Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology

Summary

The present protocol describes the techniques for the systematic assessment of skull specimens to characterize anatomical and developmental variations and abnormalities of the teeth, periodontal disease, endodontal disease, and temporomandibular joint pathology.

Abstract

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental pathology, and anatomic variations in many mammalian species. Studying the teeth and jaws of an array of species can present a challenge requiring attention to detail and understanding of a species' normal anatomy. In the present article, a systematic and precise protocol for examining skull specimens is discussed that has been applied to a variety of mammals to define characteristic diseases in the oromaxillofacial region. The procedure outlined is simultaneously precise, repeatable, and adaptable to the highly differing skull and tooth shapes and anatomy across species. Specifically, specimens are examined for missing teeth, periodontal disease, endodontal disease, TMJ pathology, and anatomical variations. Results gleaned from research on museum specimens may reflect the natural history, health, and disease status of individuals and species. Furthermore, these data can inform ecological and conservation research efforts, as well as the care of captive individuals.

Introduction

The development of jaws and teeth marks a critical time point in the evolution and development of vertebrates. While jaws initially developed as part of a mechanism of respiration in aquatic and marine species, teeth offered a new manner of apprehending and processing prey items^1,2. Since the development of jaws and teeth, organisms have evolved innumerable variations in anatomy that correspond to their function and reflect the ecologic role to which they belong. Due to their mineralized nature, teeth and skulls represent a bounty of information that persists in the environment and fossil record and can offer myriad insights into the ecology, health status, and behavior of individuals and, by extension, species.

The acquisition of information pertaining to the teeth and jaws of animals and characterizing form and pathology has many benefits. Recognizing common disease processes can improve conservation efforts of wild species and optimize the care of captive animals^3,4,5. For example, information gleaned from museum skull specimens has been used to make inferences on the exposure of the Baltic grey seal (Halichoerus grypus) and harbor seals (Phoca vitulina) to environmental pollutants such as organochlorines over time^6,7, although a causative relationship between orofacial lesions and pollutants has not been confirmed. Furthermore, diseases of the oral cavity are some of the most prevalent diseases in domestic species, and understanding the oral health status of wild species may advance the clinical medicine and management of domestic species^8,9.

As animals have developed such variation in normal craniofacial shape and dentition, it can be challenging to characterize and compare these aspects between species. Understanding the ecology and natural behavior of an organism, as well as its typical environment, is imperative before attempting to examine its skull. Doing so will drive the formation of questions and hypotheses about a particular species' dentition and inevitably enrich the conclusions from data analysis. For example, recognizing that the typical diet of the Southern sea otter (Enhydra lutris nereis) includes hard-shelled mollusks, crustaceans, and echinoderms is essential to contextualize the degree and effect of attrition and/or abrasion of the teeth^10,11. Although one can assume the likelihood that an individual of a species will develop certain dental diseases, it is critical to have a systematic, precise, and reproducible protocol for evaluating dental pathology. This should include an appraisal of occlusion, anatomical and developmental findings, periodontal disease, endodontal findings, and temporomandibular joint (TMJ) pathology. Developing such a protocol with similar statistical analysis will allow for a detailed comparison of dental and TMJ disease from species to species. A systematic method has been utilized to characterize dental and temporomandibular joint pathology in many mammalian species and has proven to be translatable to organisms with diverse forms^{11,12,13,14,15,16,17,18,19,20,21,22,23,24}.

To compare future data on additional species, it is important to have an accepted method for assessing diseases of the teeth and jaws that can be applied to a variety of species. This article aims to detail a standardized and organized approach for assessing the dental and TMJ pathology of skull specimens.

Protocol

The present study was conducted using specimens from the Department of Ornithology and Mammalogy, California Academy of Sciences, San Francisco, the Museum of Vertebrate Zoology, University of California, Berkeley, and the Museum of the North, University of Alaska, Fairbanks. Permission to examine skull specimens and publish works from the data was obtained from the museums that own and manage each collection.

1. Specimen selection and documentation

1. Document specimen information, including identification numbers, species, sex, and location of origin.
   NOTE: The number of specimens held within a certain collection, as well as the specimen details, may be available through the Arctos Collaborative Collection Management Solution (see Table of Materials). For the present study, skulls from the Northern elephant seal (Mirounga angustirostris), California bobcat (Lynx rufus californicus), grey fox (Urocyon cinereoargenteus), Northern fur seal (Callorhinus ursinus), Southern sea otter (Enhydra lutris nereis), California mountain lion (Puma conolor cougar), and kit fox (Vulpes macrotis) are considered.
2. Examine the skull for completeness of the anatomical structures. Do not include severely fragmented skulls such that normal anatomical structures are unrecognizable without extensive reconstruction.
3. If possible, estimate the specimen's age at the time of death based on the closure of cranial sutures. Consult pertinent literature for the time of cranial suture closure as this varies for each target species.
4. Replace loose teeth with their corresponding alveoli. Use published anatomical descriptions of study species to help recognize the tooth type of each loose tooth^{25,26,27,28,29,30}.

2. Anatomical and developmental findings

1. Successionally inspect each dental quadrant and record the presence or absence of teeth.
2. Classify the loss of each tooth as congenitally absent versus acquired tooth loss versus artifactually absent. Examine the area of the missing tooth for a smooth margin of bone (congenital), an empty alveolus with remodeling alveolar bone (acquired), or an empty but sharply outlined alveolus (artifactual).
3. Document any persistent deciduous teeth or supernumerary teeth (Figure 1).
4. Examine each tooth crown's shape and any visible root structure. Document the number of roots by examining the loose teeth out of their alveoli.
   NOTE: For teeth that cannot be removed from their alveolus, supernumerary roots can be identified by an additional protuberance, usually on the palatal or lingual aspect of the tooth, or via dental radiographs (Figure 2).
5. Document the presence of enamel hypoplasia, characterized by thinning or absence of the tooth's white, reflective enamel layer, revealing the rougher, tan-yellow dentine surface.

3. Periodontal status

1. Use a metal or plastic periodontal probe and explorer (see Table of Materials) to ascertain the texture of the alveolar bone for evidence of periodontitis³¹.
   NOTE: There are no soft tissues, so gingivitis cannot be diagnosed, but increased vascularization, as evidenced by a larger number of vascular foramina, indicates early periodontitis (Figure 3).
2. Test for the presence of furcation involvement or exposure by attempting to insert the periodontal probe between the roots of each multirooted tooth. Depending on the number of roots, multiple areas may be needed to test for furcation.
3. Use a standardized protocol to identify the stages of progressively worsening periodontitis (Table 1)³².

4. Fractured teeth and periapical lesions

1. Examine each tooth for fracture, indicated by the loss of tooth substance with sharp edges (Figure 4).
2. Determine if each fracture is complicated or uncomplicated by attempting to insert the explorer into the pulp chamber from the fractured site. Complicated fractures are indicated by the explorer tip falling into the pulp chamber.
3. Record each fracture type based on a standardized classification system (Table 2)³³.
4. Examine the teeth for evidence of periapical lesions, characterized by expansion of the alveolar bone in the region of the apex of the tooth root with evidence of increased vascularization (Figure 5). Fenestration over the expansion may or may not be present.

5. Attrition/abrasion

1. Examine each tooth for attrition/abrasion, indicated by the loss of tooth substance with a smooth, glassy appearance and rounded edges (Figure 6).
2. Use the explorer to determine pulp chamber exposure from the abraded region by attempting to insert the explorer tip into the pulp chamber.
3. Classify the degree of attrition/abrasion on each tooth using a standardized classification system (Table 3)¹¹.
   ​NOTE: Some species may be more susceptible to attrition/abrasion than others due to their natural behavior. As such, staging the severity of attrition/abrasion may be indicated, or simply recording the presence or absence of attrition/abrasion may suffice.

6. Temporomandibular joint pathology

1. Inspect the bone components of the TMJ, including the head of the mandible of the condylar process, and the mandibular fossa of the squamous part of the temporal bone, for evidence of temporomandibular joint pathology, ruling out artifacts such as post-mortem trauma (e.g., "drawer damage" or preparation artifacts)³¹ (Figure 6).
2. Independently inspect the mandibular head and fossa on both sides and use a semiquantitative scoring system for osteoarthritis (OA) to classify the lesions associated with each bone (Table 4)³⁴.

7. Trauma

1. Examine the skull for any evidence of traumatic injury.
   ​NOTE: Chronic traumatic injury may be differentiated from acute traumatic injury based upon the sharpness of the fracture edges and any evidence of osseous remodeling. Also, note that some wild species are subject to gunshot injuries, and entry/exit wounds can be recognized, as well as remnants of projectiles.

8. Checking of other parameters

1. Depending on the species, examine the skull for additional abnormalities.
2. Check for tooth resorption or dental hard tissue loss due to idiopathic odontoclastic destruction, which is most commonly seen in felids and can be diagnosed radiographically by a loss of radiodensity with or without a loss of periodontal ligaments space^19,21,35.
   NOTE: Tooth resorptive lesions can also be felt with a dental explorer as a roughness at the cervical region of the tooth, but radiographic confirmation is necessary for diagnosis.
3. Check for echinochromasia, dark purple staining of hard tissues of the skull, since the consumption of certain species of pigmented echinoderms can be seen in otters¹¹.
4. Check for dental caries, which are decay of the tooth surface linked to the metabolism of dietary carbohydrates from acidogenic bacteria³⁶.
   NOTE: Carious lesions can be discovered by probing the occlusal surface of the teeth with a dental probe to reveal any pitting or irregularities^17,18.
5. Document evidence of osteomyelitis or neoplasia, characterized by irregular productive/destructive bone lesions³⁷. Record these data descriptively and include objective measurements of any lesions.

Results

The current protocol results in a combination of objective and semi-subjective data, and the positive outcome depends on the accurate and repeatable assessment of specimens. Multiple observers with knowledge of the normal anatomy of the target species and an understanding of general dental and maxillofacial pathology ideally must be present to assess each specimen to minimize bias systematically. The assessment of each specimen must be discussed, and a consensus needs to be obtained. No threshold for the number of specim...

Discussion

The anatomy of the teeth and jaws is a quintessential example of divergent evolution and is a true reflection of a species' natural history, behavior, and health status. An individual's oral health may play directly into their survival and fitness. The current study outlines a systematic, reproducible, and detailed manner of assessing the dental health and TMJ abnormalities of museum specimens that may reflect pathology in live populations.

Despite dental diseases universally affecting...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Arctos Collaborative Collection Management Solution	https://arctosdb.org
Disposible Nitrile Gloves
Double-Ended Dental Explorer/Probe, #2 Handle	Hu Friedy	541-5860
High resolution digital camera
Light source
Magnifying glass (Optional)
Surgical Magnification Loupes (Optional)	Surgitel	EVC00TTL