Differentiation of chondrogenic precursor cells during the regeneration of articular cartilage.
OBJECTIVE
Full-thickness defects that penetrate articular cartilage are filled by fibrous, or fibrocartilaginous tissue and, to a very limited extent, also by hyaline cartilage. In rabbits, small full-thickness defects (to < or =3 mm in diameter) are capable of regenerating surfacing hyaline cartilage. However, chondrogenic differentiation does not occur in larger defects (> or =5 mm in diameter). We studied the involvement of fibroblast growth factor-2 (FGF-2) in the cartilaginous repair response in full-thickness defects of articular cartilage in vivo, and attempted to facilitate cartilaginous repair of the defects by the local administration of FGF-2.


METHODS
The right knee joint of male adolescent Japanese white rabbits was entered through a medial parapatellan approach, and the patella was dislocated laterally to expose the articular surface of the femoral trochlea. Full-thickness defects were created in the weight-bearing area of the femoral trochlea with a hand-drill (the 5-mm diameter defects in 80 rabbits and the 3-mm diameter defects in 40 rabbits). The animals were fitted with an osmotic pump connected to silastic medical grade tubing, and a length of the tubing about 5 mm long was introduced into the articular knee cavity. The 5-mm-diameter defects received FGF-2 (50 pg/h) or sterile saline via an osmotic pump for the initial 2 weeks. Five animals each were sacrificed after 1, 2, 4, 8, or 24 weeks after creation of defects. The 3-mm diameter defects received a neutralizing monoclonal antibody against FGF-2 (50 ng/h) or pre-immune mouse IgG (50 ng/h) for the initial 2 weeks. Five animals each were sacrificed after 2, 3, or 4 weeks after creation of defects. The distal portion of each femur was removed, fixed, decalcified, and embedded in paraffin for the subsequent histological analysis. Sections were cut in the transverse plane, and histologically examined.


RESULTS
The administration of FGF-2 (50 pg/h) resulted in successful regeneration of articular cartilage and the subchondral bone within 8 weeks after creation of 5-mm diameter defects. In these defects, undifferentiated mesenchymal cells initiated chondrogenic differentiation coupled with replacement by subchondral bone, resulting in the resurfacing of the defects by hyaline cartilage and the recovery of subchondral bone up to the original bone-articular cartilage junction. In contrast, the administration of a neutralizing monoclonal antibody against FGF-2 clearly interfered with the action of endogenous FGF-2 in 3-mm diameter defects, which were filled with fibrous tissue. None of the antibody-treated defects were covered with cartilage. We then assessed the proliferative capacity of the undifferentiated mesenchymal cells in the defects by immunostaining the proliferating cell nuclear antigen (PCNA) at 1 week after creation of defects. The capacity of reparative tissue to form cartilage was well correlated with the occurrence in the defects of a cell population that was PCNA-positive, undifferentiated, and capable of self-renewal.


CONCLUSIONS
The local administration of FGF-2 resulted in the successful resurfacing of large (5 mm in diameter) defects by hyaline cartilage. Prechondrogenic mesenchymal cells were the likely targets of FGF-2, which probably promoted the formation of cartilage by stimulating a selective expansion of chondroprogenitor cells. Thus, activation of FGF-2 signalling is critically important for the induction of cartilaginous repair response in full-thickness articular cartilage.
Hiraki
Y
Y
Department of Molecular Interaction and Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Japan. hiraki@frontier.kyoto-u.ac.jp
Shukunami
C
C
Iyama
K
K
Mizuta
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology; D002358:Cartilage, Articular / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D019902:Chondrocytes / Q000166:cytology*; D016222:Fibroblast Growth Factor 2 / Q000502:physiology; D006614:Hindlimb; D008297:Male; D008648:Mesoderm / Q000166:cytology; D010012:Osteogenesis / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000502:physiology; D011817:Rabbits; D012038:Regeneration / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology*
D000911:Antibodies, Monoclonal; D018809:Proliferating Cell Nuclear Antigen; D016222:Fibroblast Growth Factor 2