Partially Melted Ti6Al4V Particles Increase Bacterial Adhesion and Inhibit Osteogenic Activity on 3D-printed Implants: An In Vitro Study

Abstract

Background A porous Ti6Al4V implant that is manufactured using selective laser melting (SLM) has broad potential applications in the field of orthopaedic implants. The pore structure of the SLM porous Ti6Al4V implant allows for cell migration and osteogenic differentiation, which is favorable for bone ingrowth and osseointegration. However, it is unclear whether the pore structure and partially melted Ti6Al4V particles on a SLM porous Ti6Al4V implant will increase bacterial adhesion and, perhaps, the risk of implant-related infection. Questions/purposes (1) Is there more bacterial adhesion and colonization on SLM porous Ti6Al4V implants than on polished orthopaedic implants? (2) Do partially melted Ti6Al4V particles on SLM porous Ti6Al4V implants reduce human bone mesenchymal stem cells (hBMSCs) adhesion, viability, and activity? Methods To determine bacterial adhesion and biofilm formation, we incubated five different Ti6Al4V discs (polished, grit-blasted, plasma-sprayed, particle SLM porous, and nonparticle SLM porous discs) with methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli. Bacterial coverage on the surface of the five different Ti6Al4V discs were evaluated based on scanning electron microscopy (SEM) images quantitatively. In addition, a spread-plate method was used to quantitatively evaluate the bacterial adhesion on those implants. The biofilm formation was stained with crystal violet and semi-quantitatively determined with a microplate reader. The morphology and adhesion of hBMSCs on the five Ti6Al4V discs were observed with SEM. The cell viability was quantitatively evaluated with a Cell Counting Kit-8 assay. In addition, the osteogenic activity was determined in vitro with a quantitatively alkaline phosphatase activity assay and alizarin-red staining. For semiquantitative analysis, the alizarin-red stained mineralized nodules were dissolved and determined with a microplate reader. Results The polished discs had the lowest MRSA adhesion (8.3% ± 2.6%) compared with grit-blasted (19.1% ± 3.9%; p = 0.006), plasma-sprayed (38.5% ± 5.3%; p < 0.001), particle (23.1% ± 2.8%; p < 0.001), and nonparticle discs (15.7% ± 2.5%; p = 0.003). Additionally, when comparing the two SLM discs, we found that particle discs had higher bacterial coverage than nonparticle discs (23.1% ± 2.8% versus 15.7% ± 2.5%; p = 0.020). An E. coli analysis showed similar results, with the higher adhesion to particle SLM discs than to nonparticle discs (20.7% ± 4.2% versus 14.4% ± 3.6%; p = 0.011). In addition, on particle SLM porous discs, bacterial colonies were localized around the partially melted Ti6Al4V particles, based on SEM images. After a 7-day incubation period, the cell viability in the particle group (optical density value 0.72 ± 0.05) was lower than that in the nonparticle groups (optical density value: 0.87 ± 0.08; p = 0.003). Alkaline phosphatase activity, as a marker of osteogenic differentiation, was lower in the particle group than in the nonparticle group (1.32 ± 0.12 U/mL versus 1.58 ± 0.09 U/mL; p = 0.012). Conclusion Higher bacterial adhesion was observed on SLM porous discs than on polished discs. The partially melted Ti6Al4V particles on SLM porous discs not only enhanced bacterial adhesion but also inhibited the osteogenic activity of hBMSCs. Postprocessing treatment is necessary to remove partially melted Ti6Al4V particles on an SLM implant before further use. Additional studies are needed to determine whether an SLM porous Ti6Al4V implant increases the risk of implant-related infection in vivo. Clinical Relevance As implants with porous Ti6Al4V made using SLM are being designed, our preliminary findings suggest that postprocessing treatment is needed to remove partially melted Ti6Al4V particles before further use. In addition, the depth of the porous structure of the SLM implant should not exceed the maximum depth of bone ingrowth because the host immune defense cannot prevent bacterial adhesion without integration.

Susceptibility to biofilm formation on 3D-printed titanium fixation plates used in the mandible: a preliminary study

“…polishing. Xie et al showed that discs cut from Ti6Al4V implant manufactured by using selective laser melting (SLM), had higher bacterial adhesion than polished ones [79]. It has also been suggested that metallic implants produced by laser powder-bed fusion should be polished or coated [46,47] since coating medical devices with different kinds of active agents may be equally successful [80][81][82].…”

Mediation of mechanically adapted TiCu/TiCuN/CFR-PEEK implants in vascular regeneration to promote bone repair in vitro and in vivo

“…Additionally, the rods prepared by extrusion were cut to obtain the wafers used in the experiment, which consequently caused uneven structures of the disc surface, as observed from the SEM images. Many studies have demonstrated that a rough structure is advantageous for cell adhesion and proliferation [ [24] , [25] , [26] ]. The SEM scanning results demonstrated that the TiCu/TiCuN coating was distributed in a granular form on the surface of the CFR-PEEK substrate, uniformly covering the PEEK matrix and the carbon fibre part.…”

Characterization of dimensional, morphological and morphometric features of retrieved 3D-printed acetabular cups for hip arthroplasty

“…This falls under the material 'contact guidance' principle, whereby the cell movement and development is affected by the surface morphology of the implant [37]. However, a recent in vitro study by Xie et al [38] suggested that the presence of the partially molten particles not only enhanced bacterial adhesion but also inhibited osteogenic activity of human bone mesenchymal stem cells, thus recommending the particles to be removed. The potential release of these particles cannot also be excluded, as suggested by Matouskova et al [18]; this may lead to an increased risk of inflammatory reactions or decreased hemocompatibility [18], or to raised metal (titanium) levels in the blood.…”