For histological examination, the specimens were fixed in 10% neutral buffered formalin for 7 days, decalcified with 0.25 mol/l EDTA in phosphate-buffered saline (pH 7.4), dehydrated in graded ethanol, and embedded in paraffin wax. Sagittal sections (6 μm thick) were then cut, stained with safranin-O/fast green and haematoxylin and eosin, and examined microscopically. All sections were observed and evaluated by three authors. Histological evaluation of plug cartilage was performed using the modified Mankin's score [38 (link)] (original score proposed by Mankin and coworkers [39 (link)]). The grading system was composed of four categories – cartilage structure (6 points), cartilage cells (3 points), staining (4 points) and tidemark integrity (2 points) – with a highest score of 14 points; normal cartilage scored 0 (Table 1) [38 (link)]. When we observed newly formed tissue that covered the plug cartilage, the finding was counted as 'pannus and surface irregularities' (2 points).