Introduction
The root canal treatment aims to eliminate bacteria from the infected root canal, prevent reinfection of the tooth and promote healing of periapical tissues (Möller et al. 1981; Cook et al. 2007). The success of endodontic treatment is multifactorial, with each distinct procedural step (mechanical: instrumentation, chemical: irrigation and obturation) playing a significant role and contributing to the overall therapeutic result (Friedman et al. 2003; de Chevigny et al. 2008). Following chemo-mechanical preparation of the dentinal walls, a root canal treatment is completed by creating a bacteria- and fluid-tight seal into the root canal system with the use of filling materials (Möller et al. 1981; Guldener 1993; Wesselink 1995; Cook et al. 2007).
The most common and well-known root canal filling material is gutta-percha. Despite its high degree of volumetric stability, gutta-percha cannot adequately seal the root canal space by itself and does not adhere to the dentinal walls (Skinner & Himel 1987; Tagger et al. 1994; Li et al. 2014). Therefore, it is used as a core root canal filling material in combination with adjacent root canal filling materials, the endodontic sealers, which aim to create a homogenous interface between the dentinal walls and gutta-percha.
Endodontic sealers play an important role in hermetic obturation of the root canal system especially at the sealer–dentin interface, sealing accessory root canals and dentinal tubules (Heidemann 1989). Sealers entomb residual bacteria, prevent leakage of nutrients and ideally possess antimicrobial properties (Ørstavik 1988; Donnermeyer et al. 2019). Different types of root canal sealers, such as zinc oxide eugenol-, resin-, silicone-, calcium silicate- based materials with different setting formations are used in endodontics (Ørstavik 2014). These materials should ideally offer many biological and physiochemical properties such as antimicrobial activity, remain unaffected by the irrigating solutions, keep a long-term dimensional and physicochemical stability inside the root canal space (Ørstavik et al. 2001; Williamson et al. 2005; Camilleri & Mallia 2011; Gopikrishna 2014), remain insoluble, and not induce cytotoxic effects to surrounding periapical tissues (Dahl 2005).
Low cytotoxicity of sealers is important since extrusion of these materials to the periapical area may occur and lead to an inflammatory reaction, which may retard the healing process and even degenerate the periapical tissues (Nair 2004; Huang et al. 2005). Moreover, contact between tissue fluids or irrigation liquids and sealers may cause leaching of constituents from the sealer. Leachates could potentially migrate to exposed dentinal tubules, lateral canals or to periapical tissues through the bulk of filling materials or the dentine-sealer interface (Kazemi et al. 1993; Geurtsen & Leyhausen 1997; Ørstavik 2005; Elyassi et al. 2019). Eluates and further constituents from sealers’ degradation processes may be in long-term contact with periapical tissues and thereby induce cytotoxic effects.
Leachates of endodontic materials have attracted the attention with regard to antimicrobial properties and cytotoxicity (Arias-Moliz et al. 2017). The leachable compounds ideally should be biocompatible and hence prevent cytotoxic effects to the periapical tissues as this may jeopardize the clinical success of root canal therapies (Barbosa et al. 1993; Dahl 2005). A recent literature review on standardization of antimicrobial testing of dental materials highlights the importance of biocompatibility testing along with characterization of elution/degraded materials (Camilleri et al. 2020).
Both the biological and the physicomechanical properties of sealers depend on the chemistry of the root canal sealers. Hence, root canal sealers need to possess good physicochemical properties along with low cytotoxicity (Huang et al. 2002). Most of the endodontic root canal sealers may have some toxic properties or exhibit different levels of cytotoxicity on tissues, which would result in prolonged wound healing, inflammation, and bone resorption (Kaur et al. 2015; Silva et al. 2021). To date, there is scant scientific data about the biocompatibility of root canal filling materials of various chemistry on the periapical area. The present study aimed to investigate the effects of five different root canal sealers and their eluates on human alveolar osteoblasts as representatives of periapical bone tissue in terms of cell proliferation, adhesion, morphology and gene expression in vitro.
Materials and Methods
Root canal filling materials
An epoxy resin-based sealer, AH Plus (Dentsply International Inc, York, PA, USA), a zinc oxide eugenol-based sealer, Tubli-Seal (SybronEndo, Kerr, Romulus, MI, USA), a methacrylate resin-based sealer, Real Seal SE (SybronEndo, Kerr, Romulus, MI, USA), EndoRez (Ultradent Products, South Jordan, UT, USA), a methacrylate resin-based sealer, Apexit Plus (Ivoclar Vivadent, Schaan, Lichtenstein), and BeeFill (VDW, Munich, Germany), a gutta-percha, were tested. The materials were mixed according to manufacturer’s instructions.
Preparation of eluates and pellets from root canal filling materials
The root canal filling materials were transferred into the plastic lids of sterile Eppendorf microcentrifuge tubes and incubated for 24 hours (h) in a humidified atmosphere of 5 % CO2 at 37 °C, yielding solid sealer discs (diameter 4.5 mm, 15.89 mm2 surface area, height 2.5 mm) that were subsequently rinsed with 70 % ethanol, washed with phosphate buffered saline (PBS, Life Technologies, Darmstadt, Germany), and exposed to UV light (60 min, VL-6.L., LTF-Labortechnik GmbH & Co. KG, Wasserburg, Germany) for sterilization purposes. For eluate preparation, 1 disc/well was placed in 24-well plates containing 1 ml growth medium each, and incubated for 24 h or 72 h, respectively, at 37 °C with 5 % CO2. For quality assessment, the surface morphology of the remaining discs was analyzed by scanning electron microscopy (SEM).
Cell culture and exposure to root canal filling materials
All experiments were carried out in accordance to the guidelines of the World Medical Association Declaration of Helsinki and were approved by the Committee of Ethics of the Medical Faculty of the Albert Ludwigs-University Freiburg, Germany (vote number 516/12). All primary human alveolar osteoblasts were derived from operative biopsies of n=3 donors with informed and written consent and kindly provided by Prof. Thorsten Steinberg, Centre for Dental Medicine, University Medical Centre Freiburg, Dpt. of Oral Biotechnology. Osteoblasts were cultured in MEMalpha (Life Technologies, Darmstadt, Germany) supplemented with 10 % fetal bovine serum (Biochrom, Berlin, Germany), 1% glutamax (Life Technologies), 1% amphotericin and 1% antibiotics. Upon confluency, cells were trypsinized and passaged up to 6-9 times. For gene expression analysis, 5 × 103 cells/well were incubated in eluate medium for 24 h and 72 h, respectively. For morphology analysis, 1 × 103 cells / cm2 were cultured in microtiter plates for 3 days (d), 7 d and 14 d. For morphology testing, 2 × 103 cells/well were cultured in microtiter plates for 3 d, 7 d and 14 d at the surface of root canal filling material pellets or incubated with eluates, respectively.
Scanning Electron Microscopy (SEM)
The specimens were fixed in 8 % formaldehyde and dehydrated in an ascending ethanol series (50 %, 70 %, 80 %, 90 % once each and twice in 100 % for 1 h). Afterwards, critical point drying (Critical Point Dryer CPD 030; Bal-Tec, Wallruf, Germany) was performed, and the samples were sputter-coated with a gold palladium for 60 sec at 60 mA (SCD 050; Bal-Tec) and scanned by a Zeiss Leo 435 VP scanning electron microscope at 10 kV (Zeiss, Oberkochen, Germany).
Fluorescence microscopy (FM)
For actin staining after 3 d, 7 d and 14 d, respectively, the cells were washed with phosphate buffered saline (PBS, Biochrom AG, Berlin, Deutschland), fixed in 4 % paraformaldehyde (Sigma-Aldrich, Munich, Germany) and permeabilized with 1 % Triton X-100 (Life Technologies GmbH, Darmstadt, Germany) for 3-5 min. Non-specific binding sites were blocked with ImageIt FX® signal enhancer (Life Technologies, Darmstadt, Germany) for 30 min, and the cells were incubated with Alexa Fluor 488-conjugated phalloidine (1:40; Abcam, Cambridge, UK) at room temperature for 30 min in the dark. After intense rinsing, cell nuclei were counter-stained with 4',6-diamidino-2-phenylindole (DAPI, Merck, Darmstadt, Germany), and cells were mounted with an aqueous permanent mounting medium (Fluoromount, Southern Biotech, Birmingham, UK). Fluorescent images were acquired by a fluorescence microscope equipped with a CCD camera (BZ-9000, Keyence, Neu-Isenburg, Germany) and analyzed with BZ image analysis application software (KEYENCE, Neu-Isenburg, Germany).
qPCR assays
Total cellular RNA was purified using a guanidium–thiocyanate method (RNeasy Mini kit; Qiagen, Hilden, Germany) and stored at -80 °C. Genomic DNA contamination was eliminated by DNase digestion according to manufacturer’s instructions (RNase free DNase kit; Qiagen, Hilden, Germany). The RNA integrity and quantity were verified using the Experion RNA StdSens chip microfluidic technology according to manufacturer’s instructions (Bio-Rad, Munich, Germany), and cDNA was synthesized from 200 ng of total RNA each by using the RT2 PreAmp cDNA synthesis kit (Qiagen, Hilden, Germany), preceded by a genomic DNA elimination step at 42 °C for 5 min, in a C1000 Thermal Cycler (Bio-Rad) at 42 °C for 30 min followed by 95 °C for 5 min. Signal intensity and specificity were enhanced pre-amplifying cDNA templates by multiplex PCR by using the RT2 Pathway Primer Mix (Qiagen) in a 25 µl volume, and cycling conditions were 95 °C for 10 min followed by 12 cycles of 95 °C for 15 sec and 60 °C for 120 sec for all specific array primer sets. For real-time qPCR array, pre-amplified templates were amplified in 25 µl reaction mixture using RT2 SYBR Green qPCR mastermix (Qiagen) in a CFX96 cycler (Bio-Rad), according to manufacturer’s instructions. The products’ specificity of each amplicon was checked by examining the melting temperatures (heating at 0.05 °C/s to 95 °C). Negative reverse transcription, negative genomic DNA and negative template controls were included in all PCR runs. Data were collected with CFX96 Manager Software version 1.0 (Bio-Rad), and relative quantities of the respective genes of interest were normalized to the relative quantity of hydroxymethylbilane synthase, ribosomal protein L13a and ubiquitin as references, which were validated for Ct-value consistency.
Statistical analysis
To evaluate the effect of groups (control, root filling materials), alveolar osteoblast donors (1, 2, 3), and time, a Repeated Measures Analysis was performed for the outcomes of interest. Therefore, a linear model was fitted within which the structure of data, i.e. repeated measurements, is accounted for (repeated measurement per example, several examples per 'donor' and several materials per donor). An interaction term between material and donor is included in the model to detect inter-individual differences between the materials. The group effects and differences of least-square (ls) means are calculated with their 95 % confidence intervals. Several multiple comparisons of ls means in relevant donor and material combinations are done. P-values are adjusted by the method of Tukey. All calculations were performed with the statistical software SAS 9.1.2. using PROC MIXED.
Results
Microscopy analysis
Regarding control, the actin filaments of osteoblasts (Fig. 1Ai) were evenly distributed in the cytoplasm and showed an intact, characteristic actin cytoskeleton. The fibers appeared linear and were arranged in parallel bundles longitudinal to the cell axis. The cells displayed star- to spindle-like shapes. The blue stained DNA of the round-oval cell nuclei was abundant and evenly stained. In Fig. 1Aii and iii, a clear increase in the number of cells is recognizable. The spindle- to dumbbell-shaped cells were evenly distributed and had long filaments.
For AH Plus (Fig. 1B), an increase in cell number was evident in FM images (i), (ii), and (iii). In image (i), compared to the control (Fig. 1Ai), the cell number seemed to be lower after 3 d and the cells showed spacing from each other. In images (ii) and (iii) there was a clear increase in cell number and the cells were densely packed next to each other. The cellular actin filaments stained in green by phalloidin were even less directed in (i) than in (ii) and (iii) images. In the latter, the actin filaments were arranged in parallel to each other, indicating a flat shape of the cells. Initially, the cells were rather star-shaped (Fig. 1Bi), while they exhibited spindle- after 7d (Fig. 1Bii) and dumbbell-like shapes after 14d of culture (Fig. 1Biii). The cell nuclei, stained blue by DAPI nuclei were clearly visible in cells attached to AH Plus at each time point of examination. In SEM, cell proliferation was also evident with prolonged culture time. A dense cell layer could be detected on the surface of the specimens. The osteoblasts exhibited typical characteristics of polymorphic cells with their flattened and spindle-shaped shape, a fact which indicates good adherence to the sealer surface. The cell layer had already been very dense after 3 d (iv) and increased over time at 7 d (v) and 14 d (vi). The cells did not show any damage such as membrane defects or rounding.
The FM images (Fig. 1C) showed a lower number of osteoblasts growing close to Apexit Plus compared to matched controls. The green-stained actin filaments of the osteoblasts showed star-shaped cells with short cell filaments after 3 d of culture (i). The cytoskeleton was less structured compared to the control group and fragmented filamentous actin could be detected. After 7 d (ii), only a slight increase in the number of cells was observed and the cytoskeleton was also not well developed. The cell nuclei were partially detached from the cell bodies. Further after 7 d (ii), cell variations could be identified. The actin filaments of the spindle- to dumbbell-shaped cells were arranged in parallel. The cell nuclei were abundant and clearly stained. In the SEM micrographs (iv)-(vi), no intact cells are present on the surface of the specimens even after only 3 d of incubation. The osteoblasts, which could be found in isolated cases, had a round, apoptotic shape. This observation could be indicative of a cytotoxic effect of Apexit Plus. The differing results in immunofluorescent stainings compared to SEM images could either be attributed to the fact that cell shapes differed in various microscopic regions, meaning that both living and dead cells were detectable, or merely be an artefact resulting from different experimental procedures to fix the cells.
When osteoblasts were grown on Tubli-Seal pellets (Fig. 1Di-iii), a lower increase in cell number was observed over time compared to the control group. After 3 d, only few cells with short projections were detectable (i), and the cellular actin filaments were fragmented. After 7 d of culture (ii), the cells had a flattened and wide-spread cell body with a structured cytoskeleton. However, the cytoskeleton of the cells was poorly organized and appeared partially disrupted after 14 d (iii), while the morphology varied from star-shaped to dumbbell-like shapes. In SEM images (iv) and (v), only round cells were present on the sample surfaces, indicative of cell apoptosis. The osteoblasts were dislocated and lacked extensions. After 7 d (v), the typical apoptotic spiny shape of the cell membrane was obvious, while only cell remnants were visible on the sample surfaces at 14 d (vi).
Alveolar osteoblasts grown on the surface of Real Seal SE pellets (Fig. 1E), showed elongating filaments after 3 d (i). After 7 d of incubation (ii), the cell number was increased, and the cell bodies were flat and displayed a dumbbell-like morphology. Compared to the control group, the actin fibres did not run in rectified bundles and were less evenly distributed over the cytoplasm. The nuclei were numerous and clearly stained. Fewer osteoblasts were observed after 14 d (iii) than after 7 d (ii). The cytoskeleton of the cells was less elongated than in image (ii), suggesting small, compact cells. In the SEM images at 3 d (iv), only few osteoblasts with single long filaments were visible on the sample surface. Cells with spindle-shaped morphology could not be found in direct contact with the material, while inactive, rounded osteoblasts were shown. After 7 (v) and 14 d (vi), only apoptotic cells were visible, clearly indicating a cytotoxic effect of Real Seal SE.
Regarding EndoRez, the FM image (i) after 3 d of incubation, showed small cells in size. After 7 d (ii), a higher number of cells could be detected. The actin filaments were, however, less aligned compared to the control group in Figure 1A. In image (iii) after 14 d, fewer osteoblasts could be identified and the actin filaments were also superimposed and fragmented. The osteoblasts appeared more rounded and did not show extensions. The SEM image (iv) showed a few round osteoblasts on the sample surface after 3 d of incubation. After 7 d (v) and 14 d (vi), only cell debris was present on the sample surface.
Based on the FM images (Fig. 1G), BeeFill had a stimulative effect on cell number with increasing culture. After 3 d (i), short, spindle-shaped osteoblasts with extending filaments were visible. After 7 (ii) and 14d (iii), only in the BeeFill group of all sealers under study, the cell number was comparable to that of the control group (Fig. 1A). There were evenly distributed, spindle-shaped cells with oval nuclei. The actin filaments seemed to overlap partially and showed a parallel arrangement. In SEM image (iv) a large number of round cells could be detected on the surface of the sample.
Gene expression analysis
Next, the expression of selected genes for inflammation (IL-6, MMP1, MMP3; Fig. 2), proliferation (Hist3H3; Fig. 3A) and apoptosis (Casp3; Fig. 3B) was measured, all normalized to a pool of three house-keeping genes. Due to insufficient RNA amounts, the analysis of the EndoREZ and Tubli-Seal samples was not feasible.
With regard to inflammation markers, no statistically significant difference was found in the expression of IL-6 in the 24-hour, nor in the 72-hour samples (Fig. 2A). However, the expression of IL-6 was increased over time for Apexit Plus. No statistically significant differences could be detected in the expression of MMP1 after 24 hours, both between different sealer groups and compared to controls. However, after 72 hours, osteoblasts expressed significantly more MMP1 RNA in the AH Plus group compared to controls (p=0.009, Fig. 2B). An increasing trend in expression of the inflammation marker MMP1 was observed over time with Apexit Plus and Real Seal SE. At 24 hours, MMP3 was expressed to a significantly higher degree compared to controls in cells cultured with eluates from Apexit Plus (p=0.001) and BeeFill (p=0.001). In addition, cells in the BeeFill group expressed significantly more MMP3 RNA than cells in the Apexit group (p=0.004). Interestingly, there were no significant differences for the 72-hour samples. On the other hand, over time, there was a decreasing trend in the expressed inflammation markers in all samples. With regard to gene expression of the proliferation marker Hist3H3 (Fig. 3A), osteoblasts cultured in the presence of Real Seal SE showed a significantly lower expression compared to controls SE (p=0.017). Interestingly, after 72 hours, the opposite effect was noticed, because cells in the Real Seal SE group expressed significantly higher HistH3 RNA levels than controls (p=0.0001). Similarly, cells incubated with eluates from Apexit Plus expressed more HistH3 than cells in the control group (p=0.063; Fig. 3A). Over time, an increase in the expression of Hist3H3 was observed for Apexit Plus and BeeFill, whereas for AH Plus and the control group the expression of Hist3H3 tended to decrease. As for apoptosis (Fig. 3B) in the 24-hour samples, no significant difference was found between the material groups. After 72h, osteoblasts in the AH Plus (p=0.0003) and in the Apexit Plus (p<0.0001) groups expressed significantly less Casp3 RNA than cells in the Real Seal SE group. Further, cells in the Apexit Plus group expressed significantly less Casp3 RNA than cells in the AH Plus group (p=0.026). There was not significant difference in gene expression between the sealer groups and controls, respectively, which presumably results from a high variation of Casp3 expression in control. Real Seal SE and BeeFill showed an increasing trend in gene expression of Casp3 over time. In contrast, there was a decreasing trend in Casp3 expression for AH Plus.
Discussion
In root canal treatments, the use of sealers in combination with gutta-percha is the standard procedure. Based on the established ISO 10993 series, a medical device should aim at the protection of humans from potential biological risks. The root canal filling materials (endodontic sealers, gutta-percha) can be categorized to externally communicating dental medical devices (non-implantable) as they are in contact with tissue, bone or pulp/dentin systems. Thus, the biological evalution of endodontic sealers is crucial to assess the safety of these materials for clinical use. A series of tests for genotoxicity, in vitro cytotoxicity, systemic toxicity, potential degradation and leachables is suggested (ISO 10993-1:2018). The physical and biological properties of dental materials have been and are being investigated in vitro, in vivo and in clinical studies. The results of our study render valuable insights on the cytotoxicity of endodontic sealers, because we investigated primary human cells isolated from the target tissue, i.e. alveolar bone, which reflect the clinical situation much more realistically than animal tissue-unspecific or immortalised cells (Huang & Chang 2002).
Five different sealers, as well as BeeFill as a representative of gutta-percha material group were tested following setting for their cytotoxicity with primary human osteoblasts. It is well documented that set materials are less cytotoxic than freshly mixed as they release less components (Camargo et al. 2014). Since the filling material affects the periapical tissues by releasing components (Tronstad et al. 1988), but also by direct contact if extrusion occurs, both material surfaces and eluates were investigated. Scanning electron micrographs were used to investigate the cell morphology of osteoblasts seeded on the surface of sealer samples. Through surface receptors and adhesion links, osteoblasts attach to materials (Hong et al. 2003). As a result, cell adhesion, as a prerequisite for proliferation and growth of the cells, is dependent on the chemical and physical properties of the material surfaces (Wang et al. 2013).
In direct contact with the sample surface, alveolar osteoblasts in the AH Plus group showed good adhesion after 3 d and an increase in cell number over time. Together with the gene expression data in the AH Plus group, our results do not reflect a strong initial inflammatory reaction, as observed in an implantation study in porcine bone (Sousa et al. 2006).
On the surface of the Real Seal SE samples after 3 d, a few spindle-shaped cells could be detected next to dislocated, apoptotic cells. After 7 and 14 d, few living cells were detectable. The cytotoxic effect of Real Seal SE on osteoblasts has already been shown in a previous study (Scelza et al. 2012) and can be presumably attributed to the release of toxic monomers of incompletely polymerized metacrylate (Bouillaguet et al. 2006). With regard to Apexit Plus, EndoREZ and Tubli-Seal samples, only inactive round cells were observed. The initial toxic effect of these materials has already been reported in various studies (Zmener et al. 2005; Eldeniz et al. 2007; Kangarloo et al. 2009); however, our study is the first to show that the cells do not recover from this initial cyotoxic effect. On the surface of the BeeFill samples, apoptotic cells were also visible after 3 d. A toxic effect of gutta-percha caused by zinc oxide and zinc ions has already been demonstrated in 1990 by means of a chromium release assay with mouse fibroblasts (Pascon & Spangberg 1990). Nevertheless, osteoblasts nicely recovered from the initial BeeFill cytotoxicity, as shown by FM, SEM and qPCR results.
For the morphological examination of the osteoblasts by FM and for cell analysis by quantitative qPCR, sample eluates were prepared. Sealers are supposed to have a certain solubility, thus release of components may have a biological effect on the surrounding tissue (Geurtsen & Leyhausen 1997; Schafer & Zandbiglari 2003). In FM, cells are fixed in their vital state, which allows the morphological examination of cells in their natural behavior and their protein metabolism. Cell functions, such as adhesion strength, differentiation and proliferation, are closely related to cell shape (Chen et al. 1997). It is a well-established concept that human osteoblasts proliferate rapidly in vitro within the first few days (Auf'mkolk et al. 1985; Robey & Termine 1985). After incubation of the cells in the material eluates for 3, 7 and 14 d, a reduced cell number and a change in the morphology could be detected, which varied among the different materials. The AH Plus samples affected the cell morphology after 3 d of incubation while the cells appeared to proliferate over time. The cell density and the morphology of the cells were similar to the control group after 7 and 14 d. An initial toxic effect for AH Plus has already been demonstrated by Sousa et al. (2006) (Sousa et al. 2006), in an implantation study and is probably due to the release of formaldehyde (Leonardo et al. 1999; Cohen et al. 2000). Low cytotoxicity of AH Plus was found in 2007 in a study with human gingival fibroblasts (Eldeniz et al. 2007). The same study showed a significantly more toxic effect of Apexit Plus and EndoREZ compared to AH Plus, a result that is consistent with the morphological examination of cells in the present study exhibiting a reduced cell density at all three time points. Similar results were obtained with Tubli-Seal and Real Seal SE, and showed an even more pronounced effect. Different studies on the biocompatibility of Real Seal SE have produced conflicting results so far (Kim et al. 2010). On the one hand, low cytotoxicity was demonstrated in an indirect test with human gingival fibroblasts (Scotti et al. 2008), whereas other studies showed a strong toxic effect of the materials (Scelza et al. 2012). Morphological examination also indicated a toxic effect, which is demonstrated by a reduced cell number of the osteoblasts and an effect on the actin cytoskeleton. A possible cause for this may be the release of toxic monomers, which occurs due to a non-complete polymerization of the methacrylate (Bouillaguet et al. 2006). A study by Chang et al. (2010) showed a toxic effect of Tubli-Seal on periodontal ligament fibroblasts due to the eugenol contained in the material (Chang et al. 2010). Contrary to the results of Pascon et al. (1990) (Pascon & Spangberg 1990), the morphological examination of BeeFill samples revealed an initial interaction with the osteoblasts after 3 d. However, after 7 and 14 d, the cell density, morphology of the cells were comparable to those of the control group.
In this study, the behavior of human osteoblasts after cultivation with different sealer eluates was analyzed on a molecular level using the gene expression of specific genes. The most impressive result was that no RNA at all could be retrieved from cells cultured in eluates from EndoREZ and Tubli-Seal. Thus, both materials seem to exhibit a massive toxicity on osteoblasts so that the amount of RNA extracted after 24 and 72 h was either too low or non-existent, indicating the absence of any living cells. This interpretation is substantiated by our SEM findings of round and apoptotic-like cells in both groups after prolonged culture periods. In line with our results, this toxicity has been documented in several studies (Eldeniz et al. 2007; Chang et al. 2010; Diomede et al. 2014).
In line with scientific evidence, our data reveal a clear cytotoxic effect of Tubli-Seal, which is supposed to result from its component eugenol (Briseno & Willershausen 1990; Gulati et al. 1991). Al-Awadhi et al. (2004) documented increased apoptosis activity in primary osteoblasts in the presence of eugenol-containing sealer (Al-Awadhi et al. 2004). Futhermore, human PDL fibroblasts in contact with the eugenol-containing sealer endomethasone and N2 showed decreased metabolic activity (Schwarze et al. 2002). The other sealer that proved highly cell damaging in our study was EndoREZ. Its cytotoxicity may presumably attributed to the release of urethane dimethacrylate (UDMA), which was already recognized as a toxic substance by Hikage et al. 1999 (Hikage et al. 1999). The results of several in vivo and in vitro studies substantiate clear toxic properties of the material (Zmener 2004; Bouillaguet et al. 2006).
Besides apoptosis, we aimed at measuring the inflammatory reaction of alveolar osteoblasts exposed to the different endodontic sealers. Interleukins are peptide hormones from the cytokine group and are well-known immunomdulators. The cytokine IL-6 is a marker for tissue injuries and infections (Hirano et al. 1990; Kishimoto et al. 1992) and plays a significant role in the activation of the immune system through the differentiation of B cells, as well as IL-6 acts as initiator of bone resorption (Ishimi et al. 1990). In addition, various studies have demonstrated a link between the progression of inflammatory reactions in the periapical tissue and the expression of IL-6 (Bando et al. 1993; Euler et al. 1998; Honma et al. 1998). In a study by Huang et al. (2005), the effect of AH Plus on the expression of IL-6 and IL-8 genes of human osteoblasts was investigated. There is evidence that AH Plus triggers the gene expression of IL-6 and even further of IL-8, which was interpreted as a result of the sealer release of formaldehyde (Huang et al. 2005). In the present study, no sealer significantly affected the expression of IL-6 in aleoar osteoblasts. Matrix metalloproteinases (MMPs) are members of the zinc-dependent endopeptidase family, and regulate tissue homeostatsis, bone remodelling and wound healing (Chen 1992). Nevertheless, they are also involved in pathological processes such as tumor growth and metastasis, rheumatoid arthritis or oral diseases, (Stetler-Stevenson et al. 1996; Basset et al. 1997). The interstitial collagenase MMP1 degrades fibrillar and non-fibrillar collagen (types I, II, III, VI and IX) and gelatin (Bhuvarahamurthy et al. 2006). Stromelysin (MMP3) is involved in the degradation of proteoglycans, laminin, fibronectin, collagen (types III, IV, V and IX), gelatin and pro-MMP1 and plays an important role in the physiology of inflammatory processes and wound healing (Chen 1992). A dysregulation of MMP gene expression or activation of MMPs can lead to various clinical conditions such as chronic inflammation (Rodriguez et al. 2010). A study by Paula-Silva et al. (2010), found significantly increased MMP expression and degeneration of the periapical tissue as a result of bacterial invasion in untreated apical periodontitis (Paula-Silva et al. 2010). In our hands, the significantly increased expression of the MMP3 gene in the Apexit Plus, BeeFill and Real Seal SE samples after 24 hours, suggests a similar inflammatory reaction. After 72 hours, increased expression of the MMP1 gene was observed in cells incubated with Apexit Plus eluates and slightly in the AH Plus group. From these results, it can be concluded that exposure of the periapical tissues to endodontic materials after the root canal treatment could induce an inflammatory reaction, which may undermine the therapy success.
Histone 3H3 (Hist3H3) from the histone family is involved in chromosome organization in the cell nucleus. The expression for these proteins takes place primarily during the replication of DNA and is thus a sign of cell proliferation (Peterson & Laniel 2004). The significantly lower expression of the Hist3H3 gene in the Real Seal SE group compared to the control group after 24 hours indicates a low proliferation of osteoblasts, which increased slightly at 72-hours. The same tendency was also observed for Apexit Plus. The low proliferation in Real Seal SE group can be explained by the time-dependent cytotoxic effect of unbound monomers as already described by Scelza et. al. (2012) (Scelza et al. 2012).
In this study, the effect of Real Seal SE on the cellular behavior of human osteoblasts at two time points (1 and 3 d) was tested. It was shown that the freshly prepared samples had a high toxic effect on the cells 24 hours after mixing. After 72 hours, this effect was also detectable, albeit to a lesser extent (Scelza et al. 2012). The reduced proliferation of the Real Seal SE samples, which is presumably caused by monomer release, also appears to be time-dependent, since a slight increase in cell proliferation was observed after 72 hours.
Caspase 3 (Casp3) is a cysteine protease that, as a component of an enzyme cascade, is involved in the initiation of cell apoptosis (Kurokawa & Kornbluth 2009). As a result of the activated cascade, there is dissolution of the nuclear envelope, disruption of transcription and translation, chromatin condensation and DNA cleavage through increased nuclease activity (Scaffidi et al. 1998). In this study, an increased expression of the Casp3 gene in Apexit Plus, AH Plus and Real Seal SE samples after 72 hours was observed. Interestingly, the toxic effect of Real Seal SE seemed to be the most pronounced one, while the apoptosis stimulus was highest among the materials tested. Real Seal SE contains calcium hydroxide, barium sulphate, barium glass and silicates as fillers. There are reports in which the filler content of the material is up to 70% (Versiani et al. 2006). One possible explanation for the cytotoxic effect of this sealer is the release of filler particles or monomers as mentioned above. The toxic effect of the calcium hydroxide-based sealer Apexit Plus was not confirmed in previous studies (Beltes et al. 1995; Miletic et al. 2000). The reason for our contradictory results could be the use of different cell lines (mouse fibroblasts, PDL fibroblasts). However, a study by Eldeniz et al. (2007), showed similar results showing a high cytotoxicity of Apexit Plus on mouse and human gingival fibroblasts (Eldeniz et al. 2007).
Conclusions
In the present study, it was shown that the sealers investigated from different material groups, as well as BeeFill as a representative of the material group gutta-percha, have varying degrees of cytotoxic effect on human osteoblasts. The toxic substances released into the periapical area causes reduced density of the cells, have an inflammatory effect and are able to induce apoptosis. In inter-group comparison, EndoREZ and Tubli-Seal performed the worst, followed by Apexit Plus and Real Seal SE. BeeFill and AH Plus proved to have low cytotoxicity.
The in vitro tests carried out are very helpful in investigating the biological properties of sealers. However, they cannot directly simulate the clinical scenario. Nevertheless, such investigations are necessary in the search for sealers with all the required chemical, physical and biological properties. In everyday dental practice, the human immune system may restore healthy tissues and inflammatory reactions can be compensated for a long period of time. However, practitioners should take special care to avoid iatrogenic periapical inflammation caused by root canal filling materials, to ensure complete healing of the apical and periapical tissues.
Acknowledgments and Conflicts of Interest.
All authors declare that they do not have any conflict of interest.
Zusammenfassung
Einleitung: Das periapikale Gewebe kann infolge einer Wurzelkanalbehandlung mit Wurzelkanalfüllungsmaterialien in Berührung kommen. Eine geringe Zytotoxizität von Wurzelkanalsealer ist wichtig, da es zu einer Extrusion dieser Materialien in den periapikalen Bereich und zu einer Fremdkörperreaktion kommen kann, die den Heilungsprozess verzögern und sogar zu einer Degeneration des periapikalen Gewebes führen kann. Es gibt nur wenige wissenschaftliche Daten über die Biokompatibilität von Wurzelkanalfüllungsmaterialien unterschiedlicher Chemie im periapikalen Bereich. Ziel dieser Studie war es, die Auswirkungen verschiedener Wurzelkanalsealer und ihrer Eluate auf menschliche Alveolar-Osteoblasten in Bezug auf Zellproliferation, Adhäsion, Morphologie und Genexpression in vitro zu untersuchen.
Material und Methoden: Fünf Wurzelkanalsealer (AH Plus®, Apexit® Plus, Tubli Seal®, Real Seal SE®, EndoRez®) und ein Guttapercha-Obturationsmaterial (BeeFill®) wurden getestet. Humane Alveolar-Osteoblasten von 3 verschiedenen Spendern wurden nach Inkubation mit 24- und 72-stündigen Sealer-Eluaten mittels qPCR (Genexpression) untersucht. Die morphologischen Reaktionen der alveolären Osteoblasten wurden visualisiert, nachdem die Zellen für 72 Stunden, 7 Tage und 14 Tage kultiviert wurden, gefolgt von Rasterelektronenmikroskopie (Morphologie, Adhäsion) und Fluoreszenzmikroskopie (Morphologie des Aktinzytoskeletts, Proliferation). Es wurde eine Analyse mit wiederholten Messungen durchgeführt und die p-Werte wurden mit Tukey angepasst.
Resultate: Während alle Wurzelkanalsealer die Zellmorphologie und die Genexpression beeinflussten, die mit Apoptose (Casp3), Proliferation (Histon H3) und Entzündung (Interleukin-6 und Matrix-Metalloproteinasen 1 und 3) in Verbindung gebracht werden, sorgten vor allem AH Plus® und Apexit® Plus für ein regelmäßiges Aktinzytoskelett und gutes Genexpressionsmuster. Was die Zelladhäsion betrifft, nur AH Plus® unterstützte die ordnungsgemäße Verankerung der alveolären Osteoblasten.
Diskussion: In der vorliegenden Studie konnte gezeigt werden, dass die untersuchten Sealer aus verschiedenen Materialgruppen, sowie BeeFill als Vertreter der Materialgruppe Guttapercha, Toxizität auf die Zellen aufweisen. Im Zytotoxizitätsvergleich schnitten EndoREZ und Tubli Seal am schlechtesten ab, gefolgt von Apexit Plus und Real Seal SE. Am besten schnitten BeeFill und AH Plus ab, die eine vergleichsweise geringe Zytotoxizität aufweisen. Unsere Ergebnisse belegen die Biokompatibilität von Wurzelkanalsealer auf Epoxidharzbasis, d.h. AH Plus®, während sich andere Wurzelkanalsealer als zytotoxisch für alveoläre Osteoblasten erwiesen. Weitere Studien sind erforderlich, um die Reaktionen der Knochenzellen nach einer Wurzelkanalbehandlung zu verstehen und die klinische Entscheidung über den Wurzelkanalsealer der Wahl zu treffen.
Résumé
Objectif : Les tissus périapicaux peuvent être exposés aux matériaux d'obturation canalaire à la suite d'un traitement canalaire. La faible cytotoxicité des produits d'obturation est importante car l'extrusion de ces matériaux dans la zone périapicale peut se produire et entraîner une réaction à corps étranger qui peut retarder le processus de guérison et même dégénérer les tissus périapicaux. Il existe peu de données scientifiques sur la biocompatibilité des matériaux d'obturation des canaux radiculaires de différentes compositions chimiques sur la zone périapicale. Cette étude visait à étudier les effets de différents matériaux d'obturation canalaire et de leurs éluats sur les ostéoblastes alvéolaires humains en termes de prolifération cellulaire, d'adhésion, de morphologie et d'expression génétique in vitro.
Matériels et méthodes : Cinq scellants endodontiques (AH Plus®, Apexit®, Tubli-Seal®, Real Seal SE®, EndoRez®) et un matériau d'obturation à base de gutta-percha (BeeFill®) ont été testés. Les ostéoblastes alvéolaires humains dérivés de 3 donneurs différents après incubation avec des éluats de scellement de 24 et 72 heures ont été étudiés au moyen de la qPCR (expression génétique). Les réactions morphologiques des ostéoblastes alvéolaires ont été mesurées en cultivant les cellules pendant 72 h, puis 7 et 14 jours, respectivement, et en procédant à une microscopie électronique à balayage (morphologie, adhésion) et à une imagerie par fluorescence du cytosquelette d'actine (morphologie, prolifération). Une analyse de mesures répétées a été réalisée et les valeurs p ont été ajustées par Tukey.
Résultats : Alors que tous les scellants endodontiques ont influencé la morphologie cellulaire et l'expression des gènes associés à l'apoptose (Casp3), à la prolifération (histone H3) et à l'inflammation (interleukine-6 et métalloprotéinases matricielles 1 et 3), AH Plus® et Apexit® ont principalement produit un cytosquelette d'actine régulier et des modèles d'expression génétique bénéfiques. En ce qui concerne l'adhésion cellulaire, seul AH Plus® a permis un ancrage correct des ostéoblastes alvéolaires.
Discussion : Dans la présente étude, il a été démontré que les scellants étudiés provenant de différents groupes de matériaux, ainsi que BeeFill en tant que représentant du groupe de matériaux gutta-percha, ont des degrés variables d'effet cytotoxique sur les cellules. Dans la comparaison de la cytotoxicité, EndoREZ et Tubli-Seal ont obtenu les pires résultats, suivis par Apexit Plus et Real Seal SE. BeeFill et AH Plus se sont avérés être les meilleurs, et ont donc comparativement une faible cytotoxicité. Nos résultats fournissent des preuves de la biocompatibilité des scellants endodontiques à base de résine époxy, c'est-à-dire AH Plus®. D'autres études sont nécessaires pour comprendre les réactions des cellules osseuses après un traitement endodontique et la prise de décision clinique concernant le scellant de choix pour les obturations canalaires.