Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum‐free organ culture sternal model that recapitulates normal development and maintains the three‐dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (α2, α3, or β1). In the presence of anti‐β1 integrin (25 μg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti‐α2 (10 μg/ml, clone P1E6) or anti‐ α3 (10 μg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation. Dev. Dyn. 1997;210:249–263. © 1997 Wiley‐Liss, Inc.

Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum-free organ culture sternal model that recapitulates normal development and maintains the three-dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (alpha2, alpha3, or beta1). In the presence of anti-beta1 integrin (25 microg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti-alpha2 (10 microg/ml, clone P1E6) or anti-alpha3 (10 microg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation.

Parathyroid hormone (PTH) regulates calcium and phosphate homeostasis through the endocrine system. Parathyroid hormone-related peptide (PTHrP) is a heterogeneous polypeptide with sequence homology to PTH in its first 13 amino acid residues. Both bind and activate a common receptor, the type 1 PTH/PTHrP receptor (PTH1R). Activation of this Gprotein-coupled receptor by PTHrP has been shown to regulate chondrogenesis in a manner that attenuates chondrocyte hypertrophy. Here, we report the dose-response (10 Ϫ7 to 10 Ϫ15 M) effects of PTH on chondrogenesis using an avian sternal organ culture model. PTH increased cartilaginous tissue length and downregulated the deposition of type X collagen and its mRNA expression. In addition, PTH increased chondrocyte cell diameter in prehypertrophic and proliferative regions while decreasing chondrocyte apoptosis in the hypertrophic zone. In conclusion, these experiments demonstrate that PTH regulates cartilage growth, chondrocytic apoptosis, deposition of type X collagen protein, and expression of type X collagen mRNA. Type X collagen mRNA expression was downregulated by PTH in this organ culture model, but cell size, another marker for terminal differentiation, increased. © 2004 Wiley-Liss, Inc.Key words: hyaline cartilage; apoptosis; parathyroid hormone; parathyroid hormone-related peptide; chondrogenesis; parathyroid hormone receptor; type X collagen Parathyroid hormone (PTH) is one of the hormones and growth factors that regulate hyaline cartilage development. Classically, PTH is an 84-amino acid protein that regulates calcium and phosphate homeostasis primarily through actions on specific receptors in kidney and bone (Juppner et al., 1991;Juppner and Schipani, 1996;Bro and Olgaard, 1997). Native 1-84 PTH and 1-34 PTH fragments stimulate adenylate cyclase through a G-proteincoupled receptor (GPCR) with equal potency (Abou-Samra et al

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