Activating macrophages for enhanced osteogenic and bactericidal performance by Cu ion release from micro/nano-topographical coating on a titanium substrate

In the field of orthopaedics, inflammation-modulatory biomaterials are receiving increasing attentions due to their abilities to regulate innate immune response and mediate wound healing. In the current work, a Cu-containing micro/nano-topographical bio-ceramic surface (Cu-Hier-Ti surface) was employed as material model to explore the role played by Cu2+ release or material surface in regulating macrophage polarization as well as macrophage-mediated osteogenic and bactericidal effect. A Cu-free micro-topographical surface (Micro-Ti surface) generated by micro-arc oxidation was employed as control. The results showed that Cu2+ supplemented directly into the culture medium or released from Cu-Hier-Ti surface could polarize macrophages to pro-inflammatory M1 phenotype by activating Cu-transport signaling (copper transporter 1 (CTR1) and ATP7A) in macrophages, while the material characteristics exhibited anti-inflammatory effect to some extent by regulating integrin (α5, αM, β1 and β2) and TLR (TLR-3, TLR-4, Myd88 and Ticam-1/2) signaling. Macrophages grown on Cu-Hier-Ti surface or treated by Cu2+ could create a favorable inflammatory microenvironment for osteoblast-like SaOS-2 cell proliferation and differentiation. Moreover, Cu-Hier-Ti surface promoted macrophage capacity to engulf and kill bacteria, even though it did not show direct bactericidal effect against Staphylococcus aureus. In vivo results showed that Cu-Hier-Ti surface could lead to promoted osteointegraion and enhanced expression levels of M1 surface marker CD11c, growth factor BMP-6 and osteogenic makers including osteocalcin (OCN) and Runx-2 at the biomaterial/bone tissue interface in a rat model. The results indicate that Cu could be employed as a promising inflammation-modulatory agent to activate macrophages for enhanced osteogenic and bactericidal effect. The next generation of bone biomaterials should be active to regulate the local inflammatory environment such that it favors bone regeneration. For the design and development of Cu-containing inflammation-modulatory biomaterials, it is of great importance to recognize the exact role played by Cu2+ release or material surface characteristics. So far, relatively little is known about the regulatory role of Cu2+ or micro/nano-topographical surface on macrophages. The results in the current work suggest that Cu2+ release and material surface characteristics of Cu-containing micro/nano-topographical coating could activate distinct signaling pathways in macrophages. The activated M1 macrophages exhibited stimulatory effect on osteoblast maturation and enhanced bactericidal capacity against Staphylococcus aureus. This study might provide new thoughts for the development of multi-functional Cu-containing biomaterials.