INTRODUCTION

Animal models are fundamental in dental research, especially in the field of Endodontics, as many aspects of periapical tissues and their response and healing impose an ethical barrier for their assessment in humans, and they are better explored in animal models that allow histological evaluation. These models include different species, from rodents ^1-4 to animals higher up in the phylogenetic scale such as dogs ^5-7 and primates ⁸.

Wistar rats were selected in the present study because they can be easily housed and handled, and enable histological sections to be obtained relatively fast, at a lower acquisition and maintenance cost, with fewer breeding, management and ethical concerns. Furthermore, the reaction of their pulpal³ and periapical tissues ⁹ is similar to that of humans, which makes them a suitable option for experimental models in Endodontics.

Albino rats lack primary dentition -they are monophyodont- and present four permanently-growing incisors with a wide-open apex separated from the three molars in each quadrant by a large diastema. Interestingly, rat molars present developmental, anatomical, histological and physiological features similar to those of human teeth ¹⁰. The mandibular first molar of the rat is the largest and has four roots-one mesial, one distal and two smaller central ones on the buccal and lingual side, respectively ¹⁰. In 1967, the research group led by Erausquin at the School of Dentistry of the University of Buenos Aires described a protocol for performing the root canal treatment of the mesial root of this molar, in accordance with the existing endodontic procedures, in order to study the reaction of the periapical tissues in response to different root canal sealers ⁴. However, to date, few research groups have used the rodent model of experimental endodontics in vivo in their lines of research, which can range from periapical repair^1,2 to post-endodontic pain ¹¹. In fact, when it comes to evaluating endodontic sealers, subcutaneous implantation models are preferred^12,13. It is likely that the scant research using this model is largely due to the difficulty of the technique and the lack of studies describing a standardised protocol according to age and size of the animals, as well as to the lack of information on the morphology of the root canal system of the rat molar. Such information is vital to predictable endodontic treatment ¹⁴, since what is not known cannot be adequately treated.

Given the need to present relevant data regarding the reproducibility of the model, the aim of the present study was to describe the anatomic and histologic characteristics of the mandibular first molar of the Wistar rat and to develop a standardised model for experimental root canal treatment of this tooth.

MATERIALS AND METHODS

The experimental protocol and use of animals in the present study was authorised by the Institutional Committee for the Care and use of Laboratory Animals of the School of Dentistry, University of Buenos Aires (CD No. 012/2016 CICUAL - ODON/ FOUBA) and all procedures were performed in accordance with the 2011 8^th edition of the National Research Council Guide for the Care and Use of Laboratory Animals. Animals were housed in cages in groups of two or three per cage at the animal facility in the Department of Histology and Embryology ofthe School of Dentistry of University of Buenos Aires under 12:12 h light/dark cycles, and allowed ad libitum access to food (standard chow, Cooperación, Buenos Aires, Argentina) and water.

Root canal treatment procedure in experimental animals

For the second stage of this study, aimed at standardising the endodontic procedure, 50- to 60-day-old healthy female Wistar rats (n=5) weighing about 180 g were used. Animals were anaesthetised with a combination of 50 mg/kg of ketamine (Ketamid; Holliday-Scott S.A., Beccar, Buenos Aires, Argentina) and 10 mg/kg of xylazine (Kensol; König S.A., Avellaneda, Buenos Aires, Argentina) administered i.p. in order to attain deep levels of anaesthesia. Procedures were carried out using a stretcher that incorporated original modifications to the one described by Erausquin in 1967 ⁴: once anaesthetised, the animal is laid in dorsal position and its head immobilised by the upper incisors, which are placed through a metal wire attached to the headrest. Mouth opening, tongue separation and mandibular first molar isolation are achieved by an ad hoc aluminium clamp with projections that grip the first molar and an orifice for securing the lower incisors. This makes the mandibular first molar readily accessible (Fig. 1) and enables its isolation, enhanced by the placement of light-cured resin barrier. All procedures can thus be performed without difficulty. Prior to endodontic access, the surface of the isolated molar was cleaned using 0.5% sodium hypochlorite. The mesial pulp horn was then exposed by drilling a small hole through the roof of the pulp chamber using a size 1/4 round dental bur mounted on an electric engine-driven micromotor and placed on the mesial pit with a disto-mesial inclination. Using the same bur, the distal pulp horn was immediately exposed and a size 1/2 round dental bur was used to refine the access until all four root canal orifices were visible. While performing these procedures, it is important to bear in mind that the cusp tips are separated from the pulp chamber floor by only 1.5-2 mm, so care is required to avoid perforation (Fig. 2).

Fig. 1 A metal wire holds the upper incisors, immobilising the head, while a specially designed clamp integrated into the stretcher fixes the position of the lower jaw, keeping the mouth open and displacing the tongue and cheek, thus making the intact mandibular first molar clearly visible and accessible for access and further preparation and obturation. 

Fig. 2 a) Diagram of the occlusal and pulp chamber topography of the mandibular first molar and the size of the crown. Adequate refinement of the endodontic access enables visualisation of the mesial, distal, and the smaller buccal and lingual root canal orifices. M: mesial, L: lingual. Grey dots mark the starting location for penetration of the pulp chamber roof. b) Diagram of the internal anatomy of the mandibular first molar. The distance between the molar cusp tips and the pulp chamber floor is 1.5 mm and from this point to the root apex, 2.5 mm. Note the scarce dentine thickness separating the pulp chamber from the furcal area, resulting in high risk of perforation during the access phase. Buccal and lingual roots are not depicted. c) Radiograph of a 55-day-old rat hemimandible (ex vivo) showing files placed in the mesial and distal canals at a working length of 4 mm. 

Initial catheterisation was performed using a #08 K-file (Dentsply Sirona, Ballaigues, Switzerland) and a working length of 4 mm (Fig. 2), and confirmed using an electronic apex locator (Root ZX mini; J Morita corp., Tokyo, Japan). Root canal enlargement was performed using K-files #10 to #25 with in and out and clockwise and counter-clockwise rotation motions. At each change of instrument, canals were irrigated with 0.3 ml of freshly-prepared 2.5% sodium hypochlorite delivered by means of a carpule dental syringe and an extra-short 30G needle (Misawa Medical Industry, Kasama City, Ibaraki Prefecture, Japan) while aspirating it with a micro-cannula (Roeko SurgiTip-Endo; Coltene/ Whaledent, Altstätten, Switzerland). In central canals -buccal and lingual- only #08 and #10 K-files were used in order to achieve pulp removal without further enlargement, given the thinness of their walls.

Canals were then dried using sterile paper points #25 and filled with a #25 gutta-percha master cone, accessory FF cones (Meta Biomed; Cheongju, North Chungcheong, South Korea) -both previously decontaminated in 2.5% sodium hypochlorite- and a ZOE-based sealer (Grossman's Root Canal Sealer; Farmadental, Buenos Aires, Argentina) using cold lateral condensation technique and paying special attention to the distinctive morphology of each canal. After sectioning the filling material to the cervical level with an adapted heated #40 digital plugger (VDW, München, Germany) and vertically compacting it, the pulp chamber was filled with light-cured glass-ionomer cement (SDI Riva, Victoria, Australia).

After recovery from anaesthesia, animals were monitored for signs of discomfort or pain, including alteration of feeding and hydration patterns and behaviour, to detect any unexpected post-treatment reaction. If any of the animals showed signs of severe pain according to the score suggested by Wolfensohn and Lloyd ¹⁵, or any signs of infection, they were to be removed from the experimental group and administered antibiotic and/or analgesic medication. However, this was not necessary, as all operated animals returned to normal behaviour, including feeding and social interaction, after the first day post-treatment. After two weeks, animals were euthanised by i.p. injection of a lethal dose of pentobarbital sodium (Euthanyle; Brouwer S.A., Buenos Aires, Argentina) at approximately the same time of the day in order to minimise the effect of changes owed to circadian rhythm. Right and left hemimandibles were dissected and fixed in buffered formalin (pH 7.3-7.5) for 48 h.

RESULTS

Histomorphometric description of the mandibular first molar

The histologic characteristics of the roots of the mandibular first molar of 30, 55 and 75 day old rats were analysed. In 30 day old animals, root development was still incomplete, with thin root canal walls, a wide root canal, a wide-open apex and the presence of Hertwig's Epithelial Root Sheath (HERS) (Fig. 3a). Animals aged 55 days had a completely developed apex with no evidence of HERS (confirming that the radicular dentine had reached its maximum length), cementum lining the external surface and increased root canal wall thickness (Fig. 3b). In older animals (75 days old) further cellular cementum deposition occurred at the apex, sometimes invading the apical third of the canal, thus increasing radicular length and altering apical morphology (Fig. 3c). The length of the mesial and distal roots did not differ significantly. However, mean root length increased progressively and significantly from 30 days (1.9±0.24mm), to 55 days (2.5±0.18mm) and up to 75 days of age (3.1±0.11mm) (Fig. 4).

Fig. 3 Microphotographs of HE stained histological sections of the distal root of the mandibular first molar depicting different stages of root development. In 30-day-old animals a) root development is still incomplete and Hertwig's Epithelial Root Sheath (HERS) can still be observed. By 55 days of age b), radicular dentine has been completely laid down, with cementum laid parallel and externally to it and no evidence of HERS. In 75-day-old animals c) greater amounts of cellular cementum can be seen at the root apex, increasing radicular length and often invading the apical third of the root canal, thereby altering canal morphology. Bars represent 100 gm. 

Fig. 4 The graph shows the mean length and SD (in mm) of the mesial and distal roots of the mandibular first molar No differences were found between roots within each age group but root length was found to increase significantly with age (p<0.0001). 

These histological observations led us to rule out the 30- and 75-day-old groups from further endodontic protocol experiments and to select the 55-day-old group for the rest of the radicular analysis and experimental root canal treatment. In these animals, in addition to root length, root canal width and wall thickness of both mesial and distal root canals were measured in both orientations (MD and BL) and all three radicular thirds (cervical, middle and apical).

The mean width (gm) of the mesial root canal was significantly greater in the MD direction (MD: 293±120 vs BL: 165±54, _p<0.0001), while the distal root canal was found to be wider in the BL direction (MD: 192±69 vs BL: 398±147, ^<0.0001) (Fig. 5). The analysis of all four root canal walls (furcal, external, buccal and lingual) showed that the relationship between dentine and cementum is inversely proportional in each of the three thirds as the greatest thickness corresponds to dentine in the cervical third and cementum in the apical third. Considering these parameters, we can state that the total wall thickness (gm) remains constant (without significant differences between thirds) showing a progressive decrease in dentine and increase in cementum thickness from the cervical to the apical third (p<0.0001 between thirds of each root canal wall) (Fig. 5 and Table 1).

Fig. 5 Microphotographs of HE stained histological sections of the mesial and distal roots of the mandibular first molar Bucco-lingual sections of both roots show an even thickness of buccal and lingual walls while mesio-distal sections reveal that furcationfacing walls are notably thinner. B: buccal, L: lingual, E: external, F:furcal Original magnification 40X. Bars represent 100 µ. 

Table 1 Root canal wall thickness 

Since preserving dentine is an important aspect of endodontic shaping procedures, dentine thickness (gm) was analysed within each root (MR and DR) in each of the three thirds (C, M and A) and considering all four walls (F, E, B and L). The furcal wall was found to be the thinnest in both roots, and this was significantly more noticeable in the cervical third of the mesial root (MRC: F: 154±27, E: 256±33, L: 233±32, B: 237±19, _p<0.0001) and the middle third of the distal root: F: 98±10, E: 176±20, L: 237±43, B: 184±42, p<0.0001) (Fig. 5 and Table 1).

Post-endodontic observations

The average time needed to perform each root canal treatment was 40 minutes, from the time of drilling for endodontic access to placing the coronal seal. When the animals were euthanised (2 weeks posttreatment), all coronal seals were intact and in place. Mesial and distal root canal fillings showed satisfactory radiographic or micro-tomographic three-dimensionality and adequate length, and there was no evidence of surrounding alveolar bone loss (Fig. 6). Histological observations were consistent with the aforementioned. Furthermore, no pulpal remnants were found in treated animals and the apical limit was confirmed to be correct (Fig. 7), demonstrating satisfactory achievement of the physical and chemical goals of chemomechanical preparation. The presence of remaining dentine surrounding the filling material proved that the calibre of the preparation was in accordance with the internal anatomy.

Fig. 6 Periapical radiograph of an endodontically treated a) molar depicts achievement of adequate working length and proper filling of both distal and medial root canals. This was confirmed by micro-tomographic slices that also enabled more detailed assessment of the condition of the surrounding bone. Sagittal slices b) confirmed that no stripping of the canal walls had occurred, which is consistent with an intact interradicular bone, while coronal slices of the distal c) and mesial d) roots showed correct adaptation of the obturation mass to the root canal walls and only small pores within it, even in the distal root c) which is fairly wide in the bucco-lingual plane. In some cases, a minor extrusion of sealer was seen through the foramen of both distal and mesial roots, but it seems not to have caused loss of apical bone. 

Fig. 7 Microphotographs of HE stained histological sections of a control a, c, d) and an endodontically treated molar b, e, f). Comparison shows that the preparation technique achieved satisfactory elimination of dental pulp tissue at an adequate working length and an apical size in accordance with the anatomy of each root. Original magnification 20X (a-b) and 100X (c-f). GP: guttapercha, PDL: periodontal ligament, D: dentine, C: cellular cementum, B: bone, →: dentine debris, ×: extruded root canal sealer. Bars represent 100 µm. 

A moderate amount of lymphocytic infiltrate was observed in the periapical tissues, as well as the presence of small fragments of eosinophilic material with a tubular pattern resembling dentine, possibly consisting of debris or, in absence of this pattern, root canal sealer extruded during the shaping and obturation phases, respectively (Fig. 7). No areas of necrosis were observed within the adjacent periodontal ligament or cellular cementum.

DISCUSSION

Correctly designed animal models using rodents have been extensively used in dental research^1-4,9,16,17 because these animals can be easily housed and handled and enable histological sections to be obtained relatively fast. In the field of Endodontics, larger animals such as dogs, whose teeth have a single canal and are more comparable in size to human teeth, have been preferred for studying root canal treatment procedures ^5-7. However, in addition to dogs being more expensive and often drawing opposition from public opinion ¹⁸, it has been reported that their apical anatomy is complex and invariably presents an apical delta^19,20, making them a less than ideal model for understanding the repercussions of root canal chemomechanical debridement and obturation on periapical tissues. Although scarce, some studies using the rat as a model for endodontic treatment are available in literature, the vast majority using the mandibular first molar: some have focused on treating either the mesial^1,4 or distal^21,22 root and even fewer have addressed both^2,23. Sadly, the careful dissection of some of the endodontic treatments reported reveals suboptimal results regarding working length or excessive tapers which turn out to be inadequate for the root canal anatomy present in the rat. These problems are probably owed to the lack of studies describing a standardised protocol for root canal treatment in accordance with animal age, size and root canal morphology, as well as the adequate age for treatment. Moreover, to our knowledge, there is no study addressing the anatomic and histologic characteristics of the mandibular first molar of the Wistar rat.

According to early published data, in albino rats, molar crown formation is completed between postnatal days 11-21, and root development in first, second and third mandibular molar is completed by days 25, 28 and 40, respectively ¹⁰. Nevertheless, the data presented in our study revealed that by day 25, root development of the mandibular first molar is not yet complete and Hertwig's Epithelial Root Sheath (HERS) can still be observed. In our study, root development of the mandibular first molar was found to be complete at around day 55, which is in agreement with recent data ². From this age onwards, root length increases as a result of the continuous deposition of cellular cementum at the root apex. Interestingly, in adult rats, up to one third of the total root length can be composed of cementum alone. This mechanism, termed hypercementosis, is considered to be physiological, continuous ²⁴ and necessary to compensate for the attrition resulting from mastication and diet characteristics ¹⁰, and potentially complicates the achievement of patency and satisfactory endodontic debridement and obturation. Taking all this into account, we decided to use Wistar rats aged 55 days which, according to histological observations, have a completely developed root (no HERS is observed) and cementum -including the cellular variety- lining the external surface of radicular dentine, sometimes extending slightly apically to it, and maintaining a relatively stable root length of 2.5 mm and a radicular canal that is accessible for instrumentation and the establishment of an apical stop.

Our findings regarding the distinctive anatomy of the mesial and distal canals revealed that both are oval, as within each sample the diameter in one of the directions -either MD or BL- was two times greater than the diameter in the other direction ²⁵. Furthermore, while the mesial canal was found to be wider in the mesio-distal plane, the opposite occurred with the distal canal. These characteristics should be taken into account when pondering the question of which type of preparation and obturation techniques to use. In terms of preparation, attention should be paid to cleaning and shaping the canal entirely in order to eliminate inflamed or necrotic pulp. Most rotary instruments produce a round preparation^26,27, leaving unprepared recesses in oval-shaped canals ²⁸. Even if this problem can be partially surmounted by the use of laterally directed brushing working strokes ²⁹, unfortunately this would not be feasible in the rat, given that the restricted angle for instrumentation and the short length of the roots limit the amplitude

and control of movements. We therefore consider manual circumferential instrumentation to be more appropriate, as previously suggested for this type of anatomy in human teeth ²⁵.

In this regard, it is important to consider dentine thickness in each radicular third in order to preserve as much as microbiologically admissible, since excessive loss of dentine may contribute to increased susceptibility to fracture ³⁰. Even though micro-CT technology could prove to be a powerful non-destructive method for understanding the anatomy of the rat molar, segmentation of dentine and cementum based on pixel grey level values would be very difficult -if not practically impossible when considering acellular cementum-and require very high resolutions, resulting in time-consuming scanning protocols (estimated resolution of 2 pm or less, and >10 h). These difficulties are caused by the small size of the sample and the fact that dentine and cementum have similar mineral densities ³¹. We therefore decided to use HE stained histological sections, which enabled us to identify and measure the thickness of each tissue ³². We thus found furcation-facing walls to be thinner than their external counterparts, especially in the coronal third. This is analogous to what is observed in human molars, in which the limited amount of dentine associated with the concavity and root curvature makes them danger zones³³ where overzealous filing must be avoided in order to prevent strip perforations. An analysis of the diameters of different NiTi instruments with equal tip size (i.e. 0.25 mm) over the first 3 mm of their active portion -including one used in a recent study proposing a model for root canal treatment in the rat ²- based on the premise that they produce centred preparations ³⁴ reveals that all of them would cause excessive thinning of dentine. In the direction in which each canal attains its minimum diameter -BL and MD for the mesial and distal canals respectively- this could take up from 33% to 97% of the dentine wall in the cervical and middle thirds of the root, depending on the system, while the use of K files of equal diameter would cause a decrease in thickness of no more than 39%. This shows that more conservative tapers (i.e. 2%) should be preferred to larger tapers (i.e. 4%, 6%, 7%, 8%). As for the obturation phase, a single cone technique would not be likely to satisfactorily fill the entire canal space, which is why we consider cold lateral condensation with accessory guttapercha points to be a better choice.

Another important aspect is final restoration of the access cavity, which has been shown to have impact on the rate of success of endodontic therapy ³⁵ and is important in preserving the treatment throughout experiment. Materials such as amalgam, as proposed by other groups ²², tend to result in lack of retentiveness due to the shallowness of the access cavity (1.5 mm in depth) or cracks/fracture of the walls upon compaction. The use of composites, on the other hand, demands a strict adhesive technique and, once cured, can result in premature contacts that would lead to a state of inflammation of the periodontal ligament, in addition to muscle fatigue and/or temporomandibular joint disorders ³⁶. Furthermore, their high elastic modulus could complicate the obtention of histological sections using a microtome once the hemimandible has been decalcified. We therefore suggest the use of type II restorative glass-ionomer cement, given its compressive and tensile strength, as well as its excellent adhesion to dentine and enamel and hardness that permits surface wear when in occlusion with the antagonist tooth ³⁷. Other groups have also used this material^1,21,23. In our experience, a thorough removal of excess gutta-percha from the pulp chamber is key to guaranteeing sufficient contact of the material with the walls and to ensure greater longevity of the restorations.

In view of achieving operator training and carrying out a successful treatment, we have considered the methodological steps followed and prepared the following protocol and recommendations for experimental root canal treatment in Wistar rats:

- Select appropriately aged animals in order to avoid intraoperative complications. We found 55-day-old rats to be optimal models, but consider the interval of 50 to 60 days of age to be adequate.

- Attain deep levels of general anaesthesia for up to 40-60 minutes.

- Use a stretcher that provides proper positioning of the animal's head and enough mouth opening in order to ensure clear visualization view of the molar and operator's access to perform shaping,

cleaning and filling procedures.

- Use a specially designed clamp and an aspiration micro-cannula to enable soft-tissue separation and provide a thoroughly dry field while maintaining oropharynx permeability and avoiding ingestion and/or obstruction of the respiratory airway.

- Position the dental bur on the mesial pit with a disto-mesial inclination so as to enter the mesial pulp horn and avoid perforation of the pulp chamber floor.

- Perform catheterisation of the canals using #08 K-files and confirm working length (estimated in 4 mm) using an electronic apex locator.

- Shape and enlarge mesial and distal canals using manual circumferential instrumentation because of their oval shape and using the minimum taper available (i.e. 2%) because of the thinness of their walls.

- Fill canals using a cold lateral condensation technique instead of a single cone technique, given the oval shape of the canals.

- Remove excess gutta-percha from the pulp chamber and seal it with type II restorative glass-ionomer cement.

- Throughout the procedure, hydrate the animal's eye and monitor its breathing and heartbeat, in order to provide immediate care should any emergency or complication arise.

- Monitor animals until after anaesthesia has worn off. The postoperative recovery will depend upon basic sanitary care, place (cage) in which animals are being kept, proper ventilation and hygiene.

- Consider rodents to be living beings whose biological responses are similar to humans', but faster, hence, whenever this model is used, results and their extrapolation to humans should be interpreted carefully.

To conclude, the experimental protocol presented herein enables the study of biological responses that would otherwise be ethically inaccessible in humans and could not be replicated effectively by using extracted teeth. We consider that this model for experimental root canal treatment could contribute to future research, unravelling a variety of aspects related to endodontic treatment.