In vitro dentin barrier cytotoxicity testing of some dental restorative materials

Abstract

Objectives

To investigate the cytotoxicity of four dental restorative materials in three-dimensional (3D) L929 cell cultures using a dentin barrier test.

Methods

The cytotoxicities of light-cured glass ionomer cement (Vitrebond), total-etching adhesive (GLUMA Bond5), and two self-etching adhesives (GLUMA Self Etch and Single Bond Universal) were evaluated. The permeabilities of human dentin disks with thicknesses of 300, 500, and 1000 μm were standardized using a hydraulic device. Test materials and controls were applied to the occlusal side of human dentin disks. The 3D-cell scaffolds were placed beneath the dentin disks. After a 24-h contact with the dentin barrier test device, cell viabilities were measured by performing MTT assays. Statistical analysis was performed using the Mann–Whitney U test.

Results

The mean (SD) permeabilities of the 300-μm, 500-μm, and 1000-μm dentin disks were 0.626 (0.214), 0.219 (0.0387) and 0.089 (0.028) μl min −1 cm −2 cm H 2 O −1 . Vitrebond was severely cytotoxic, reducing the cell viability to 10% (300-μm disk), 17% (500 μm), and 18% (1000 μm). GLUMA Bond5 reduced the cell viability to 40% (300 μm), 83% (500 μm), and 86% (1000 μm), showing moderate cytotoxicity (300-μm) and non-cytotoxicity (500-μm and 1000-μm). Single Bond Universal and GLUMA Self Etch did not significantly reduce cell viability, regardless of the dentin thicknesses, which characterized them as non-cytotoxic.

Conclusions

Cytotoxicity varied with the materials tested and the thicknesses of the dentin disks.

Clinical significance

The tested cytotoxicity of materials applied on 300-, 500-, and 1000-μm dentin disks indicates that the clinical use of the test materials (excepting self-etching adhesives) in deep cavities poses a potential risk of damage to the pulp tissues to an extent, depending on the thickness of the remaining dentin.

Introduction

Biocompatibility has been described as the reaction of a living system to exogenous materials . It is necessary to evaluate the biocompatibility of dental materials for safety before clinical use, and the main means for biocompatibility testing are in vitro, animal, and clinical tests . Cytotoxicity tests are convenient, provide repeatable results, do not require hurting animals, and are commonly used in vitro; however, the testing procedures of some traditional in vitro cytotoxicity tests, such as the agar or filter diffusion tests or extract tests, do not mirror clinical practice . In these traditional tests, dental cements, adhesives, and composite resins have been reported to elicit varying degrees of chemical toxicity to cultured cells, which mainly depends on the content of unpolymerized monomers, such as bis-GMA, HEMA, TEGDMA, UDMA, glutaraldehyde, and camphorquinone . However, some of these materials were reported to have no effect on pulp tissue in vivo . This discrepancy may have arisen because traditional methods combined with two-dimensional (2D) monolayer cell culture behave too sensitively, in comparison to complex in vivo conditions.

The dentin barrier test was developed to screen for chemical toxicity to pulp tissue by dental restorative materials, especially those used in direct contact with dentin . This method is used instead of traditional in vitro models, as it closely mimics clinical practice and provides results that more accurately reflect in vivo conditions . The design of this method, which simulates the contact process after materials are applied to teeth, enables the implementation of various other clinical procedures, such as etching, desensitization, and laser treatment, allowing it to replace animal experiments .

Diffusion of substances from dental materials to the pulp is influenced by dentin thickness due to the diffusion process through the dentinal tubules and the adsorption on dentin components . Consequently, the permeability of dentin can strongly affect the transfer of cytotoxic components from dental materials . The permeability of human dentin varies greatly, as human teeth develop independently and can be affected by various factors such as aging, carious lesions, and other external stimuli . This heterogeneity may interfere with test results in a dentin barrier test. Many in vitro studies have employed bovine dentin instead of human dentin . Although bovine dentin is thought to show less permeability variation than human dentin , human dentin disks better resemble the characteristic of human target tissue in vivo than does bovine dentin. In addition, the permeability of dentin has been shown to be inversely related to its thickness . However, to our knowledge, no study has investigated the effect of the thickness of human dentin disks on the cytotoxicity of materials in a dentin barrier test, and only one study was conducted to investigate this effect with bovine dentin disks, which showed that the cytotoxicity of materials decreased when increasing thickness of the bovine dentin disk; however, this effect was not always observed and was material-dependent . Many dentin barrier cytotoxicity studies have used 500-μm-thick dentin disks to represent the remaining dentin beneath a deep cavity . Thinner or thicker slices may be used to stimulate deeper or shallower clinical cavities .

It is important to use a dentin barrier testing method based on conditions that are as similar as possible to clinical conditions when evaluating the cytotoxicity of currently used dental restorative materials, especially those used in direct contact with dentin such as cements and adhesives, so as to help determine the chemical toxicity of these materials to pulp tissue in specific application in a cavity. Such a method should incorporate human dentin thickness as a critical parameter for simulating clinical cavities of various depths. Standardization of human dentin permeability is required for this testing method to produce consistent results .

The aim of this study is to evaluate, in vitro, the cytotoxicity of four dental restorative materials to three-dimensional (3D) cultures of fibroblasts in a dentin barrier test device, using human dentin disks of varying thickness.

Materials and methods

This study was approved by the Institutional Review Board of Peking University School and Hospital of Stomatology. Informed consent from patients was not required by the ethics committee.

Materials and sample preparation

The materials used in this study are listed in Table 1 . All materials were applied directly to the dentin, according to the manufacturer’s instructions. Non-toxic medical-grade silicone blocks, Φ 6 mm × 2 mm, were used as the negative control (100% cell viability).

Table 1
Materials used in the study.
Material Classification Manufacturer Lot. No. Main components
Vitrebond Light-cured glass ionomer cement 3M EPSE Dental Products N621444 Powder: glass powder, diphenyliodonium chloride
Liquid: copolymer of acrylic and itaconic acid, water, HEMA
GLUMA Bond5 Total-etching adhesive Heraeus Kulzer GmbH 010301 UDMA, 4-meta, HEMA, glutaraldehyde (trace), silica (trace), ethanol, camphorquinone (trace), water
GLUMA Self Etch Self-etching adhesive Heraeus Kulzer GmbH 010705 Acetone, water, UDMA, 4-meta, camphorquinone (trace), silica (trace)
Single Bond Universal Self-etching adhesive 3M EPSE Dental Products 528361 HEMA, bis-GMA, ethyl alcohol, MDP, silanized silica, water, camphorquinone
Positive control Peking University School of Stomatology Powder: glass powder, polyacrylic acid, diphenyliodonium chloride
Liquid: camphorquinone, ethyl-4-dimethylaminobenzoate, HEMA, water
Negative control Ji’nan Medical Silicone Rubber Products Factory 050701 Medical-grade silicone

Dentine disks

In total, 120 human third molars collected from adult patients aged 18–40 years were used. After extraction for orthodontic reasons, the teeth were cleaned by removing the debris and soft tissues, stored in 0.5% chloramine T solution in deionized water at 4 °C, and used within 2 months. Before use, the teeth were soaked in 70% ethanol for 15 min.

Dentin disks were obtained by cutting the teeth perpendicular to the long axis using a low-speed saw (Isomet-Buehler, Lake Bluff, IL, USA). The first cut was made at the cementoenamel junction to remove the root. Using the same cutting angle, disks of (300 ± 50)-μm, (500 ± 50)-μm, and (1000 ± 50)-μm thicknesses were obtained after removal of the entire pulp cavity, including the pulp horn. Only the disk next to the pulp cavity was used and, therefore, only one disk was sampled from each crown.

Assessment of dentin permeability

The hydraulic conductance of the dentin disks was measured before use in cytotoxicity tests. The equipment, made in-house according to the hydraulic-conductance model described by Outhwaite and Pashley , consisted of a water bath, a steel chamber, and a micropipette ( Fig. 1 ). The water bath, filled with deionized water, provided a pressure of 32 cm H 2 O (3.14 kPa) to the pulp side of the dentin disk. To remove the smear layer, the dentin disks were acid-etched on both sides with 35% phosphoric acid for 30 s, rinsed with deionized water, and cleaned in an ultrasonic cleanser (Kudos, Shanghai, China) at 53 kHz for 5 min. The dentin disks were fixed in the middle of the chamber by the steel inserts, with pressure applied from the pulp side to the occlusal side. A measurement area of 0.28 cm 2 was delineated by a pair of rubber “O” rings with an inner diameter of 6 mm, and only the area in the middle of the disks was used. In the dentin barrier test, the materials were applied to the same area. After the equipment chamber was filled with deionized water from the water bath, it was sealed, and then the whole system was filled with deionized water. A small air bubble was introduced into the micropipette. Experiments were performed at room temperature after the air bubble showed stable motion for 5 min. Before every measurement, a glass disk with a size similar to that of the dentin disks was tested to ensure a good seal. The scale of the micropipette was 100 μl and the division value was 5 μl.

Fig. 1
The hydraulic permeability device.

The volume of water filtering through the dentin disk was measured by displacement of the air bubble over a defined period of certain time (10 min for the 300-μm and 500-μm disks, 20 min for the 1000-μm disks). The dentin permeability was calculated using the following equation:

L p = J v /(A × t × P)

where L p is the hydraulic conductance of dentin (μl min −1 cm −2 cm H 2 O −1 ), J v is the volume of water filtering through the dentin disks during the observation time (μl), A is the measurement area (cm 2 ), t is the observation time (min), and P is the pressure applied to the dentin disks (cm H 2 O).

Thirty dentin disks with closer permeability to the mean value (n = 40) were selected from 40 dentin disks for each thickness and grouped randomly into four test material groups and two control groups (five disks per group). Before the application of cements and self-etching adhesives for the dentin barrier test, the smear layer of the dentin disks was rebuilt by grinding the disks with 400-grit sandpaper for 15 s under the same pressure. The prepared dentin disks were sterilized by soaking in 70% ethanol for 15 min and then thoroughly rinsed with deionized water, as described in the guidelines published by the International Organization for Standardization . The disks were stored in 0.9% sodium chloride solution at 4 °C and used within one week.

Three-dimensional cell culture

L929 mouse fibroblasts (ATCC CCL1) were maintained in minimum essential medium (MEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 IU/ml penicillin, 150 μg/ml streptomycin, and 2.2 mg/ml sodium bicarbonate. Cells at the end of the exponential growth phase were used.

Polystyrene scaffolds with 4 fiber layers (8-mm diameter, 3D Biotek, New Jersey, USA), a 150-μm fiber diameter, and 150-μm fiber spacing were used for 3D cell culture. The scaffolds were placed on the inserts of 6-well tissue culture plates with 2 ml of growth medium per well and seeded with 40 μl of L929 cell suspension (1.5 × 10 5 cells/ml). After a 48-h incubation (37 °C, 5% CO 2 ), the scaffolds were transferred to 24-well plates and incubated for 14 ± 2 d. The growth medium was changed three times per week, and the plates were changed once per week.

Dentin barrier test

After 14 ± 2 d, the scaffolds were introduced into a cell culture perfusion system (3D Biotek), which was partially customized. The polycarbonate split chamber, the main component of this system, was comprised of a cylindrical cavity with an inner diameter of 6 mm and a height of 25 mm ( Fig. 2 ). The dentin disks were placed on top of the scaffolds (occlusal side facing upward), such that the chamber was separated into two compartments by the dentin disks. The upper compartment simulated the tooth cavity, and the lower one simulated the pulp cavity.

Fig. 2
Diagram (A) and photograph (B) of the split chamber.

The lower compartment was perfused with 0.3 ml of assay medium (growth medium with 6 g/l HEPES buffer) per h for 24 h at 37 °C. The assay medium was pumped into the chamber inlet and out via the outlet. In the lower compartment, the fluid covered the cell scaffolds to just below the dentin disks. After 24 h, perfusion was stopped and the test materials were placed into the upper compartment in direct contact with the occlusal side of the dentine disk. This treatment lasted for 24 h at 37 °C. Cell viability was determined by performing an MTT assay.

The scaffolds were removed from the split chambers and placed into 24-well plates containing 1 ml of pre-warmed MTT solution (Sigma, St. Louis, MO, USA; 1 mg MTT/ml in MEM without phenol red). The scaffolds were incubated with MTT at 37 °C under 5% CO 2 for 2 h and washed two times with phosphate-buffered saline solution. The blue formazan precipitate was extracted by adding 0.5 ml of dimethyl sulfoxide and then shaking the plates at room temperature for 30 min. This solution (200 μl) was transferred to a 96-well plate, and the absorbance at 540 nm (OD 540 ) was determined spectrophotometrically.

Statistical analysis

Five replicates were used for each dental material and control, and each test was performed in duplicate. The results are expressed as a percentage of the negative control. The non-parametric Mann–Whitney U test (α = 0.05) was performed for statistical comparisons between groups, using SPSS software, version 20.0 (SPSS, Chicago, IL, USA).

Materials and methods

This study was approved by the Institutional Review Board of Peking University School and Hospital of Stomatology. Informed consent from patients was not required by the ethics committee.

Materials and sample preparation

The materials used in this study are listed in Table 1 . All materials were applied directly to the dentin, according to the manufacturer’s instructions. Non-toxic medical-grade silicone blocks, Φ 6 mm × 2 mm, were used as the negative control (100% cell viability).

Table 1
Materials used in the study.
Material Classification Manufacturer Lot. No. Main components
Vitrebond Light-cured glass ionomer cement 3M EPSE Dental Products N621444 Powder: glass powder, diphenyliodonium chloride
Liquid: copolymer of acrylic and itaconic acid, water, HEMA
GLUMA Bond5 Total-etching adhesive Heraeus Kulzer GmbH 010301 UDMA, 4-meta, HEMA, glutaraldehyde (trace), silica (trace), ethanol, camphorquinone (trace), water
GLUMA Self Etch Self-etching adhesive Heraeus Kulzer GmbH 010705 Acetone, water, UDMA, 4-meta, camphorquinone (trace), silica (trace)
Single Bond Universal Self-etching adhesive 3M EPSE Dental Products 528361 HEMA, bis-GMA, ethyl alcohol, MDP, silanized silica, water, camphorquinone
Positive control Peking University School of Stomatology Powder: glass powder, polyacrylic acid, diphenyliodonium chloride
Liquid: camphorquinone, ethyl-4-dimethylaminobenzoate, HEMA, water
Negative control Ji’nan Medical Silicone Rubber Products Factory 050701 Medical-grade silicone
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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on In vitro dentin barrier cytotoxicity testing of some dental restorative materials
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