A distal portion of the femur was resected and fixed in a 10% neutral buffered formalin solution for 3 days, decalcified with 50 mM EDTA for 3 weeks, and then cast in a paraffin block. The femur was sectioned parallel to the longitudinal axis, and stained with hematoxylin-eosin (HE) and Safranin-O. For immunohistochemical evaluations, monoclonal antibody (anti-hCL (II), purified IgG, Fuji Chemical Industries Ltd, Toyama, Japan) was used as the primary antibody. Immunostaining was carried out according to the manufacturer instructions using the Envision immunostaining system (DAKO Japan, Kyoto, Japan). Finally, the sections were counterstained with hematoxylin.
The histology of the tissue regenerated in the defect was quantitatively evaluated with the scoring systems reported by the O’Driscoll [
25]. Histology was evaluated for
cellular morphology (hyaline articular cartilage, 4 points; incompletely differentiated mesenchyme, 2 points; and fibrous tissue or bone, 0 points),
Safranin O staining of the matrix (normal or nearly normal, 3 points; moderate, 2 points; slight, 1 point; and none, 0 points),
surface regularity (smooth and intact, 3 points; superficial horizontal lamination, 2 points; fissures of 25–100% of thickness, 1 point; and severe disruption including fibrillation, 0 points),
structural integrity (normal, 2 points; slight disruption including cysts, 1 point; and severe disintegration, 0 points),
thickness (100% of normal adjacent cartilage, 2 points; 50–100% of normal cartilage, 1 point; and 0–50% of normal cartilage, 0 points),
bonding to the adjacent cartilage (bonded at both ends of the graft, 2 points; bonded at one end or partially at both ends, 1 point; and not bonded, 0 points),
hypocellularity (normal cellularity, 3 points; slight hypocellularity, 2 points; moderate hypocellularity, 1 point; and severe hypocellularity, 0 points),
chondrocyte clustering (no clusters, 2 points; <25% of the cells, 1 point; and 25–100% of the cells, 0 points), and
adjacent cartilage degradation (normal, no clusters, and normal staining, 3 points; normal cell, mild clusters, and moderate staining, 2 points; mild/moderate hypocellularity and slight staining, 1 point; severe hypocellularity, 0 points). The maximum total score was 24 points.