## 3 Results  
### Polymucleotides Increase Cell Number  
As a first step, we decided to assess the best PN dose for our cell model and thus stimulated primary gingival fibroblasts with increasing doses of PN, up to 100 \(\mu\)g/mL (Figure 11). After 96 h of stimulation, we observed that PN had significantly increased cell number in all treated groups vs. the vehicle-treated, control group, with the difference becoming more significant as the dose increased. Concentrations higher than 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL did not cause further effects in cell number (data not shown). The concentration of 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL was therefore chosen for the subsequent experiments. To investigate the effects of PN with or without HA, we stimulated primary gingival fibroblasts with vehicle or 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of PN in the absence or in the presence of HA for 96 h or 1 week of culture (Figure 1). We observed that PN significantly increased cell number as compared to control at both time points (\(p\) < 0.0001) and that addition of HA further increased cell number as compared to both control (\(p\) < 0.0001 both at 96 h and 1 week) and PN alone (\(p\) = 0.087 at 96 h and \(p\) = 0.0014 at 1 week), as shown in Figure 1A, and captured quite visibly also by the microphotographs of the transmission microscope (Figure 1E,G,I). The microphotographs highlight that cells in the treated wells were more numerous, but that they also started to stack along multiple planes of growth in the presence of HA (Figure 1I). Though cells mostly exhibited a parallel or fan-like alignment in the control (Figure 1E) and PN-only groups (Figure 1G), they grew in orthogonal direction in the PN + HA group, and apparently superimposed onto one another (Figure 1I). We also assessed cell growth by evaluating their viability using two commercially available assays, CellTiter-GLO, which measures cells' ATP content, and MTT assay, which is based on cells' redox capability (Figure 1B,C). Though both these assays confirmed that PN increased the assay signal in a visible and significant way, PN + HA failed to further increase cell growth as compared to PN alone, at all time-points with the only exception of 1 week of culture in the CellTiter assay (Figure 1B, \(p\) < 0.0001). Figure 1F,H,J show  
Figure 1: **(A)** A Coulter Counter was used to determine cell number at 96 h and 1 week of culture. PN and PN + HA significantly increased cell number (****\(p\) < 0.001 control vs. either stimulated group). **(B)** Cell Titer GLO assay was used to assay cell viability in the control, PN or PN + HA groups at 96 h and 1 week of culture. PN or PN + HA significantly increased viability vs. the control at both time-points. (****\(p\) < 0.001 control vs. PN and PN + HA). **(C)** MTT viability assay was carried out at 96 h and 1 week of culture. PN, with or without HA, significantly increased cell viability at 96 h and 1 week of culture. ****\(p\) < 0.01 control vs. PN or PN + HA groups. **(D)** Lowry assay was also used to determine the protein control, PN or PN + HA groups after 96 h and 1 week of culture. Protein levels were significantly higher in the presence of PN or PN + HA at 96 h and 1 week of culture. ****\(p\) = 0.0068 control vs. PN, \(p\) = 0.0023 control vs. PN + HA; ****\(p\) = 0.0006 and \(p\) = 0003 control vs. PN and PN + HA, respectively. **(E,G,I)** Microphotographs of primary human fibroblasts at 1 week of culture in the control group **(E)** or in PN **(G)** or PN + HA **(I)** groups, at phase contrast microscopy. Magnification = 10\(\times\) (Bar = 100 \(\mathrm{\SIUnitSymbolMicro m}\)). **(F,H,J)** Microphotographs of primary human fibroblasts at 1 week of culture in control group **(F)** or in PN **(H)** or PN + HA **(J)** groups, after adding MTT, which generates blue needle-shape precipitates upon reduction to Formazan. Cells appeared more numerous in the presence of PN **(G,H)**, but even more so with the addition of HA, where cells appear stacked in multi-layered nodules **(I,J)**.