*3.5. Cytotoxicity Assay*

In vitro cytotoxicity was evaluated on human osteoblast-like MG-63 cells after 72 h. No significant changes in cell viability were observed in all tested series. As shown in Figure 7, the decrease in cell survival after treatment for all tested samples was assessed to be not higher than 12%.

**Figure 7.** Cytotoxicity test results represented as survivability of cells (in %).

The most significant cytotoxicity (ca. 12%) showed only LDPE substrate after deposition of N-DLC layer on the top (series PE\_2). This elevated cytotoxicity can be explained by a high content of N (ca. 8 at.%) in the modified surface.

Additionally, detailed analysis based on cellular morphology observation under fluorescent microscopy (Figure 8) did not reveal any significant changes in mitochondrial shape and size, and no apoptotic bodies were formed after this long incubation time.

**Figure 8.** Live (green) and dead (red) human osteoblast-like MG-63 cells after 72 h of treatment. Cells were stained with acridine orange and propidium iodide, indicating viable and dead necrotic or late apoptotic cells, respectively.

Additionally, PE\_3 modification (with the deposition of N-DLC/DLC layers) resulted in comparable biocompatibility to the control (untreated MG-63 cells) with significant improvement of mechanical properties (vide supra Figure 6).

The proposed DLC, N-DLC, or Si-DLC coatings on polymeric substrate were less cytotoxic than studied by us previously (DLC layers doped with N and Si atoms and deposited on titanium alloy). The latter coatings influenced the viability of the treated MG-63 cells, decreasing the cell surviving fraction by even up ca. 29% (modification C; after nitriding process and Si/N-DLC deposition) [51]. It can be concluded that the application of N-DLC or Si-DLC resulted in lower cytotoxic effect than the addition of N and Si atoms to the DLC structure during coating deposition.
