Root canal dressings for revascularization influence in vitro mineralization of apical papilla cells

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP – ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.


Introduction
Endodontics is the branch of dentistry that deals with the treatment of diseases affecting the dental pulp and apical tissues. The treatment of permanent immature teeth with necrotic pulp remains as one of the most important challenges for contemporary Endodontics 1 . For many decades, dentists have used calcium hydroxide to aid in the formation of a calcified barrier in the apical region of an immature root 2  Currently, revascularization has been proposed as a new treatment protocol for immature teeth with pulpal necrosis 6 . In this context, a blood clot is induced into the pulpal cavity after root canal disinfection with intracanal antimicrobials. This treatment is known for leading to an increase in root thickness and eventually in length as well, resulting in a better prognosis for the structural outcome of these teeth 4,6 . The main mechanism involved in these procedures is suggested as the migration of mesenchymal cells from apex surrounding tissues into the canal lumen, giving rise to the mineralized tissue formation 7 . Nevertheless, the success of this kind of therapy depends simultaneously on the maximum disinfection of pulpal cavity and host cells function maintenance 8 .
Since root canal walls are not mechanically cleaned during revascularization treatment, irrigation and intracanal dressings have a pivotal role in pulpal cavity disinfection. The induced blood clot then brings the apical tissue cells into the disinfected pulpal cavity, which, in turn, differentiates and results in hard tissue deposition inside the dentinal walls 7 . Due to its high pH, calcium hydroxide was initially discouraged as an intracanal dressing for revascularization treatment 9 , and triple antibiotic paste was proposed as an interappointment dressing instead 6 . However, in vitro studies have demonstrated higher biocompatibility of calcium hydroxide compared to other antibiotic combinations in both direct 10 and indirect cytotoxicity 11,12 . On the other hand, in a clinical study comparing the triple antibiotic paste with calcium hydroxide, Bose, et al. 13 (2009) found higher increase in dentin wall thickness for TAP when compared to CH.
These data suggest the need to understand not only cytotoxicity, but also the late effect of the medications on the mineralization potential of APCs. TAP 14 was proposed as the first choice for intracanal dressing at revascularization protocols 6 . However, cytotoxicity studies pointed to a significant toxic effect of antimicrobials on APC in vitro 10,15 , specially due to minocycline 16 , which, in turn, is also responsible for the discoloration caused by TAP 17 . Additionally, TAP has already been observed to negatively modulate SCAP differentiation in vitro, even at low concentrations 15 .
Considering these issues together, the proposed formula, which replaces minocycline with cefaclor 10,18 , was used in this study (modified TAP -mTAP). Despite  Contrastingly, in periodontal ligament fibroblasts, TAP was observed to be more cytotoxic than CH, and it was also able to induce interleukin-6 mRNA in a pro-inflammatory context 16  In summary, when considered together, these data suggest that CH and antibiotics containing pastes might differentially modulate inflammatory mediators and growth factors that, in turn, would control the differentiation rate of APCs in vitro. The role of these substances on the milieu of growth factors released by this cell population is an issue that requires further investigations.
In conclusion, our study demonstrated that, while CH induces cellular proliferation and improves early in vitro mineralization, mTAP was found to be more cytotoxic and to decrease the differentiation potential of APCs in vitro.