Hirsch M S, Lunsford L E, Trinkaus-Randall V, Svoboda K K
Department of Anatomy and Neurobiology, Boston University School of Medicine, Massachusetts 02118, USA.
Dev Dyn. 1997 Nov;210(3):249-63. doi: 10.1002/(SICI)1097-0177(199711)210:3<249::AID-AJA6>3.0.CO;2-G.
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.
鸡胸骨特定区域的软骨细胞具有不同的发育命运。头部软骨细胞会肥大，并在骨沉积之前向细胞外基质分泌X型胶原蛋白。中部和尾部软骨细胞在整个发育过程中保持软骨状态，并继续分泌II型、IX型和XI型胶原蛋白。整合素受体与细胞外基质分子的相互作用已被证明会影响其他细胞类型的细胞骨架组织、增殖、分化和基因表达。我们推测软骨细胞的存活和分化，包括X型胶原蛋白沉积到间质基质中，可能是由整合素受体介导的。为了验证这一假设，我们建立了一种无血清器官培养胸骨模型，该模型可重现正常发育过程并维持组织的三维关系。我们通过五个参数来检测软骨细胞的分化：X型胶原蛋白沉积到间质基质中、胸骨生长、肌动蛋白分布、细胞形状和细胞直径变化。使用DNA片段分析对额外的胸骨进行凋亡分析。将胸骨与针对整合素亚基（α2、α3或β1）的阻断抗体进行器官培养。在存在抗β1整合素（25微克/毫升，克隆W1B10）的情况下，X型胶原蛋白沉积到间质基质中和胸骨生长受到显著抑制。此外，所有软骨细胞都显著变小，肌动蛋白被破坏，并且整个标本中的凋亡显著增加。添加抗α2（10微克/毫升，克隆P1E6）或抗α3（10微克/毫升，克隆P1B5）整合素可部分抑制X型胶原蛋白沉积到间质基质中；然而，胸骨生长和细胞大小显著减小。这些数据是首次从完整组织中获得的，表明软骨细胞与细胞外基质的相互作用是软骨细胞存活和分化所必需的。
