Subchondral bone thickening, or sclerosis, is one of the hallmarks of osteoarthritis, both in animal models and in humans. Currently, the severity of histologic subchondral bone thickening is mostly determined by visual estimation based on semi-quantitative grading systems. This study aim to develop a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus.
We created a mouse model for knee osteoarthritis by surgical destabilization of the medial meniscus which is performed under surgical microscope and the sterile condition. The histological process of mouse knee joints include tissue fixation, decalcification, trimming, processing, embedding, sectioning, and staining, followed by microscopic imaging. The image on the screen shows mouse knee osteoarthritis with articular cartilage lesions, osteophyte formation, and subchondral bone thickening Quantitative measurement of osteoarthritic subchondral bone with ImageJ software.
Download ImageJ from imagej.nih.gov/ij. Open ImageJ. Click the File tab and then click Open option to open the histologic image.
Find the file directory address, select the picture file, and click Open. Use the straight line tool to sketch one unit of length, a micrometer, and click Analyze, then Set Scale. Set the known distance and the pixel aspect ratio to one and click Ok.ImageJ can convert the pixel length to the unit length, a micrometer.
Set the measured factor to area. Click Analyze then Set Measurement and check Area and Limit to Threshold box under the new window. This step sets ImageJ to measure parameter area within selected threshold.
Defined total subchondral bone area of interest as shown in radio which covers the subchondral cortical plate and a portion of the underlying trabecular bone adjacent to cortical plate. Sketch the outline of total subchondral bone area by using the selection tool under the main window of image. The selection tools gives the system a threshold to limit the measurement after threshold being selected.
Click Analyze then Measure. A result window with area measurement will open. Click Edit then Clear Outside to exclude the area outside total subchondral bone area.
Only total subchondral bone area is visible after clicking Clear Outside option. Click Image then Adjust then Color Threshold to open the threshold color window. Click Original at the bottom of threshold color window to restore picture to original status.
Use selection tools to draw a small box in the bone substance region. Click Sample Option at the bottom of window to define the bone substance area. Selected bone substance area will turn to red.
Click Select at the bottom of threshold color window to create area measurement threshold. Click Analyze then Measure in ImageJ main menu. The bone substance area measurement result will show on result window.
Save the data of total subchondral bone area and bone substance area. The inter and intraobserver variability and the reproducibility were determined by Pearson's correlation coefficient analysis. The significance of difference between study groups were determined by using Student t-test or one-way ANOVA, followed by post-hoc test.
This figure shows upper and lower panels. The upper panel shows photomicrographs of histologic images from Sham and DMM groups for visual estimate-based subchondral bone grading. The lower panels show photomicrographs of histologic images from Sham and DMM groups for ImageJ-assisted, quantitative subchondral bone measurement.
The boxes outlined with a dotted yellow line made with Adobe Illustrator defines the total subchondral bone area of interest. The area of bone substance within the boxes is highlighted in orange. Remarkable subchondral bone thickening in the medial femoral condyle and medial tibial plateau can be quantified using ImageJ software.
In this figure, interobserver variation tests indicate a high reproducibility between observers for both the first and second measurements of subchondral bone thickness in the medial tibial plateau and the medial femoral condyle regions of interest. In this figure, intraobserver variation tests indicate a high reproducibility between the first and second subchondral bone thickness measurements in the medial tibial plateau and medial femoral condyle regions of interest. Table 1 shows comparative analysis of reproducibility between the visual grading and ImageJ-assisted quantitative measurement of subchondral bone thickness.
Correlation coefficient test suggests that the quantitative measurement was relatively more reproducible than the visual grading system. This figure shows comparative sensitivity analysis of visual grading and ImageJ-assisted quantitative measurement of subchondral bone thickness in the medial femoral condyle and the medial tibial plateau. The histologic images for visual estimate grading were divided into three groups.
The quantitative subchondral bone thickness value from all three observers for the DMM images with a zero visual score were significantly higher than that of the Sham images with a zero visual score, indicating that the quantitative measurement is more sensitive than the visual grading to mild subchondral bone thickening. The newly developed protocol for quantitative measurement of osteoarthritic subchondral bone thickness is more sensitive to mild subchondral bone changes than the widely used visual grading systems. And this protocol can be used for detecting early osteoarthritic subchondral bone changes and for assessing in vivo efficacy of osteoarthritis treatments in concert with osteoarthritis cartilage grading.
