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 &amp; Craniofacial Research of the National Institutes of Health under Award Number K08DE025914 and by the American Association of Orthodontic Foundation to Sumit Yadav.

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 i...

<ol>
	<li>LeResche, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Temporomandibular+Disorders:+Implications+for+the+Investigation+of+Etiologic+Factors.">Epidemiology of Temporomandibular Disorders: Implications for the Investigation of Etiologic Factors.</a> Crit Rev Oral Biol Med. 8 (3), 291-305 (1997).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+functional+loading+causes+differential+effects+in+the+subchondral+bone+and+condylar+cartilage+in+the+temporomandibular+joint+from+young+mice.">Altered functional loading causes differential effects in the subchondral bone and condylar cartilage in the temporomandibular joint from young mice.</a> Osteoarthr Cartil. 17 (3), 354-361 (2009).</li><li>Pirttiniemi, P., Kantomaa, T., Sorsa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+decreased+loading+on+the+metabolic+activity+of+the+mandibular+condylar+cartilage+in+the+rat.">Effect of decreased loading on the metabolic activity of the mandibular condylar cartilage in the rat.</a> Eur J Orthod. 26 (1), 1-5 (2004).</li><li>Chavan, S. J., Bhad, W. A., Doshi, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+temporomandibular+joint+changes+in+Twin+Block+and+Bionator+appliance+therapy:+a+magnetic+resonance+imaging+study.">Comparison of temporomandibular joint changes in Twin Block and Bionator appliance therapy: a magnetic resonance imaging study.</a> Prog Orthod. 15 (57), (2014).</li><li>Dutra, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+Matrix+Response+of+the+Mandibular+Condylar+Cartilage+to+Botulinum+Toxin.">Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.</a> PLoS ONE. 11 (10), 0164599 (2016).</li><li>Benjamin, M., Ralphs, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+fibrocartilage+cells.">Biology of fibrocartilage cells.</a> Int Rev Cytol. 233, 1-45 (2004).</li><li>Shen, G., Darendeliler, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Adaptive+Remodeling+of+Condylar+Cartilage-+A+Transition+from+Chondrogenesis+to+Osteogenesis.">The Adaptive Remodeling of Condylar Cartilage- A Transition from Chondrogenesis to Osteogenesis.</a> J Dent Res. 84 (8), 691-699 (2005).</li><li>Utreja, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+matrix+response+of+temporomandibular+cartilage+to+mechanical+loading.">Cell and matrix response of temporomandibular cartilage to mechanical loading.</a> Osteoarthr Cartil. 24 (2), 335-344 (2016).</li><li>Kaul, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Altered+Loading+on+Mandibular+Condylar+Cartilage.">The Effect of Altered Loading on Mandibular Condylar Cartilage.</a> PLoS ONE. 11 (7), 0160121 (2016).</li><li>Dyment, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput,+Multi-Image+Cryohistology+of+Mineralized+Tissues.">High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.</a> J Vis Exp. , e54468 (2016).</li><li>Kawamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+new+adhesive+film+for+the+preparation+of+multi-purpose+fresh-frozen+sections+from+hard+tissues,+whole-animals,+insects+and+plants.">Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.</a> Arch Histol Cytol. 66 (2), 123-143 (2003).</li><li>Hayman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tartrate-resistant+acid+phosphatase+(TRAP)+and+the+osteoclast/immune+cell+dichotomy.">Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy.</a> Autoimmunity. 41 (3), 218-223 (2008).</li></ol>

The authors have no competing financial interests.

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...

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 the interactions between its constituents, including water, the collagen network, and densely packed proteoglycans. The MCC has four distinct cellular zones that express different types of collagen and non-collagen proteins: 1) the superficial or articular zone; 2) the proliferative zone, composed of undifferentiated mesenchymal cells and that responds to loading demands; 3) the prehypertrophic zone, composed of mature chondrocytes expressing collagen type 2; and 4) the hypertrophic zone, the region where the hypertrophic chondrocytes expressing collagen type 10 die and undergo calcification. The non-mineralized region is rich in proteoglycans which provide resistance to compressive forces6.
There is continuous mineralization at the hypertrophic zone of the MCC, where the transition from chondrogenesis to osteogenesis occurs, guaranteeing the robust mineral structure of the subchondral bone of the mandibular condyle7. Cellular changes in the unmineralized and mineralized regions ultimately lead to morphological and structural changes in the mandibular condyle and mandible. Maintenance of the homeostasis of all cellular regions of the MCC and the mineralization of the subchondral portion are essential to the health, load-bearing capacity, and integrity of the TMJ.
The multiple collagen transgenic mouse model (as described by Utreja et al.)8 is a great tool to use to understand changes in collagen expression because all transgenes are expressed in the MCC. For an in-depth histological evaluation, histological stains are used to study matrix deposition, mineralization, cell proliferation, and apoptosis, as well as protein expression at the different cell layers of the MCC.
In this manuscript, histological and morphometric analyses are used to evaluate cellular and structural changes in the MCC and subchondral bone of the mandibular condyle of mice. In addition, a cell quantification method, for analyzing fluorescent histological images and for mapping light microscope slides, is described. The compressive static TMJ loading method, which causes cellular and morphological changes at the MCC and subchondral bone9, is also illustrated to validate our methods.
The methods described here can be used to determine morphometric and histological changes in the mandibular condyle and mandible of rodents or to analyze other regions of endochondral ossification and the morphology of additional mineralized tissues.

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			<td height="21" style="height:21px;">ClickiT EdU Alexa Fluor 594 HCS kit</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">C10339&#160;</td>
			<td>includes EdU (5-ethynyl-2&#39;-deoxyuridine)&#160;</td>
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			<td height="21" style="width:392px;height:21px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:219px;">Thermo Scientific</td>
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			<td height="21" style="width:392px;height:21px;">Sodium phosphate dibasic&#160;</td>
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			<td>S3264</td>
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			<td height="21" style="width:392px;height:21px;">Sodium phosphate monobasic&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">71505</td>
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			<td height="21" style="width:392px;height:21px;">Toluidine Blue O&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3260</td>
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			<td height="25" style="width:392px;height:25px;">Adobe Photoshop&#160;</td>
			<td style="width:219px;">Adobe Systems Incorporated</td>
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			<td height="24" style="width:392px;height:24px;">Phosphate buffered saline tablets (PBS)</td>
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			<td>Ortho Organizers, Inc.</td>
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			<td>GraphPad Software, Inc.</td>
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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.

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, ...

Watch this Scientific Journal Video about A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle at JoVE.com

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

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 ...

The overall goal of this experiment is to observe the effects of compressive loading in the mandibular condylar cartilage of mice using Morphometric measurements in radiographs and histological analysis. These metrics can help answer key questions regarding the structural and cellular changes within the mandible condyle of rodents. The main advantage of the technique shown here is that they could be used to analyze other small born or participated animals.For this protocol, have isolated, dissected mouse mandibles ready for use. On a Petri dish, transfer the mandibles into an x-ray cabinet system. Then, take radiographs at 26 kilovolts for five seconds.Now, open the images in the analysis software to make Morphometric measurements. First, use the unit button to setup the scale bar. Next, select six anatomical points using marker style 2.First, select the most posterior points of the mandibular condyle as point one. Next, select the incisored process for point two. Then, select the deepest point at the sigmoid notch for point three.Next, select the deepest point in the concavity of the mandibular ramus, point four and then, select the most anterior point of the condylar articular surface, point five. Now, select the most posterior point of the condylar articular surface as point six and proceed with taking length measurements using the length tool. Next, use the perpendicular line tool from points three to four to measure the condyle head length, which is the length of the perpendicular, from point one to the line between points three and four.Then, measure the mandibular length, between points one and two. Lastly, measure the condyle head width, which is between points five and six. To save the data, copy the measurement list on the right side of the screen.Before embedding the fixed, but not decalcified mandibles, soak them overnight in 30 percent sucrose in PBS. The next day, dissect away any excess soft tissue and carefully cut the mandibular condyle. Now, place the samples in plastic molds, with the medial surface of the condyle against the base of the mold and the sample parallel to the bottom of the mold.Then, immerse them in embedding resin and cool the resin with a piece of dry ice. Next, top off the molds with more embedding resin. Transfer them into cold two methyl butane, cooled by a freezer or by dry ice.Once chilled, store the specimens at negative 20 degrees Celsius or at negative 80 degrees Celsius for sectioning. To quantify Col10a1 expression in the MCC of sagittal sections of condyles, open the histological images in an image analysis software package and select the area of interest using the lasso tool. In this case, the MCC region is selected.Take note of the number of pixels in the area, which is reported in the histogram box. Next, select the Col10a1 red pixels by adjusting the color range. Use the eye dropper tool to select a red pixel from the image and click OK.Then, record the number of red pixels in the area, as reported in the histogram box.The fraction of red pixels in the area represents the amount of Col10a1 expression. Now, using the same basic technique, quantify the TRAP activity in the MCC and subchondral bone. First, select the cartilage in subchondral bone regions as the areas to analyze and take note of the total pixels.Then, select the yellow pixels generated by the ELF97 substrate. Take note of the number of positive pixels and calculate the fraction of TRAP positive pixels. Now, quantify the EDU positive cells that are counter-stained in blue by DEPI.Again, select the area of interest and then quantify the blue pixels within it. Also use this method to determine the number of the EDU positive pixels in the same region. Morphometric measurements of mice, subjected to compressive TMJ loading, showed that the loading significantly increased mandible and condyle head length.Nevertheless, loading did not result in a significant change in condyle width. Quantification of the Collagen distribution revealed a significant increase in Col10a1 expression and the MCC of the condyles of loaded animals. On the other hand, Col2a1 expression was not much different from control.TRAP activity increased in the subchondral region of condyles and loaded mice as did sub proliferation. EDU staining denotes number of proliferative cells. Proteoglycan distribution in the MCC was quantified by evaluating the Toluidine blue stained area and the distance map.There was a significant increase in distance mapping in the MCC of condyles of the loaded group. However, the proteoglycan stained area was not statistically different between groups. After watching this video, you should have a good understanding of how to analyze the cellular and the structural changes in the mandibular condyle of rodents.

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8.1K visualizações.  University of Connecticut Health Center. The overall goal of this experiment is to observe the effects of compressive loading in the mandibular condylar cartilage of mice using Morphometric measurements in radiographs and histological analysis. These metrics can help answer key questions regarding the structural and cellular changes within the mandible condyle of rodents. The main advantage of the technique shown here is that they could be used to analyze other small born or participated animals.For this protocol, have isolat...