There are three causes of vital pulp exposure: caries, trauma, and mechanical sources. Mechanical exposure occurs during the preparation of a cavity without caries. Mechanical exposure of the pulp is typically due to a misadventure during tooth preparation.¹ Pulp exposure initially leads to reversible changes, irreversible, and partial necrosis of the tissue and eventually completes necrosis in the tissue. Tissue necrosis is accompanied by structural changes in the pulp and periapical tissues. These changes include decreasing the number of odontoblast cells and eventually destroying them in different areas of the canal walls.²,³,⁴ Odontoblast cells are responsible for making dentin. If the root dentin is destroyed and odontoblast cells are destroyed, Hertwig's Epithelial Root Sheath (HERS) sends signals to the dental papilla, and under these signals, dental pulp the stem cells differentiate into odontoblast-like cells, and then they start to build up a tertiary dentin at the site of the decay.⁵,⁶,⁷,⁸ Generally, the rate of restorative dentin formation depends on the amount of primary dentin degradation. The immature tooth is characterized by an open apex and thin dentin walls. Embryonic tooth formation is a result of the interaction between the ectodermal coating and the underlying mesodermal tissues. During tooth growth, various molecules, including FGF, TGF-β, BMP4, and BMP2 send signals to the ectodermal and mesodermal tissues.⁹,¹⁰ This interaction at different evolutionary stages results in the formation of the tooth and its retaining tissues. Root formation is the last stage of tooth formation that starts from the cervical arch. The cervical arch is where the internal and external enamel epithelium collide. Cells in this region multiply in the shape of a bilayer epithelial sheath termed HERS that acts as a template for root formation.¹¹,¹² Inducement and regulation of root formation are among the different functions attributed to these cells, including the size, shape, and number of roots. If the HERS disappears during infection, inflammation, or necrosis, it may lead to a nonevoluted root in future. The HERS is in full contact with pulp cells in healthy teeth. However, with more severe inflammation and necrosis, the integrity between the apical papilla and the HERS is compromised, and the HERS is discontinuously seen around the pulp. As this process continues, the HERS is destroyed, and as a result, root apical evolution does not occur.² Suzuki et al. studied the function of the HERS cells during root formation. Their results showed that the formation of HERS is due to the invasion of dental follicle cells. Differentiation of papilla dental cells into odontoblast cells occurs following their attachment to the HERS.¹³

Ricucci et al. in their study on the teeth with irreversible pulpitis found that there was severe inflammation with small pieces of necrosis in the pulp tissue of the tooth. They showed that large parts of the canal walls lacked odontoblast cells, and the apical papilla was significantly reduced; in addition, the HERS was either disrupted or completely obliterated.²

Given that the treatment of immature teeth with open apex is a problem, on the other hand, defects in the composition of dentin and the thin walls of the roots have made immature teeth very sensitive to breakage. Therefore, immature apex has caused problems in the endodontic treatment of these teeth.¹⁴,¹⁵ The description of pathological changes in the pulp and immature apical structures as a consequence of caries in immature teeth is not available in the articles. The answer to the question of what changes occurs in the HERS and tooth apical region as caries progresses is debatable. Therefore, this study aimed to evaluate the morphological changes that occurred in the dental pulp after mechanical exposure and restoration with reinforced zinc oxide-eugenol in immature teeth in the sheep, as an animal model.

In this experimental study, six male adult sheep with immature teeth weighing between 30 and 40 kg with the age of 1 year old and Merino race were used in this study. Of these, four sheep were used as the experimental group, and two sheep remained intact as controls (to avoid killing more animals only two intervals were chosen).

Two central mandibular incisors with immature teeth (total number: 12) were used from each sheep. After anesthesia with the muscular injection of 0.01 mg/kg xylazine, lidocaine hydrochloride 2% with 1:80000 epinephrine (Persocaine, Darou Paksh, Iran) for local anesthesia. The isolation was performed using the split dam so that the incisor teeth were outside of the rubber dam. Teeth disinfected with 0.2% chlorhexidine. An access cavity was created at the lingual surface of the tooth, using a high-speed diamond fissure bur (Tizcavan, Tehran, Iran) with copious water irrigation. The pulp was mechanically exposed. The size of pulp exposure in all teeth was similar and was 1.1.1 mm.

Initial hemorrhaging was controlled by placing sterile cotton pellets moisturized with saline over the pulp stump using slight pressure and waiting 5 min for hemostasis. Then, the pulp was covered with reinforced zinc oxide eugenol cement (Kemdent, United Kingdom). Cement was prepared according to the manufacturer's instructions and then inserted into the cavity. Using a dry sterile cotton pellet, the cement was gently adapted on the blood clot-free pulpal wound and dentinal walls, and the coronal portion of the cavity was permanently filled with amalgam (Cinalux plus, Iran). Central mandibular incisors from the control group remained intact.

Inclusion criteria

Male sheep had not received any medicine or antibiotics in the past 2 weeks; the permanent teeth of the mandibular incisors had just erupted, and the access cavity preparation site was located above the gums, the apex of the mandibular incisor was open in the radiograph, and the diameter of the apical region of the canal was about 2–3 mm in all samples.

Exclusion criteria

Preparation of the access cavity with any accident, failure to stop pulp bleeding after 5 min of packing with cotton soaked in normal saline, teeth restoration defects during the study period, crown fracture in the studied teeth, and occurrence of systemic diseases in animals during the study period.

The ARRIVE recommendations were considered for conducting this animal research (https://arriveguidelines.org/arrive-guidelines). This study was approved by the Ethics Committee of Zahedan University of Medical Sciences (IR.ZAUMS.REC.1397.054).

In the experimental group, animals were sacrificed in the 2^nd, 4^th, 6^th, and 8^th weeks (named: E2, E4, E6, and E8, respectively). Immediately after sacrificing the animals, central mandibular incisors were resected along with the surrounding supporting tissues and alveolar bone. In the control group, the teeth were removed in the interval of 2 and 8 weeks (named: C2 and C8).

The samples were placed in 10% formalin saline for at least 14 days for tissue fixation. Then, they were cleaned and embedded in 10% formic acid, 9.2% citric acid, and 8.1% trisodium citrate solution for decalcification for about 21 days at the room temperature. To verify full decalcification, teeth radiography was done. Then, the samples were dehydrated in a graded series of ethanol, cleared in xylene, and impregnated in paraffin wax using a Leica TP 1010 tissue processor (Leica, Germany). Paraffin tissue blokes were prepared as the samples were placed longitudinally along the long axis of the tooth. Then, sectioning was done using the fully automated Leica RM2255 instrument (Leica, Germany). 4 μm serial sections were prepared from each specimen, and the samples were mounted on glass slides.

Sections were stained with routine H and E staining, and histological and morphometric studies were done.

To calculate the morphometric parameters, the images of the relevant slides were transferred to the computer using the camera transfer (CME ×50, Euromex), and by considering the magnifications of the images, the morphometric calculations for each sample were examined in five areas, comprising the apical foramen region and compared with the parallel slides from the control group using the image analyzing software of the photomicroscope (Leica, Germany).

In addition, each sample was observed under the microscope for the presence or absence of tertiary dentin in the root canal.

The present study focused on the histologic qualitative interpretation of changes in the root canal at different time durations. Histology of apical and periapical area, thickness of odontoblast layer, presence or absence of tertiary dentin and its thickness, and HERS changes such as size and shape of the inner enamel epithelium (IEE) and outer enamel epithelium (OEE) cells were studied.

Statistical analysis

The Kruskal–Wallis and Mann–Whitney U tests were used to analyze the data and compare the changes at different times between the experimental and control groups. P < 0.05 was considered statistically significant.

Table 1 shows the morphometric information and the changes between the case and control groups. The changes in the histologic status of the periapical tissue in the case groups showed that the connective tissue was denser and its fibers became thicker than the control group. Furthermore, in the experimental group, from the 2^nd week to the 8^th week, there was a trend of progressive increase in density and thickness of connective tissue fibers. However, bone tissue in this area appears to be gradually diminishing.{Table 1}

A comparison of changes in the odontoblast cells in the experimental and control groups is shown in Figure 1. There was a significant difference in the thickness of the odontoblast cell layer between the experimental and control groups. The highest thickness of the odontoblast layer was observed in the E4 group and the lowest thickness was observed in the C2 group. In the other groups, the thickness of the odontoblast layer was similar.Figure 1

Thickness reduction of the odontoblastic layer cells in the second week of the control group (a) compared to 4^th week (b) of the study after mechanical pulp exposure in the experimental group (arrows indicate odontoblastic layer cells); (hematoxylin-eosin stained sections)

The present study showed that in response to mechanical exposure, the thickness of the odontoblastic layer in the C2 group was the lowest and in the E4 group was the highest amount. In the case groups, tertiary dentin was formed and its thickness increased during the time of the study. The diameter of the apical foramen in the E8 was decreased significantly compared to control groups. Our study results showed that there was the highest OEE in the E6 group and the lowest was observed in the control groups.

The results of this study showed that pulp stimulation causes the formation of connective tissue with denser and thicker fibers in the apical region. This is one of the differences between the stimulated and the unstimulated pulp in the experimental and control groups. In addition, the bones of the periapical area degenerate over time. Ricucci et al. showed that even with moderate inflammation in the pulp, secretion of proinflammatory mediators and cytokines and their entry into the apical region causes periapical bone resorption. They also have shown that in cases of irreversible pulpitis, the number of cells in the apical area is drastically reduced, and in some areas, there are no cells. Furthermore, the pulp tissue showed an abundance of dilated vessels and nerves, and in some areas, the pulp exhibited fewer cells and an abundance of fibers, this tissue showed an abundance of dense collagen bundles. All of these findings can indicate the inflammatory mechanism of pulp.²

Our results showed that in the control group, the cells of the odontoblast layer in the coronal region were higher with more specific odontoblastic appendages than in the apical region. In the experimental groups, the odontoblastic layer was similar to the control groups, except for the exposed areas of the pulp, where no odontoblasts were cells due to cavity formation. Odontoblast cells form the outermost layer of the dental pulp and they are like a barrier between the dentin and the vital tissues of the tooth. Odontoblast cells protect the dentin and living pulp against mild stimuli by reactive dentin deposition. When bacteria invade deep dentin and dentin tubules, odontoblast cells are the first pulp cells to encounter them. In very severe and long-lasting inflammations, odontoblast cells break down the pulp by producing pro-inflammatory mediators such as cytokines. Furthermore, immune cells that present in the pulp produce proteases, such as metalloproteinase, and various enzymes to combat invading pathogens. However, these molecules can also cause damage to dental pulp cells and lead to host cell death or reduce their number.¹⁶,¹⁷

We found that the level of tertiary dentin in the case group was significantly higher than the control group. Because no stimulation was performed on the teeth in the control group, no tertiary dentin was formed in this group. However, in the case group, tertiary dentin formed at the site of mechanical pulp exposure and its amount increased over time. So that the lowest amount of tertiary dentin was seen in the E2 group and then with an upward trend in other groups, the highest amount of tertiary dentin was seen in the E8 group. In our study, the tertiary dentin formed at the apex of the pulp chamber; under the exposed area and then progressed toward the canal walls. The amount of tertiary dentine produced is proportionally equal to the amount of dentin destroyed previously.⁷,¹⁸ Our study is similar to the results of Zhang et al. they found a large strip of tertiary dentin beneath the area involved in the decay. They also showed that the number of odontoblast cells in the vicinity of tertiary dentin had decreased and showed that in response to pulp exposure, stem cells were differentiated into odontoblast cells and formed tertiary dentin.¹⁹

The results of this study also showed that by increasing tertiary dentin deposition and by continuing to make dentin, the diameter of the root canal in the experimental groups was significantly reduced compared to the control groups, so that, the lowest diameter of the root channel was observed in the E8 week of the case group.

It has been shown that the formation of hard tissue begins immediately after pulp exposure and is a pulp protective mechanism against local stimuli. It also protects the pulp from reinfection. Although the formation of hard tissue in an uncontrolled manner is a complication of this defense mechanism, the subsequent closure and narrowing of the apical foramen can also be part of the pulp defense mechanisms in the experimental group compared to the controls.²⁰,²¹ Although, we did not find any studies that show what changes occur in the condition of apical foramen inflammation. However, the present study showed that mechanical exposure accelerated the closure of the apical foramen.

The results of this study showed that the predentin layer in the experimental group was thinner than the control group. However, these differences were not statistically significant. The highest thickness of predentin was observed in the E6.

The results of our study showed that the apical papilla in the experimental group was histologically similar to the control group. In this area, the connective tissue was denser, and the blood vessels were narrower than in other areas of the papilla. These results are similar to the results of Ricucci et al.² In the advanced stages of root development, the volume of the dental papilla is significantly reduced. This reduction is observed in all parts of the dental papilla except the apical part of the root. This part of the root is called the apical papilla. Stem cells are located in the apical region of the papilla, which differentiate into odontoblast cells and form the dentin of the root region. Therefore, the dental papilla is the part of the dental pulp that helps teeth form.²²,²³

The present results also showed that the cells of the HERS became smaller in the experimental group, especially in the E6 week, and their intercellular space decreased. In addition, it seems like these cells become denser and more atrophic. In the Ricucci et al.'s study, the HERS was observed discontinuous and in some cases did not exist at all.² Luan et al. studied the evolution of the HERS in rat molar teeth. They believed that the HERS continued to grow in the early stages of root development and that this growth was due to the proliferation of HERS cells. In the later stages of root development, the distance between the HERS cells and the root surface increases. Eventually, with the final evolution of the root, the proliferation of HERS cells decreases and they undergo apoptosis. Furthermore, some of these cells migrate from the root surface to form the epithelial sheath of Malassez.²⁴ In another study, Wentz et al. showed that as the primary dentin formed, at the same time, the HERS cells were fragmented and then removed from the root surface to form the epithelial remnants of Malassez.²⁵

After differentiating odontoblast cells from dental papilla cells, these cells begin to secrete predentin. With the onset of predentin formation, the HERS begin to fragment. These cells then attach to the surface of the root and could be recognized as Malassez epithelial cells. If the integrity of HERS is disrupted prematurely, differentiation of root odontoblasts and the formation of predentin is impaired. Anyway any disturbance in the formation of the HERS leads to malformations affecting root shape, number, length, structure, and other features.²⁶

The use of reinforced zinc oxide-eugenol for mechanical exposure of the pulp can lead to severe and progressive morphometric damage. These changes can include an increment of the density of connective tissue and destruction of odontoblast cells that then may lead to tertiary dentin formation and shrinkage and atrophy of HERS cells. Understanding the histology and pathology of the dental pulp can be helpful in the timely diagnosis and treatment of pulp injuries. Future studies will help us gain a better understanding of the premature damage to the root of the tooth and the resulting changes.

Acknowledgments

The Deputy of Research of Zahedan University of Medical Sciences financially supported this research (Grant No. 8663).

Ethics approval

This study was approved by the Ethics Committee of Zahedan University of Medical Sciences (IR.ZAUMS.REC.1397.054).

Financial support and sponsorship

The Deputy of Research of Zahedan University of Medical Sciences financially supported this research (Grant No. 8663).

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.