# Evaluation of the Bond Strength and Cytotoxicity of Alkasite Restorative Material
## 4 Discussion
Despite their limitations, the advantages of in-vitro cytotoxicity tests include the control of the environmental conditions of cells, and the precise measurement of the response of the cells to restorative materials [21]. The biological effect of restorative materials can be evaluated by several methods [21]; for example, cell viability tests can be used to evaluate cytotoxicity of materials [47]. In such tests, certain cell lines should be used to mimic the clinical conditions [48]. HGFCs can be exposed to resin monomers released from RBC restorations [49; 50]; thus, this cell line was used in this study. To evaluate the cytotoxic effect of material thickness, both materials were tested at 2 and 4 mm. RBC discs were sterilized or decontaminated with an UV-light, despite its possible adverse effects on mechanical and biological properties of RBC materials [51]. Cell viability was assessed using the AlamarBlue(r) test,as fluorescence intensity could be related to the number and activity of cells [25]. The cell viability results showed that cells were viable, with constant growing over the seven-day period, which indicates that both materials are biocompatible at thicknesses of 2 and 4 mm. However, the cell viability for the 2- and 4-mm-thick CN was significantly lower than that of the 2-mm-thick BF-RBC at days 1 and 3. Moreover, at day 1, more prominent cell morphological changes were noted for CN groups. Therefore, the second hypothesis was partially rejected. The higher cell viability for the 2-mm-thick BF-RBC may be explained by the improved degree of conversion and the depth of cure at this thickness due to better penetration of the curing light. One of the basic characteristics of CN is its ion releasing property due to presence of alkaline fillers. CN releases fluoride amounts comparable to those of RMGIC [18]. This can, in part, explain the difference between the two materials. The cytotoxic effect of fluoride-releasing materials can be correlated to the concentration of fluoride ions released [52], as they can induce inflammation and apoptosis of HGFCs [53]. The degree of conversion of CN could be comparable to that of conventional RBCs [18]. Cytotoxicity of BF-RBCs can be affected by the depth of cure [28], and the BF-RBCs show variable degree of conversion rates and depth of cure at different thicknesses [5, 54]. Dual-cure RBCs may have less cytotoxic effects based on monomer release [55], as they combine various initiation systems that enhance the degree of conversion [56]. It is noteworthy that the difference between the cell viability of the 2- and 4-mm-thick CN was statistically insignificant over seven-day period. This can be attributed to their similar polymerization kinetics at the two thicknesses [12]. In addition, this might be also attributed to the similar levels of ion release at both thicknesses; however, further investigations should be performed to confirm this speculation. The thermocycling protocol, short-term aging, and the use of one BF-RBC for comparison with CN are of the limitations of this study. Further long-term studies using different universal adhesives and self-curing activators to compare CN to dual-cure RBCs or hybrid restorative material of regular viscosity are suggested. Moreover, although HGFCs primary cells were used in this study; they were used in a two-dimensional culture system, which might have affected their behavior.