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Dental pulp exposure, periapical inflammation and suppurative osteomyelitis of the jaws in juvenile Baltic grey seals (Halichoerus grypus grypus) from the late 19th century

  • Uwe Kierdorf ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    uwe.kierdorf@uni-hildesheim.de

    Affiliation Department of Biology, University of Hildesheim, Hildesheim, Germany

  • Morten T. Olsen,

    Roles Resources, Writing – review & editing

    Affiliation Natural History Museum of Denmark, Department of Biology, University of Copenhagen, Copenhagen, Denmark

  • Patricia Kahle,

    Roles Formal analysis, Investigation, Resources, Visualization, Writing – review & editing

    Affiliation Department of Biology, University of Hildesheim, Hildesheim, Germany

  • Catharina Ludolphy,

    Roles Formal analysis, Investigation, Resources, Visualization, Writing – review & editing

    Affiliation Department of Biology, University of Hildesheim, Hildesheim, Germany

  • Horst Kierdorf

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Visualization, Writing – review & editing

    Affiliation Department of Biology, University of Hildesheim, Hildesheim, Germany

Dental pulp exposure, periapical inflammation and suppurative osteomyelitis of the jaws in juvenile Baltic grey seals (Halichoerus grypus grypus) from the late 19th century

  • Uwe Kierdorf, 
  • Morten T. Olsen, 
  • Patricia Kahle, 
  • Catharina Ludolphy, 
  • Horst Kierdorf
PLOS
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Abstract

The systematic analysis of museum collections can provide important insights into the dental and skeletal pathology of wild mammals. Here we present a previously unreported type of dental defect and related skull pathology in five juvenile Baltic grey seals that had been collected in the course of a seal culling program along the Danish coast in 1889 and 1890. All five skulls exhibited openings into the pulp cavities at the crown tips of all (four animals) or two (one animal) canines as well as several incisors and (in one animal) also some anterior premolars. The affected teeth showed wide pulp cavities and thin dentin. Pulp exposure had caused infection, inflammation, and finally necrosis of the pulp. As was evidenced by the extensive radiolucency around the roots of the affected teeth, the inflammation had extended from the pulp into the periapical space, leading to apical periodontitis with extensive bone resorption. Further spreading of the inflammation into the surrounding bone regions had then caused suppurative osteomyelitis of the jaws. The postcanine teeth of the pathological individuals typically had dentin of normal thickness and, except for one specimen, did not exhibit pulp exposure. The condition may have been caused by a late onset of secondary and tertiary dentin formation that led to pulp exposure in anterior teeth exposed to intense wear. Future investigations could address a possible genetic causation of the condition in the studied grey seals.

Introduction

The systematic analysis of mammalian skeletons from museum collections can provide important information on the spectrum and prevalence of anomalies and diseases of bones and teeth in the studied species [14]. The findings of such investigations are not only of interest with respect to comparative pathology, but also helpful in the assessment of population health and its changes over time. The results can thereby contribute to the identification of emerging health threats and to an effective population management.

Several studies reported dental anomalies and lesions, alveolar bone pathology, and degenerative changes of the temporomandibular joint in pinnipeds from a comparative point of view [514]. Other studies addressed the prevalence and severity of skull lesions in marine mammals, including polar bears (Ursus maritimus) [3], harbor seals (Phoca vitulina) [2, 15, 16], and grey seals (Halichoerus grypus) [17, 18], in relation to levels of exposure to persistent organic pollutants.

The grey seal is a member of the family Phocidae with a cold temperate to subarctic distribution in North Atlantic waters over the continental shelf [19]. As is typical for the amphibious lifestyle of pinnipeds, it spends parts of its life on land for breeding, molting and thermoregulation. Total population size of the grey seal has more recently been estimated at 632,000 individuals with an increasing trend [19]. The species exhibits a pronounced sexual dimorphism, with adult males from the Eastern North Atlantic weighing between 170 and 310 kg, while the body mass of adult females ranges between 100 and 190 kg [20]. The skull of adult males differs from that of females by its elevated fronto-nasal region [21]. Females reach sexual maturity at 3 to 5 years of age, males at 6 years [21]. Maximum life expectancy in the wild has been estimated at around 35 years for females and 25 years for males [22]. The grey seal is a generalist predator that mainly feeds on a variety of fish species [19, 23]. It uses pierce feeding, i.e., the prey is captured with a piercing bite and then swallowed whole [24].

There exist three geographically separated populations of grey seals, present, respectively, in the western North Atlantic, the Eastern North Atlantic and the Baltic Sea [19, 21]. Currently, two subspecies of the grey seal are recognized, viz. the Atlantic grey seal (Halichoerus grypus atlantica) and the Baltic grey seal (H. g. grypus) [25, 26].

The population of Baltic grey seals in the early 1900s was estimated at 88,000 to 100,000 individuals, but intensive hunting in the course of a multi-national culling program led to a decline to about 20,000 animals in the 1940s [27, 28]. After cessation of hunting, the population did not recover as expected, but further declined to less than 4,000 individuals in the late 1970s [28]. This further decline was linked to impaired reproductive performance of females caused by exposure to high levels of organochlorines [27, 28]. Declining concentrations of these pollutants in their prey species during recent decades led to an improvement of the health situation of the Baltic grey seals and to an increase in reproductive success and population size, which in 2014 was estimated at between 40,000 and 53,000 individuals [28]. Pupping dates differ among the populations in the Northwest Atlantic, the Northeast Atlantic and the Baltic Sea [19]. Peak pupping in the latter occurs in late February to early March [28]. The period of milk-feeding lasts about 15 to 18 days, and weaning is abrupt. First molting occurs around the time of weaning [28].

As a reaction to an intensifying seal-fishery conflict, a seal culling program was started in Denmark in October 1889 that lasted until 1927 [29, 30]. A bounty system was introduced, and in the years 1889 and 1890 hunters were required to send the skulls of culled seals to the Zoological Museum in Copenhagen (now the Natural History Museum of Denmark) to collect their bounty. Following the introduction of the bounty system, the numbers of grey seals in Danish waters rapidly declined and all breeding colonies disappeared before 1900 [30, 31]. Analysis of mitochondrial DNA indicated that historically the grey seals inhabiting inner Danish waters were part of the large Baltic Sea population [32]. The grey seals currently recolonizing the Kattegat primarily originate from the North Sea population, whereas those recolonizing the southwestern Baltic Sea primarily originate from the population in the central Baltic Sea [32].

Thus far, only few studies have addressed the occurrence of dental anomalies in grey seals [11, 33, 34]. In addition, a recent paper described the dentition of a hybrid between a grey seal and a ringed seal (Pusa hispida) [35].

The permanent dentition of the grey seal comprises 34–36 teeth, the dental formula being I 3/2, C 1/1, P 4/4, M 1−2/1 [I: incisors (dentes incisivi), C: canines (dentes canini), P: premolars (dentes praemolares), M: molars (dentes molares] (Fig 1). In the literature on pinnipeds, premolars and molars are often collectively referred to as postcanine teeth [21, 36, 37]. The number of molars can differ between maxilla and mandible, as a maxillary second molar is rather frequently present either uni- or bilaterally. The crowns of the brachydont premolars and molars are of a uniform shape with a large conical central cusp and small secondary cusps (Fig 1). The deciduous teeth of the grey seal are already shed prenatally. Development of all permanent teeth is well advanced at birth, and they erupt a few days after birth [22]. At the end of the short suckling period, the permanent dentition is thus fully erupted and functional.

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Fig 1. Permanent dentition of a Baltic grey seal (Halichoerus grypus grypus; specimen ZMUC CN 1157, juvenile male).

(a) Maxillary dentition. (b) Mandibular dentition.

https://doi.org/10.1371/journal.pone.0215401.g001

The bulk of a typical mammalian tooth is formed by dentin that in the crown is covered by enamel and in the root by cementum [37, 38]. While enamel is only formed prior to tooth eruption, deposition of dentin and cementum continues in an incremental fashion during the life of a tooth. In seals, the growth layers in dentin and cementum are used for age estimation [19, 22]. Dentin is formed by odontoblasts that are arranged in a layer at the interface with the dental pulp [38]. The dentin is permeated by dentinal tubules that extend through the entire thickness of the tissue, each dentinal tubule harboring a long odontoblast process [37, 38].

The dentin formed prior to (full) tooth eruption and completion of the apical tooth region (in brachydont teeth) is referred to as primary dentin. It includes the initially formed mantle dentin and the outer zone of the subsequently deposited circumpulpal dentin [39, 40]. Secondary dentin is the regular circumpulpal dentin deposited at a slower rate during the later life of a tooth. [39, 40]. The tubules in secondary dentin are largely continuous with those of the primary dentin, suggesting that both dentin types are formed by the same odontoblasts. Frequently, the direction of the dentinal tubules changes from primary to secondary dentin, and the tubular pattern of secondary dentin is somewhat less regular than that of primary dentin [41]. Tertiary dentin is a more or less irregular tissue formed locally as a pulpal reaction to strong stimuli, such as excessive wear, mechanical, chemical or thermal stresses [3941]. It has fewer tubules than primary or secondary dentin or may be largely atubular. Tertiary dentin has been classified into reactionary dentin, whose matrix is secreted by remaining postmitotic odontoblasts, and reparative dentin, whose matrix is produced by newly differentiated pulpal cells in response to stronger stimuli following the death of the original odontoblasts [40, 41].

During dentinogenesis first an unmineralized organic matrix, the predentin, is secreted that is subsequently mineralized. The mineral initially forms globular structures referred to as calcospherites that gradually enlarge and thereby become confluent [37, 38, 41]. During later phases of dentinogenesis, when dentin deposition slows down, a more linear pattern of mineralization can often be seen [38]. When the calcospherites fail to fuse, the dentin exhibits unmineralized or hypomineralized areas referred to as interglobular dentin [37, 38].

In the course of a research project on the health of marine mammals from the North Sea and the Baltic Sea, the skulls of grey seals held in the skull collection of the Natural History Museum of Denmark were systematically inspected for dental anomalies and lesions. Here we describe a previously unreported type of dental pathology and its sequelae in the skulls of grey seals collected in the southwestern Baltic region in the period 1889−1890.

Materials and methods

Three complete and two fragmentary skulls of Baltic grey seals from the skull collection of the Natural History Museum of Denmark, University of Copenhagen, exhibited a similar spectrum of pathological dental and osseous changes. None of the other 204 grey seal skulls from the collection that were inspected in the course of our study showed comparable lesions.

The five pathological skulls belonged to individuals that had been collected in the Southwestern Baltic in either 1889 (months October to December) or 1890 (Table 1, Fig 2). In four of the specimens, this information was given both on the skulls and the associated tag. In the fifth specimen (ZMUC 303), this was not the case. However, the specimen’s low ID number, the fact that the skull showed the same type of ink labeling as the other four specimens, and the observation that the handwriting on the piece of paper found in the plastic bag containing the skull was the same as in the case of one of the other four skulls (ZMUC 139, collected in 1890) is taken as evidence that specimen ZMUC 303 was also collected in the said period (probably in 1890). All five skulls are therefore considered to have originated from individuals that had been obtained during the first two years of the aforementioned Danish seal culling program. It is not stated in the records whether the animals had been hunted or were found dead. None of the complete skulls showed evidence of projectile or blunt force (clubbing) trauma. However, two pathological skulls were fragmentary with the entire braincase (ZMUC 139) or large portions of it (ZMUC 303) missing, which would be consistent with a trauma to the skull. Given the origin of the 19th century skulls it is assumed that all originate from grey seals that had been killed in the course of the culling program.

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Fig 2. Map indicating the localities (asterisks) where the Baltic grey seals (Halichoerus grypus grypus) had been collected in 1889–1890.

Five skulls belonged to animals culled on Rødsand sandbank (locality 1), one to an animal culled near Faxe on Sjaelland (locality 2), and one to an animal from Christiansø on Bornholm (locality 3).

https://doi.org/10.1371/journal.pone.0215401.g002

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Table 1. List of Baltic grey seals (Halichoerus grypus grypus) skulls from the collection of the Natural History Museum of Denmark, University of Copenhagen (ZMUC) analyzed in the present study.

All teeth present in the specimens belonged to the permanent dentition, and no persistent deciduous teeth were recorded. The labeling is given as it appears on the skulls and the tags (Nobr = November, Dcbr = December).

https://doi.org/10.1371/journal.pone.0215401.t001

For comparison, three complete skulls of Baltic grey seals that were free of the lesions seen in the five pathological skulls were also studied (Table 1). Two of the three control skulls (ZMUC 713 and ZMUC 719) had also been collected in 1889 and originated from the same locality (Rødsand, for location see Fig 2) as three of the specimens (ZMUC 155, ZMUC 424, ZMUC 693) exhibiting pathological changes. The third control skull (ZMUC CN 1157) was that of a modern juvenile male grey seal (S1 Fig). No information on the sex of the other seven grey seals was available. As sexual dimorphism in skull size or canine length is marked only in older individuals [22], sex of the juvenile grey seals from the 19th century could not be established. Condylobasal length was measured in the six complete skulls (not possible in the fragmentary skulls ZMUC 139 and ZMUC 303). Values ranged between 190 mm and 202 mm in the five complete skulls from 1889, while condylobasal length in specimen CN 1157 was 214 mm (Table 1).

Based on an assessment of the degree of fusion of sutures and synchondroses, applying the scoring system by Sivertsen [42], condylobasal length (in the complete skulls) and the size of the canines [22], all eight studied skulls were classified as those of juvenile individuals. Three of the pathological skulls (ZMUC 155, ZMUC 424, and ZMUC 693) and two of the control skulls (ZMUC 713 and ZMUC 719) belonged to animals collected in the months October to December (see Table 1 for collection dates). These skulls were classified as those of young-of-the-year. The other three skulls (two pathological, one control) belonged to older juveniles. Specimen ZMUC 139 originated from an individual collected in June 1890 and was characterized by pronounced wear of incisors, canines and anterior premolars. In specimens ZMUC 303 and ZMUC CN 1157, a higher age of the individuals was indicated by the larger size of the (fragmentary or complete) skulls compared to those of the five young-of-the-year. Moreover, in ZMUC CN 1157 the sphenooccipital synchondrosis was already fused over more than 50% of its length, while it was open in all young-of-the-year. Due to the fragmentary condition of the skulls, this synchondrosis could not be assessed in specimens ZMUC 139 and ZMUC 303.

All teeth present in the studied skulls belonged to the permanent dentition, and no persistent deciduous teeth were found. Information on the teeth available for inspection in the specimens is given in Table 1.

The eight skulls were first studied macroscopically. Photos were obtained with a digital camera, and radiographs were taken of all specimens. Subsequently, the right maxillary C (length 33.9 mm) and the right M1 of specimen ZMUC 693, the left maxillary C (length 41.2 mm) of specimen ZMUC CN 1157, and the right M1 of specimen ZMUC 719 were extracted, photographed, and embedded in epoxy resin (Biodur, Biodur Products, Heidelberg, Germany or Epofix, Struers, Ballerup, Denmark). Canine length of ZMUC 693 falls within the range (32.3 to 38.5 mm) given for 6 to 8 month-old grey seals [22]. The embedded teeth were sectioned longitudinally in the labio-palatal (canines) or the bucco-lingual (molars) plane. The cut surfaces of the resulting halves were smoothed and polished using a series of silicon carbide papers (grits 320−4,000), followed by further sequential polishing on a motorized polisher (Labopol 5, Struers, Ballerup, Denmark) with diamond suspensions of 3 and 1 μm particle size, respectively. Backscattered electron (BSE) imaging of the (uncoated) polished cut surfaces was performed in a scanning electron microscope (SEM, Zeiss Evo Ma 15, Carl Zeiss AG, Oberkochen, Germany), operated in a low pressure mode at 20 kV. Following BSE imaging and hardness testing in the canines (see below), the cut surfaces of the teeth were etched (34% phosphoric acid for 5 seconds) and subsequently repeatedly rinsed with deionized water and propanol. The dried blocks were sputter-coated with gold-palladium (Leica EM ACE200, Leica Microsystems, Wetzlar, Germany), and viewed again in the SEM (at 10 kV) using a secondary electron (SE) detector.

To analyze whether the abnormal condition of the teeth in the five pathological skulls was related to a hypomineralization of their enamel and/or coronal dentin, following BSE imaging and prior to etching we performed hardness testing on the polished cut surfaces of the two extracted canines (from control specimen ZMUC CN 1157 and pathological specimen ZMUC 693). Testing was performed in labial enamel and coronal dentin with a Vickers indenter (load of 25 p, dwell time 20 sec.) in a Dura-Scan 20 hardness tester (Struers, Ballerup, Denmark). The probed surfaces were oriented perpendicular to the tip of the diamond pyramid. In each of the two teeth, eight measurements were performed in enamel (four in the inner half and four in the outer half of the enamel layer) and another eight in dentin (four at a distance of 50 μm and four at a distance of 200 μm from the enamel-dentin junction (EDJ). To improve the visibility of the indentations, prior to testing the probed surfaces were sputter-coated with a very thin (15 nm) layer of gold-palladium. Data for enamel and dentin hardness were compared between the two teeth using the Mann-Whitney U-test, with the level of significance set at p < 0.05.

Results

On macroscopic and radiographic inspection, the five pathological grey seal skulls showed a similar spectrum of dental and bone lesions. All specimens exhibited openings into the pulp cavity at the crown tips of incisors and canines, and in one case (ZMUC 139) also of three anterior premolars (Table 1, Fig 3). Except for one specimen (ZMUC 155, both maxillary C unaffected), exposure of the pulp cavity through an opening at the crown tip was observed in all mandibular and maxillary canines from the five pathological skulls. In the affected teeth, both the tip of the enamel cap and the dentin overlying the pulp horn had thus been lost soon after eruption due to wear. No indication of fracturing of the tooth tips was observed. Except for specimen ZMUC 139, no exposure of the pulp cavity was recorded in postcanine teeth.

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Fig 3. Exposure of the pulp cavities through openings at the crown tips in permanent maxillary teeth of juvenile Baltic grey seals (Halichoerus grypus grypus) from the 19th century.

(a) Ventral view of the incisive and anterior maxillary region of specimen ZMUC 424. Note openings at the crown tips of all six maxillary I and both maxillary C. (b) Ventral view of the incisive and anterior maxillary region of specimen ZMUC 693. Note openings at the crown tips of all six I (very small in both I1) and both maxillary C. (c) Ventral view of the incisive and anterior maxillary region of specimen ZMUC 139. Note openings at the crown tips of both I3 and maxillary C, the right P1 and P2, and the left P2 and increased wear of these teeth. Both I1, both I2, the left P1 and the right P3 are missing due to postmortem loss, as is evidenced by the unfilled and sharp-edged alveoli of these teeth. (d) Radiograph (lateromedial projection) of the bones of the anterior facial region of specimen ZMUC 139. Note thin walls and wide apical foramen of the right maxillary C as well as marked radiolucency around its root apex (asterisk) indicative of osteolysis in the course of a periapical inflammation.

https://doi.org/10.1371/journal.pone.0215401.g003

In the skulls of the young-of-the-year, radiographic inspection demonstrated that canines with openings at the crown tips had thinner dentin than the unaffected canines of the two controls (Fig 4).

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Fig 4. Radiographs (dorsoventral projections) of the anterior portions of the mandibles of four juvenile Baltic grey seals (Halichoerus grypus grypus).

Note thin walls of the mandibular C exhibiting exposed pulp cavities through openings at the crown tips in the two specimens shown in (a) [ZMUC 424] and (b) [ZMUC 303] compared to thicker canine walls in the unaffected “control” specimens shown in (c) [ZMUC 713] and (d) [ZMUC CN 1157]. Arrows: radiolucent areas around the roots of the canines, indicative of pronounced periapical osteolysis. Arrowheads: radiolucency around the root apices of incisors.

https://doi.org/10.1371/journal.pone.0215401.g004

Thickness of the canine walls in the control skull (ZMUC CN 1157; Fig 4D) from the older juvenile was markedly higher (due to progressive dentin apposition) than in the skulls of the young-of-the-year (Fig 4A–4C). In contrast, in pathological specimen ZMUC 139, likewise belonging to an older juvenile, the canine walls were still thin (Fig 3D), denoting a lack of dentin deposition. The canines of the young-of-the-year exhibited wide apical foramina typical for that age (Fig 4A–4C). In the older control specimen (ZMUC CN 1157), the apical foramina of the canines were markedly narrower (Fig 4D), while this was not the case in pathological specimen ZMUC 139 (Fig 3D). The above findings indicate that, following the formation of an initial layer of dentin, no further dentin apposition had occurred in the canines of the pathological skulls. Corresponding changes to those seen in the canines were also observed in the incisors that exhibited openings into the pulp cavity at their crown tips.

In addition to the changes described above, specimen ZMUC 139 showed abnormally intensified wear on all incisors and canines available for inspection as well as on four anterior premolars (right P1, both P2 and left P1). The crowns of the affected teeth had been completely worn away, with only the roots remaining (Fig 3C and 3D). In the pathological skulls (except for specimen ZMUC 139, see above), dentinal thickness of premolars and molars was markedly higher than that of the incisors and canines with exposed pulp cavities (Fig 4A and 4B; Fig 5B).

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Fig 5.

Dorsal view (a) and radiograph (b) of the anterior mandibular region of a juvenile Baltic grey seal (Halichoerus grypus grypus) from the 19th century (specimen ZMUC 155). Note opening into the pulp cavity at the crown tip of the right mandibular C (small arrow in (a)) and radiolucency indicative of pronounced osteolysis around the root of this tooth (asterisk in (b)). In the right mandible, the P1 is missing, most likely due to intravital loss. In the left mandible, the C, P1 and P2 are missing due to intravital loss and the alveoli of these teeth have been filled with bone (large arrows in (a) and (b)).

https://doi.org/10.1371/journal.pone.0215401.g005

Radiographic inspection demonstrated pronounced radiolucency around the root apices of the teeth with exposed pulp cavities (Figs 3D, 4A and 4B, 5B), indicating intense bone resorption in the course of an inflammatory process. In the left mandible of specimen ZMUC 155, the canine and the P1 and P2 were missing (intravital loss), and the alveoli of these teeth had become partially (C) or completely (P1 and P2) filled with new bone (re-ossification of alveoli) (Fig 5).

Occurrence of the extended osteolytic lesions around the root apices was associated with a spectrum of other pathological changes in the incisive, maxillary, and mandibular bones (Figs 6 – 8). These changes included areas of extensive cortical bone destruction and openings of draining tracts for pus discharge in the cortex (Fig 6). In addition, the bilateral presence of sequestra and involucra was observed in the mandibles of two of the pathological specimens (ZMUC 424 and ZMUC 303) (Figs 7 and 8). In specimen ZMUC 303, an incomplete pathological fracture had occurred in the left mandibular corpus along the demarcation line around the sequestrum (Fig 7B).

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Fig 6. Osteolytic lesions in the skulls of juvenile Baltic grey seals (Halichoerus grypus grypus).

(a) Dorsal view of the anterior cranium of a juvenile grey seal from the 19th century (specimen ZMUC 303) showing numerous openings of draining tracts in the incisive bones. (b) Dorsal view of the anterior cranium of a modern juvenile grey seal (specimen ZMUC CN 1157) free of such lesions. (c) Left lateral view of the anterior cranium of a juvenile grey seal from the 19th century (specimen ZMUC 693) showing openings of draining tracts and eroded surface of the maxilla indicative of extended cortical bone resorption. Arrow: root tip of left maxillary C.

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Fig 7.

Dorsal (a) and ventral (b) views of the anterior portion of the mandibles of a juvenile Baltic grey seal (Halichoerus grypus grypus) from the 19th century (specimen ZMUC 303) showing signs of suppurative osteomyelitis. (a) Note opening of a draining tract in the left mandible (arrow), and presence of a large (entirely detached) sequestrum (S) and an involucrum (I) in the right mandible. Arrowheads: openings into the pulp cavities at the tips of both mandibular C. The right P1 is missing, most likely due to intravital loss. (b) Note presence of a large sequestrum (asterisk), demarcated by a dividing line but still in place, in the left mandible, and occurrence of an involucrum (I) in the right mandible. An incomplete pathological fracture of the left mandible had occurred along the demarcation line.

https://doi.org/10.1371/journal.pone.0215401.g007

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Fig 8. Signs of periapical abscesses and suppurative osteomyelitis in the mandibles of a juvenile Baltic grey seal (Halichoerus grypus grypus) from the 19th century (specimen ZMUC 424).

(a) Dorsal view of anterior mandibular region. Note openings into the pulp cavities at the crown tips of both I1 and both mandibular C, openings of draining tracts (arrowheads), large resorption cavity around the root of the right canine and presence of a large sequestrum in each mandible (asterisks). The loose right P2 was removed to show the resorption cavity. (b) Completely detached sequestrum that was removed from the right mandible. The surface of the sequestrum shows traces of intense resorption.

https://doi.org/10.1371/journal.pone.0215401.g008

The anterior portions of the mandibles of the five pathological specimens were enlarged due to the apposition of porous periosteal new bone (Fig 9). This is evidence of a proliferative reaction of the periosteum to the inflammatory process. Porous bone apposition was also noted on the walls in several of the widened alveoli of incisors and canines (Fig 9A).

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Fig 9. Lateral views of the anterior regions of the left mandibles of two juvenile Baltic grey seals (Halichoerus grypus grypus) from the 19th century.

(a) Specimen ZMUC 139; I1, I2, C and P1 are missing, most likely due to intravital loss. Note widened alveolus of the mandibular C and enlargement of the mandible due to apposition of periosteal new bone with numerous small vascular foramina. Porous bone has also been deposited on the wall of the enlarged alveolus. Some of the mental foramina appear enlarged. The changes are indicative of a severe inflammatory process in the anterior mandible. (b) Specimen ZMUC 719, showing the normal condition of the mandible for comparison.

https://doi.org/10.1371/journal.pone.0215401.g009

The two extracted maxillary canines showed a similar position of the crown-root border (Fig 10A). While the enamel cover of the maxillary canine of the control specimen ZMUC CN 1157 was still present, it had been worn away in the incisal area of the maxillary canine of specimen ZMUC 693 (Fig 10A, Fig 11). Following the wearing away also of the incisal primary dentin, pulp exposure had occurred in the former tooth.

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Fig 10. Permanent maxillary canines of juvenile Baltic grey seals (Halichoerus grypus grypus).

(a) Left maxillary C of specimen ZMUC CN 1157 (top) and right maxillary C of specimen ZMUC 693 (bottom). Root development is more advanced in the canine of ZMUC CN 1157. Arrows: crown-root border; arrowhead: opening into the pulp cavity at the crown tip of the right maxillary C of ZMUC 693. Insert: apical foramina of the canines of ZMUC CN 1157 (left) and ZMUC 693 (right). (b) SEM-BSE image of polished cut surface of the longitudinally sectioned left maxillary C of specimen ZMUC 693, showing external root resorption affecting cementum (Ce) and dentin (D). Asterisk: cementum-dentin-junction. (c) SEM-BSE image showing interglobular dentin (areas where the calcospherites have failed to fuse; these areas appear black in the image) in the root of the left maxillary C of specimen ZMUC 693. Ce: cementum; D: dentin; asterisk: cementum-dentin-junction.

https://doi.org/10.1371/journal.pone.0215401.g010

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Fig 11. Longitudinally sectioned right maxillary C of specimen ZMUC 693.

Exposure of the pulp cavity through an opening at the crown tip. D: dentin; E: enamel; arrowheads: crown-root border. Resin-embedded tooth; labial side directed to upper-left corner of image.

https://doi.org/10.1371/journal.pone.0215401.g011

SEM-BSE imaging revealed that in the juxtapulpal dentin overlying the pulp cavity in the canine of specimen ZMUC 693 many dentinal tubules had become occluded by mineral deposits (Fig 12A), while this was not the case in the canine of control specimen ZMUC CN 1157 (Fig 12B). The overall density and arrangement of the dentinal tubules in coronal dentin did, however, not differ between the two teeth. SEM-BSE imaging further demonstrated signs of external and internal root resorption in the canine of specimen ZMUC 693. The marked external root resorption had affected both cementum and dentin (Fig 10B). Signs of internal resorption were present in the dentin of the root canal near the root apex. Contrary to the control canine, the root dentin of the canine of ZMUC 693 also exhibited rather extended areas of interglobular dentin (Fig 10C). In contrast to the canine, the analyzed M1 of ZMUC 693 exhibited both secondary and tertiary dentin in its crown (Fig 13) that still possessed a complete enamel covering. The tertiary dentin had been deposited in the tip of the pulp cavity and was largely atubular. Corresponding findings were made in the studied M1 of specimen ZMUC 719.

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Fig 12. SEM-BSE images of polished cut surface showing the dentin overlying the pulp cavity in permanent maxillary canines of juvenile Baltic grey seals (Halichoerus grypus grypus).

(a) Right maxillary C of specimen ZMUC 693. Note occlusion of many dentinal tubules in juxtapulpal dentin by mineral deposits. As evidenced by its higher brightness, the material occluding the dentinal tubules is more highly mineralized than the surrounding intertubular dentin. PC: resin-filled pulp cavity. (b) Left maxillary C of specimen ZMUC CN 1157. No occlusion of dentinal tubules. D: dentin; PC: resin-filled pulp cavity.

https://doi.org/10.1371/journal.pone.0215401.g012

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Fig 13. SEM-BSE image of polished cut surface of the bucco-lingually sectioned M1 of specimen ZMUC 693 showing the dentin overlying the pulp cavity.

Three types of dentin can be distinguished. The primary dentin (PD) is characterized by regularly arranged, straight dentinal tubules, whereas the tubules in the secondary dentin (SD) are curved and arranged somewhat more irregularly. The tertiary dentin (TD) that has been deposited in the pulp horn exhibits only very few tubules. PC: resin-filled pulp cavity. The stippled line indicates the approximate border between secondary and tertiary dentin.

https://doi.org/10.1371/journal.pone.0215401.g013

SEM-SE imaging of etched cut surfaces revealed normal prismatic structure and presence of Hunter-Schreger bands in the enamel of all four analyzed teeth (Fig 14). Hardness values of enamel and coronal dentin did not differ significantly between the two studied canines (Table 2).

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Fig 14. Enamel structure in longitudinally sectioned teeth of specimen ZMUC 693; SEM-SE images of etched polished cut surface.

(a) buccal enamel of the right M1. The enamel is prismatic and shows Hunter-Schreger bands, i.e., alternating zones of more longitudinally and more transversely sectioned prisms. D: dentin; EDJ: enamel-dentin junction, OES: outer enamel surface; R: resin. (b) labial enamel of the right maxillary C showing zones with transversely (arrowheads) and longitudinally (arrows) sectioned prisms. The outer enamel surface is located to the left of the field of view.

https://doi.org/10.1371/journal.pone.0215401.g014

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Table 2. Vickers hardness of enamel and coronal dentin in maxillary canines of two juvenile Baltic grey seals, Halichoerus grypus grypus (left maxillary C of specimen ZMUC CN 1157, right maxillary C of specimen ZMUC 693).

https://doi.org/10.1371/journal.pone.0215401.t002

Discussion

In the five grey seals exhibiting pathological skull changes, pulp exposure of incisors, canines and, in one skull also anterior premolars had occurred following the loss of the enamel layer covering the crown tip and the wearing away of the subjacent thin dentin layer overlying the pulp cavity. Normally, pulp exposure due to tooth wear is prohibited by the lifelong apposition of dentin that causes a progressive narrowing of the pulp cavity with age [22].

The fact that, except for specimen ZMUC 139, only incisors and canines showed pulp exposure might be linked to the feeding mode of the grey seal. During the piercing bite used for prey capturing, the anterior teeth are likely exposed to more intense wear than the posterior ones. It is presently unclear, why in some anterior teeth (e.g., the canine of skull ZMUC 693) the enamel covering the crown tip was lost early, while in others (e.g., the canine of skull ZMUC CN 1157) it was still present. As was demonstrated by SEM imaging, the enamel of both teeth showed a normal microstructure, which corresponded to that reported for other pinniped species [43]. Hardness testing on the two grey seal canines revealed no significant differences in the degree of mineralization of their enamel and dentin. Thus, the quality of their enamel did apparently not differ between the two teeth. Possible factors responsible for an early local loss of enamel in anterior teeth include individual or sex-related variation in enamel thickness and variation in the composition (and related “abrasiveness”) of the animals’ diet.

According to Hewer [22], at birth the canines of grey seals possess a thin cone of prenatally formed primary dentin, which this author refers to as “fetal dentin”. In the first three to four postnatal weeks, while the canines considerably increase in length, a second dentin cone, referred to as “pup dentin”, is laid down onto the prenatally deposited dentin, and subsequently further dentin layers are added in an incremental fashion [22]. Given the early eruption of the permanent teeth in the grey seal, at least the later-formed pup dentin has to be classified as secondary dentin.

In the five pathological specimens, no or only little dentin had apparently been laid down in anterior teeth following the deposition of an initial (primary) dentin layer. This failure to deposit (sufficient) secondary and/or tertiary dentin in response to wear led to exposure, infection, inflammation (pulpitis) and, finally, necrosis of the pulp in these teeth. In contrast, the postcanine teeth of the pathological skulls typically had thick dentinal walls and showed no pulp exposure. Furthermore, contrary to the studied canine from this skull, the analyzed M1 of specimen ZMUC 693 exhibited deposition of secondary and tertiary dentin at the tip of the pulp cavity, even though the coronal dentin of this tooth was still covered by enamel. The thinner dentin in the canines of the three pathological skulls from young-of-the-year compared to those of the two control skulls from this age group indicates a rapid death of the pulp following its early exposure in the former that then precluded any further dentin formation.

The higher dentinal thickness in postcanine teeth of the pathological skulls suggests that the reason for the observed pathological condition may have been a late onset of secondary and tertiary dentin formation rather than the absolute inability to produce these dentin types. According to this view, for posterior teeth this was compatible with maintaining a viable pulp and ongoing dentin apposition, whereas it proved fatal for anterior teeth in which more intense wear led to pulp exposure and necrosis.

In human dentistry, occlusion of dentinal tubules by mineral deposits, as was observed in the juxtapulpal coronal dentin of the maxillary canine of specimen ZMUC 693, is a typical response of the pulpodentinal complex to caries lesions, particularly slowly progressing and arrested ones [44]. Tubular occlusion causes a reduction in dentin permeability, thereby slowing down further lesions progression [44]. Another potential stimulus that can cause occlusion of dentinal tubules is increased dental attrition [45, 46]. Rapid wear of the grey seal teeth with only thin dentinal walls was substantiated in the present study, and it is suggested that the occlusion of dentinal tubules occurred as a response to this stimulus.

In teeth of juvenile grey seals (≤ 1 year), formation of interglobular dentin has not been reported [22]. In the harp seals (Phoca groenlandica), layers of interglobular dentin are frequently part of later-formed annual growth layer groups, but not in that representing the first year of growth [47]. The interglobular dentin present in the canine of specimen ZMUC 693, which was formed in the year of birth, is therefore considered a pathological feature.

In the five pathological grey seals, the inflammation had spread from the pulp into the periapical space, causing severe lytic lesions with extensive destruction of periapical bone, radiographically visible as extended radiolucent spaces around the tooth roots, and the formation of draining tracts for pus discharge. Especially via the large, deeply inserted canines with their still very wide apical foramina (typical for juvenile seals) the inflammation could easily spread into the jaws and cause the formation of periapical abscesses. The findings in the maxillary canine from specimen ZMUC 693 indicate pronounced external resorption on the tooth root situated in the abscess cavity, the resorptive process penetrating from the cementum into the dentin. The canine also showed signs of internal root resorption in the course of the inflammatory process, which is a common finding in teeth with periapical lesions [48].

From the periapical space the inflammation extended into the surrounding bone regions, leading to suppurative osteomyelitis of the jaws. The bone lesions formed in the course of the osteomyelitis include perforations and extensive subperiosteal resorption of cortical bone, periosteal new bone formation due to proliferative periostitis, and the formation of sequestra and involucra in the mandibles of two animals. Formation of sequestra, i.e., of pieces of bone that have become necrotic due to ischemia and are separated from the surrounding living bone, is a typical sign of osteomyelitis [4952]. An involucrum is a layer of living bone formed around a sequestrum by periosteal activity [49, 50]. The young age of the pathological specimens indicates a rapid progression of the disease process once pulp exposure of the teeth had occurred. It is concluded that the affected individuals had suffered from severe septicemia, which would probably have resulted in their early death.

Periapical abscesses associated with (mostly maxillary) canines and maxillary second incisors have been reported in Weddell seals (Leptonyches weddellii) following exposure of the pulp cavity by excessive wear [53]. This excessive wear was related to the use of the teeth for abrading sea ice to keep breathing holes open during winter. In contrast to the situation in the grey seals studied by us, exposure of the pulp cavities of canines and incisors in the Weddell seals occurred in older animals in which dentin formation had progressed normally [53]. Periapical abscesses and osteomyelitis of the jaws secondary to exposure of the pulp cavities can also occur if teeth are rapidly worn down due to a hypomineralization of their enamel, as has for instance been observed in wild red deer (Cervus elaphus) exhibiting dental fluorosis [54]. In the grey seals, hardness testing indicated no significant difference in the degree of mineralization of enamel and coronal dentin between the pathological and control canine.

Defective dentinogenesis characterizes different dental disorders, and it may therefore be asked, whether the condition seen in the pathological grey seal teeth can be related to specific disorders known in dental pathology. Odontodysplasia is a rare, non-hereditary developmental disturbance of uncertain etiology in humans [5558]. As the anomaly is usually of a localized nature, affecting mostly teeth of only one quadrant of the dentition, it is mostly referred to as regional odontodysplasia. A corresponding condition has also been described in a maxillary canine of a domestic dog [59]. Affected teeth show wide pulp cavities and thin dentin with occurrence of interglobular dentin [5558]. However, in odontodysplasia also the enamel is abnormal, exhibiting both hypoplasia and hypomineralization [5558]. In contrast, the enamel of the affected canine of specimen ZMUC 693 was normally mineralized, and none of the teeth of the pathological grey seal skulls exhibited enamel hypoplasia. The pathological changes in the grey seal teeth are therefore not consistent with a diagnosis of regional odontodysplasia.

In humans, teeth with enlarged pulp cavities and only thin dentin layers, referred to as shell teeth, have been reported from individuals with dentinogenesis imperfecta type III (DGI-III), a heritable dentin defect with an autosomal dominant mode of inheritance [6064]. The condition is associated with a high frequency of pulp exposure and occurrence of periapical radiolucencies. SEM observations on permanent teeth of patients with DGI-III revealed normal enamel structure, while the dentin showed structural changes [61]. DGI-III is associated with mutations in the DSPP gene that encodes dentin sialophosphoprotein (DSPP), a non-collagenous extracellular matrix protein [6265]. The DSPP produced by the odontoblasts is proteolytically cleaved into fragments, thereby converting the inactive precursor into active proteins involved in the formation and mineralization of dentin [6367].

DSPP-/- knockout mice (Mus musculus) exhibit dental defects similar to those seen in humans with DGI-III. The affected mouse teeth have enlarged pulp cavities, pulp exposure and degeneration, periapical bone resorption and abnormally intensified wear [65]. Light and scanning electron microscopy revealed a marked reduction in the thickness of mineralized dentin, a widened predentin zone, an irregular dentinal mineralization front, and the frequent occurrence of interglobular dentin in the teeth of the DSPP-/- mice [65]. Similar changes and a marked drop in dentinal mineral apposition rate compared to controls were also observed in mice in which the proteolytic processing of DSPP had been blocked [67].

Contrary to the presentation of DGI-III in humans and the dental changes seen in the DSPP-/- knockout mice, in the pathological grey seals only anterior teeth showed thin dentin and enlarged pulp cavities. This is in our view not consistent with the diagnosis of a pinniped equivalent of DGI-III. However, it could be speculated that the condition in the grey seals was related to a less severe disturbance in the formation, secretion, or processing of DSPP that caused a delay in dentin apposition following the formation of an initial primary dentin layer.

The reported dental and skull pathology has thus far been observed only in juvenile grey seals collected in 1889/1890 and within a relatively narrow area of the subspecies, which might be seen as circumstantial evidence for a heritable dentin defect in the animals. Future studies are needed to clarify a possible genetic causation of the reported condition in the Baltic grey seals. The fact that the condition is not known from modern grey seals could mean that it does not exist in today’s populations, although the possibility exists that it is only unreported. Therefore, further studies addressing dental pathology in other grey seal populations are encouraged.

It has been experimentally demonstrated in rats (Rattus norvegicus) that certain persistent organic pollutants can disturb tooth development and impair dentinogenesis [6872]. However, in the late 19th century these substances were not yet existing and the Baltic Sea was still unpolluted [73]. Therefore, pollutant exposure is excluded as a factor involved in the formation of the lesions observed in the grey seals from that period.

In conclusion, the present study has demonstrated the occurrence of a specific and previously unreported form of dental defect of unknown etiology and its sequelae (pulp exposure and inflammation, periapical abscesses and osteomyelitis of the jaws) in Baltic grey seals from the 19th century. It is hypothesized that a late onset or reduced rate of secondary and tertiary dentin formation led to pulp exposure in anterior teeth that were exposed to more intense wear than posterior ones. The present study underscores the importance of systematic studies of museum collections for a broadening of our knowledge of pathological dental and skeletal conditions in wild mammals.

Supporting information

S1 Fig. Right lateral view of the skull of a juvenile male Baltic grey seal (Halichoerus grypus grypus; specimen ZMUC CN 1157).

https://doi.org/10.1371/journal.pone.0215401.s001

(TIF)

Acknowledgments

We gratefully acknowledge the financial support of our study by the Volkswagen Foundation (Az. 89 911) within the framework of the program “Research in Museums”. One of the authors (UK) furthermore received funding from the European Union’s Seventh Framework Program under grant agreement No 228506 (SYNTHESYS; DK-TAF-5825). The Natural History Museum of Denmark, University of Copenhagen, is thanked for access to collection of grey seal skulls. We further thank the two reviewers for their very helpful comments on an earlier version of the manuscript.

References

  1. 1. Miles AEW, Grigson C. Colyer’s Variations and Diseases of the Teeth of Animals. Revised edition. Cambridge; Cambridge University Press. 1990.
  2. 2. Mortensen P, Bergmann A, Bignert A, Hansen HJ, Härkönen T, Olsson M. Prevalence of skull lesions in harbor seals (Phoca vitulina) in Swedish and Danish museum collections: 1835−1988. Ambio. 1992; 21:520−524.
  3. 3. Sonne C, Rigét FF, Dietz R, Wiig O, Kirkegaard M, Born EW. Skull pathology in East Greenland and Svalbard polar bears (Ursus maritimus) during 1892 to 2002 in relation to organochlorine pollution. Sci Total Environ. 2007; 372:554–561. pmid:17156821
  4. 4. Azorit C, Oya A, Tellado S, Carrasco R, Moro J. Mandibular osteomyelitis in red deer (Cervus elaphus hispanicus) and fallow deer (Dama dama): occurrence and associated factors in free-living populations in southern Spain. J Wildl Dis. 2012; 48:77–86. pmid:22247376
  5. 5. Drehmer CJ, Fabián ME, Menegheti JO. Dental anomalies in the Atlantic population of South American sea lion, Otaria byronia (Pinnipedia, Otariidae): Evolutionary implications and ecological approach. Latin Am J Aquat Mamm. 2004; 3:7–18.
  6. 6. Abbott C, Verstraete FJM. The dental pathology of northern elephant seals (Mirounga angustirostris). J Comp Pathol. 2005; 132:169–178. pmid:15737343
  7. 7. Sinai NL, Dadaian RH, Kass PH,Verstraete FJM. Dental pathology of the California sea lion (Zalophus californianus). J Comp Pathol. 2014; 151:113–121. pmid:24725510
  8. 8. Aalderink MT, Nguyen HP, Kass PH, Arzi B, Verstraete FJM. Dental and temporomandibular joint pathology of the northern fur seal (Callorhinus ursinus). J Comp Pathol. 2015a; 152:325–334.
  9. 9. Aalderink MT, Nguyen HP, Kass PH, Arzi B, Verstraete FJM. Dental and temporomandibular joint pathology of the Eastern Pacific harbour seal (Phoca vitulina richardii). J Comp Pathol. 2015b; 152:335–344.
  10. 10. Arzi B, Leale DM, Sinai NL, Kass PH, Lin A, Verstraete FJM. The temporomandibular joint of California sea lions (Zalophus californianus): Part 2–osteoarthritic changes. Arch Oral Biol. 2015; 60:216−222. pmid:25451465
  11. 11. Drehmer CJ, Sanfelice D, Loch C. Dental anomalies in pinnipeds (Carnivora. Otariidae and Phocidae): occurrence and evolutionary implications. Zoomorphology. 2015; 134:325–338.
  12. 12. Winer JN, Arzi B, Leale DM, Kass PH, Verstraete FJM. Dental and temporomandibular joint pathology of the walrus (Odobenus rosmarus). J Comp Pathol. 2016b; 155:242–253.
  13. 13. Kahle P, Ludolphy C, Kierdorf H, Kierdorf U. Dental anomalies and lesions in Eastern Atlantic harbor seals, Phoca vitulina vitulina (Carnivora, Phocidae), from the German North Sea. PLoS ONE. 2018; 13(10):e0204079. pmid:30281623
  14. 14. Ludolphy C, Kahle P, Kierdorf H, Kierdorf U. Osteoarthritis of the temporomandibular joint in the Eastern Atlantic harbour seal (Phoca vitulina vitulina) from the German North Sea: a study of the lesions seen in dry bone. BMC Vet Res. 2018; 14:150. pmid:29716601
  15. 15. Schandorff S. Developmental stability and skull lesions in the harbor seal (Phoca vitulina) in the 19th and 20th centuries. Ann Zool Fennici. 1997; 34:151−166.
  16. 16. Pertoldi C, Jensen LF, Alstrup AKO, Munk OL, Pedersen TB, Sonne C, et al. Prevalence of skull pathologies in European harbor seals (Phoca vitulina) during 1981−2014. Mamm Res. 2018; 63:55−63.
  17. 17. Bergman A, Olsson M, Reiland S. Skull-bone lesions in the Baltic grey seal (Halichoerus grypus). Ambio. 1992; 21:517–519.
  18. 18. Lind PM, Bergman A, Olsson M, Örberg J. Bone mineral density in male Baltic grey seal (Halichoerus grypus). Ambio. 2003; 32: 385–388. pmid:14627366
  19. 19. Bowen D. Halichoerus grypus. The IUCN Red List of Threatened Species 2016: e.T9660A45226042. 2016. http://dx.doi.org/10.2305/IUCN.UK.2016-1.RLTS.T9660A45226042.en; last accessed 2 January 2019.
  20. 20. Hall A, Thompson D. Gray seal Halichoerus grypus. In: Perrin WF, Würsig B, Thewissen JGM (Eds) Encyclopedia of Marine Mammals. Second Edition. Amsterdam; Elsevier-Academic Press; 2009. pp. 500–503.
  21. 21. Anderson SS. Halichoerus grypus (Fabricius, 1791)–Kegelrobbe. In: Niethammer J, Krapp F (Eds) Handbuch der Säugetiere Europas. Band 6: Meeressäuger. Teil II: Robben–Pinnipedia. Wiesbaden; Aula-Verlag; 1992. pp. 97−115.
  22. 22. Hewer HR. The determination of age, sexual maturity, longevity and a life-table in the grey seal (Halichoerus grypus). Proc Zool Soc London. 1964; 142:593−624.
  23. 23. Scharff-Olsen CH, Galatius A, Teilmann J, Dietz R, Andersen SM, Jarnit S, et al. Diet of seals in the Baltic Sea region: a synthesis of published and new data from 1968 to 2013. ICES J Marine Sci. 2018;
  24. 24. Kienle SS, Berta A. The better to eat with you: the comparative feeding morphology of phocid seals (Pinnipedia, Phocidae). J Anat. 2016; 228:396−413. pmid:26646351
  25. 25. Olsen MT, Galatius A, Biard V, Gregersen K, Kinze CC. The forgotten type specimen of the grey seal [Halichoerus grypus (Fabricius, 1791)] from the island of Amager, Denmark. Zool J Linnean Soc. 2016; 178:713−720.
  26. 26. Society for Marine Mammalogy, Committee on Taxonomy. List of marine mammal species and subspecies. 2017. Available at www.marinemammalscience.org; last accessed on 2 January 2019.
  27. 27. Harding KC, Härkönen T, Helander B, Karlsson O. Status of Baltic grey seals: Population assessment and extinction risk. NAMMCO Sci Publ. 2007; 6:33−56.
  28. 28. Härkönen T. Halichoerus grypus (Baltic Sea subpopulation). The IUCN Red List of Threatened Species 2016: e.T74491261A74491289. 2016. http://dx.doi.org/10.2305/IUCN.UK.2016-1.RLTS.T74491261A74491289.en; last accessed 2 January 2019.
  29. 29. Joensen AH, Søndergaard NO, Hansen EB. Occurrence of seals and seal hunting in Denmark. Danish Rev Game Biol. 1976; 10:1−20.
  30. 30. Olsen MT, Galatius A, Härkönen T. The history and effects of seal-fishery conflicts in Denmark. Mar Ecol Progr Ser. 2018; 595:233−243.
  31. 31. Härkönen T, Brasseur S, Teilmann J, Vincent C, Dietz R, Abt K, et al. Status of grey seals along mainland Europe from the Southwestern Baltic to France. NAMMCO Sci Publ. 2007; 6:57−68.
  32. 32. Fietz K, Galatius A, Teilmann J, Dietz R, Frie AK, Klimova A, et al. Shift of grey seal subspecies boundaries in response to climate, culling and conservation. Mol Ecol. 2016; 25:4097–4112. pmid:27616353
  33. 33. Könemann S, van Bree PJH. Gebißanomalien bei nordatlantischen Phociden (Mammalia, Phocidae). Z Säugetierkunde 1997; 62:71−85.
  34. 34. Cruwys L, Friday A. Visible supernumerary teeth in pinnipeds. Polar Rec. 2006; 220:83−85.
  35. 35. Savriama Y, Valtonen M, Kammonen JI, Rastas P, Smolander OP, Lyyski A, et al. Bracketing phenogenotypic limits of mammalian hybridization. R Soc Open Sci. 2018; 5:180903. pmid:30564397
  36. 36. Jefferson TA, Leatherwood S, Webber MA. Marine Mammals of the World. FAO Species Identification Guide. Rome; Food and Agriculture Organization of the United Nations (FAO). 1993.
  37. 37. Hillson S. Teeth. Second edition. Cambridge; Cambridge University Press. 2005.
  38. 38. Nanci A. Ten Cate’s Oral Histology. Ninth edition. St. Louis; Elsevier. 2018.
  39. 39. Frank RM, Nalbandian J. Structure and ultrastructure of dentine. In: Oksche A, Vollrath L (Eds) Handbook of Microscopic Anatomy, Vol V/6: Teeth. Berlin; Springer; 1989. pp. 173−247.
  40. 40. Smith AJ. Formation and repair of dentin in the adult. In: Hargreaves KM, Goodis HE, Tay FR (Eds.) Seltzer and Bender’s Dental Pulp. Second edition. Chicago; Quintessence; 2012. pp. 27−46.
  41. 41. Berkovitz BKB, Holland GR, Moxham BJ. Oral Anatomy, Histology and Embryology. Fifth edition. Edinburgh; Elsevier. 2018.
  42. 42. Sivertsen E. A survey of the eared seals (family Otariidae) with remarks on the Antarctic seals collected by M/K “Norvegia” in 1928−1929. Scientific Results of the Norwegian Antarctic Expeditions 1927−1928 et SQQ., Instituted and Financed by Consul Lars Christensen. No. 36. Oslo: Det Norske Videnskaps-Akademi i Oslo. 1954.
  43. 43. Loch C, Boessenecker RW, Churchill M, Kieser J. Enamel ultrastructure of fossil and modern pinnipeds: evaluating hypotheses of feeding adaptations in the extinct walrus Pelagiarctos. Sci Nat. 2016; 103: 44.
  44. 44. Tay FR, Messer H, Schwartz R. Caries, restorative dentistry, and the pulp. In: Hargreaves KM, Goodis HE, Tay FR (Eds.) Seltzer and Bender’s Dental Pulp. Second edition. Chicago; Quintessence; 2012. pp. 323−348.
  45. 45. Schroeder HE. Pathobiologie oraler Strukturen. Second edition. Basel; Karger; 1991.
  46. 46. Appleton J, Chesters J, Kierdorf U, Kierdorf H. Changes in the structure of dentine from cheek teeth of deer chronically exposed to high levels of environmental fluoride. Cells Tissues Organs. 2000; 167:266−272. pmid:11014916
  47. 47. Bowen D, Sergeant DE, Øritsland T. Validation of age estimation in the harp seal, Phoca groenlandica, using dentinal annuli. Can J Fish Aquat Sci. 1983; 40:1430−1441.
  48. 48. Levin LG. Root resorption. In: Hargreaves KM, Goodis HE, Tay FR (Eds.) Seltzer and Bender’s Dental Pulp. Second edition. Chicago; Quintessence; 2012. pp. 397−419.
  49. 49. Resnick D, Niwayama G. Osteomyelitis, septic arthritis, and soft tissue infection: Mechanisms and situations. In: Resnick D (Ed.) Diagnosis of Bone and Joint Disorders. Third edition. Volume 4. Philadelphia; WB Saunders; 1995. pp. 2325−2418.
  50. 50. Ortner DJ. Identification of Pathological Conditions in Human Skeletal Remains. Second edition. Amsterdam; Elsevier; 2003.
  51. 51. Baltensperger MM, Eyrich GKH. Osteomyelitis of the jaws: Definition and classification. In: Baltensperger MM, Eyrich GKH (Eds) Osteomyelitis of the Jaws. Berlin; Springer; 2009. pp. 5−56.
  52. 52. Craig LE, Dittmer KE, Thompson KG. Bones and joints. In: Maxie MG (Ed.) Pathology of Domestic Animals. Sixth edition. Volume 1. Amsterdam; Elsevier; 2016. pp. 16−163.
  53. 53. Stirling I. Tooth wear as a mortality factor in the Weddell seal (Leptonychotes weddelli). J Mamm. 1969; 50:559–565.
  54. 54. Schultz M, Kierdorf U, Sedlacek F, Kierdorf H. Pathological bone changes in the mandibles of wild red deer (Cervus elaphus L.) exposed to high environmental levels of fluoride. J Anat. 1998; 193:431−442. pmid:9877298
  55. 55. Pindborg JJ. Pathology of the Dental Hard Tissues. Philadelphia; Saunders; 1970.
  56. 56. Tervonen SA, Stratmann U, Mokrys K, Reichart PA. Regional odontodysplasia: a review of the literature and report of four cases. Clin Oral Invest. 2004; 8:45−51.
  57. 57. Slootweg PJ. Dental Pathology: A Practical Introduction. Second edition. Heidelberg; Springer; 2013.
  58. 58. Alotaibi ON. Regional odontodysplasia: an overview. Pak Oral Dent J. 2015; 35:175−178.
  59. 59. Schamberger G, Marretta SM, Dubielzig R, Finn M. Regional odontodysplasia in a juvenile dog. J Vet Dent. 2010; 27:98–103.
  60. 60. Shields ED, Bixler D, El-Kafrawy . A proposed classification for heritable human dentine defects with a description of a new entity. Arch Oral Biol. 1973; 18:543–553. pmid:4516067
  61. 61. Levin LS, Leaf SH, Jelmini RJ, Rose JJ, Rosenbaum KN. Dentinogenesis imperfecta in the Brandywine isolate (DI type III): Clinical, radiologic, and scanning electron microscopic studies of the dentition. Oral Surg Oral Med Oral Pathol. 1983; 56:267–274. pmid:6579461
  62. 62. MacDougall M, Dong J, Acevedo A. Molecular basis of human dentin diseases. Am J Med Genet Part A. 2006; 140A:2536–2546.
  63. 63. Hart PZ, Hart TC. Disorders of human dentin. Cells Tissues Organs. 2007; 186:70−77. pmid:17627120
  64. 64. de La Dure-Molla M, Fournier BJ, Berdal A. Isolated dentinogenesis imperfecta and dentin dysplasia: revision of the classification. Eur J Hum Genet. 2015; 23: 445−451. pmid:25118030
  65. 65. Sreenath T, Thyagarajan T, Hall B, Longenecker G, D’Souza R, Hong S, et al. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III. J Biol Chem. 2003; 278:24874–24880. pmid:12721295
  66. 66. Yamakoshi Y, Simmer JP. Structural features, processing mechanism and gene splice variants of dentin sialophosphoprotein. Jap Dent Sci Rev. 2018; 54:183–196.
  67. 67. Zhu Q, Gibson MP, Liu Q, Liu Y, Lu Y, Wang X, et al. Proteolytic processing of dentin sialophosphoprotein (DSPP) is essential to dentinogenesis. J Biol Chem. 2012; 287:30426–30435. pmid:22798071
  68. 68. Alaluusua S, Lukinmaa PL, Pohjanvirta R, Unkila M, Tuomisto J. Exposure to 2,3,7,8-tetrachlorodibenzo-para-dioxin leads to defective dentin formation and pulpal perforation in rat incisor tooth. Toxicology. 1993; 81:1–13. pmid:8367879
  69. 69. Kattainen H, Tuukanen J, Simanainen U, Tuomisto JT, Kovero O, Lukinmaa PL, et al. In utero/lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure impairs molar tooth development in rats. Toxicol Appl Pharmacol. 2001; 174:216–224. pmid:11485382
  70. 70. Gao Y, Sahlberg C, Kiukkonen A, Alaluusua S, Pohjanvirta R, Tuomisto J, et al. Lactational exposure of Han/Wistar rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin interferes with enamel maturation and retards dentin mineralization. J Dent Res. 2004; 83:139–144. pmid:14742652
  71. 71. Long PH, Herbert RA, Nyska A. Hexachlorobenzene-induced incisor degeneration in Sprague-Dawley rats. Toxicol Pathol. 2004; 32:35–40. pmid:14713546
  72. 72. Guo H, Zhang L, Wei K, Zhao J, Wang Y, Jin F, et al. Exposure to a continuous low dose of tetrachlorodibenzo-p-dioxin impairs the development of the tooth root in lactational rats and alters the function of the apical papilla-derived stem cells. Arch Oral Biol. 2015; 60:199–207. pmid:25455135
  73. 73. Olsson M, Bignert A, Eckhéll J, Jonsson P. Comparsion of temporal trends (1940s–1990s) of DDT and PCB in Baltic sediment band biota in relation to eutrophication. Ambio. 2000; 29:195–201.