Histochemical quantification of collagen content in articular cartilage

Background Articular cartilage (AC) is mainly composed of water, type II collagen, proteoglycans (PGs) and chondrocytes. The amount of PGs in AC is routinely quantified with digital densitometry (DD) from Safranin O-stained sections, but it is unclear whether similar method could be used for collagens. Objective The aim of this study was to clarify whether collagens can be quantified from histological AC sections using DD. Material and methods Sixteen human AC samples were stained with Masson's trichrome or Picrosirius red. Optical densities of histological stains were compared to two commonly used collagen parameters (amide I and collagen CH2 side chain peak at 1338cm(-1)) measured using Fourier Transform Infrared (FTIR) spectroscopic imaging. Results Optical density of Modified Masson's trichrome staining, which included enzymatic removal of PGs before staining, correlated significantly with FTIR-derived collagen parameters at almost all depths of cartilage. The other studied staining protocols displayed significant correlations with the reference parameters at only few depth layers. Conclusions Based on our findings, modified Masson's trichrome staining protocol is suitable for quantification of AC collagen content. Enzymatic removal of PGs prior to staining is critical as us allows better staining of the collagen. Further optimization of staining protocols may improve the results in the future studies.