Resin-based dental materials are widely used in restorative dentistry, owing to their many desirable qualities, including excellent esthetic outcome, easy handling, favorable mechanical properties, and improved bonding efficiency [1
]. However, concerns remain over the biocompatibility of unreacted resin monomers following incomplete polymerization of such light-cured materials [4
]. Extracts from resin composites have been reported to have genotoxic, mutagenic, and estrogenic effects [7
]. Therefore, toxicity levels need to be experimentally determined to clarify the safety of resin-based dental materials in clinical settings [7
There have been contradictory reports concerning the biocompatibility of resin-based materials. Ruey-Song Chen et al. [14
] reported the cytotoxicity of three dentin bonding agents on human dental pulp cells. In contrast, Alexander Franz et al. [15
] reported no significant cytotoxic effects of dental bonding substances on L929 cells. Another study tested the same resin-based materials and reported very different results [16
]. However, these studies differed in sample preparation methods, cell lines, application methods, and types of products. Perhaps most importantly, different products undergo different degrees of conversion and, thus, produce different amounts of monomer extract, thereby leading to large variations in cytotoxicity [10
]. Therefore, standardized sample preparation and curing procedures are necessary to assess the actual toxicity of dental resin-based materials.
The international standard ISO 7405, which addresses the biocompatibility of dental materials and devices, states that biocompatibility tests should be performed on materials in an “as-used state.” In the case of light-curing materials, the standards recommend that light curing be performed in the presence of oxygen, reflecting the conditions that are experienced in clinical use [20
]. According to the recommendations, light-curable composites should be polymerized using a light-curing unit for 20 s and then used in biocompatibility tests. However, complete curing is not always possible in clinical practice because of the existence of saliva or anatomical problems [21
]. The incomplete curing thus leads to the release of cytotoxic leachable monomers. Hence, toxicity should be tested in vitro and in vivo to elucidate the actual effects of dental materials.
Accordingly, in the present study, we evaluated the cytotoxicity of two types of light-curing resin under four different conditions, including uncured, direct light-cured, post-cured, and post-cured samples, with the removal of the oxygen-inhibition layer. We aimed to assess the cytotoxicity of resin-based dental materials under conditions that closely mimic those encountered in clinical practice.
A standardized protocol for biocompatibility evaluation is essential to assess the safety of dental materials. Several studies have investigated the cytotoxicity of resin-based materials and found that unreacted dental resin monomers are toxic to human gingival fibroblasts and keratinocytes. These monomers can be released from the final product, which can also be cytotoxic itself, according to in vitro studies [6
]. Geurtsen et al. [9
] reported that the bisphenol-A-glycidyl methacrylate (Bis-GMA) monomer has the strongest cytotoxicity, followed sequentially by UDMA, TEGDMA, and HEMA. In addition, all resin monomers exhibited a dose-dependent genotoxicity [7
]. Furthermore, it has been reported that unreacted resin-based materials may release substances into an aqueous environment for extended periods, possibly causing cell damage and pulpal inflammation [8
]. These findings suggest that a cytotoxicity test is suitable for the evaluation of basic biocompatibility [24
There are numerous techniques available for cytotoxicity evaluation, such as the MTT assay, agar diffusion test, filter diffusion test, and pulp and dentine usage test [20
]. Two test methods were used in this study: the agar diffusion test and MTT assay. In the agar diffusion test, samples were separated from the cells by an agar layer mimicking the mucosal membrane, whereas in the MTT assay, extracts of the samples were used, mimicking constituents leaching into the saliva. The endpoint of the agar diffusion test is membrane integrity, and that of the MTT assay is mitochondrial activity. The methods address different aspects of cytotoxicity, and the results collectively provide an overview of the cytotoxic potential of the samples [31
]. Previous in vitro studies have yielded contradictory findings regarding the cytotoxicity of resin-based dental materials [6
]. These differences likely arise because in vitro cytotoxicity tests do not accurately reflect the clinical situation. In the clinic, resin-based materials are inserted into the oral cavity in a freshly mixed, incompletely polymerized stage; local responses are provoked by unreacted or only partially reacted components. After polymerization, the surface is usually polished to remove the oxygen inhibition layer, which may cause the release of toxic constituents from the material [8
]. To optimize cytotoxicity evaluation, suitable sample preparation methods are important. In this study, the effect of these different conditions on the sample preparation of resin based materials was investigated. We applied the various conditions shown in Table 2
. The results showed similar trends in both the agar diffusion and MTT tests; however, there was a significant difference in cell viability according to different sample preparation methods for the same materials. This was also confirmed in the images obtained using confocal laser microscopy. Uncured samples had the highest cytotoxicity, while the samples retaining the oxygen-inhibition layer had a higher degree of toxicity than those that underwent the polishing process. In this respect, the removal of the inhibition layer was found to be a crucial factor for increased cell viability.
In this study, the degree of conversion was assessed using FTIR spectroscopy (Table 3
). The degree of conversion would indicate the proportion of polymerized products from their original monomer state through free radical polymerization reactions. In other words, the lower the degree of conversion, greater the proportion of unpolymerized monomers available to cells during the cytotoxicity tests. The results indicated that the value for the degree of conversion was approximately 75% and 49% for direct-cured composite resin and adhesive resin, respectively. The values then increased as post-cured samples were analyzed (approximately 88% and 61% for composite resin and adhesive resin, respectively). These values were, in fact, in agreement with previous studies that used either the same or similar products [33
]. However, the degree of conversion further increased to approximately 95% for both products after removal of the oxygen inhibition layer, which is an extremely high value―such a result has not been previously reported in the literature. It was clear that the degree of conversion of the two materials, in descending order, was samples with unreacted layer removed > post-cured > directly cured > uncured.
The results were then compared with the cytotoxicity tests. The agar diffusion test indicated that both directly cured and uncured samples were more cytotoxic than both post-cured and those with the unreacted layer removed. Additionally, Figure 3
and Figure 4
indicated that the order of cell viability following MTT assay was unreacted layer removed > post-cured > directly cured > uncured, for both bonding resin and composite resin―an order equivalent to the degree of conversion. This finding was further confirmed by fluorescence imaging in the Live/Dead Assay®
. It is well known that oxygen inhibits free radical polymerization and yields polymers with uncured surfaces [35
]. Hence, it may be the reason for the extremely high degree of conversion value for samples with the unreacted layer removed. Removal of such a layer would have increased the degree of conversion and, consequently, the adequate polymerization would have influenced the biocompatibility of the restoration [36
As shown in Figure 3
and Figure 4
, there was a difference in cytotoxicity between composite and bonding resins. Generally, composite resin has a higher filler content than bonding resin, which contains water, alcohol, or acetone, although the contents differ depending on the product [19
]. These hydrophilic components may affect the solubility of bonding resin, i.e., cells may be more easily affected by toxic materials from bonding resins. However, as the dilutions increase, the effects of hydrophilic resin monomers, such as Bis-GMA, UDMA, TEGDMA or camphorquinone, have a critical impact on total cell viability in the MTT assay. It has also been reported that filler contents appear to influence polymerization [37
]. The results shown in Figure 3
and Figure 4
can be explained by these differences in released components and filler contents.
Although the MTT assay revealed differences between dilutions and resin types, these differences were not observed in the agar diffusion test. Because an agar overlay test only quantitatively demonstrates the decolorization zone and lysis index, cytotoxicity is usually confirmed by other qualitative methods such as an MTT assay.
This study showed that different methods of sample preparation led to different cytotoxicity levels of dental resin. Because the present study was conducted on mouse fibroblast cells, as recommended by international standards, conclusions regarding the possible toxicity in vivo are limited [24
]. A major concern regarding in vitro test data is the relatively poor correlation among these tests. The International Standards Organization (ISO) recommends the use of established cell lines, such as L929 mouse fibroblasts, for cytotoxicity tests. Because L929 cells are easy to prepare and culture, they are commonly used for cell culture-based standardization of cytotoxicity studies [20
]. In addition, L929 cells are highly sensitive to the lytic action of cytotoxins, and exhibit a greater decrease in cell viability than other cell lines [31
]. This enables greater sensitivity in assessing the degree of cytotoxicity.
Dental materials, such as resin composites and bonding agents, can harm teeth and the surrounding soft tissues, and lead to hypersensitivity or other symptoms when applied clinically. Therefore, tests methods that mimic in vivo conditions, such as a dentin barrier test will, no doubt, be more clinically relevant. Although our protocol does not reflect clinical conditions as well as an extended dentin barrier test or a long-term in vivo study, it is economical and easily available. Further studies comparing and correlating cytotoxicity results with a dentin barrier test or in vivo test will produce more clinically relevant results. Despite these limitations, the present study indicated that the selection of an improper method may lead to false-negative cytotoxicity results. Hence, careful consideration in selecting the sample preparation is required.
Furthermore, our findings may have implications for the selection of sample preparation method. More specifically, clinicians should remove unreacted monomers on the resin surface immediately after restoring teeth with light-curing resin to limit cytotoxic effects.