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.