Page 348 - IJB-10-4

International Journal of Bioprinting Stiffness of scaffold-mediated immune response

fluorescence) and dead cells (red fluorescence). Three for 7 or 14 days, the mice were sacrificed, and the scaffolds
independent visual fields under the microscope were with skin were harvested for further assessment. After
randomly selected for Live/Dead calculation. Each group being fixed in 4% paraformaldehyde for 6 h, all the samples
was conducted with three parallel samples. were dehydrated and embedded in paraffin for further
slicing and hematoxylin and eosin (H&E) staining. For
2.6. Cell harvesting from the scaffolds immunostaining, antigen retrieval was first performed on
The strategy for cell harvesting was based on our previous paraffin sections. The scaffolds were prepared following the
study. Sodium citrate (55 mM), 20 mM EDTA (Sigma- protocol in section 2.3. The rest of the steps were the same
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Aldrich, USA), and 150 mM sodium chloride (Sigma- as the protocol described in section 2.7.
Aldrich, USA) were added into 500 mL ddH O. The
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solution was shaken in a shaker until it was completely 2.9. RNA-seq analysis
dissolved. The scaffolds were dissolved in solution for 15 The total RNA of scaffolds and mouse back skin was
min while gently shaking the centrifugal tube for better extracted using TRIzol (Invitrogen). RNA sequencing
dissolving. The cell suspensions were centrifuged at 1500 analysis was performed with HiSeq 2500 (Illumina, USA).
rpm for 10 min, and the supernatant liquid was removed Gene with P < 0.05 and fold change >1.2 were considered
to harvest cells for further analysis. to be significant. Bioinformatic analysis was performed
using tools obtained from online sources: https://www.
2.7. Macrophage polarization in vitro omicstudio.cn/tool, https://www.bioinformatics.com.cn,
The M1 and M2 polarization markers of macrophage and https://metascape.org.
were detected by immunofluorescence staining. The
harvested cells were resuspended with a solution of 4% 2.10. Statistical analysis
paraformaldehyde, then transferred onto adhesive slides, The data are expressed as means ± standard deviation and
and dried at 60°C for 15 min. The slides were infiltrated were processed using the statistical software GraphPad
with 0.3% Triton X-100 (Sigma-Aldrich, USA) for 15 min Prism 8.0. One-way analysis of variance (ANOVA) was
and washed three times with PBS. To block non-specific performed to compare the differences between multiple
protein interaction, the slides were incubated in 5% goat groups at the same time point. Two-way ANOVA was
serum (Zsbio, China) prepared in PBS for 1 h. After the performed to compare the differences between multiple
slides were incubated with anti-iNOS antibody (1:500, groups at the different time point. Statistical significance
ab178945, Abcam, USA), anti-TNF alpha antibody (1:1000, was considered when *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001,
ab66579, Abcam, USA), anti-Liver Arginase antibody and ****P ≤ 0.0001.
(1:500, ab96183, Abcam, USA), and anti-IL-10 antibody
(1:300, ab133575, Abcam, USA) for 15 h at 4°C. After being 3. Results
washed three times with PBS, the slides were immersed in
the CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L) 3.1. Physical properties of bioinks with
(1:200, SA00013–4, Proteintech, USA) for 2 h in the dark different stiffness
at room temperature. Nuclei were counterstained with In this study, we used alginate–gelatin bioinks (named
DAPI Fluoromount-G (0100-20, Southern Biotech, USA). bioinks S1, S2, and S3), which were commonly used in 3D
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Fluorescence images were collected using a laser scanning bioprinting research. During the preparation process, we
confocal microscope (Leica, SP8 FALCON, Germany). found that at room temperature (25°C) and 37°C, Bioink S1
had good fluidity, while bioink S3 appeared as a thick liquid
2.8. Immunoreaction evaluation of the 3D-bioprinted (Figure 1A). Rheological tests indicated that the viscosity
scaffold in vivo of all bioinks decreased with the shear rate increasing
All animal experiments were approved by the Institutional at bioprinting temperature (10°C), demonstrating the
Animal Care and Use Committee of Chinese PLA General shear-thinning property of the bioinks (Figure 1B). The
Hospital (approval number SCXK(BJ)2017–0019). To compression modulus of bioinks was tested and found to
explore the immune response induced by hydrogel be consistent with our previous study. The compression
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scaffolds with different stiffness in vivo, female C57BL/6 modulus and stiffness increased with the solute
mice aged 8 weeks were obtained from SPF Biotechnology concentration in bioink (S1 < S2 < S3; Figure 1C and E).
Company (China) and served as subcutaneous In addition, the result of SEM showed that the bioinks had
implantation subjects. After the general anesthesia, a 1.5 the same interconnected porous structures (Figure 1D).
cm subcutaneous incision was cut on the mouse’s back. A Meanwhile, the pore size decreased (S1 > S2 > S3) with the
subcutaneous cavity was formed by blunt separation and increase of stiffness (P < 0.05, Figure 1F). However, there
then implanted with different scaffolds. Each group was was a slight difference in porosity among the three bioinks
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conducted with three parallel samples. After implantation with no statistical significance (Figure 1G).

Volume 10 Issue 4 (2024) 340 doi: 10.36922/ijb.2874