Identification of the Tidemark Line of Calcification in Osteoarthritic Cartilage Using a Stain for Alkaline Phosphatase

Abstract

Inflammatory response and calcification are strongly implicated in osteoarthritis (OA) progression. Key inflammatory biomarkers present throughout the process of OA have been established but an association with calcification has not been clarified. A faint line, tidemark, exists between the subchondral bone and the articular surface in knee joints that is presumed to be the line of demarcation between calcified and uncalcified cartilage. This study shows that the tidemark is where calcification is actively occurring. Mouse knees (n=8) were stained for both Alkaline Phosphatase (ALP) and Safranin-O/Fast Green. ALP, active during tissue calcification, was used to identify the true location of calcification. Images from both stains were overlaid for comparison of complementary tissues. The cartilage area above the tidemark was then compared to the area above the ALP line of calcification. Because the two areas proved to be nearly statistically identical, the conclusion is that the tidemark observed in the Safranin-O/Fast Green stain is indeed the line of calcification for articular cartilage. The role of calcification in OA was further examined by comparing NPP1, calcification marker, with HtrA1. We demonstrate a reciprocal correlation between HtrA1 and NPP1, suggesting a link between pathological calcification and inflammation in joints experiencing OA.

Osteoarthritis-Like Changes in Bardet–Biedl Syndrome Mutant Ciliopathy Mice (Bbs1M390R/M390R): Evidence for a Role of Primary Cilia in Cartilage Homeostasis and Regulation of Inflammation

“…This was done to measure the area between the apical surface of the cartilage and where the articular cartilage interfaces with subchondral bone. The technique was perfected in our laboratory and previously reported ( Black et al, 2015 ). To help standardize the human measurement error, two different lab members independently measured the tissue samples.…”