Identification of the tidemark line of calcification in osteoarthritic cartilage using a stain for alkaline phosphatase

      Purpose: While osteoarthritis (OA) has traditionally been classified as a “wear and tear” disease, recent studies have shown that this disease is due to an inflammatory response. Our lab has made significant strides in identifying key biomarkers that are present throughout the progression of OA. In past histochemical staining, we and others have noticed a faint line located between the subchondral bone and the articular surface in knee joints, presumed to be the tidemark or line of demarcation between calcified and uncalcified cartilage. This would suggest that cartilage in osteoarthritic joints is lost in part due to a metabolic calcification process. The purpose of this study is to prove quantitatively that the faint line is the tidemark and not simply an artifact of the staining process.
      Methods: Mouse knees (n = 8) were embedded and stained for both Alkaline Phosphatase (ALP) and Safranin-O/Fast Green (Saf-O, FG). ALP, a metallo hydrolase enzyme that is active during tissue calcification, was used to identify the true location of the tidemark. Using Adobe Photoshop, pictures from the two stains were overlaid for tidemark comparison. A standard-sized box was cropped over the two pictures and the “lasso” tool was used to outline the area between the articular surface and the tidemark in each stain. The area of each of the outlined pictures was then calculated in order to compare the tidemark location between the two stains. To ensure that the anatomy was nearly identical between samples and that specific anatomical landmarks would match up when overlaid, the comparison was only made between consecutive slides taken from the same sample. Data were analyzed using a paired T-test.
      Results: A comparison of the areas outlined in both stains resulted in a T-value of 0.51 that equated to a P-value greater than 0.6 with a standard deviation of 5.87%. Thus, we can conclusively say that the thin line observed in the Safranin-O/Fast Green stain is indeed the tidemark line of calcification we see in the Alkaline Phosphatase stain.
      Conclusions: Proving that the thin line observed between subchondral bone and the articular surface after Saf-O, FG staining is the tidemark demarcation of calcification and not an artifact is important. It allows us and others to compare the progression of the tidemark over multiple age groups and/or models using the simple Saf-O, FG stain. It lends support to the idea that OA is not simply a “wear and tear” disease.