprinter (Shangpu Boyuan Company, cells were grown on 48-well plates. Then, the CCK-8 kit
China). Sprague Dawley rats were obtained from Top was used to measure the number of cells at different time
Biotech Biotechnology Co. Ltd. (Shenzhen, China) or points.
Changzhou Cavens Biological Technology Co. Ltd. and
allowed to acclimatize for 1 week in the laboratory. 2.6. Subcutaneous implantation assay

2.2. 3D printing of the scaffolds All the animal experiments have been approved by the
ethics committee of Shenzhen Top Biotech Co. Ltd.
Gelatin and rColIII were mixed with the ratios of 1:0, (no. TOP-IACUC-2020-09-0007). Hydrogel samples
1:0.05, 1:0.1, 1:0.15, and 1:0.2 to form the hydrogel. were implanted subcutaneously in rats to evaluate
A model was built by 3D CAD file (.stl file format). biocompatibility in vivo. After the rats were anesthetized
We set the wire spacing of the model at 1.5 mm and with 1.5% isoflurane, the dorsal region was disinfected,
the layer height at 0.25 mm. Rectangular parallelepiped and an incision of about 2 cm was made. A sterile
specimens with dimensions of 20 mm × 20 mm × 3 mm hydrogel cube of 1 cm in length and width and 3 mm
(length × width × height) were used. Briefly, gelatin and in thickness was implanted. The incision was sutured
rColIII were sterilized by Co60 radiation. The gelatin with 4-0 sutures. After implantation for 4 weeks, the rats
powder was dissolved in ultrapure water (1 g gelatin were anesthetized and killed. The back hydrogel and
and 10 ml ultrapure water are mixed to make a 10% surrounding tissues were taken out together and fixed
solution, heated to 40°C, and stirred for 30 min), and with 4% paraformaldehyde. After dehydration, they were
rColIII was added in different proportions (0 g, 0.05 g, embedded in paraffin and cut into pathological sections.
0.10 g, 0.15 g, and 0.20 g). Before 3D printing, 1 ml The sections were stained with hematoxylin and eosin
of 10% transglutaminase aqueous solution was added (HE), and immunohistochemical staining was applied
for enzymatic crosslinking for 5 min. Then, 3D porous to quantify the inflammatory factors such as CD68,
hydrogel scaffolds with a size of 20 mm × 20 mm × interleukin (IL)-10, and tumor necrosis factor-alpha
3 mm were printed by a CPD1 3D printer. (TNF-α). Immunohistochemical staining was performed
as previously reported . The primary antibodies used
[39]
2.3. Porosity measurement in this experiment were anti-collagen I, anti-collagen
Porosity is defined as the volume of the pores over the III, anti-IL-10, anti-TNF-α, and anti-CD68 antibodies
total volume of the scaffold. Ethanol occupation was (Abcam, Cambridge, MA, UK).
used to estimate the volumes. The 3D scaffolds were
lyophilized for 48 h, and the dry weight was measured. 2.7. Cell live-dead staining
The scaffolds were then immersed in absolute ethanol and NIH-3T3 cells were inoculated into the hydrogel for 1,
placed under a vacuum until all the pores were filled. The 4, and 7 days in separate experiments. After inoculation,
weight of ethanol that occupies the pores is calculated as cells were then stained with calcein AM (2 μM) and
M . The weight of ethanol that corresponds to the total propidium iodide (3 μM) for 20 min at 37°C before
p
volume of the scaffold is calculated as M . Porosity is imaging under a fluorescence microscope.
T
then calculated as M /M . The samples were measured in
p
T
triplets. The moisture content is calculated based on the 2.8. Animal wound treatment
weight loss of the stent after freeze-drying. The swelling Briefly, male adult Sprague Dawley rats (180–220 g) were
curve of the material was calculated by measuring the kept in polypropylene cages with free access to water and
water absorption of the freeze-dried scaffold at different food. Animals were divided into four groups, and each
time points. group contained six animals: A, PBS control; B, GRH
0
2.4. SEM analysis group; C, GRH group; and D, GRH group. All rats
10
20
were anesthetized with sodium phenobarbitone before
Scaffolds were sliced into samples of 4 mm × 4 mm × 3 mm surgery. Then, the dorsal surface of rats was surgically
sizes, fixed in 2.5% glutaraldehyde solution, dehydrated manipulated to remove one full-thickness skin wound
gradually from 50% up to 100% ethanol, displaced with (diameter = 2 cm) under aseptic conditions. A 2 ml
isopropyl acetate, and freeze-dried for 48 h before being hydrogel materials were then applied to the surface of
processed for SEM imaging. the wounds and bandage, and the dressing was changed

International Journal of Bioprinting (2022)–Volume 8, Issue 2 15