Abstract
Introduction
The present study reported the histological events that occurred in the radicular pulp of human mature teeth in the presence of medium/deep untreated caries lesions, and those teeth with restorations or direct pulp capping, with particular emphasis on the morphology of the canal wall dentine and the odontoblast layer.
Methods
Sixty-two teeth with medium/deep caries lesions, extensive restorations or after application of a direct pulp capping procedure were obtained from 57 subjects. Fourteen intact mature teeth served as controls. Stained serial sections were examined for the pulp conditions of the coronal pulp. The teeth were classified as those with pulpal inflammation, or those with healed pulps. Histological changes that occurred in the roots at the pulp-dentine junction were investigated in detail.
Results
All teeth (100%) in the experimental group showed pathologic changes in the radicular pulp, with varying amounts of tertiary dentine on the canal walls and absence of odontoblasts. These changes were identified from different portions of the canal wall surface. Non-adherent calcifications in the pulp tissue were observed in more than half of the specimens. Changes that deviate from classically-perceived histological relationships of the pulp-dentine complex were also observed in the radicular pulps of 33.7% of the control teeth.
Conclusion
When challenged by bacteria and bacterial by-products invading dentinal tubules, odontoblasts in the radicular pulp may undergo cell death, possibly by apoptosis. This phenomenon may be caused by progressive root-ward diffusion of bacterial by-products, cytokines or reactive oxygen species through the pulp connective tissue.
Clinical significance
Although the vitality of the dental pulp in teeth with deep dentinal caries may be maintained with direct pulp capping or pulpotomy, the repair tissue that is formed resembles mineralised fibrous connective tissues more than true tubular dentine.
1
Introduction
Odontoblasts are long-lived, terminally-differentiated post-mitotic cells that are not replaced during the lifetime of an individual [ ]. After secreting primary dentine during the period of active tooth development, these cells continue to deposit secondary dentine as a slower rate throughout life in the absence of pathophysiological insults that challenge their integrity. Traditionally it has been held that reactionary dentine is produced in the presence of mild injurious challenges by the original surviving odontoblasts as well as genetically-committed daughter cells that reside in the subodontoblastic cell-rich zone [ ]. In the presence of more severe challenges, reparative dentine is produced by recruitment and proliferation of pulp-derived stem/progenitor cells that differentiate into mineralised dentine matrix-producing odontoblast-like cells [ ]. A recent histological study challenged the generally-accepted view that stem cells are recruited to produce a new generation of odontoblast-like cells that produce reparative dentine [ ]. In that study, the authors demonstrated that under deep caries lesions and following capping of pulp exposures, an amorphous, atubular calcified tissue was deposited with no evidence of morphologically identifiable odontoblast-like cells. The stem/progenitor cells may not have received adequate epigenetic signals, such as the lack of cross-talk with cells derived from the ectoderm [ ]. Hence, after their full differentiation, the secondary odontoblasts possess a phenotype that differs from the primary odontoblasts [ ]. Although the newly-formed tertiary dentine-producing cells may express several genes that are present in the primary odontoblasts [ ], these cells do not exhibit the typical elongated polarized shape, and do not produce a tubular matrix that resembles the morphological characteristics of primary, secondary or reactionary dentine [ ].
Histological investigations of the pulp response to medium and deep caries were almost exclusively performed on caries-affected dentine and the adjacent tissues in the pulp chamber [ ]. Detailed morphologic changes in the dentine structure had been reported, including the formation of reactionary or reparative tertiary dentine, uniformity of the predentine and the odontoblast layer, changes in the sub-odontoblastic areas, as well as the accumulation of inflammatory cells in the vicinity of the pulp chamber where the dentinal tubules were affected by caries [ ]. It is generally assumed that pulp reactions only occur in the pulp subjacent to the carious lesion and that no morphological changes occur in the radicular pulp.
Contrary to these findings, a recent study identified that histopathological and histobacteriological changes occurred in the radicular pulp of immature teeth affected by deep caries [ ]. These changes were identified in areas that were remote from the site of infection in the coronal dentine. To date, information is lacking on the changes that may occur in the radicular pulp of mature human teeth. Accordingly, the objective of the present study was to examine the histological events that occurred in the radicular pulp of human mature teeth in the presence of medium/deep untreated caries lesions, and those teeth with restorations or direct pulp capping, with particular emphasis on the morphology of the canal wall dentine and the odontoblast layer. Knowledge of the radicular pulp tissue response in these conditions is important in understanding and predicting the healing mechanisms of treatment procedures targeted at maintaining the vitality of all or part of the dental pulp.
2
Materials and methods
The materials for the present study consisted of 62 mature human teeth with caries or amalgam/resin composite restorations, or teeth that had been subjected to direct pulp capping (48 molars, 11 premolars, 3 canines). Those teeth were obtained from a single dental practice from 57 patients (34 females, 23 males) aged between 16 and 75 (mean age 40.1 years). From this pool, 40 teeth had untreated carious lesions, 18 teeth had amalgam or resin composite restorations and 4 teeth had received direct pulp capping with calcium hydroxide paste (calcium hydroxide powder mixed with distilled water).
The carious lesions and the restorations were clinically classified as shallow, medium or deep, as determined from periapical radiographs [ ]. Shallow caries/restorations were those involving less than one-quarter of the dentine thickness. Medium caries/restorations were those involving approximately one-quarter to three-quarters of the dentine thickness. Deep caries/restorations were those involving more than three quarters of the dentin thickness. Only teeth with medium and deep caries or restorations were included in the present study. Teeth with periodontal pockets exceeding 5 mm were excluded. The teeth were clinically categorised as having “normal pulp”, “reversible pulpitis” or “irreversible pulpitis” based on pre-established criteria [ ]. The teeth were extracted for prosthetic or orthodontic reasons, or because of the patient́s desire in not having the tooth treated.
Fourteen intact mature human teeth (two premolars extracted for orthodontic reasons and 12 third molars extracted for pericoronitis) obtained from 10 patients (six females, four males) aged between 13 and 37 (mean age 23.6 years), were used as controls. The study was approved by the institutional review board for the use of extracted teeth in research. Written consent was received from all patients (or from their legal guardians in case of minors) for histologic analysis.
Immediately after extraction the following approaches were undertaken to facilitate proper fixation of the pulp tissues and correct orientation of the specimen in the paraffin block. The teeth were shaved under magnification with high speed diamond burs using water spray on the mesiodistal or the buccolingual plane until one or two pulp horns were encountered. In some maxillary and mandibular molars, the roots were separated 2 to 3 mm apically to the root canal orifices before shaving the crowns. The teeth were immersed in 10% neutral buffered formalin for 48 h. Demineralization was performed using an aqueous solution consisting of a mixture of 22.5% (v/v) formic acid and 10% (w/v) sodium citrate for 3 to 4 weeks, with the end point of demineralization determined radiographically. All specimens were subsequently washed in running tap water for 48 h, dehydrated in ascending grades of ethanol, cleared in xylene, infiltrated and embedded in paraffin (melting point 56 °C) according to standard procedures of tissue processing. With the microtome set at 4–5 μm, meticulous longitudinal serial sections were taken of the whole tooth, for those teeth that were processed in one piece, until the pulp tissue was exhausted. For some of the roots that were separated from their crowns, longitudinal serial sections were taken, until no more radicular pulp tissues were encountered. Cross sections were prepared for the rest of the roots. Approximately 200 sections were cut at the transition from the coronal to middle third, and another 200 sections at the transition from the middle to apical third of each root. Every fifth section was stained with haematoxylin and eosin for screening purpose and evaluation of the reactions. Those sections were used to locate the areas with the most severe reactions. Based on this initial evaluation, all slides adjacent to the location with the most severe reactions were stained. In addition, a modified Brown and Brenn technique for staining bacteria [ ] was used for the selected slides. Cover slips were then placed on the slides, and the sections were examined using a light microscope. The worst histologic condition observed was recorded for each pulp. Two evaluators examined the sections independently. In the event that disagreement occurred, it was resolved by involving the third evaluator.
Irrespective of the clinical diagnosis, the teeth were histologically classified into four categories according to a slight modification of previously-reported criteria [ ]:
- a)
Reversible histological pulpal changes . This group included specimens with evidence of moderate or severe chronic inflammation confined to the coronal pulp. Lymphocytes and plasma cells were identified in varying concentrations beneath the deepest areas of caries penetration. No coagulation or liquefaction necrosis was evident. Bacteria were seen in large numbers in the carious dentine, but were absent from the pulpal tissues.
- b)
Irreversible histological pulpal changes . At least one area (even if it was very small) of the coronal pulp tissue had undergone liquefaction or coagulation necrosis (i.e. pulpal abscess), surrounded by masses of live and dead polymorphonuclear neutrophils (PMNs). Bacterial aggregations/biofilms were observed colonising the necrotic pulp tissues or the adjacent dentine walls. Peripherally, concentrations of chronic inflammatory cells (lymphocytes, plasma cells and macrophages) formed a dense halo around the central zones of the abscess.
- c)
Normal, intact pulp . Pulps with no changes in the dentin/predentine/odontoblast complex in the crown. Dentinal tubules could be seen running parallel to each other through the dentine and predentine, with no reduction in numbers. There was no reduction of the odontoblast layer or reduction in the odontoblast cell size. Tertiary dentine or other calcifications were also absent. In addition, there were no inflammatory cell accumulations or dilated vessels, and no bacteria.
- d)
Healed pulp . Pulps with no evident accumulation of inflammatory cells, but deviations from normal histologic findings were present. That is, a variable amount of tubular/atubular tertiary dentine, or free/adherent pulp stones could be identified in the pulp chamber. Scattered inflammatory cells were present, together with hyperaemic vessels, fibrosis, and a reduced odontoblastic layer.
In addition to these features which were related only to the coronal portion of the tooth, the following aspects, related to the radicular portion, were investigated and recorded in the present investigation:
- 1.
Changes in the regularity of the odontoblast layer on the root canal walls;
- 2.
Tertiary dentine formation on the root canal walls;
- 3.
Isolated, non-adherent calcifications in the radicular pulpal tissues.
Because quantification of the surface of the root canal wall involved in these changes was impossible using serial sections, these features were recorded qualitatively as being present or absent. Accordingly, no statistical analysis was performed in the present study.
2
Materials and methods
The materials for the present study consisted of 62 mature human teeth with caries or amalgam/resin composite restorations, or teeth that had been subjected to direct pulp capping (48 molars, 11 premolars, 3 canines). Those teeth were obtained from a single dental practice from 57 patients (34 females, 23 males) aged between 16 and 75 (mean age 40.1 years). From this pool, 40 teeth had untreated carious lesions, 18 teeth had amalgam or resin composite restorations and 4 teeth had received direct pulp capping with calcium hydroxide paste (calcium hydroxide powder mixed with distilled water).
The carious lesions and the restorations were clinically classified as shallow, medium or deep, as determined from periapical radiographs [ ]. Shallow caries/restorations were those involving less than one-quarter of the dentine thickness. Medium caries/restorations were those involving approximately one-quarter to three-quarters of the dentine thickness. Deep caries/restorations were those involving more than three quarters of the dentin thickness. Only teeth with medium and deep caries or restorations were included in the present study. Teeth with periodontal pockets exceeding 5 mm were excluded. The teeth were clinically categorised as having “normal pulp”, “reversible pulpitis” or “irreversible pulpitis” based on pre-established criteria [ ]. The teeth were extracted for prosthetic or orthodontic reasons, or because of the patient́s desire in not having the tooth treated.
Fourteen intact mature human teeth (two premolars extracted for orthodontic reasons and 12 third molars extracted for pericoronitis) obtained from 10 patients (six females, four males) aged between 13 and 37 (mean age 23.6 years), were used as controls. The study was approved by the institutional review board for the use of extracted teeth in research. Written consent was received from all patients (or from their legal guardians in case of minors) for histologic analysis.
Immediately after extraction the following approaches were undertaken to facilitate proper fixation of the pulp tissues and correct orientation of the specimen in the paraffin block. The teeth were shaved under magnification with high speed diamond burs using water spray on the mesiodistal or the buccolingual plane until one or two pulp horns were encountered. In some maxillary and mandibular molars, the roots were separated 2 to 3 mm apically to the root canal orifices before shaving the crowns. The teeth were immersed in 10% neutral buffered formalin for 48 h. Demineralization was performed using an aqueous solution consisting of a mixture of 22.5% (v/v) formic acid and 10% (w/v) sodium citrate for 3 to 4 weeks, with the end point of demineralization determined radiographically. All specimens were subsequently washed in running tap water for 48 h, dehydrated in ascending grades of ethanol, cleared in xylene, infiltrated and embedded in paraffin (melting point 56 °C) according to standard procedures of tissue processing. With the microtome set at 4–5 μm, meticulous longitudinal serial sections were taken of the whole tooth, for those teeth that were processed in one piece, until the pulp tissue was exhausted. For some of the roots that were separated from their crowns, longitudinal serial sections were taken, until no more radicular pulp tissues were encountered. Cross sections were prepared for the rest of the roots. Approximately 200 sections were cut at the transition from the coronal to middle third, and another 200 sections at the transition from the middle to apical third of each root. Every fifth section was stained with haematoxylin and eosin for screening purpose and evaluation of the reactions. Those sections were used to locate the areas with the most severe reactions. Based on this initial evaluation, all slides adjacent to the location with the most severe reactions were stained. In addition, a modified Brown and Brenn technique for staining bacteria [ ] was used for the selected slides. Cover slips were then placed on the slides, and the sections were examined using a light microscope. The worst histologic condition observed was recorded for each pulp. Two evaluators examined the sections independently. In the event that disagreement occurred, it was resolved by involving the third evaluator.
Irrespective of the clinical diagnosis, the teeth were histologically classified into four categories according to a slight modification of previously-reported criteria [ ]:
- a)
Reversible histological pulpal changes . This group included specimens with evidence of moderate or severe chronic inflammation confined to the coronal pulp. Lymphocytes and plasma cells were identified in varying concentrations beneath the deepest areas of caries penetration. No coagulation or liquefaction necrosis was evident. Bacteria were seen in large numbers in the carious dentine, but were absent from the pulpal tissues.
- b)
Irreversible histological pulpal changes . At least one area (even if it was very small) of the coronal pulp tissue had undergone liquefaction or coagulation necrosis (i.e. pulpal abscess), surrounded by masses of live and dead polymorphonuclear neutrophils (PMNs). Bacterial aggregations/biofilms were observed colonising the necrotic pulp tissues or the adjacent dentine walls. Peripherally, concentrations of chronic inflammatory cells (lymphocytes, plasma cells and macrophages) formed a dense halo around the central zones of the abscess.
- c)
Normal, intact pulp . Pulps with no changes in the dentin/predentine/odontoblast complex in the crown. Dentinal tubules could be seen running parallel to each other through the dentine and predentine, with no reduction in numbers. There was no reduction of the odontoblast layer or reduction in the odontoblast cell size. Tertiary dentine or other calcifications were also absent. In addition, there were no inflammatory cell accumulations or dilated vessels, and no bacteria.
- d)
Healed pulp . Pulps with no evident accumulation of inflammatory cells, but deviations from normal histologic findings were present. That is, a variable amount of tubular/atubular tertiary dentine, or free/adherent pulp stones could be identified in the pulp chamber. Scattered inflammatory cells were present, together with hyperaemic vessels, fibrosis, and a reduced odontoblastic layer.
In addition to these features which were related only to the coronal portion of the tooth, the following aspects, related to the radicular portion, were investigated and recorded in the present investigation:
- 1.
Changes in the regularity of the odontoblast layer on the root canal walls;
- 2.
Tertiary dentine formation on the root canal walls;
- 3.
Isolated, non-adherent calcifications in the radicular pulpal tissues.
Because quantification of the surface of the root canal wall involved in these changes was impossible using serial sections, these features were recorded qualitatively as being present or absent. Accordingly, no statistical analysis was performed in the present study.
3
Results
3.1
Control teeth
Descriptive statistics of the control teeth are included in Table S1. Only the dental pulps of the control intact teeth could be classified as normal, intact pulps. These pulps contained uninflamed connective tissues in the pulp chamber, with abundant cells and neurovascular bundles, and a peripheral palisading layer of odontoblasts ( Fig. 1 ). The predentine was of uniform thickness and the course of the dentine tubules could be easily identified, with no interruption through dentine and predentine ( Fig. 1 ). In the radicular pulp, the same uniformity in the interrelationship between dentine, predentine and the odontoblast layer could only be observed in 9 of the 14 (64.3%) of the control teeth. In the 9 teeth in which there was no disruption in the interrelationship between the dentine, predentine and the odontoblast layer, the only identifiable difference was in the shape of odontoblasts. The odontoblasts were mostly cuboidal or spindle-shaped in the apical direction ( Fig. 1 D–E). No interruption or other disturbances could be seen in the course of the dentinal tubules from all serial sections derived from the same tooth ( Fig. 1 F). There was no sign of tertiary dentine formation along the canal walls ( Fig. 1 F).
In the remaining 5 teeth, deviation from classically-described dentine morphology could be observed ( Figs. 2 and 3 ). These deviations were minimal in general and consisted of restricted areas in which the canal walls appeared to be devoid of odontoblasts, despite the presence of dentinal tubules ( Fig. 2 C–F). In 4 of those 5 teeth with loss of odontoblasts, atubular tertiary dentine could be identified along the canal walls ( Fig. 3 B, D). In 3 of those 5 teeth, small, non-adherent pulp stones that were completely segregated from the predentine could be identified in the vicinity of the neurovascular bundles ( Fig. 1 E, G).
3.2
Experimental teeth
Irrespective of whether the teeth were untreated or treated, all teeth from the experimental group, including those that had received direct pulp capping, showed changes in the morphology of the radicular pulp-dentine complex to varying extents. These morphologic changes, which included the loss of odontoblasts and the presence of tertiary dentine, were observed irrespective of the overall clinical diagnosis of reversal or irreversible pulpitis or histological manifestation of reversible/irreversible pulpal morphologic features or those characteristic of a healed dental pulp (Tables S2–S4).
Of the 31 teeth histologically that were presented with irreversible pulpal changes, non-adherent calcifications were observed in the radicular pulps of 21 teeth (67.7%). These calcified masses were present in 10 of the 18 teeth diagnosed with reversible pulpitis (55.5%), and in 7 of the 13 teeth diagnosed with healed pulps (53.8%) (Tables S2–S4).
3.2.1
Teeth with irreversible pulpal changes
The most severe and diffuse reactions in the radicular pulp/dentine complex were seen in untreated and treated teeth presented with the histologic features of irreversible pulpal changes ( Figs. 4–6 ). Irregular calcified tissues, hereby referred to as tertiary dentine, were present extensively on the root canal walls. These calcified tissues had reduced, irregular dentinal tubules or were entirely atubular, and were separated from the secondary dentine by a dark “calciotraumatic line” (Figs. 4 J–M; 5 G–H; 6 D–I). The tertiary dentine were variable in thickness, sometimes featuring scattered lacunae containing cells with projecting canaliculi that resembled cellular cementum ( Fig. 4 H–I). In a few cases, these tissues were discontinuous, being interrupted by secondary dentine in close proximity with large multinucleated cells (odontoclasts) ( Fig. 4 J–K). The tertiary dentine appeared highly irregular and unevenly stained in some areas, and was present in successive layers ( Fig. 6 E). On the pulpal side, the tertiary dentine showed relatively regular, darkly-stained profiles ( Fig. 5 G–H) and was occasionally presented with irregular edges ( Fig. 6 I). In some instances, the pulpal margins appeared highly eosinophilic, with dense collagen bundles inserted into the tertiary dentine at varying angles, resembling the periodontal ligament ( Fig. 4 L–M). These changes were predominantly present in the middle and apical thirds of the root (Figs. 4 G–M; 5 G–K). Paradoxically, they were often absent in the coronal third of the roots, where the most severe inflammatory reactions occurred. It was not infrequent to observe normal relationship among the dentine, predentine and the odontoblasts in the canal orifice and coronal third of the roots (Figs. 4 E–F; 5 C).
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