Introduction

The developing dentition is at risk for pulpal necrosis due to trauma and developmental dental anomalies such as dens evaginatus [1–7]. Loss of an immature permanent tooth in young patients with mixed dentition can be devastating, leading to loss of function, malocclusion, and inadequate maxillofacial development. These teeth traditionally have

A.R. Diogenes, DDS, MS, PhD (*)

N.B. Ruparel, MS, DDS, PhD

Department of Endodontics, University

of Texas Health Center at San Antonio,

San Antonio, TX, USA

e-mail: Diogenes@uthscsa.edu

been treated with apexification procedures using either long-term calcium hydroxide treatment [8, 9] or immediate placement of a mineral trioxide aggregate (MTA) apical plug [10]. Although these treatments often result in the resolution of signs and symptoms of pathosis, they provide little to no benefit for continued root development [11]. Thus, immature teeth treated with these procedures are considered in a state of “arrested development,” and no further root growth, normal pulpal nociception, and immune defense should be expected.

Regenerative endodontic procedures (REPSs) have emerged as an alternative treatment for these teeth that, in addition to healing of apical periodontitis, aims to promote normal pulpal physiologic functions. These include continued root

development, immunocompetency, and normal nociception, as seen in some published cases [12]. Thus, the ultimate goal of these procedures is to regenerate the components of the pulp-dentin complex. A significant number of case reports and case series have been published since the first reported case in 2001 [12]. These published cases document: 1) commonly observed clinical outcomes such as continued root development and sometimes normal nociceptive responses to vitality testing, 2) commonly found challenges such as technical pitfalls and unwanted adverse reactions such as coronal staining, and 3) great variability in treatment protocols [12]. Despite the lack of randomized clinical trials, these published clinical observations support the hypothesis that patients with otherwise limited treatment options could benefit from these procedures.

In 2011, a study demonstrated that a substantial number of undifferentiated mesenchymal stem cells are delivered into root canal systems following REPS [13]. This finding represented a turning point because treatment protocols previously used in REPS aimed to provide maximum disinfection without consideration for their impact on stem cells. Contemporary regenerative endodontics acknowledges and follows principles of bioengineering regarding the interplay between stem cells, scaffolds, and growth factors [14]. Since stem cells represent one of the pillars of REPS, a series of translational studies evaluating effect of disinfection on stem cell fate have been conducted. These studies have contributed to the foundational framework for the currently American Association of Endodontists (AAE) recommended regenerative endodontic treatment protocol [15]. The present chapter will focus primarily on shedding light on the studies evaluating the role of various irrigants on the survival, differentiation, and other properties of stem cells that are key to an optimal regenerative outcome.

Chemical Debridement

in Regenerative Procedures

Clinicians often face the challenge of adequately debriding large infected root canals in REPS. In these procedures, similar to conventional endodontic therapy, microbial control is crucial.

These canals with compromised fragile underdeveloped dentinal walls represent a contraindication for mechanical instrumentation; thus, chemical debridement remains the main form of disinfection in REPS. Sodium hypochlorite (NaOCl) is the most widely used agent for chemical debridement in endodontic procedures, including REPS [12]. It has several desirable characteristics including: 1) excellent bactericidal efficacy [16–18], 2) tissue dissolution capacity [19–21], and 3) effective lubrication for endodontic instruments. The first two beneficial properties are crucial for the disinfection of immature teeth in regenerative endodontic procedures, which typically involve minimal to no mechanical preparation. However, what are the effects of NaOCl on stem cells?

A study evaluated the survival of stem cells of apical papilla (SCAP) cultured in an organotype root canal model previously irrigated with various combinations of commonly used chemical agents [22]. It was found that dentin conditioning with 17 % ethylenediaminetetraacetic acid (EDTA) promoted greater survival of SCAP, whereas the use of 6 % NaOCl had a profound detrimental effect on SCAP survival. Importantly, the use of EDTA following 6 % NaOCl attenuated its undesirable effects [22] (Fig. 18.1).

Another ex vivo study by Galler et al. [23] evaluated the effects of full-strength (5.25 %) NaOCl compared to 17 % EDTA on dentin surface. Dentin cylinders used as cell carriers were subjected either to 5.25 % NaOCl or 17 % EDTA. Dental pulp stem cells (DPSCs) with biodegradable hydrogel scaffold enhanced with bioactive molecules such as heparinbinding growth factors vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGF-β1), and fibroblast growth factor-2 (FGF-2) were loaded into the cylinders which were in turn implanted into immunodeficient mice. The histological results of the study clearly demonstrated that dentin treated with 5.25 % NaOCl leads to resorption and clastic cellular activity along the dentinal walls. On the other hand, dentin conditioned with 17 % EDTA promoted the formation of pulp-like tissue with blood vessels and polarized cells that often extended processes into dentinal tubules and expressed the odontoblastic marker dentin sialoprotein (DSP) (Fig. 18.2). One study evaluated the effects of 5.25 % NaOCl or 17 % EDTA dentin

Fig. 18.1 EDTA promotes SCAP survival. Organotype immature teeth root canal models were irrigated with 20 ml of irrigant for 1 min followed by thorough rinsing with Hanks’ Balanced Salt Solution for 7 days. SCAPs were then seeded with platelet-rich plasma (PRP) scaffold into the root segments. The percentage of viable cells (vimentin positive) of total cells (TO-PRO-3 positive) were determined by confocal microscopy for each group after 21 days. 17 % EDTA group demonstrated the maximum number of viable cells followed by EDTA/NaOCl. No viable cells were seen for the EDTA/CHX and NaOCl/ EDTA/NaOCl/isopropyl alcohol (IPA)/CHX groups (Modified from Trevino et al. [22]). *** P < .001

conditioning on stem cell expression of odontoblastic markers [24]. Dentin disks were treated (conditioned) with 5.25 % NaOCl or 17 % EDTA. The expression of odontoblastic markers such as matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP) was evaluated using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). This study demonstrated that tooth slices subjected to 5.25 % NaOCl showed no expression of the abovementioned markers. However, the ones treated with 17 % EDTA showed significant increase in these markers in vitro (Fig. 18.3). Moreover, when tooth slices were implanted into the dorsum of immunodeficient mice, similar results were obtained at 14 and 28 days. It is also noteworthy that this prolonged effect of dentin conditioning with NaOCl was detected long after the irrigant had been removed, suggesting that NaOCl has a profound indirect effect on stem cell toxicity [25]. Thus, dentin conditioning with sodium hypochlorite at its maximum used clinical concentration leads to greatly diminished stem cell survival and loss of odontoblast-like cell differentiation.

Another recent study was conducted to evaluate whether other clinically used concentrations of NaOCl were conducive for stem cell (SCAP) survival and differentiation [26]. Standardized root canals were prepared in extracted human teeth. The prepared teeth were then irrigated with NaOCl at the concentrations of 6, 3, and 1.5 %. Approximately half of the samples received a second irrigation with 17 % EDTA, whereas all samples received a copious final flush with saline to remove any residual chemical from the canal space. SCAP in a hyaluronic acid hydrogel were seeded in all canals and cultured for 7 days. The number of viable cells was assessed using a luminescence assay, while the level of DSPP was assessed by real-time RT-PCR. It was found that dentin conditioning with NaOCl decreases both SCAP survival and differentiation in a concentra- tion-dependent manner. However, the concentration of 1.5 % of NaOCl was found to have minimal effects on the survival and differentiation. In addition, it was demonstrated that a final irrigation with 17 % EDTA reverses the deleterious effects of NaOCl (Fig. 18.4). Thus, this study agrees with other studies that dentin conditioning with 6 % NaOCl has a negative effect, while 17 % EDTA has a positive effect on the survival and differentiation of stem cells subsequently cultured in contact with the conditioned dentin [22, 26, 27]. The negative effects of NaOCl do not appear to be directly related to residual NaOCl in the dentinal tubules resulting in direct toxicity since neutralization with sodium thiosulfate (5 %) did not reverse this effect [26]. Thus, NaOCl has a profound effect on dentin resulting in diminished stem cell survival and differentiation. These effects can be minimized by using 1.5 % NaOCl followed by 17 % EDTA [19]. Collectively, all studies mentioned here point to the detrimental effects of fullstrength NaOCl and the beneficial effects of 17 % EDTA on dentin.

Irrigants and Dentin Matrix Growth

Factors

Important biologically active growth factors are trapped in the dentin matrix during dentinogenesis. Some of these growth factors such as VEGF

Fig. 18.2 EDTA promotes pulp-like tissue formation and DSP expression. Dentin cylinders were irrigated with 5.25 % NaOCl or 17 % EDTA. DPSCs mixed with hydrogel scaffold were loaded into the cylinders. Dentin cylinders were then implanted into immunodeficient mice. Hematoxylin and eosin staining and tartrate-resis- tant acid phosphatase (TRAP) done at 6 weeks show the presence of disorganized fibrous connect tissue and presence of large multinucleated giant cells/odontoclasts in

the NaOCl group (panels a, c, g) in the NaOCl group. Well-organized vascularized connective tissue with cells at the cell-dentin interface that appear flat and are in close contact with the dentin wall (panels b, d). Immunohistochemistry for DSP demonstrates that cells adjacent to the dentin surface stain positive for DSP, which indicates that these cells have differentiated into an odontoblast-like phenotype (panel h) (Modified from Galler et al. [23])

a

7 days

Tooth slice/scaffold

MEPE

DMP-1

DSPP

GAPDH

28 days

b

MEPE

DMP-1

DSPP

GAPDH

Fig. 18.3 EDTA promotes odontoblastic differentiation of stem cells. Scaffold without tooth slice was used as a negative control. Tooth slices were treated with 5.25 % NaOCl for 5 days (to denature dentin proteins), left untreated, or treated with 17 % EDTA for 1 min (to mobilize dentin proteins). Markers of odontoblastic differentiation, i.e., DSPP, DMP-1, and MEPE, were evaluated by RT-PCR. For in vivo studies, tooth slices were treated with the same irrigation protocol and were then loaded

with scaffold and SHED cells. They were then implanted subcutaneously into the dorsum of immunodeficient mice. After 14 or 28 days, markers of odontoblastic differentiation (i.e., DSPP, DMP-1, and MEPE) were evaluated by RT-PCR. Both studies demonstrated increased expression of all markers at in the untreated and 17 % EDTA groups whereas no expression was observed in the scaffold only and 5.25 % NaOCL groups (panels a, b) (Modified from Casagrande et al. [24])

[28] and TGFB1 [29] are known to have a robust effect on the differentiation and/or proliferation of mesenchymal stem cells. These growth factors appear particularly efficacious in promoting the proliferation of mesenchymal stem cells and directing them toward an odontoblast-like phenotype [30, 31]. Irrigants, especially NaOCl in high concentration, are known to denature these dentin-derived growth factors [32]. In an in vivo study, dental pulp stem cells (DPSCs) prolifer-

ated at higher rates and expressed higher levels of odontoblastic markers in a tooth slice model compared to DPSCs placed in scaffold only [27]. These findings suggest that morphogens, such as the many growth factors known to be present in dentin, are sufficient to promote the survival, proliferation, and importantly the differentiation of dental stem cells. EDTA is known to solubilize and mobilize these growth factors from dentin, thereby increasing their bioavailability [33, 34].

Fig. 18.4 Sodium hypochlorite decreases SCAP survival and differentiation in a concentration-dependent manner. Organotype immature teeth root canal models were irrigated with different concentrations of NaOCl following a standardized protocol that included a final wash of saline or EDTA. SCAPs were seeded into the root segments and cultured in vitro for 7 days. The percentage of viable cells were determined by a luminescence assay. NaOCl concentration-dependent decrease in SCAP survival is partially reversed by a final irrigation

with 17 % EDTA (panel a). In addition, real-time qRTPCR was used to determine the expression of the odonto- blast-like cell marker dentin sialophosphoprotein (DSPP) mRNA. NaOCl decreases DSPP expression in a concentration-dependent manner with no expression seen in the group treated with 6 %. In addition, EDTA partially reversed the negative effect of NaOCl on DSPP expression (panel b). Data are presented by % of maximum observed effect on the EDTA only-treated group (control) (Modified from Martin et al. 2014 [26])

Thus, its use may allow clinicians to harness the inductive properties of dentin-derived morphogens and growth factors normally present in dentin [35]. Therefore, the indirect negative effect of NaOCl and positive effect of EDTA on stem cell proliferation and differentiation appear to be directly related to the denaturing and solubilizing effects of these irrigants, respectively, on dentin matrix proteins. Astute clinicians must use the best available evidence to choose the combinations and concentrations of irrigants to achieve the greatest antimicrobial effect while minimizing stem cells death and loss of differentiation potential.

Stem cell survival, proliferation, and differentiation are also known to be dictated by the surface on which the cells grow [36–38]. Stem cells attach to a specific surface such as a target organ during organogenesis, or repair, via the interaction of specific cell-adhesion molecules such as integrins expressed on the plasma membrane of these cells. The effect of the substrate on stem cell behavior is best illustrated by the effect of the stem cell niche that in addition to growth factors (discussed above) pro-

vide attachment signals resulting in cell arrestment in a quiescent state [39, 40]. Cells released from their niche become “activated” and start proliferating and undergoing differentiation. The process of culturing tooth-derived stem cells such as DPSCs or SCAP is a good example of cells leaving their inhibited state in the niche (dental pulp or apical papilla, respectively) and displaying remarkable proliferative and differentiation potentials. This information has strong clinical implications since the dentin matrix composition (stem cell substrate) is altered by chemical treatment during the process of chemical debridement. NaOCl is known to cause changes in dentin matrix composition with decrease in carbon and nitrogen content and demineralization when used at high concentrations [41]. In contrast, the concentration of 1 % NaOCl does not cause any significant changes in dentin composition or mechanical properties. The property of attachment to a substrate has been evaluated using various other irrigants as well [42]. Ten treatment groups with different combinations of irrigants were used to evaluate attachment of stem cells from