Regulation of macrophage polarization and osteoblastic cellular behaviours on titanium surface through the design of surface pattern that facilitates implant-to-bone osteointegration

Abstract

Successful osteointegration at the interface between host bone and orthopaedic implants determines the outcome of implantation. Among different implant surface modification strategies, surface topography designs have aroused much attention due to their feature of noninvasiveness and providing stable physical cues for cell-to-implant interaction. Although efforts have been made to develop different surface topographies, the underlying mechanism of how surface topography cues orchestrate the osteogenesis process remains unclear. Hence, in this study, four scales of novel TiO2 honeycomb-like surface structures ranging from the nanometer scale to the micrometer scale were fabricated on titanium surfaces using a customized interfacial lithography method. The obtained TiO2 honeycomb-like surface structures provided a platform with different physical topographies but similar chemical composites and wettability. Thus, this experimental setup enables us to isolate the modulatory role of surface topography in the osteointegration process on implant surfaces. In the first part of this study (Chapter 1), the cellular behaviors of mouse embryo osteoblast precursor cells (MC3T3-E1) in response to different TiO2 honeycomb-like surface structures were investigated. It was found that different TiO2 honeycomb-like surface structures influenced the formation of filopodia and cytoskeleton arrangement, which could modulate cell behaviors through signal transduction and gene expression. The effects of TiO2 honeycomb-like surface structures on osteogenesis, including cellular behaviors of osteoblasts in vitro and implant-to-bone integration in vivo were investigated. The in vitro results suggested that the osteogenic differentiation and mineralization of osteoblasts could be up-regulated by TiO2 honeycomb-like surface structures at different degrees. Specifically, the 90 nm TiO2 honeycomb-like structures could induce rapid osteogenic differentiation of osteoblasts. The in vivo results further demonstrated that the 90 nm TiO2 honeycomb-like surface structures could promote bone-to-implant integration with a higher amount of new bone formation and bone-to-implant contact ratio. In addition to promoting osteogenic differentiation of osteoblastic cells, a proper osteoimmune microenvironment is essential to successful implant-to-bone integration. In the osteoimmunomodulatory process, macrophages are one of the earliest arrivals to initiate immune responses. Based on the TiO2 honeycomb-like surface structures, the immunomodulatory effect of surface topography cues on macrophages and the subsequent implant-to-bone integration were investigated in the second part of this study (Chapter 5). It was found that the regulation effect of TiO2 honeycomb-like surface structures on the polarization of macrophages was closely related to the scale of honeycomb-like structures, and the smallest honeycomb-like structure (90 nm) can induce the polarization of M2 macrophages and simultaneously secrete a large amount of the pro-regenerative cytokines. Subsequently, the role of osteoimmune microenvironment generated by different samples in regulating osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and osteointegration in vivo were investigated. The results demonstrated that cytokines collected from honeycomb-like structures (90 nm) could induce the highest degree of osteogenic differentiation of MSCs and implant-to-bone integration. Moreover, the transcriptomic analysis showed that the smallest TiO2 honeycomb-like surface structures (90 nM) promoted filopodia formation in macrophages and up-regulated the expression levels of RhoA, Rac1 and Cdc42, regulating the polarization of macrophages through activation of the RhoA/Rho kinase signaling pathway.