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Composite Scaffolds for Skin Repair
5, 8, 11, and 24. The scaffolds were changed at each DE microparticles were <20 μm in length with highly
time point. The area of wound sites was measured using regular nanoscale pore structure (Figure 1A and B).
ImageJ (NIH, USA), an image processing software. According to the X-ray diffraction (XRD) pattern,
The formula to calculate the relative wound area is as the peaks of 22.0° and 26.6° of DE could be indexed
follows: _ into SiO with the cristobalite (PDF No. 39-1425) and
2
Relative wound area (%) = A /A × 10 0 % quartz (PDF No. 46-1045) structure (Figure 1C). The
0
t
Where, A indicates the value of the wound bed area peaks of cristobalite and quartz between 20° and 40° are
_
t
at some point in time A and indicates the value of initial typical for DE [27,28] . In addition, the microparticles with
0
wound area (0 d). the concentrations of dispersion ≤50 μg/ml exhibited
After 14 days, the skin samples were obtained low cytotoxicity to HDFs and human umbilical
after sacrificing all of the mice and then fixed in 4% vascular endothelial cells (HUVECs) culturing for
paraformaldehyde. 5 days (Figure 1D and E), which confirmed the good
biocompatibility of DE.
2.11. Histological analysis of the wound healing
study 3.2. Characterization of DE-containing
biomaterial inks and 3D-printed DE-containing
The skin samples harvested from in vivo experiment composite scaffolds
were immersed in 4% paraformaldehyde for at least
24 h, then dehydrated, embedded in paraffin, and cut With the aim to develop an inorganic/organic composite
into sections with thickness of 6 μm. After drying, wound dressing for skin repair, GelMA pre-gel solution
the sections were baked at 60°C for 1 h, followed by was selected to serve as the matrix of the biomaterial
deparaffinizing to water and staining with hematoxylin ink for 3D printing. After that, DE microparticles
and eosin (H&E) (Sigma-Aldrich, USA), Masson’s were incorporated into the 6% (v/v) GelMA solution
Trichrome (Beyotime, China), and immunofluorescence in different ratios of 0, 5%, 10%, 20%, and 30% to
for histological analysis. For immunofluorescent CD31 obtain GelMA, 5%DE-GelMA, 10%DE-GelMA,
protein staining, the sections were immersed in 0.01 20%DE-GelMA, and 30%DE-GelMA composite inks.
M citrate buffer solution with pH of 6.0 at 99°C for Based on the previous studies, GelMA was defined
20 min for antigen retrieval. After cooling, the non- as a temperature-sensitive hydrogel material, which
specific binding was blocked by dropping 5% (v/v) could form reversible physical cross-linking at low
[29,30]
bovine serum albumin on the sections. Next, sliced temperature . From this perspective, modulus-
tissues were incubated with a diluted (1:200) CD31 temperature test was performed to determine the
primary antibody (ab28364, Abcam) solution overnight appropriate temperature for the fabrication of 3D-printed
at 4°C, and then, solution of green fluorescent secondary scaffolds with structure stability. The results showed
antibody was dropped and incubated for 1 h in the dark. that GelMA inks with/without DE changed from liquid
The observation of stained sections was performed using to gelatinous status when the temperature decreasing
a microscope (Model DMi8 S, Leica, Germany) and a below 15°C (Figure 2A). Therefore, the inks with ideal
formability were anticipated to allow for extrusion 3D
confocal laser scanning microscope (Model TCS SP8, printing at near 10°C. Besides, according to the result of
Leica, Germany). Moreover, the statistics of collagen flow behavior test, the decrease in viscosity with shear
area and vessel number were conducted using ImageJ rate varying from 0.1 to 10 s indicated the obvious
-1
(NIH, USA). shear thinning performance of the inorganic/organic
2.12. Statistical analysis composite inks, which made them suitable for extrusion
3D printing (Figure 2B).
The numerical data in this study are expressed as mean ± Next, the five inks were utilized to print 3D
standard deviation and analyzed by one-way analysis of porous scaffolds with different concentrations of DE,
variance using Origin 2017 software (OriginLab, USA). which were denoted as Gel, 5DE-Gel, 10DE-Gel,
Significant differences were denoted with *P < 0.05, 20DE-Gel, and 30DE-Gel, respectively (Figure 2C).
**P < 0.01, and ***P < 0.001. With the increasing of DE content, the transparency
of the scaffold gradually decreased. Then, the inside
3. Results and discussion morphology of these scaffolds was characterized by

3.1. Characterization of DE microparticles SEM. As shown in Figure 2D, the DE microparticles
were embedded in the scaffolds uniformly without
DE microparticles with small particle size were obvious aggregation. The incorporation of DE led to the
segregated through a dry sieving method using a 500 change of the inner walls of pore structure from smooth
mesh sieve. As observed by the SEM, the obtained to rough. In addition, the elemental mapping observed

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