Effects of surface-modified 3D printed titanium bone implants on human macrophages

Titanium orthopaedic biomaterials to replace degenerated joint surface, improve bone regeneration and fixation are studied and used worldwide. However, not always biomaterial implantation is successful, and the main causes of implant failure remain implant associated infections (IAI) and poor osseointegration. In the recent years, additive manufacturing and titanium surface modification have been extensively researched in order to develop bone implants able to enhance bone formation at bone/material interface while reducing the risk of microbial adhesion. New bone formation at the implant site is an inflammation-driven process, since the implantation of a biomaterial in the human body is always followed by an immune response. Among the inflammatory cells involved in the inflammation stage following initial hemostasis after surgery, macrophages play a crucial role in regulating subsequent bone formation and remodelling secreting a wide range of factors. Therefore, in this study, human macrophage response to 3D printed Ti-6Al-4V surfaces was investigated. Ti-6Al-implants were modified by plasma electrolytic oxidation (PEO), during which an oxide layer containing electrolyte components were generated on implant surfaces. In order to generate an antimicrobial coating, silver nano-particles (Ag NPs) were also incorporated into the surface. Surface morphology and chemical composition were analysed by SEM. Human macrophages were cultured in the presence of SLM implants in a transwell culture or directly on the surface for 4 days. Macrophage morphology and viability were assessed with scanning electron microscopy (SEM) and fluorescence microscopy. Cell secreted factors were analysed performing an ELISA assay while gene expression analysis was performed using qPCR. Human mesenchymal stem cell (hMSC) response in terms of morphology and viability was also evaluated after 4 days of culture. PEO modification on SLM surfaces resulted in an additional TiO2 layer containing electrolyte components (Ca, P and Ag) and interconnected porosity. The ion release from SLM surfaces did not have an effect on human macrophage polarisation, while when cells where cultured directly on the implants, they secreted and expressed different amounts of pro- and anti-inflammatory factors. SLM NT (not PEO-treated) surfaces up-regulated macrophage pro-inflammatory cytokines compared to SLM PEO surfaces. However, cells cultured on SLM NT up-regulated also anti-inflammatory factors. SLM surfaces containing Ag NPs were cytotoxic for human macrophages but not for hMSCs. In general, both SLM NT and SLM PEO surfaces expressed anti-inflammatory and tissue repair-related macrophage factors, suggesting that either implant geometry achieved by 3D printing and surface biofunctionalization may have beneficial impact on promoting macrophage anti-inflammatory secretion and subsequent osteogenesis around the implant