PA-Zn@TiNPs Promote Osteogenesis by Inducing M2 Macrophage Polarization and Creating an Osteogenic Microenvironment

PA-Zn@TiNPs Promote Osteogenesis by Inducing M2 Macrophage Polarization and Creating an Osteogenic Microenvironment

During the resolution phase of inflammation, establishing a favorable mineralization and osteoimmune microenvironment at the bone-implant interface is crucial for osseointegration (24, 40). Additionally, successful osseointegration is vital in preventing implant-associated infection (IAI) (13). This study investigated the efficacy of bioactive ion-incorporated titanium nanoparticle coatings (PA-Zn@TiNPs) in promoting osteogenesis by inducing M2 macrophage polarization and fostering a pro-osteogenic microenvironment.

To assess the mineralization-inducing capability of the engineered materials, we immersed the samples in simulated body fluid (SBF) and monitored the formation of bone-like hydroxyapatite. On day 7, the bare Ti plate exhibited sparse distribution of point-like crystals that did not increase even by day 14 (Fig. 6A). In contrast, PA-Zn@TiNPs and CaO2/PA-Zn@TiNPs plates displayed abundant lath-like crystals that were uniformly distributed on day 7, with further growth and formation of a dense mineralized layer by day 14. EDS analysis revealed a Ca/P mole ratio of 1.51, which closely approximated the theoretical ratio of 1.67 for hydroxyapatite (Fig. 6B). Intriguingly, a substantial quantity of mineralized crystals was also observed on the TiNPs plate by day 14. This observation led us to speculate that the grooves present in the 3D structure of the TiNPs plate acted as reactors that facilitated ion-to-ion interactions.

We devised a hydrophilic surface with nanoscale roughness and the release of biologically active zinc ions (Zn2+), with the aim of promoting the activation of M2 macrophages (40, 41). LPS-stimulated macrophages were incubated on the surfaces of engineered implants and then macrophage M1/M2 polarization balance was studied (Fig. 6D). Immunofluorescence staining and flow cytometric analysis revealed an abundance of CD206-positive cells on PA-Zn@TiNPs, providing evidence that PA-Zn@TiNPs induced M2 macrophage polarization (Figs. 6, E and F). Enzyme-linked immunosorbent assay (ELISA) validation confirmed that PA-Zn@TiNPs augmented the secretion of the anti-inflammatory cytokine IL-10 while suppressing the pro-inflammatory cytokine TNF-α (fig. S12). The aforementioned findings strongly suggest that the Zn2+ ions released from PA-Zn@TiNPs, known for their anti-inflammatory properties (40), potentially play a crucial role in promoting M2 macrophage polarization.

Subsequently, RNA-seq transcriptomics analysis was conducted to elucidate the underlying mechanisms. The volcano plot diagram revealed the downregulation of a total of 267 differentially expressed genes (DEGs) and upregulation of 144 DEGs (fold change > 2.0 and p < 0.05) (fig. S13A). From the top 20 enriched pathways obtained through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the mitogen-activated protein kinase (MAPK) signaling pathway, which plays a key role in activating inflammatory responses, was significantly downregulated (fig. S13B). Western blot analysis further confirmed the significant reduction in both expression levels and phosphorylation status of the MAPK family (ERK1/2, JNK1/2, and P38) (fig. S13D). The results indicated that the downregulation of the MAPK pathway was associated with PA-Zn@TiNPs-induced M2 macrophage polarization. Enrichment of Gene Ontology (GO) terms revealed the involvement of the identified 411 DEGs in processes such as focal adhesion, protein binding, and inflammatory response. KEGG pathway analysis assigned the upregulated DEGs to terms associated with focal adhesion and the actin cytoskeleton, which aligned with the findings from the GO analysis (fig. S14A).

Furthermore, SEM micrographs revealed that cells on the PA-Zn@TiNPs plate exhibited a shuttle shape, displaying more peripheral filopodia compared to those on the TiNPs plate. In contrast, cells on the bare Ti plate appeared nearly spherical with smooth boundaries (fig. S14B). These morphological changes suggested that PA-Zn@TiNPs had the potential to promote M1-to-M2 macrophage transition (41).

Subsequently, we investigated whether a macrophage-conditioned medium could modulate bone marrow mesenchymal stem cells (BMSCs) towards osteogenic differentiation. qRT-PCR assay revealed upregulation of osteogenic genes, including ALP, osteopontin (OPN), and osteocalcin (OCN), in the PA-Zn@TiNPs group compared to other materials (Fig. 6G). Consistently, ARS staining demonstrated that neo-calcific nodules were more pronounced in the PA-Zn@TiNPs group than in other groups (Fig. 6H). Both the optical density acquired after alizarin red S (ARS) dissolution and the alkaline phosphatase (ALP) activity assay indicated a higher level of mineralization in the PA-Zn@TiNPs group compared to other groups (Fig. 6I).

In conclusion, the results above indicated that PA-Zn@TiNPs facilitated the transition of M2 macrophages, establishing an osteogenic microenvironment for BMSCs and ultimately leading to rapid and intensive osteogenesis. This study highlights the potential of PA-Zn@TiNPs as a promising biomaterial for enhancing bone regeneration and improving osseointegration of implants.