Page 158 - IJB-8-4

Recombinant Human Collagen for 3D Bioprinting of Skin Equivalent
10 (1:100, Abcam). After washing with PBS 3 times 3. Results and discussion
with 10 min between each wash, we added secondary
antibodies (goat-anti-mouse antibody 1:300 and goat- 3.1. Determining the suitable concentration
anti-rabbit antibody 1:400, Servicebio) and incubated the of GelMA for engineering the in vitro dermal
sections for 1 h at room temperature followed by PBS constructs
washing 3 times. Finally, the slides were counterstained We first optimized the concentration of GelMA, the base
with DAPI for 10 min at room temperature and then bioink content, regarding the construction of the dermal
washed with PBS 3 times before observation under a layer. We encapsulated HDFs in GelMA hydrogels at
confocal microscope. The K14 and K10 fluorescent varying concentrations (5, 7.5, and 10 wt%) and evaluated
intensity levels of the immunohistochemistry sections the activity of HDFs in vitro. The LIVE/DEAD
TM
were quantitatively analyzed using FIJI software. staining indicated that HDFs viability in all groups
was maintained over 90% throughout the 7-day culture
2.12. In vivo experiment (Figure 2A). The majority of HDFs presented a spherical
Eight-week-old SD rats were used, and all animal morphology at day 3 among all groups while displaying
experiments were performed according to the protocols a more elongated spreading morphology at day 7
approved by the National Institutes of Health Guide (Figure 2A). The CCK-8 assay revealed the proliferation
for the Care and Use of Laboratory Animals, China. of HDFs, and we found that 7.5 wt% GelMA supported
All experiments and procedures were approved by a significantly higher growth rate at day 7 (Figure 2B),
Laboratory Animal Research Center, Tsinghua University which was 1.3 and 1.5 fold of those obtained by 5 and
(SYXK 2019-0037). Intraperitoneal injection of 10% 10 wt% GelMA, respectively. This is in accordance with
chloral hydrate (3 mg/kg) was applied to anesthetize the the previous studies reporting that cellular processes
rat. Then, the hairs on the dorsal surface were shaved, and are highly affected by physical cues provided by the
[32]
the skin was sterilized with 75% alcohol. Full-thickness surrounding ECM . As supporting evidence, SEM
excisional wounds were created on the back of each rat images of lyophilized GelMA hydrogels demonstrated
by a 5 mm diameter biopsy punch. Bioinks were then that the pore size decreased with the increase of GelMA
carefully added to the trauma through injection, which concentration (Figure 2C and Figure S1), in agreement
[33]
was followed by UV crosslinking to gel the inks. Wounds with the previous studies . It should be noted that such
without any treatment were set as controls. All samples microporosity in the lyophilized state does not represent
were carefully covered with medical gauze and secured the nanoporosity generated by cross-linked polymer
[34]
with Tegaderm™ dressing (3M Health Care, Germany) mesh in the hydrogel state . The stiffness of the
immediately to protect them from dryness and self- GelMA hydrogels has been found to be another crucial
grooming damage. At days 3, 7, and 14 after the operation, parameter in regulating the proliferation of the HDFs.
photos of wound sites were taken, and the wound areas The mechanical compression tests demonstrated that 10
were determined using Matrix Laboratory (MATLAB wt% GelMA showed much higher compression modulus
R2019a). To determine the healing progress, we sacrificed (22.6 kPa) than 7.5 and 5 wt% GelMA did (7.5 and
all rats at day 14 and carefully excised wound sites with 3.5 kPa, respectively) (Figure S2). Too stiff hydrogel has
the surrounding skin. About 4% paraformaldehyde was been demonstrated to hinder cell activity, while too soft
used to fix the tissues overnight, and later, the tissues were hydrogel might face challenges in supporting prolonged
cell growth
. Similar to our results, a recent study
[35,36]
dehydrated in graded ethanol and embedded in paraffin. reported that, compared to 5 and 10 wt% GelMA
The embedded block was sectioned with 10 μm of hydrogel, HDFs cultured in 7.5 wt% GelMA hydrogel
thickness and stained with H&E and Masson’s trichrome showed the highest proliferation activities . Another
[37]
(MT) staining (Beyotime Institute of Biotechnology, study revealed increased remodeling and proliferation
China). Images of the H&E-stained tissue were taken activities in the 5 wt% GelMA group compared to GelMA
with microscopy (Eclipse TS100 Nikon) for further study.
hydrogels (10 and 15 wt%) with much higher stiffness .
[38]
2.13. Statistical analysis We further investigated the gene expression of
HDFs cultured in 5, 7.5, and 10 wt% GelMA hydrogels.
Unless otherwise stated, the data are presented as the Particularly, the expression of relevant genes, such as
mean ± standard deviation with triplicate or more for type I collagen (Col-I), TGF-β, vimentin, and α-SMA,
each condition throughout this study using GraphPad was examined by PCR analysis after culturing for 14 days
Prism 6. Statistical significance was assessed with a (Figure 2D). Col-I is the fundamental ECM structure
Student’s t-test, and significance was indicated with protein that plays critical roles, such as supporting and
*P < 0.05, **P < 0.01, and ***P < 0.001, where P ≥ 0.05 maintaining the integrity of the dermal architecture
was considered not statistically significant. in skin tissue . TGF-β and vimentin are known as
[39]
150 International Journal of Bioprinting (2022)–Volume 8, Issue 4