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Dentin Conditioning with Bioactive Molecule Releasing Nanoparticle System Enhances Adherence, Viability, and Differentiation of Stem Cells from Apical Papilla
Basic Research—Biology
Dentin Conditioning with Bioactive Molecule Releasing Nanoparticle System Enhances Adherence, Viability, and Differentiation of Stem Cells from Apical Papilla Suja Shrestha, MSc, PhD, Calvin D. Torneck, DDS, MS, and Anil Kishen, BDS, MDS, PhD Abstract Introduction: Temporal-controlled bioactive molecule (BM) releasing systems allow the delivery of appropriate concentration of BM to enhance the interaction of stem cells to dentin matrix and subsequent odontogenic differentiation in regenerative endodontics. Objectives: The goal of this study was to evaluate the effect of dentin conditioning with 2 variants of dexamethasone (Dex) releasing chitosan nanoparticles (CSnp), (1) DexCSnpI (slow releasing) and (2) Dex-CSnpII (rapid releasing), on adherence, viability, and differentiation of stem cells from apical papilla (SCAP) on root dentin exposed to endodontic irrigants. Methods: Slab-shaped dentin specimens were prepared parallel to the root canal and treated with 5.25% sodium hypochlorite (NaOCl) for 10 minutes and/or 17% EDTA for 2 minutes. Dentin was then conditioned accordingly by (1) no nanoparticle treatment, (2) CSnp, (3) Dex-CSnpI, and (4) Dex-CSnpII. The effect of nanoparticle conditioning on SCAP viability was determined by cell count and a circularity index. SCAP adherence and viability on dentin were assessed by fluorescence and scanning electron microscopy and odontogenic differentiation by immunofluorescence. Results: SCAP on dentin treated with NaOCl alone or NaOCl as the last irrigant showed the least adherence, minimal cytoplasmic extensions, and higher circularity. SCAP adherence and viability on Dex-CSnpI and Dex-CSnpII conditioned dentin were increased and had a welldeveloped cytoplasmic matrix and significantly lower circularity (P < .05). SCAP cultured in Dex-CSnpII group expressed higher levels for DSPP and DMP-1 than in CSnp or Dex-CSnpI groups. Conclusions: Dex-CSnpI and Dex-CSnpII conditioning of dentin enhanced SCAP adherence and viability. Temporal-controlled release of Dex from Dex-CSnpII enhanced odontogenic differentiation of SCAP. This study highlighted the ability of dentin conditioning with temporal-controlled BM releasing nanoparticles to improve the local environment in regenerative endodontics. (J Endod 2016;-:1–7)
Key Words Cell adherence, chitosan nanoparticles, dentin, dexamethasone, odontogenic differentiation, regenerative endodontic procedures, stem cells from apical papilla
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egenerative endodontic procedures are biologically based procedures designed to replace damaged, diseased, or missing structures such as dentin and cells of pulp-dentin complex (1). Chemical disinfection of root canal and placement of intracanal medicaments are an essential prerequisite for regenerative endodontic procedures (2). However, their cytotoxicity and deleterious effects they have on dentin and apical tissue are a major concern. Root dentin exposed to endodontic irrigants or intracanal medicaments may alter the bioactivity of dentin and compromise the survival, adherence, proliferation, and differentiation of dental stem cells (3). Although root canal irrigation with EDTA appears to improve stem cell adherence and survival, irrigation with sodium hypochlorite (NaOCl) after EDTA negates its positive effect and accelerates dentinal erosion (4). Such changes to the structure of dentin may further compromise the interaction between dentin and neotissue to successful pulp regeneration. Chitosan is a cationic natural polymer with broad-spectrum antibacterial property, excellent biocompatibility and biodegradability characteristics (5). Its molecular structure is similar to extracellular matrix component and possesses a large number of free hydroxyl and amino groups that facilitate its chemical modification (5). The incorporation of chitosan nanoparticles (CSnp) and its derivatives on dentin matrix appears to enhance the mechanical properties of dentin and its resistance to bacterial enzymatic degradation (6). CSnp also possesses certain physicochemical characteristics such as nanoscale sizes, large surface area/mass ratio, and increased chemical reactivity that make it useful in local delivery of bioactive molecules (BMs) in regenerative procedures (7). Temporal-controlled release of bovine serum albumin from CSnp, for example, has been shown to increase the viability and alkaline phosphatase activity of stem cells from apical papilla (SCAP) (8). More recently, temporal-controlled release of dexamethasone (Dex) from CSnp was shown to enhance the odontogenic differentiation of SCAP in vitro. DexCSnpII (rapid releasing) variant has also been shown to be more effective in increasing biomineralization and expression of odontogenic markers in cultured SCAP than the Dex-CSnpI (slow releasing) variant (9). Strategies to improve the successful tissue engineering should allow (1) favorable interaction between stem cells and dentin matrix and (2) availability of BMs at an optimal time and concentration. It has been hypothesized that dentin conditioned with a temporal-controlled Dex releasing CSnp system can neutralize the detrimental effect of root canal irrigants on dentin and provide a bioactive extracellular matrix
From the Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Anil Kishen, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON M5G 1G6, Canada. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.01.026
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Figure 1. Representative images of SCAP adherence on chemically untreated/treated dentin specimens. SCAP were cultured on dentin specimens for 24 hours and stained with calcein-AM (bar: 100 mm) and phalloidin-TRITC/DAPI (bar: 20 mm). Few cells with cytoplasmic extensions were present in NaOCl/EDTA group. Significantly less numbers of cells with rounded morphology and devoid of cytoplasmic extensions were observed in NaOCl and NaOCl/EDTA/NaOCl groups.
that promotes SCAP adherence, viability, and differentiation. The purpose of this study was to evaluate the effect of a slow Dex releasing nanoparticle system (Dex-CSnpI) and a rapid Dex releasing nanoparticle system (Dex-CSnpII) in promoting SCAP adherence, viability, and odontogenic differentiation that was seeded on dentin exposed to endodontic irrigants.
Materials and Methods Chitosan, sodium tripolyphosphate, Dex, a-minimum essential medium, and EDTA were purchased from Sigma-Aldrich Inc (St Louis, MO). EDTA was used as a 17% solution. Fetal bovine serum, L-glutamine solution, and antibiotic:antimycotic solution were obtained from Gibco (Carlsbad, CA). NaOCl was purchased from Lavo Inc
Figure 2. Representative images of SCAP adherence on NaOCl/EDTA-treated dentin specimens without/with nanoparticle conditioning. SCAP were cultured on dentin specimens for 24 hours and stained with calcein-AM (bar: 100 mm) and phalloidin-TRITC/DAPI (bar: 10 mm). SCAP ultrastructure was also observed under scanning electron microscope (SEM). Cell adherence and morphology were improved by nanoparticle conditioning on dentin specimens. Unconditioned: NaOCl/EDTA-treated specimens.
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Basic Research—Biology (Montreal, Quebec, Canada) and used as a 5.25% solution. All the chemicals were of analytical grade (purity $ 95%).
Teeth Collection and Dentin Specimen Preparation Freshly extracted caries-free human teeth were collected for use in the study in accordance with University of Toronto Ethical Guidelines. After extraction all soft tissue was removed, and teeth were cleaned before storage in deionized water at 4 C. The crown of a tooth selected for use was removed at the level of cementoenamel junction, and the root was shortened by grinding to allow for the creation of uniform root sections 5 mm in length. Root dentin sections 1 mm in thickness were prepared from either side of the root canal by using a slow speed diamond-wafering blade (Buehler, Coventry, UK) under continuous water spray (10). Sections were then reduced by using wet emery paper, grit sizes 400, 800, and 4000, under a continuous water spray to produce dentin slabs 5 5 0.5 mm in size. Specimens were stored in fresh deionized water at 4 C until used. Chemical Treatment Protocol Dentin specimens were autoclaved for 20 minutes at 121 C before use, and all experimentation was performed under a laminar flow hood to assure asepsis (11). A total of 30 specimens were treated with NaOCl and/or EDTA to simulate clinical condition (12) by using 5 protocols (n = 6/group): group 1, no treatment (control); group 2, 10-minute treatment with NaOCl (NaOCl); group 3, 2-minute treatment with EDTA (EDTA); group 4, 10-minute treatment with NaOCl followed by 2-minute treatment with EDTA (NaOCl/EDTA); and group 5, 10minute treatment with NaOCl followed by 1-minute treatment with EDTA followed by 1-minute treatment with NaOCl (NaOCl/EDTA/ NaOCl). Specimens were thoroughly rinsed with sterile deionized water after each treatment protocol (3, 13, 14).
(PBS) and stained for fluorescence microscopy. Similarly, SCAP was cultured on 9 dentin specimens from each nanoparticle conditioned group and stained for fluorescence microscopy and prepared for scanning electron microscopy. SCAP Adherence and Viability on Nanoparticle Conditioned Dentin. Three specimens in each group were stained with calcein-AM (1 mmol/L in PBS; Life Technologies, Grand Island, NY) by directly adding the staining solution to cultured cells (18) and examined under a fluorescence microscope (Carl Zeiss, Gottingen, Germany). The number of cells adherent to dentin was calculated from 12 microscopic fields at 20 magnification. Images were captured and processed by using ImageJ software (National Institutes of Health, Bethesda, MD) and then saved as a 32-bit jpeg file. Cell counts were made from 8-bit images normalized to 50–500 pixels by using ImageJ. Cell viability was calculated as a percentage relative to the total number of cells attached to control dentin that was designated as 100% viability. SCAP Spreading on Nanoparticle Conditioned Dentin. Cell circularity was determined by analyzing cells in the same specimens that were stained with calcein-AM by using ImageJ. Circularity was assessed as 1, a perfect sphere or rounded cell, to 0, representing a
Nanoparticle Conditioning Nanoparticles used for dentin conditioning were synthesized as previously described (8, 9). Physical characterization and release kinetics of 2 variants Dex-CSnpI (slow releasing) and Dex-CSnpII (rapid releasing) have been previously published (9). Seventy-two of NaOCl/EDTA-treated dentin specimens were divided into 4 groups (n = 18) and treated accordingly with 300 mg/mL nanoparticles (8, 9) in deionized water (1 mL) for 12 hours. The 4 groups were designated as (1) unconditioned, (2) CSnp conditioned, (3) DexCSnpI conditioned, and (4) Dex-CSnpII conditioned. SCAP Culture A previously characterized SCAP cell line was used in all experiments (15). Cells were cultured and expanded by adding single-cell suspensions (1 105 cells) to a-minimum essential medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and 100 units/mL antibiotic:antimycotic solution. Cells were allowed to expand in culture to 70%–80% confluency and then released with 0.05% trypsin (Gibco, Carlsbad, CA). Cells from the third to fifth passages (15–17) were used in all experiments. Characterization of SCAP on Nanoparticle Conditioned Dentin Six dentin specimens from each chemical treatment group were placed in a 24-well plate, and 1 mL SCAP at a concentration of 1.0 105 was introduced into each well. The cells were allowed to grow at 37 C in a humidified incubator in an atmosphere of 5% CO2 for 24 hours. Specimens were washed with phosphate-buffered saline JOE — Volume -, Number -, - 2016
Figure 3. (A) Relative viability of SCAP in NaOCl/EDTA-treated dentin specimens without/with nanoparticle conditioning. Groups conditioned with nanoparticles demonstrated no significant change in viability relative to the unconditioned group. (B) Circularity of cells cultured in NaOCl/EDTAtreated dentin specimens without/with nanoparticle conditioning. Cells in the unconditioned group demonstrated 3-fold increase in circularity as compared with the control group and 2-fold increase in value as compared with the CSnp, Dex-CSnpI, or Dex-CSnpII conditioned groups. Data are presented as mean standard deviation of the mean (n = 3). **P < .05 versus the control group. ##P < .05 versus unconditioned group.
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Figure 4. (A) Scanning electron microscopic micrographs showing morphology and biomineralization of SCAP cultured for 2 weeks on NaOCl/EDTA-treated dentin specimens with/without nanoparticle conditioning. CSnp conditioned specimen without cells was studied as negative control to determine the effect of CSnp on mineralization. Mineralization was present in the conditioned groups, with the greatest degree present in the Dex-CSnpII conditioned group. Immunofluorescence localization of DSPP (B) and DMP-1 (C) during odontogenic differentiation. Fluorescein isothiocyanate (FITC)–conjugated antibody was used to detect the localization of the protein (green signal); all samples were counterstained with DAPI (blue signal). Scale bar = 25 mm.
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Basic Research—Biology non-circular or highly spread cell (19). Ten cells in each microscopic view were analyzed and averaged to assign circularity values. In addition, SCAP present on 3 specimens from each group were fixed for 5 minutes in 10% normal buffered formalin and thoroughly washed with PBS. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 20 minutes and washed again with PBS. Cells were stained with DAPI (Roche, Basel, Switzerland) for 5 minutes and then fluorescent phalloidin-tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC; Sigma-Aldrich) solution in PBS for 40 minutes at room temperature. They were washed several times with PBS to remove the unbound phalloidin conjugate. The excitation and emission wavelengths for Phalloidin-TRITC are 540 nm and 570 nm, respectively. The specimens were examined under a fluorescent microscope to visualize cytoskeletal actin. SCAP Ultrastructure on Nanoparticle Conditioned Dentin. Three specimens from each group were fixed overnight in 2.5% glutaraldehyde. These specimens were then dehydrated in graded solutions of ethanol and dried with hexamethylene disiloxane. After palladium coating, specimens were examined under a scanning electron microscope (Hitachi S-2500, Ibaragi, Japan) to assess SCAP ultrastructure.
SCAP Differentiation on Nanoparticle Conditioned Dentin The remaining 9 specimens in each nanoparticle conditioned group were placed in an 8-chamber polystyrene treated vessel of a tissue culture glass slide (BD Biosciences, Bedford, MA) and seeded with 2 104 cells in standard culture medium containing 50 mg/ mL ascorbic acid, 10 mmol/L b-glycerolphosphate, and 1.8 mmol/ L potassium dihydrogen phosphate (KH2PO4). Cultures were maintained at 37 C in 5% CO2 humidified incubator for 2 weeks. Comparisons were made between the 2 variants, Dex-CSnpI (slow releasing) and Dex-CSnpII (rapid releasing). All experiments were performed in triplicate before analysis. SCAP Biomineralization on Nanoparticle Conditioned Dentin. After 2 weeks, 3 specimens from each group were prepared for scanning electron microscopy as described earlier to assess biomineralization (20). Ten images were analyzed in a sequential manner for each sample. The effect of CSnp on biomineralization was studied by incubating CSnp conditioned dentin under the same conditions/duration without cells (negative control). Immunofluorescence Analysis on Nanoparticle Conditioned Dentin. At the end of 2 weeks, specimens were washed in PBS and fixed with 4% paraformaldehyde in PBS containing 0.1% Triton X-100 at 4 C for 30 minutes. After blocking the fixed specimens in 2.5% bovine serum albumin for 30 minutes at room temperature, 3 specimens from each group were incubated with the following primary antibodies, mouse anti-DMP-1 (Santa Cruz Biotechnologies, Santa Cruz, CA) or mouse anti-DSPP (Santa Cruz Biotechnologies), diluted 1:50 in blocking reagent at 37 C for 2 hours. After 3 washes in PBS/Tween 20, specimens were incubated with secondary antibody goat anti-mouse immunoglobulin G fluorescein isothiocyanate conjugate (Santa Cruz Biotechnologies), diluted 1:1500 in PBS at 37 C, for 1 hour. After rinsing, specimens were counterstained with DAPI and examined by using confocal laser scanning microscopy (Leica Microsystems, Richmond, IL). Statistical Analysis Viability and circularity results were subjected to one-way analysis of variance with Dunnett test. The difference between 2 groups was considered statistically significant if P < .05. JOE — Volume -, Number -, - 2016
Results Effect of Nanoparticle Conditioning on SCAP Characteristics SCAP Adherence. Figure 1 shows the typical fluorescence microscopic images of SCAP adherence on dentin. Numerous cells with a fibroblast-like morphology were homogenously distributed on the untreated dentin (control group) in a unidirectional manner. The number and morphology of cells present on EDTA only treated dentin were similar to those present in the control group, but cells were more flattened. Few cells with cytoplasmic extensions were present on dentin treated with NaOCl/EDTA. Significantly less numbers of cells with rounded morphology and devoid of cytoplasmic extensions were observed in NaOCl and NaOCl/EDTA/NaOCl groups. The morphology of cells after F-actin staining with phalloidin-TRITC was concurrent with the green fluorescent images stained with calcein-AM. Cells on dentin treated with NaOCl and NaOCl/EDTA/NaOCl displayed less ctytoplasmic F-actin compared with the rest of the groups. Nanoparticle conditioning on NaOCl/EDTA treated dentin resulted in significant increase in number of SCAP adherence compared with unconditioned dentin as shown in Figure 2. Cells in all the conditioned groups were uniformly distributed and displayed well-developed lamellipodia and filopodia. SCAP Ultrastructure. Cells on unconditioned dentin were round and devoid of cell extensions (Fig. 2). Cells on dentin conditioned with nanoparticles were flattened, appeared more numerous and more attached, and had well-developed cytoplasmic extensions, similar to what was seen in these same groups under fluorescence microscopy. SCAP Viability and Circularity. The quantitative relative viability of cells on dentin varied among groups (Fig. 3A). The viability of cells in the unconditioned group was decreased by 50% when compared with the untreated control group. Groups conditioned with nanoparticles demonstrated no significant change in viability relative to the unconditioned group. Cells in the unconditioned group demonstrated a 3-fold increase in circularity as compared with the control group (P < .05) and a 2-fold increase in value as compared with the CSnp, DexCSnpI, or Dex-CSnpII conditioned groups (P < .05) (Fig. 3B). Effect of Nanoparticle Conditioning on SCAP Differentiation SCAP Biomineralization. No evidence of mineralization could be seen under scanning electron microscopy in the unconditioned and negative control groups (Fig. 4A). Mineralization was present in the nanoparticle conditioned groups, with the greatest degree present in the Dex-CSnpII conditioned group. SCAP Immunofluorescence Localization of Dentin Matrix Protein-1 and Dentin Sialophosphoprotein. As shown in Figure 4B and C, expressions of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) were not observed in unconditioned group. Cells in the rest of the groups demonstrated a higher signal for cytoplasmic DSPP expression than for DMP-1 expression. Higher signals for DSPP and DMP-1 expression were seen in the cells of the Dex-CSnpI and Dex-CSnpII conditioned groups compared with the CSnp conditioned group, and signals for DSPP and DMP-1 were markedly higher in cells of the Dex-CSnpII group than in cells of the Dex-CSnpI group.
Discussion A treatment protocol that promotes adherence of dental stem cells to root canal dentin and enhances their odontogenic differentiation is essential in reestablishing dentinogenesis in a pulpless environment
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Basic Research—Biology (21). Dex has been identified as an odontogenic stimulant for SCAP in regenerative endodontics. Ideally this stimulation works best when the dentin is undamaged (13). Many commonly used root canal irrigants, especially those used in root canal disinfection, have a potential to damage dentin and therefore impede cell adherence and new tissue formation (22, 23). NaOCl, a highly effective root canal antimicrobial agent for example, alters the surface characteristics of dentin and impedes the odontogenic differentiation of pulp stem cells (22, 24, 25). Conditioning with CSnp or Dex-CSnp, as shown in this study, allowed cells to adhere and retain their viability on NaOCl/EDTA-treated dentin. Of the numerous chemicals currently used in root canal irrigation, only EDTA appears to promote adherence and cell viability on dentin (22, 23). These findings were evident in this study and have been cited several times in the literature (4, 13, 22, 23). It has been suggested that this effect is related to an increase in the surface wettability of dentin (26) and the release of dentinogenic BMs from the matrix, when dentin is chelated (27). The effect was noted in this study and supported by findings in previous studies. Furthermore, EDTA as a last irrigant would remove the smear layer and expose organic substrate for interaction with nanoparticles. The actual adherence of stem cells to dentin has been attributed to fibronectin, an adhesion protein that is present in the dentin matrix and preferentially adsorbed on a hydrophilic surface (28). Nanoparticle conditioning of NaOCl/EDTA-treated dentin appears to provide that hydrophilic surface (5) and hence has the ability to enhance the degree of adherence normally seen. In the current study, biomineralization and signals from biomarkers such as DSPP and DMP-1 were distinctly higher in SCAP cultured on Dex-CSnpI and Dex-CSnpII conditioned dentin than in SCAP cultured on CSnp conditioned dentin alone. DSPP is a matrix phosphoprotein strongly expressed in odontoblasts and a marker of biomineralization (29). DMP-1 is a non-collagenous matrix protein expressed in odontoblasts and regulates biomineralization during tooth development and repair (30). Because DSPP and DMP-1 are expressed in odontoblasts in the early stages of odontogenesis (31), the elevated expression of these 2 odontogenic proteins in Dex-CSnpI and DexCSnpII conditioned specimens suggested that Dex has been made available to the cells in an appropriate concentration and at a critical time. Furthermore, because cells conditioned with Dex-CSnpII (rapid release) showed higher expression of these odontogenic markers than cells conditioned with Dex-CSnpI (slow release), a temporal system of BM release may be advantageous when developing a regenerative endodontic protocol (9). The usefulness of temporal-controlled BM release in regenerative endodontics has been previously discussed (8, 9). The key advantages of temporal-control release over static availability are that it enhances stability of the BMs and allows release of a desired concentration of BM during a predetermined period of time. When used as a vehicle to deliver BMs, CSnp adds an antimicrobial and antibiofilm effect and greater hydrophilicity to the dentin, making it an ideal nanoparticle for use in regenerative procedures (32). Release of endogenous dentin matrix components, such as transforming growth factor–b, bone morphogenic protein–2, platelet-derived growth factor, has been shown to be advantageous in promoting stem cell proliferation and differentiation (27, 33, 34) and can potentially be delivered in a temporal manner. Growth factors which regulate specific components of the healing process that are currently being used as therapeutic agents to enhance repopulation of root surfaces denuded by periodontal disease (35) have a potential that can be readily applied to endodontic regenerative procedures. In conclusion, results of this investigation demonstrate that conditioning of dentin with CSnp, Dex-CSnpI, or Dex-CSnpII has a potential to 6
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reverse the deleterious effect of NaOCl treatment on stem cell viability and adherence to dentin in regenerative procedures. This may allow it to be used as a root canal disinfectant when regenerative procedures are contemplated. The study has also demonstrated that controlling the duration and concentration of essential BMs from nanoparticles enhanced odontogenic differentiation of SCAP, and such a manner of release may lead to a more favorable outcome when regenerative procedures are undertaken.
Acknowledgments The authors gratefully acknowledge the support of Dr Anibal Diogenes in this study. This study was supported by the University Start Up Fund, University of Toronto, Ontario, Canada and a Research Grant from the American Association of Endodontists (AAE) Foundation(2014). The authors deny any conflicts of interest related to this study.
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Basic Research—Biology 21. Huang GT, Yamaza T, Shea LD, et al. Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 2010;16:605–15. 22. Trevino EG, Patwardhan AN, Henry MA, et al. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. J Endod 2011;37:1109–15. 23. Ring KC, Murray PE, Namerow KN, et al. The comparison of the effect of endodontic irrigation on cell adherence to root canal dentin. J Endod 2008;34:1474–9. 24. Fu J, Wang YK, Yang MT, et al. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat Methods 2010;7:733–6. 25. Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–89. 26. Huang X, Zhang J, Huang C, et al. Effect of intracanal dentine wettability on human dental pulp cell attachment. Int Endod J 2012;45:346–53. 27. Begue-Kirn C, Smith AJ, Ruch JV, et al. Effects of dentin proteins, transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro. Int J Dev Biol 1992;36:491–503. 28. Wei J, Igarashi T, Okumori N, et al. Influence of surface wettability on competitive protein adsorption and initial attachment of osteoblasts. Biomed Mater 2009;4:045002.
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29. Qin C, Brunn JC, Cadena E, et al. The expression of dentin sialophosphoprotein gene in bone. J Dent Res 2002;81:392–4. 30. Almushayt A, Narayanan K, Zaki AE, et al. Dentin matrix protein 1 induces cytodifferentiation of dental pulp stem cells into odontoblasts. Gene Ther 2006;13: 611–20. 31. Ye L, MacDougall M, Zhang S, et al. Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 2004;279:19141–8. 32. Kishen A, Shi Z, Shrestha A, et al. An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. J Endod 2008;34: 1515–20. 33. Zhao S, Sloan AJ, Murray PE, et al. Ultrastructural localisation of TGF-beta exposure in dentine by chemical treatment. Histochem J 2000;32:489–94. 34. Roberts-Clark DJ, Smith AJ. Angiogenic growth factors in human dentine matrix. Arch Oral Biol 2000;45:1013–6. 35. Gamal AY, Mailhot JM. The effect of local delivery of PDGF-BB on attachment of human periodontal ligament fibroblasts to periodontitis-affected root surfaces– in vitro. J Clin Periodontol 2000;27:347–53.
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Dentin Conditioning with Bioactive Molecule Releasing Nanoparticle System Enhances Adherence, Viability, and Differentiation of Stem Cells from Apical Papilla Suja Shrestha, MSc, PhD, Calvin D. Torneck, DDS, MS, and Anil Kishen, BDS, MDS, PhD Abstract Introduction: Temporal-controlled bioactive molecule (BM) releasing systems allow the delivery of appropriate concentration of BM to enhance the interaction of stem cells to dentin matrix and subsequent odontogenic differentiation in regenerative endodontics. Objectives: The goal of this study was to evaluate the effect of dentin conditioning with 2 variants of dexamethasone (Dex) releasing chitosan nanoparticles (CSnp), (1) DexCSnpI (slow releasing) and (2) Dex-CSnpII (rapid releasing), on adherence, viability, and differentiation of stem cells from apical papilla (SCAP) on root dentin exposed to endodontic irrigants. Methods: Slab-shaped dentin specimens were prepared parallel to the root canal and treated with 5.25% sodium hypochlorite (NaOCl) for 10 minutes and/or 17% EDTA for 2 minutes. Dentin was then conditioned accordingly by (1) no nanoparticle treatment, (2) CSnp, (3) Dex-CSnpI, and (4) Dex-CSnpII. The effect of nanoparticle conditioning on SCAP viability was determined by cell count and a circularity index. SCAP adherence and viability on dentin were assessed by fluorescence and scanning electron microscopy and odontogenic differentiation by immunofluorescence. Results: SCAP on dentin treated with NaOCl alone or NaOCl as the last irrigant showed the least adherence, minimal cytoplasmic extensions, and higher circularity. SCAP adherence and viability on Dex-CSnpI and Dex-CSnpII conditioned dentin were increased and had a welldeveloped cytoplasmic matrix and significantly lower circularity (P < .05). SCAP cultured in Dex-CSnpII group expressed higher levels for DSPP and DMP-1 than in CSnp or Dex-CSnpI groups. Conclusions: Dex-CSnpI and Dex-CSnpII conditioning of dentin enhanced SCAP adherence and viability. Temporal-controlled release of Dex from Dex-CSnpII enhanced odontogenic differentiation of SCAP. This study highlighted the ability of dentin conditioning with temporal-controlled BM releasing nanoparticles to improve the local environment in regenerative endodontics. (J Endod 2016;-:1–7)
Key Words Cell adherence, chitosan nanoparticles, dentin, dexamethasone, odontogenic differentiation, regenerative endodontic procedures, stem cells from apical papilla
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egenerative endodontic procedures are biologically based procedures designed to replace damaged, diseased, or missing structures such as dentin and cells of pulp-dentin complex (1). Chemical disinfection of root canal and placement of intracanal medicaments are an essential prerequisite for regenerative endodontic procedures (2). However, their cytotoxicity and deleterious effects they have on dentin and apical tissue are a major concern. Root dentin exposed to endodontic irrigants or intracanal medicaments may alter the bioactivity of dentin and compromise the survival, adherence, proliferation, and differentiation of dental stem cells (3). Although root canal irrigation with EDTA appears to improve stem cell adherence and survival, irrigation with sodium hypochlorite (NaOCl) after EDTA negates its positive effect and accelerates dentinal erosion (4). Such changes to the structure of dentin may further compromise the interaction between dentin and neotissue to successful pulp regeneration. Chitosan is a cationic natural polymer with broad-spectrum antibacterial property, excellent biocompatibility and biodegradability characteristics (5). Its molecular structure is similar to extracellular matrix component and possesses a large number of free hydroxyl and amino groups that facilitate its chemical modification (5). The incorporation of chitosan nanoparticles (CSnp) and its derivatives on dentin matrix appears to enhance the mechanical properties of dentin and its resistance to bacterial enzymatic degradation (6). CSnp also possesses certain physicochemical characteristics such as nanoscale sizes, large surface area/mass ratio, and increased chemical reactivity that make it useful in local delivery of bioactive molecules (BMs) in regenerative procedures (7). Temporal-controlled release of bovine serum albumin from CSnp, for example, has been shown to increase the viability and alkaline phosphatase activity of stem cells from apical papilla (SCAP) (8). More recently, temporal-controlled release of dexamethasone (Dex) from CSnp was shown to enhance the odontogenic differentiation of SCAP in vitro. DexCSnpII (rapid releasing) variant has also been shown to be more effective in increasing biomineralization and expression of odontogenic markers in cultured SCAP than the Dex-CSnpI (slow releasing) variant (9). Strategies to improve the successful tissue engineering should allow (1) favorable interaction between stem cells and dentin matrix and (2) availability of BMs at an optimal time and concentration. It has been hypothesized that dentin conditioned with a temporal-controlled Dex releasing CSnp system can neutralize the detrimental effect of root canal irrigants on dentin and provide a bioactive extracellular matrix
From the Discipline of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Anil Kishen, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, ON M5G 1G6, Canada. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.01.026
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Figure 1. Representative images of SCAP adherence on chemically untreated/treated dentin specimens. SCAP were cultured on dentin specimens for 24 hours and stained with calcein-AM (bar: 100 mm) and phalloidin-TRITC/DAPI (bar: 20 mm). Few cells with cytoplasmic extensions were present in NaOCl/EDTA group. Significantly less numbers of cells with rounded morphology and devoid of cytoplasmic extensions were observed in NaOCl and NaOCl/EDTA/NaOCl groups.
that promotes SCAP adherence, viability, and differentiation. The purpose of this study was to evaluate the effect of a slow Dex releasing nanoparticle system (Dex-CSnpI) and a rapid Dex releasing nanoparticle system (Dex-CSnpII) in promoting SCAP adherence, viability, and odontogenic differentiation that was seeded on dentin exposed to endodontic irrigants.
Materials and Methods Chitosan, sodium tripolyphosphate, Dex, a-minimum essential medium, and EDTA were purchased from Sigma-Aldrich Inc (St Louis, MO). EDTA was used as a 17% solution. Fetal bovine serum, L-glutamine solution, and antibiotic:antimycotic solution were obtained from Gibco (Carlsbad, CA). NaOCl was purchased from Lavo Inc
Figure 2. Representative images of SCAP adherence on NaOCl/EDTA-treated dentin specimens without/with nanoparticle conditioning. SCAP were cultured on dentin specimens for 24 hours and stained with calcein-AM (bar: 100 mm) and phalloidin-TRITC/DAPI (bar: 10 mm). SCAP ultrastructure was also observed under scanning electron microscope (SEM). Cell adherence and morphology were improved by nanoparticle conditioning on dentin specimens. Unconditioned: NaOCl/EDTA-treated specimens.
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Basic Research—Biology (Montreal, Quebec, Canada) and used as a 5.25% solution. All the chemicals were of analytical grade (purity $ 95%).
Teeth Collection and Dentin Specimen Preparation Freshly extracted caries-free human teeth were collected for use in the study in accordance with University of Toronto Ethical Guidelines. After extraction all soft tissue was removed, and teeth were cleaned before storage in deionized water at 4 C. The crown of a tooth selected for use was removed at the level of cementoenamel junction, and the root was shortened by grinding to allow for the creation of uniform root sections 5 mm in length. Root dentin sections 1 mm in thickness were prepared from either side of the root canal by using a slow speed diamond-wafering blade (Buehler, Coventry, UK) under continuous water spray (10). Sections were then reduced by using wet emery paper, grit sizes 400, 800, and 4000, under a continuous water spray to produce dentin slabs 5 5 0.5 mm in size. Specimens were stored in fresh deionized water at 4 C until used. Chemical Treatment Protocol Dentin specimens were autoclaved for 20 minutes at 121 C before use, and all experimentation was performed under a laminar flow hood to assure asepsis (11). A total of 30 specimens were treated with NaOCl and/or EDTA to simulate clinical condition (12) by using 5 protocols (n = 6/group): group 1, no treatment (control); group 2, 10-minute treatment with NaOCl (NaOCl); group 3, 2-minute treatment with EDTA (EDTA); group 4, 10-minute treatment with NaOCl followed by 2-minute treatment with EDTA (NaOCl/EDTA); and group 5, 10minute treatment with NaOCl followed by 1-minute treatment with EDTA followed by 1-minute treatment with NaOCl (NaOCl/EDTA/ NaOCl). Specimens were thoroughly rinsed with sterile deionized water after each treatment protocol (3, 13, 14).
(PBS) and stained for fluorescence microscopy. Similarly, SCAP was cultured on 9 dentin specimens from each nanoparticle conditioned group and stained for fluorescence microscopy and prepared for scanning electron microscopy. SCAP Adherence and Viability on Nanoparticle Conditioned Dentin. Three specimens in each group were stained with calcein-AM (1 mmol/L in PBS; Life Technologies, Grand Island, NY) by directly adding the staining solution to cultured cells (18) and examined under a fluorescence microscope (Carl Zeiss, Gottingen, Germany). The number of cells adherent to dentin was calculated from 12 microscopic fields at 20 magnification. Images were captured and processed by using ImageJ software (National Institutes of Health, Bethesda, MD) and then saved as a 32-bit jpeg file. Cell counts were made from 8-bit images normalized to 50–500 pixels by using ImageJ. Cell viability was calculated as a percentage relative to the total number of cells attached to control dentin that was designated as 100% viability. SCAP Spreading on Nanoparticle Conditioned Dentin. Cell circularity was determined by analyzing cells in the same specimens that were stained with calcein-AM by using ImageJ. Circularity was assessed as 1, a perfect sphere or rounded cell, to 0, representing a
Nanoparticle Conditioning Nanoparticles used for dentin conditioning were synthesized as previously described (8, 9). Physical characterization and release kinetics of 2 variants Dex-CSnpI (slow releasing) and Dex-CSnpII (rapid releasing) have been previously published (9). Seventy-two of NaOCl/EDTA-treated dentin specimens were divided into 4 groups (n = 18) and treated accordingly with 300 mg/mL nanoparticles (8, 9) in deionized water (1 mL) for 12 hours. The 4 groups were designated as (1) unconditioned, (2) CSnp conditioned, (3) DexCSnpI conditioned, and (4) Dex-CSnpII conditioned. SCAP Culture A previously characterized SCAP cell line was used in all experiments (15). Cells were cultured and expanded by adding single-cell suspensions (1 105 cells) to a-minimum essential medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and 100 units/mL antibiotic:antimycotic solution. Cells were allowed to expand in culture to 70%–80% confluency and then released with 0.05% trypsin (Gibco, Carlsbad, CA). Cells from the third to fifth passages (15–17) were used in all experiments. Characterization of SCAP on Nanoparticle Conditioned Dentin Six dentin specimens from each chemical treatment group were placed in a 24-well plate, and 1 mL SCAP at a concentration of 1.0 105 was introduced into each well. The cells were allowed to grow at 37 C in a humidified incubator in an atmosphere of 5% CO2 for 24 hours. Specimens were washed with phosphate-buffered saline JOE — Volume -, Number -, - 2016
Figure 3. (A) Relative viability of SCAP in NaOCl/EDTA-treated dentin specimens without/with nanoparticle conditioning. Groups conditioned with nanoparticles demonstrated no significant change in viability relative to the unconditioned group. (B) Circularity of cells cultured in NaOCl/EDTAtreated dentin specimens without/with nanoparticle conditioning. Cells in the unconditioned group demonstrated 3-fold increase in circularity as compared with the control group and 2-fold increase in value as compared with the CSnp, Dex-CSnpI, or Dex-CSnpII conditioned groups. Data are presented as mean standard deviation of the mean (n = 3). **P < .05 versus the control group. ##P < .05 versus unconditioned group.
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Figure 4. (A) Scanning electron microscopic micrographs showing morphology and biomineralization of SCAP cultured for 2 weeks on NaOCl/EDTA-treated dentin specimens with/without nanoparticle conditioning. CSnp conditioned specimen without cells was studied as negative control to determine the effect of CSnp on mineralization. Mineralization was present in the conditioned groups, with the greatest degree present in the Dex-CSnpII conditioned group. Immunofluorescence localization of DSPP (B) and DMP-1 (C) during odontogenic differentiation. Fluorescein isothiocyanate (FITC)–conjugated antibody was used to detect the localization of the protein (green signal); all samples were counterstained with DAPI (blue signal). Scale bar = 25 mm.
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Basic Research—Biology non-circular or highly spread cell (19). Ten cells in each microscopic view were analyzed and averaged to assign circularity values. In addition, SCAP present on 3 specimens from each group were fixed for 5 minutes in 10% normal buffered formalin and thoroughly washed with PBS. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 20 minutes and washed again with PBS. Cells were stained with DAPI (Roche, Basel, Switzerland) for 5 minutes and then fluorescent phalloidin-tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC; Sigma-Aldrich) solution in PBS for 40 minutes at room temperature. They were washed several times with PBS to remove the unbound phalloidin conjugate. The excitation and emission wavelengths for Phalloidin-TRITC are 540 nm and 570 nm, respectively. The specimens were examined under a fluorescent microscope to visualize cytoskeletal actin. SCAP Ultrastructure on Nanoparticle Conditioned Dentin. Three specimens from each group were fixed overnight in 2.5% glutaraldehyde. These specimens were then dehydrated in graded solutions of ethanol and dried with hexamethylene disiloxane. After palladium coating, specimens were examined under a scanning electron microscope (Hitachi S-2500, Ibaragi, Japan) to assess SCAP ultrastructure.
SCAP Differentiation on Nanoparticle Conditioned Dentin The remaining 9 specimens in each nanoparticle conditioned group were placed in an 8-chamber polystyrene treated vessel of a tissue culture glass slide (BD Biosciences, Bedford, MA) and seeded with 2 104 cells in standard culture medium containing 50 mg/ mL ascorbic acid, 10 mmol/L b-glycerolphosphate, and 1.8 mmol/ L potassium dihydrogen phosphate (KH2PO4). Cultures were maintained at 37 C in 5% CO2 humidified incubator for 2 weeks. Comparisons were made between the 2 variants, Dex-CSnpI (slow releasing) and Dex-CSnpII (rapid releasing). All experiments were performed in triplicate before analysis. SCAP Biomineralization on Nanoparticle Conditioned Dentin. After 2 weeks, 3 specimens from each group were prepared for scanning electron microscopy as described earlier to assess biomineralization (20). Ten images were analyzed in a sequential manner for each sample. The effect of CSnp on biomineralization was studied by incubating CSnp conditioned dentin under the same conditions/duration without cells (negative control). Immunofluorescence Analysis on Nanoparticle Conditioned Dentin. At the end of 2 weeks, specimens were washed in PBS and fixed with 4% paraformaldehyde in PBS containing 0.1% Triton X-100 at 4 C for 30 minutes. After blocking the fixed specimens in 2.5% bovine serum albumin for 30 minutes at room temperature, 3 specimens from each group were incubated with the following primary antibodies, mouse anti-DMP-1 (Santa Cruz Biotechnologies, Santa Cruz, CA) or mouse anti-DSPP (Santa Cruz Biotechnologies), diluted 1:50 in blocking reagent at 37 C for 2 hours. After 3 washes in PBS/Tween 20, specimens were incubated with secondary antibody goat anti-mouse immunoglobulin G fluorescein isothiocyanate conjugate (Santa Cruz Biotechnologies), diluted 1:1500 in PBS at 37 C, for 1 hour. After rinsing, specimens were counterstained with DAPI and examined by using confocal laser scanning microscopy (Leica Microsystems, Richmond, IL). Statistical Analysis Viability and circularity results were subjected to one-way analysis of variance with Dunnett test. The difference between 2 groups was considered statistically significant if P < .05. JOE — Volume -, Number -, - 2016
Results Effect of Nanoparticle Conditioning on SCAP Characteristics SCAP Adherence. Figure 1 shows the typical fluorescence microscopic images of SCAP adherence on dentin. Numerous cells with a fibroblast-like morphology were homogenously distributed on the untreated dentin (control group) in a unidirectional manner. The number and morphology of cells present on EDTA only treated dentin were similar to those present in the control group, but cells were more flattened. Few cells with cytoplasmic extensions were present on dentin treated with NaOCl/EDTA. Significantly less numbers of cells with rounded morphology and devoid of cytoplasmic extensions were observed in NaOCl and NaOCl/EDTA/NaOCl groups. The morphology of cells after F-actin staining with phalloidin-TRITC was concurrent with the green fluorescent images stained with calcein-AM. Cells on dentin treated with NaOCl and NaOCl/EDTA/NaOCl displayed less ctytoplasmic F-actin compared with the rest of the groups. Nanoparticle conditioning on NaOCl/EDTA treated dentin resulted in significant increase in number of SCAP adherence compared with unconditioned dentin as shown in Figure 2. Cells in all the conditioned groups were uniformly distributed and displayed well-developed lamellipodia and filopodia. SCAP Ultrastructure. Cells on unconditioned dentin were round and devoid of cell extensions (Fig. 2). Cells on dentin conditioned with nanoparticles were flattened, appeared more numerous and more attached, and had well-developed cytoplasmic extensions, similar to what was seen in these same groups under fluorescence microscopy. SCAP Viability and Circularity. The quantitative relative viability of cells on dentin varied among groups (Fig. 3A). The viability of cells in the unconditioned group was decreased by 50% when compared with the untreated control group. Groups conditioned with nanoparticles demonstrated no significant change in viability relative to the unconditioned group. Cells in the unconditioned group demonstrated a 3-fold increase in circularity as compared with the control group (P < .05) and a 2-fold increase in value as compared with the CSnp, DexCSnpI, or Dex-CSnpII conditioned groups (P < .05) (Fig. 3B). Effect of Nanoparticle Conditioning on SCAP Differentiation SCAP Biomineralization. No evidence of mineralization could be seen under scanning electron microscopy in the unconditioned and negative control groups (Fig. 4A). Mineralization was present in the nanoparticle conditioned groups, with the greatest degree present in the Dex-CSnpII conditioned group. SCAP Immunofluorescence Localization of Dentin Matrix Protein-1 and Dentin Sialophosphoprotein. As shown in Figure 4B and C, expressions of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) were not observed in unconditioned group. Cells in the rest of the groups demonstrated a higher signal for cytoplasmic DSPP expression than for DMP-1 expression. Higher signals for DSPP and DMP-1 expression were seen in the cells of the Dex-CSnpI and Dex-CSnpII conditioned groups compared with the CSnp conditioned group, and signals for DSPP and DMP-1 were markedly higher in cells of the Dex-CSnpII group than in cells of the Dex-CSnpI group.
Discussion A treatment protocol that promotes adherence of dental stem cells to root canal dentin and enhances their odontogenic differentiation is essential in reestablishing dentinogenesis in a pulpless environment
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Basic Research—Biology (21). Dex has been identified as an odontogenic stimulant for SCAP in regenerative endodontics. Ideally this stimulation works best when the dentin is undamaged (13). Many commonly used root canal irrigants, especially those used in root canal disinfection, have a potential to damage dentin and therefore impede cell adherence and new tissue formation (22, 23). NaOCl, a highly effective root canal antimicrobial agent for example, alters the surface characteristics of dentin and impedes the odontogenic differentiation of pulp stem cells (22, 24, 25). Conditioning with CSnp or Dex-CSnp, as shown in this study, allowed cells to adhere and retain their viability on NaOCl/EDTA-treated dentin. Of the numerous chemicals currently used in root canal irrigation, only EDTA appears to promote adherence and cell viability on dentin (22, 23). These findings were evident in this study and have been cited several times in the literature (4, 13, 22, 23). It has been suggested that this effect is related to an increase in the surface wettability of dentin (26) and the release of dentinogenic BMs from the matrix, when dentin is chelated (27). The effect was noted in this study and supported by findings in previous studies. Furthermore, EDTA as a last irrigant would remove the smear layer and expose organic substrate for interaction with nanoparticles. The actual adherence of stem cells to dentin has been attributed to fibronectin, an adhesion protein that is present in the dentin matrix and preferentially adsorbed on a hydrophilic surface (28). Nanoparticle conditioning of NaOCl/EDTA-treated dentin appears to provide that hydrophilic surface (5) and hence has the ability to enhance the degree of adherence normally seen. In the current study, biomineralization and signals from biomarkers such as DSPP and DMP-1 were distinctly higher in SCAP cultured on Dex-CSnpI and Dex-CSnpII conditioned dentin than in SCAP cultured on CSnp conditioned dentin alone. DSPP is a matrix phosphoprotein strongly expressed in odontoblasts and a marker of biomineralization (29). DMP-1 is a non-collagenous matrix protein expressed in odontoblasts and regulates biomineralization during tooth development and repair (30). Because DSPP and DMP-1 are expressed in odontoblasts in the early stages of odontogenesis (31), the elevated expression of these 2 odontogenic proteins in Dex-CSnpI and DexCSnpII conditioned specimens suggested that Dex has been made available to the cells in an appropriate concentration and at a critical time. Furthermore, because cells conditioned with Dex-CSnpII (rapid release) showed higher expression of these odontogenic markers than cells conditioned with Dex-CSnpI (slow release), a temporal system of BM release may be advantageous when developing a regenerative endodontic protocol (9). The usefulness of temporal-controlled BM release in regenerative endodontics has been previously discussed (8, 9). The key advantages of temporal-control release over static availability are that it enhances stability of the BMs and allows release of a desired concentration of BM during a predetermined period of time. When used as a vehicle to deliver BMs, CSnp adds an antimicrobial and antibiofilm effect and greater hydrophilicity to the dentin, making it an ideal nanoparticle for use in regenerative procedures (32). Release of endogenous dentin matrix components, such as transforming growth factor–b, bone morphogenic protein–2, platelet-derived growth factor, has been shown to be advantageous in promoting stem cell proliferation and differentiation (27, 33, 34) and can potentially be delivered in a temporal manner. Growth factors which regulate specific components of the healing process that are currently being used as therapeutic agents to enhance repopulation of root surfaces denuded by periodontal disease (35) have a potential that can be readily applied to endodontic regenerative procedures. In conclusion, results of this investigation demonstrate that conditioning of dentin with CSnp, Dex-CSnpI, or Dex-CSnpII has a potential to 6
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reverse the deleterious effect of NaOCl treatment on stem cell viability and adherence to dentin in regenerative procedures. This may allow it to be used as a root canal disinfectant when regenerative procedures are contemplated. The study has also demonstrated that controlling the duration and concentration of essential BMs from nanoparticles enhanced odontogenic differentiation of SCAP, and such a manner of release may lead to a more favorable outcome when regenerative procedures are undertaken.
Acknowledgments The authors gratefully acknowledge the support of Dr Anibal Diogenes in this study. This study was supported by the University Start Up Fund, University of Toronto, Ontario, Canada and a Research Grant from the American Association of Endodontists (AAE) Foundation(2014). The authors deny any conflicts of interest related to this study.
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