Alginate/chitosan modified immunomodulatory titanium implants for promoting osteogenesis in vitro and in vivo

The essentiality of macrophages for biomaterial-mediated osteogenesis has been increasingly recognized. However, it is still unclear what is the specific role and molecular mechanisms of macrophages and material properties in the regulation of osteogenesis. As an interdisciplinary field exploring the cross-talk between immune and skeletal systems, osteoimmunology has shifted the perspective of bone substitute materials from immunosuppressive materials to immunomodulatory materials. To fabricate an immunomodulatory Ti implant, alginate/chitosan multilayer films were fabricated on the surface of titania nanotubes (TNTs) to control the release of an anti-inflammatory cytokine interleukin (IL)-4 according to our previous work. The osteogenic effects and regulation mechanisms of the immunomodulatory Ti implants were investigated in vitro in different BMSCs culture modes. Alginate/chitosan multilayer-coated samples (with or without IL-4 loading) showed better direct osteogenic ability than TNTs by promoting biomineralization and up-regulating osteogenic gene expression (BMP1α, ALP, OPN, OCN) of BMSCs. Notably, material-induced macrophage polarization, M1 and M2, enhanced early and mid-stage osteogenesis of BMSCs via distinct pathways: M1 activated both BMP6/SMADs and Wnt10b/β-catenin pathways; while M2 activated TGF-β/SMADs pathway. Material surface properties dominated in regulating late osteogenesis probably due to the surface chemical composition (alginate, chitosan and Ca²⁺, etc.). Due to synergistic effects of material-induced inflammatory microenvironment and material surface properties, IL-4-loaded samples exhibited superior osteogenic capability through co-activation of three signaling pathways. The in vivo studies in rat bone defect model revealed that IL-4-loaded immunomodulatory implants successfully achieved macrophage phenotypic transition from pro-inflammatory M1 to anti-inflammatory M2 and subsequently improved new bone formation.