2.1

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 .

2.2

Cytotoxicity assay

2.2.1

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.

2.2.2

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.

2.2.3

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.

2.2.4

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.

2.2.5

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).

2.2.6

Statistical evaluation of cell culture experiments

2.2.6.1

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.

2.2.6.2

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.

2.3

Spectroscopic measurement of degree-of-conversion

2.3.1

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.

2.3.2

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.

2.1

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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