(I-356) Intracellular Magnetic Force-Induced Modulation of Macrophages Phenotype for Bone Regeneration and Fracture Healing

Introduction:: Persistent inflammation is a crucial cause of the pathogenesis of musculoskeletal diseases such as fragility, fracture, and nonunion. Studies have shown that modulating the immune phenotype of macrophages from pro-inflammatory to pro-healing mode can heal recalcitrant bone defects. Macrophages adopt discrete functional phenotypes at different stages of fracture healing, playing an indispensable role in bone regeneration. However, relatively less known is how physical cues in the fracture microenvironment might modulate macrophage polarization. Current therapeutic strategies predominantly apply biochemical cues, which often lack target specificity, and controlling their release kinetics in-vivo is challenging spatially and temporally. Here, we have developed a magnetic iron-oxide nanocomplexes (MNC)-based immunomodulatory strategy targeting macrophage for fracture healing. The MNC-loaded macrophages, when coupled with an external magnetic field, can exert an intracellular magnetic force on the cytoskeleton, promoting their functional phenotype from pro-inflammatory to prohealing. Biophysical manipulation through engineered MNC can elicit transcriptional control of macrophage phenotype by regulating cytoplasm to nuclear translocation of actin cytoskeleton-dependent transcription factors (MRTF-A) and epigenetic markers (HDAC3), which drives the expression of inflammatory genes. These findings may facilitate efforts to develop MNC-based resolution-centric therapeutic intervention to direct macrophage phenotype and function towards healing to supplement or replace the currently used anti-inflammatory therapies (Fig.1A).


Materials and Methods:: Magnetite nanocrystals were synthesized by thermos-decomposition of iron acetylacetonate. Water-dispersible MNCs were generated by coating 15 nm magnetite nanocrystals with phospholipid-PEG copolymers. Then the MNCs were labeled with a fluorophore, DiI. Electron micrographs were used to determine the size distribution of MNC. The macrophage uptake kinetics was quantified through ferrozine assay. The IC-21 murine peritoneal macrophage cell line was used in this study. LPS/IFN and Il-4/Il-13 were added to media to polarize macrophage to pro-inflammatory (M1-like) and Prohealing (M2-like) phenotypes, respectively. The cellular uptake rate of MNC by macrophage immunotypes was quantified by flow cytometry. The subcellular localization of MNC was analyzed by Lysotracker live staining, and their cytocompatibility was characterized by Presto blue assay. To access the ROS production by MNC, we measured the nitric oxide levels in macrophages cultured with and without MNC using the Griess assay. A magnetic field gradient was created using an array of cylindrical neodymium magnets (3.175 mm), and the field strength was determined by numerical simulation. Then macrophage phenotype with and without an external magnetic field was investigated through mRNA quantification. Cytoskeletal dynamics and protein expression profile of macrophages were characterized using immunofluorescent staining. A micropatterned substrate with fibronectin coating was developed to enrich cells in the areas of maximum magnetic force gradient. The cells subjected to intracellular magnetic force were then analyzed using qPCR for phenotype changes. For mechanistic validation studies, Latrunculin- A and RGFP966 were used to inhibit actin polymerization and HDAC3, respectively.

Results, Conclusions, and Discussions:: The MNCs were rapidly internalized by the macrophages when supplemented in culture media within a few minutes, and the rate of uptake varied with their immunophenotype (Fig.1B). The MNCs accumulated in lysosomes around the nucleus and did not affect cell viability or metabolism of macrophages even at high concentrations (100 μg/mL). The ferrozine assay showed that macrophages can uptake more than 1x10⁶ MNC within two hours of incubation. Our numerical simulation showed that 1x10⁶ MNC could effortlessly exert an intracellular force of 0.1 - 0.7 nN, which is sufficient to modulate the cytoskeleton of macrophages.

When coupled with an external magnetic field, we observed an elongated morphology along the magnetic gradient (Fig.1C). The intercellular magnetic force redistributed MNCs that accumulated in the cytoplasmic vesicles perturbing the actin cytoskeleton, which was evident from a significant reduction in F/G-actin levels in the cytoplasm (Fig.1D). Further, this change in cytoskeletal dynamics reduced the nuclear translocation of the transcription factor MRTF-A (Fig.1E), and the chromatin remodeling enzyme HDAC3 (Fig.1F). To study the functional implications of the changes causes by MNCs coupled with an external magnet, we stamped fibronectin on a hydrophobic substrate and enriched cells in areas of high magnetic field gradient for transcriptomic analysis. The analysis of the total RNA transcripts showed that compared to the control with no magnet, Nos2, a prominent M1 marker, was significantly downregulated in macrophages subjected to an intracellular magnetic force (Fig.1G). Moreover, upregulation of Tgf-b (p=0.09) expression in macrophages with an external magnet was also observed.

To mechanistically confirm these phenotypic changes, we used small molecule inhibitors of actin polymerization, which significantly increased circularity, and Bmp2 expression in macrophages. On the other hand, inhibition of HDAC3 significantly reduced circularity and increased anti-inflammatory genes, Pparg and Chil3, along with Bmp2. Although these studies did not directly reflect MNC-mediated phenotype modulation, they confirmed the suppression of the pro-inflammatory phenotype through cytoskeletal modifications.

Overall, MNCs are a potent tool to manipulate actin cytoskeleton-driven transcription machinery to modulate inflammation. Our work provides crucial insights into the mechanobiology of macrophages and yields a potent immunomodulatory therapy for recalcitrant wounds.


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