Biocompatibility Evaluation of Four Dentin Adhesives Used as Indirect Pulp Capping Materials.

BACKGROUND: In many cases, the indirect pulp treatment (IPT) is an acceptable treatment for deciduous teeth with reversible pulp inflammation. Various medicaments have been used for IPT, ranging from calcium hydroxide and glass ionomers to dentin adhesives.
OBJECTIVE: This in vitro trial aimed to measure cytotoxicity in a cell culture, comparing the following four adhesives: Xeno® V (XE), Excite® F DSC (EX), Adhese® OneF (AD) and Prime & Bond NT (PB).
MATERIALS AND METHODS: The adhesives were prepared according to the manufacturer's instructions. After 24 hours of exposure, the cell viability was evaluated using a photometrical test (MTT test). Data were subjected to analysis of variance (ANOVA).
RESULTS: Adhesives, the main component of which was 2-hydroxyethyl methacrylate (HEMA), were found to be less cytotoxic, while those that included the monomer urethane dimethacrylate (UDMA were the most cytotoxic) in their composition. The effects on cell viability assay varied between the adhesives assayed with statistically significant differences.
CONCLUSIONS: The results may support the argument that Adhese® OneF is the least cytotoxic of the adhesives assayed, and may be considered as an adhesive agent for indirect pulp treatment. However, Prime and Bond NT showed a reduced biocompatibility under the same conditions.

INTRODUCTION

Pulp repair is possible whenever reversible pulpitis occurs, whether caused by caries, restoration, or trauma (). The objective of indirect pulp treatment (IPT) is to preserve vitality of pulpally involved teeth challenged by either reversible pulpitis or deep caries without pulp exposure (). To ensure the success of IPT, it is important to eliminate caries from the dentino-enamel junction and from the walls of the lateral cavity to obtain the best possible seal between tooth and restoration, thus preventing microfiltration (-). In this procedure, calcium hydroxide and glass ionomers are used as liner materials with good results.

Adhesive resin systems offer another IPT option. Etching before applying the adhesive will facilitate dentin dissolution, releasing growth factors that stimulate osteoblast activity. This causes the formation of sclerotic/tertiary dentin deposits, reducing dentinal permeability (). According to Falster (2002), () acid etching at a 10% concentration, has similar bacteriostatic and bactericidal effects as calcium hydroxide. Although this concentration was lower than that usually used, Falster found that this did not compromise its bacteriostatic and bactericidal effects.

The use of either self-etching adhesive systems or adhesives with previous acid etching reduces marginal microfiltration and caries recurrence (, ).

In IPT, however, good clinical outcomes depend not only on the physical and chemical properties of the product used but also on the biocompatibility of the adhesive system (). Biological compatibility must be a basic property of any dental material, and this is particularly relevant for adhesives used in cases involving proximity to dental pulp.

Clinical research has revealed relatively few adverse biological effects derived from applying adhesives directly to dentin. But numerous in vitro studies (, ) have found that the components of adhesive resins can have cytotoxic effects on fibroblasts. The pulp tissues may suffer pathological alteration when they come into contact with resin-composite adhesives, since uncured monomers can penetrate the dentinal tubules and thus reach the pulp (). Resin composite materials contain cytotoxic components such as monomers and co-monomers in their organic matrix (). Restoration materials commonly include two-hydroxyethyl methacrylate (HEMA), triethyleneglycol dimethacrylate (TEGDMA), bisphenol A-glycidyl-methacrylate (Bis-GMA), and urethane dimethacrylate (UDMA), all of which have been found in aqueous extracts taken from the cured restoration materials (, ). It has been shown that dentin adhesives present differing levels of cytotoxicity after exposure times of 24 and 72 hours as follows (from most to least toxic): Bis-GMA>UDMA>TEGDMA>HEMA (). HEMA and TEGDMA would appear to present less cytotoxicity in vitro than Bis-GMA or UDMA, which are more hydrophobic (, ). Adhesive systems contain a range of components, hence interactions between these may lead to varying levels of cytotoxicity that may be higher or lower than the individual substances alone (, ).

HEMA is a frequent constituent of dentin adhesive agents, and is present at concentrations that vary between 30 and 55%, playing a key role in the process of dentin impregnation (). Due to its low molecular weight and relative hydrophilicity, HEMA can spread through residual dentin, which may have harmful effects on odontoblast vitality, as well as physiological activity of the pulp ().

Polymerized dental resins release TEGDMA into aqueous media in large quantities causing a high proportion of their unreacted double bonds (). TEGDMA makes up 25-50% of the content of dentin adhesives (). Because of its lipophilic characteristics, TEGDMA has a capacity of penetrating the cytosol and membrane lipid compartments of mammalian cells with a number of cytotoxic effects ().

Another common component of dentinal adhesives – camphoroquinone (CQ) – is a photoinitiator that was released following the polymerization (, ). CQ is not a constituent of the polymer chain; hence a proportion of the component not involved in polymerization can provoke oxidative stress, DNA damage, and cytotoxicity ().

In this way, the cytotoxicity of adhesives may vary depending on the proportions of these components and their potential to penetrate the dentin.

This in vitro study used indirect contact testing to evaluate the potential cytotoxic effects of four recently developed adhesives in different cell culture dilutions.

MATERIALS AND METHODS

The trial utilized the L929 fibroblast line (European Collection of Cell Cultures) in a culture medium (Dulbecco’s Modified Eagle’s Medium (DMEM)) combined with 10% of fetal calf serum (FCS) and antibiotics (penicillin 100 U/ml and streptomycin 100 µg/ml).

Methyl methacrylate (Merck, Darmstadt, Germany) was used as positive control, while the culture medium was used as negative control.

An indirect method (based on extracts) was used following ISO 10993-5 norms (). A previous test was carried out to confirm the suitability of the methodology employed and the validity of the prototypes. After thawing the cell line, centrifugation was carried out at 200 g for 10 minutes, followed by cell counting and seeding in a 75 cm3 culture flask, which in turn was incubated under 7.5% CO2. The test sensitivity range was evaluated, and a growth curve plotted. Based on the results obtained, it was decided to culture 5000 cells per cell for 24 hours.

Adhesive procedures

Experimental procedures were carried out in triplicate with 6 wells per variable. Materials were used following the manufacturer’s instructions:

Material 1: Xeno® V

Material 2: Excite® F DSC

Material 3: Adhese® One F

Material 4: Prime & Bond® NT

These materials, their compositions, and the manufacturer of each material are listed in Table 1. Each material was placed on a Petri plate, light-cured, and allowed to set for two hours. The samples were covered with 2-8 μl of culture medium, without phenol red, at a surface-to-unit volume ratio of 64 mm2 / 200 μl, and were kept in the CO2 incubator for 24 hours. After this period of time, the pH of the extracts was determined; all yielded a pH of 8.5. Afterwards, the extract of each material was aspirated with a sterile syringe and filtered through a pore diameter of 0.45 μm.

Table 1

Materials, manufacturer and composition.

Dentin adhese	Manufactured	Components
Xeno V (XE)	DENTSPLY De Trey GmbH (Konstanz, Baden-Württemberg, Germany)Self-adhesive	Bifunctional acrylate, acidic acrylate, functionalized phosphoric acid ester, acrylic acid, water, dl-camphorquinone, tertiary butane, stabilizer.
Excite F DSC	Ivoclar Vivadent, Schaan, LiechtensteinTwo-step adhesive	HEMA, phosphonic acid acrylate, Bis-GMA, dirue thacrylates, silica, ethanol, catalysts, stabilizers.
Adhese One F	Ivoclar Vivadent, Schaan, LiechtensteinSelf-adhesive	Primer: acrylic ether phosphonic acid, bisacrylamide, water, Camphoroquinone, stabilizers.Bonding: Bis-GMA, GDMA, HEMA, fumedsilice, CQ, tertiary amine, stabilizers.
Prime & Bond NT/ NRC	DENTSPLY De Trey (Konstanz, Germany)Two-step adhesive	Adhesive: PENTA, UDMA, cetylamine hydrofluoride, acetone, nanofiller (amorphous silicon dioxide 8 nm), stabilizers.

With culture medium without phenol red and the corresponding extract, 1/1 (100% extract), 1/2, 1/4, 1/8 and 1/16 dilutions were prepared for each material, and the osmolarity of the dilutions was measured. These dilutions in turn were added to the cells 24 hours after seeding of the latter in 96-well culture plates. Methyl methacrylate dilutions of 10%, 5%, 2.5% and 1.25% were also added and used as positive controls. To assess the influence of pH upon cell viability, wells containing culture medium without phenol red were included, which served as negative controls, together with other wells containing culture medium prepared at pH 8. The plates were then incubated under 7.5% CO2 for 24 hours, and methyl thiazol tetrazolium (MTT) cytotoxicity assay was performed (MTT; Sigma Chemical Co. St. Louis, MO, USA), measuring absorbance in a plate reader at 570 nm, using a wavelength of 690 nm as reference. After 24 hours, the extracts’ pH was measured: all presented a pH of 8.

The results were interpreted by a technician blinded as to which materials were involved in different samples. Cytotoxicity was analyzed both quantitatively (% viability with respect to control) and qualitatively (cell morphology and viability).

Statistical analysis

Data underwent two-way univariate analysis of variance (ANOVA), supplemented by equality of matched pairs testing, using the least significant difference (LSD) method, with Bonferroni correction.

RESULTS

Figure 1 shows percentages of pulp fibroblast cell viability. Quantitative cytotoxicity results (% viability in comparison with the control) obtained for each material are expressed in Figure 1. For all materials, cell viability decreased as the concentration of extracts was increased. No significant differences were identified between concentrations 1, 2, and 3. Nor were differences obtained between 4 and 5, with the exception of Prime and Bond.

Figure 1

Cell viability values for all materials.

The least cytotoxic of the adhesives tested was Adhese, followed by Excite, Xeno and Prime and Bond (the most cytotoxic). Their effects on cell viability varied with statistically significant differences (p<0.001).

It was observed that pH 8 reduced cell viability, which was reduced by 40% in comparison with the control (Figure 2).

Figure 2

Cell viability pH values.

In the qualitative evaluation of cytotoxicity (compared with controls), methyl methacrylate had an effect evidenced by cell rounding and the disappearance of the cell nucleus. In the case of the negative control (culture medium), the cells were seen to maintain their characteristic elongated shape and the nucleus remained intact. In general, the materials tested included some rounded cells undergoing degeneration. (Figures 3 and 4).

Figure 3

Cell culture after exposition. Material: Adhese® One F 1/16 dilution.

Figure 4

Cell culture after exposition. Material: Prime & Bond® NT 1/1 dilution.

DISCUSSION

Biocompatibility must be a fundamental property of any dental material and this is of particular relevance in the case of dentin adhesives in close proximity to the pulp. Resinous monomers and other components of adhesive systems can cause varying levels of cell damage due to differences in chemical composition (). Interactions between these components and dentin will lead to varying pulp tissue responses (). In this way, the evaluated adhesives produced different levels of cytotoxicity, probably due to variations in chemical composition, physical properties, and the method of application.

When evaluating cytotoxicity, research has employed different methods of cell-to-material contact (). For cytotoxicity testing, The International Organization for Standardization (ISO), () recommends the use of established cell lines including L-929, Balb/3T3 and WI-38. These offer homogeneous morphology and growth characteristics and so facilitate reproducibility in in vitro cytotoxicity testing (). The present study selected the L-929 cell line as it is readily available, has been widely used in similar research, and behaves efficiently in in vitro situations.

Indirect pulp capping materials do not enter into direct contact with the pulp; therefore, indirect contact testing was used, since it could provide more realistic in vitro conditions for testing the cytotoxicity of the adhesives. The toxic effects on cells were evaluated using the MTT assay. This assay reduces methyl thiazol tetrazolium metabolically to colored formazan; the color reacts to the factors inhibiting dehydrogenase activity (, ).

Chen et al. () observed that adhesives might cause cytotoxicity in pulp cells when they came into close contact for 24 hours, which depended on their dilution, hence cytotoxicity increased in proportion to concentration of the adhesive. In this way, cytotoxic effects decreased when materials were more diluted due to the reduced concentration of toxic constituents.

Dentin adhesives contain different combinations and different concentrations of four methacrylate monomers: Bis-GMA, HEMA, UDMA and PENTA. Therefore, variations in concentration affect the toxicity of each material. Evaluations of the cytotoxicity of acrylates and methacrylates in dental materials display varying levels of cytotoxicity that is dependent on structure (). TEGDMA, Bis-GMA, and UDMA all present moderate levels of cytotoxicity (, ). Ratanasathien et al. () evaluated the cytotoxicity of the constituents of dentin adhesives and rated toxicity levels from highest to lowest as follows: Bis-GMA>UDMA>TEGDMA>HEMA after 24 hours and 72-hours exposure. Kusdemir et al. () also reported that the primer used with HEMA-based two-step self-etching adhesives presented lower levels of cytotoxicity than one-step bond materials containing monomers of higher molecular weight.

Previous studies have shown that typical components of adhesive and restoration resins, such as HEMA and TEGDMA are able to spread through dentin tubules, thus reaching the pulp tissue at concentrations that fall within the millimolar range (, ). It has been demonstrated in vitro that even at non-toxic levels; these monomers can disrupt the normal differentiation processes of pulp fibroblasts (, ). This finding concurs with another study that affirmed that HEMA and TEGDMA are detrimental to odontogenic differentiation of pulp stem/progenitor cells, an effect that would negatively affect the pulp tissue homeostasis and repair (-). Applied to deep cavities, these residual monomers can reach the pulp by diffusion, and penetrate more easily when the dentin has been etched. At certain concentrations they exert a toxic effect on pulp cells, resulting in inflammation and tissue disorganization. Pulp reactions vary in severity depending on additional factors including the composition of the material and clinical techniques employed ().

In the present study, Adhese showed low toxicity, a finding that concurs with other research (). This is due to the presence of HEMA in its composition. Bis-GMA shows the highest toxicity among components of Adhese but has less capacity to penetrate the dentin due to its higher molecular weight (228.29). However, bis-GMA is subject to hydrolysis, generating the water-soluble metabolite methacrylic acid (MAA). MAA is a source of cytotoxicity as it can stimulate TNF-α release, or alter the lipid layer of cell membranes, thus influencing the permeability of the membrane ().

UDMA is more toxic to cells than HEMA. Huang and Chang () observed a higher cytotoxicity with Prime and Bond and argued that this is caused by the presence of UDMA in its composition. Indeed, in the present study, adhesives containing UDMA were found to be more cytotoxic, with Prime and Bond the most cytotoxic of all. This result agrees with other studies that have shown that Prime and Bond is initially highly cytotoxic ().

The present study found no significant difference between Xeno and Excite. This finding is not in agreement with other authors (), who have observed that Xeno shows less cytotoxicity as it has a lesser tendency to degrade, has more stable molecules and does not contain HEMA or bis-GMA.

Camphoroquinone (CQ) may be another cause of dentin adhesive cytotoxicity, being the most frequently used photoinitiator (). This substance was present in all the materials that were evaluated (Table 1) and could affect cell metabolism, a possible mechanism provoking negative clinical and subclinical responses ().

With regard to control samples, pH was seen to be a non-specific variable that influenced the total cell viability. Thus, viability was seen to reduce due (partly) to this factor rather than the specific toxicity of the material; for this reason, the results obtained could achieve greater reliability by controlling this variable.

The thickness of the Dentin can have an effect on both the concentration and quantity of the adhesive reaching the pulp area. Hamid and Hume () investigated into the influence of dentin thickness on the level of penetration by the resin monomers in bonding agents after 24 hours incubation, testing dentin slices of 0.4-3.6 mm thickness. The diffusion rate was inversely proportional to the area of dentin consisting of dentinal tubules. Toxicity decreases as dentin thickness increases; if it is greater than 0.5 mm, toxicity is reduced by 75%, and if greater than 1 mm, toxicity falls by 90% () Therefore, dentin thickness in IPC is a determining factor for controlling the toxicity of adhesive systems.

CONCLUSIONS

Both self-etching and two-step adhesive systems show high cytotoxicity, which decreases as dilution increases.

Adhese presented the highest biocompatibility among the adhesives that were evaluated, and the lowest cytotoxicity. Next in order was Excite, found to present moderate cytotoxicity. Xeno presented high cytotoxicity, but Prime and Bond were found to display the highest cytotoxicity, as UDMA is its main component. Further studies are needed to determine which of the components of the material are responsible for harmful effects on cells. Such studies will need to take into account other physical and chemical properties of adhesives, which could affect the successful treatment.