The authors thank 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, for making their collections available for this research.

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>If possible, estimate the specimen&#39;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.</li>
	<li>Replace loose teeth with their corresponding alveoli. Use published anatomical descriptions of study species to help recognize the tooth type of each loose tooth25,26,27,28,29,30.</li>
</ol>
2. Anatomical and developmental findings
<ol>
	<li>Successionally inspect each dental quadrant and record the presence or absence of teeth.</li>
	<li>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).</li>
	<li>Document any persistent deciduous teeth or supernumerary teeth (Figure 1).</li>
	<li>Examine each tooth crown&#39;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).</li>
	<li>Document the presence of enamel hypoplasia, characterized by thinning or absence of the tooth&#39;s white, reflective enamel layer, revealing the rougher, tan-yellow dentine surface.</li>
</ol>
3. Periodontal status
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Use a standardized protocol to identify the stages of progressively worsening periodontitis (Table 1)32.</li>
</ol>
4. Fractured teeth and periapical lesions
<ol>
	<li>Examine each tooth for fracture, indicated by the loss of tooth substance with sharp edges (Figure 4).</li>
	<li>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.</li>
	<li>Record each fracture type based on a standardized classification system (Table 2)33.</li>
	<li>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.</li>
</ol>
5. Attrition/abrasion
<ol>
	<li>Examine each tooth for attrition/abrasion, indicated by the loss of tooth substance with a smooth, glassy appearance and rounded edges (Figure 6).</li>
	<li>Use the explorer to determine pulp chamber exposure from the abraded region by attempting to insert the explorer tip into the pulp chamber.</li>
	<li>Classify the degree of attrition/abrasion on each tooth using a standardized classification system (Table 3)11. 
	&#8203;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.</li>
</ol>
6. Temporomandibular joint pathology
<ol>
	<li>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., &#34;drawer damage&#34; or preparation artifacts)31 (Figure 6).</li>
	<li>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.</li>
</ol>
7. Trauma
<ol>
	<li>Examine the skull for any evidence of traumatic injury. 
	&#8203;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.</li>
</ol>
8. Checking of other parameters
<ol>
	<li>Depending on the species, examine the skull for additional abnormalities.</li>
	<li>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 space19,21,35. 
	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.</li>
	<li>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.</li>
	<li>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 irregularities17,18.</li>
	<li>Document evidence of osteomyelitis or neoplasia, characterized by irregular productive/destructive bone lesions37. Record these data descriptively and include objective measurements of any lesions.</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diets+high+in+fruits+and+low+in+gum+exudates+promote+the+occurrence+and+development+of+dental+disease+in+pygmy+slow+loris+(Nycticebus+pygmaeus).">Diets high in fruits and low in gum exudates promote the occurrence and development of dental disease in pygmy slow loris (Nycticebus pygmaeus).</a> Zoo Biology. 34 (6), 547-553 (2015).</li><li>Kahle, P., Ludolphy, C., Kierdorf, H., Kierdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+anomalies+and+lesions+in+Eastern+Atlantic+harbor+seals,+Phoca+vitulina+vitulina+(Carnivora,+Phocidae),+from+the+German+North+Sea.">Dental anomalies and lesions in Eastern Atlantic harbor seals, Phoca vitulina vitulina (Carnivora, Phocidae), from the German North Sea.</a> PLoS One. 13 (10), 1-25 (2018).</li><li>Ludolphy, C., Kahle, P., Kierdorf, H., Kierdorf, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> BMC Veterinary Research. 14 (1), 1-14 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

The anatomy of the teeth and jaws is a quintessential example of divergent evolution and is a true reflection of a species&#39; natural history, behavior, and health status. An individual&#39;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 mammals of a wide variety of genera, this topic remains underexplored. Understanding dental and craniofacial disorders in wild, captive, and domestic species has implications for conservation efforts, zoological husbandry, companion animal healthcare, and animal-related industry3,32,33,40,41. Therefore, studies on dental diseases could be key for improving and understanding animal health as a whole.
Critical steps in the current assessment algorithm include research into a species&#39; natural behavior prior to data collection, understanding the target species&#39; normal anatomy, and systematically and precisely assessing each tooth and anatomical landmark. It is also imperative to report results in a systematic and detailed manner so that comparisons between species can be made. Failure to complete these steps in an organized manner can lead to missing or inaccurate data or a misinterpretation of findings.
The system presented successfully characterizes many species, but the method has limitations. First, the examination is performed on museum specimens, and the quality of the specimens is subject to their preparation, care, and storage method. Therefore, artifacts may arise and could be inadvertently interpreted as true pathology present ante mortem. Additionally, the study population is inevitably biased toward individuals found in regions that humans could access easily. Furthermore, studies conducted in such a manner could overestimate the prevalence of dental diseases or craniofacial abnormalities that truly exist in a species. These abnormalities could reduce an individual&#39;s fitness. In contrast, the study design cannot evaluate diseases of the oral and facial soft tissues. It is also important to consider how individuals in a collection died and were collected, as this may impact the observed frequency of pathology. Individuals dispatched in large-scale hunting or trapping events may show increased tooth fractures or skull trauma prevalence compared with the general population22. In contrast, populations that experienced acute or subacute large-scale mortality events that do not have a known effect on the craniofacial region may provide a more accurate representation of dental diseases and TMJ abnormalities at a given time point42,43. Finally, while the current study method offers a comprehensive analysis of possible dental and TMJ abnormalities in a given species, one cannot conclude how the abnormalities discovered may impact the fitness of an individual or the evolutionary pressures to which a species is subject.
In conclusion, a systematic review of museum specimens has proven to be effective in characterizing dental anomalies, dental disease, and TMJ and craniofacial pathology in many species. The current methods offer a non-invasive means of data collection that can reflect the potential for disease in wild populations. The continuation of research into the dental pathology of wild and captive species is necessary to understand the health status and biological niche of these species and offer evidence-based means of optimizing management.

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 items1,2. 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 animals3,4,5. 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 time6,7, 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 species8,9.
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&#39; 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 teeth10,11. 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)&#160;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 forms11,12,13,14,15,16,17,18,19,20,21,22,23,24.
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&#160;pathology of skull specimens.

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systematic assessment of mammalian skull specimens for dental and temporomandibular joint pathology

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&#39; 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.

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 specimens has been suggested to ensure a representative sample, but published studies have included 76 to 1,205 individuals11,20. Additionally, a specimen free of any detectable pathology or minimal disease or anatomic variation must be found and documented to act as a standard for comparison (Figure 7). Furthermore, a successful outcome depends on the differentiation of pathology from artifactual damage. Often, specimens may incur artifactual damage during processing or storage. For this reason, for accurate data collection, the researcher must familiarize themselves with the general anatomy and pathophysiology of the maxillofacial region to understand the evidence of true chronic and acute disease processes. If there is evidence supporting that a finding is artifactual, such as evidence of post-mortem tampering with human tools or injuries to teeth or structures that would be biomechanically impossible in life, these findings must not be recorded as true pathology. If radiographs are utilized for specimen assessment, appropriate radiographic views need to be obtained for diagnostic quality38,39. Finally, pathology and anatomic anomalies must be documented with precise descriptions and/or high-resolution photographs.
Results may not be representative if normal anatomy is interpreted as pathology or if data are noted to have a high degree of variation between observers. Specimens must be examined carefully to preserve their quality as diligently as possible to allow for accurate repeat assessment in the future if necessary. Additionally, if meticulous record-keeping is not maintained, pathology or anatomical anomalies may be assigned to incorrect specimens, and the conclusions drawn from correlations from the data will be of questionable accuracy.
Data should be statistically analyzed to determine if there are differences in the frequency of abnormalities associated with the age of the individual at the time of death and sex. The frequency of dental abnormalities can be compared between different subspecies, geographic locations, and historic time points. Finally, data can be compared with previously published data on species of different genera.
The current protocol has proven to be an effective means of characterizing dental and TMJ abnormalities in various species11,12,13,14,15,16,17,18,19,20,21,22,23,24. The presentation of the results of future reports must follow a similar pattern to that of the previous manuscripts to maintain the completeness and to be able to compare results easily.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64223/64223fig01.jpg" /> 
Figure 1: Persistent deciduous teeth in the Northern elephant seal (Mirounga angustirostris).&#8203; Shown are persistent deciduous right mandibular first, second, and third premolar teeth (arrows) mesial to the corresponding permanent successor teeth. This figure has been modified from Abbott et al.12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64223/64223fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64223/64223fig2v2.jpg" /> 
Figure 2: Supernumerary roots in the California bobcat (Lynx rufus californicus). (A) Radiographical and (B) gross appearance of a left maxillary third premolar tooth with an extra root (arrowed). This figure has been modified from Aghashani et al.19. Scale bar = 5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64223/64223fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64223/64223fig3v2.jpg" /> 
Figure 3: Stages of periodontitis in the grey fox (Urocyon cinereoargenteus). (A) Stage 2 associated with the right maxillary fourth premolar tooth, characterized by increased vascularization of the alveolar bone (arrow). (B) Stage 3 associated with the right mandibular second premolar tooth, characterized by moderate bone loss and furcation involvement (arrow). (C) Stage 4 associated with the left maxillary premolar teeth, characterized by severe alveolar bone loss (arrows). This figure has been modified from Evenhuis et al.22. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64223/64223fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64223/64223fig04.jpg" /> 
Figure 4: Fractured teeth in the Northern fur seal (Callorhinus ursinus). Root fractures of the right mandibular third and fourth premolar teeth (white arrows) and stage 2 periodontitis around the right mandibular first&#160;molar tooth (black arrow)13. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64223/64223fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64223/64223fig05.jpg" /> 
Figure 5: Periapical lesions in the Southern seal otter&#160;(Enhydra lutris nereis). Periapical disease associated with complicated crown root fractures of the right mandibular fourth premolar and first and second molar teeth in an adult female specimen. This figure has been modified from Winer et al.24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64223/64223fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64223/64223fig06.jpg" /> 
Figure 6: TMJ osteoarthritis in the California mountain lion (Puma concolor cougar). (A) Image of the irregular articular surface, subchondral bone exposure, and porosity of the mandibular condylar processes (open arrows). (B)&#160;The right TMJ exhibits no significant periarticular proliferation at the retroarticular process. (C) The left TMJ exhibits periarticular bony proliferation at the retroarticular process (closed arrows), partially encircling the mandibular head, resulting in partial ankylosis. This figure has been modified from Aghashani et al.21. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64223/64223fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64223/64223fig07.jpg" /> 
Figure 7: Normal dentition of the kit fox (Vulpes macrotis). (A) Right lateral view. (B) Rostral view. (C) Ventral view of the maxilla (left) and dorsal view of the mandibles (right). This figure has been modified from Yanagisawa et al.23. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64223/64223fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Degree of Periodontitis</td>
			<td colspan="3">Clinical Features</td>
		</tr>
		<tr>
			<td>Periodontitis stage 2</td>
			<td colspan="3">Evidence of increased vascularity at the alveolar margin (more prominent vascular foramina in, and slightly rougher texture of, the bone of the alveolar margin)</td>
		</tr>
		<tr>
			<td>Periodontitis stage 3</td>
			<td colspan="3">Rounding of the alveolar margin; moderate horizontal or vertical bone loss</td>
		</tr>
		<tr>
			<td>Periodontitis stage 4</td>
			<td colspan="3">Widening of the periodontal space; severe horizontal or vertical bone loss; tooth mobile in the alveolus; furcation exposure</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of clinical features of progressive stages of periodontitis.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fracture Type</td>
			<td colspan="2">Description</td>
		</tr>
		<tr>
			<td>Enamel fracture</td>
			<td colspan="2">A chip fracture or crack of the enamel only.</td>
		</tr>
		<tr>
			<td>Uncomplicated crown fracture</td>
			<td colspan="2">A fracture involving enamel and dentine, but not exposing the pulp.</td>
		</tr>
		<tr>
			<td>Complicated crown fracture</td>
			<td colspan="2">A fracture involving enamel and dentine, with pulp exposure.</td>
		</tr>
		<tr>
			<td>Uncomplicated crown&#8211;root fracture</td>
			<td colspan="2">A fracture involving enamel, dentine and cementum, but not exposing the pulp.</td>
		</tr>
		<tr>
			<td>Complicated crown&#8211;root fracture</td>
			<td colspan="2">A fracture involving enamel, dentine and cementum, with pulp exposure.</td>
		</tr>
		<tr>
			<td>Root fracture</td>
			<td colspan="2">A fracture affecting dentine, cementum and the pulp.</td>
		</tr>
	</tbody>
</table>
Table 2: Summary of clinical features of tooth fracture types.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stage of Attrition/abrasion</td>
			<td colspan="2">Description</td>
		</tr>
		<tr>
			<td>Attrition/abrasion stage 1</td>
			<td colspan="2">Mild wear of enamel, without dentine exposure</td>
		</tr>
		<tr>
			<td>Attrition/abrasion stage 2</td>
			<td colspan="2">Exposure of dentine on the cuspal tip, without tertiary dentine formation</td>
		</tr>
		<tr>
			<td>Attrition/abrasion stage 3</td>
			<td colspan="2">Exposure of dentine on the cuspal tip, with tertiary dentine formation</td>
		</tr>
		<tr>
			<td>Attrition/abrasion stage 4</td>
			<td colspan="2">Pulp cavity exposure secondary to attrition/abrasion</td>
		</tr>
	</tbody>
</table>
Table 3: Summary of clinical features of tooth attrition and abrasion.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Severity of Osteoarthritis (OA)</td>
			<td colspan="3">Clinical Features</td>
		</tr>
		<tr>
			<td>Mild OA</td>
			<td colspan="3">There is any evidence of early lesions of periarticular new bone formation/osteophytes with minimal or no subchondral bone changes.</td>
		</tr>
		<tr>
			<td>Moderate OA</td>
			<td colspan="3">There is periarticular new bone formation and/or subchondral bone changes are more pronounced.</td>
		</tr>
		<tr>
			<td>Severe OA</td>
			<td colspan="3">All previously described signs are present and more pronounced, or if subchondral bone lysis is present. Partial or complete ankylosis may be observed.</td>
		</tr>
	</tbody>
</table>
Table 4: Summary of clinical features of TMJ osteoarthritis.

Watch this Scientific Journal Video about Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology at JoVE.com

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

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.

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental ...

systematic-assessment-mammalian-skull-specimens-for-dental

Nanyang Technological University

Research

JoVE Journal

Biology

1.3K Views.  University of California-Davis. We werken al geruime tijd samen met de California Academy of Sciences en andere musea om de tandheelkundige pathologie van zoogdieren in het westen van de Verenigde Staten te documenteren, inclusief de beren, de zeezoogdieren, de katachtigen en de canids, en het voordeel van deze techniek dat het reproduceerbare resultaten over de hele soort mogelijk maakt. Ons protocol is een uitstekend middel om de wetenschappelijke methode vast te leggen. Eerdere projecten zijn vormend geweest voor veterin...

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

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Bioengineering

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Developmental Biology

Inflammatory TMJ Pain Mouse Model: Bite Force and Von Frey Filament Assessments

Temporomandibular Joint Pain Measurement by Bite Force and Von Frey Filament Assays in Mice

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms driving diseases-related pain are poorly understood, and current treatments fail to provide effective and long-term therapeutic effects. Additionally, pain assessment in research, particularly orofacial pain, poses several challenges that complicate studies in both clinical and basic science settings. Therefore, we have established an inflammatory TMJ pain mouse model via intra-articular injection of CFA (Complete Freund's Adjuvant) and evaluated pain behaviors by bite force measurement and the von Frey filament test. Mice with CFA injection exhibited orofacial pain behaviors compared to PBS injection, including reduced bite force and head withdrawal threshold in the von Frey filament test. These methods are relatively easy to execute to have reproducible results and can be potentially extended to pain studies for other disease models related to TMJ disorders. Together, bite force, and the von Frey filament tests are reliable in measuring orofacial pain, as demonstrated in CFA injection-induced painful TMJOA mouse models.

This protocol describes how measuring bite force and head withdrawal threshold can capture pain behavior in Complete Freund's Adjuvant (CFA)-induced inflammatory temporomandibular joint (TMJ) pain.

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms ...

Behavior

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

High-Speed Human Temporal Bone Sectioning for the Assessment of COVID-19-Associated Middle Ear Pathology

Histopathologic analysis of human temporal bone sections is a fundamental technique for studying inner and middle ear pathology. Temporal bone sections are prepared by postmortem temporal bone harvest, fixation, decalcification, embedding, and staining. Due to the density of the temporal bone, decalcification is a time-consuming and resource-intensive process; complete tissue preparation may take 9-10 months on average. This slows otopathology research and hinders time-sensitive studies, such as those relevant to the COVID-19 pandemic. This paper describes a technique for the rapid preparation and decalcification of temporal bone sections to speed tissue processing.
Temporal bones were harvested postmortem using standard techniques and fixed in 10% formalin. A precision microsaw with twin diamond blades was used to cut each section into three thick sections. Thick temporal bone sections were then decalcified in decalcifying solution for 7-10 days before being embedded in paraffin, sectioned into thin (10 &#181;m) sections using a cryotome, and mounted on uncharged slides. Tissue samples were then deparaffinized and rehydrated for antibody staining (ACE2, TMPRSS2, Furin) and imaged. This technique reduced the time from harvest to tissue analysis from 9-10 months to 10-14 days. High-speed temporal bone sectioning may increase the speed of otopathology research and reduce the resources necessary for tissue preparation, while also facilitating time-sensitive studies such as those related to COVID-19.

This article describes a technique for rapid human temporal bone sectioning that utilizes a microsaw with twin diamond blades to generate thin slices for rapid decalcification and analysis of temporal bone immunohistochemistry.

Histopathologic analysis of human temporal bone sections is a fundamental technique for studying inner and middle ear pathology. Temporal bone sections ...

Immunology and Infection

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were euthanized, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&#38;E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

This protocol demonstrates step-by-step details of how to extract the mandibular first molar in the mouse. It provides an alternative method for researchers focusing on jawbone healing and regeneration.

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone ...

Investigating Orthodontic Tooth Movement and Bone Adaptation Mechanisms in Mice

Studying Orthodontic Tooth Movement in Mice

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at tension sites, orchestrated by osteoclasts and osteoblasts, respectively. This mechanism serves as a valuable model for studying various aspects of bone adaptation, including root resorption and the cellular response to mechanical force stimuli. The protocol outlined here offers a straightforward approach to investigate OTM, establishing 0.35 N as the optimal force in a mouse model employing a nickel-titanium (NiTi) coil spring. Utilizing micro-computed tomography analysis, we quantified OTM by assessing the discrepancy in the linear distance at the cement-enamel junction. The evaluation also included an analysis of orthodontic-induced inflammatory root resorption, assessing parameters such as root mineral density and the percentage of root volume per total volume. This comprehensive protocol contributes to advancing our understanding of bone remodeling processes and enhancing the ability to develop effective orthodontic treatment strategies.

Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at ...

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

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