Page 405 - IJB-9-2

International Journal of Bioprinting 3D-printed skin substitute accelerates wound healing in vivo

diameter of 200 μm, pneumatic pressure: 0.8 – 1.2 bar, the sections were deparaffinized, immersed in potassium
moving speed: 3.2 – 5.6 mm/s, scaffold diameter: 8 mm, dichromate, stained with Ponceau staining solution,
string distance: 800 μm, layer height: 180 μm, and layer washed in phosphomolybdic acid, stained with aniline
number: 4 layers. The scaffold was exposed to 405 nm UV blue, and washed in 1% acetic acid. An optical microscope
irradiation for 4 – 5 s to make GelMA and HAMA polymer (Nikon Eclipse E100, Japan) was used for observation and
photo-crosslink after each layer was printed. dECM- the area percentage of collagen was analyzed by ImageJ
GelMA-HAMA skin substitute loaded with hADSCs were software.
temporarily immersed in DMEM/F12 supplemented with
10% (v/v) FBS and 1% (v/v) penicillin-streptomycin and 2.5.3. Immunohistochemistry staining
stored at 37°C in a 5% CO incubator for later use. CD-31 primary antigen used for immunohistochemistry
2
was purchased from Abcam (Cambridge, United Kingdom).
2.5. In vivo experiment After deparaffinization, repairing antigen and quenching
2.5.1. Wound healing model endogenous peroxidase, the sections were sealed with 3%
All animal experiments and surgical procedures were bovine serum albumin and incubated with CD-31 (diluted
approved by the animal ethics committee and performed in a ratio of 1:4000) primary antibody and secondary
following the code of practice for animal experimentation. antibody. 3, 3’-diaminobenzidine (G1211; ServiceBio Inc.,
Twenty-four 6-week-old male Balb/c mice were purchased Wuhan, China) was used for visualization. Cell nuclei
from experimental animal center of Chinese People’s were counterstained with hematoxylin. Five fields from
Liberation Army General Hospital. After anesthesia with 1% each sample were randomly selected and the number of
pentobarbital sodium (40 mg/kg), two circular excisional capillaries was counted.
full-thickness skin defect model with a diameter of 8 mm 2.5.4. Laser Doppler perfusion imaging
was established on the back of mice by trephine. Rubber
rings with the inner diameter of 8 mm was sutured with Laser Doppler perfusion imaging is a commonly used,
6-0 nylon around the wound to reduce skin contraction. noninvasive, repeatable method for assessing blood flow.
The mice were divided into four groups randomly: (A) full- The machine emits collimated laser beams to hit moving
thickness skin graft treatment group, (B) 3D-bioprinted red blood cells, so that the Doppler shifts in frequency.
skin substitute treatment group as the experimental group, A small percentage of these Doppler-shifted and non-
(C) microskin graft treatment group, and (D) control shifted beams are scattered backwards and processed into
group. The wounds were covered with semipermeable a color Doppler flow imaging proportional to blood flow.
membrane and bandaged, and dressings were placed on Measurements are expressed in perfusion units. At days
the wounds in each group to prevent dessication. Mice 7 and 14 after operation, the mice in each group were
were fed separately after operation. Wound images and anesthetized with 1% pentobarbital sodium and placed
wounds area were measured by ImageJ software on days 0, under the probe of laser Doppler perfusion imaging
7, 10, and 14 after the operation. The wound healing rate (PeriCam PSI, Perimed, Sweden). PIMSoft software was
was calculated by the following formula: used to select the wound site and record wound blood flow.
Wound healing rate = 2.6. Statistical Analysis
Wound area of day 0 − Wound area of day N ×1 00% The data are expressed as mean ± standard deviation. SPSS
Wound area of day 0 26.0 was used for statistical analysis. The Student’s t test
was used between two groups if the data were normally
2.5.2. Hematoxylin-eosin (HE) staining and Masson distributed and met the assumptions of homogeneity of
staining variance. One-way analysis of variance (ANOVA) was
used for comparison between multiple groups. P<0.05 was
On days 7 and 14 after the operation, two mice in considered statistically significant: *P < 0.05, **P < 0.01,
each group were sacrificed, and wound specimen was ***P < 0.001, ****P < 0.0001.
removed and fixed in 4% paraformaldehyde, dehydrated,
embedded in paraffin and cut into 5-μm-thick sections 3. Results
and HE staining was performed to observe the histological
structure and the quality of wound healing following the 3.1. hADSCs culture
staining steps. Masson staining was performed to observe The passage time of cell was 3 – 4 days. The hADSCs
the production and arrangement of collagen following the exhibited adherent growth ability, morphology of
staining instructions. The Masson staining was performed fibroblast-like cells and spindle-shaped appearance. The
using a Masson staining kit (HT15, Sigma, USA). Briefly, cell concentration was about 1 × 10 /mL.
8

Volume 9 Issue 2 (2023) 397 https://doi.org/10.18063/ijb.v9i2.674