<i>In vitro</i> cytotoxicity and oxidative stress of Ti6Al4V and 316L stainless steel powders used in additive manufacturing

Additive manufacturing (AM) is the fabrication of three-dimensional objects by successively joining layers of feedstock material. Metal AM is the fastest-growing segment. Alloy powders containing different metals are used as feedstock material. Despite the benefits of metal AM, workers may be exposed to powder particles when performing manual tasks. Limited studies have investigated the toxic potency of alloy powders. The aim of this study was to investigate the toxicological potential of virgin (V) and used (U)-Ti6Al4V, and virgin 316L stainless steel (316L SS) powders on human cell lines, including nasal septum (RPMI-2650), lung (A549), and intestinal (HuTu-80), and rat liver (H4IIE-<i>luc</i>) cells. Laser diffraction was employed to determine particle size distribution. Powder dispersions (1 mg/ml) were diluted in culture medium to obtain exposure concentrations of 1, 5, 10, 20, 50, 100, or 200 µg/ml to assess cytotoxicity, reactive oxygen species (ROS) generation, and DNA damage. Bulk powders were 22.1 to 90.15 µm in physical diameter, while sonicated powder dispersions ranged between 21.85 and 84.49 µm. This approximated to an aerodynamic diameter of 46.41-180.3 µm and 45.89-168.98 µm, for bulk powders and dispersions, respectively. For toxicity evaluations, V-Ti6Al4V was non-cytotoxic. Exposure to 200 µg/ml U-Ti6Al4V induced cytotoxicity in intestinal cells. 316L SS was cytotoxic to all cell lines, exhibiting concentration-dependent cytotoxicity in HuTu-80 and H4IIE-<i>luc</i> cells. Metal powders significantly increased generation of ROS in nasal septum (50-100 µg/ml V-Ti6Al4V, 100 µg/ml U-Ti6Al4V, and 20 µg/ml 316L SS) and liver (20 µg/ml 316L SS) cells. None of the powders induced genotoxicity. Our findings indicate that a portion of Ti6Al4V and 316L SS powders contain particles with an aerodynamic diameter <100 µm, and that the powder dispersions initiate significant toxicity <i>in vitro</i>.