Neutral density filters

The 3 µm sections were stained with Safranin-O and measured with quantitative DD system to determine PG distribution^{26 (link)}. The system consists of a light microscope (Nikon Microphot-FXA, Nikon Co., Tokyo, Japan), equipped with a monochromatic light source and a 12-bit CCD camera (ORCA-ER, Hamamatsu Photonics K.K., Hamamatsu, Japan). The system was calibrated with neutral density filters (Schott, Mainz, Germany) covering optical density (OD) range from 0 to 3.0. The samples were imaged with a 4.0x objective resulting in a pixel size of 1.56 × 1.56 μm².

After synchrotron microCT imaging, the frozen samples were thawed and cut in half. The first half was fixed in 10% formalin and then dehydrated in ascending series of ethanol for the reference histological analysis. After dehydration, the samples were decalcified in EDTA and embedded in paraffin in order to cut 3 µm thick sections. Then, the paraffin was removed, and Safranin-O staining was performed to study the spatial FCD (i.e. PG) distribution in the cartilage. After staining, quantitative digital densitometry measurements were conducted to study the optical density (OD, e.g., PG distribution) in cartilage using a light microscope (Nikon Microphot-FXA, Nikon Co., Japan) equipped with a monochromatic light source and a 12-bit CCD camera (ORCA-ER, Hamamatsu Photonics K.K., Japan). System was calibrated using neutral density filters (Schott, Germany) covering OD range from 0 to 2.6. Histological images of Safranin-O stained sections were also imaged with a light microscope (Leica MZ75, Leica Microsystems Ltd., Switzerland) fitted with a CCD camera (Nikon digital sight DS-Fi2, Nikon Co., Japan). The depth-wise PG content within each cartilage sample was determined as an average of three sections. From the second half, water content was determined by calculating the difference between the wet weight and dry weight after freeze-dying the samples for 20 h.