Multilayered coating of titanium implants promotes coupled osteogenesis and angiogenesis in vitro and in vivo

We used surface-modified titanium (Ti) substrates with a multilayered structure composed of chitosan-catechol (Chi-C), gelatin (Gel) and hydroxyapatite (HA) nanofibers, which were previously shown to improve osteogenesis, as a platform to investigate the interaction of osteogenesis and angiogenesis during bone healing. Combined techniques of Transwell co-culture, wound healing assay, enzyme linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemical staining were used to evaluate adhesion, morphology and migration of adipose-derived mesenchymal stem cells (Ad-MSCs) and human umbilical vein endothelial cells (HUVECs) grown on different Ti substrates. We investigated the effect of substrates on the osteogenic differentiation of Ad-MSCs and reciprocal paracrine effects of Ad-MSCs on HUVECs or vice versa. The multilayered Ti substrates directly regulated the cellular functions of Ad-MSCs and angiogenic HUVECs and mediated communication between them by enhancing paracrine effects via cell–matrix interactions in vitro. The in vivo results showed that the change of microenvironment induced by surface-modified Ti implants promoted the adhesion, recruitment and proliferation of MSCs and facilitated coupled osteogenesis and angiogenesis in bone healing. The study proved that multilayer-film-coated Ti substrates positively mediated cellular biological function in vitro and improved bone healing in vivo. Recent studies have revealed that osteogenesis and angiogenesis are coupled, and that communication between osteoblasts and endothelial cells is essential for bone healing and remodeling processes; however, these conclusions only result from in vitro studies or in vivo studies using transgenic murine models. Relatively little is known about the communication between osteoblasts and endothelial cells in peri-implants during bone healing processes. Our results revealed the cellular/molecular mechanism of how multilayered Ti substrates mediate reciprocal paracrine effects between adipose-derived mesenchymal stem cells and human umbilical vein endothelial cells; moreover, the interactions between the cell-matrix and peri-implant was proven in vivo with enhanced bone healing. This study contributes to our understanding of the fundamental mechanisms of angiogenesis and osteogenesis that affect peri-implantation, and thus, provides new insights into the design of future high-quality orthopedic implants.