## 3 Results and Discussion
### Discussion  
Mg-based biodegradable orthopedic implants dissolve in the physiological environment of the body, producing corrosion products [3; 4]. Studies suggest that the degradation behavior of the Mg-based implants varies due to different corrosive environments of in vitro and in vivo studies [5], which makes it challenging to establish Mg as a next-generation biodegradable material [3; 7]. Therefore, in this study, we used a real-time ECIS system to mimic the in vivo environment for assessing the cytotoxicity of different concentrations (0, 1, 5, 10, and 50 mM) of MgO/Mg(OH)\({}_{2}\) NPs in hFOB 1.19 cells [7]. Based on our findings by real-time ECIS study, we propose the development of sustainable, biodegradable Mg-based alloys as the implant materials for orthopedic applications.  
Osteoblast differentiation progresses by the process of cell proliferation, matrix maturation, and mineralization [30]. In the case of the in vitro environment, the complete confluence of the cells increases matrix maturation and mineralization [31]. Magnesium homeostasis and regulation of cell functions such as cell proliferation, energy metabolism, and the apoptosis process in healthy and diseased cells are also reported [32]. Therefore, in our study, we investigated the confluence of the hFOB 1.19 cells for 24, 48, 72, and 96 h [7] before ECIS experiments. Results show that after 96 h, cells were approximately 70-80% confluent with a negligible number of dead cells when compared to 72 (50-60% confluence), 48 (30-40% confluence), and 24 h (20-30% confluence). The maximum convergence of hFOB 1.19 cells significantly contributes to avoiding any false-positive results with dead cells that might otherwise interfere with the ECIS impedance upon exposure to MgO/Mg(OH)\({}_{2}\) NPs [7].  
The ECIS impedance result reveals that 1 mM concentrations of MgO NPs provide a similar range of impedance as the control group (0 mM), which indicates magnesium homeostasis, as shown in Figure 4. However, at the subsequent higher concentrations, such as 5, 10, and 50 mM concentrations of MgO NPs [7], the impedance drops significantly, indicating cytotoxicity by inhibiting cell proliferation. Comparative impedance results of MgO and Mg(OH)\({}_{2}\) NPs [7] in Figures 4 and 5, respectively, show that impedance is lower at 50 mM concentrations of Mg(OH)\({}_{2}\) NPs [7] when compared to MgO NPs [7], which indicates high cytotoxicity of Mg(OH)\({}_{2}\) at 50 mM. These results have been confirmed by the images of live and dead cells on microfabricated electrodes in Figures 2 and 3, respectively.  
Studies suggest that extracellular pH and oxygen tension alters the bone hemostasis [29]. Besides, the skeleton serves as the reservoir of alkaline mineral (hydroxyapatite), which releases in case of metabolic acid-base imbalance [33]. Also, reports on bone cells suggest that at acidic pH, osteoblast activity inhibits, which results in inhibition of mineral deposition [29; 33]. However, the bone resorption activity of osteoclast cells gets activated at acidic pH [33]. The changes in pH levels of the possible corrosion products are not entirely understood. Therefore, with this study, insight into the change in pH of different concentrations of MgO/Mg(OH)\({}_{2}\) NPs upon exposure to hFOB 1.19 cells [7] has also been gained.  
Little evidence of the in vitro test to study the cytotoxicity of the corrosion products is reported [7]. However, in this study, we provide real-time in vitro ECIS results for an allowable concentration of MgO/Mg(OH)\({}_{2}\) NPs in hFOB 1.19 cells. With the results obtained from our study, we propose the development of sustainable, biodegradable Mg-based alloys for future in vivo testing for orthopedic implant applications.