2.1

Cell cultures

Primary human gingival fibroblasts (HGFs) were obtained from the gingiva of permanent molars from a healthy patient (with informed consent). HGFs were grown in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose, 25 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethane sulfonic acid (HEPES), 4 mM l -glutamine, 3.7 g/L NaHCO ₃ , 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B, supplemented with 10% fetal bovine serum (FBS, all from Biochrom KG, Berlin, Germany) at 37 °C and 10% CO ₂ in a humidified atmosphere. Cells were subcultured by trypsinization with trypsin/ethylenediaminetetraacetic acid (EDTA, 0.25% trypsin, 0.02% EDTA; GIBCO/Invitrogen, Darmstadt, Germany) when the confluency reached approximately 90%.

The immortalized human keratinocyte cell line OKF6/TERT2 was obtained from Dr. J. Rheinwald (Harvard University) [ ]. Keratinocytes were cultured in serum-free medium (ker-sfm) supplemented with 25 μg/mL bovine pituitary extract (BPE), 0.2 ng/mL epidermal growth factor (EGF, all from GIBCO/Invitrogen, Darmstadt, Germany), 0.3 mM CaCl ₂ , 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B, at 37 °C and 5% CO ₂ in a humidified atmosphere. Trypsin/EDTA (0.125% trypsin, 0.01% EDTA) was used to subculture the cells and trypsin was inactivated by adding one volume of Dulbecco’s modified Eagle medium/Ham’s F-12 medium (DMEM/F-12, Biochrom KG, Berlin, Germany) supplemented with 10% FBS. When a confluency of about 30% was reached, OKF6/TERT2 cells were supplied with medium with a higher nutrient concentration: 1:1 mixture of DMEM/F-12 and ker-sfm supplemented with 25 μg/mL BPE, 0.2 ng/mL EGF, 0.2 mM CaCl ₂ , 0.75 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B (HD-ker).

2.2

3D co-culture model

3D co-culture models were prepared as described previously with some adjustments [ ]. Briefly, HGF-containing collagen gels (3 mL) were cast on top of an acellular basis (1 mL) in 6-well plates. The cellular gel was prepared from 0.84 mL H ₂ O, 0.25 mL FBS, 0.3 mL of 10x DMEM (Biochrom KG, Berlin, Germany), 0.03 mL l -glutamine (200 mM), 0.3 mL 10x reconstitution buffer (20 mM HEPES, 22 mg/mL sodium bicarbonate in 20 mL 0.062 M Sodium hydroxide), 1.18 mL of type 1 rat tail collagen (2 mg/mL in 0.1% acetic acid; SERVA Electrophoresis GmbH, Heidelberg, Germany) and 0.1 mL DMEM with 2 × 10 ⁵ HGFs per gel. After polymerization, collagen gels were covered with 3 mL of DMEM + 10% FBS and cultured at 37 °C and 10% CO ₂ in a humidified atmosphere. The HGF-containing collagen gels were continuously observed microscopically and their contraction was measured to ensure consistent seeding and viability of HGFs. 5 × 10 ⁵ OKF6/TERT2 cells in 1.5 mL HD-ker were seeded on a cell culture insert with a pore diameter of 0.4 μm (ThinCert 657641, Greiner-Bio-One, Kremsmuenster, Austria). 2.6 mL HD-ker was added underneath the cell culture insert and the cells were cultivated at 37 °C and 5% CO ₂ in a humidified atmosphere. HGFcontaining collagen gels and OKF6/TERT2 cells were cultivated separately, until 3D-CCMs were assembled seven days after seeding. For that, both components were transferred to a new 6-well plate and supplied with 2.6 mL of DMEM/F-12 medium supplemented with 25 μg/mL BPE, 0.2 ng/mL EGF, 0.2 mM CaCl ₂ , 1.5 mM l -glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B and 5% FBS (DF-K medium) in the lower compartment and 1.5 mL of DFK with the test substance as described in chapter 2.4 in the upper compartment. Assembled 3D-CCMs were cultivated at 37 °C and 5% CO ₂ in a humidified atmosphere. Lucifer Yellow assays were carried out using extra cell culture inserts with OKF6/TERT2 cells before the assembly to ensure that keratinocytes reached the desired confluency in every experiment [ ]. Briefly, the strongly hydrophilic fluorescent dye Lucifer Yellow was added to the upper compartment, above OKF6/TERT2 cells. The amount of Lucifer Yellow that was able to pass the cell layer was measured in the lower compartment and calculated relative to the theoretical equilibrium concentration. A threshold of 8% Lucifer Yellow of the equilibrium concentration was defined previously as a threshold for a maximal confluency of OKF6/TERT2 cells on cell culture inserts, which was optically supported by microscoscopy [ ].

2.3

CQ Treatment of 3D co-culture models

The photoinitiator CQ was supplied by VOCO (Cuxhaven, Germany) and solved in ethanol. 3D-CCMs were treated only in the upper compartment (supra-epithelial treatment) at two time points. 2.5 mM CQ in 1.5 mL DF-K medium was added to the upper compartment and 2.6 mL DFK medium to the lower compartment when the 3D-CCMs were assembled. After 24 h, half of the 3D-CCMs were harvested and the treatment solution of remaining models was exchanged with fresh treatment solution in the upper compartment and DFK medium in the lower compartment. The second treatment was carried out with 50% of the initial CQ concentration (1.25 mM). After additional 48 h, the remaining 3D-CCMs were harvested (72 h time point). The concentration of the solvent (ethanol) was equal in every treated sample at all phases of the experiment. In one variant, the treatment solution was irradiated with blue light two times for 10 s directly after the addition to the upper compartment using a dental curing light (800 mW/cm ² ; Elipar II, Espe, Seefeld, Germany). 0.25% ethanol (C1), 0.25% ethanol with blue light irradiation (C1 + BL) and culture medium (C2) served as controls. A schematic presentation of the model and the experimental setup is shown in Fig. 1 .

2.4

Gene expression analyzes

RNA isolation, cDNA synthesis, and gene expression analysis of genes related to oxidative stress and extracellular proteases was carried out as described previously [ , ]. The QuantiTect Primer assays used are listed in Supplemental file 1 (Qiagen, Hilden, Germany). We applied geNorm to evaluate the stability of six candidate housekeeping genes for both cell types independently (18S ribosomal RNA [ 18S rRNA ], Actin beta [ ACTB ], Beta-2-microglobulin [ B2M ], Glyceraldehyde-3-phosphate dehydrogenase [ GAPDH ], Succinate dehydrogenase complex subunit A flavoprotein [ SDHA ] and Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta [ YWHAZ ]) [ ]. B2M and YWHAZ were identified as most stable housekeeping genes for OKF6/TERT2 cells and GAPDH and YWHAZ for HGFs. Normalized ratios between samples and the untreated control (C2) were calculated using the efficiency adjusted delta delta ct method according to Pfaffl [ ]. Three independent sets of experiments were performed.

2.5

Western blot

Isolation of total cellular proteins and western blots were carried out as described previously [ ]. The following primary antibodies were used: anti-GAPDH (rabbit; 1:10,000 in PBST with 0.5% milk powder; Sigma-Aldrich, St. Louis, USA), anti-HO-1 (rabbit; 1:2,000; Enzo Life Sciences; Farmingdale, USA), anti NQO1 (goat; 1:1,000; Abcam, Cambridge, UK) and anti-CAT (rabbit; 1:16,000; Merck, Darmstadt, Germany). Goat anti-rabbit or rabbit anti-goat, conjugated with horseradish peroxidase were used as secondary antibodies (1:10,000 in PBST with 0.5% milk powder, DakoCytomation, Hamburg, Germany). Polyvinylidene difluoride membranes (PVDF, Millipore, Burlington, USA) were developed with an enhanced chemiluminescence substrate (SuperSignal West Pico chemi-luminescent substrate, ThermoFisher, Waltham, USA). The chemiluminescence signal was captured using an imaging system (Fusion SL-4-400WL, Vilber, Eberhardzell, Germany) and quantified with the software Bio1D (Vilber Lourmat). Experiments were carried out three times independently.

2.6

GC/MS measurement of CQ

Cell culture inserts with OKF6/TERT2 cells were treated with 2.5 mM CQ or 0.25% ethanol (C1) as described in 2.3. 1 mL medium was taken from the upper and the lower compartment at both time points (24 h and 72 h) and briefly centrifuged. The supernatant was used for GC/MS measurements. For a more detailed analysis of the kinetic of the CQ allocation within the first 24 h, six inserts with OKF6/TERT2 cells were treated with 2.5 mM CQ. 1 mL medium was taken from each compartment at standardized time points (0.5 h, 1 h, 3 h, 6 h 12 h 24 h) and prepared as described above. Two inserts with OKF6/TERT2 cells were treated with 0.25% ethanol and sampled at the time points 0.5 h and 24 h as solvent controls (C1). Additionally, an aliquot of the 2.5 mM CQ treatment solution was incubated in 6-well plates without cells at 37 °C and 5% CO ₂ in a humidified atmosphere to evaluate the influence of time and temperature on the CQ degradation in medium. Eight calibrators were prepared by consecutively diluting the 2.5 mM CQ treatment solution 1:2 with DFK medium supplied with 0.25% ethanol to keep the ethanol concentration constant. CQ was extracted by adding one volume of ethyl acetate (Merck, Darmstadt, Germany), supplemented with 1.25 mM caffeine (SigmaAldrich, St. Louis, USA) as internal standard, followed by 10 min of mixing. Additional 0.5 vol of ethyl acetate was added afterwards. After 2 min of vortexing, the mixture was centrifuged at 5445× g (5 min, 4 °C). 50 μL of the resulting upper phase were transferred to an amber glass vial with inserts (f N11-1 HP; MachereyNagel, Düren, Germany) and 200 μL ethyl acetate was added. 1 μL of each extract was injected via a 7693 ALS Autosampler (Agilent Technologies, Santa Clara, USA) into the Agilent 7890B GC System equipped with a HP-5MS UI column, 30 m × 0.25 mm ID, 0.25 μm film thickness coupled to an Agilent 7000C GC/MS triple quadrupole mass spectrometer. Helium gas with a constant flow of 1.2 mL/min was used as carrier gas. The following temperature program was used for the separation in the MS: 50 °C initial temperature for 1 min followed by a temperature-ramp of 25 °C/min to 150 °C and a second ramp of 15 °C/min to 300 °C, held for 1 min. The resulting retention time of CQ was 5.84 min. CQ was identified by MS via its m / z 69.1 (quantifier ion) and m / z 86.9 (qualifier ion) mass fragments. Three independent sets of experiments were performed.

2.7

Proteome Profiler array

The Proteome Profiler array ‘Human Protease Array Kit’ from R&D Systems (Minneapolis, USA) was used to quantify extracellular proteases in supernatants of 3D-CCMs that were used in experiments for western blot analysis. Samples were taken from both compartments of 3D-CCMs that were treated with 0.25% ethanol (solvent control; C1) or with CQ without blue light irradiation at the second harvest time point, 72 h after beginning of the treatment (described in Section 2.3 ). The cell culture supernatant was taken before the cells were harvested for protein analysis and briefly centrifuged to pellet floating cells and cell debris (5 min; 800× g ). The resulting supernatant was transferred to a new tube and stored at −80 °C. The Proteome Profiler array was performed according to the manual, using 400 μL of each supernatant. The chemiluminescence was captured using a Fusion SL-4-400WL imaging system. The spot intensities were quantified based on pixel densities with the software Bio1D. The signals were normalized by background subtraction and quantified in relation to the positive controls of the membranes. Spots that showed signal intensities only slightly above the background and also spots that were influenced by very bright spots next to them were discarded from the analysis. Experiments were carried out three times independently. Representative membranes are shown in Supplemental file 2.

2.8

Statistics

Fold changes from qRT-PCR and western blot analyzes ( Fig. 2 and 4 ) were calculated relative to the medium control (C2) and log2transformed to enable statistical testing. The values from treated samples were tested against their corresponding solvent control (C1) from the according time point (24 h/72 h: CQ vs C1 and CQ + BL vs C1 + BL) via two-tailed t-test with a significance level of 0.05. The same test was applied when testing the results from GC/MS analyzes comparing CQ concentrations between the upper and lower compartment ( Fig. 3 ). For Proteome Profiler arrays ( Fig. 5 ) and the comparison of results from Proteome Profiler arrays to the gene expression analysis of MMPs ( Fig. 6 ), ratios from normalized signal intensities were calculated relative to the solvent control (C1). Ratios were log2-transformed and tested by one sample t -test to be significantly different from 0 (not regulated) in consideration of the variance from 3 biological replicates. The correlation between the regulation of the transcription of genes from both cell types and the protein quantities of extracellular proteases in the cell culture supernatants from both compartments ( Fig. 6 ) were calculated using the Pearson correlation coefficient (based on the linear values).

Relative mRNA (a) and protein quantities (b) of oxidative stress-related genes in keratinocytes and HGFs in CQ-treated 3D co-culture models. 3DCCMs were treated by adding 2.5 mM CQ in the upper compartment ± blue light (BL) for 24 h (first harvest time point). Afterwards, 50% of the initial CQ concentration (1.25 mM) was reapplied (± blue light) and remaining 3D-CCMs were treated for additional 48 h (72 h total). Relative mRNA quantities were analyzed by qRT-PCR, normalized to two housekeeping genes per cell type and standardized relative to the untreated medium control (C2, not shown). Protein quantities were analyzed by western blots. Chemiluminescence signals were quantified by Bio1D and normalized to the protein amount of GAPDH and C2. Data are shown as mean ± SD of log2-transformed fold changes from three independent experiments. Values on the Y-axis were transformed to linear values for a better view. Asterisks above the error bars indicate significant differences (p < 0.05) in comparison to the respective solvent control at particular time points (C1 ± BL at 24 h and 72 h; two-tailed t-test).

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

1. Evaluation of translucency, Marten’s hardness, biaxial flexural strength and fracture toughness of 3Y-TZP, 4Y-TZP and 5Y-TZP materials
2. Titanium dioxide nanotubes with triazine-methacrylate monomer to improve physicochemical and biological properties of adhesives
3. Behaviour of different bioactive glasses incorporated in polydimethylsiloxane endodontic sealer
4. The effect of chlorhexidine and dimethyl sulfoxide on long-term sealing ability of two calcium silicate cements in root canal
5. Nonlinear viscoelastic properties of human dentin under uniaxial tension
6. Does implant surface hydrophilicity influence the maintenance of surface integrity after insertion into low-density artificial bone?

Stay updated, free dental videos. Join our Telegram channel

Join