The response of costal cartilage to mechanical injury in mice.

BACKGROUND: The healing potential of articular cartilage in response to injury is poor, because articular cartilage lacks blood vessels or perichondrium. Costal cartilage is covered with a vascularized perichondrium, which is known to have chondrogenic potential. The purpose of this study was to determine whether costal cartilage can heal in response to mechanical injury. METHODS: Sixty-five ICR mice were used. Under anesthesia, the left tenth costal cartilage was dissected using microscissors. At 1 day and at 1, 2, 3, and 12 weeks after injury, the mice were killed and paraffin sections were prepared. Safranin O staining, in situ hybridization of type II collagen, and immunostaining for CD44 were performed. Localization of cell proliferation was performed using immunohistochemistry with bromodeoxyuridine monoclonal antibody. In situ detection of apoptosis (deoxynucleotidyl transferase-mediated dUTP nick end labeling) was performed using an Apop Tag Kit. RESULTS: From 1 to 2 weeks after costal cartilage injury, bromodeoxyuridine-positive cells were observed in the perichondrium. Two weeks after injury, the dissected cartilage fragments had combined with newly formed safranin O-positive tissue. Type II collagen mRNA was strongly expressed in the cells of the newly formed tissue. Apoptosis was detected in newly formed cartilaginous tissue at 1 and 2 weeks after injury. The cartilage fragments failed to unite at 12 weeks after injury. CD44 immunoreactivity was detected on the surface of the cavity between the cartilage fragments. CONCLUSION: Although the dissected fragments of costal cartilage can combine with newly formed cartilaginous tissue temporarily, they fail to unite ultimately.

Developmental abnormalities in rat embryos leading to tibial ray deficiencies induced by busulfan.

BACKGROUND: Little is known about the developmental changes associated with tibial ray deficiencies. The aim of this study was to detect cell death, proliferation, and gene expression that result in tibial ray deficiencies. METHODS: We induced tibial ray deficiencies in rat embryos using a teratogenic agent (busulfan) and observed the developmental changes in 1126 hindlimbs. We performed Nile blue staining, whole mount in situ hybridization for fibroblast growth factor 8 (Fgf8), bone morphogenetic protein 4 (Bmp4) and Sonic hedgehog (Shh), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and assessment of cell proliferation by 5-bromo-2'-deoxy-uridine (BrdU)/anti-BrdU immunohistochemistry. RESULTS: In situ hybridization showed reductions in Fgf8 and Bmp4 expression. Histological examination showed a delay of mesenchymal condensation, increased mesenchymal cell death, decreased mesenchymal cell proliferation, and a reduction in the number of mesenchymal cells. These abnormalities may cause hypoplasia of the limb. Bmp4 expression was markedly reduced in the anterior mesenchyme. Shh was expressed in the posterior mesenchyme. We suggest that the posterior skeletal elements may be fully formed owing to Shh expression, but the anterior skeletal elements may be underdeveloped owing to an intense reduction of Bmp4 expression in the anterior mesenchyme, causing hypoplasia of the tibial ray. CONCLUSIONS: The combined effects of increased cell death, decreased cell proliferation, reduction of Fgf8 expression, and intense reduction of Bmp4 expression in the anterior mesenchyme may play an important role in the development of tibial ray deficiency induced by busulfan.

Developmental failure of phalanges in the absence of growth/differentiation factor 5.

Growth/differentiation factor 5 (GDF5) is a member of the bone morphogenetic protein (BMP) family, which has been implicated in several skeletogenic events including cartilage and bone formation. To study the role of GDF5, we analyzed digit development in brachypodism (bp) mice, which carry functional null mutations of the Gdf5 gene and exhibit a reduction in the length of digit bones and loss of the middle phalanges. In situ detection of apoptosis and whole-mount detection of cell death showed abnormal apoptosis in the developing phalanges of bp mice. In situ hybridization in bp mice showed overexpression of Gdf5 mRNA in the developing phalanges where apoptotic cells were increased. In addition, bp mice exhibited excessive apoptosis in the interdigital regions. The condensed mesenchymal cells were progressively decreased in the developing phalanges and failed to form cartilage models of the middle phalanges. These findings show that excessive apoptosis in the absence of GDF5 results in developmental failure of the phalanges. We conclude that GDF5 is essential for maintenance and growth of the developing phalanges.