# Metal Release and Cell Viability of 316L Stainless Steel Sputter-Coated with N-Doped a-C:H Coatings
## 3 Results and Discussion
### Cytotoxicity Evaluation
#### 3.3.3. Coculture  
Finally, statistically significant differences were found for the MTT results of the coulture between the following groups: saliva and CH_0N (_p_ < 0.05) and CH_6N (_p_ < 0.01); and between SS and CH_0N and CH_6N (both \(p\) < 0.01), Figure 5c.  
The cell viability results confirm that the extracts released from the SS were the least biocompatible, especially compared with both CH_0N and CH_6N (Figure 5c). A higher dispersion was assessed for the results of CH_9N, but the median value was between those of CH_0N and CH_9N. Moreover, all the groups concerning coated samples showed: (i) median MTT values above the SS, as found for isolated monocultures of macrophages; and (ii) nonsignificant differences among them.  
The results suggest that the SS extracts decrease cell viability even though the difference between the SS and saliva groups was nonsignificant. The impact of these extracts thus seems to be lower for the coulture when compared with the monocultures. The coulture assays are expected to better mimic the in vivo scenario, in which different types of cells simultaneously interact in a collaborative manner. For instance, fibroblasts can synthesize and release growth factors [61] that can enhance the survival of macrophages, whereas this phagocytic cell type can improve the quality of the culture medium by removing adverse chemical stimuli [62]. Additionally, the cell viability overestimation effect mentioned for macrophages may also be present in the coculture experiment (and not in the fibroblast monoculture). This may help explain the abnormal increase in the median MTT values for the groups of coated samples (CH_0N, CH_6N, and CH_9N). Nonetheless, it was absent for SS, which indicates a poorer in vitro biocompatibility of the 316L medical-grade alloy when uncoated.  
As in all in vitro studies, this research work presents some limitations which do not enable absolute extrapolations to more complex real applications of the coated 316L SS, such as orthodontics. On one hand, extracts for the cell viability assays were produced after a 30-day release of metallic ions, while longer contact with the intraoral tissues occurs in the in vivo situations. In fact, standard treatment for dental malocclusion may last for approximately 2 years. Moreover, the cleansing effect of saliva and the possible uptake by oral micro-organisms may limit the cell exposure to metallic ions. On the other hand, additional factors of the intraoral cavity, such as dynamic changes in chemical composition, temperature, and pH, further enhanced by wear mechanisms (e.g., on the bracket/archive contact) and Microbiologically Induced Corrosion (MIC), can increase metal release from fixed appliances. Nonetheless, the current study showed that the biocompatibility of medical-grade SS alloys can be further enhanced by using surface functionalization strategies, such as coating with a-C:H-based materials.  
Finally, the present research revealed that both the metal release and cell viability do not follow the good results regarding the superior bacterial inhibition properties of coatings with an N-doping level of \(\sim\)6 at.% [13].