Page 351 - IJB-10-4

International Journal of Bioprinting Stiffness of scaffold-mediated immune response

There was no significant difference in cell viability among fluorescence intensity of Arg1 was the lowest (Figure 5C).
the scaffolds at day 1 and day 7, respectively. However, the As the time of scaffold implantation increased, the
number of dead cells increased at day 7 compared to that fluorescence intensity of iNOS in the S3 group remained
at day 1 (P < 0.05). In general, the biocompatibility of all the strongest, and the fluorescence intensity of Arg1 was
scaffolds was favorable. the lowest among the three groups of scaffolds (Figure 6C).
The results suggested that the scaffold with high stiffness
3.3. In vitro immunoreaction of 3D-bioprinted triggered a more significant pro-inflammatory phenotype
scaffolds with different stiffness macrophage-related immune response.
It had been proved that stiffness had an impact on cell
fate. 28,33 In this part, the immunomodulatory activity To further explore the regulatory effect and potential
of 3D-bioprinted scaffolds with different stiffness mechanism of the scaffolds in vivo, sequencing of RNA
on macrophage polarization was tested in vitro by obtained from the subcutaneously implanted scaffold
immunofluorescence staining of iNOS and TNF-α comprising surrounding tissue in situ was performed.
(representing M1 pro-inflammatory phenotype), and Arg1 Principal component analysis (PCA) revealed that the
and IL-10 (representing M2 anti-inflammatory phenotype). genes of the three scaffolds interacted at day 7 after
As shown in Figure 3C, the Arg1 and IL-10 expression in implantation; the three scaffolds were better separated at
three scaffolds at day 1 after printing has no significant day 14 after implantation (Figure 7A). The result showed
difference, while the iNOS expression in scaffold S3 was that there were 2045 upregulated differentially expressed
higher than S1 and S2 (P < 0.05) and the TNF-α expression genes (DEGs) and 1013 downregulated DEGs when
in scaffold S3 was higher than S1 (P < 0.05), indicating that scaffold S3 was compared with S1 at day 14 (Figure 7B).
macrophage polarized into pro-inflammatory phenotype Venn diagram demonstrated that group S3 had the most
as scaffold’s stiffness increased. On the contrary, there was DEGs, which implied that the most heterogeneity existed
no significant difference in iNOS expression in all scaffolds between scaffolds S1 and S3 at day 14 after implantation
at day 3 after printing, while the TNF-α expression in (Figure 7C). In the volcano plot, we found a significant
scaffold S3 was higher than S1 (P < 0.05). But the Arg1 and elevation of the inflammatory factors IL-1β, IL-6, Myd88,
IL-10 expression in scaffold S1 was higher than in S3 (P and iNOS in the high-stiffness (S3) group, which are
< 0.05). In summary, macrophages at day 1 after printing involved in the innate immune response, and a similarly
showed a predominantly pro-inflammatory phenotype, high expression of the IL-17ra (Figure 7D). The gene
while macrophages with anti-inflammatory phenotype ontology (GO) analysis of biological processes in scaffold
increased significantly at day 3. S3 showed the high expression of “immune system process,”
“innate immune response,” “inflammatory response,”
3.4. In vivo immunoreaction of 3D-bioprinted and “immune response,” indicating the stronger innate
scaffolds with different stiffness immune response by high-stiffness scaffold implantation
To investigate the immune response of 3D-bioprinted (Figure 7E). Meanwhile, KEGG analysis also demonstrated
scaffolds in vivo, mouse subcutaneous implantation model the highly active pathways associated with the innate
was established. Figure 4A shows the experimental process. immune response, such as NOD-like receptor signaling
After 14 days of implantation, the three scaffolds were not pathway, Toll-like receptor signaling pathway, JAK-
completely degraded (Figure 4B). Meanwhile, the size of STAT signaling pathway, and NF-κB signaling pathway
the scaffolds in each group at day 14 was smaller than that (Figure 7F). In summary, RNA-seq showed that the innate
at day 7 (Figure S4 in Supplementary File). Otherwise, the immune response dominated the immune response in the
fibrous capsule of scaffolds had grown into the inner pore high-stiffness group at day 14 after implantation, possibly
with unclear boundaries at day 7 (Figure 4C). Then, CD68 triggered by the promotion of high pro-inflammatory
immunofluorescence staining was performed to observe factor expression through the NF-κB signaling pathway
macrophage infiltration in scaffolds (Figure 4D). There was and JAK-STAT signaling pathway (Figure 8).
no significant difference in the macrophages recruited by
the scaffolds at day 7 after implantation. However, at day 4. Discussion
14, the number of recruited macrophages was significantly
higher in scaffold S3 than in the other two groups In recent years, the field of tissue engineering has made
(P < 0.05, Figure 4E). iNOS, Arg1, and F4/80 co-staining significant advancements in wound healing and tissue
was detected by immunofluorescence for macrophage regeneration. However, one major challenge that remains is
polarizations (Figure 5A and B; Figure 6A and B). At day 7 the immune response triggered by implanted materials. The
after implantation, the fluorescence intensity of iNOS in the use of 3D-bioprinted scaffolds has provided a promising
S3 group was higher than that in the S1 and S2 groups, but the solution by creating a suitable microenvironment for cells

Volume 10 Issue 4 (2024) 343 doi: 10.36922/ijb.2874