# Metal Release and Cell Viability of 316L Stainless Steel Sputter-Coated with N-Doped a-C:H Coatings
## 3 Results and Discussion
### Cytotoxicity Evaluation
#### 3.3.1 Macrophages  
Concerning the MTT results from the macrophage monocultures, statistically significant differences were found between saliva and SS (\(p\) < 0.01), CH_0N (\(p\) < 0.001), CH_6N (\(p\) < 0.001), and CH_9N (\(p\) < 0.01); SS and CH_0N (\(p\) < 0.001), CH_6N (\(p\) < 0.001), and CH_9N (\(p\) < 0.001); and between CH_6N and CH_9N (\(p\) < 0.05), as presented in Figure 5a. The SS group shows the lowest cell viability, with statistically significant differences regarding all the other groups of coated samples (Figure 5a). These findings seem to follow the overall reduction in the metal release rates upon coating with the DLC, as presented in Figure 4.  
Significant attention has been given to the cytotoxicity of nickel (Ni) [43]. In fact, the International Agency for Research on Cancer (IARC) classifies nickel compounds as carcinogenic to humans (Group I), while metallic nickel is potentially carcinogenic [5]. In a recent study, [PERSON] et al. [44] investigated the in vitro inflammatory effects over THP-1 monocytes and macrophages after exposure to separate solutions of Cr(III) and Ni(II) ions with successively higher concentrations (10-500 uM, found in periprosthetic tissues). The authors reported that Cr(III) did not significantly influence the cell viability of both types of cells for the tested concentration range; conversely, Ni(II) ions induced cytotoxic effects that were concentration-dependent. Moreover, low concentrations of bivalent ions were found to trigger the release of inflammatory mediators, especially from macrophages. For Ni(II) solutions with the highest concentrations (100 and 500 uM), however, the reactive oxygen species (ROS) significantly increased in the supernatant compared to the control. ROS are produced by innate immune system cells to deal with pathogens; nonetheless, for  
Figure 4: Average Ni, Cr, and Fe release for the uncoated and DLC-coated 316L SS substrates after the 30-day corrosion test in acidic artificial saliva. high production, ROS can accumulate and induce oxidative cell stress, leading to oxidation of cellular biomolecules and possibly cell damage or death [44], which may be occurring in this study. Regarding chromium, it is accepted that its release during corrosion occurs in its trivalent ionic form, despite evidence of the in vivo release of Cr(IV), which is rapidly reduced to Cr(III) in cells [45]. While Cr's toxicity is usually assigned to its hexavalent oxidation state, other researchers pointed out \"the potential for Cr(III) to cause toxic, carcinogenic, genotoxic, mutagenic, and epigenetic effects in biological systems\" [46]. Nevertheless, Cr average release rates were similar for all tested samples. Thus, the contents of both Ni(II) and Cr(II) ions may help explain the statistically significant differences found between the uncoated and coated SS groups. Interestingly, standardized MTT average values above the SL line (y = 1) were found when macrophages were incubated with the culture medium conditioned using the extracts from the coated samples (CH_0N, CH_6N, and CH_9N), but that was not the case for the saliva group (i.e., macrophages grown with medium conditioned with artificial saliva  
Figure 5: MTT cell viability results of (**a**) macrophages (5 days), (**b**) fibroblasts (6 days), and (**c**) co-culture (6 days) after the test in culture medium conditioned with saliva or with extracts from SS, CH_0N, CH_6N, and CH_9N. The MTT values are expressed as a percentage of each mean value of each experimental control. SL: standardization line. 70%: refers to 70% of the control. Statistical significances: * \(p<0.05\); ** \(p<0.01\)**; and *** \(p<0.001\)**. without corrosion extracts), Figure 5a. Explaining this result is challenging. One potential justification could be the activation of the macrophages, namely regarding the release of Fe and their fundamental role in vivo [47, 48].