**2. Results**

#### *2.1. Human MNNG/HOS Osteosarcoma Cell Line*

#### 2.1.1. Effect of Scandium Alone on Cell Proliferation and Viability

Figure 1 shows the effect of scandium alone on MNNG/HOS osteosarcoma cell proliferation as well as cell viability. Concerning the cell index (CI) measured by xCELLigence RTCA, the software employed transformed the CI of all wells at all time points to the normalized cell index (NCI), which was considered the CI value of individual wells at the same time point (base-time). This transformation made the NCI more comparable between the wells [23].

**Figure 1.** Normalized cell index plot of MNNG/HOS cells after 160 h of contact with Sc3+ at 50 μg mL−<sup>1</sup> (\*\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*\*\*) \* = *p* value ≤ 0.0001, \*\* = *p* value ≤ 0.005, \*\*\* = *p* value ≤ 0.05.

The NCI plot shown in Figure 1 depicts two different kinds of curves. The ascending bi-exponential curve of the control is due to the fact that, in its well, the space to grow up was not saturated yet. The saturation was reflected by a plateau. Normally, two scenarios can occur once the plateau is set: (1) the plateau continues until starting a definitive descending evolution characteristic of damaged cells that are not attached to the surface of the well; (2) the plateau continues until starting a momentary decrease followed by a brief rise, that is characteristic of the replacement of damaged cells by new healthy cells. The curves for scandium concentrations at 50 and 100 μg mL-<sup>1</sup> showed a plateau that reflected no more MNNG/HOS cells proliferation but the detachment from the well was not observed yet. For the scandium concentration at 200 μg mL-1, the detachment of cells slightly began at 140 h. Unfortunately, the time frame of 160 h was not sufficient to see if the decrease was definitive or kept oscillating. The MTT assay confirmed that the concentration of 200 μg mL−<sup>1</sup> displayed a reduction of cell viability of around 25% after 160 h. For lower concentrations (i.e., 50 μg mL−<sup>1</sup> and 100 μg mL−1), an increase of cell viability was noticed.

#### 2.1.2. Effect of EPS and Heparin Alone

The effect of both EPS-DR and -DRS and heparin on cell proliferation and cell viability is showed in Figure 2. It is noticed that NCI plot of EPS-DRS is very different from the ones of heparin and EPS-DR. For EPS-DRS, a second round of xCELLigence experiment was performed, leading to a higher number of MNNG/HOS cells (i.e., 5000 cells) compared to the first one (i.e., 3000 cells). This is why the control of EPS-DRS reached the peak of proliferation sooner than the two other ones, while for heparin and EPS-DR cells were still proliferating. This allowed evidencing the effect of the not modulated by the addition of heparin whatever the concentration considered. It was noticed that for 50 μg mL−<sup>1</sup> of EPS-DR, the lowest sulfated EPS of this study, 100% viability was reached compared to the control.

**Figure 2.** To the left, normalized cell index plot of MNNG/HOS cells after 160 of contact with (**a**) heparin alone at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*); (**b**) EPS DRS alone at 50 μg mL−<sup>1</sup> (\*\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*); (**c**) EPS-DR alone at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*).

2.1.3. Effect of Polysaccharide:Scandium Complexes Heparin:Scandium (Hep:Sc) Complex

Figure 3 shows the NCI plot from xCELLigence RTCA of MNNG/HOS cells after 160 h of contact. The Hep:Sc complexes were prepared for four different metal-to-ligand ratios. For a metal-to-ligand ratio of 1:0.5 (Figure 3a), with a total concentration of heparin of 50 μg mL−1, there was no effect on cell proliferation. After 100 h of contact, for the same metal-to-ligand ratio but with higher concentrations of heparin (i.e., 100 and 200 μg mL−<sup>1</sup> respectively), there was a significant decrease of the cell proliferation rates.

**Figure 3.** Normalized cell index Plot of MNNG/HOS cells after 160 h of contact with different stoichiometric ratios L:M: (**a**) heparin:scandium (Hep:Sc) 1:0.5 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−1, and 200 μg mL−1; (**b**) Hep:Sc 1:1 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*); (**c**) Hep:Sc 1:2 at 50 μg mL−1, 100 μg mL−<sup>1</sup> (\*\*), and 200 μg mL−1; (**d**) Hep:Sc 1:4 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*).\* = *p* value ≤ 0.0001, \*\* = *p* value ≤ 0.005.

With a metal-to-ligand ratio Hep:Sc of 1:1 (Figure 3b), the decrease in cell proliferation was well displayed and dose-dependent. For metal-to-ligand ratio of Hep:Sc of 1:2 (Figure 3c), there was no further exponential growth after 90 h of contact for all the concentrations of heparin considered. Finally, for a metal-to-ligand ratio of Hep:Sc of 1:4 (Figure 3d), there were the same proliferation rates between the control and treated cells until 100 h of incubation followed by a temporary decrease of cell proliferation.

If those results are compared to the ones obtained on cell viability, the metal-to-ligand ratio ratios of 1:0.5 and 1:4 exhibited higher percentages of cell viability. In contrast, for a constant metal-to-ligand ratio of 1:1, a significant decrease of almost 40% is observed when increasing the total concentration of heparin. The only drop on cell viability is observed for a metal-to-ligand ratio of 1:2 and for a concentration of heparin of 200 μg mL−1; at these conditions, there is no more viable cells present in the well after 160 of contact.

## EPS-DRS:Scandium (EPS-DRS:Sc) Complex

Figure 4 shows the NCI plot of MNNG/HOS cells after 160 h of contact with four metal-to-ligand ratio of EPS-DRS:Sc complex. Regarding the four metal-to-ligand ratio of the EPS-DRS:Sc complexes, the shape are very close to each other, meaning that neither the metal to ligand ratio, nor the total concentration of EPS-DRS play a key role in the cell proliferation. Only one difference could be noticed, a slight reduction of cell proliferation, in the range 10–15 to 5–10 of NCI. For an EPS-DRS concentration of 200 μg mL−1, the cell proliferation decreases suddenly after 150 h, after than a plateau has been reached. This has been noticed for the four metal-to-ligand ratios considered. This seems stopping the oscillating kinetics to very low and stable NCI values, comparable to those of heparin complexes (see Figure 3). No significant changes in cell viability for the four metal-to-ligand ratios considered, was observed, even at for the concentration of 200 μg mL−<sup>1</sup> in EPS-DRS.

**Figure 4.** Normalized cell index plot of MNNG/HOS cells after 160 h of contact with different stoichiometric ratios L:M: (**a**) EPS-DRS:Sc 1:0.5 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−1 (\*), and 200 μg mL−<sup>1</sup> (\*); (**b**) EPS-DRS:Sc 1:1 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*\*); (**c**) EPS-DRS:Sc 1:2 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*\*\*), and 200 μg mL−1; (**d**) EPS-DRS:Sc 1:4 at 50 μg mL−<sup>1</sup> (\*), 100 μg mL−<sup>1</sup> (\*), and 200 μg mL−<sup>1</sup> (\*).\* = *p* value ≤ 0.0001, \*\* = *p* value ≤ 0.005, \*\*\* = *p* value ≤ 0.05.
