This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.
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Method Article
A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle
In This Article
Summary
Abstract
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.
Introduction
The TMJ is a unique load-bearing joint located in the craniofacial region and is formed of fibrocartilage. The MCC of the TMJ is essential for joint function, including unhindered jaw movement while speaking and masticating, but it is commonly affected by degenerative diseases, including osteoarthritis1. The TMJ has the capacity to adapt to external stimuli and loading alterations, leading to structural and cellular changes to the components of the MCC2,3,4,5. The load-bearing properties of the MCC can be explained by....
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Protocol
The institutional animal care committee of the University of Connecticut Health Center approved all animal procedures.
1. Compressive Static TMJ Loading: Mouth Forced Open
Note: Four-week-old transgenic mice harboring fluorescent reporters for collagen (Col2a1XCol10a1), kindly provided by Dr. David Rowe (University of Connecticut), were used for the experiments described in this manuscript (n = 8; 4 males and 4 females). The Col2a1 cyan (blue) transgene is expressed in cells at the prehypertrophic zone of the MCC, while the Col10a1 cherry (red) cells are present in the hypertrophic region8 (....
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Results
Descriptive statistics were performed to examine the distribution of morphometric measurements (mandibular length, condylar length, condylar width) and histological analyses. Outcomes were compared between the loaded group (i.e., mice subjected to compressive loading with the beta titanium spring) and the control group (i.e., matching control mice that did not receive any procedure). Statistically significant differences between means were determined by unpaired t-test, .......
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Discussion
This manuscript described methods for the morphometric measurement and cellular analysis of murine mandibular condyles and mandibles. The radiographic morphometric measurements can also be used to analyze other bones from small experimental animals. In addition, the cellular analysis (cell quantification and cartilage distance mapping) are not limited to the rodent mandibular condyle, but can be used to quantify histological sections of numerous tissues.
Transgenic mouse models expressing flu.......
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Disclosures
The authors have no competing financial interests.
Acknowledgements
The authors would like to thank Dr. David Rowe for kindly providing the transgenic mice and Li Chen for the histological assistance.
The research reported in this publication was supported by the National Institute of Dental & Craniofacial Research of the National Institutes of Health under Award Number K08DE025914 and by the American Association of Orthodontic Foundation to Sumit Yadav.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| MX20 Radiography System | Faxitron X-Ray LLC | ||
| Digimizer Image software | MedCalc Software | ||
| Shandon Cryomatrix embedding resin | Thermo Scientific | 6769006 | |
| Manual microscope Axio Imager Z1 | Carl Zeiss | 208562 | |
| yellow fluorescent protein filter - EYFP | Chroma Technology Corp | 49003 | |
| cyan fluorescent protein filter - ECFP | Chroma Technology Corp | 49001 | |
| red fluoresecent protein filter - Cy5 | Chroma Technology Corp | 49009 | |
| sodium acetate anhydrous | Sigma-Aldrich | S2889 | |
| sodium L-tartrate dibasic dihydrate | Sigma-Aldrich | 228729 | |
| sodium nitrite | Sigma-Aldrich | 237213 | |
| ELF97 substrate | Thermo Fisher Scientific | E6600 | |
| ClickiT EdU Alexa Fluor 594 HCS kit | Life Technologies | C10339 | includes EdU (5-ethynyl-2'-deoxyuridine) |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Scientific | D1306 | |
| Sodium phosphate dibasic | Sigma-Aldrich | S3264 | |
| Sodium phosphate monobasic | Sigma-Aldrich | 71505 | |
| Toluidine Blue O | Sigma-Aldrich | T3260 | |
| Adobe Photoshop | Adobe Systems Incorporated | ||
| Phosphate buffered saline tablets (PBS) | Research Products International | P32080-100T | |
| CNA Beta III Nickel-Free Archwire | Ortho Organizers, Inc. | ||
| GraphPad Prism | GraphPad Software, Inc. |
References
- LeResche, L. Epidemiology of Temporomandibular Disorders: Implications for the Investigation of Etiologic Factors. Crit Rev Oral Biol Med. 8 (3), 291-305 (1997).
- Chen, J., et al.
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