## 3 Experimental Results
### In Vitro Experiment  
Figure 7 shows the microbial viability assays (WST) for three of the four substrates, compared with those of bulk silicon nitride and an untreated reference. As the experiments were conducted at different times and because cell culture conditions (number of cells, viability, etc.) vary in each experiment, there is no way to directly compare results from different materials. It can be observed that Si\({}_{3}\)N\({}_{4}\) reference samples usually displayed the lowest optical density at 48 h, with the exception of the Ti6 Al4V substrate where the performance is comparable to that of the coated sample. Previous literature reports that the biological effects of silicon nitride are time-dependent and reach their maximum after about 12 h of treatment [46]. In the case of LDPE substrates, the trend observed for Si\({}_{3}\)N\({}_{4}\) is the same, but on polymer and composite substrates, it changed completely. Si\({}_{3}\)N\({}_{4}\) bulk is the only class of sample which showed how the bacterial viability decreased over time. The LDPE substrates exhibit a growth of more than 60% in OD between 24 and 48 h possessing the highest bacterial amount in both the times. Despite the lowest OD value presenting at 24 h, the Si\({}_{3}\)N\({}_{4}\)-coated samples showed a slight variation, even if lower than that of the negative control [39]. In the case of Ti6 Al4V, the microbial viability assay at 24 h showed a higher bacterial count on the Ti6 Al4V uncoated sample than on statistically identical uncoated or coated Si\({}_{3}\)N\({}_{4}\) samples. At 48 h, a statistically validated decreasing trend of OD was detected on all substrates. However, the Si\({}_{3}\)N\({}_{4}\)-coated substrate had the lowest OD value when compared with the two bulk sample classes in which nearly all bacteria underwent lysis [36]. For zirconia samples (ZTA and Y-TZP show similar results), the silicon nitride coated samples treated with _S. epidermidis_ have the highest 24-h living bacteria count, when compared to both references. However, the efficiency increases progressively with time. At 48 h, the optical density of the bacteria is 60% higher than on silicon nitride, but lower than on Y-TZP monoliths. Similar results have been reported in previous literature [37]. Figure 7: Biological assay of _Staphylococcus epidermidis_ as an indicator of bacterial exposure time for all substrates. Figures 8-10 show the results of the fluorescence microscopy testing with markers for nuclei (blue), osteoclast (green), and osteopontin (red). The fluorescence microscopy images obtained for various samples show increased bone tissue production for the laser cladded surfaces when compared to the uncoated substrates, as evidenced by both osteocalcin and osteopontin distributions. It must be noted that while both proteins are associated with the presence of bone tissue and their effect is considered synergetic, osteoclast [47] is mainly observed in mineralized tissue while osteopontin [48] is often associated with bone remodeling. Figure 8 shows the fluorescence microscopy results obtained on the LDPE samples. Unlike zirconia and Ti6 Al4V, the uncoated LDPE substrates appear to be colonized by cells and coating application does not significantly affect the amount or distribution of either cell nuclei or bone tissue. Figure 9 shows the fluorescence microscopy results obtained on the Ti6 Al4V alloy substrates. It can be observed that cell nuclei are not homogeneously distributed on the sample surface, forming agglomerates at the center where bone tissue (osteopontin and osteocalcin) is also formed. Coated samples, on the other hand, are more homogeneously covered. Figure 8: LDPE fluorescence micrograph samples after exposure to SaOS-2 cells. [PERSON] and red stains identify osteoclast and osteopontin, respectively, while blue shows cell nuclei. Figure 9: Ti6 Al4V fluorescence micrograph samples after exposure to SaOS-2 cells. [PERSON] and red stains identify osteoclast and osteopontin, respectively, while blue shows cell nuclei.