## 4 Discussion
Another drawback is that live/dead microscopy can only depict the current status of cells and does not provide information about the underlying mechanisms or long-term effects of cellular damage. For these reasons, it is recommended that other complementary tests are performed to gain a more comprehensive understanding of cytotoxic effects. The safety of 3D-printed acrylic resins highly depends on their degree of conversion. The incomplete polymerization may lead to a higher concentration of free monomers known to cause cytotoxicity [31]. Previous in vitro studies comparing heat-polymerized acrylic resins to auto-polymerized resins also found that the latter tend to exhibit higher cytotoxicity due to incomplete polymerization, leading to increased monomer release [32; 33]. The duration of resin exposure to biological tissues also plays a critical role in cytotoxicity, with prolonged exposure correlating with heightened adverse effects on cell cultures [32]. To reduce the number of uncured monomers from 3D-printed devices, washing protocols are essential [20; 28]. Usually, the rinsing liquid is a 90% or higher concentration of ethylic or isopropyl alcohol (IPA) [24; 25], or tripropylene glycol monomethyl ether (TPM) [20; 21]. There are no statistical differences in biocompatibility regarding the washing solutions [20]. The washing method was also investigated, and no differences in cytotoxicity were found between the rotatory wash and the ultrasonic bath [21]. The washing time presents conflicting data. While some studies do not present differences in washing time biocompatibility results, ranging from 5 min to 1 h [25; 28], others demonstrate a significant improvement in cell viability related to an increase in washing time, reaching their best results at 20 min [20; 21]. Surface treatment of resin-based 3D-printed dental devices is recommended to reduce surface roughness, which can minimize biofilm formation and enhance the longevity of prosthetic appliances. For a long time, it has been known that the surface roughness of materials plays a critical role in bacterial adhesion, and smoother surfaces exhibit a significantly lower risk of biofilm development [34]. A novel approach involves applying unpolymerized 3D printing resin as a coating agent to the device's surface, which can be particularly advantageous when physical modifications, such as abrasive polishing, are not desired--for example, in the intaglio surface of dentures. This technique has been shown to improve surface smoothness and mechanical properties without altering the physical structure of the prosthetic. Additionally, in vitro studies using the L929 mouse fibroblast line have demonstrated that this method does not induce significant cytotoxic effects, as determined by LDL assay (ISO 10993-5 standards) [23; 24]. Special attention must be paid to the post-curing treatment, which is essential to properly polymerize the coating resin [18]. On the other hand, when no coating is applied, mechanical polishing of 3D-printed devices with low-grit silicon carbide instruments is essential to reduce their cytotoxic effects. After a proper polishing procedure, 3D resin-based printed occlusal devices perform similarly regarding cytotoxic effects in human gingival fibroblast cell assays compared to milled and conventionally processed ones [14]. Post-curing protocols vary in the literature. The time of post-curing is the most studied variable, ranging from 1 min to 120 min [9; 10; 12; 13]. An increase in post-curing time enhanced cell viability and biocompatibility in human periodontal ligament fibroblasts [9] and human gingival fibroblasts [10; 13]. Data show that a post-curing time of around 20 to 30 min is necessary to reduce the cytotoxicity of 3D printable resins to clinically acceptable levels [9; 10; 12; 13]. The temperature of the post-curing protocol ranges from 40 \({}^{\circ}\)C to 80 \({}^{\circ}\)C [13].