## 4 Discussion  
CS-based treatments have been proposed in dentistry to eliminate bacteria and promote dentin and bone remineralization [17]. Biological substances seeded in the scaffolds are released and implanted in the body, directly influencing the behavior of ingrowing cells and thereby increasing the amount and quality of tissue neogenesis [18]. Thus, we fabricated CS scaffolds, with and without SIM, and investigated their outcome when in \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**AB**} & \multicolumn{2}{c}{**ALP**} \\ \cline{2-5}
**Group** & **Day 1** & **Day 14** & **Day 1** & **Day 14** \\  & **Mean (SD)** & **Mean (SD)** & **Mean (SD)** & **Mean (SD)** \\ \hline Negative & 124.67 (31.13) & 1.03 (0.21) & 0.46 (0.27) & 1.19 (0.26) \\ Collacote-Positive & 167.83 (29.63) & 378 (132.55) & 0.64 (0.19) & 1.05 (0.36) \\ CS alone & 198.67 (79.96) & 179.67 (15.24) & 0.51 (0.29) & 0.52 (0.27) \\ CS/SIM 0.01 & 125.33 (7.74) & 1.05 (0.07) & 0.30 (0.19) & 0.37 (0.05) \\ CS/SIM 0.03 & 124.83 (17.33) & 1.06 (0.12) & 0.25 (0.07) & 0.62 (0.52) \\ CS/SIM 0.05 & 125.33 (15.06) & 1.12 (0.06) & 0.74 (0.74) & 0.80 (0.41) \\ \hline \hline \end{tabular}  
* AB: AlamarBlue, ALP: Alkaline Phosphatase, SD: Standard deviation, CS: Chitosan, SIM: Simvastatin.  
\end{table} 
Table 1: AB and ALP data in days 1 and 14. Figure 4: Alkaline phosphate activity for the SIM dose response on day 1 and 14. ALP, alkaline phosphate activity; CS, chitosan; SIM, simvastatin. Figure 5: Alizarin red S staining images of the SIM dose response on (**A**) day 1 and (**B**) day 14. SIM, simvastatin. Arrows indicate mineralized matrix formation. contact to cells regarding their growth, osteoblast differentiation, and matrix mineralization. Previous studies have shown that both CS and SIM promote proliferation and osteoblastic/odontoblastic differentiation of mesenchymal stem cells, supported by ALP production, mineralization, and bone sialoprotein expression [5; 16; 19]. Furthermore, the incorporation of SIM into scaffolds including CS in previous in vitro studies showed no adverse effect on cell viability, enhanced cell proliferation, and mineralization [20; 21]. Our results are in agreement with these studies in using CS alone, where the cells were able to proliferate and present differentiation patterns on the scaffold structure while SIM did not promote cell proliferation within all groups and only supported cell differentiation. The range of SIM doses is broad in the literature and varied values have been reported as optimum concentrations; 0.01-1 umol/L SIM was shown to have a stimulatory effect on osteoblastic/odontoblastic differentiation of mesenchymal stem cells (MSC) [14], 0.2 um SIM was found to be the ideal concentration for mouse bone marrow MSC proliferation [22], \(10^{-8}\) M was reported to be the optimum dose for periodontal ligament cell osteoblastic differentiation [23] and recently 0.1 umol/L SIM was selected as the bioactive dose for dental pulp cell migration and odontoblastic differentiation [5]. Doses above 0.1 um were reported cytotoxic [24] and in other studies, 1 um and 5 um were reported as the cytotoxic doses that significantly inhibited MSC proliferation [24; 25]. Furthermore, SIM concentrations higher than 0.5 mM showed a reduction in cell proliferation [22]. These different findings can be explained in that statins induce their beneficial effect on cell proliferation only at specific concentrations and can be highly cytotoxic if above the optimum concentration level. In addition to concentration, statins' hydrophilicity or hydrophobicity can regulate their cellular toxicity [26; 27]. SIM molecule hydrophobicity enables passive penetration into cell membranes causing its cytotoxicity thus inhibiting cell proliferation [26]. While we chose to evaluate lower doses, based on the nearest aims of these studies to our research purpose [5], we can still assume that they had some cytotoxic effects.