## 4 Discussion  
Overall, the studies reviewed here indicated that acrylic resins used in prosthodontic appliances can exhibit varying levels of cytotoxicity, with significant differences observed across different resin formulations. Residual monomers, such as methyl methacrylate (MMA), are a primary source of cytotoxicity, as these compounds are released over time and negatively affect cellular viability [29]. Moreover, we observed that the cytotoxic response seems to be influenced by factors such as the curing process, resin composition, and storage conditions. Cytotoxicity analysis of dental materials is of utmost importance as it ensures the safety and biocompatibility of these materials when in contact with human tissues. By evaluating the potentially toxic effects of dental materials on cells, such as pulp cells or gingival fibroblasts, cytotoxicity analysis provides valuable insights into their potential adverse effects on oral tissues. This analysis aids in identifying any potential risks or side effects associated with dental materials, allowing dentists and dental professionals to make informed decisions when selecting suitable materials for various dental procedures. Ultimately, cytotoxicity analysis plays a crucial role in promoting patient safety and optimizing the long-term success of dental treatments. According to International Standard Organization (ISO) 10993-5 [30], the in vitro cytotoxicity test plays a vital role in assessing the biocompatibility of biomedical materials. It serves as the first step in determining the safety of these materials before further tests, including those involving laboratory animals. In vitro techniques offer numerous advantages compared to in vivo methods. These include controlling experimental variables, obtaining relevant data more efficiently, and often having shorter testing periods. Although there is a challenge in extrapolating in vitro data to clinical applications of biomaterials, this can be addressed using suitable reference materials currently utilized in clinical settings. Overall, by establishing the non-toxicity of a material through in vitro cytotoxicity analysis, researchers can proceed with confidence in studying its overall biocompatibility, which will include other materials' biological properties. ISO 10993-5 also establishes methodologies and cell types for cytotoxicity analysis. In this review, we observed that most of the studies used different methods and cell types compared to the suggested by ISO. According to ISO 10993-5, if viability drops below 70%, the material is considered to have cytotoxic potential. ISO suggests the MTT test, which uses the reagent (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl) tetrazolium bromide) to indicate cell metabolism. Many studies included in this review applied analyses based on this cellbehavior, which differs from cell viability. Despite being widely applied, the MTT assay has some disadvantages compared to viability tests such as the sulforhodamine B (SRB) assay, which are also found in the studies along with this review. The SRB method calculates cell enumeration based on protein content, minimizing the interference of endogenous and exogenous substances, which strongly influence the MTT outcomes. For this reason, despite not being the suggested ISO method, the SRB test has presented superior predictive power than the MTT assay and is encouraged to be performed. Microscopy analyses with Live/Dead kit were performed in some of the selected studies. This methodology is a valuable tool for assessing the cytotoxicity of materials due to its ability to visualize the cell damage directly. One major advantage of live/dead microscopy is its real-time assessment, enabling the observation of cellular responses immediately after exposure to a material. This provides valuable information regarding the speed and extent of cell death or damage. However, despite the name, live/dead kits do not necessarily stain live and dead cells. In contrast, they stain cells with or without membrane damage.