Background: Despite the considerable development of the field of orthopedic implants in the past century, complications including poor bone ingrowth and implant associated infection (IAI) persist to this day, causing a huge burden to millions of patients and the healthcare systems worldwide. Additive manufacturing (AM) and the subsequent surface biofunctionalization and antibacterial element incorporation are promising techniques to achieve dual antibacterial and osteogenic functionalities within a bone implant. In addition, macrophages are an immune cell type that are known to be essential for the implant success in the body, by having an intimate crosstalk with mesenchymal stem cells (MSCs) in the process of new bone formation. However, the behaviour of these immune cells is affected by environmental cues, including the implant surface properties. Therefore, this work investigated for the first time the effects of AM titanium implants biofunctionalized via plasma electrolytic oxidation (PEO) and incorporated with silver nanoparticles (AgNPs) on the human mesenchymal stem cells (hMSCs) co-cultured in vitro with human macrophages. Specifically, the paracrine effects of immune cells on the hMSCs osteogenic differentiation were studied by the development of an indirect co-culture model.
Materials and methods: AM Ti-6Al-4V implants were biofunctionalized via PEO and AgNPs incorporation and the surface characterization was performed by a scanning electron microscope (SEM) and ion release analysis. The effects of such implants on the hMSCs osteogenic differentiation in vitro were subsequently evaluated by measuring the mineralization and osteogenic gene expression. In addition, a macrophage-hMSCs indirect co-culture system was developed in order to study the effects of the macrophage polarization induced by the implants on the hMSCs osteogenic differentiation by means of a paracrine communication. The macrophage polarization was characterized by measuring the cytokine secretion pattern with an ELISA assay and gene expression.
Results: PEO modification of AM implants created TiO₂ surfaces with interconnected micro and nanoporosities and the incorporation of AgNPs. The single-culture of hMSCs on PEO and PEO + Ag implants revealed their ability to promote the osteogenic differentiation and no detrimental effects were observed in this process by the presence of AgNPs. The immunological evaluation of the co-culture system revealed similar polarization patterns when macrophages were cultured on PEO and PEO + Ag surfaces. In addition, factors secreted by polarized macrophages did not elicit a paracrine effect on the co-cultured hMSCs, neither enhancing nor inhibiting their osteogenic differentiation.
Discussion and conclusions: Based on the findings gathered in this study, the incorporation of AgNPs in the PEO layers, under the conditions used in this work, did not compromise the osteogenic differentiation and mineralization of the hMSCs. In addition, PEO + Ag surfaces did also not cause any detrimental effects on the paracrine communication of human macrophages on hMSCs. Therefore, further investigations regarding the osteoimmunomodulatory potential of these biofunctionalized AM implants are worth performing, in an attempt to achieve an important additional biofunctionality next to their proven osteogenic and antibacterial activity. These future researches should include further optimization of the PEO surface morphology and chemistry as well as the co-culture model used in this study.

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.