An ideal endodontic sealer requires several characteristics: adhesion to dentin preventing leakage from the interface between the core material and dentin, low viscosity, good wettability to flow into the irregularities on the walls of the root canal, good physicochemical properties and biocompatibility, and stimulating the reparative and biological sealing by mineralized tissue deposition in the apical foramen 10,28,29. Most conventional root canal sealers have demonstrated inadequate biological activity and cytotoxicity in cultures, especially after fresh mix 30–32. Ever since the development of MTA and its widespread use in dentistry, several studies have concluded that MTA can be used as an endodontic sealer because of its excellent sealing properties and biological properties 33,34. Several modifications in the original formulation of MTA have been suggested in order to improve its characteristics as a root canal sealer; however, the developed MTA sealers have not yet satisfied all the ideal requirements 34,35. In our previous studies, incorporation of ELP improved the physical properties of MTA in terms of compressive strength, bonding property, wettability and flow rate 19,21. Considering these advantageous characteristics, we expected that ELP-incorporated MTA could be used as an endodontic sealer. Therefore, this study was designed to compare the physical properties of the ELP-based experimental MTA sealer with those of commercial MTA sealers, and to investigate the optimal proportion of ELP in experimental MTA sealer.
In the present study, the ELP-based experimental MTA sealer showed superior physical properties compared to commercial MTA sealers. In particular, the experimental sealer with 0.3 and 0.4 L/P ratio exhibited a higher push-out bond strength than the other groups (Fig. 1). V125E8 was selected as the functional additive ELP of the experimental sealer in this study. It has the octa-glutamic acid (E8) in the N-terminus, which is genetically constructed as a hydroxyapatite binding motif. The multi-glutamic acids make this peptide a negative charge, which increases the binding affinity to calcium ions on the dentin surface. This characteristic might have improved the push-out bond strength of the ELP-based experimental sealer. On the other hand, the 05ELP group showed significantly lower bond strength than groups 03ELP and 04ELP, and exhibited no significant difference from commercial MTA sealers. This might be due to the greater number of micro-bubbles in the fresh mixture of 05ELP compared to 03ELP and 04ELP. It is well known that more liquid generates more air bubbles in the mixtures 36. In fact, more micro-bubbles were observed in the SEM samples of 05ELP compared to 03ELP and 04ELP in this study (Fig. 4)
Increased flow rate is one of the changes in the physical properties of freshly mixed MTA caused by ELP incorporation 21. The amount of penetration through the dentinal tubules appeared to be unaffected by the increased flow rate. Group 05ELP presented lower penetration through the dentinal tubule, although it had the highest flow rate among groups. ES showed higher sealer penetration despite its lowest flow rate (Figs. 2 and 3). The penetrability of sealers through the tubules depends on many factors such as the presence of the smear layer, the chemical and physical properties of sealers, and root dentin permeability. Therefore, penetration of the sealer cannot be explained only by the flow rate. Among the ELP-incorporated MTA sealers, ELP proportion in MTA mixtures did not exhibit a direct relationship with penetration through the dentinal tubules. The 04ELP mixture showed better penetration through the dentinal tubules compared to 03ELP and 05ELP mixtures. This might be because the 03ELP mixture is too viscous and 05ELP has many micro-bubbles, which may have interfered with the penetration through the dentinal tubule despite its increased flow rate. ELP-based MTA sealers showed a small gap interface with dentin compared to commercial MTA sealers in this study (Fig. 4). The increased flow rate and chemical affinity to calcium ion of dentin hydroxyapatite may have caused more intimate contact with the dentin wall. The increased adhesion by V125E8 ELP incorporation revealed in our previous study is also considered to contribute to the enhanced contact with the dentin wall 21. The optimally high flow rate prior to setting might help the endodontic sealer to have a stable adhesion, by the quality of sealer tag formed inside the dentinal tubule and filling up of the irregularities of the root canal wall.
The ELP-based experimental sealers with various L/P ratios showed a distinguished washout resistance compared to commercial MTA sealers used in this study. In particular, at 0.3 and 0.4 L/P ratio, a superior anti-washout property was observed (Fig. 5). This result might be caused by the reversible transition phase of V125E8 based on a transition temperature (Ts) between 31° and 33 °C. V125E8 exists in a non-aggregated and highly flowable form at ambient temperature below the Ts, while it shows aggregate form and viscous characteristics above the Ts 22. In the washout resistance test, the testing temperature was set as the body temperature using a 37 °C HEPES buffer solution. At this temperature, the V125E8 solution exists in aggregate form, so that freshly mixed MTA particles are constrained in the aggregated peptide solution and would have shown notable washout resistance. Washout resistance is an important property among the requirements of an ideal sealer, because unset sealers may reach the periapical tissue beyond the apex due to flushing by body fluids, causing harmful effects on healing of inflamed periapical tissue, and breaking the hermetic seal in the apical foramen 28. The excellent washout resistance of ELP-based MTA sealer is believed to be highly beneficial in preventing leakage and healing the apical tissue, especially, when it is set at body temperature.
In this study, the experimental ELP-based MTA sealers were compared with commercial MTA sealers for few characteristics, among the various requirements for endodontic sealer. More research is needed to investigate whether the ELP-based MTA sealer meets other ideal requirements. The experimental sealer contains polypeptides as a component, therefore, it is necessary to check the immune response of living tissue, although ELP is known to be biocompatible and less immunogenic 37. In addition, in vivo tests and clinical trials will be necessary for clinical application of ELP-based MTA sealer.
Regarding the question of optimal ELP ratio in novel experimental MTA sealers, the 0.4 L/P ratio is considered to be the best among the three ratios in this study. 04ELP mixture showed the highest bond strength to dentin and presented a greater flow rate and penetration in the dentinal tubule. Specifically, 04ELP mixture exhibited remarkable washout resistance. 03ELP mixture showed higher performance in washout resistance, but lower performance in bond strength, flow rate, and penetration in dentinal tubules compared to 04ELP. 05ELP mixture showed less performance in most of the experimental outcomes of this study, except for presenting the best flow rate. Production of more air bubbles may also be another disadvantage in 05ELP mixture. The 0.4 L/P ratio might be an appropriate proportion to obtain the best properties for the requirement of an endodontic sealer.