Comparison between normal and loose fragment chondrocytes in proliferation and redifferentiation potential

Abstract

Purpose: Loose fragments in osteochondritis dissecans (OCD) of the knee require internal fixation. On the other hand, loose fragments derived from spontaneous osteonecrosis of the knee (SONK) are usually removed. However, the difference in healing potential between OCD- and SONK-related loose fragments has not been elucidated. In this study, we investigated proliferative activity and redifferentiation potential of normal cartilage-derived and loose fragment-derived chondrocytes.

Methods: Cells were prepared from normal articular cartilages and loose fragment cartilages derived from knee OCD and SONK. Cellular proliferation was compared. Redifferentiation ability of pellet-cultured chondrocytes was assessed by real-time PCR analyses. Mesenchymal differentiation potential was investigated by histological analyses. Positive ratio of a stem cell marker CD166 was evaluated in each cartilaginous tissue.

Results: Normal and OCD chondrocytes showed a higher proliferative activity than SONK chondrocytes. Chondrogenic pellets derived from normal and OCD chondrocytes produced a larger amount of safranin O-stained proteoglycans compared with SONK-derived pellets. Expression of chondrogenic marker genes was inferior in SONK pellets. The CD166-positive ratio was higher in normal cartilages and OCD loose fragments than in SONK loose fragments.

Conclusions: The OCD chondrocytes maintained higher proliferative activity and redifferentiation potential compared with SONK chondrocytes. Our results suggest that chondrogenic properties of loose fragment-derived cells and the amount of CD166-positive cells may affect the repair process of osteochondral defects.

Fig. 1

Normal chondrocytes and OCD loose fragment chondrocytes showed a higher proliferative activity than SONK loose fragment chondrocytes in cell proliferation assays. Normal chondrocytes (a). OCD-derived chondrocytes (b). SONK-derived chondrocytes (c). *p < 0.05

Fig. 2

Normal and OCD chondrogenic pellets produced the larger amount of safranin O-stained proteoglycans compared with SONK pellets (a–c, red). Lipid vacuoles were observed in normal and OCD chondrocytes after adipogenic induction (a and b, red). Calcium depositions were induced by osteogenic treatments in normal cartilage- and OCD-derived cells (a and b, brown). On the other hand, neither adipogenesis nor osteogenesis was induced in SONK-derived cells (c). The Bern pellet histology score was higher in normal and OCD pellets than in SONK pellets (d). Staining density of oil red O and von Kossa was lower in SONK-derived cells than in normal and OCD chondrocytes after the treatment of adipogenic and osteogenic induction (e and f). Bars, 100 μm. *p < 0.05

Fig. 3

Expression pattern of chondrogenic marker genes. Expression of COL2A1 and SOX9 was more activated in normal and OCD chondrogenic pellets than in SONK pellets (a and b). However, the difference among normal, OCD, and SONK chondrogenic pellets was not observed in SOX5/6 expression (c and d). *p < 0.05

Fig. 4

CD166, a MSC surface marker, was detected in the cells of normal and loose fragment cartilages (a–c, brown in the transitional zone of articular cartilage). Normal cartilage (a). OCD loose fragment (b). SONK loose fragment (c). Negative control in the absence of an anti-CD166 antibody (d). CD166-positive cell ratio was higher in normal cartilages and OCD fragments than in SONK fragments (e). Normal cartilages and OCD fragments showed higher staining densities of CD166 compared with SONK loose fragment cartilages (f). Bars, 100 μm. *p < 0.05