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International Journal of Bioprinting 3D-bioprinted macrophage inflammation model

stiffness, mechanical force, pore size, availability of specific genes, namely IL-1β, IL-6, TNF-α, iNOS, and IL10,
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49
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oxygen (related to the diffusion coefficient of hydrogel), was significantly increased compared to that of untreated
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and cell-binding motifs, can stimulate M1 or M2 M0 cells. Based on gene expression, cytokine and surface
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polarization. Therefore, the influence of the 3D-bioprinted marker profile, and an optimized LPS dose of 1000 ng/mL, a
microenvironment toward the M1/M2 polarization of 3D-bioprinted model for the activation and polarization of
M0 macrophages was examined. Gene expression of THP-1 cells toward the M1 phenotype was demonstrated,
M1 markers (IL-1β, IL-6, iNOS, and TNF-α) and M2 with potential application to the study of pro-inflammatory
markers (CD206 and DC-SIGN) in M0 cells present in the systems and anti-inflammatory interventions.
3D-bioprinted constructs at 6 h, and days 1, 2, and 4 post-
printing was compared with the gene expression of M0 3.5. Anti-inflammatory properties of Ibu on
cells in 2D culture (Figure 2a). The expression of IL-1β and 3D-bioprinted model
iNOS in 3D-bioprinted macrophages was downregulated Ibu is a non-steroidal anti-inflammatory drug that
compared to 2D culture, while IL-6, TNF-α, CD206, and decreases cyclooxygenase activity and inhibits
DC-SIGN expression remained comparable to that in 2D prostaglandins, such as PGE , and other pro-
2
cultures, indicating that neither the bioprinting process inflammatory cytokines. 53-55 The efficacy of Ibu in
nor the 3D construct microenvironment stimulates reducing inflammation in the optimized 3D-bioprinted
macrophage polarization. As such, the findings show that “inflammatory M1” model was evaluated using qPCR,
M0 macrophage retains this phenotype, and the model is cytokine, and surface marker analyses described above.
suitable for activation studies. The M1-polarized constructs were incubated with 10 µg/
mL (Ibu1) and 15 µg/mL Ibu (Ibu2) (Figure 4). While no
3.4. Optimization of LPS dose for M1 polarization significant change in IL-1β and TNF-α gene expression
LPS has been shown to stimulate M1 polarization of THP- was observed in the first 24 h, both concentrations of Ibu
1 cells in 2D and 3D environments. 35,38,42 However, data significantly reduced iNOS, IL-6, IL-1β, and TNF-α gene
on the influence of LPS on 3D-bioprinted macrophages expression by day 3 of culture (Figure 4a–d). Notably,
are limited. Here, polarization of M0 cells to the M1 downregulated gene expression of iNOS at this time
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phenotype within 3D-bioprinted constructs was assessed point was comparable to iNOS expression in M0 controls
by measuring secreted levels of the pro-inflammatory (Figure 4c). The anti-inflammatory action of Ibu on
marker IL-6 in the presence of increasing concentrations bioprinted M1 cells was further demonstrated by flow
of LPS over 3 days in culture (Figure 2b). The increase cytometry targeting the cell surface marker CD80, with
in LPS dose resulted in an increased IL-6 release. LPS the percentage of CD80 cells in the CD11b CD33 CD80
+
+
+
+
(1000 ng/mL) treatment resulted in a sharp increase in (M1) population reduced approximately 2-fold
the release of IL-6 compared to the lower concentrations in the presence of Ibu, compared to M1 controls
(0–500 ng/mL). Secreted IL-6 levels in constructs (Figure 4e and f).
exposed to LPS concentrations over 1000 ng/mL were not Conditioned media from 3D-bioprinted constructs
significantly different statistically compared to constructs cultured in the presence or absence of Ibu were
in the presence of 1000 ng/mL. The expression of iNOS assayed by ELISA to determine the concentration of
by polarized cells was visualized using immunostaining, PGE and cytokines (IL-6, IL-1β, and TNF-α) released
with constructs treated with 1000 ng/mL comprising by 3D-bioprinted M0/M1 cells (Figure 5b–d). The
2
more iNOS-expressing cells (green color in Figure 2c), presence of Ibu in the culture medium significantly
confirming the successful polarization of cells toward the reduced PGE expression to levels lower than the M0
M1 phenotype at this LPS dose, within the 3D construct. 2
control at all three time points (Figure 5a). A similar
The expression of M1 surface marker CD80 was effect of both Ibu concentrations was observed for
quantified using flow cytometry (Figure 3). An increase in TNF-α and IL-6 release, although levels of these
LPS dose saw an associated increase in the percentage of cytokines remained above that of M0 controls (Figure
CD80 cells (Figure 3a and b). The highest concentrations 5b and d). Levels of IL-1β in conditioned media from
+
of 5 µg/mL and 10 µg/mL showed a similar CD80 M1 constructs exposed to Ibu after 2 and 3 days were
expression as that of 1000 ng/mL; therefore, the lower dose significantly lower than M1 without Ibu, although IL-
was selected for M1 polarization in the subsequent 3D 1β release remained markedly higher than M0 control
construct study. constructs (Figure 5c).
The effect of the selected LPS dose (1000 ng/mL) on the The efficacy of Ibu against inflammation stimulated
expression of M1 phenotype-associated genes at selected by the LPS produced by a different pathogenic bacterium
time points is shown in Figure 3c–g. Expression of M1- was also evaluated. P. gingivalis, an anaerobic gram-

Volume 10 Issue 2 (2024) 405 doi: 10.36922/ijb.2116