Cytotoxicity of post and core composites as a function of environmental conditions

Abstract

Objectives

In the revised version of ISO 7405 there are so far no detailed recommendations concerning temperature and humidity during specimen production for light curing and chemically setting dental materials. The main objective of the present study was to observe if different environmental conditions during specimen production influence cytotoxicity and degree of conversion of four post and core composite materials and to investigate if cytotoxicity of post and core materials is influenced by their corresponding bonding substances.

Methods

Specimens of four different post and core composite materials (LuxaCore – Dual, Core X-Flow, Flow White and MultiCore Flow) were produced in a climate test chamber at 23 °C/50% relative humidity or 37 °C/95% relative humidity and were dual-cured or self-cured, with or without their corresponding bonding substances. Specimens were added to cell cultures immediately after production or after preincubation for 7 days. Specimens were incubated with L-929 fibroblasts for 72 h and cell numbers determined by a flow cytometer. FTIR spectroscopic measurements of post and core materials were performed at the same temperature conditions as for the cytotoxicity assay (23 °C or 37 °C).

Results

Dual-cured specimens of all post and core composites exhibited less cytotoxicity under both environmental conditions than self-cured specimens. All self-cured specimens manufactured at 37 °C/95% showed less cytotoxicity than specimens produced at 23 °C/50%. All dual-cured specimens showed similar cytotoxicity at both environmental conditions. After 7 days of preincubation most dual-cured specimens produced at 23 °C/50% showed less cytotoxicity than self-cured specimens (with the exception of Flow White). Compared to fresh specimens, 7-day aged specimens of most materials showed reduced cytotoxicity. Materials already showing low cytotoxicity as fresh specimens did not further reduce their cytotoxicity after 7 days of preincubation. For dual-cured materials the degree of conversion was higher compared to self-cured materials.

Significance

Different temperatures during specimen production have an impact on cytotoxicity and degree of conversion of dual-curing composite materials. Detailed recommendations for standardization concerning environmental conditions during specimen production are required.

Introduction

Biocompatibility of dental materials has gained increasing interest among dentists and patients during recent decades. Cytotoxicity is one important aspect of biocompatibility and has been tested with varying protocols and therefore results are not generally comparable between laboratories . It has been shown that, for example, the color of specimen molds and different ratios of specimen sizes to cell culture parameters produce different results . Therefore in the revised version of ISO 7405 specimen production has been identified as a critical factor regarding the comparability of results. Recommendations for specimen production have been introduced. For light curing materials and chemically setting materials specific recommendations exist concerning light and oxygen exposure, but not for temperature and humidity.

We have shown earlier that the degree of conversion of dentin bonding substances is significantly influenced by an air inhibition layer which is generated when specimens are cured in the presence of oxygen . A lower degree of conversion of dentin bonding agents results in increased cytotoxicity . Therefore in ISO 7405 recommendations were introduced to avoid air inhibition during specimen production to ensure internationally comparable results for cytotoxicity testing .

It has been known for a long time that temperature and relative humidity influence the quality of composite restorations. Some studies indicate that the bond strength is influenced by temperature and relative humidity. It has been shown that bond strength increases with increased temperature and decreases with increased relative humidity . However, contradictory results have been found in that some studies report that relative humidity does not influence bond strengths of self-etch adhesives . The influence of environmental conditions on the cytotoxicity of composite materials has not been studied so far, although in the recently revised version of the standard ISO 7405, dealing with the evaluation of biocompatibility of medical devices used in dentistry, it is recommended that temperature and humidity should be taken into account during specimen production .

Other factors that might influence cytotoxicity of composite materials are: (i) aging of specimens, e.g. fresh specimens vs. 7-day preincubated specimens , (ii) curing mode of specimens and (iii) bonding substances . With regard also to these criteria the following null-hypotheses were formulated with respect to conversion and cytotoxicity of post and core materials:

  • (1)

    The ambient temperature during specimen production does not influence degree of conversion of post and core materials.

  • (2)

    Fresh specimens are not cytotoxic (i.e. cell cultures incubated with fresh specimens do not show reduced cell numbers compared to negative controls).

  • (3)

    Bonding substances do not influence the cytotoxicity of post and core materials.

  • (4)

    Additional light curing (i.e. dual curing) of post and core materials does not influence cytotoxicity compared to chemically setting only.

  • (5)

    Certain environmental conditions (i.e. combinations of temperature and relative humidity) during specimen production do not influence cytotoxicity.

  • (6)

    7-days preincubated post and core specimens do not show reduced cytotoxicity in comparison to fresh specimens.

Materials and methods

Post and core materials

The following four post & core materials: LuxaCore – Dual (LXC), Core X-Flow (CXF), Flow White (FW) and MultiCore Flow (MCF) and their corresponding bonding substances are listed in Table 1 .

Table 1
Materials investigated: post & core materials, bonding materials and positive control.
Material a Manufacturer Lot Number Formulation
A LuxaCore – Dual DMG Chemisch-Pharmazeutische Fabrik GmbH, Hamburg, Germany 600811 Barium glass and pyrog. silica in a Bis-GMA based matrix of dental resins. Filler volume: 72% by weight = 49 percent by volume (0.02–4 μm)
A LuxaBond
Pre-bond
DMG Chemisch-Pharmazeutische Fabrik GmbH, Hamburg, Germany 604691 No information available
A LuxaBond
Bond A
A LuxaBond
Bond B
B Core X-Flow
Base and catalyst
Dentsply DeTrey GmbH, Konstanz, Germany 080418 Urethane dimethycrylate, di-& tri-functional methacrylates, barium boron fluoroaluminosilicate glass, camphorqhinone, photoaccelerators, silicon dioxide, benzoyl peroxide
B XP Bond Dentsply DeTrey GmbH, Konstanz, Germany 0803001932 No information available
C Flow White Cumdente GmbH, Tübingen, Germany 7811889 Mixture of Bis-GMA-based resins, highly dispersed silicon dioxide in prepolymer, silanized barium glass fillers, initiators, stabilizers and pigments
C Cumdente activator Cumdente GmbH, Tübingen, Germany 5803621 Solution of methacrylates, catalysts and stabilizers in ethanol.
C Cumdente adhesive Cumdente GmbH, Tübingen, Germany 5807860 Modified polyacrylic acids, methacrylates, ethanol, initiators and stabilizers
D MultiCore flow Ivoclar Vivadent, Schaan, Lichtenstein M24376 Dimethacrylates and fillers. The monomer
matrix consists of Bis-GMA, urethane dimetacrylate and triethylene glycol dimethacrylate.
The inorganic fillers are barium glass, Ba-Al-fluorosilicate glass, silicon dioxide and ytterbium
trifluoride
D ExciTE DSC Ivoclar Vivadent, Schaan, Lichtenstein M12980 No information available
Durelon carboxylate cement
Triple size powder
3M ESPE AG, Seefeld, Germany 322116 Zinc oxide
Durelon carboxylate cement
Triple size liquid
238723 Poly(acrylic acid), aq

a Post & core materials and their corresponding bonding substances are indicated with the same capital letter.

Cytotoxicity assay

Preparation of specimens

All specimens were produced in a climate test chamber (type VC 0018, Vötsch Industrietechnik GmbH, Balingen-Frommern, Germany) at two different environmental conditions (23 °C and 50% relative humidity [23/50] and 37 °C and 95% relative humidity [37/95]) and were dual-cured (dc) or self-cured (sc), with or without the corresponding bonding substance. Cylindrical specimens were prepared in Teflon blocks containing 5 mm diameter cylindrical holes (cylinder height 2 mm), covered with a polyethylene foil (Hostaphan ® , Mitsubishi Polyester Film GmbH, Wiesbaden, Germany; film thickness 75 μm). Dual-cured specimens (dc) were light cured from one end according to the manufacturers’ instructions. Self-cured specimens (sc) were produced in the same manner as described for dual-cured specimens but instead of light curing the specimens they were allowed to set for 7 min.

In a second series of experiments, specimens were combined with the corresponding bonding materials. Bonding agents were applied on a Hostaphan ® foil, in one or two layers, light cured or not light cured, depending on the manufacturers’ instructions. Subsequently, Teflon molds were placed on top of the bonding materials and composites prepared as described above. After unidirectional light curing (dc) from the top of the cylindrical holes or self-curing for 7 min (sc), visual confirmation of adherence at the base was obtained. All dual-cured materials were hardened with a DEMI™ curing light (Kerr Corporation, Middleton, WI, USA; light irradiance >800 mW/cm 2 ) according to the manufacturers’ instructions [LX (40 s), CXF (20 s), FW (20 s), MCF (40 s)].

All specimens were then sterilized by UV-radiation for 40 min from both sides using the UV lamp (UV tube 3FT 30 W) of a class II biosafety cabinet (Esco Technologies, Inc., Hatboro, PA, USA) and were added to the cultures immediately after production (“fresh”) or after preincubation for 7 days under cell-culture conditions. Specimens were incubated with L-929 fibroblasts for 72 h and cell numbers determined by flow cytometry.

Controls

Glass disks of 5 mm diameter and 2 mm height were used as negative controls, carboxylate cement (Durelon, Triple size powder, 3 M ESPE AG, Seefeld, Germany – see Table 1 ) was the positive control. Glass specimens were cut out of a glass blank using a diamond precision saw (IMPTECH PC 10, Boksburg, Republic of South Africa). For each positive control three specimens of Durelon carboxylate cement resembling test specimens in size and shape were fabricated.

Preincubation of specimens

Specimens were either used immediately after production (fresh specimens) or preincubated in cell culture medium (one specimen in 10 ml of Dulbecco’s Modified Eagle Medium [DMEM; Sigma, Germany]) at 37 °C, pH 7.2 for 7 days (7-day preincubated specimens) . The culture medium was then removed and specimens used for experiments.

Culture of L-929 fibroblasts

The murine fibroblast cell line L-929 was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). L-929 cells were cultivated in Costar 162 cm 2 flasks (Costar, Corning Incorporated Life Sciences, Tewksbury, MA, USA) in DMEM supplemented with 10% fetal calf serum (PAA Laboratories GmbH, Pasching, Austria), 1% glutamine and 1% penicillin/streptomycin at 37 °C in a fully humidified air atmosphere containing 5% CO 2 and were passaged by trypsinization. Fibroblasts (5 ml aliquots, containing 3 × 10 4 cells/ml) were exposed to freshly prepared or 7-day preincubated specimens or (positive or negative) controls in Costar 6-well tissue culture plates (Costar, Corning Incorporated Life Sciences, Tewksbury, MA, USA) for 72 h at 37 °C/5% CO 2 . Cells were then harvested with trypsin (2.5% in Ca 2+ and Mg 2+ free Hanks balanced salt solution; JRH Biosciences, KA, USA), centrifuged and resuspended in 500 μl DMEM.

Flow cytometry

Cells were counted in a volume of 500 μl DMEM over a fixed time (30 s) with a flow cytometer (FACSCalibur, Becton Dickinson, San José, CA, USA) equipped with an argon laser tuned at 488 nm. Cell numbers after exposure to test specimens were compared to negative controls (cultures without specimens) and standardized as the percent cell count (ratio between test cell counts and negative-control cell counts; note that negative controls were set to 100% and that lower values of the standardized cell numbers indicated higher cytotoxicity).

Statistical evaluation of cell culture experiments

Two-stage design

To keep the width of the confidence interval small even in cases of larger observed standard deviations, a two-stage plan was applied, as described previously . In brief, if the estimated standard deviation of each of the tested substances was equal to or smaller than 0.15 the trial was terminated after the first 18 samples (first stage). A second stage (second sequence of experiments) was performed if the observed standard deviation after the first stage exceeded 0.15. As a result of this statistical model for 19 groups the final sample size was n = 18, for 13 groups a second stage was necessary for which the sample size ranged between n = 21 and n = 54 samples.

Comparisons between settings

To evaluate the differences between the different settings, at each point of preincubation time (fresh and 7 days), for each of the four groups (LXC, CXF, FW and MCF) an analysis of variance was performed accounting for the different settings (bonding [with or without the corresponding bonding], curing [dc or sc] and type of climate [23/50 or 37/95]).

To adjust for the multiple test groups, all p -values smaller than 0.006 (=0.05/8, Bonferroni adjustment for the 8 performed setting comparisons for each groups) were considered as statistically significant. Mean values and Bonferroni adjusted confidence intervals for the different settings were performed. Furthermore, the comparison between 0 and 7 days was performed using t -tests.

All calculations were performed with SAS© Release 9.2.

Spectroscopic measurement of degree-of-conversion

Degree of conversion assay (only fresh specimens)

The spectroscopic measurements of degree of conversion were performed on a Fourier Transform Infrared (FTIR) spectrometer (Spectrum One, Perkin Elmer Instruments, MA, USA) equipped with an ATR Zinc Selenide device measuring surface reflection. Measurements were made on at least three samples of each material and temperature. For this purpose the spectrometer was equipped with a custom designed incubator box to set the temperature at either 23 °C or 37 °C.

Baseline correction and integration of peak areas were made using the software Spectrum v10.01.00 (Perkin Elmer Instruments, MA, USA). The degree of conversion (DC) was calculated from characteristic peaks of the reactive (meth)acrylate groups in the material and reference signals that remained unchanged during curing. For the material MultiCore Flow the disappearance of the characteristic signal at 1637 cm −1 relative to the carbonyl band at 1716 cm −1 was followed. Peak deconvolution was used for accurate quantification of the signals . DC was calculated as the ratio between the peak areas of the (meth)acrylate peaks in the cured and the uncured sample. The mean value and the standard deviation for DC were calculated from at least three measurements for each material and test condition.

Statistical evaluation of degree of conversion experiments

For each of the four groups (LXC, CXF, FW and MCF) an analysis of variance was performed accounting for the different settings (curing [dc or sc] and type of climate [23 °C or 37 °C]). All p -values smaller than 0.006 (Bonferroni adjusted for multiple testing) were considered as statistically significant.

Materials and methods

Post and core materials

The following four post & core materials: LuxaCore – Dual (LXC), Core X-Flow (CXF), Flow White (FW) and MultiCore Flow (MCF) and their corresponding bonding substances are listed in Table 1 .

Table 1
Materials investigated: post & core materials, bonding materials and positive control.
Material a Manufacturer Lot Number Formulation
A LuxaCore – Dual DMG Chemisch-Pharmazeutische Fabrik GmbH, Hamburg, Germany 600811 Barium glass and pyrog. silica in a Bis-GMA based matrix of dental resins. Filler volume: 72% by weight = 49 percent by volume (0.02–4 μm)
A LuxaBond
Pre-bond
DMG Chemisch-Pharmazeutische Fabrik GmbH, Hamburg, Germany 604691 No information available
A LuxaBond
Bond A
A LuxaBond
Bond B
B Core X-Flow
Base and catalyst
Dentsply DeTrey GmbH, Konstanz, Germany 080418 Urethane dimethycrylate, di-& tri-functional methacrylates, barium boron fluoroaluminosilicate glass, camphorqhinone, photoaccelerators, silicon dioxide, benzoyl peroxide
B XP Bond Dentsply DeTrey GmbH, Konstanz, Germany 0803001932 No information available
C Flow White Cumdente GmbH, Tübingen, Germany 7811889 Mixture of Bis-GMA-based resins, highly dispersed silicon dioxide in prepolymer, silanized barium glass fillers, initiators, stabilizers and pigments
C Cumdente activator Cumdente GmbH, Tübingen, Germany 5803621 Solution of methacrylates, catalysts and stabilizers in ethanol.
C Cumdente adhesive Cumdente GmbH, Tübingen, Germany 5807860 Modified polyacrylic acids, methacrylates, ethanol, initiators and stabilizers
D MultiCore flow Ivoclar Vivadent, Schaan, Lichtenstein M24376 Dimethacrylates and fillers. The monomer
matrix consists of Bis-GMA, urethane dimetacrylate and triethylene glycol dimethacrylate.
The inorganic fillers are barium glass, Ba-Al-fluorosilicate glass, silicon dioxide and ytterbium
trifluoride
D ExciTE DSC Ivoclar Vivadent, Schaan, Lichtenstein M12980 No information available
Durelon carboxylate cement
Triple size powder
3M ESPE AG, Seefeld, Germany 322116 Zinc oxide
Durelon carboxylate cement
Triple size liquid
238723 Poly(acrylic acid), aq
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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Cytotoxicity of post and core composites as a function of environmental conditions
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