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

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.


Figure S3 :
Figure S3: Cumulative and individual release at each collection time point from 3D printed scaffolds in 10mL PBS.(A) Cumulative release from 3D printed scaffolds of PCL, PCL/L31 (70:30), and PCL/L31 (60:40) loaded with 0.17% wt/wt of DEX-CYD.(B) Cumulative release over 1 day.(C) Individual release at each collection time point.Release medium was collected every two days after day 1.All data represent mean ± SD (n=3).

Figure S4 :
Figure S4: Cumulative and individual release at each collection time point from 3D printed scaffolds in 10mL PBS.(A) Cumulative release percentage of 3D printed scaffolds of PCL, PCL/Span80 (70:30), and PCL/Span80 (60:40) loaded with 0.17% wt/wt of DEX-CYD.(B) Cumulative release percentage over 1 day.(C) Individual release at each collection time point.Release medium was collected every two days after day 1.All data represent mean ± SD (n=3).

Figure S5 :
Figure S5: The polarisation of M0, M1 and M2 macrophages derived from THP-1 monocytes and M0, M1, and M2 cytokine expression.(A) Immunostaining of macrophages polarised at days 3 and 7 in RPMI single-media.The scale bar is 40 µm, respectively.(B) Fluorescence intensity was quantified by measuring the integrated density of Calprotectin (M1) and Mannose (M2) positive cells; this was normalized against the total number of cells (DAPI) by ImageJ.(C) pro-inflammatory cytokines TNF-α, IL-6, and anti-inflammatory IL-10 quantification of macrophages polarised in RPMI single media were quantified by ELISA and normalized to DNA content.All data represent mean ± SD (n=3).Statistical analysis was performed by oneway analysis of variance with Tukey's post hoc test indicated statistical differences with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, respectively.

Table S1 :
Parameters determined by DSC for different ink formulations.

Table S4 :
(Figure 6in the main text) One-way ANOVA Pairwise comparison of the effect of dexamethasone concentration on IL1β production from macrophage.THP-1 cells were first differentiated into M0 macrophages.LPS, GM-CSF and dexamethasone were added to the media (Except the M0 control) at days 3 and 7.

Table S5 :
(Figure 6in the main text) One-way ANOVA Pairwise comparison of the effect of dexamethasone concentration on IL-6 production from macrophage.THP-1 cells were first differentiated into M0 macrophages.LPS, GM-CSF and dexamethasone were added to the media (Except the M0 control) at days 3 and 7.

Table S6 :
(Figure 6in the main text) One-way ANOVA Pairwise comparison of the effect of dexamethasone concentration on TNF-α production from macrophage.THP-1 cells were first differentiated into M0 macrophages.LPS, GM-CSF and dexamethasone were added to the media (Except the M0 control) at days 3 and 7.

Table S8 :
(Figure 7in the main text) One-way ANOVA Pairwise comparison of the effect of dexamethasone concentration on TNF-α production from macrophage.THP-1 cells were first differentiated into M0 macrophages.LPS, GM-CSF and PCL/DEX-CYD or PCL/SAIB (60:40)/DEX-CYD were added to the media (Except the M0 control) at days 3 and 7.