Our protocol addresses an unmet need for a robust and reproducible method for quantitative analysis of joint changes in osteoarthritis development and progression. Our technique outlines specific and reproducible parameters for the quantitative analysis of changes in multiple joint tissues in osteoarthritis. As this technique allows for quantitative analysis of osteoarthritis, it can be employed to compare the effects of specific treatments on multiple joint tissues in order to evaluate their therapeutic efficacy.
Our protocol can also be applied to other osteoarthritis models or areas of musculoskeletal research in which quantification of cartilage and bone is needed. Although the system is straightforward and intuitive, training sessions are still necessary to practice obtaining measurements in a repeated manner to achieve reproducibility. Vengadeshprabhu Karuppagounder, a postdoctoral scholar from our laboratory will now be demonstrating our procedure.
Begin by turning on the microscope, camera, computer, and touch screen monitor. Select the 4X objective and click the software icon to launch the software program. Place the slide on the microscope stage and center the sample within the measurement window ensuring that the measurement grid is set to the proper magnification to match the objective in that both the tibial and femoral articular surface and tibial subchondral bone are included within the image regions of interest.
Then under the File menu, select Read Settings. In the Settings window, select the appropriate settings file for the parameters to be measured. To measure the total cartilage area, select the Tib TC function and trace along the superior edge of the tibial cartilage surface where the cartilage meets the joint space and along the chondro-osseous junction where calcified cartilage meets the subchondral bone.
To measure the calcified cartilage area select the Tib CC function and trace a line along the tide mark, a naturally occurring line separating the calcified and uncalcified regions of cartilage, and along the chondro-osseous junction where the calcified cartilage meets the subchondral bone. To determine the uncalcified cartilage area, subtract the calcified cartilage area from the total cartilage area. To determine the chondrocyte number, create separate count functions within the settings in the histomorphometry software to distinguish the matrix producing and matrix non-producing chondrocytes.
Then use the stylus to make a small dot over each chondrocyte expressing the specified phenotype to determine the number of matrix producing or not producing chondrocytes. To measure the total tibial articular surface parameter, trace a line across the surface of the tibial cartilage to carefully define the individual fibrillations. Then draw a second line along the tide mark to serve as an internal control for the plane of each section and divide the tibial articular surface parameter by the tide mark parameter to calculate the tibial articular surface fibrillation index.
To measure the subchondral marrow space area, select the Marrow Area function. Then draw lines around the parameter of these areas to determine the total area of bone marrow space within the tibial subchondral bone. To measure the total subchondral area, select the Subchondral Bone function and outline the region between the chondro-osseous junction in the superior border of growth plate extending laterally to the regions of the anterior and posterior osteophytes.
Then subtract the subchondral bone marrow space area from the total subchondral area to calculate the subchondral bone area. To measure the osteophyte area, select the anterior osteophyte function and trace the anterior osteophyte of the proximal tibia. Then select posterior osteophyte and trace the posterior osteophyte of the proximal tibia.
To measure the anterior femoral synovial thickness, click Anterior Femur Synovium Meniscus and draw one line from the inner insertion point of the anterior synovium on the femur towards its attachment point on the meniscus and one line from the outer insertion of the synovium on the femur towards its attachment to the meniscus. Then divide the total synovial area by the synovial parameter to calculate the synovial thickness. DMM induced osteoarthritis results in an increased Osteoarthritis Research Society International score compared to sham mice.
Distinctly characterized by surface erosion and cartilage lost. This histomorphometry protocol can be used to detect several osteoarthritis associated changes including a decrease in the total cartilage and uncalcified cartilage areas. A reduction in the total chondrocyte number and importantly, a loss in the number of matrix producing chondrocytes.
Changes to the articular surface, indicative of the severity of erosion, are also observed, including an overall increase in the fibrillation index. Osteoarthritis also includes an increase in the subchondral bone area and a reduction in the area of the bone marrow space in DMM mice indicating subchondral bone sclerosis. Both the anterior and posterior osteophyte areas also increased in DMM mice, suggesting an ongoing subchondral bone remodeling that acts as a compensatory mechanism to handle changes in joint loading at the site of injury.
Histomorphometric analysis of the synovium shows an increase in the synovial thickness in DMM mice. A typical outcome of osteoarthritis associated synovial inflammation and the diffusion of inflammatory cytokines into the joint space. In addition, no significant inter-user variability between the histomorphometric analysis of the uncalcified cartilage area in the Osteoarthritis Research Society International score is observed.
After histomorphometric analysis, sequential slides and immunofluorescence staining can be used to examine changes in protein expression between samples.
