Highlights
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Osteoinductive properties of Bioroot™ RCS was compared to Kerr’s Pulp Canal Sealer™ using the mouse pulp-derived stem cell line A4, which have an osteo/odontogenic potential in vitro .
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Trypan Blue exclusion assay was done for cytotoxicity evaluation, immunocytochemical staining was done for SIBLINGs (BSP, DMP 1, Collagen Type 1), and Von Kossa was done for mineralization evaluation.
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Bioroot™ RCS was not found cytotoxic as PCS. It did not recruit the pulpal stem cells toward differentiation but preserve their osteo-odontogenic intrinsic properties.
Abstract
Objective
To evaluate the biocompatibility and osteoinductive properties of Bioroot™ RCS (BR, Septodont, France) compared to Kerr’s Pulp Canal Sealer™ (PCS, Kerr, Italy) using the mouse pulp-derived stem cell line A4, which have an osteo/odontogenic potential in vitro and contribute to efficient bone repair in vivo .
Methods
A4 cells were cultured at the stem cell stage in the presence of solid disks of BR or PCS, whereas untreated A4 cells were used as control. After 3, 7, 10 days of direct contact with the sealers, cell viability was quantified using Trypan Blue exclusion assay. Immunolabelings were performed to assess the expression of odontoblast markers i.e. type 1 collagen, DMP1 or BSP. Finally, sealer-treated cells were induced toward osteo/odontogenic differentiation to assess the impact of the sealers on mineralization by Von Kossa staining. Statistical significance was evaluated by one-way analysis of variance and t -test ( p < 0.05).
Results
BR did not alter the viability and morphology of A4 pulpal cells compared to control group ( p > 0.05); however, living cell percentage of PCS was significantly lower compared to control and BR groups ( p < 0.05). BR preserved the intrinsic ability of A4 cells to express type 1 collagen, DMP1 or BSP at the stem cell stage. It did not alter the integrity of collagen fibers surrounding the cells and promoted overexpression of BSP and DMP1 at the cell surface. In contrast to PCS, BR did not compromise the mineralization potential of pulpal A4 stem cells.
Significance
Bioroot™ RCS was not as cytotoxic as PCS. It did not recruit the pulpal stem cells toward differentiation but preserve their osteo-odontogenic intrinsic properties. Bioroot™ RCS might provide more suitable environment to induce stem cells for hard tissue deposition.
1
Introduction
Root canal filling aims to eradicate bacterial infection and protects the tooth against re-contaminations. One of the goals of endodontic therapy is also to induce periapical repair by recruiting surrounding osteogenic and/or odontogenic cells of apical tissues. Gutta-percha cones and an endodontic sealer are used as root canal filling materials, which are essential components of root canal obturation to establish a fluid-tight seal. Mainly, it is expected that irregularities and minor discrepancies between the gutta-percha cones and canal walls, accessory canals, and multiple foramina will be filled with root canal sealers. The ideal root canal sealers must have certain characteristics, including adequate marginal sealing quality, antimicrobial activity, dimensional stability, radio-opacity, the ability to promote periapical bone repair and biocompatibility . Biocompatibility is the ability of a material to perform with an appropriate host response in a specific application and biomaterials need to be in contact with a living system without producing an adverse effect . One of the factors that mainly influence the clinician’s choice of filling material in root canal treatment is the biocompatibility of the material. As sealers are assumed to directly interact with periapical tissues or through diffusion of components within the living surrounding tissues, biocompatibility of root canal sealers is of key importance to positively influence cell reparative responses in apical foramen and thereby the outcome of the endodontic treatment .
There are many different root canal sealers, now used in clinical practice, including zinc oxide–eugenol cements, glass ionomer cements, epoxy resins, mineral trioxide aggregate- and calcium silicate-based cements but all of them have substantial limitations and still there is no current root canal sealer that meets all the appropriate requirements . Calcium containing root canal sealers exert antimicrobial effects and might enhance the mineralization process by elevating the pH levels. Chen et al. showed that exposure of human dental pulp primary cells to calcium silicate cement enhances their proliferation and the mRNA expression levels of alkaline phosphatase (TNAP) and bone sialoprotein (BSP). The release of calcium and hydroxyl ions from the sealer also results in the formation of hydroxyapatites in the presence of phosphate-containing fluids that may promote mineral deposition by matrix-forming cells . However, in vivo and in vitro studies reported controversial results for sealers regarding bone repair .
Bioroot™ RCS (BR) is a bioactive mineral root canal sealer based on innovative mineral micro-aggregate chemistry named “active biosilicate technology” ( http://www.septodontusa.com/sites/default/files/BioRoot-IFU.pdf ). It is resin- and eugenol-free sealer, which makes it different from conventional eugenol and resin based root canal sealers. BR powder based on tricalcium silicate, zirconium oxide and excipients is mixed with an aqueous solution of calcium chloride and excipients. It has hydrophilic properties and allows increase of pH values more than 11 ( http://www.septodontusa.com/sites/default/files/BioRoot-Brochure.pdf ). It is important to investigate the biocompatibility and the impact of root canal sealers on extracellular matrix mineralization before clinical use.
One approach to test the biological compatibility of root canal sealers is to use an in vitro model to monitor cellular responses. A few cell lines such as MRC-5 human fetal lung fibroblasts , human tooth germ stem cells , 3T3 mouse fibroblasts , MO6-G3 dental papilla mesenchymal cells immortalized by SV40 , have been used in dental material biocompatibility testing. In the present study, a multipotent mouse pulp-derived stem cell line was introduced to evaluate the biological effects of exposure to BR. The A4 cell lines were derived from embryonic ED18 first molars of mouse transgenic for a recombinant plasmid adeno-SV40 . A4 cells display stem cell properties maintaining a stable and undifferentiated phenotype under long-term standard culture conditions . Upon induction, A4 cells behave as multipotent mesoblastic stem cells with the ability to undergo osteogenic, chondrogenic, adipogenic or odontogenic differentiation . Implantation of A4 cells in a mouse incisor or rat molar promotes efficient dentin repair . Notably, A4 cells have been shown to also contribute to bone repair of a critical-size defect of the calvaria . Such a potential to promote dentin repair as well as bone repair upon implantation in vivo provides a rationale to use the A4 cell line for biocompatibility testing of root canal sealers.
Since it is of key importance that root canal sealers are not harmful for osteogenic and/or odontogenic stem cells located close to the apex to induce periapical repair, the stem cell properties of A4 cells were exploited to evaluate the biocompatibility of a new root canal sealer. BR was compared to the conventional, well-known zinc oxide eugenol based root canal sealer (PCS, Kerr’s Pulp Canal Sealer™), which has been widely used as a comparative sealer in several studies , at the stem cell stage. Moreover, A4 cells were also exposed to these sealers during their kinetics of differentiation to evaluate whether the sealers impact on the differentiating cell ability to form a mineralized matrix.
2
Materials and methods
2.1
Dental pulp cells: cell culture and differentiation
A4 pulpal cells, which have been derived from first molar tooth germs of E18 mouse embryos transgenic for a recombinant plasmid adeno-SV40, were cultured in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal calf serum (FCS) as described by Priam et al. . The growth medium was changed every 3 or 4 days. A4 cells were plated (3 × 10 5 cells) in the presence of 1 to 4 solid disks of Bioroot™ RCS (BR, Septodont, Saint-Maur-des-fossés, France) or Kerr’s Pulp Canal Sealer™ (PCS, Kerr, Salerno, Italy) and grown for 3, 7 and 10 days. A4 cells were grown to confluence in DMEM supplemented with 10% FCS in absence of sealers (control, Group 1) or presence of 1 solid disk BR (Group 2) or PCS (Group 3) and were used to evaluate cytotoxicity, odontoblast markers expression and to test whether these sealers have potential to induce mineralization in the absence of inducers by Von Kossa staining.
Confluent A4 stem cells were switched to DMEM supplemented with 1% FCS containing β-glycerophosphate (β-GP) (5 mM), ascorbic acid (AA) (50 μg/mL) and dexamethasone (DEX) (10 −7 M) in a 37 °C humidified incubator in an atmosphere of 5% CO 2 to induce the odontogenic program , for evaluating whether these sealers have an impact on osteo-odontogenic differentiation potential of pulpal A4 stem cells via Von Kossa staining. Untreated steady state cells were negative controls, while untreated cells induced toward the odontogenic program were positive controls. The chemicals were from Sigma (Sigma–Aldrich, St. Louis, MO, USA) unless otherwise specified. All experiments were run in triplicates.
2.2
Composition and preparation of the root canal sealers
The BR powder is based on tricalcium silicate, zirconium oxide and excipients. The powder phase of BR was mixed with liquid phase consisting of calcium chloride and excipients. PCS was composed from the mixture of 41.25% ZnO, 20.25% silver, 3.71% thymol iodide and 19.54% eugenol. Silicone molds were used to standardize the weight of prepared disks. To synchronize the setting times of sealers, the silicone molds were stored in an incubator with a humidified 5% CO 2 and 95% air atmosphere for 24 h at 37 °C. After setting, the mean weights of the BR or PCS disks were between 0.22 and 0.23 g. Various numbers of disks (1, 2, 3 and 4) were first tested to determine the dose effect on cell viability. Based on the first data, the 1 disk was finally retained for the following experiments. Disks were washed twice with phosphate-buffered solution, dried under laminar flow for 24 h at room temperature, and sterilized by ultraviolet (UV) light for 1 h before being added to cell cultures.
2.3
Trypan Blue (TB) exclusion assay
To evaluate cell viability in the three experimental groups at day 7 and 10, Trypan Blue colorimetric test was used as described in previous study .
2.4
Immunocytochemical and Von Kossa experiments
For cell surface detection of type 1 collagen, bone sialoprotein (BSP) and dentin matrix protein-1 (DMP1), cells grown on glass coverslips were washed with cold phosphate buffered saline (PBS) supplemented with 1 mM Ca 2+ and 1 mM Mg 2+ (PBS/Ca/Mg) and fixed with 3.6% formaldehyde in PBS. Cells were incubated for 1 h at room temperature with the primary rabbit polyclonal antibodies for BSP, DMP1 (10 μg/ml from Dr Larry Fischer, NIH, Bethesda, USA) and type I collagen (Chemicon, Temecula, CA, USA) in blocking buffer (PBS enriched with 2% fetal calf serum) and then with the secondary antibody AlexaFluor488-conjugated goat-anti-rabbit immunoglobulins (1 μg/ml; Molecular Probes, Eugene, OR, USA). Cell preparations were mounted under coverslips with Fluoromount G (Fisher Scientific, Pittsburgh, PA, USA) and analyzed by wide-field indirect immunofluorescence using a Nikon Eclipse TE-2000 E-inverted microscope ×40 oil-immersion objective (NA = 1.3; Nikon, Tokyo, Japan), and a black and white charge-coupled device (CCD) Photometrics CoolSnap HQ2 camera (Photometrics, Tucson, AZ) controlled by NIS elements.
Matrix mineralization was evaluated by Von Kossa staining as described previously to evaluate whether these sealers have potential to induce mineralization in the absence of inducers and have effect on osteo-odontogenic differentiation potential of pulpal A4 stem cells in the presence of inducers. Stained phosphate deposits were observed and pictured using a light microscope (Nikon TS100, Melville, NY, USA).
2.5
Statistical analysis
All experiments were repeated at least 3 times. Cytotoxicity data displayed normal distribution according Shapiro–Wilk test and statistical significance ( p < 0.05) was evaluated by one-way analysis of variance and Student’s t -test using SPSS software (version 21.0; SPSS, Chicago, IL).
2
Materials and methods
2.1
Dental pulp cells: cell culture and differentiation
A4 pulpal cells, which have been derived from first molar tooth germs of E18 mouse embryos transgenic for a recombinant plasmid adeno-SV40, were cultured in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal calf serum (FCS) as described by Priam et al. . The growth medium was changed every 3 or 4 days. A4 cells were plated (3 × 10 5 cells) in the presence of 1 to 4 solid disks of Bioroot™ RCS (BR, Septodont, Saint-Maur-des-fossés, France) or Kerr’s Pulp Canal Sealer™ (PCS, Kerr, Salerno, Italy) and grown for 3, 7 and 10 days. A4 cells were grown to confluence in DMEM supplemented with 10% FCS in absence of sealers (control, Group 1) or presence of 1 solid disk BR (Group 2) or PCS (Group 3) and were used to evaluate cytotoxicity, odontoblast markers expression and to test whether these sealers have potential to induce mineralization in the absence of inducers by Von Kossa staining.
Confluent A4 stem cells were switched to DMEM supplemented with 1% FCS containing β-glycerophosphate (β-GP) (5 mM), ascorbic acid (AA) (50 μg/mL) and dexamethasone (DEX) (10 −7 M) in a 37 °C humidified incubator in an atmosphere of 5% CO 2 to induce the odontogenic program , for evaluating whether these sealers have an impact on osteo-odontogenic differentiation potential of pulpal A4 stem cells via Von Kossa staining. Untreated steady state cells were negative controls, while untreated cells induced toward the odontogenic program were positive controls. The chemicals were from Sigma (Sigma–Aldrich, St. Louis, MO, USA) unless otherwise specified. All experiments were run in triplicates.
2.2
Composition and preparation of the root canal sealers
The BR powder is based on tricalcium silicate, zirconium oxide and excipients. The powder phase of BR was mixed with liquid phase consisting of calcium chloride and excipients. PCS was composed from the mixture of 41.25% ZnO, 20.25% silver, 3.71% thymol iodide and 19.54% eugenol. Silicone molds were used to standardize the weight of prepared disks. To synchronize the setting times of sealers, the silicone molds were stored in an incubator with a humidified 5% CO 2 and 95% air atmosphere for 24 h at 37 °C. After setting, the mean weights of the BR or PCS disks were between 0.22 and 0.23 g. Various numbers of disks (1, 2, 3 and 4) were first tested to determine the dose effect on cell viability. Based on the first data, the 1 disk was finally retained for the following experiments. Disks were washed twice with phosphate-buffered solution, dried under laminar flow for 24 h at room temperature, and sterilized by ultraviolet (UV) light for 1 h before being added to cell cultures.
2.3
Trypan Blue (TB) exclusion assay
To evaluate cell viability in the three experimental groups at day 7 and 10, Trypan Blue colorimetric test was used as described in previous study .
2.4
Immunocytochemical and Von Kossa experiments
For cell surface detection of type 1 collagen, bone sialoprotein (BSP) and dentin matrix protein-1 (DMP1), cells grown on glass coverslips were washed with cold phosphate buffered saline (PBS) supplemented with 1 mM Ca 2+ and 1 mM Mg 2+ (PBS/Ca/Mg) and fixed with 3.6% formaldehyde in PBS. Cells were incubated for 1 h at room temperature with the primary rabbit polyclonal antibodies for BSP, DMP1 (10 μg/ml from Dr Larry Fischer, NIH, Bethesda, USA) and type I collagen (Chemicon, Temecula, CA, USA) in blocking buffer (PBS enriched with 2% fetal calf serum) and then with the secondary antibody AlexaFluor488-conjugated goat-anti-rabbit immunoglobulins (1 μg/ml; Molecular Probes, Eugene, OR, USA). Cell preparations were mounted under coverslips with Fluoromount G (Fisher Scientific, Pittsburgh, PA, USA) and analyzed by wide-field indirect immunofluorescence using a Nikon Eclipse TE-2000 E-inverted microscope ×40 oil-immersion objective (NA = 1.3; Nikon, Tokyo, Japan), and a black and white charge-coupled device (CCD) Photometrics CoolSnap HQ2 camera (Photometrics, Tucson, AZ) controlled by NIS elements.
Matrix mineralization was evaluated by Von Kossa staining as described previously to evaluate whether these sealers have potential to induce mineralization in the absence of inducers and have effect on osteo-odontogenic differentiation potential of pulpal A4 stem cells in the presence of inducers. Stained phosphate deposits were observed and pictured using a light microscope (Nikon TS100, Melville, NY, USA).
2.5
Statistical analysis
All experiments were repeated at least 3 times. Cytotoxicity data displayed normal distribution according Shapiro–Wilk test and statistical significance ( p < 0.05) was evaluated by one-way analysis of variance and Student’s t -test using SPSS software (version 21.0; SPSS, Chicago, IL).
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