Regenerative endodontics model in immature infected rat molars using two step protocol

Aim: to develop a model for regenerative endodontics using newly-weaned Wistar rats immature molars with pulp necrosis to histologically describe the evolution of apical tissues following treatment with a bi-antibiotic paste, induced bloodclot formation and MTA. Methods: Ten 25-day-old female Wistar rats were divided into an initial control group (Ci) and two experimental groups in which pulp necrosis was experimentally induced on the left mandibular first molar by exposing the pulp chamber and leaving it open to the oral environment. One of the experimental groups was left untreated (E1) while the other was submitted to a protocol of regenerative endodontics 10 days thereafter (E2). Fifteen days after placement of a bi-antibiotic paste, bleeding was induced into the root canal space and MTA was placed upon. Animals were euthanized 30 days later. Right mandibular first molars served as an 80-day-old final control group (Cf). Each hemimandible was histologically processed to analyse parameters associated with root development. Statistical analysis was carried by means of ANOVA; p values below 0.05 were considered statistically significant. Results: baseline (i.e. 25-days old) mean root length and apical diameter of the distal root canal were 1.84±0.25 and 0.38±0.02mm respectively. Following the regenerative endodontic protocol, cells lining the walls of the root canal and significant increase to both length (2.37±0.22mm) and diameter (0.32±0.03 mm) were observed. Conclusions: newly-weaned Wistar rats serve as a suitable model to evaluate regenerative endodontic protocols. However, further research is needed in order to disclose the nature of the cells and/or cell mediators involved

Methodological considerations for a model of endodontic treatment in Wistar rats / Consideraciones metodológicas para el modelo de tratamiento endodóntico en ratas Wistar

ABSTRACT The use of correctly designed animal models is a fundamental step prior to clinical trials in humans. Although rats are easy to house and handle, and have molars that resemble those of humans, very few researchers use them as a model for root canal treatment, probably due to their small size and the lack of relevant data necessary to reproduce the model. Our aims were to describe the anatomic and histologic characteristics of the mandibular first molar of the Wistar rat and present a standardised model for its experimental endodontic treatment. Twenty female rats were used. The characteristics of the mesial and distal roots were described histologically and the quality of the results achieved following the treatment protocol presented herein was assessed by means of digital radiographs, micro-CT and histological sections. The age of 55 days was found to be the most adequate for performing this technique, but we consider the interval of 50 to 60 days to be suitable. Both canals are oval, although in opposite planes, and the furcating-facing walls present the minimum dentine thickness. It was essential to become familiar with these aspects in order to decide upon the most appropriate instrumentation and obturation techniques that would enable replication of this model in basic science research.

RESUMEN El uso de modelos animales correctamente diseñados es un paso fundamental previo al desarrollo de ensayos clínicos en humanos. A pesar de resultar fáciles de criar y manipular y de poseer molares que se asemejan a los humanos, muy pocos grupos utilizan a la rata como modelo experimental para el tratamiento endodóntico probablemente debido a su pequeño tamaño y a la escasa información disponible para poder aplicar los modelos existentes. Nuestros objetivos fueron describir las características anatómicas e histológicas del primer molar inferior de la rata Wistar y presentar un modelo estandarizado para el tratamiento endodóntico experimental de esta pieza. Se utilizaron 20 ratas hembra. Las características de las raíces mesial y distal fueron descritas histológicamente y los resultados obtenidos fueron evaluados mediante radiografías digitales, microCTy cortes histológicos. La edad de 55 días demostró ser la más adecuada para ejecutar la técnica, pero consideramos que el intervalo de 50 a 60 días puede resultar apropiado. Se observó que ambos canales presentan una morfología oval, aunque en direcciones opuestas, y que las paredes furcales resultaron ser las que presentan el menor espesor de dentina. Familiarizarse con estos aspectos de la anatomía e histología del molar de la rata resultó fundamental para decidir sobre las técnicas de preparación y obturación más apropiadas que permitieran la replicación de este modelo en el campo de las ciencias básicas.

Methodological considerations for a model of endodontic treatment in Wistar rats.

The use of correctly designed animal models is a fundamental step prior to clinical trials in humans. Although rats are easy to house and handle, and have molars that resemble those of humans, very few researchers use them as a model for root canal treatment, probably due to their small size and the lack of relevant data necessary to reproduce the model. Our aims were to describe the anatomic and histologic characteristics of the mandibular first molar of the Wistar rat and present a standardised model for its experimental endodontic treatment. Twenty female rats were used. The characteristics of the mesial and distal roots were described histologically and the quality of the results achieved following the treatment protocol presented herein was assessed by means of digital radiographs, micro- CT and histological sections. The age of 55 days was found to be the most adequate for performing this technique, but we consider the interval of 50 to 60 days to be suitable. Both canals are oval, although in opposite planes, and the furcating-facing walls present the minimum dentine thickness. It was essential to become familiar with these aspects in order to decide upon the most appropriate instrumentation and obturation techniques that would enable replication of this model in basic science research.

Este estudo in vitro teve objetivo de avaliar o efeito de protocolos clareadores sobre a rugosidade de superfície (Ra), alteração de cor e micromorfologia de resina bulk-fill de baixa viscosidade (Filtek Bulk Fill Flow, 3M ESPE), alta viscosidade (Filtek Bulk Fill, 3M ESPE) e de uma resina composta nanoparticulada (controle) (Filtek Z350 XT, 3M ESPE). Quarenta amostras de cada resin composta (discos de 5 mm de diâmetro e 2 mm de espessura) foram aleatoriamente divididas em quatro grupos , de acordo com protocolo clareador (n=10): a) Gel de peróxido de carbamida (Opalescence, Ultradent Products) (2 horas/dia, por 14 dias); b) Gel de peróxido de hidrogênio (Opalescence Boost, Ultradent Products) (3 sessões de clareamento, uma por semana, 45 min/sessão); enxaguatório clareador (Listerine Whitening Extreme, Johnson & Johnson) (2 min/dia, por 14 dias); d) água destilada (controle). As amostras foram submetidas a leituras, em triplicata (Ra e cor (parâmetros CIELab) antes e depois do contato com os protocolos clareadores. A micromorfologia de superfície foi conduzida em microscópio eletrônico de varredura (MEV). Ra e parâmetros de cor (ΔL, Δa, Δb e ΔE) foram analisados por modelos lineares generalizados (α=0.05). A Ra da resina bulk-fill de alta viscosidade foi significantemente superior do que para os outros compósitos (p<0.05). A Ra aumentou significantemente (p<0.05) e a superfície ficou mais irregular (MEV) para todos os compósitos, independente do protocolo clareador (p<0,05). A resina bulk-fill de alta viscosidade obteve menor ΔE (p<0.05) do que a resina composta nanoparticulada, imersa em água destilada. Pode-se concluir que as características de cada resina composta influenciaram de forma mais significativa a Ra do que o protocolo clareador. A resina bulk fill de alta viscosidade apresentou menor alteração de cor.

Effect of periapical inflammation on calcium binding proteins and ERK in the trigeminal nucleus.

Peripheral inflammation induces plastic changes in neurons and glia which are regulated by free calcium and calcium binding proteins (CaBP). One of the mechanisms associated with the regulation of intracellular calcium is linked to ERK (Extracellular Signal-Regulated Kinase) and its phosphorylated condition (pERK). ERK phosphorylation is important for intracellular signal transduction and participates in regulating neuroplasticity and inflammatory responses. The aim of this study is to analyse the expression of two CaBPs and pERK in astrocytes and neurons in rat trigeminal subnucleus caudalis (Vc) after experimental periapical inflammation on the left mandibular first molar. At seven days post-treatment, the periapical inflammatory stimulus induces an increase in pERK expression both in S100b positive astrocytes and Calbindin D28k positive neurons, in the ipsilateral Vc with respect to the contralateral side and control group. pERK was observed coexpressing with S100b in astrocytes and in fusiform Calbindin D28k neurons in lamina I. These results could indicate that neural plasticity and pain sensitization could be maintained by ERK activation in projection neurons at 7 days after the periapical inflammation.

La inflamación periférica induce cambios plásticos en las neuronas y en la glía, los cuales están regulados por el calcio libre y las proteínas fijadoras calcio (CaBP). Uno de los mecanismos asociados con la regulación del calcio intrace-lular está vinculado con la fosforilación de la pro teína quinasa ERK. Asimismo, ERK fosforilado es importante para la trans-ducción de señales intracelulares y participa en la regulación de la neuroplasticidad y las respuestas inflamatorias. El objetivo de este estudio es analizar la expresión de dos CaBPs y pERK en astrocitos y neuronas del subnúcleo caudal del trigémino (Vc) después de una inflamación periapical experimental en el primer molar inferior izquierdo en ratas. A los siete días posteriores al tratamiento, el estímulo inflamatorio periapical induce un aumento en la expresión de pERK, en el número de astrocitos positivos para la proteína marcadora astroglial S100b y en neuronas positivas para Calbindina D28k, en el Vc ipsilateral respecto del lado contralateral y el grupo de control. Además, se observó coexpresión de pERK tanto en astrocitos S100b positivos, como en neuronas fusiformes Calbindin D28k positivas, de la lámina I. Estas observaciones podrían indicar que la neuroplasticidad y la sensibilización al dolor podrían mantenerse mediante la activación de ERK en las neuronas de proyección a los 7 días de la inflamación periapical.

Effect of periapical inflammation on calcium binding proteins and ERK in the trigeminal nucleus / Efecto de la inflamación periapical sobre las proteínas fijadoras de calcio y ERK en el núcleo trigeminal

Peripheral inflammation induces plastic changes in neurons and glia which are regulated by free calcium and calcium binding proteins (CaBP). One of the mechanisms associated with the regulation of intracellular calcium is linked to ERK (Extracellular Signal-Regulated Kinase) and its phosphorylated condition (pERK). ERK phosphorylation is important for intracellular signal transduction and participates in regulating neuroplasticity and inflammatory responses. The aim of this study is to analyse the expression of two CaBPs and pERK in astrocytes and neurons in rat trigeminal subnucleus caudalis (Vc) after experimental periapical inflammation on the left mandibular first molar. At seven days post-treatment, the periapical inflammatory stimulus induces an increase in pERK expression both in S100b positive astrocytes and Calbindin D28k positive neurons, in the ipsilateral Vc with respect to the contralateral side and control group. pERK was observed coexpressing with S100b in astrocytes and in fusiform Calbindin D28k neurons in lamina I. These results could indicate that neural plasticity and pain sensitization could be maintained by ERK activation in projection neurons at 7 days after the periapical inflammation.

La inflamación periférica induce cambios plásticos en las neuronas y en la glía, los cuales están regulados por el calcio libre y las proteínas fijadoras calcio (CaBP). Uno de los mecanismos asociados con la regulación del calcio intrace-lular está vinculado con la fosforilación de la pro teína quinasa ERK. Asimismo, ERK fosforilado es importante para la trans-ducción de señales intracelulares y participa en la regulación de la neuroplasticidad y las respuestas inflamatorias. El objetivo de este estudio es analizar la expresión de dos CaBPs y pERK en astrocitos y neuronas del subnúcleo caudal del trigémino (Vc) después de una inflamación periapical experimental en el primer molar inferior izquierdo en ratas. A los siete días posteriores al tratamiento, el estímulo inflamatorio periapical induce un aumento en la expresión de pERK, en el número de astrocitos positivos para la proteína marcadora astroglial S100b y en neuronas positivas para Calbindina D28k, en el Vc ipsilateral respecto del lado contralateral y el grupo de control. Además, se observó coexpresión de pERK tanto en astrocitos S100b positivos, como en neuronas fusiformes Calbindin D28k positivas, de la lámina I. Estas observaciones podrían indicar que la neuroplasticidad y la sensibilización al dolor podrían mantenerse mediante la activación de ERK en las neuronas de proyección a los 7 días de la inflamación periapical.

Nicotinamide adenine dinucleotide phosphate/neuronal nitric oxide synthase-positive neurons in the trigeminal subnucleus caudalis involved in tooth pulp nociception.

Sensory information on facial structures, including teeth pulp, periodontium, and gingiva, is relayed in the trigeminal complex. Tooth pulp inflammation constitutes a common clinical problem, and this peripheral injury can induce neuroplastic changes in trigeminal nociceptive neurons. There is considerable evidence that the trigeminal subnucleus caudalis (Vc) is the principal relay for trigeminal nociceptive information as well as modulation of the painful stimuli. Glutamatergic primary afferents innervating the tooth pulp project to the most superficial laminae of the Vc. N-methyl-D-aspartate receptor stimulation leads to the activation of the enzyme nitric oxide synthase (NOS), which synthesizes the free radical nitric oxide (NO). This enzyme is expressed mainly in lamina II interneurons, and in a small number of cells in lamina I as well as in deep laminae projection neurons of Vc. In the present study, we analyzed the temporal changes in neuronal NOS (nNOS) in Vc local circuitries after unilateral intermediate molar pulp injury. Our results demonstrate that a peripheral dental pulp injury leads to neuroplastic changes in the relative amount and activity of nNOS enzyme. Moreover, after a period of time, the nitrergic system shifts to the initial values, independently of the persistence of inflammation in the pulp tissues.

Pulpar tooth injury induces plastic changes in S100B positive astroglial cells in the trigeminal subnucleus caudalis.

Pain in humans constitutes a complex perception that involves peripheral tissues, nerves and the central nervous system. The glia is thought to be involved in the propagation and modulation of painful stimuli, as well as for the neurotransmission and plastic changes. In this work, an intermediate inflammatory nociceptive stimulus was generated after the exposure of the pulp in the left mandibular first molar. We analyzed the relationship between this tooth injury and the S100B expression in the trigeminal subnucleus caudalis. A significant difference of S100B immunoreactivity between the ipsilateral and the contralateral side could be observed at the 4th postoperative day. Besides, S100B positive-astrocytes located in the ipsilateral side showed an increase in their arborization, but not in the level of expression of the S100B protein. Our findings suggest that a unilateral peripheral nociceptive stimulus produces modifications in S100B positive-astrocytes in both sides of the trigeminal subnucleus caudalis.