# 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
Iron is an essential trace metal for living organisms, taking part in multiple vital functions and metabolic pathways, such as oxygen transport throughout the body and DNA synthesis [47, 49]. For 19-50-year-old men and women (nonpregnant, not breastfeeding), recommended dietary allowances (RDA) of \(\sim\)8 and 18 mg/day of Fe were estimated [50], respectively, even though only a portion will be biologically accessible. After absorption in the duodenal mucosa cells via a divalent metal transporter-1 (DMT-1), a strictly regulated and complex homeostasis of this metallic element occurs, in which macrophages play an important role [47, 48]. The tight Fe regulation is justified due to the capacity of \"free\" iron to donate or accept electrons from other molecules and to consequently produce ROS [48, 51, 52, 53]. This is the case of ferrous Fe(II) ions, which are commonly released during metallic corrosion processes. Due to the potential cytotoxicity of its ions, Fe does not move freely throughout the body. Instead, it is maintained \"captive\", either bonded to other molecules (e.g., ferritin) or compounds, and/or kept inside the cells [47, 48]. Macrophages are cells from the innate immune system that play a pivotal role in dealing with this metal [47, 53]. For instance, specialized spleen and liver macrophages phagocytize damage or senescent red blood cells to collect iron that is found in the heme groups of hemoglobin. Moreover, differently polarized M1 or M2 macrophages can sequestrate or release iron, respectively. M1-like macrophages maximize their ability to retain Fe inside the cells to restrict its availability to pathogens (e.g., during bacterial infections) or under an inflammatory response. Conversely, the fast iron release promoted by M2-like macrophages induces tissue repair and regeneration, angiogenesis, and inflammation resolution [47, 48, 53]. In the current study, the increased MTT values for macrophages incubated with culture medium conditioned with corrosion extracts from coated samples--when compared to the SL and the saliva group--may be related to their higher activity. According to the abovementioned role in Fe homeostasis they play, macrophages may be importing these ions from the extracts present in the culture medium. A higher activation of the naive macrophages based upon this factor could explain the increased MTT colorimetric assay values for cultures conditioned with the corrosion extracts [54], whereas the same would not occur for the controls or the saliva group, which do not have such concentration in Fe. Despite the remarkable ability of macrophages to adapt to external stimuli, a decrease in cell viability was found between the CH_6N and CH_9N groups (\(p<0.05\)), Figure 5a. Data suggests an overall decrease trend in cell viability for macrophages cultured with corrosion extracts as follows: CH_0N \(\simeq\) CH_6N \(>\) CH_9N \(>\) SS. Particular attention should be given to macrophages cultured with medium conditioned with the eluates from the uncoated SS, which showed a statistically significant decrease in cell viability, evidencing it is poorer in vitro biocompatibility when compared with the coated samples. Overall, the best cell viability results obtained for macrophages were obtained for the CH_0N and CH_6N coatings.