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 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.
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.
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 (Figure 1). Mice were equally divided into two groups: 1) the loaded group, where mice were subjected to compressive static TMJ loading (described in step 2) and 2) the control group, where mice received no intervention.
Figure 1.Representative sagittal of the condyle of a double-collagen fluorescent reporter mouse (Col2a1XCol10a1). Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 2. Compressive static TMJ loading: mouth forced open model. (A) Spring fabricated of 0.017 x 0.025 beta titanium alloy archwire. (B) Loaded mouse with spring. (C) Radiograph of loaded and control mice showing differences in the positioning of the mandible. Please click here to view a larger version of this figure.
2. Mandible Dissection and Fixation
3. X-ray Imaging and Morphometric Measurements
Figure 3. Representation of morphometric measurements of the mandible. (A) Use the scale bar of the radiograph to determine the unit (circled in red, scale bar: 10 mm). (B) Select the anatomical points using the "marker style 2" (circled in red). 1) Condylion; 2) Incisor process; 3) Deepest point at the sigmoid notch; 4) Deepest point in the concavity of the mandibular ramus; 5) Most anterior point of the condylar articular surface; 6) Most posterior point of the condylar articular surface. Scale bar: 10 mm.(C) Perform measurements with the "length" and "perpendicular" tools (circled in red). Measurements from point 1 to 2: mandibular length; from point 5 to 6: condylar width; perpendicular from point 1 to 4 – 3: condylar head length. Save measurements from the "measurement list." Scale bar = 10 mm. Please click here to view a larger version of this figure.
4. Condyle Embedding
NOTE: After taking the radiographic images, the mandibles can be embedded and sectioned for histological analysis.
5. Condyle Sagittal Sectioning and Slide Preparation
6. Histological Staining and Microscopic Imaging
Note: Most of the histological staining is performed as described in the histological section of the paper by Dyment et al10.
7. Fluorescent Histological Quantification
Figure 4. Representation of transgene Col10a1 quantification. (A) Select the area of interest with the "Lasso Tool" (L). For Col10a1-positive cells, select the whole mandibular cartilage. Save the number of pixels from the "histogram" box. (B) Select the pixel of interest, in this case, the red fluorescent Col10a1 pixels. Note that only the red pixels within the area of interest will be selected. Save the number of red pixels from the "histogram" box. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 5. Representation of fluorescent TRAP quantification. (A) Select the area of interest (mandibular cartilage and subchondral bone) and save the number of pixels of this region. (B) Select the yellow fluorescent pixels, representing TRAP activity. Note that only TRAP-positive pixels will be select. Save the number of selected pixels. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 6. Representation of EdU quantification. (A) Select the proliferative region of the MCC (the outer layer of the cartilage). Select DAPI-positive pixels and save the number of pixels. (B) Select EdU-positive pixels (yellow fluorescent) and save the number of pixels. Scale bar = 200 µm. Please click here to view a larger version of this figure.
8. Quantification of Cartilage Thickness and Proteoglycan Distribution
Figure 7: Representation of proteoglycan distribution quantification. (A) Use the scale bar of the histological image to determine the unit by clicking on the "unit" button (circled in red, unit selected: 500 µm). (B) Measure thickness of the cartilage in different locations by using the "length" tool (circled in red). Save the measurements from the "measurement list" in the upper right panel. The software also provides "statistics" in the lower right panel, so the mean and SD of the measurements can be obtained directly. (C) Measure the toluidine blue-stained area by using the "area" tool (circled in red). Circle the area of interest and save the measurement from the "measurement list." Please click here to view a larger version of this figure.
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, and a p-value of <0.05 was deemed to be statistically significant.
Morphometric measurements, as described in step 4 and Figure 3, were performed in the mandibles of the loaded and control groups. The loaded group presented with significantly increased mandible length (loaded: 16.62 ± 0.23 mm versus control: 16.21 ± 0.2 mm; p < 0.05) and condyle head length (loaded: 4.6 mm, SD 0.08 mm versus control: 4.4 mm; SD 0.06; p < 0.05) in comparison to the control group. Nevertheless, there was no significant difference in condyle width between groups (loaded: 3.06 ± 0.12 mm versus control: 2.9 ± 0.11; p = 0.09).
Quantification of the collagen distribution using the method described in step 8 revealed significantly increased Col10a1 expression in the MCC of the condyles of the loaded group in comparison to the control (loaded: 21 % ± 4.46% versus control: 7.50% ± 2.03%; p < 0.005; Figure 8A and D). On the other hand, there was no statistically difference in Col2a1 expression between the loaded and control groups (loaded: 4.85% ± 1.95% versus control: 2.92% ± 1.89%; p = 0.13; Figure 8B and E). In addition, the analysis of TRAP activity showed increased TRAP-positive regions in the subchondral region of the condyles of loaded mice (loaded: 5.28% ± 1.45% versus control: 2.41% ± 1.39%; p < 0.05; Figure 8C and F). Similarly, we observed increased cell proliferation, as indicated by increased EdU-positive cells in the MCC of the loaded group in comparison to control mice (loaded: 6.23% ± 1.89% versus control: 1.90% ± 1.03%; p < 0.05; Figure 9A and C).
Proteoglycan distribution in the MCC of loaded and control mice was quantified by evaluating the toluidine blue-stained area and the distance map, as described in step 9 and Figure 7. We found significantly increased distance mapping in the MCC of condyles of the loaded group in comparison to control (loaded: 210.22 µm ± 4.11 µm versus control: 187.36 µm ± 8.64 µm; p < 0.005; Figure 9B and D). However, the proteoglycan-stained area was not statistically different between the loaded and control groups (loaded: 203,897.93 µm2 ± 10,171.00 µm2 versus control: 202,875.09 µm2 ± 33,419.09 µm2; p = 0.94; Figure 9B and E)
Figure 8: Representative results: (A) Col10a1-positive cells in the sagittal sections of the MCC of condyles from control and loaded mice. There are increased numbers Col10a1-positive cells in the loaded group (D). (B) Col2a1-positive cells in control and loaded mice. There is no difference in Col2a1-positive cells between groups (E). (C) TRAP staining in condyles of control and loaded mice. Increased TRAP activity in the loaded group in comparison to control (F). The histograms (D-F) represent the means ± SD for n = 4 per group. ** Significant difference between the control and loaded groups (p < 0.005). Scale bar = 200µm. Please click here to view a larger version of this figure.
Figure 9:Representative results: (A) EdU staining in condyles of control and loaded mice. Increased cell proliferation in the loaded group, as represented by increased EdU-positive cells (C). (B) Toluidine blue staining in the condyles of the control and loaded mice. Increased cartilage thickness in the loaded mice, as shown by increased distance mapping in the experimental group (D). No difference in proteoglycan-stained area between the control and loaded groups (E). Histograms (C-E) represent the means ± SD for n = 4 per group. ** Significant difference between the control and loaded groups (p < 0.005). Scale bar = 200 µm. Please click here to view a larger version of this figure.
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 fluorescent reporters are excellent tools to visually understand changes in gene expression. The double-collagen fluorescent reporter mouse model (Col2a1XCol10a1) used in this report was especially suitable for the study of the MCC, since the transgenes are expressed in the prehypertrophic and hypertrophic region of the MCC so that collagen distribution could be evaluated. If the researcher does not have access to a transgenic mouse model expressing fluorescent reporters, other methods of histological protein expression analysis, such as immunofluorescence, can be used.
For the morphometric measurements, the critical step before taking the cabinet radiograph images is to remove all soft tissues of the mandible or of the bones to be analyzed. Excessive muscle or soft tissue attached to the bones could mask the real measurements of the structures. It is important to be aware of the limitations of the cabinet radiograph system. Like every x-ray, it is a 2-dimensional representation of a 3-dimensional structure, and overlap of structures and artifacts should be carefully interpreted. In addition, the positioning of the bones in the radiographic cabinet should be consistent.
The cellular quantification by means of counting the number of positive pixels is an efficient method for histological quantification in a cell-rich region such as the MCC, but the limitation of this approach is that it does not provide the precise number of cells. In addition, since it quantifies only the pixels selected, it is recommended to quantify all images of interest using the same “sampled” pixel to avoid the quantification of pixels of different intensities when analyzing different images.
A general piece of advice when quantifying histological sections is to analyze multiple serial sections of each sample (in this manuscript, three serial sections were analyzed for each condyle), since variations within each sample are usually observed.
The methodology mentioned in our experiments is simple, easy to use, and can be used in any mineralized tissue study in which reporter mice are being used. With the existing methodology, it is possible to visualize the cells that are stained for TRAP (Col2a1 or Col10a1) or EdU (Col1a1 or Col2a1) by visualizing the co-localization of cells.
The authors have nothing to disclose.
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.
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. |