2.1

Chemicals

The monomers/comonomers triethyleneglycoldimethacrylate (TEGDMA; CAS-No. 109-16-0), bisphenol-A-glycidylmethacrylate (BisGMA; CAS-No. 1565-94-2), hydroxyethylenemethacrylate (HEMA; CAS-No. 868-77-9), and urethanedimethacrylate (UDMA; CAS-No. 72869-86-4) were obtained from Evonik Röhm (Essen, Germany).

HEMA and TEGDMA were directly dissolved in medium. BisGMA and UDMA were dissolved in dimethyl sulfoxide (DMSO, 99% purity, Merck, Darmstadt, Germany) and diluted with medium (final DMSO concentration: 0.20%). Control experiments contained DMSO (0.20%) in medium only.

2.2

Cell culture

The human gingival fibroblast (HGF) cultures used in this study were produced by Provitro on the base of human tissues and obtained from Cell-Lining, Berlin, Germany, Cat-No.: 1210412. The HGFs (passage 9) were grown on 175 cm ² cell culture flasks to approximately 75–85% confluence and maintained in an incubator with 5% CO ₂ atmosphere at 100% humidity and 37 °C. Quantum 333 medium supplemented with l -glutamine and 1% antibiotic/antimycotic solution (10,000 Units/mL penicillin, 25 mg/mL streptomycin sulfate, 25 μg/mL amphotericin B) was used in the experiments. After reaching confluence the cells were washed with Dulbecco’s phosphate buffered saline (PBS), detached from the flasks by a brief treatment with trypsin/EDTA. Quantum 333, antibiotic–antibiotic solution, PBS and trypsin/EDTA were purchased from PAA Laboratories GmbH, Cölbe, Germany.

2.3

Instrumentation

2.3.1

xCELLigence system

The xCELLigence system was used according to the instructions of the supplier (Roche Applied Science and ACEA Biosciences) . The xCELLigence system consists of four main components: the RTCA analyzer, the RTCA SP station, the RTCA computer with integrated software, and disposable E-plate 96. The RTCA SP station fits inside a standard tissue-culture incubator, while an analyzer and laptop computer with software will be on the outside. The core of the xCELLigence system is the E-plate 96: this is a single use, disposable device used for performing cell-based assays on the RTCA SP instrument, which has similar application like commonly used 96-well microtiter plate. However the E-plate 96 differs from standard 96-well microtiter plates vastly with its incorporated gold cell sensor arrays in the bottom, which contributes cells inside each well to be monitored and assayed. The E-plate 96 has a low evaporation lid design : the bottom diameter of each well is 5.0 mm ± 0.05 mm; with a total volume of 243 ± 5 μL, approximately 80% of the bottom areas of each well is covered by the circle-on-line-electrodes, which is designed to be used in an environment of +15 to +40 °C, relative humidity 98% maximum without condensation .

The electronic impedance of sensor electrodes is measured to allow monitoring and detection of physiological changes of the cells on the electrodes. The voltage applied to the electrodes during RTCA measurement is about 20 mV (RMS) . The impedance measured between electrodes in an individual well depends on electrode geometry, ion concentration in the well and whether or not cells are attached to the electrodes . In the absence of cells, electrode impedance is mainly determined by the ion environment both at the electrode/solution interface and in the bulk solution. In the presence of cells, cells attached to the electrode sensor surfaces will act as insulators and thereby alter the local ion environment at the electrode/solution interface, leading to an increase in impedance . Thus, the more cells that are growing on the electrodes, the larger the value of electrode impedance. The RTCA associated software allows users to obtain parameters such as: average value, maximum and minimum values, standard deviation (SD), half maximum effect of concentration (EC ₅₀ ), half maximum inhibition of concentration (IC ₅₀ ), cell index (CI), and in addition graphics. The data expressed in CI units can be exported to Excel for any type of mathematical analysis .

2.3.2

Derivation of cell index (CI)

An unit-less parameter termed cell index (CI) is derived to represent cell status based on the measured relative change in electrical impedance that occurs in the presence and absence of cells in the wells , which is calculated based on the following formula: CI = ( Z _i − Z ₀ )/15, where Z _i is the impedance at an individual point of time during the experiment and Z ₀ is the impedance at the start of the experiment . Impedance is measured at 3 different frequencies (10, 25 or 50 kHz) and a specific time . Impedance change can occur depending on mainly two factors :

• (1)

  The number of cells attached to the electrodes : When there are no cells on an electrode surface, the sensor’s electronic feature will not be affected and the impedance change will be 0 ( Fig. 1 A). Attaching of one cell onto the electrodes, this value will be 1 ( Fig. 1 B). When more cells attach onto the electrodes, the value will further increase ( Fig. 1 C). All the factors that increase the number of attached cells on the electrodes, e.g., attachment from solution or cell proliferation leads to a higher CI value. However cell death or toxicity induces cell-detachment, which will lead to a decreased CI value.

  Scheme of impedance measurement.

  Baseline impedance : There are no cells on an electrode surface (A).

  Impedance : A cell labels to the electrode surface and blocks partially the electrical current in the circuit, inducing an increase in the electrode impedance (B).

  Impedance doubly : Two cell labels to the electrode surface and reduce even further the electrical current, as compared with B inducing to doubly increased impedance (C).

  Impedance further : Two cell labels to the electrode surface with more extension, which induce much more impedance in comparison with C (D).

• (2)

  The dimensional change of the attached cells on the electrodes : Despite the same cell numbers, dimensional changes of the attached cells on the electrodes will lead to change the CI, e.g., an increase in cell adhesion or cell spread will lead to a higher CI value ( Fig. 1 D) . Toxicity can induce cells to spread or cluster thereby leading to a larger cell surface/sensor contact, which in turn can increase the CI value . On the other hand, toxic compounds can induce cells to round up and/or to detach leading to a decrease in CI .

2.4

Cell growth and proliferation assay using xCELLigence system

HGF cells were grown and expanded in tissue-culture flasks. After reaching ∼75% confluence, the HGFs (passage 9) were washed with PBS, afterwards detached from the flasks by a brief treatment with trypsin/EDTA. Subsequently, 50 μL of cell culture media at room temperature was added into each well of E-plate 96. After this the E-plate 96 was connected to the system and checked in the cell culture incubator for proper electrical-contacts and the background impedance was measured during 24 s. Meanwhile, the cells were resuspended in cell culture medium and adjusted to 400,000, 200,000, 100,000, and 50,000 cells/mL. 100 μL of each cell suspension was added to the 50 μL medium containing wells on E-plate 96, in order to determine the optimum cell concentration. After 30 min incubation at room temperature, E-plate 96 was placed into the cell culture incubator. Finally, adhesion, growth and proliferation of the cells was monitored every 15 min for a period of up to 18 h via the incorporated sensor electrode arrays of the E-Plate 96. The electrical impedance was measured by the RTCA-integrated software of the xCELLigence system as a dimensionless parameter termed CI.

2.5

Cytotoxicity assay using xCELLigence system

First, the optimal seeding concentration for proliferation experiments of the HGFs was determined. After seeding the respective number of cells in 100 μL medium to each well of the E-plate 96, the proliferation, attachment and spreading of the cells was monitored every 15 min by the xCELLigence system. Approximately 18 h after seeding, when the cells were in the log growth phase, the cells were exposed to 50 μL of medium containing the following substances: BisGMA (0.01, 0.3, 1, 30 mM), HEMA (0.01, 0.3, 1, 30 mM), TEGDMA (0.03, 0.1, 3, 10 mM), and UDMA (0.001, 0.01, 0.1, 1 mM). Controls received either medium only, or medium + DMSO with a final concentration of 0.20%. All experiments were run for 24 h.

2.6

Statistics

All calculations were obtained using the RTCA-integrated software of the xCELLigence system. The RTCA software performs a curve-fitting of selected “sigmoidal dose–response equation” to the experimental data points and calculates logarithmic half maximum effect of concentration (log [EC ₅₀ ]) values at a given time point based on log of concentration producing 50% reduction of CI value relative to solvent control CI value (100%), expresses as log EC ₅₀ (M/L), which was converted into EC ₅₀ (mmol/L) in our results. Data are represented as mean [mmol/L] ± S.E.M. ( n = 5).

The statistical significance ( p < 0.05) of the differences between the experimental groups was checked using the t -test, corrected according to Bonferroni-Holm-modification preferred by Forst et al. .

2.1

Chemicals

The monomers/comonomers triethyleneglycoldimethacrylate (TEGDMA; CAS-No. 109-16-0), bisphenol-A-glycidylmethacrylate (BisGMA; CAS-No. 1565-94-2), hydroxyethylenemethacrylate (HEMA; CAS-No. 868-77-9), and urethanedimethacrylate (UDMA; CAS-No. 72869-86-4) were obtained from Evonik Röhm (Essen, Germany).

HEMA and TEGDMA were directly dissolved in medium. BisGMA and UDMA were dissolved in dimethyl sulfoxide (DMSO, 99% purity, Merck, Darmstadt, Germany) and diluted with medium (final DMSO concentration: 0.20%). Control experiments contained DMSO (0.20%) in medium only.

2.2

Cell culture

The human gingival fibroblast (HGF) cultures used in this study were produced by Provitro on the base of human tissues and obtained from Cell-Lining, Berlin, Germany, Cat-No.: 1210412. The HGFs (passage 9) were grown on 175 cm ² cell culture flasks to approximately 75–85% confluence and maintained in an incubator with 5% CO ₂ atmosphere at 100% humidity and 37 °C. Quantum 333 medium supplemented with l -glutamine and 1% antibiotic/antimycotic solution (10,000 Units/mL penicillin, 25 mg/mL streptomycin sulfate, 25 μg/mL amphotericin B) was used in the experiments. After reaching confluence the cells were washed with Dulbecco’s phosphate buffered saline (PBS), detached from the flasks by a brief treatment with trypsin/EDTA. Quantum 333, antibiotic–antibiotic solution, PBS and trypsin/EDTA were purchased from PAA Laboratories GmbH, Cölbe, Germany.

2.3

Instrumentation

2.3.1

xCELLigence system

The xCELLigence system was used according to the instructions of the supplier (Roche Applied Science and ACEA Biosciences) . The xCELLigence system consists of four main components: the RTCA analyzer, the RTCA SP station, the RTCA computer with integrated software, and disposable E-plate 96. The RTCA SP station fits inside a standard tissue-culture incubator, while an analyzer and laptop computer with software will be on the outside. The core of the xCELLigence system is the E-plate 96: this is a single use, disposable device used for performing cell-based assays on the RTCA SP instrument, which has similar application like commonly used 96-well microtiter plate. However the E-plate 96 differs from standard 96-well microtiter plates vastly with its incorporated gold cell sensor arrays in the bottom, which contributes cells inside each well to be monitored and assayed. The E-plate 96 has a low evaporation lid design : the bottom diameter of each well is 5.0 mm ± 0.05 mm; with a total volume of 243 ± 5 μL, approximately 80% of the bottom areas of each well is covered by the circle-on-line-electrodes, which is designed to be used in an environment of +15 to +40 °C, relative humidity 98% maximum without condensation .

The electronic impedance of sensor electrodes is measured to allow monitoring and detection of physiological changes of the cells on the electrodes. The voltage applied to the electrodes during RTCA measurement is about 20 mV (RMS) . The impedance measured between electrodes in an individual well depends on electrode geometry, ion concentration in the well and whether or not cells are attached to the electrodes . In the absence of cells, electrode impedance is mainly determined by the ion environment both at the electrode/solution interface and in the bulk solution. In the presence of cells, cells attached to the electrode sensor surfaces will act as insulators and thereby alter the local ion environment at the electrode/solution interface, leading to an increase in impedance . Thus, the more cells that are growing on the electrodes, the larger the value of electrode impedance. The RTCA associated software allows users to obtain parameters such as: average value, maximum and minimum values, standard deviation (SD), half maximum effect of concentration (EC ₅₀ ), half maximum inhibition of concentration (IC ₅₀ ), cell index (CI), and in addition graphics. The data expressed in CI units can be exported to Excel for any type of mathematical analysis .

2.3.2

Derivation of cell index (CI)

An unit-less parameter termed cell index (CI) is derived to represent cell status based on the measured relative change in electrical impedance that occurs in the presence and absence of cells in the wells , which is calculated based on the following formula: CI = ( Z _i − Z ₀ )/15, where Z _i is the impedance at an individual point of time during the experiment and Z ₀ is the impedance at the start of the experiment . Impedance is measured at 3 different frequencies (10, 25 or 50 kHz) and a specific time . Impedance change can occur depending on mainly two factors :

• (1)

  The number of cells attached to the electrodes : When there are no cells on an electrode surface, the sensor’s electronic feature will not be affected and the impedance change will be 0 ( Fig. 1 A). Attaching of one cell onto the electrodes, this value will be 1 ( Fig. 1 B). When more cells attach onto the electrodes, the value will further increase ( Fig. 1 C). All the factors that increase the number of attached cells on the electrodes, e.g., attachment from solution or cell proliferation leads to a higher CI value. However cell death or toxicity induces cell-detachment, which will lead to a decreased CI value.

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