Melatonin-doped polymeric nanoparticles reinforce and remineralize radicular dentin: Morpho-histological, chemical and biomechanical studies
Graphical abstract
Introduction
The concept of apical periodontitis involves an inflammatory disease that affects the peri-radicular tissues and bacterial presence in dental pulp. It is the most common sequel of untreated tooth decay and usually origins tooth loss. Nevertheless, apical periodontitis poses an important host defense reaction destined to confine root canal bacteria and avoid them from spreading into adjacent oral tissues [1]. The interest for strengthening the radicular dentin after periapical periodontitis, abscesses and pulpitis that may indicate endodontic treatment, has increased in the last years [2]. The structure and treatment of radicular dentin gather several peculiarities [3]. An endodontically-treated tooth is prone to fracture because the tooth structure damage caused by carious infection and pulpitis, preparation of cavity and canal instrumentation [4]. Besides, the composition of dentin is not the same across its anatomy, but it changes from the cervical down to the apical zones in root length [5].
Even when eliminating microorganisms from the root canal system before canal filling is the principal aim of endodontic treatment; sealing and remineralization should also be considered [3,6]. Indicated materials as endodontic sealers have ranged from the employ of silicate aluminum based cements [7], glass-ionomer [8], to epoxy resins for radicular canal sealers such as AH-Plus [9], but most of them have presented different clinical limitations [4]. Non-resorbable polymeric NPs doped with antimicrobials and reinforcing agents have successfully been tested in advance [10], but combined anti-inflammatory therapy has not been considered yet. The role of melatonin (ML) in hard tissues has attracted attention [11]. ML (N-acetyl-5-metoxy-tryptamine) is an indoleamine that is synthesized and secreted by the pineal gland in a circandian pattern [12]. Melatonin is also formed in perhaps all organs in quantities by orders of magnitude higher than in the pineal gland and in the circulation [13]. ML may be involved in the development of hard tissues, as bone and teeth [14]. ML increases alkaline phosphatase activity, dentin sialoprotein expression and mineralized matrix formation, all of them involved in dentin remineralization. ML may also regulate dentin formation and tooth development [15]. The generation of reactive oxygen species is always associated with inflammation. It has been demonstrated that, in several endodontic pathologies such as periapical abscess and pulpitis, oxidative stress is an important pathogenetic mechanism [1]. The free radicals that generate reactive oxygen or nitrogen species are considered to be highly destructive, but directly neutralized by ML [12].
As stated, ML has been used because of its anti-inflammatory, antioxidant and free-radical-scavenger properties [16,17] and cytoprotective actions [18,19]. A decline in the production of inflammatory mediators, by regulating NF-kB activity, occurs when there is high concentration of ML and contributes to signaling pathway. Although the beneficial effects of ML on periodontal regeneration has been demonstrated in gingival fibroblasts and in vivo and in vitro animal models [18], its effects in radicular dentin regeneration have not been reported yet. The circulating half-life of ML is approximately 23 min [20]. Hence, few authors have recommended the use of carriers in ML in order to release it gradually and increase the half-life in the tissues. Constant release of ML by the use of poly-lactic-co-glycolic acid microspheres has been demonstrated to differentiate human mesenchymal stem cells into osteoblasts. Novel ML delivery systems such as ML microspheres and bone-regenerating scaffolds have shown great promise for use in regenerative medicine and dentistry, specifically bone-grafting procedures, to inspire new bone formation [21], but the release kinenics in all these studies is unknown. Controlled delivery of drugs is determinant for the radicular dentin treatment trying to reinforce the tissue [22]. Currently, this can be attained as a result of the increased use of nanoparticles (NPs) and the rapid advances of nanotechnology [23,24]. Novel polymeric NPs with anionic carboxylate (i.e., COO−) groups placed along the backbone of the polymer, that may be loaded with calcium (Ca-NPs), zinc (Zn-NPs) or doxycycline (D-NPs) have been previously synthesized to favor dentin remineralization of endodontically treated teeth [4]. However, in some cases in which apical periodontitis exists it may be valuable to consider the anti-inflammatory, antioxidant and possible remineralizing effects exerted by ML.
Histomorphometric, physical, chemical and mechanical analysis of dentin performance under the administration of exogenous melatonin would be very helpful to tentatively elucidate and confirm the therapeutic role of melatonin in dentin strengthening. Thereby, the present study was aimed to examine the possibility that melatonin might exert its influence on root dentin remineralization. Hence, the purpose of this investigation was to evaluate remineralization and dentin permeability after dentin infiltration with experimental ML-loaded NPs before the endodontic sealer placement. The tested null hypothesis was that melatonin-doped NPs application did not affect, at the short term or overtime, dentin micropermeability, neither remineralization of the endodontically treated radicular dentin.
Section snippets
Nanoparticles fabrication
PolymP-n Active nanoparticles (NPs) (NanoMyP, Granada, Spain) were obtained through polymerization precipitation. In order to control the process of precipitation, a thermodynamic approach has been used: the Flory-Huggins model based on the Hansen solubility parameters. The model was developed based on the growth of polymeric chains and solvent molecules interactions by hydrogen-bonding, polar and dispersion forces [25]. The NPs are designed with 2-hydroxyethyl methacrylate as the backbone
Melatonin liberation profile
The melatonin liberation profile curve is shown in Fig. S2. It corresponded with the following amount of melatonin expressed in mg/mL at each time point: 24 h, 1.85; 48 h, 0.65; 3 d, 0.37; 7 d, 0.13; 14 d, 0.13; 28 d, 0.033.
Sealing ability trough the fluid filtration system
The fluid filtration rate (μL min−1) at distinct storage time for all groups is shown in Fig. 1. Significant differences (P < 0.05) were found between groups. All groups showed the same microleakage values after 24 h and 1 w of storage. Control samples promoted the highest
Discussion
ML-NPs achieved root dentin sealing and remineralization, as they occlude dentinal tubules and attain reinforcement of the radicular dentin structure, but did not affect fluid filtration at the dentin interface. Therefore, the null hypothesis that ML-NPs application did not affect, at the short term or overtime, dentin micropermeability neither remineralization of the endodontically treated radicular dentin, must be partially rejected.
NPs application (undoped or ML-doped) reduced liquid flow if
Conclusions
Sealing and remineralization of the radicular dentin was not efficient at the control group, as a high quantity of the dentin tubules remained open and the highest microleakage was obtained over time. The use of melatonin promoted total occlusion of dentinal tubules and an almost complete reduction of fluid flow, exhibiting the highest sealing ability among groups. Non-functional remineralization was produced at the control group, based on the new deposits of immature and imperfect
Acknowledgements
This work was funded by: 1) the Ministry of Economy and Competitiveness and European Regional Development Fund (MAT2017-85999P MINECO/AEI/FEDER/UE).
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