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. 2023 Nov 28;15(49):56623-56638.
doi: 10.1021/acsami.3c09774. Online ahead of print.

Sustained Release of Dexamethasone from 3D-Printed Scaffolds Modulates Macrophage Activation and Enhances Osteogenic Differentiation

Affiliations

Sustained Release of Dexamethasone from 3D-Printed Scaffolds Modulates Macrophage Activation and Enhances Osteogenic Differentiation

Majed Majrashi et al. ACS Appl Mater Interfaces. .

Abstract

Enhancing osteogenesis via modulating immune cells is emerging as a new approach to address the current challenges in repairing bone defects and fractures. However, much remains unknown about the crosstalk between immune cells and osteolineage cells during bone formation. Moreover, biomaterial scaffold-based approaches to effectively modulate this crosstalk to favor bone healing are also lacking. This study is the first to investigate the interactions between macrophages and mesenchymal stem cells (MSCs) in co-cultures with the sustained release of an anti-inflammatory and pro-osteogenesis drug (dexamethasone) from three-dimensional (3D)-printed scaffolds. We successfully achieved the sustained release of dexamethasone from polycaprolactone (PCL) by adding the excipient-sucrose acetate isobutyrate (SAIB). Dexamethasone was released over 35 days in the 17-163 nM range. The osteogenic differentiation of MSCs was enhanced by M1 macrophages at early time points. The late-stage mineralization was dominated by dexamethasone, with little contribution from the macrophages. Besides confirming BMP-2 whose secretion was promoted by both dexamethasone and M1 macrophages as a soluble mediator for enhanced osteogenesis, IL-6 was found to be a possible new soluble factor that mediated osteogenesis in macrophage-MSC co-cultures. The phenotype switching from M1 to M2 was drastically enhanced by the scaffold-released dexamethasone but only marginally by the co-cultured MSCs. Our results offer new insight into macrophage-MSC crosstalk and demonstrate the potential of using drug-release scaffolds to both modulate inflammation and enhance bone regeneration.

Keywords: 3D printing; controlled-release; dexamethasone; immunomodulation; macrophages; mesenchymal stem cells; tissue engineering.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
SEM images representing (A) PCL scaffold, (B) PCL/DEX-CYD, (C) PCL/SAIB (70:30)/DEX-CYD, and (D) PCL/SAIB (60:40)/DEX-CYD. (E) Strut diameter and (F) pore sizes (between struts). Results are presented as mean ± SD (n = 8).
Figure 2
Figure 2
Cumulative and individual release at each collection time point in 10 mL of PBS. (A–C) 3D-printed PCL scaffolds loaded with 0.05, 0.1, and 0.25 wt % of dexamethasone. (D–F) 3D-printed scaffolds of PCL/DEX-CYD, PCL/ SAIB (70:30)/DEX-CYD, and PCL/SAIB (60:40)/DEX-CYD with 0.17 wt % dexamethasone. LOD, limit of detection. LOQ, limit of quantification. At each collection point, the whole release medium was replaced. Release medium was collected every 2 days after day 1. All data represent mean ± SD (n = 3).
Figure 3
Figure 3
Dexamethasone distribution within polymer struts analyzed by using ToF-SIMS. (A, B) Spectra showing the two dexamethasone ion peaks. (C) Image of a strut before and after depth profiling. (D) Ion images of different individual struts. (E–G) Depth profiling of dexamethasone molecular ion C22H27FO5 (orange) and fragment C21H26FO4 (blue), F (gray), PCL marker C6H9O2 (yellow), and SAIB marker C34H53O17 (green) in three different scaffolds (E-PCL + dex; F-PCL + dex + SAIB; G-PCL + SAIB).
Figure 4
Figure 4
Cell viability and proliferation of THP-1-derived macrophages in RPMI and co-culture media (both media supplemented with LPS + GM-CSF) with and without scaffolds in the media. All data represent mean ± SD (n ≥ 3).
Figure 5
Figure 5
Viability and proliferation of MSCs in α-MEM and co-culture media with or without drug-loaded scaffolds. DNA fold change was normalized to day 7. All data represent mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
Effect of dexamethasone concentration on macrophage polarization and viability. THP-1 cells were first differentiated into M0 macrophages with PMA. LPS, GM-CSF, and dexamethasone were then added together to the media (except the M0 control). All data represent mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Figure 7
Figure 7
Quantification of pro-inflammatory cytokines in macrophage culture with the presence of 3D-printed PCL, PCL/DEX-CYD, and PCL/SAIB (60:40)/DEX-CYD scaffolds for 3 and 7 days. The concentrations of TNF-α and IL-6 were quantified by ELISA and normalized to cell DNA content. All data represent mean ± SD (n ≥ 3). ** p < 0.01, **** p < 0.0001.
Figure 8
Figure 8
(A) ALP activity in different cultures over 7, 14, and 21 days (B) Alizarin red staining showing the mineralization in different cultures. Scale bar-100 μm. (C) Quantification of Alizarin red staining. (D) Gene expression of osteogenic marker RUNX2. (E) BMP-2 levels in different cultures. All data represent mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 9
Figure 9
Macrophage activation status in co-cultures. (A) Immunostaining of M1 marker calprotectin (red) and M2 marker mannose receptor (green) and cell nuclei (blue). Scale bar, 100 μm. (B) Quantified fluorescence intensity of calprotectin and mannose receptors. (C) Quantifying pro-inflammatory (IL-6) and anti-inflammatory cytokines (TGF-β1 and IL-10). All data represent mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 10
Figure 10
Responses of co-cultured macrophages and MSCs in 3D-printed scaffolds over 14 days. (A) Cell seeding efficiency in the porous scaffolds. (B) IL-6 and (C) TGF-β1 secretion from co-cultured cells at different times. All data represent mean ± SD (n ≥ 3). Comparison between drug-releasing scaffolds and drug-free scaffolds at each time point was made using Student’s t test. * p < 0.05, ** p < 0.01.

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