## 3 Results
### Cytotoxicity (CCK-8 Assay)  
Application of an indirect method provided experimental assessments for the biocompatibility assays. Potential detrimental effects on host cells of Mg alloy materials and treatments were tested in vitro using the international standards ISO-10993-5 and ISO-10993-12, which are commonly used to evaluate Magnesium alloys [45; 46; 47; 48; 49; 50; 51; 52; 53].  
The SBF corrosion test solutions (which we will name \"extraction fluids\") of the PHB coated and uncoated AZ31 alloy samples, which contained the corrosion byproducts after 1 day of exposure, were used to perform cytotoxicity trials at different dilutions, namely, 25%, 50%, and 100% of the initial concentrated extracted medium.  
HUVEC viability in these two types of extraction fluids at different dilutions after 1 day, 3 days, and 5 days, respectively, is summarized in Figure 5 as percentage of viability in standard control fluid (RPMI1640 medium).  
After 1 day of exposure, significant, high differences in cell proliferations compared with the control were observed for 100% AZ31 extraction fluid and its 50% dilution, and for exposure to the 100% PHB-AZ31 extraction fluid. No significant differences with the control were observed for the other groups.  
After 3 days of culture, significant differences for cell proliferation compared to the control were observed in 100% AZ31 extract fluid, in its 25% dilution, and in the 25% dilution of the PHB-AZ31 extraction fluid.  
Finally, after 5 days of culture, only the 100% AZ31 extraction fluid had a significant effect on cell proliferation, while there were no significant differences between the other groups and the control.  
It can be inferred that many favorable and adverse factors jointly influenced cell viability. The presence of Mg ions is one of these factors; it can act positively at lower concentrations (day 1), while cell growth adverse factors may arise at higher Mg ion concentrations (day 5). This point is to be further considered in future investigations.  
The extraction fluid of the PHB-coated magnesium alloy, which was characterized by a significantly lower Mg ion concentration (see Figure 3b) had a significant effect on cell proliferation only in the early stage of the culture (1 day).  
Table 3 includes the details of the relative growth rate (RGR) and cytotoxicity levels for the AZ31 and PHB-coated AZ31 extraction fluids and their dilutions. Levels of cytotoxicity were grade 0 for all extraction fluids and their dilutions when cultured for 1 day. After 3 days of culture, two of the three concentrations (25 and 100%) of the AZ31 extraction fluid exhibited a cytotoxicity level of grade 2, while the 50% was grade 1; all cytotoxicity levels for the PHB-coated samples were of grade 1. After 5 days of cell culture, the cytotoxicity  
Figure 5: Proliferation of HUVECs cultured on extraction fluids after 1, 3, and 5 days. Data are reported as % of viability in standard control fluid. * Significant difference (\(p<0.05\)); ** highly significant difference (\(p<0.01\)).  
levels of the AZ31 sample fluids were all of grade 1, while two concentrations (25 and 50%) of the PHB-coated sample fluids showed a cytotoxicity of grade 0 and the other of grade 1.  
It can be noticed that the PHB-coated AZ31 magnesium alloy extraction fluid has a less toxic behavior and the capacity to promote cell proliferation. This peculiar behavior derives from the lower concentration of corrosion Mg\({}^{2+}\) ions that directly originates from the diffusion control exerted by the polymeric coating films on the fluxes of the dissolved ionic species (both from the metal surface to the solution and _vice versa_).