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Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization
Regenerative Endodontics
Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization Matthias Widbiller, DDS,*† Riyadh I. Althumairy, DDS,* ‡ and Anibal Diogenes, DDS, MS, PhD* Abstract Introduction: Several irrigants have been used for disinfection in regenerative endodontic procedures including chlorhexidine (CHX). In this context, the antibacterial properties of disinfectants are mainly in focus of research even though they may have an undesirable impact on the fate of stem cells. In this study, we hypothesized that CHX has both a direct effect when applied to stem cells of the apical papilla (SCAPs) and an indirect effect when SCAPs are exposed to dentin previously conditioned with CHX. Methods: Cell toxicity was evaluated in vitro using the CellTox green fluorescence assay (Promega, Madison, WI) and CellTiter-Glo (Promega) after SCAPs were exposed directly to a dynamic concentration range of CHX; apical papilla explant cultures were stained with ApopTag (Merck Millipore, Billerica, MA) after culture with CHX. Furthermore, standardized slabs from human dentin were treated with CHX and consecutively rinsed in EDTA, L-a-lecithin (Sigma-Aldrich, St Louis, MO), or L-a-lecithin followed by EDTA. After that, SCAPs were cultured on the slabs for 5 days, and cellular viability was determined (indirect effect). Data were treated nonparametrically and analyzed using the Krukal-Wallis test (P # .05). Results: Direct exposure of SCAPs to CHX highly affected cell viability at concentrations above 10 3%, whereas lower concentrations had no adverse effect. During the initial 60 minutes, concentrations of 10 2% CHX or higher resulted in early pronounced toxicity with a maximum effect within 15 minutes after exposure. Likewise, CHX-conditioned dentin slabs were detrimental to SCAP survival; however, the deleterious effects were completely reversed by neutralization with L-a-lecithin. Conclusions: Chlorhexidine is toxic to SCAPs when applied directly or indirectly via conditioned dentin. If applied for a short time and neutralized by L-a-lecithin, it can be a gentle and cell-preserving disinfectant before endodontic regeneration. (J Endod 2019;45:156–160)
Key Words Chlorhexidine, dentin, endodontic regeneration, lecithin, stem cells, toxicity
P
ulpal necrosis in Significance immature teeth is a Residual biofilm or bacterial endotoxins have been challenging clinical situashown to be critical for the outcome of regenerative tion. The ability to perform endodontic procedures. Thus, thorough disinfecroot canal treatment of tion of infected root canals is warranted; however, such teeth as well as the irrigants like chlorhexidine can be deleterious for long-term prognosis are apical papilla stem cells at the same time. Optimicompromised by the preszation of clinical disinfection protocols and neutralence of a wide-open apex. ization of the cytotoxic effects are necessary to Traditionally, these teeth provide a regenerative environment that allows were treated by apexificasurvival, proliferation, and differentiation of newly tion with several rounds introduced cells. of calcium hydroxide to create an apical barrier for ease of obturation (1). Mineral trioxide aggregate was also used as an apical plug, which reduced the need for several dental appointments, resulting in better patient compliance (2, 3). More recently, regenerative endodontic procedures (REPs) have become a recognized alternative for the treatment of such teeth, and several case reports were published showing successful outcomes (4). The new approach uses chemical disinfection in the first appointment with irrigants followed by placement of an intracanal medicament with minimal mechanical instrumentation. After resolution of clinical signs and symptoms, the treatment continues in the second appointment with removal of the medicament, initiation of bleeding into the root canal, a coronal seal with a calcium silicate cement, and adhesive restoration. Besides the resolution of clinical symptoms and healing of apical periodontitis, regenerative cases offer continued root growth with responsiveness to vitality tests in some cases. Therefore, this innovative approach has the potential to improve the clinical outcome of compromised teeth otherwise susceptible to fracture (5, 6). Stem cells of the apical papilla (SCAPs) are thought to be the primary source of stem cells in REPs. Initiation of bleeding from the periapical tissues transports stem cells into the canal system (7). Maintaining stem cell viability in addition to promoting proliferation and determining their differentiation are crucial for the clinical outcome. This can only be achieved when the environment in the root canal is optimized before the introduction of stem cells. An optimized protocol of chemical disinfectants is important to determine the fate of stem cells (8–10).
From the *Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas; †Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany; and ‡Department of Restorative Dental Sciences, King Saud University, Riyadh, Saudi Arabia. Address requests for reprints to Dr Anibal R. Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2018 American Association of Endodontists. https://doi.org/10.1016/j.joen.2018.11.012
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Regenerative Endodontics Irrigation of the root canal system as well as intracanal medications could have both direct and indirect effects on cells (Fig. 1A). Direct effects result from the contact of SCAPs to the irrigant or medicament during or after their application. Indirect effects are caused by changes in the environment that could affect stem cells even after removal of the disinfectant. Although a previous study has shown the direct negative effect of chlorhexidine (CHX) on stem cell survival (10), no study has evaluated the concentration response for this toxicity. This is particularly important because CHX has been used clinically in REPs at the concentrations of either 0.12% or 2% (11). Lastly, the neutralization of this negative effect on SCAPs has never been investigated. Thus, this study aimed to evaluate both the direct and indirect effects of different concentrations of CHX on SCAP survival as well as the possibility to neutralize possible indirect cytotoxic effects using a dentin slab model.
Materials and Methods Patient Recruitment This study was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio. After verbal and written communication for informed consent, extracted teeth were immediately rinsed in sterile phosphate-buffered saline (PBS) and stored in a container with sterile 1 PBS. Apical papilla samples were dissected from immature third molars with less than two thirds of the root being formed and used in the ex vivo toxicity experiment. Additionally, extracted healthy third molars were collected and used to generate standardized dentin slabs as described later. SCAP Survival after Direct Exposure to CHX A characterized SCAP cell line was used in this study (10). Cells were seeded in a 24-well plate with each well containing 25,000 cells in 800 mL media. CHX was diluted in media in the following concentrations: 2%, 1%, 0.5%, 0.25%, 0.12%, 10 2 %, 10 3 %, 10 4 %, 10 5 %, 10 6 %, and 10 7 % (Endo-CHX; Essential Dental Systems, South Hackensack, NJ). SCAPs were exposed to either media (control) or media with different CHX concentrations and incubated at 37 C and 5% CO2 for 3 days. On day 3, SCAPs reached approximately 80% confluency in the control group. Media were removed, and SCAPs were washed with 1 PBS (Gibco/Thermo Fisher Scientific, Waltham, MA) followed by application of 200 mL CellTiter-Glo reagent (Promega, Madison, WI) to each well and incubation at room temperature for 10 minutes. To determine background luminescence, media without cells were mixed with CellTiter-Glo reagent. Relative luminescence for each group was detected in a FlexStation 3 Benchtop Multi-Mode microplate reader (Molecular Devices, San Jose, CA). To evaluate cytotoxicity on SCAPs in their original environment, apical papillae were collected and cultivated in either media only (control) or media containing 2% CHX for 3 days. After the culture period, explants were rinsed in 1 PBS and fixed with 4% paraformaldehyde, embedded in Neg-50 (Richard-Allan, Thermo Fisher Scientific), and serially sectioned with a cryostat (20 mm). Sections were placed onto Superfrost Plus slides (Thermo Fisher Scientific), air dried, and stained with the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA). Z-stack images were taken on a Nikon Eclipse 90i microscope (Nikon, Minato, Japan) equipped with a Nikon C1si laser scanning confocal imaging system using standardized settings at 20 magnification and processed in ImageJ (Fiji) software (National Institutes of Health, Bethesda, MD) (12). Kinetics of Direct CHX-evoked Toxicity on SCAPs SCAPs were seeded in a 96-well plate with each well containing 15,000 cells in 200 mL of different concentrations of CHX or media only (control); 100 mL CellTox reagent (Promega) was added to each well, and plates were incubated at 37 C and 5% CO2. During incubation, cell toxicity was evaluated using CellTox green fluorescence assay at 2, 5, 15, 30, and 60 minutes. To determine background fluorescence, media without cells were mixed with CellTox reagent. The relative fluorescence for each group was detected in a FlexStation 3 Benchtop Multi-Mode microplate reader.
Figure 1. Disinfecting agents exert direct and indirect effects on stem cells during REPs. (A) The schematic illustration shows that irrigants or medicaments do not only affect SCAPs by direct contact but also have indirect effects on introduced stem cells through contamination of dentin. (B) Direct cytotoxicity of CHX on SCAPs is concentration dependent, and concentrations above 10 3% significantly affected cell survival as indicated by asterisks (P # .05). JOE — Volume 45, Number 2, February 2019
SCAP Survival after Exposure to Irrigated Dentin Slabs (Indirect Effect) Dentin slabs were prepared similarly to previous protocols (13). Briefly, gingival and periodontal tissues were removed from the tooth surface with a sterile surgical blade. The teeth were then washed in Chlorhexidine and Survival of SCAPs
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Figure 2. Apical papilla samples were cultured in media without (control) and with supplementation of CHX (2%) for 3 days. (A) ApopTag stained hardly any apoptotic nuclei in the control samples. (B) CHX induced apoptosis throughout the papilla but especially in proximity to the surface (dotted line).
cold Hank’s Balanced Salt Solution (HBSS, Sigma-Aldrich). An IsoMet 1000 precision saw (Buehler, Lake Bluff, IL) with a diamond blade was used to generate dentin slabs (400 mm in thickness and 20 mm2 surface area) by axial cuts above the pulp chambers of molar teeth. The tooth slices were then washed twice with cold sterile HBSS, blotted dry, gas sterilized, and stored at 4 C until usage. Dentin slabs were rehydrated in HBSS for 5 minutes and irrigated with saline (control), 2% CHX, EDTA (Endo-Cleanse 17% EDTA Solution; Roydent Dental Products, Johnson City, TN), or L-a-lecithin (0.07 % L-a-Lecithin/Soybean and 0.5% Tween 80, Sigma-Aldrich) for 5 minutes. Additionally, the following irrigation sequences were established with incubation times of 5 minutes: CHX/L-a-lecithin, CHX/EDTA, and CHX/L-a-lecithin/EDTA. Slabs were then washed with saline for 5 minutes, placed in 24-well plates with 50,000 cells in 500 mL media per well, and incubated at 37 C and 5% CO2 for 5 days. After the culture time, dentin slabs were gently transferred to another plate to eliminate cells present at the well bottom outside the dentin slabs; 200 mL CellTiter-Glo reagent (Promega) was added to each well and incubated at room temperature
for 10 minutes. To determine background luminescence, media without cells were also mixed with the reagent. The relative luminescence for each group was detected in a FlexStation 3 Benchtop Multi-Mode microplate reader.
Data Analysis The relative luminescence and fluorescence units were calculated for each group after background subtraction. Data were depicted as means with standard deviations and treated nonparametrically. Results were analyzed statistically by the Krukal-Wallis test at a significance level of P # .05 using GraphPad Prism 7 (GraphPad Software, La Jolla, CA).
Results SCAP Survival after Direct Exposure to CHX CHX concentrations between 2% and 10 3% led to a significant decline in the survival of SCAPs (Fig. 1B). Lower concentrations did not show any detrimental effects or slightly induced survival and proliferation (eg, 10 6% and 10 7%) at a nonsignificant level.
Figure 3. Kinetic assessment of direct CHX toxicity on SCAPs over the initial 60 minutes revealed concentration-dependent effects, which were significant at 0.01% and above (the gray area includes nonsignificant results). Cytotoxicity was pronounced early and reached its maximum within 15 minutes after exposure.
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Figure 4. Indirect cytotoxic effects of irrigants in the dentin slab model. (A) L-a-lecithin or EDTA did not affect SCAP viability, whereas CHX conditioning of dentin significantly reduced cell survival. (B) CHX-induced cytotoxicity was neutralized by subsequent irrigation with L-a-lecithin, whereas subsequent EDTA irrigation was futile. Cytotoxic effects of CHX-conditioned dentin were also reversed when slabs were subsequently rinsed with L-a-lecithin followed by EDTA. Asterisks indicate significant differences to the untreated control (P # .05).
In the explant cultures, cells of the apical papilla became apoptotic when cultured in media containing 2% CHX, whereas papilla samples cultured without CHX showed no signs of apoptosis (Fig. 2A and B). Nuclei with DNA fragmentation predominated in the superficial regions of the papillae but were found throughout the tissue. Compared with the in vitro system, cells embedded in the apical papilla seemed more resistant to toxicity by 2% CHX.
Kinetics of Direct CHX-evoked Toxicity on SCAPs No CHX toxicity on SCAPs was detected after 2 minutes. Concentrations below 10 2% had no significant cytotoxic impact during the 60 minutes of observation, but higher amounts were detrimental to SCAPs in a concentration-dependent manner (Fig. 3). At 5 minutes postexposure, cell damage by 0.1%–2% CHX attained almost 50% of the maximum toxicity and steadily increased over time with a plateau after 30 minutes. SCAP Survival after Exposure to Irrigated Dentin Slabs (Indirect Effect) Although dentin slabs conditioned with 0.07% L-a-lecithin or 17% EDTA did not affect SCAP survival, 2% CHX treatment induced a significant decline in cell viability (Fig. 4A). This indirect cytotoxicity was fully reversed when the dentin slabs were rinsed with lecithin subsequent to CHX (Fig. 4B). However, subsequent irrigation with EDTA did not weaken CHX toxicity; cell viability was still reduced significantly. When CHX-pretreated dentin slabs were neutralized by L-a-lecithin, a final rinse with EDTA did not alter the neutralizing effect significantly.
Discussion Stem cells are an integral part of pulp tissue engineering, which aims to regenerate tissue in form and function. For a positive clinical outcome of REPs, it is crucial to establish an environment that allows stem cell survival, proliferation, and differentiation (14, 15). Currently applied protocols for regenerative endodontics successfully lead to resolution of clinical signs and symptoms as well as continued root development (5, 6). Several histologic studies described ectopic mineralized components in regenerated tissues such as cementum and osteodentin (16, 17). Lately, residual biofilm or bacterial endotoxins have been shown to be critical for the outcome of regenerative procedures (18) because they can alter the fate of stem cells and lead to an osteoblast phenotype (19, 20). Thus, further investigation and reevaluation of applied clinical protocols are needed in terms of microbial reduction and detoxification in infected root canals. JOE — Volume 45, Number 2, February 2019
Several irrigants and intracanal medicaments have been used for disinfection before regenerative procedures including CHX in concentrations of 0.12%–2% (21–23); 2% CHX in liquid form had a similar microbial performance against several microorganisms as 5.25% sodium hypochlorite (24, 25); however, it does not dissolve organic compounds. CHX solution (2%) inhibited bacterial growth after a contact time of only 15 seconds (24). Although sodium hypochlorite reportedly affected regenerative outcome (15), CHX is less toxic and malodorous (10, 24, 25) and facilitated a significantly higher release of growth factors from dentin (26). As shown previously, no viable stem cells were detected after irrigation with CHX at a concentration of 2% (10). Likewise, CHX has been shown to be toxic on SCAPs in this study at concentrations of 10 3% or higher. Cytotoxicity came into effect after a contact time of 5 minutes and increased over 30 minutes. When apical papilla explants were exposed to media with 2% CHX for 3 days, apoptosis occurred throughout the tissue with most apoptotic nuclei in proximity to the surface. The remaining viable cells, especially in the core, gave the impression that SCAPs are more resistant to CHX when encapsulated in the papilla tissue compared with the in vitro situation. In a clinical protocol, CHX is used as the root canal irrigant and comes in contact with SCAPs. Limitation of the contact time (<5 minutes) and a thorough rinse with saline are necessary to provide an environment where SCAPs can resist and survive. Once stem cells are transported into the canal space and reside in the blood clot, they are indirectly confronted with irrigants or medicaments. Residual particles or dentin-bound molecules like CHX can exert toxic effects and influence cell fate as well as the outcome of regenerative procedures. This indirect toxicity was assessed with dentin slabs conditioned with CHX for 5 minutes. Although slabs were finally rinsed with saline, cytotoxicity endured because of the substantivity of cationic CHX molecules. As shown in a previous study, the remaining CHX was detectable for up to 12 weeks after irrigating a root canal system with a 2% solution (27). This study did not evaluate the potential long-lasting effect of CHX conditioning because it tested the immediate contact of the cells with the conditioned dentin surface as seen in the clinical setting after evoked bleeding. Interestingly, the indirect cytotoxicity of CHX was reversed by a subsequent rinse with L-a-lecithin presumably because of binding of the cationic CHX to the negatively charged scavenger molecule (28). Indeed, a solution containing a detergent and a negatively charged protein has been used previously to reverse the antibacterial effects of CHX (29). Likewise, in this study, a solution of Tween and L-a-lecithin resulted in complete neutralization of CHX and abolishment of its deleterious effects on SCAPs. In fact, this approach has been successfully used to
Chlorhexidine and Survival of SCAPs
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Regenerative Endodontics reduce the carryover effect in a case series of REPs (30). Even the final irrigation with EDTA, as recommended for regenerative procedures, did not alter its neutralizing effect. Therefore, despite its robust cytotoxicity, CHX can be effectively neutralized even when used at 2% as is routinely done clinically. In conclusion, CHX used in clinically relevant concentrations affects SCAP survival directly and indirectly. These effects can be overcome by limitation of irrigation time (direct effects) and subsequent neutralization with L-a-lecithin (indirect effects). Therefore, CHX might be a promising and cell-friendly disinfection agent before REPs, and further studies are needed to determine the regenerative outcome after CHX disinfection in an in vivo situation.
Acknowledgments The study was funded by the University of Texas Health at San Antonio. The authors deny any conflicts of interest related to this study.
References 1. Frank AL. Therapy for the divergent pulpless tooth by continued apical formation. J Am Dent Assoc 1966;72:87–93. 2. Shabahang S, Torabinejad M. Treatment of teeth with open apices using mineral trioxide aggregate. Pract Periodontics Aesthet Dent 2000;12:315–20. quiz322. 3. Witherspoon DE, Small JC, Regan JD, Nunn M. Retrospective analysis of open apex teeth obturated with mineral trioxide aggregate. J Endod 2008;34:1171–6. 4. Diogenes AR, Ruparel NB, Shiloah Y, Hargreaves KM. Regenerative endodontics: a way forward. J Am Dent Assoc 2016;147:372–80. 5. Lin J, Zeng Q, Wei X, et al. Regenerative endodontics versus apexification in immature permanent teeth with apical periodontitis: a prospective randomized controlled study. J Endod 2017;43:1821–7. 6. Nagy MM, Tawfik HE, Hashem AAR, Abu-Seida AM. Regenerative potential of immature permanent teeth with necrotic pulps after different regenerative protocols. J Endod 2014;40:192–8. 7. Lovelace TW, Henry MA, Hargreaves KM, Diogenes AR. Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod 2011;37:133–8. 8. Martin DE, De Almeida JFA, Henry MA, et al. Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation. J Endod 2014;40:51–5. 9. Ruparel NB, Teixeira FB, Ferraz CC, Diogenes AR. Direct effect of intracanal medicaments on survival of stem cells of the apical papilla. J Endod 2012;38:1372–5. 10. Trevino EG, Patwardhan AM, 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.
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11. Kontakiotis EG, Filippatos CG, Tzanetakis GN, Agrafioti A. Regenerative endodontic therapy: a data analysis of clinical protocols. J Endod 2015;41:146–54. 12. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9:676–82. 13. Widbiller M, Lindner SR, Buchalla W, et al. Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements. Clin Oral Investig 2016;20: 237–46. 14. Diogenes AR, Hargreaves KM. Microbial modulation of stem cells and future directions in regenerative endodontics. J Endod 2017;43:S95–101. 15. Galler KM, D’Souza RN, Federlin M, et al. Dentin conditioning codetermines cell fate in regenerative endodontics. J Endod 2011;37:1536–41. 16. Martin G, Ricucci D, Gibbs JL, Lin LM. Histological findings of revascularized/ revitalized immature permanent molar with apical periodontitis using platelet-rich plasma. J Endod 2013;39:138–44. 17. Becerra P, Ricucci D, Loghin S, et al. Histologic study of a human immature permanent premolar with chronic apical abscess after revascularization/ revitalization. J Endod 2014;40:133–9. 18. Verma P, Nosrat A, Kim JR, et al. Effect of residual bacteria on the outcome of pulp regeneration in vivo. J Dent Res 2017;96:100–6. 19. Vishwanat L, Duong R, Takimoto K, et al. Effect of bacterial biofilm on the osteogenic differentiation of stem cells of apical papilla. J Endod 2017;43: 916–22. 20. Widbiller M, Eidt A, W€olflick M, et al. Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells. Int Endod J 2018;51:877–88. 21. Petrino JA, Boda KK, Shambarger S, et al. Challenges in regenerative endodontics: a case series. J Endod 2010;36:536–41. 22. Shivashankar VY, Johns DA, Vidyanath S, Kumar MR. Platelet rich fibrin in the revitalization of tooth with necrotic pulp and open apex. J Conserv Dent 2012; 15:395–8. 23. Soares Ade J, Lins FF, Nagata JY, et al. Pulp revascularization after root canal decontamination with calcium hydroxide and 2% chlorhexidine gel. J Endod 2013;39:417–20. 24. Vianna ME, Gomes BP, Berber VB, et al. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79–84. 25. Gomes BP, Vianna ME, Zaia AA, et al. Chlorhexidine in endodontics. Braz Dent J 2013;24:89–102. 26. Galler KM, Buchalla W, Hiller K-A, et al. Influence of root canal disinfectants on growth factor release from dentin. J Endod 2015;41:363–8. 27. Rosenthal S, Sp angberg L, Safavi K. Chlorhexidine substantivity in root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:488–92. 28. Babich H, Wurzburger BJ, Rubin YL, et al. An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol 1995;11: 79–88. 29. Zamany A, Sp angberg LSW. An effective method of inactivating chlorhexidine. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;93:617–20. 30. Nagata JY, Soares AJ, Souza-Filho FJ, et al. Microbial evaluation of traumatized teeth treated with triple antibiotic paste or calcium hydroxide with 2% chlorhexidine gel in pulp revascularization. J Endod 2014;40:778–83.
JOE — Volume 45, Number 2, February 2019
Direct and Indirect Effect of Chlorhexidine on Survival of Stem Cells from the Apical Papilla and Its Neutralization Matthias Widbiller, DDS,*† Riyadh I. Althumairy, DDS,* ‡ and Anibal Diogenes, DDS, MS, PhD* Abstract Introduction: Several irrigants have been used for disinfection in regenerative endodontic procedures including chlorhexidine (CHX). In this context, the antibacterial properties of disinfectants are mainly in focus of research even though they may have an undesirable impact on the fate of stem cells. In this study, we hypothesized that CHX has both a direct effect when applied to stem cells of the apical papilla (SCAPs) and an indirect effect when SCAPs are exposed to dentin previously conditioned with CHX. Methods: Cell toxicity was evaluated in vitro using the CellTox green fluorescence assay (Promega, Madison, WI) and CellTiter-Glo (Promega) after SCAPs were exposed directly to a dynamic concentration range of CHX; apical papilla explant cultures were stained with ApopTag (Merck Millipore, Billerica, MA) after culture with CHX. Furthermore, standardized slabs from human dentin were treated with CHX and consecutively rinsed in EDTA, L-a-lecithin (Sigma-Aldrich, St Louis, MO), or L-a-lecithin followed by EDTA. After that, SCAPs were cultured on the slabs for 5 days, and cellular viability was determined (indirect effect). Data were treated nonparametrically and analyzed using the Krukal-Wallis test (P # .05). Results: Direct exposure of SCAPs to CHX highly affected cell viability at concentrations above 10 3%, whereas lower concentrations had no adverse effect. During the initial 60 minutes, concentrations of 10 2% CHX or higher resulted in early pronounced toxicity with a maximum effect within 15 minutes after exposure. Likewise, CHX-conditioned dentin slabs were detrimental to SCAP survival; however, the deleterious effects were completely reversed by neutralization with L-a-lecithin. Conclusions: Chlorhexidine is toxic to SCAPs when applied directly or indirectly via conditioned dentin. If applied for a short time and neutralized by L-a-lecithin, it can be a gentle and cell-preserving disinfectant before endodontic regeneration. (J Endod 2019;45:156–160)
Key Words Chlorhexidine, dentin, endodontic regeneration, lecithin, stem cells, toxicity
P
ulpal necrosis in Significance immature teeth is a Residual biofilm or bacterial endotoxins have been challenging clinical situashown to be critical for the outcome of regenerative tion. The ability to perform endodontic procedures. Thus, thorough disinfecroot canal treatment of tion of infected root canals is warranted; however, such teeth as well as the irrigants like chlorhexidine can be deleterious for long-term prognosis are apical papilla stem cells at the same time. Optimicompromised by the preszation of clinical disinfection protocols and neutralence of a wide-open apex. ization of the cytotoxic effects are necessary to Traditionally, these teeth provide a regenerative environment that allows were treated by apexificasurvival, proliferation, and differentiation of newly tion with several rounds introduced cells. of calcium hydroxide to create an apical barrier for ease of obturation (1). Mineral trioxide aggregate was also used as an apical plug, which reduced the need for several dental appointments, resulting in better patient compliance (2, 3). More recently, regenerative endodontic procedures (REPs) have become a recognized alternative for the treatment of such teeth, and several case reports were published showing successful outcomes (4). The new approach uses chemical disinfection in the first appointment with irrigants followed by placement of an intracanal medicament with minimal mechanical instrumentation. After resolution of clinical signs and symptoms, the treatment continues in the second appointment with removal of the medicament, initiation of bleeding into the root canal, a coronal seal with a calcium silicate cement, and adhesive restoration. Besides the resolution of clinical symptoms and healing of apical periodontitis, regenerative cases offer continued root growth with responsiveness to vitality tests in some cases. Therefore, this innovative approach has the potential to improve the clinical outcome of compromised teeth otherwise susceptible to fracture (5, 6). Stem cells of the apical papilla (SCAPs) are thought to be the primary source of stem cells in REPs. Initiation of bleeding from the periapical tissues transports stem cells into the canal system (7). Maintaining stem cell viability in addition to promoting proliferation and determining their differentiation are crucial for the clinical outcome. This can only be achieved when the environment in the root canal is optimized before the introduction of stem cells. An optimized protocol of chemical disinfectants is important to determine the fate of stem cells (8–10).
From the *Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas; †Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Regensburg, Germany; and ‡Department of Restorative Dental Sciences, King Saud University, Riyadh, Saudi Arabia. Address requests for reprints to Dr Anibal R. Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2018 American Association of Endodontists. https://doi.org/10.1016/j.joen.2018.11.012
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Regenerative Endodontics Irrigation of the root canal system as well as intracanal medications could have both direct and indirect effects on cells (Fig. 1A). Direct effects result from the contact of SCAPs to the irrigant or medicament during or after their application. Indirect effects are caused by changes in the environment that could affect stem cells even after removal of the disinfectant. Although a previous study has shown the direct negative effect of chlorhexidine (CHX) on stem cell survival (10), no study has evaluated the concentration response for this toxicity. This is particularly important because CHX has been used clinically in REPs at the concentrations of either 0.12% or 2% (11). Lastly, the neutralization of this negative effect on SCAPs has never been investigated. Thus, this study aimed to evaluate both the direct and indirect effects of different concentrations of CHX on SCAP survival as well as the possibility to neutralize possible indirect cytotoxic effects using a dentin slab model.
Materials and Methods Patient Recruitment This study was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio. After verbal and written communication for informed consent, extracted teeth were immediately rinsed in sterile phosphate-buffered saline (PBS) and stored in a container with sterile 1 PBS. Apical papilla samples were dissected from immature third molars with less than two thirds of the root being formed and used in the ex vivo toxicity experiment. Additionally, extracted healthy third molars were collected and used to generate standardized dentin slabs as described later. SCAP Survival after Direct Exposure to CHX A characterized SCAP cell line was used in this study (10). Cells were seeded in a 24-well plate with each well containing 25,000 cells in 800 mL media. CHX was diluted in media in the following concentrations: 2%, 1%, 0.5%, 0.25%, 0.12%, 10 2 %, 10 3 %, 10 4 %, 10 5 %, 10 6 %, and 10 7 % (Endo-CHX; Essential Dental Systems, South Hackensack, NJ). SCAPs were exposed to either media (control) or media with different CHX concentrations and incubated at 37 C and 5% CO2 for 3 days. On day 3, SCAPs reached approximately 80% confluency in the control group. Media were removed, and SCAPs were washed with 1 PBS (Gibco/Thermo Fisher Scientific, Waltham, MA) followed by application of 200 mL CellTiter-Glo reagent (Promega, Madison, WI) to each well and incubation at room temperature for 10 minutes. To determine background luminescence, media without cells were mixed with CellTiter-Glo reagent. Relative luminescence for each group was detected in a FlexStation 3 Benchtop Multi-Mode microplate reader (Molecular Devices, San Jose, CA). To evaluate cytotoxicity on SCAPs in their original environment, apical papillae were collected and cultivated in either media only (control) or media containing 2% CHX for 3 days. After the culture period, explants were rinsed in 1 PBS and fixed with 4% paraformaldehyde, embedded in Neg-50 (Richard-Allan, Thermo Fisher Scientific), and serially sectioned with a cryostat (20 mm). Sections were placed onto Superfrost Plus slides (Thermo Fisher Scientific), air dried, and stained with the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA). Z-stack images were taken on a Nikon Eclipse 90i microscope (Nikon, Minato, Japan) equipped with a Nikon C1si laser scanning confocal imaging system using standardized settings at 20 magnification and processed in ImageJ (Fiji) software (National Institutes of Health, Bethesda, MD) (12). Kinetics of Direct CHX-evoked Toxicity on SCAPs SCAPs were seeded in a 96-well plate with each well containing 15,000 cells in 200 mL of different concentrations of CHX or media only (control); 100 mL CellTox reagent (Promega) was added to each well, and plates were incubated at 37 C and 5% CO2. During incubation, cell toxicity was evaluated using CellTox green fluorescence assay at 2, 5, 15, 30, and 60 minutes. To determine background fluorescence, media without cells were mixed with CellTox reagent. The relative fluorescence for each group was detected in a FlexStation 3 Benchtop Multi-Mode microplate reader.
Figure 1. Disinfecting agents exert direct and indirect effects on stem cells during REPs. (A) The schematic illustration shows that irrigants or medicaments do not only affect SCAPs by direct contact but also have indirect effects on introduced stem cells through contamination of dentin. (B) Direct cytotoxicity of CHX on SCAPs is concentration dependent, and concentrations above 10 3% significantly affected cell survival as indicated by asterisks (P # .05). JOE — Volume 45, Number 2, February 2019
SCAP Survival after Exposure to Irrigated Dentin Slabs (Indirect Effect) Dentin slabs were prepared similarly to previous protocols (13). Briefly, gingival and periodontal tissues were removed from the tooth surface with a sterile surgical blade. The teeth were then washed in Chlorhexidine and Survival of SCAPs
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Figure 2. Apical papilla samples were cultured in media without (control) and with supplementation of CHX (2%) for 3 days. (A) ApopTag stained hardly any apoptotic nuclei in the control samples. (B) CHX induced apoptosis throughout the papilla but especially in proximity to the surface (dotted line).
cold Hank’s Balanced Salt Solution (HBSS, Sigma-Aldrich). An IsoMet 1000 precision saw (Buehler, Lake Bluff, IL) with a diamond blade was used to generate dentin slabs (400 mm in thickness and 20 mm2 surface area) by axial cuts above the pulp chambers of molar teeth. The tooth slices were then washed twice with cold sterile HBSS, blotted dry, gas sterilized, and stored at 4 C until usage. Dentin slabs were rehydrated in HBSS for 5 minutes and irrigated with saline (control), 2% CHX, EDTA (Endo-Cleanse 17% EDTA Solution; Roydent Dental Products, Johnson City, TN), or L-a-lecithin (0.07 % L-a-Lecithin/Soybean and 0.5% Tween 80, Sigma-Aldrich) for 5 minutes. Additionally, the following irrigation sequences were established with incubation times of 5 minutes: CHX/L-a-lecithin, CHX/EDTA, and CHX/L-a-lecithin/EDTA. Slabs were then washed with saline for 5 minutes, placed in 24-well plates with 50,000 cells in 500 mL media per well, and incubated at 37 C and 5% CO2 for 5 days. After the culture time, dentin slabs were gently transferred to another plate to eliminate cells present at the well bottom outside the dentin slabs; 200 mL CellTiter-Glo reagent (Promega) was added to each well and incubated at room temperature
for 10 minutes. To determine background luminescence, media without cells were also mixed with the reagent. The relative luminescence for each group was detected in a FlexStation 3 Benchtop Multi-Mode microplate reader.
Data Analysis The relative luminescence and fluorescence units were calculated for each group after background subtraction. Data were depicted as means with standard deviations and treated nonparametrically. Results were analyzed statistically by the Krukal-Wallis test at a significance level of P # .05 using GraphPad Prism 7 (GraphPad Software, La Jolla, CA).
Results SCAP Survival after Direct Exposure to CHX CHX concentrations between 2% and 10 3% led to a significant decline in the survival of SCAPs (Fig. 1B). Lower concentrations did not show any detrimental effects or slightly induced survival and proliferation (eg, 10 6% and 10 7%) at a nonsignificant level.
Figure 3. Kinetic assessment of direct CHX toxicity on SCAPs over the initial 60 minutes revealed concentration-dependent effects, which were significant at 0.01% and above (the gray area includes nonsignificant results). Cytotoxicity was pronounced early and reached its maximum within 15 minutes after exposure.
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Figure 4. Indirect cytotoxic effects of irrigants in the dentin slab model. (A) L-a-lecithin or EDTA did not affect SCAP viability, whereas CHX conditioning of dentin significantly reduced cell survival. (B) CHX-induced cytotoxicity was neutralized by subsequent irrigation with L-a-lecithin, whereas subsequent EDTA irrigation was futile. Cytotoxic effects of CHX-conditioned dentin were also reversed when slabs were subsequently rinsed with L-a-lecithin followed by EDTA. Asterisks indicate significant differences to the untreated control (P # .05).
In the explant cultures, cells of the apical papilla became apoptotic when cultured in media containing 2% CHX, whereas papilla samples cultured without CHX showed no signs of apoptosis (Fig. 2A and B). Nuclei with DNA fragmentation predominated in the superficial regions of the papillae but were found throughout the tissue. Compared with the in vitro system, cells embedded in the apical papilla seemed more resistant to toxicity by 2% CHX.
Kinetics of Direct CHX-evoked Toxicity on SCAPs No CHX toxicity on SCAPs was detected after 2 minutes. Concentrations below 10 2% had no significant cytotoxic impact during the 60 minutes of observation, but higher amounts were detrimental to SCAPs in a concentration-dependent manner (Fig. 3). At 5 minutes postexposure, cell damage by 0.1%–2% CHX attained almost 50% of the maximum toxicity and steadily increased over time with a plateau after 30 minutes. SCAP Survival after Exposure to Irrigated Dentin Slabs (Indirect Effect) Although dentin slabs conditioned with 0.07% L-a-lecithin or 17% EDTA did not affect SCAP survival, 2% CHX treatment induced a significant decline in cell viability (Fig. 4A). This indirect cytotoxicity was fully reversed when the dentin slabs were rinsed with lecithin subsequent to CHX (Fig. 4B). However, subsequent irrigation with EDTA did not weaken CHX toxicity; cell viability was still reduced significantly. When CHX-pretreated dentin slabs were neutralized by L-a-lecithin, a final rinse with EDTA did not alter the neutralizing effect significantly.
Discussion Stem cells are an integral part of pulp tissue engineering, which aims to regenerate tissue in form and function. For a positive clinical outcome of REPs, it is crucial to establish an environment that allows stem cell survival, proliferation, and differentiation (14, 15). Currently applied protocols for regenerative endodontics successfully lead to resolution of clinical signs and symptoms as well as continued root development (5, 6). Several histologic studies described ectopic mineralized components in regenerated tissues such as cementum and osteodentin (16, 17). Lately, residual biofilm or bacterial endotoxins have been shown to be critical for the outcome of regenerative procedures (18) because they can alter the fate of stem cells and lead to an osteoblast phenotype (19, 20). Thus, further investigation and reevaluation of applied clinical protocols are needed in terms of microbial reduction and detoxification in infected root canals. JOE — Volume 45, Number 2, February 2019
Several irrigants and intracanal medicaments have been used for disinfection before regenerative procedures including CHX in concentrations of 0.12%–2% (21–23); 2% CHX in liquid form had a similar microbial performance against several microorganisms as 5.25% sodium hypochlorite (24, 25); however, it does not dissolve organic compounds. CHX solution (2%) inhibited bacterial growth after a contact time of only 15 seconds (24). Although sodium hypochlorite reportedly affected regenerative outcome (15), CHX is less toxic and malodorous (10, 24, 25) and facilitated a significantly higher release of growth factors from dentin (26). As shown previously, no viable stem cells were detected after irrigation with CHX at a concentration of 2% (10). Likewise, CHX has been shown to be toxic on SCAPs in this study at concentrations of 10 3% or higher. Cytotoxicity came into effect after a contact time of 5 minutes and increased over 30 minutes. When apical papilla explants were exposed to media with 2% CHX for 3 days, apoptosis occurred throughout the tissue with most apoptotic nuclei in proximity to the surface. The remaining viable cells, especially in the core, gave the impression that SCAPs are more resistant to CHX when encapsulated in the papilla tissue compared with the in vitro situation. In a clinical protocol, CHX is used as the root canal irrigant and comes in contact with SCAPs. Limitation of the contact time (<5 minutes) and a thorough rinse with saline are necessary to provide an environment where SCAPs can resist and survive. Once stem cells are transported into the canal space and reside in the blood clot, they are indirectly confronted with irrigants or medicaments. Residual particles or dentin-bound molecules like CHX can exert toxic effects and influence cell fate as well as the outcome of regenerative procedures. This indirect toxicity was assessed with dentin slabs conditioned with CHX for 5 minutes. Although slabs were finally rinsed with saline, cytotoxicity endured because of the substantivity of cationic CHX molecules. As shown in a previous study, the remaining CHX was detectable for up to 12 weeks after irrigating a root canal system with a 2% solution (27). This study did not evaluate the potential long-lasting effect of CHX conditioning because it tested the immediate contact of the cells with the conditioned dentin surface as seen in the clinical setting after evoked bleeding. Interestingly, the indirect cytotoxicity of CHX was reversed by a subsequent rinse with L-a-lecithin presumably because of binding of the cationic CHX to the negatively charged scavenger molecule (28). Indeed, a solution containing a detergent and a negatively charged protein has been used previously to reverse the antibacterial effects of CHX (29). Likewise, in this study, a solution of Tween and L-a-lecithin resulted in complete neutralization of CHX and abolishment of its deleterious effects on SCAPs. In fact, this approach has been successfully used to
Chlorhexidine and Survival of SCAPs
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Acknowledgments The study was funded by the University of Texas Health at San Antonio. The authors deny any conflicts of interest related to this study.
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