Histological analysis

At four weeks postoperatively, each graft-tibia specimen was harvested. All specimens were fixed in 10% buffered formalin solution at room temperature for 48 hours. After fixation, specimens were decalcified in Kalkitox (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 7 days, embedded in paraffin, and serially sliced in the coronal plane at a thickness of 4 μm. The slides were stained with hematoxylin and eosin (H&E), safranin O, and picrosirius red and toluidine blue (P&T). Digital images of the stained slides were obtained (Olympus System Microscope Model BX53 LED and a cellSens Standard Version 1.17; Olympus Co., Tokyo, Japan). H&E and safranin O staining were evaluated under unpolarized light microscopy, while P&T staining was evaluated under polarized light microscopy. Picrosirius red alone was used to stain type 1 and type 3 collagen fibers, but not type 2; therefore, the fibrocartilaginous tissue at the tendon-bone junction was not stained. Because toluidine blue can stain type 2 collagen fiber, double staining with toluidine blue staining was applied to P&T staining to evaluate the continuous formation of type 1 to 3 collagen fibers under polarized light microscopy. Histological analysis was performed to observe the formation of chondrocytes by H&E staining, appearance of type 2 collagen by safranin O, and continuity between the graft tendon and fibrocartilage tissue (containing chondrocytes and type 2 collagen) by P&T double staining.