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International Journal of Bioprinting Stiffness of scaffold-mediated immune response

studies focused on macrophage implantation onto the that M2 macrophages predominated around the scaffolds
surface of the hydrogel. 15,38 To better emulate the migration in the S1 group at days 7 and 14 after implantation, whereas
of macrophages into the scaffold after in vivo implantation, M1 macrophages predominated around the scaffolds in the
we chose to incorporate the cells directly into the hydrogel S3 group. From the above experiments, we clarified that
and subsequently print them. This approach provides a more macrophages tend to differentiate into a pro-inflammatory
realistic representation of the in vivo scenario and enables phenotype on higher-stiffness substrates and into an anti-
us to study the dynamic interaction between macrophages inflammatory phenotype on softer substrates.
and the 3D-bioprinted scaffolds. To further investigate the To further explore the mechanism behind the high
impact of stiffness on macrophages, we conducted Live- immune response triggered by stiffer scaffolds, RNA-Seq
Dead staining experiments and observed no significant analysis was conducted. PCA was performed to detect the
difference in macrophage survival across different stiffness correlation of the samples. There was an interaction of gene
conditions. However, we consistently observed that expression among the three groups at day 7, but the groups
macrophages exhibited a small, rounded morphology in were better separated at day 14. Differentially expressed
softer hydrogels while becoming larger and more irregular genes were mainly observed between the S1 and S3 groups
as stiffness increased. This morphological change suggests at day 14. Combined with the experimental results of
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that macrophages respond to different stiffness levels by the in vivo study, we mainly analyzed the differentially
altering their shape and size. Immunofluorescence staining expressed genes in the S3 and S1 groups at day 14. Notably,
was employed to evaluate macrophage polarization the S3 group exhibited significant upregulation of pro-
in response to stiffness. We found that macrophages inflammatory genes compared to the S1 group, including
cultured on softer matrices showed reduced polarization IL-1β, IL-6, TLR4, Myd88, and iNOS. IL-1β and iNOS
toward the M1 pro-inflammatory phenotype at an early are well-known pro-inflammatory factors that modulate
stage compared to those on stiffer matrices. Additionally, both innate and adaptive immune responses. 41,42 The top
macrophages in the softer hydrogels appeared to undergo two entries of GO analysis were immune system processes
an earlier conversion to the M2 anti-inflammatory and innate immune response, suggesting that the S3
phenotype. These results indicate that substrate stiffness not group mainly triggered the innate immune response. The
only affects macrophage morphology but also influences pattern recognition receptor (PRR) is an important part
their phenotypic polarization. Macrophages cultured of the innate immune response system that recognizes
on soft substrates maintained a round morphology and biomolecules with pathogen-associated molecular
exhibited an anti-inflammatory phenotype with increased pattern (PAMP) or damage-associated molecular pattern
phagocytic activity. Conversely, macrophages on high- (DAMP). PRRs consist of five families including Toll-like
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stiffness substrates displayed a spread-out morphology receptors (TLRs), RIG-I-like receptors (RLRs), nucleotide-
and shifted toward a pro-inflammatory phenotype with binding oligomerization domain (NOD)-like receptors
impaired phagocytic capabilities. Overall, our experiments (NLRs), C-type lectin receptors (CLRs), and DNA
established in vitro evidence indicating that substrate sensors. In this study, KEGG analyses revealed that NLR,
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stiffness plays a crucial role in macrophage behavior and TLR, TNF-α, NF-κB, and JAK-STAT signaling pathways
supporting the notion that macrophages tend to adopt an were significantly activated in the S3 group. TLR activation
anti-inflammatory phenotype on softer substrates and a is a host defense mechanism against infection and tissue
pro-inflammatory phenotype on harder substrates.
damage, Myd88 is a connector protein downstream of
To investigate the biological effects of scaffolds TLR4, and NF-κB signaling pathway plays a crucial role
with different stiffnesses after implantation in vivo, we in cell proliferation and differentiation as well as in the
utilized the mouse subcutaneous implantation model. regulation of immune and inflammatory responses. 45,46
A foreign body reaction and subsequent fibrous capsule When the NF-κB signaling pathway is activated, it induces
encapsulation are common occurrences following the production of pro-inflammatory factors such as IL-1β,
implantation. However, when an absorbable scaffold IL-6, and TNF-α, leading to an inflammatory response and
3,40
degrades, the fibrous capsule may eventually be absorbed restoration of damaged tissues. Subsequently, these pro-
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to achieve tissue repair. In the present study, we discovered inflammatory factors in turn activate the NF-κB signaling
that fibrous capsules formed after 7 days of implantation pathway and maintain the immune response. Therefore,
regardless of stiffness. After analyzing the macrophage we speculate that the mechanism of triggering higher
infiltration around the scaffolds in each group, we found immune response in S3 was mainly achieved through the
that the macrophage infiltration around the scaffolds TLR4/Myd88/NF-κB signaling pathway. In addition, the
increased with increasing scaffold stiffness at days 7 and 14 JAK-STAT signaling pathway was also activated in the S3
after implantation. Further typing of macrophages showed group, and IL-6, one of the activators of this pathway, was
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Volume 10 Issue 4 (2024) 350 doi: 10.36922/ijb.2874