In Vitro Characterization of In Situ Alloyed Ti6Al4V(ELI)-3 at.% Cu Obtained by Laser Powder Bed Fusion

Abstract

The intensive cytotoxicity of pure copper is effectively kills bacteria, but it can compromise cellular behavior, so a rational balance must be found for Cu-loaded implants. In the present study, the individual and combined effect of surface composition and roughness on osteoblast cell behavior of in situ alloyed Ti6Al4V(ELI)-3 at.% Cu obtained by laser powder bed fusion was studied. Surface composition was studied using scanning electron microscopy, energy dispersive spectroscopy, and X-ray diffraction. Surface roughness measurements were carried out using confocal microscopy. In vitro osteoblast performance was evaluated by means of cell morphology observation of cell viability, proliferation, and mineralization. In vitro studies were performed at 1, 7, and 14 days of cell culture, except for cell mineralization at 28 days, on grounded and as-built (rough) samples with and without 3 at.% Cu. The addition of 3 at.% Cu did not show cell cytotoxicity but inhibited cell proliferation. Cell mineralization tends to be higher for samples with 3 at.% Cu content. Surface roughness inhibited cell proliferation too, but showed enhanced cell mineralization capacity and therefore, higher osteoblast performance, especially when as-built samples contained 3 at.% Cu. Cell proliferation was only observed on ground samples without Cu but showed the lowest cell mineralization.

Keywords: Ti–Cu alloys; implants; in-vitro tests; laser powder bed fusion; surface roughness.

Figure 1

SEM and surface topography micrographs of (a) as-built L-PBF Ti6Al4V(ELI), (b) Ti6Al4V(ELI)-3 at.% Cu surfaces, and (c) after grinding process (same surface topography was obtained for both samples with and without 3 at.% Cu).

Figure 2

SEM BSE micrographs of the central area of L-PBF Ti6Al4V(ELI)-3 at.% Cu specimen, (a) illustrating general microstructure, (b) XRD (with L-PBF Ti6Al4V(ELI) as reference), (c) Cu-rich region and (d) Cu map of (c).

Figure 3

Fluorescence images of a LIVE/DEAD assay of osteoblast cells cultured for 1, 7, and 14 days (from left to right) of cell culture onto (a–c) ground and (d–f) as-built L-PBF Ti6Al4V(ELI) and, (g–i) ground and (j–l) as-built L-PBF Ti6Al4V(ELI)-3 at.% Cu surfaces (n = 3).

Figure 4

MTS assay at 1, 7, and 14 days of cell culture onto ground and as-built L-PBF Ti6Al4V(ELI) and Ti6Al4V(ELI)-3 at.% Cu surfaces. (a) Normalized MTS values in each tested time and, (b) non-normalized MTS results per studied materials (n = 3; * p-values < 0.05).

Figure 5

Phalloidin staining at 1 day of cell culture onto (a) ground and (b) as-built L-PBF Ti6Al4V(ELI) and (c) ground and (d) as-built L-PBF Ti6Al4V(ELI)-3 at.% Cu surfaces (n = 3). White arrows show the cell cytoplasm covering several surface peaks formed due to the attachment of un-melted or partially melted particles of as-built surfaces.

Figure 6

Phalloidin staining at 14 days of cell culture onto (a) ground and (b) as-built L-PBF Ti6Al4V(ELI) and (c) ground and (d) as-built L-PBF Ti6Al4V(ELI)-3 at.% Cu surfaces (n = 3).

Figure 7

Cell mineralization assay at 28 days of cell culture onto ground and as-built L-PBF Ti6Al4V(ELI) and Ti6Al4V(ELI)-3 at.% Cu (n = 3; * p-values < 0.05).