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Effects of two fast-setting pulp-capping materials on cell viability and osteogenic differentiation in human dental pulp stem cells: An in vitro study
Archives of Oral Biology 100 (2019) 100–105
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Effects of two fast-setting pulp-capping materials on cell viability and osteogenic differentiation in human dental pulp stem cells: An in vitro study Yan Suna,b,1, Jun Liuc,1, Tao Luoa, Ya Shend, Ling Zoua,
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a State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Conservative Dentistry and Endodontics, West China School and Hospital of Stomatology, Sichuan University, Chengdu, 610041, China b Department of Conservative Dentistry and Endodontics, School & Hospital of Stomatology, Wenzhou Medical University, Wenzhou, 325027, China c State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China School and Hospital of Stomatology, Sichuan University, Chengdu, 610041, China d Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, Canada
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Keywords: Biodentine (BD) iRoot fast set root repair material (FS) Pulp capping Cell viability Osteogenic differentiation Human dental pulp stem cells (hDPSCs)
Objective: The purpose of this study was to compare the effects of two fast-setting pulp-capping materials, Biodentine (BD) and iRoot Fast Set (FS) root repair material, on the attachment, viability, migration, and differentiation of human dental pulp stem cells (hDPSCs). Methods: A comparative study was conducted between BD and FS material disks. Scanning electron microscope (SEM) images were used to observe the attachment of hDPSCs on the disks. A live/dead assay was used to assess the cell viability. Transwell assay was performed to study cell migration. Cell differentiation was determined by quantitative real-time polymerase chain reaction (qRT-PCR) for the analysis of osteogenic differentiation gene expression: alkaline phosphatase (ALP), collagen type I (COL1) and osteocalcin (OCN). Results: SEM images indicated that hDPSCs showed a well-spreading morphology on both BD and FS disks. FS significantly increased the proliferation and migration of hDPSCs on day 7 (P＜0.05). Neither BD nor FS promoted the expression of osteogenic genes during the observation period. Conclusions: BD and FS both were beneficial to hDPSC attachment, and they had similar effects on cell osteogenic differentiation, whereas FS performed better than BD on hDPSCs proliferation and migration.
1. Introduction Direct capping of exposed, vital, painless pulps aims to maintain pulpal health, thereby allowing patients to retain teeth longer and at lower costs than alternative, more invasive interventions, such as root canal treatment (Schwendicke & Stolpe, 2014). Factors influencing the potential prognosis of directly capped pulps may be the state of the exposed pulp and its subsequent reaction as well as a potential bacterial contamination of (peri-)pulpal tissues (Schwendicke, Brouwer, Schwendicke, & Paris, 2016). Moreover, the capping materials play significant roles in the success of this treatment. Calcium hydroxide (CH) was the gold standard for pulp capping before mineral trioxide aggregate (MTA) was introduced as a dental material. The disadvantage of CH is its dissolution and failure to provide a long-term biological seal against bacterial infection (Komabashi, Zhu, Eberhart, & Imai, 2016). MTA has been recognised as a bioactive material with antibacterial
properties, good sealing ability and biocompatibility (Roberts, Toth, Berzins, & Charlton, 2008; Torabinejad & Chivian, 1999), but the discoloration potential, the poor-handling property and the long setting time may be its drawbacks in application (Parirokh & Torabinejad, 2010a, 2010b). Thus the novel materials and strategies that can optimize the performance without compromising the bioactivity of cements attract wide attention (Dubey, Rajan, Bello, Min, & Rosa, 2017). Biodentine (BD) was formulated by MTA-based technology and improved the handling properties to some extent (Malkondu, Kazandaǧ, & Kazazoǧlu, 2014). It is noteworthy that BD presented a relatively short setting time (initial setting time is 9–12 minutes) compared with MTA (initial setting time is 2 h and 45 min) (Malkondu et al., 2014; Torabinejad, Hong, McDonald, & Pitt Ford, 1995). This can be partially explained by the presence of calcium carbonate in BD, which acts as a nucleation site for calcium silicate hydrate, thereby reducing the duration of the induction period (Camilleri, Sorrentino, & Damidot,
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Corresponding author. E-mail addresses: [email protected] (Y. Sun), [email protected] (J. Liu), [email protected] (T. Luo), [email protected] (Y. Shen), [email protected] (L. Zou). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.archoralbio.2019.02.014 Received 16 October 2018; Received in revised form 31 January 2019; Accepted 19 February 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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2013). Since the first reporting of BD in 2008, several studies on its clinical application have been published (Laurent, Camps, De Méo, Déjou, & About, 2008). In 2013, Villat, Grosgogeat, Seux, and Farge (2013) performed a partial pulpotomy by BD, and there was no pain or other discomfort complaints throughout the 6-month follow-up. In addition, a dentin-bridge was formed and accompanied by a continuous root development. BD appears to be of great value in clinical operation. Another calcium silicate-based material, iRoot Fast Set (FS), was also introduced as a dental material to reduce chair-side time. FS is an insoluble, radiopaque, and aluminium-free material that does not shrink during setting (U.S. Food and Drug Administration, FDA). Previous studies found that the cells (L929 cells, MG63 cells or MC3T3E1, human periodontal ligament cells (hPDLCs)) attached to the surface of FS displayed extended cell morphology (Jiang, Zheng, Zhou, Gao, & Huang, 2014; Luo, Liu, Sun, Shen, & Zou, 2018; Lv et al., 2017). Moreover, iRoot FS eluate presents a favourable biocompatibility for promoting cellular proliferation and mineralization (Jiang et al., 2014; Luo et al., 2018; Lv et al., 2017; Sun et al., 2017). Apart from the biological characteristics, FS exhibited a similar sealing ability with MTA (Shi, Zhang, Chen, Bao, & Guo, 2015). Guo et al. (2016) reported that iRoot FS had a faster setting time (initial setting time is 15.7–20.9 minutes) and hydrating process than the other bioceramic cements tested, including Endosequence Root Repair Material Putty (ERRM Putty), Gray MTA(G-MTA), White MTA(W-MTA), and its mechanical properties were similar to G-MTA and W-MTA. Understanding the cell-material interfacial activity is important as pulp-capping material comes into contact with the pulp cells or tissue (Lee et al., 2014; Zhang, Yang, & Fan, 2013). Although our previous study proved that eluates of iRoot FS could promote the proliferation, migration, and osteogenic differentiation of human dental pulp stem cells (hDPSCs) (Sun et al., 2017), there is no evidence on the potential effect of iRoot FS on hDPSCs by cell-material direct contact tests, which could mimic the clinical practice of direct pulp capping to a great extent. Besides, the cytotoxicity data are also needed for the complete risk assessment of these novel pulp-capping materials. For these reasons, we conducted this study to compare the cell viability and osteogenic differentiation of the two fast-setting materials in hDPSCs.
(FBS; Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO), and then incubated at 37 °C in 5% CO2. When the cell colony reached 80% confluence, the cells were digested for subculture. Then, the medium was changed every 3 days. At passage 6 hDPSCs at passage 6 were identified by flow cytometry and were used for the following experiments. All the following experiments were repeated three times. 2.3. Flow cytometry analysis As briefly described in our published paper (Sun et al., 2017), the hDPSCs were collected, and we performed a flow-cytometric analysis of specific antigens (CD29, CD44, CD105, CD31, CD34, and CD45). The samples were tested on a flow cytometer (Beckman Coulter, FC500, FL), and the data were analyzed using FlowJo software (Tree Star, San Carlos, CA). 2.4. Cell attachment To observe the morphology of cell attachment, hDPSCs were seeded onto the disks (n = 3) at an initial density of 1.5 × 105 cells/well. hDPSCs were grown for 48 h, then the cell-adhered samples were fixed with 2.5% glutaraldehyde overnight. After this period the samples were treated with a series of graded ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15 min each and examined by scanning electron microscope ([SEM]; FEI, Hillsboro, OR). 2.5. Live/dead assay A live/dead Viability Kit (Invitrogen) was used to study cell attachment and viability. Cell suspensions were seeded on BD and FS disks (n = 3) at 1.5 × 105 cells/well for 1, 3, and 7 days. The culture medium was refreshed every 3 days. The samples were incubated with ethidium homodimer (4 μM) and calcein-AM (2 μM) for 20 min at room temperature. Live cells were stained green, and dead cells were stained red through fluorescence microscopy (OlympusBX63, Japan). Six pictures were acquired for each sample, and they were analysed by ImagePro Plus software. In each image, live cells (Nl) and dead cells (Nd) were counted. The live cell density is the number of live cells (Nl) per image area. The live cell percentage was calculated as Nl /(Nl+ Nd).
2. Materials and methods 2.1. Material preparation
2.6. Transwell migration assay BD (Septodont, France) and iRoot FS (Innovative Bioceramix, Canada), were prepared according to the manufacturer’s instructions under sterile conditions. The premixed cement was placed into a sterile custom-made plastic cylindrical mold (1-mm thickness and 10-mm diameter) at 37 °C under 100% humidity until it was completely solidified. The samples were processed according to the previous study with modification (Jiang et al., 2014). The disks were briefly placed in 24-well tissue culture polystyrene (TCPS) plates (Corning Inc., Corning, NY) and immersed in Dulbecco phosphate-buffered saline (DPBS) for 2 weeks; the medium was refreshed every day. Before cell seeding, the disks were immersed in Dulbecco modified Eagle medium (DMEM) for 1 day.
A two-chamber Transwell system (8 μM pore size and 6.5 mm diameter; Corning) was used for cell migration assay as previously described (D’Antò et al., 2010). BD and FS disks (n = 5) were placed in a lower chamber with 700 μL of serum-free DMEM for 24 h. hDPSCs (5 × 104 cells/well) suspended in 100 μL of serum-free DMEM were seeded into the upper chamber. In the control group, only serum-free DMEM was put into the lower chamber. After 24 h, the migrated cells were stained with 0.1% crystal violet for 20 min. 2.7. Real time reverse-transcriptase polymerase chain reaction (RT-PCR) The osteogenic differentiation-related genes, alkaline phosphatise (ALP), collagen type I (COL1), and osteocalcin (OCN), were analysed. hDPSCs were seeded on the disks (n = 5) and induced with osteogenic medium consisting of the growth medium plus 100 nM of dexamethasone, 10 mM of β-glycerophosphate, and 0.28 mM of ascorbic acid (Sigma-Aldrich). Cells with osteogenic medium only were blank controls. The medium was changed every 3 days. At days 1, 3, and 7 the ribonucleic acid (RNA) was collected and extracted for SYBR Green qRT-PCR (Takara, Otsu, Japan). The total RNA was extracted from cells using Trizol reagent (Life Technologies Inc, Gaithersburg, MD) as manufacturer’s instructions. After deoxyribonucleic acid (DNA) contamination was removed by PrimeScript reverse transcription (RT)
2.2. Cell culture This study was approved by the Institutional Review Board of West China Hospital of Stomatology of Sichuan University. The isolation of hDPSCs was performed according to the previously described methods (Gronthos, Mankani, & Brahim, 2000). Healthy pulp tissue was collected from extracted premolars for orthodontics. The dental pulps were isolated, minced and digested with 1 mg/mL type I collagenase (Gibco, Thermo Fisher Scientific, Waltham, MA) for 60 min in the shaking bath at 37℃. The digested pulp tissue was centrifuged and paved in a flask with DMEM (Hyclone), supplemented with 10% fetal bovine serum 101
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during the experimental period. The live cell density on FS disks was higher than that on BD disks on day 7 (P < 0.05).
Reagent Kit with genomic DNA Eraser (Perfect Real Time) (Takara Bio Inc., Otsu, Japan), the reverse transcription (RT) was conducted according to the kit instructions by incubating reaction mixture at 37℃ for 15 min and 85℃ for 5 s. The PCR cycles were performed as follows: 1 cycle at 95℃ for 30 s, and then 40 cycles at 95℃ for 10 s and 60℃ for 20 s). The gene expression levels were normalized to the level of glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The primer sequence is as follows (GAPDH as a reference gene): ALP,5'-GGACCATTCCCACGTCTTCAC-3' (forward) 5′−CCTTGTAGCCAGGCCCATTG-3′ (reverse); COL1,5′−CCCGGGTTTCAGAGACAACTTC-3′ (forward) 5′-TCCACATGCTTTATTCCAGCAATC-3′ (reverse); OCN,5′−CCCAGGCGCTACCTGTATCAA-3′ (forward) 5′-GGTCAGCCAACTCGTCACAGTC-3′ (reverse); GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse).
3.3. Cell migration Transwell assay was used to evaluate the effect of BD and FS on the mobility of hDPSCs (Fig. 3). Both FS and BD promoted cell migration compared with the blank control. FS enhanced cell migration more significantly than BD (P < 0.05). 3.4. Expression of osteogenic genes The expressions of osteogenic genes measured by qRT-PCR were plotted in Fig. 4.On day 1, COL1 and OCN were upregulated in the FS group compared with the BD group (P < 0.05). No difference was detected in the expression of the COL1 and OCN genes between BD, FS and the blank control group on day 7.The expression of ALP in the FS group was similar to that of the BD group and lower than that of the blank control group at examination time (P < 0.05).
2.8. Statistical analysis The data were analysed using the one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test with SPSS 21.0 (SPSS Inc., Chicago, IL), and P < 0.05 was considered statistically significant.
4. Discussion As mentioned previously, these cell-material direct contact tests were conducted to compare the cell viability and osteogenic differentiation of BD and FS in hDPSCs. Our flow cytometry results verified that hDPSCs used in the present study possess the characteristics of stem cells (Sun et al., 2017). SEM imaging was adopted to observe the material surface structure and cell morphology. The surfaces of BD and FS disks presented a crystalline structure that was consistent with previous studies (Attik et al., 2014; Jiang et al., 2014; Zhou et al., 2013). Calcium silicon-based materials require a moist environment to solidify and activate the bioactivity of the materials(such as forming crystals) (Gandolfi, Taddei, Tinti, & Prati, 2010; Gandolfi et al., 2011; Prati & Gandolfi, 2015). Both the physical structure and chemical elements influence cell attachment (Hamilton & Brunette, 2007). A previous study confirmed that calcium silicon-based materials immersed in DPBS showed remarkable bioactivity. This may due to the use of a phosphate solution, which makes the surface structure of the materials rich in apatite crystals (Gallego, Higuita, Garcia, Ferrell, & Hansford, 2008; Tay, Pashley, Rueggeberg, Loushine, & Weller, 2007). Calcium silicon-based materials with crystals could promote protein absorption, osteoblast adhesion and proliferation (Gandolfi, Taddei et al., 2010; Gandolfi et al., 2008). Although some studies pointed out that fresh materials boosted cell viability more than the aged counterpart, the materials disks were immersed in DPBS for 2 weeks in the present study to alleviate the toxicity of some harmful elements (Gandolfi et al., 2009; Gandolfi, Ciapetti et al., 2010). The favourable cell attachment morphology in our study is also similar to that of previous studies (Corral Nuñez, Bosomworth, Field, Whitworth, & Valentine, 2014; Jiang et al., 2014). Human gingival fibroblasts and MG-63 osteosarcomas seeded on BD disks presented wellextending morphologic conditions (Attik et al., 2014; Zhou et al., 2013). Luo et al. found that the expression of adhesion molecules was upregulated when cells were cultured in 0.2 mg/mL BD eluates (Luo, Li, & Kohli, 2014). Cell attachment is an important process in cell-material interaction; it coordinates closely with cell proliferation, migration and differentiation for tissue repair (Zhu, Yang, Zhang, Lei et al., 2014; Zhu, Yang, Zhang, & Peng, 2014). To further determine cell attachment and viability, a live/dead assay was introduced. On day 7 hDPSCs were found to be growing well on both materials, and the live cell percentage of FS was higher than BD, which resulted in FS being superior to BD on hDPSC proliferation. Cell migration is the key process to form a reparative dentin bridge, which recruits more cells to repair the damage. External factors, such as the physical and molecular characteristics of the environment, influence the cell migration activity (Justus, Leffler, Ruiz-Echevarria, &
3. Results 3.1. Cell attachment On both BD and FS material disks, hDPSCs presented good attachment morphology (Fig. 1). The hDPSCs were spindle-shaped or polygonal and stretched like slender cell processes onto the surface of the materials. 3.2. Cell viability In both groups, cells gathered without extending on the first day, then spread and proliferated on material disks on days 3 and 7 (Fig. 2). The live cell density and the live cell percentage continued to increase
Fig. 1. SEM micrograghs of cell attachment (n = 3). hDPSC attachment on BD disks A (2400×), B (10,000×). hDPSC attachment on FS disks C (2400×), D (10,000×). 102
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Fig. 2. hDPSC viability on BD disks (A–C), FS disks (D–F) at 1, 3 and 7 d. The live cell density (G), percentage of live cells (H) (mean ± sd; n = 3). Values with dissimilar letters are significantly different from each other (p < 0.05).
Fig. 3. hDPSC migration determined by Transwell Assay. Migrated cells were stained with crystal violet (A–C). Migrated cells were quantified for Transwell assay (D) (mean ± sd; n = 5). Values with dissimilar letters are significantly different from each other (p < 0.05).
with hydroxide (OH-). OH- was a nucleation site for apatite formation. When immersed into simulated body fluids (SBF), calcium from the cement and phosphorus from the solution were absorbed into the material surface to form calcium phosphate apatite deposits (Prati & Gandolfi, 2015). That is to say, calcium (Ca2+), phosphorus (P), silicon (Si), and hydroxide (OH-) all play roles in cell differentiation, and all of these are closely related to the bioactivity of calcium-silicon materials. As for osteogenic differentiation on material disks, some specific genes take part in the physiological activity. In the present study, the ALP, COL1 and OCN gene expression trend of FS was similar to BD, especially on days 3 and 7. Our results also showed that ALP expression in the FS and BD groups was lower than the blank control group at the detected time. ALP is the early marker of odontoblast development and is sensitive to elevated extracellular Ca2+ and P ion. An, Ling, Gao, and
Yang, 2014). Our results showed that compared with BD, the migrated numbers of hDPSCs were higher in FS, which means that FS-enhanced hDPSCs migrate more significantly. Also, it has been reported that hDPSCs cultured in iRoot BP Plus presented greater migration ability than that in MTA (Zhu, Yang, Zhang, Lei et al., 2014; Zhu, Yang, Zhang, Peng et al., 2014). The cellular migration ability to materials is in favour of repair process. To our knowledge, most of the published studies related to the cytocompatibility of calcium silicate-based materials used eluates of these materials. Few studies adopted the cell-material direct contact method because the component and surface structure of aged-materials disks are complicated. It is well-known that calcium (Ca2+) silicon-based materials solidify through hydration and then release ions. Ca2+ ions rapidly migrate and form portlandite (Ca (OH)2). Silicate combined 103
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Fig. 4. The expression of osteogenic genes of ALP, COL1 and OCN at 1d (A), 3d (B) and 7d (C) (mean ± sd; n = 5). *indicates significant differences for one certain gene at different groups (p < 0.05).
Ethics approval
Xiao (2012) observed that ALP was downregulated, whereas mineralisation continued, followed by the increase in Ca2+ and P ions. In this condition, no positive linear relationship between ALP gene expression and the degree of extracellular mineralisation was considered. Coincidentally, Zanini, Sautier, Berdal, and Simon (2012) induced osteoblasts in mineralisation medium with BD eluate and found that the ALP expression was downregulated. They contributed the behaviour to the maturation stage and secretion activity of odontoblasts. The trend of ALP expression in our study was consistent with the two results mentioned previously. Additionally, the surface chemistry and topography of calcium silicon-based materials will change with time when immersed in SBF (Gandolfi et al., 2009). Because so many dynamic elements participate in mineralisation, the expression of osteogenic genes fluctuated. The relationship among them and the intrinsic mechanism need to be further studied. This study was conducted for only 1 week. That is, indeed, a short time. However, the cell-material interfacial activity at the early contact stage was favourable, including cell attachment, proliferation, and migration, which facilitated subsequent tissue repair. The mineralisation may need a longer observation time to understand about the whole process, and, especially, reparative dentine formation in vivo experiments need further research. In conclusion, BD and FS both were beneficial to hDPSC attachment, and they had a similar effect on cell osteogenic differentiation. Moreover, FS appeared to be better on cell viability and migration ability than BD.
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Conflict of interests The authors declare that they have no conflict of interests.
Funding This work described in this manuscript was supported by National Natural Science Foundation of China (NSFC) Grant No. 81201379 and 81470722 (JL).
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26(10), 974–992. https://doi.org/10.1016/j.dental.2010.06.002. Gandolfi, M. G., Taddei, P., Tinti, A., & Prati, C. (2010). Apatite-forming ability (bioactivity) of ProRoot MTA. International Endodontic Journal, 43(10), 917–929. https:// doi.org/10.1111/j.1365-2591.2010.01768.x. Gandolfi, M. G., Taddei, P., Tinti, A., De Dorigo, E. S., Rossi, P. L., & Prati, C. (2010). Kinetics of apatite formation on a calcium-silicate cement for root-end filling during ageing in physiological-like phosphate solutions. Clinical Oral Investigation, 14(6), 659–668. https://doi.org/10.1007/s00784-009-0356-3. Gronthos, S., Mankani, M., & Brahim, J. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America, 97, 13625–13630. Retrieved from http://www.pnas.org/ content/97/25/13625.short. Guo, Y., Du, T., Li, H., Shen, Y., Mobuchon, C., Hieawy, A., ... Haapasalo, M. (2016). Physical properties and hydration behavior of a fast-setting bioceramic endodontic material. BMC Oral Health, 16(1), 23. https://doi.org/10.1186/s12903-016-0184-1. Hamilton, D. W., & Brunette, D. M. (2007). The effect of substratum topography on osteoblast adhesion mediated signal transduction and phosphorylation. Biomaterials, 28(10), 1806–1819. https://doi.org/10.1016/j.biomaterials.2006.11.041. Jiang, Y., Zheng, Q., Zhou, X., Gao, Y., & Huang, D. (2014). A comparative study on root canal repair materials: A cytocompatibility assessment in L929 and MG63 cells. The Scientific World Journal, 2014, 463826. https://doi.org/10.1155/2014/463826. Justus, C. R., Leffler, N., Ruiz-Echevarria, M., & Yang, L. V. (2014). In vitro cell migration and invasion assays. Journal of Visualized Experiments, 88, 1–8. https://doi.org/10. 3791/51046. Komabashi, T., Zhu, Q., Eberhart, R., & Imai, Y. (2016). Current status of direct pulpcapping materials for permanent teeth. Dental Materials Journal, 35(1), 1–12. https:// doi.org/10.4012/dmj.2015-013. Laurent, P., Camps, J., De Méo, M., Déjou, J., & About, I. (2008). Induction of specific cell responses to a Ca3SiO5-based posterior restorative material. Dental Materials, 24(11), 1486–1494. https://doi.org/10.1016/j.dental.2008.02.020. Lee, B. N., Lee, K. N., Koh, J. T., Min, K. S., Chang, H. S., Hwang, I. N., ... Oh, W. M. (2014). Effects of 3 endodontic bioactive cements on osteogenic differentiation in mesenchymal stem cells. Journal of Endodontics, 40(8), 1217–1222. https://doi.org/ 10.1016/j.joen.2014.01.036. Luo, T., Liu, J., Sun, Y., Shen, Y., & Zou, L. (2018). Cytocompatibility of biodentine and iRoot FS with human periodontal ligament cells: An in vitro study. International Endodontic Journal, 1–10. https://doi.org/10.1111/iej.12889. Luo, Z., Li, D., & Kohli, M. (2014). Effect of biodentine on the proliferation, migration and adhesion of human dental pulp stem cells. Journal of Dentistry, 42(4), 490–497. https://doi.org/10.1016/j.jdent.2013.12.011. Lv, F., Zhu, L., Zhang, J., Yu, J., Cheng, X., & Peng, B. (2017). Evaluation of the in vitro biocompatibility of a new fast-setting ready-to-use root filling and repair material. International Endodontic Journal, 50, 540–548. https://doi.org/10.1111/iej.12661. Malkondu, Ö., Kazandaǧ, M. K., & Kazazoǧlu, E. (2014). A review on biodentine, a contemporary dentine replacement and repair material. BioMed Research International, 2014. https://doi.org/10.1155/2014/160951. Parirokh, M., & Torabinejad, M. (2010a). Mineral trioxide aggregate: A comprehensive literature review-part I: Chemical, physical, and antibacterial properties. Journal of Endodontics, 36(1), 16–27. https://doi.org/10.1016/j.joen.2009.09.006. Parirokh, M., & Torabinejad, M. (2010b). Mineral trioxide aggregate: A comprehensive literature review-part III: Clinical applications, drawbacks, and mechanism of action. Journal of Endodontics, 36(3), 400–413. https://doi.org/10.1016/j.joen.2009.09.009.
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Effects of two fast-setting pulp-capping materials on cell viability and osteogenic differentiation in human dental pulp stem cells: An in vitro study Yan Suna,b,1, Jun Liuc,1, Tao Luoa, Ya Shend, Ling Zoua,
T
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a State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Conservative Dentistry and Endodontics, West China School and Hospital of Stomatology, Sichuan University, Chengdu, 610041, China b Department of Conservative Dentistry and Endodontics, School & Hospital of Stomatology, Wenzhou Medical University, Wenzhou, 325027, China c State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Orthodontics, West China School and Hospital of Stomatology, Sichuan University, Chengdu, 610041, China d Division of Endodontics, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, British Columbia, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Biodentine (BD) iRoot fast set root repair material (FS) Pulp capping Cell viability Osteogenic differentiation Human dental pulp stem cells (hDPSCs)
Objective: The purpose of this study was to compare the effects of two fast-setting pulp-capping materials, Biodentine (BD) and iRoot Fast Set (FS) root repair material, on the attachment, viability, migration, and differentiation of human dental pulp stem cells (hDPSCs). Methods: A comparative study was conducted between BD and FS material disks. Scanning electron microscope (SEM) images were used to observe the attachment of hDPSCs on the disks. A live/dead assay was used to assess the cell viability. Transwell assay was performed to study cell migration. Cell differentiation was determined by quantitative real-time polymerase chain reaction (qRT-PCR) for the analysis of osteogenic differentiation gene expression: alkaline phosphatase (ALP), collagen type I (COL1) and osteocalcin (OCN). Results: SEM images indicated that hDPSCs showed a well-spreading morphology on both BD and FS disks. FS significantly increased the proliferation and migration of hDPSCs on day 7 (P＜0.05). Neither BD nor FS promoted the expression of osteogenic genes during the observation period. Conclusions: BD and FS both were beneficial to hDPSC attachment, and they had similar effects on cell osteogenic differentiation, whereas FS performed better than BD on hDPSCs proliferation and migration.
1. Introduction Direct capping of exposed, vital, painless pulps aims to maintain pulpal health, thereby allowing patients to retain teeth longer and at lower costs than alternative, more invasive interventions, such as root canal treatment (Schwendicke & Stolpe, 2014). Factors influencing the potential prognosis of directly capped pulps may be the state of the exposed pulp and its subsequent reaction as well as a potential bacterial contamination of (peri-)pulpal tissues (Schwendicke, Brouwer, Schwendicke, & Paris, 2016). Moreover, the capping materials play significant roles in the success of this treatment. Calcium hydroxide (CH) was the gold standard for pulp capping before mineral trioxide aggregate (MTA) was introduced as a dental material. The disadvantage of CH is its dissolution and failure to provide a long-term biological seal against bacterial infection (Komabashi, Zhu, Eberhart, & Imai, 2016). MTA has been recognised as a bioactive material with antibacterial
properties, good sealing ability and biocompatibility (Roberts, Toth, Berzins, & Charlton, 2008; Torabinejad & Chivian, 1999), but the discoloration potential, the poor-handling property and the long setting time may be its drawbacks in application (Parirokh & Torabinejad, 2010a, 2010b). Thus the novel materials and strategies that can optimize the performance without compromising the bioactivity of cements attract wide attention (Dubey, Rajan, Bello, Min, & Rosa, 2017). Biodentine (BD) was formulated by MTA-based technology and improved the handling properties to some extent (Malkondu, Kazandaǧ, & Kazazoǧlu, 2014). It is noteworthy that BD presented a relatively short setting time (initial setting time is 9–12 minutes) compared with MTA (initial setting time is 2 h and 45 min) (Malkondu et al., 2014; Torabinejad, Hong, McDonald, & Pitt Ford, 1995). This can be partially explained by the presence of calcium carbonate in BD, which acts as a nucleation site for calcium silicate hydrate, thereby reducing the duration of the induction period (Camilleri, Sorrentino, & Damidot,
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Corresponding author. E-mail addresses: [email protected] (Y. Sun), [email protected] (J. Liu), [email protected] (T. Luo), [email protected] (Y. Shen), [email protected] (L. Zou). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.archoralbio.2019.02.014 Received 16 October 2018; Received in revised form 31 January 2019; Accepted 19 February 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.
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2013). Since the first reporting of BD in 2008, several studies on its clinical application have been published (Laurent, Camps, De Méo, Déjou, & About, 2008). In 2013, Villat, Grosgogeat, Seux, and Farge (2013) performed a partial pulpotomy by BD, and there was no pain or other discomfort complaints throughout the 6-month follow-up. In addition, a dentin-bridge was formed and accompanied by a continuous root development. BD appears to be of great value in clinical operation. Another calcium silicate-based material, iRoot Fast Set (FS), was also introduced as a dental material to reduce chair-side time. FS is an insoluble, radiopaque, and aluminium-free material that does not shrink during setting (U.S. Food and Drug Administration, FDA). Previous studies found that the cells (L929 cells, MG63 cells or MC3T3E1, human periodontal ligament cells (hPDLCs)) attached to the surface of FS displayed extended cell morphology (Jiang, Zheng, Zhou, Gao, & Huang, 2014; Luo, Liu, Sun, Shen, & Zou, 2018; Lv et al., 2017). Moreover, iRoot FS eluate presents a favourable biocompatibility for promoting cellular proliferation and mineralization (Jiang et al., 2014; Luo et al., 2018; Lv et al., 2017; Sun et al., 2017). Apart from the biological characteristics, FS exhibited a similar sealing ability with MTA (Shi, Zhang, Chen, Bao, & Guo, 2015). Guo et al. (2016) reported that iRoot FS had a faster setting time (initial setting time is 15.7–20.9 minutes) and hydrating process than the other bioceramic cements tested, including Endosequence Root Repair Material Putty (ERRM Putty), Gray MTA(G-MTA), White MTA(W-MTA), and its mechanical properties were similar to G-MTA and W-MTA. Understanding the cell-material interfacial activity is important as pulp-capping material comes into contact with the pulp cells or tissue (Lee et al., 2014; Zhang, Yang, & Fan, 2013). Although our previous study proved that eluates of iRoot FS could promote the proliferation, migration, and osteogenic differentiation of human dental pulp stem cells (hDPSCs) (Sun et al., 2017), there is no evidence on the potential effect of iRoot FS on hDPSCs by cell-material direct contact tests, which could mimic the clinical practice of direct pulp capping to a great extent. Besides, the cytotoxicity data are also needed for the complete risk assessment of these novel pulp-capping materials. For these reasons, we conducted this study to compare the cell viability and osteogenic differentiation of the two fast-setting materials in hDPSCs.
(FBS; Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO), and then incubated at 37 °C in 5% CO2. When the cell colony reached 80% confluence, the cells were digested for subculture. Then, the medium was changed every 3 days. At passage 6 hDPSCs at passage 6 were identified by flow cytometry and were used for the following experiments. All the following experiments were repeated three times. 2.3. Flow cytometry analysis As briefly described in our published paper (Sun et al., 2017), the hDPSCs were collected, and we performed a flow-cytometric analysis of specific antigens (CD29, CD44, CD105, CD31, CD34, and CD45). The samples were tested on a flow cytometer (Beckman Coulter, FC500, FL), and the data were analyzed using FlowJo software (Tree Star, San Carlos, CA). 2.4. Cell attachment To observe the morphology of cell attachment, hDPSCs were seeded onto the disks (n = 3) at an initial density of 1.5 × 105 cells/well. hDPSCs were grown for 48 h, then the cell-adhered samples were fixed with 2.5% glutaraldehyde overnight. After this period the samples were treated with a series of graded ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15 min each and examined by scanning electron microscope ([SEM]; FEI, Hillsboro, OR). 2.5. Live/dead assay A live/dead Viability Kit (Invitrogen) was used to study cell attachment and viability. Cell suspensions were seeded on BD and FS disks (n = 3) at 1.5 × 105 cells/well for 1, 3, and 7 days. The culture medium was refreshed every 3 days. The samples were incubated with ethidium homodimer (4 μM) and calcein-AM (2 μM) for 20 min at room temperature. Live cells were stained green, and dead cells were stained red through fluorescence microscopy (OlympusBX63, Japan). Six pictures were acquired for each sample, and they were analysed by ImagePro Plus software. In each image, live cells (Nl) and dead cells (Nd) were counted. The live cell density is the number of live cells (Nl) per image area. The live cell percentage was calculated as Nl /(Nl+ Nd).
2. Materials and methods 2.1. Material preparation
2.6. Transwell migration assay BD (Septodont, France) and iRoot FS (Innovative Bioceramix, Canada), were prepared according to the manufacturer’s instructions under sterile conditions. The premixed cement was placed into a sterile custom-made plastic cylindrical mold (1-mm thickness and 10-mm diameter) at 37 °C under 100% humidity until it was completely solidified. The samples were processed according to the previous study with modification (Jiang et al., 2014). The disks were briefly placed in 24-well tissue culture polystyrene (TCPS) plates (Corning Inc., Corning, NY) and immersed in Dulbecco phosphate-buffered saline (DPBS) for 2 weeks; the medium was refreshed every day. Before cell seeding, the disks were immersed in Dulbecco modified Eagle medium (DMEM) for 1 day.
A two-chamber Transwell system (8 μM pore size and 6.5 mm diameter; Corning) was used for cell migration assay as previously described (D’Antò et al., 2010). BD and FS disks (n = 5) were placed in a lower chamber with 700 μL of serum-free DMEM for 24 h. hDPSCs (5 × 104 cells/well) suspended in 100 μL of serum-free DMEM were seeded into the upper chamber. In the control group, only serum-free DMEM was put into the lower chamber. After 24 h, the migrated cells were stained with 0.1% crystal violet for 20 min. 2.7. Real time reverse-transcriptase polymerase chain reaction (RT-PCR) The osteogenic differentiation-related genes, alkaline phosphatise (ALP), collagen type I (COL1), and osteocalcin (OCN), were analysed. hDPSCs were seeded on the disks (n = 5) and induced with osteogenic medium consisting of the growth medium plus 100 nM of dexamethasone, 10 mM of β-glycerophosphate, and 0.28 mM of ascorbic acid (Sigma-Aldrich). Cells with osteogenic medium only were blank controls. The medium was changed every 3 days. At days 1, 3, and 7 the ribonucleic acid (RNA) was collected and extracted for SYBR Green qRT-PCR (Takara, Otsu, Japan). The total RNA was extracted from cells using Trizol reagent (Life Technologies Inc, Gaithersburg, MD) as manufacturer’s instructions. After deoxyribonucleic acid (DNA) contamination was removed by PrimeScript reverse transcription (RT)
2.2. Cell culture This study was approved by the Institutional Review Board of West China Hospital of Stomatology of Sichuan University. The isolation of hDPSCs was performed according to the previously described methods (Gronthos, Mankani, & Brahim, 2000). Healthy pulp tissue was collected from extracted premolars for orthodontics. The dental pulps were isolated, minced and digested with 1 mg/mL type I collagenase (Gibco, Thermo Fisher Scientific, Waltham, MA) for 60 min in the shaking bath at 37℃. The digested pulp tissue was centrifuged and paved in a flask with DMEM (Hyclone), supplemented with 10% fetal bovine serum 101
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during the experimental period. The live cell density on FS disks was higher than that on BD disks on day 7 (P < 0.05).
Reagent Kit with genomic DNA Eraser (Perfect Real Time) (Takara Bio Inc., Otsu, Japan), the reverse transcription (RT) was conducted according to the kit instructions by incubating reaction mixture at 37℃ for 15 min and 85℃ for 5 s. The PCR cycles were performed as follows: 1 cycle at 95℃ for 30 s, and then 40 cycles at 95℃ for 10 s and 60℃ for 20 s). The gene expression levels were normalized to the level of glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The primer sequence is as follows (GAPDH as a reference gene): ALP,5'-GGACCATTCCCACGTCTTCAC-3' (forward) 5′−CCTTGTAGCCAGGCCCATTG-3′ (reverse); COL1,5′−CCCGGGTTTCAGAGACAACTTC-3′ (forward) 5′-TCCACATGCTTTATTCCAGCAATC-3′ (reverse); OCN,5′−CCCAGGCGCTACCTGTATCAA-3′ (forward) 5′-GGTCAGCCAACTCGTCACAGTC-3′ (reverse); GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse).
3.3. Cell migration Transwell assay was used to evaluate the effect of BD and FS on the mobility of hDPSCs (Fig. 3). Both FS and BD promoted cell migration compared with the blank control. FS enhanced cell migration more significantly than BD (P < 0.05). 3.4. Expression of osteogenic genes The expressions of osteogenic genes measured by qRT-PCR were plotted in Fig. 4.On day 1, COL1 and OCN were upregulated in the FS group compared with the BD group (P < 0.05). No difference was detected in the expression of the COL1 and OCN genes between BD, FS and the blank control group on day 7.The expression of ALP in the FS group was similar to that of the BD group and lower than that of the blank control group at examination time (P < 0.05).
2.8. Statistical analysis The data were analysed using the one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test with SPSS 21.0 (SPSS Inc., Chicago, IL), and P < 0.05 was considered statistically significant.
4. Discussion As mentioned previously, these cell-material direct contact tests were conducted to compare the cell viability and osteogenic differentiation of BD and FS in hDPSCs. Our flow cytometry results verified that hDPSCs used in the present study possess the characteristics of stem cells (Sun et al., 2017). SEM imaging was adopted to observe the material surface structure and cell morphology. The surfaces of BD and FS disks presented a crystalline structure that was consistent with previous studies (Attik et al., 2014; Jiang et al., 2014; Zhou et al., 2013). Calcium silicon-based materials require a moist environment to solidify and activate the bioactivity of the materials(such as forming crystals) (Gandolfi, Taddei, Tinti, & Prati, 2010; Gandolfi et al., 2011; Prati & Gandolfi, 2015). Both the physical structure and chemical elements influence cell attachment (Hamilton & Brunette, 2007). A previous study confirmed that calcium silicon-based materials immersed in DPBS showed remarkable bioactivity. This may due to the use of a phosphate solution, which makes the surface structure of the materials rich in apatite crystals (Gallego, Higuita, Garcia, Ferrell, & Hansford, 2008; Tay, Pashley, Rueggeberg, Loushine, & Weller, 2007). Calcium silicon-based materials with crystals could promote protein absorption, osteoblast adhesion and proliferation (Gandolfi, Taddei et al., 2010; Gandolfi et al., 2008). Although some studies pointed out that fresh materials boosted cell viability more than the aged counterpart, the materials disks were immersed in DPBS for 2 weeks in the present study to alleviate the toxicity of some harmful elements (Gandolfi et al., 2009; Gandolfi, Ciapetti et al., 2010). The favourable cell attachment morphology in our study is also similar to that of previous studies (Corral Nuñez, Bosomworth, Field, Whitworth, & Valentine, 2014; Jiang et al., 2014). Human gingival fibroblasts and MG-63 osteosarcomas seeded on BD disks presented wellextending morphologic conditions (Attik et al., 2014; Zhou et al., 2013). Luo et al. found that the expression of adhesion molecules was upregulated when cells were cultured in 0.2 mg/mL BD eluates (Luo, Li, & Kohli, 2014). Cell attachment is an important process in cell-material interaction; it coordinates closely with cell proliferation, migration and differentiation for tissue repair (Zhu, Yang, Zhang, Lei et al., 2014; Zhu, Yang, Zhang, & Peng, 2014). To further determine cell attachment and viability, a live/dead assay was introduced. On day 7 hDPSCs were found to be growing well on both materials, and the live cell percentage of FS was higher than BD, which resulted in FS being superior to BD on hDPSC proliferation. Cell migration is the key process to form a reparative dentin bridge, which recruits more cells to repair the damage. External factors, such as the physical and molecular characteristics of the environment, influence the cell migration activity (Justus, Leffler, Ruiz-Echevarria, &
3. Results 3.1. Cell attachment On both BD and FS material disks, hDPSCs presented good attachment morphology (Fig. 1). The hDPSCs were spindle-shaped or polygonal and stretched like slender cell processes onto the surface of the materials. 3.2. Cell viability In both groups, cells gathered without extending on the first day, then spread and proliferated on material disks on days 3 and 7 (Fig. 2). The live cell density and the live cell percentage continued to increase
Fig. 1. SEM micrograghs of cell attachment (n = 3). hDPSC attachment on BD disks A (2400×), B (10,000×). hDPSC attachment on FS disks C (2400×), D (10,000×). 102
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Fig. 2. hDPSC viability on BD disks (A–C), FS disks (D–F) at 1, 3 and 7 d. The live cell density (G), percentage of live cells (H) (mean ± sd; n = 3). Values with dissimilar letters are significantly different from each other (p < 0.05).
Fig. 3. hDPSC migration determined by Transwell Assay. Migrated cells were stained with crystal violet (A–C). Migrated cells were quantified for Transwell assay (D) (mean ± sd; n = 5). Values with dissimilar letters are significantly different from each other (p < 0.05).
with hydroxide (OH-). OH- was a nucleation site for apatite formation. When immersed into simulated body fluids (SBF), calcium from the cement and phosphorus from the solution were absorbed into the material surface to form calcium phosphate apatite deposits (Prati & Gandolfi, 2015). That is to say, calcium (Ca2+), phosphorus (P), silicon (Si), and hydroxide (OH-) all play roles in cell differentiation, and all of these are closely related to the bioactivity of calcium-silicon materials. As for osteogenic differentiation on material disks, some specific genes take part in the physiological activity. In the present study, the ALP, COL1 and OCN gene expression trend of FS was similar to BD, especially on days 3 and 7. Our results also showed that ALP expression in the FS and BD groups was lower than the blank control group at the detected time. ALP is the early marker of odontoblast development and is sensitive to elevated extracellular Ca2+ and P ion. An, Ling, Gao, and
Yang, 2014). Our results showed that compared with BD, the migrated numbers of hDPSCs were higher in FS, which means that FS-enhanced hDPSCs migrate more significantly. Also, it has been reported that hDPSCs cultured in iRoot BP Plus presented greater migration ability than that in MTA (Zhu, Yang, Zhang, Lei et al., 2014; Zhu, Yang, Zhang, Peng et al., 2014). The cellular migration ability to materials is in favour of repair process. To our knowledge, most of the published studies related to the cytocompatibility of calcium silicate-based materials used eluates of these materials. Few studies adopted the cell-material direct contact method because the component and surface structure of aged-materials disks are complicated. It is well-known that calcium (Ca2+) silicon-based materials solidify through hydration and then release ions. Ca2+ ions rapidly migrate and form portlandite (Ca (OH)2). Silicate combined 103
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Fig. 4. The expression of osteogenic genes of ALP, COL1 and OCN at 1d (A), 3d (B) and 7d (C) (mean ± sd; n = 5). *indicates significant differences for one certain gene at different groups (p < 0.05).
Ethics approval
Xiao (2012) observed that ALP was downregulated, whereas mineralisation continued, followed by the increase in Ca2+ and P ions. In this condition, no positive linear relationship between ALP gene expression and the degree of extracellular mineralisation was considered. Coincidentally, Zanini, Sautier, Berdal, and Simon (2012) induced osteoblasts in mineralisation medium with BD eluate and found that the ALP expression was downregulated. They contributed the behaviour to the maturation stage and secretion activity of odontoblasts. The trend of ALP expression in our study was consistent with the two results mentioned previously. Additionally, the surface chemistry and topography of calcium silicon-based materials will change with time when immersed in SBF (Gandolfi et al., 2009). Because so many dynamic elements participate in mineralisation, the expression of osteogenic genes fluctuated. The relationship among them and the intrinsic mechanism need to be further studied. This study was conducted for only 1 week. That is, indeed, a short time. However, the cell-material interfacial activity at the early contact stage was favourable, including cell attachment, proliferation, and migration, which facilitated subsequent tissue repair. The mineralisation may need a longer observation time to understand about the whole process, and, especially, reparative dentine formation in vivo experiments need further research. In conclusion, BD and FS both were beneficial to hDPSC attachment, and they had a similar effect on cell osteogenic differentiation. Moreover, FS appeared to be better on cell viability and migration ability than BD.
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Conflict of interests The authors declare that they have no conflict of interests.
Funding This work described in this manuscript was supported by National Natural Science Foundation of China (NSFC) Grant No. 81201379 and 81470722 (JL).
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