Abstract
Objectives
The aim of this study was the detection of putative gene expression-related effects of dental composites in conventional and interactive gingival cell systems.
Methods
Conventional monoculture (MC) and interactive cell systems (ICS) comprising human gingival fibroblast (HGF) and immortalized human gingival keratinocytes (IHGK) were exposed for 24 h and 7 days according to ISO10993-12:2012 manufactured eluates of different composites (Ceram X ® , Filtek™ Supreme XT, Filtek™ Silorane, Fusio™ Liquid Dentin, and Vertise™ Flow). qRT-PCR-based mRNA analysis for biomarkers indicating cell proliferation, differentiation, apoptosis, inflammation, and adhesion was performed. Apoptotic cells were quantified by annexin-V labeling.
Results
Due to low RNA amounts, qPCR could not be performed for Vertise™ Flow and Fusio™ Liquid Dentin at day 7. At 24 h, flowables yielded increased transcription for biomarkers of inflammation and apoptosis in IHGK, irrespective of the cell system. HGF cultures displayed lower transcription for cell adhesion markers in both cell systems. Filtek™ Supreme XT showed increased differentiation by elevated filaggrin gene expression in both cell systems for IHGK at day 7, while Filtek™ Silorane and Ceram X ® yielded elevation of inflammation biomarkers in both cell types. Annexin-V labeling revealed high apoptosis rates for both flowables and Filtek™ Supreme XT for IHGK, while low rates were detected for Filtek™ Silorane and Ceram X ® .
Significance
Among the composites evaluated, exposition of IHGK and HGF in conventional and interactive cell systems demonstrated most pronounced gene expression alterations in response to flowables, coinciding with elevated levels of apoptosis.
1
Introduction
The use of composite materials have gained popularity in dental treatment through the past decades because of their rapid polymerization, their ability to bond with tooth surfaces, their excellent mechanical properties, and their esthetic appearance. The organic resin matrix of these dental restorative materials is usually based on methacrylate monomers, such as BisGMA (bisphenol A glycol dimethacrylate), UDMA (urethane dimethacrylate), TEGDMA (triethylene glycol dimethacrylate), and BisEMA (bisphenol A ethoxylated dimethacrylate) . Recently, some new resin systems such as ormocers and silorane have been introduced as alternative to the methacrylate-based composites . Composite characteristics including reactivity, viscosity, and the polymerization shrinkage as well as the mechanical properties are determined by the chemistry of the monomer . Despite the achievements made in recent composite technology, composite polymerization is still incomplete, thereby affecting their physical properties and clinical performance .
With respect to the clinical success of dental composite materials, a critical issue is their biocompatibility. In this regard, differential release of monomers from dental composite materials after storage in water, ethanol 75% or methanol has been described for BisGMA, TEGDMA, HEMA (2-hydroxethyl methacrylate) and UDMA . Of note, induction of cytotoxicity and apoptosis in human dental pulp cells has been observed for TEGDMA, BisGMA, and HEMA . TEGDMA and HEMA have been shown to induce apoptosis in several cell types through the generation of redox balance-disturbing reactive oxygen species (ROS) , which lead to DNA damage . Substances like BisGMA, BisDMA and Bisphenol A are assumed to indirectly induce estrogen-like reactions . A further critical issue is that harmful effects on periodontal cells have been described in a study on human gingival fibroblasts (HGF) by BisGMA-induced DNA double-strand breaks , while other studies on fibroblasts revealed HEMA-associated alterations in morphology and extracellular type-I collagen protein abundance .
Until now, studies elucidating putative hazardous effects of composite-eluted substances share two major handicaps. First, frequently pure monomers in various concentrations have been used for cell exposition, while studies recruiting authentically eluted substances from the polymerized composites are rare. Several parameters might influence the elution of the substances of the composite materials. Testing the original substances that are used for the manufacturing of the composite materials does not really represent the clinical situation. Although the release of the same substances has been certified by several authors, these substances are not the only ones that are eluted from the composite materials as further degradation products might be additionally eluted. Additionally, an interaction between the eluted substances and the production of other molecules might take place. Second, the majority of the toxicity tests have been carried out on rodent cell systems, which may yield discriminative results to human target tissue cells, for instance, human gingival fibroblasts. So far available in vitro studies on periodontal cells usually assess substance-related effects on cell behavioral features in conventional monolayer cell systems . However, although the use of such conventional monolayers gives important information of the direct effect of a tested composite or its monomer on cell behavior, the translation of the elaborated results to in the in vivo situation is limited. This limitation arises from the lack of cell−cell interactions provided by the physiological, i.e. in vivo situation. Hence, the use of interactive gingival cell systems, comprising fibroblasts and keratinocytes would be beneficial in order to evaluate the effects of composite materials, due to the ability of the cells to cooperate with each other and therefore to simulate the in vivo situation. The differential cell behavior by using human gingival keratinocytes and gingival fibroblasts in an interactive cell system compared with the conventional use of the same cell types in monolayers has been stated in the past .
Therefore, the aim of the present study was to evaluate the effects of eluates of five polymerized dental composite materials with different chemistry on the gene expression of human gingival keratinocytes and human gingival fibroblasts in conventional and interactive cell systems. We hypothesized that eluates of these dental composites have impact on the gene expression of the investigated cells and that the crosstalk between the cells in an interactive cell system putatively modulate these alterations. Based on our hypothesis, the present gene expression analysis focuses on genes of cell behavioral features (i) apoptosis, (ii) inflammation, (iii) adhesion, (iv) proliferation, and (v) differentiation. In order to detect possible direct substance-related effects on cell behavior on the phenomenological level, early apoptosis was quantified in conventional cell systems.
2
Materials and methods
2.1
Composite materials
In the present study, three different composite materials were tested: a nanohybrid resin composite, Filtek™ Supreme XT (3M ESPE Dental Products, Seefeld, Germany), an Ormocer, Ceram X ® (Dentsply DeTrey GmbH, Konstanz, Germany), a composite material representing the Silorane technology, Filtek™ Silorane (3M ESPE Dental Products, Seefeld, Germany) and two self-adhering flowable composite materials, Vertise™ Flow (KERR, Orange/CA, USA) and Fusio™ Liquid Dentin (Pentron Clinical, Wallingford/CT, USA). Detailed information about the composition of the composite materials and the manufacturers are given in Table 1 .
| Material | Category | Composition a (main monomers) | Manufacturer |
|---|---|---|---|
| Ceram X ® | Ormocer | Methacrylate-modified polysiloxane, Dimethacrylate resin | Dentsply DeTrey GmbH, Konstanz Germany |
| Filtek™ Supreme XT | Nanohybrid composite | Bis-GMA, TEGDMA, Bis-EMA | 3M ESPE Dental Products, Seefeld, Germany |
| Filtek™ Silorane | Silorane | Silorane resin | 3M ESPE Dental Products, Seefeld, Germany |
| Fusio™ Liquid Dentin | self-adhering flowable composite | HEMA | Pentron Clinical, USA-Wallingford/CT |
| Vertise™ Flow | self-adhering flowable composite | Uncured methacrylate ester monomers, GPDM monomer | KERR, USA-Orange/CA |
2.2
Preparation of composite samples and eluates
From each composite material, 26 specimens (shade A3) were prepared. The samples were prepared according to ISO 10993-12:2012 using molds, allowing the production of standardized cylindrical specimens (diameter 7 and 2 mm thickness). The forms were positioned on a transparent plastic matrix strip lying on a glass plate and were filled with the composite material. The samples were built up in one increment. After inserting the material into the discs, a transparent plastic matrix strip (Kerr Hawe, Bioggio, Switzerland) was placed on top of them in order to avoid an oxygen-inhibited superficial layer. Additionally, a glass slide was used in order to flatten the surface. The samples were polymerized using a halogen unit (Elipar ® Highlight, 3M ESPE, Seefeld, Germany) with a light intensity of 803 mW/cm 2 . The spectral irradiance was determined with a visible curing light meter (Cure Rite; Dentsply, USA). The polymerization of the samples took place according to the manufacturers’ instructions. The samples of Filtek™ Supreme XT, Ceram X ® , Vertise™ Flow, and Fusio™ Liquid Dentin were cured for 20 s and the samples of Filtek™ Silorane for 40 s.
In order to achieve an adequate disinfection, the composite samples were immersed in a solution of 75% ethanol (Sigma Aldrich, St. Louis, USA) for 1 min. After that, the samples were washed three times with sterile water and then dried for 2 min. During our pilot studies, this method was shown to be most effective with the less effect on the composite samples.
Each composite sample was immersed in 1 mL FAD cell culture medium each (Ham’s F12/DMEM: mixing ratio 1:3, Biochrom, Berlin, Germany), 5% FCS, 100 μg/mL kanamycin (Sigma, Mannheim, Germany) and the supplements of KGM2 (Promocell, Heidelberg, Germany) in a 24-wells plate (Greiner-Bio-One, Frickenhausen, Germany). The samples were stored in a dark box at room temperature (21 °C). Half of them were stored for 24 h and the other half for 7 days. After the end of the storage period, the samples were removed and the media-samples (in 24-wells plate) were stored at 4 °C until the cells were prepared for the experiment. FAD-Medium stored for the same time periods was used as control.
2.3
Cell cultures: Establishment of monocultures (MC) and interactive cell systems (ICS) and exposure to composite eluates
In the present study, two different gingival cells were used: human gingival keratinocytes immortalized with the human papilloma virus type16 E6/E7 oncogenes (IHGKs) and human gingival fibroblasts (HGFs). The primary cultures of the cells were established and cultured as described in previous studies . For the use of the tissues informed consent was obtained by the patients according to the Helsinki Declaration, and the protocol was approved by the institutional ethics committee (Votum Nr. 411/08; Date: 11/20/2008). IHGKs in the passages between 90 and 95 were maintained in low-calcium keratinocyte growth medium (basal keratinocyte medium, KGM2, with provided supplements, Promocell, Heidelberg, Germany), containing 100 μg/mL kanamycin (Sigma, Mannheim, Germany). HGFs were cultured in passages 12−14 and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (PAA, Marburg, Germany) containing 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and 100 μg/mL kanamycin.
To detect both short-term and long-term effects of the composite eluates on periodontal cells, exposition periods of 24 h and 7 days were chosen. The gene expression analysis was conducted, using conventional monocultures (MC) of human gingival fibroblasts (HGF) and immortalized human gingival keratinocytes (IHGK), while interactive cell systems (ICS), comprising both cell types were employed to analyze eluate exposition under more physiological, i.e. in vivo -like conditions.
For establishment of the HGF monocultures, for the time period of 24 h, 5 × 10 4 cells were cultivated per well and for the period of 7 days 1 × 10 4 cells per well in 24-well plates (Falcon, BD Biosciences, Franklin Lakes, USA). For the monocultures of IHGK 1 × 10 5 cells per well for the shorter and 5 × 10 4 cells for the longer, eluate exposition period were cultivated on 24-well plates (Falcon, BD Biosciences, Fanklin Lakes, USA). All cells were maintained under standard cell-culture conditions: 37 °C, 97% humidity and 5% CO 2 . After 24 h, the cultivation medium was removed, replaced with the respective composite eluates and then a further incubation followed respective to the eluate exposition group they belong to (24 h or 7 days). From the incubated cell-wells, two wells per time period for each cell type and composite material were used for the determination of the apoptosis by immunofluorescence and three wells for the respective RT-PCR analysis. All groups were prepared in a biological replicate.
For establishing the IHGK/HGF interactive cell systems (ICS), the trans-well method was used as described previously . HGFs and were pre-cultivated in 24-well plates as described above for the monocultures, while 1 × 10 5 IHGKs per well were pre-cultivated separately on porous cell-culture inserts with pore sizes of 3 μm (Falcon, BD Biosciences, Franklin, USA). After 24 h pre-cultivation, the inserts with the IHGKs were placed in the wells containing the HGFs and the whole cultivation medium was replaced with the composite eluates and native FAD medium as control as described above. The ICS were incubated for further 24 h or 7 days respective to the group they belong. The ICS were used only for the evaluation of the effects of the composite on gene expression by quantitative qRT-PCR analysis. All groups were prepared in a biological replicate.
2.4
RNA isolation and quantitative PCR
Generally, for the qPCR, mRNA was extracted separately for each cell type, incubation period and exposed eluate, and cells incubated in ordinary cell culture medium served as controls. The C t -values measured for the control groups were used as reference in order to evaluate increase or decrease of the gene expression compared with the other groups.
After the respective cultivation periods, total RNA was isolated from treated and control cell cultures using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. In order to eliminate the residual genomic DNA, the RNA samples were treated with RNA-free DNase-Set (Qiagen, Hilden, Germany) during the RNA purification. The RNA concentration and integrity were determined by using an automated electrophoresis system (BioRad, Munich, Germany). First-strand cDNA was synthesized by using the C-03-first-strand-kit (SABiosciences, Qiagen, Hilden, Germany) from 500 ng total RNA. Then the cDNA concentration was quantified by PicoGreen (PicoGreen® dsDNA Assay Kit, Invitrogen, Darmstadt, Germany) with a plate reader Infinity M200 (Tecan, Crailsheim, Germany). For normalization, the cDNA concentration was adjusted to 5 ng/μL for each polymerase chain reaction (PCR). The qPCR analysis was performed with the real-time detection system CFX96 (BioRad, Munich, Germany). The SYBR Green Master Mix (SABiosciences, Qiagen, Hilden, Germany) was used for qPCR. For the qRT-PCR Analysis, different commercially available primers (SABiosciences, Qiagen, Hilden, Germany) were used: caspase 3, 8 and 9 (CASP 3, CASP 8, CASP 9), ki -67 (MKI 67), interleukin 6 and 18 (IL 6, IL 18), annexin A5 (ANX A5), involucrin (INV), filaggrin (FLG), integrin β3 (ITGB3), and primers for the housekeeping genes β-actin (ACTB) and ribosomal protein L13A (RPL13A). These genes were chosen to cover a wide range of the biological reactions of the cells, coding the proteins in the level of cell adhesion (integrin β3), proliferation (ki-67), inflammation (interleukin 6 and 18), different stages of apoptosis (caspase 3, 8 and 9 and annexin A5) and stages of differentiation (involucrin and filaggrin).
The qPCR was performed with the CFX96 real-time detection system (BioRad, Munich, Germany) and the relative mRNA gene expression was analyzed by a modification of ΔΔ C t equation using the CFX Manager Software (BioRad, Munich, Germany) and were normalized to the index C t of the non-modulated housekeeping genes actin β (ACTB) and ribosomal protein L13a (RPL13A) and referred to the relative gene expression of the control cells. An up-/down-modulation of ±1 on the amount of mRNA of interest normalized to the respective control (1 = double the amount of mRNA present compared with the control, -1 = half the amount of mRNA present compared with control) was considered as the threshold for significance and further analyzed by a Student’s t -test for unequal variance by a significance level of p < 0.01.
2.5
Determination of apoptosis by immunofluorescence
The determination of apoptosis by immunofluorescence took place only in the monocultures. After the exposure periods of 24 h and 7 days, the medium was removed from the wells intended for the detection of early apoptosis by immunofluorescence and the cells (HGFs and IHGKs) were washed once with 1 mL of annexin-V-binding buffer (Invitrogen, Darmstadt, Germany). The buffer was removed and again 500 μL Annexin-V-binding buffer containing 1 μL of annexin-V-FITC detection reagent (Invitrogen, Darmstadt, Germany) was added and the cells were incubated at room temperature for 5 min in the dark. The solution was removed from each well and the cells were washed again with annexin-V-binding buffer and then were fixed with paraformaldehyde 4% for 20 min. After that, the cells were washed once with PBS and the cell nuclei were counterstained 300 nM DAPI-solution. The cells were washed again twice with PBS, and once with distilled water and then were finally embedded in mounting medium (Fluoromount-G, Southern Biotech, Birmingham, USA) and observed by fluorescent microscopy (BZ-9000, Keyence, Neu-Isenburg, Germany).
In order to calculate the amount and the percentage of the annexin-V-positive cells, three representative images of two stained wells of two independent biological replicates for each of the tested cultivation periods were taken into account and were analyzed by the BZ-II-Analyzer Software (Keyence, Neu-Isenburg, Germany). The values were statistically analyzed by using the Student- t -Test for unequal variance, by a significance level of p < 0.01.
2
Materials and methods
2.1
Composite materials
In the present study, three different composite materials were tested: a nanohybrid resin composite, Filtek™ Supreme XT (3M ESPE Dental Products, Seefeld, Germany), an Ormocer, Ceram X ® (Dentsply DeTrey GmbH, Konstanz, Germany), a composite material representing the Silorane technology, Filtek™ Silorane (3M ESPE Dental Products, Seefeld, Germany) and two self-adhering flowable composite materials, Vertise™ Flow (KERR, Orange/CA, USA) and Fusio™ Liquid Dentin (Pentron Clinical, Wallingford/CT, USA). Detailed information about the composition of the composite materials and the manufacturers are given in Table 1 .
| Material | Category | Composition a (main monomers) | Manufacturer |
|---|---|---|---|
| Ceram X ® | Ormocer | Methacrylate-modified polysiloxane, Dimethacrylate resin | Dentsply DeTrey GmbH, Konstanz Germany |
| Filtek™ Supreme XT | Nanohybrid composite | Bis-GMA, TEGDMA, Bis-EMA | 3M ESPE Dental Products, Seefeld, Germany |
| Filtek™ Silorane | Silorane | Silorane resin | 3M ESPE Dental Products, Seefeld, Germany |
| Fusio™ Liquid Dentin | self-adhering flowable composite | HEMA | Pentron Clinical, USA-Wallingford/CT |
| Vertise™ Flow | self-adhering flowable composite | Uncured methacrylate ester monomers, GPDM monomer | KERR, USA-Orange/CA |
2.2
Preparation of composite samples and eluates
From each composite material, 26 specimens (shade A3) were prepared. The samples were prepared according to ISO 10993-12:2012 using molds, allowing the production of standardized cylindrical specimens (diameter 7 and 2 mm thickness). The forms were positioned on a transparent plastic matrix strip lying on a glass plate and were filled with the composite material. The samples were built up in one increment. After inserting the material into the discs, a transparent plastic matrix strip (Kerr Hawe, Bioggio, Switzerland) was placed on top of them in order to avoid an oxygen-inhibited superficial layer. Additionally, a glass slide was used in order to flatten the surface. The samples were polymerized using a halogen unit (Elipar ® Highlight, 3M ESPE, Seefeld, Germany) with a light intensity of 803 mW/cm 2 . The spectral irradiance was determined with a visible curing light meter (Cure Rite; Dentsply, USA). The polymerization of the samples took place according to the manufacturers’ instructions. The samples of Filtek™ Supreme XT, Ceram X ® , Vertise™ Flow, and Fusio™ Liquid Dentin were cured for 20 s and the samples of Filtek™ Silorane for 40 s.
In order to achieve an adequate disinfection, the composite samples were immersed in a solution of 75% ethanol (Sigma Aldrich, St. Louis, USA) for 1 min. After that, the samples were washed three times with sterile water and then dried for 2 min. During our pilot studies, this method was shown to be most effective with the less effect on the composite samples.
Each composite sample was immersed in 1 mL FAD cell culture medium each (Ham’s F12/DMEM: mixing ratio 1:3, Biochrom, Berlin, Germany), 5% FCS, 100 μg/mL kanamycin (Sigma, Mannheim, Germany) and the supplements of KGM2 (Promocell, Heidelberg, Germany) in a 24-wells plate (Greiner-Bio-One, Frickenhausen, Germany). The samples were stored in a dark box at room temperature (21 °C). Half of them were stored for 24 h and the other half for 7 days. After the end of the storage period, the samples were removed and the media-samples (in 24-wells plate) were stored at 4 °C until the cells were prepared for the experiment. FAD-Medium stored for the same time periods was used as control.
2.3
Cell cultures: Establishment of monocultures (MC) and interactive cell systems (ICS) and exposure to composite eluates
In the present study, two different gingival cells were used: human gingival keratinocytes immortalized with the human papilloma virus type16 E6/E7 oncogenes (IHGKs) and human gingival fibroblasts (HGFs). The primary cultures of the cells were established and cultured as described in previous studies . For the use of the tissues informed consent was obtained by the patients according to the Helsinki Declaration, and the protocol was approved by the institutional ethics committee (Votum Nr. 411/08; Date: 11/20/2008). IHGKs in the passages between 90 and 95 were maintained in low-calcium keratinocyte growth medium (basal keratinocyte medium, KGM2, with provided supplements, Promocell, Heidelberg, Germany), containing 100 μg/mL kanamycin (Sigma, Mannheim, Germany). HGFs were cultured in passages 12−14 and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (PAA, Marburg, Germany) containing 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany) and 100 μg/mL kanamycin.
To detect both short-term and long-term effects of the composite eluates on periodontal cells, exposition periods of 24 h and 7 days were chosen. The gene expression analysis was conducted, using conventional monocultures (MC) of human gingival fibroblasts (HGF) and immortalized human gingival keratinocytes (IHGK), while interactive cell systems (ICS), comprising both cell types were employed to analyze eluate exposition under more physiological, i.e. in vivo -like conditions.
For establishment of the HGF monocultures, for the time period of 24 h, 5 × 10 4 cells were cultivated per well and for the period of 7 days 1 × 10 4 cells per well in 24-well plates (Falcon, BD Biosciences, Franklin Lakes, USA). For the monocultures of IHGK 1 × 10 5 cells per well for the shorter and 5 × 10 4 cells for the longer, eluate exposition period were cultivated on 24-well plates (Falcon, BD Biosciences, Fanklin Lakes, USA). All cells were maintained under standard cell-culture conditions: 37 °C, 97% humidity and 5% CO 2 . After 24 h, the cultivation medium was removed, replaced with the respective composite eluates and then a further incubation followed respective to the eluate exposition group they belong to (24 h or 7 days). From the incubated cell-wells, two wells per time period for each cell type and composite material were used for the determination of the apoptosis by immunofluorescence and three wells for the respective RT-PCR analysis. All groups were prepared in a biological replicate.
For establishing the IHGK/HGF interactive cell systems (ICS), the trans-well method was used as described previously . HGFs and were pre-cultivated in 24-well plates as described above for the monocultures, while 1 × 10 5 IHGKs per well were pre-cultivated separately on porous cell-culture inserts with pore sizes of 3 μm (Falcon, BD Biosciences, Franklin, USA). After 24 h pre-cultivation, the inserts with the IHGKs were placed in the wells containing the HGFs and the whole cultivation medium was replaced with the composite eluates and native FAD medium as control as described above. The ICS were incubated for further 24 h or 7 days respective to the group they belong. The ICS were used only for the evaluation of the effects of the composite on gene expression by quantitative qRT-PCR analysis. All groups were prepared in a biological replicate.
2.4
RNA isolation and quantitative PCR
Generally, for the qPCR, mRNA was extracted separately for each cell type, incubation period and exposed eluate, and cells incubated in ordinary cell culture medium served as controls. The C t -values measured for the control groups were used as reference in order to evaluate increase or decrease of the gene expression compared with the other groups.
After the respective cultivation periods, total RNA was isolated from treated and control cell cultures using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. In order to eliminate the residual genomic DNA, the RNA samples were treated with RNA-free DNase-Set (Qiagen, Hilden, Germany) during the RNA purification. The RNA concentration and integrity were determined by using an automated electrophoresis system (BioRad, Munich, Germany). First-strand cDNA was synthesized by using the C-03-first-strand-kit (SABiosciences, Qiagen, Hilden, Germany) from 500 ng total RNA. Then the cDNA concentration was quantified by PicoGreen (PicoGreen® dsDNA Assay Kit, Invitrogen, Darmstadt, Germany) with a plate reader Infinity M200 (Tecan, Crailsheim, Germany). For normalization, the cDNA concentration was adjusted to 5 ng/μL for each polymerase chain reaction (PCR). The qPCR analysis was performed with the real-time detection system CFX96 (BioRad, Munich, Germany). The SYBR Green Master Mix (SABiosciences, Qiagen, Hilden, Germany) was used for qPCR. For the qRT-PCR Analysis, different commercially available primers (SABiosciences, Qiagen, Hilden, Germany) were used: caspase 3, 8 and 9 (CASP 3, CASP 8, CASP 9), ki -67 (MKI 67), interleukin 6 and 18 (IL 6, IL 18), annexin A5 (ANX A5), involucrin (INV), filaggrin (FLG), integrin β3 (ITGB3), and primers for the housekeeping genes β-actin (ACTB) and ribosomal protein L13A (RPL13A). These genes were chosen to cover a wide range of the biological reactions of the cells, coding the proteins in the level of cell adhesion (integrin β3), proliferation (ki-67), inflammation (interleukin 6 and 18), different stages of apoptosis (caspase 3, 8 and 9 and annexin A5) and stages of differentiation (involucrin and filaggrin).
The qPCR was performed with the CFX96 real-time detection system (BioRad, Munich, Germany) and the relative mRNA gene expression was analyzed by a modification of ΔΔ C t equation using the CFX Manager Software (BioRad, Munich, Germany) and were normalized to the index C t of the non-modulated housekeeping genes actin β (ACTB) and ribosomal protein L13a (RPL13A) and referred to the relative gene expression of the control cells. An up-/down-modulation of ±1 on the amount of mRNA of interest normalized to the respective control (1 = double the amount of mRNA present compared with the control, -1 = half the amount of mRNA present compared with control) was considered as the threshold for significance and further analyzed by a Student’s t -test for unequal variance by a significance level of p < 0.01.
2.5
Determination of apoptosis by immunofluorescence
The determination of apoptosis by immunofluorescence took place only in the monocultures. After the exposure periods of 24 h and 7 days, the medium was removed from the wells intended for the detection of early apoptosis by immunofluorescence and the cells (HGFs and IHGKs) were washed once with 1 mL of annexin-V-binding buffer (Invitrogen, Darmstadt, Germany). The buffer was removed and again 500 μL Annexin-V-binding buffer containing 1 μL of annexin-V-FITC detection reagent (Invitrogen, Darmstadt, Germany) was added and the cells were incubated at room temperature for 5 min in the dark. The solution was removed from each well and the cells were washed again with annexin-V-binding buffer and then were fixed with paraformaldehyde 4% for 20 min. After that, the cells were washed once with PBS and the cell nuclei were counterstained 300 nM DAPI-solution. The cells were washed again twice with PBS, and once with distilled water and then were finally embedded in mounting medium (Fluoromount-G, Southern Biotech, Birmingham, USA) and observed by fluorescent microscopy (BZ-9000, Keyence, Neu-Isenburg, Germany).
In order to calculate the amount and the percentage of the annexin-V-positive cells, three representative images of two stained wells of two independent biological replicates for each of the tested cultivation periods were taken into account and were analyzed by the BZ-II-Analyzer Software (Keyence, Neu-Isenburg, Germany). The values were statistically analyzed by using the Student- t -Test for unequal variance, by a significance level of p < 0.01.
Stay updated, free dental videos. Join our Telegram channel
VIDEdental - Online dental courses
