In this study, we assessed the cytotoxic impact of commonly utilized traditional temporary fixed materials (acrylic and composite-based) in routine dental practice, as well as temporary fixed materials generated through digital technology (CAD/CAM milling and 3D printing), which have gained prominence. Testing was conducted on mouse fibroblast cells, along with assessing residual monomer release from extracts obtained from an artificial saliva medium over durations of 24, 72, and 120 h.

Various dilutions at different time intervals showed different cytotoxic effects of the test materials. After 24 h, 72 h, and 120 h extraction, the 3D test material was found to be more cytotoxic than all other test materials. At 1:8 dilution, all materials except 3D were biocompatible at all time intervals. After 72 h extraction at 1:4 dilution, test materials except DP and 3D printing were biocompatible. Except for the 3D Printing material, cell viability rates differed between dilutions and decreased in a dose-dependent manner. In light of these findings, the first null hypothesis of the study is rejected. The digitally produced test materials had lower surface roughness values than the conventional ones. In addition, the second hypothesis of the study was partially rejected since monomer release from the test materials was not detected in the artificial saliva environment, thus showing similar findings.

Commonly employed resin-based dental materials are recognized to release various monomers following restoration placement, primarily due to incomplete polymerization, but also as a result of erosion and degradation over time [5]. UDMA, MMA, and TEGDMA are the monomers most extensively studied in the literature [7, 8, 22], and are associated with harmful biological outcomes [5].

Bandarra et al. [5] identified the release of UDMA in both ProTemp 4 (3.85 μM) and Structur 3 (83 μM), as well as the release of MMA in Tab 2000 (3.47 mM) after 24 h of immersion. In the present study, no monomer release was detected from the test materials after 24, 72, and 120 h of extraction. The difference may be attributed to the medium in which the extract was obtained. Given that the manufacturer’s SDS information does not mention the presence of UDMA in Protemp 4, the Bandarra et al. [5] UDMA detection is a remarkable finding. Manufacturers are only obliged to provide information about the main components; therefore, SDSs of dental composites do not always provide comprehensive information about the entire material composition [23].

The absence of any monomer release from the test materials in the present study may be because the resin-based material barely penetrates the polymer network when extracts are obtained in artificial saliva [24, 25]. However, ethanol has been employed by numerous researchers (due to its ability to permeate the material’s polymer network, increasing the spaces between polymer chains and promoting the gradual release of unreacted monomers [26]. An artificial saliva medium was used in this study to obtain extracts because it simulates the oral environment. Additionally, it makes sense to use this kind of solvent because provisional restorations are continuously exposed to saliva once placed. This interaction with saliva extracts and disperses various chemicals from the restorations throughout the oral cavity. Furthermore, using artificial saliva helps to standardize experimental conditions and prevent sample contamination [10].

Berghaus et al. [7] used different extraction media [water, ethanol, and ethanol/water (75/25 vol.] from CAD/CAM milling, 3D printing, and self-curing temporary crown and bridge materials and reported that there was no monomer release in the water. This finding is similar to the present study. The low tendency of the monomers to dissolve in water can be attributed to their hydrophobic nature, which is particularly evident in Bis-EMA and Bis-GMA [7].

The complete absence of monomers and polymers in the artificial saliva extracts was proven by FTIR analysis of 3D, CAD/CAM, DP and PT materials before and after 24 h, 72 h and 120 h exposure to artificial saliva. The typical absorption peaks related to the major monomers such as bis-GMA, TEG-DMA, bis-EMA and MMA were identified in the samples before extraction. These included significant peaks such as the carbonyl (C=O) stretching vibration at about 1720 cm−1, the C=C stretching vibration at about 1635 cm−1, and the aliphatic C-H stretching vibration in the range of 3000 to 3100 cm−1. However, the FTIR spectroscopy of the extracts did not show any peaks corresponding to these monomer or polymer structures, indicating that no residual monomer or polymer was released into the artificial saliva after exposure to artificial saliva. This suggests a high degree of material stability and polymerization efficiency, preventing the release of unreacted monomers.

Wei et al. [8] reported that the residual monomer released from CAD/CAM polymers (including provisional and prosthetic base polymers) is less compared to conventional ones. The increased cell proliferation observed in CAD/CAM dental polymers has been suggested to result from a reduced release of residual monomers. In the present study, CAD/CAM milling test samples (except pure and 120 h extraction time) were found to be biocompatible. However, no monomer release was detected. The reasons for the difference from the aforementioned study may be related to the chemical contents of the test materials used and the different media from which the extracts were obtained. On the other hand, CAD/CAM milling is more biocompatible than the conventional material (Dentalon Plus), which is similar to the mentioned study.

Atay et al. [11] stated in their study that one of the temporary produced by CAD/CAM milling technique (Telio CAD) was not cytotoxic and the other (Vita CAD Temp) showed mild cytotoxic. They reported that this may be due to the difference in chemical composition. In the present study, CAD/CAM milling test samples were found to be biocompatible in all subgroups (except pure and 120 h extraction time). These results partially overlap with the study of Atay et al. [11] The difference from the Atay et al.’ [11] study is the cell viability rates. The reasons for these are Atay et al. [11] categorized cytotoxicity grading as non-cytotoxic, slightly, moderately, and severely cytotoxic by the millipore filter method. They also used an XTT cell proliferation kit. In the present study, cell viability rates were determined according to ISO standards using MTT assay.

Gonçalves et al. [27] investigated the cytotoxicity of two bis-acryl composite resins (Protemp 4, Luxatemp Star) using human gingival fibroblasts. They concluded that these materials do not exhibit cytotoxic effects on human gingival fibroblasts. These findings are similar for the subgroups (except pure) of the Protemp material obtained at all time points in the present study. According to the results of the present study, Protemp material shows high cell viability rates. This can be attributed to the modification of the monomer system, which contains flexible chains instead of the rigid chains of bis-GMA homologs [11].

The primary reason for the cytotoxicity of dental resin materials arises from components that can be released, specifically, monomers and photoinitiators [12, 28]. The decreased cell viability percentage observed in the test samples generated through 3D printing in contrast to conventional and CAD/CAM milled samples may be attributed to the lower monomer-to-polymer conversion ratio in 3D-printed materials compared to other production methods. A range of factors, including the chemical makeup of the resin material, the ratio of photoinitiators, and the application method combined with light exposure, together determine the final degree of monomer conversion [28]. Short post-polymerization durations have been reported to have an adverse effect on the biological compatibility of resins [13].

In a study evaluating the cytotoxicity of temporary and permanent fixed printable materials on human gingival fibroblast cells, it was reported that lower cell viability rates may be caused by Diphenyl (2,4,6-trimethyl benzoyl) phosphine oxide photoinitiator in resins produced in DLP printers [29]. Similarly, in the present study, the 3D-printed resin showed low viability rates. The lower cell viability rates recorded in resins containing photoinitiator may explain the genotoxic and cytotoxic effects [30]. On the other hand, low cell viability may be associated with the release of by-products. As a result, by-products may show some side effects [8, 24].

In our study, cytotoxicity values were measured by obtaining extracts of test samples. Thus, the possibility of synergistic interaction between the substances was also considered. This method more accurately simulates real oral conditions compared to earlier cytotoxic studies that utilized pure substances or combinations of pure substances [5, 31].

The choice of immersion medium is a very complicated issue [32]. One study concluded that TEGDMA monomer can be released in similar amounts in saliva and 75% ethanol. However, for BisGMA, 75% ethanol was found to be more aggressive than saliva [33]. The US Food and Drug Administration (FDA) suggests a 75% ethanol/water solution, which is assumed to be a good food simulator (alcoholic beverages, fruits, and syrup) and hence clinically significant [5]. For this reason, many studies have used ethanol/water mixtures [5, 7, 24]. Nevertheless, the resins soften as a result of the nearly similar solubility characteristics of ethanol and bis-GMA. Particularly for Bis-GMA-based resins and polymers, ethanol/water solutions permeate the polymer matrix and cause irreversible degradation by creating soluble units and enlarging the area surrounding them [34]. Ethanol may not be a laboratory substitute for saliva when monomers other than TEGDMA can be eluted [33]. It was noted that acetone is very aggressive compared to all other storage media and therefore cannot be used as a clinically relevant medium [33]. As extraction solvents, both alcohol-based solutions and cell culture medium were employed; however, it has been demonstrated that albumin binding might result in false negative results, particularly when TEGDMA is detected [35]. More research with various immersion media is required.

Bandarra et al. [5] evaluated the cytotoxic effects of urethane dimethacrylate-based (Structur 3®), bis-acrylic-based (ProTemp 4™), and acrylic resin methyl methacrylate-based (Tab 2000®) used routinely in dental practice in a 3T3 mouse fibroblast cell culture model. All of the test materials caused a dose-dependent loss of cell viability, however, only Structur 3® extracts were cytotoxic against 3T3 fibroblasts, with the highest cytotoxic effect (77%) observed at 24 h incubation period, which they attributed to the releasing UDMA monomers. Ulker et al. [36] evaluated the cytotoxicity of two bisacrylic-based (Tempofit Duomix, Protemp 3 Garant) and one UDMA (Revotek LC) based temporary prosthetic materials and found that one of the bisacrylic-based materials (Tempofit duomix) was cytotoxic to L929 fibroblasts. In the present study, 3D-printed test materials were found to be cytotoxic on L929 fibroblast cells.

Cytotoxicity experiments were carried out in accordance with 10,993–5:2009 [17] (except the extraction media), which is recommended for the assessment of the biocompatibility of medical devices used in dentistry, to ascertain if the test materials were cytotoxic against fibroblast cells. The cell viability percentages of all subgroups except CAD/CAM milling 120 h 1:1, Dentalon Plus 24 and 72 h 1:1 and 72 h 1:2 dilution, Protemp material 1:1 (24, 72 and 120 h) and 3D printing are within the threshold value (70%) in ISO standards. In light of these findings, dilution rates and time intervals of the test material are also important in the cytotoxic effect.

In studies examining the cytotoxicity of temporary prosthetic materials, the media and duration of extraction differ from each other. In the studies, 24 h [6, 36,37,38,39], 72 h, [8], and 120 h extraction times [5] and cell culture media (DMEM or EMEM, etc.) [1, 5, 8], phosphate buffered saline [39] or artificial saliva [10] were used as extraction media. The reasons for the differences in cell viability rates and monomer release from the studies are that the experimental media and conditions also differ from each other.

Bandarra et al. [5] also evaluated the effect of extracts obtained at different time intervals on the cytotoxicity of the test materials and found that the longer the resin eluted, the lower the cytotoxicity. In the present study, cell viability rates between time intervals at different dilutions differed according to the test materials. No time-dependent change was observed in the cell viability rates of the 3D printing material. On the other hand, In Dentalon Plus, 72 h was more cytotoxic than the other two time intervals (1:4 dilution). For 1:2 dilution, CAD/CAM test material’s cell viability rates were ranked as 24 h > 72 h > 120 h. In Dentalon Plus, 72 h was more cytotoxic than 120 h. It is thought that the reasons for the difference from the study of Bandarra et al. [5] may be due to the difference in the cell lines used, the medium from which the extract was obtained, and the chemical composition of the test materials.

Dordevic et al. [10] evaluated the cytotoxic effects of conventionally produced PMMA-based resin extracts on rat macrophage viability under in vitro conditions. They obtained 24, 48, and 72 h extracts in an artificial saliva medium. The highest cytotoxic effect was observed in cells exposed to the highest concentrations (50, 40, and 30%) of the extracts extracted for 72 h. In the present study, for Dentalon Plus at 1:2 and 1:4 dilution, the most cytotoxic time interval was found at 72 h of extraction. In Dentalon Plus material, undiluted (pure) and 1:2 dilutions were more cytotoxic than 1:4 and 1:8 dilutions. These results are similar to the previously mentioned.

In the current research, the groups that were digitally generated exhibited substantially lower surface roughness than the conventional groups. These results were found to be consistent with Giti et al. [1]. Based on the surface roughness result of this study, it can be concluded that production methods affect the surface properties of the test materials and that digitally produced temporary materials may cause less discoloration and plaque accumulation than the conventional method. The surface roughness results are supported by the SEM images. The conventionally produced test materials exhibit a rougher structure than the digitally produced ones is similar to the SEM findings of Giti et al.[1]. Greater porosity and air bubbles are also observed in SEM images because it is challenging to achieve a relatively homogeneous mixture due to manual mixing in materials produced by the conventional method.

Shim et al. [39] evaluated the response of human gingival fibroblasts to temporary materials with different production methods and various chemical compositions and also examined the surface roughness of the materials. PMMA and bisacryl-based materials were reported to be less cytotoxic in HGF cell lines than PEMA ones. CAD/CAM milling materials have been recommended for use due to greater cell adhesion and prevention of residual monomer. PEMA and PMMA-based materials were found to be rougher than the bisacryl and CAD/CAM milling groups. In the present study, CAD/CAM milling showed a higher cell viability rate than Dentalon Plus at 24 h extraction and 1:2 dilution. This finding is similar to Shim et al. [39]. When surface roughness results are evaluated, similarly to Shim et al. [39], the CAD/CAM milling shows a smoother surface than Dentalon Plus. However, there was no difference in surface roughness between the conventional materials in the present study. The degree of polishability and smoothness is dependent on several factors, including the material’s inherent chemistry, the initiator, the makeup of the resin matrix, the presence of filler particles, and their size and distribution [40]. In the present study, Ra values ranged between 0.20 and 1.33 μm. These values were equal to or above the Ra threshold of 0.2 μm reported by Bollen et al. [41] but below the clinical undetectability limit of 10 μm defined by Kaplan et al. [42].

Chen et al. [38] evaluated the cytotoxicity of two resins for interim restorations (AA TEMP; Enlighten Materials and C&B; NextDent), which were printed using a DLP system and subjected to various post-polymerization processes. The researchers performed the cytotoxicity assay in the L929 mouse fibroblast cell model after collecting the extract for 24 h at 37 °C in cell culture medium. They found that resins not subjected to the post-polymerization procedure exhibited a reduction in cellular metabolism of over 70%. However, post-polymerization times of 1, 5, 10, 15, and 30 min resulted in only a minimal reduction in cellular metabolism. The authors indicated that the absence of post-polymerization left the resin surface unsealed, which allowed toxic substances from the inside of the material to migrate into the culture medium. With longer post-polymerization times, the surface seal improved, leading to enhanced cell viability [38]. In this study, the lower cell viability rates observed compared to the aforementioned study may be attributed to the power and parameters of the device used for post-polymerization, as well as the resin contents used. In the current study, the test samples produced by 3D printing were subjected to post-polymerization for 3 min, and considering the cell viability rates, this time should be increased. Additionally, it is essential to investigate the ideal production protocol to minimize the potential negative effects of 3D-printed temporary materials. It can be inferred that post-processing steps such as additional light curing and washing can improve the biocompatibility of printable materials.

Limitations of this study are the use of media that mimic the oral environment but also the need to incorporate thermal, chemical, and bacterial conditions to assimilate in vivo conditions, even when using human saliva. To simulate saliva flow in the mouth, the continuous change of the immersion medium must also be taken into account. In addition, it should be considered that mechanical effects on the surfaces of the temporary materials during intraoral service may affect monomer release. More studies are needed that can mimic clinical conditions by incorporating these factors into experimental conditions. On the other hand, further development of analytical methods using a combination of HPLC and mass spectrometry could provide valuable information on the identification of by-products from temporary materials.

Within the limitations of this study, the following conclusions can be drawn:

Although no residual monomer was detected in 24 h, 72 h, and 120 h extracts of temporarily fixed materials with different chemical contents in an artificial saliva medium, temporary materials produced by 3D printing were found to be cytotoxic in the fibroblast cell culture model.

Manufacturing techniques impact the surface characteristics of the test materials, with digitally produced test materials exhibiting lower surface roughness values compared to those produced by conventional methods.

CAD/CAM milling and PT materials may offer safe and effective options for temporary prosthetic restorations. Although DP showed acceptable results, it was less effective than CAD/CAM milling and PT materials. Due to their cytotoxicity, 3D-printed materials require further investigation before clinical use. Additionally, digitally produced temporary materials may result in less discoloration and plaque accumulation compared to those of conventionally produced.