In Vivo Analysis of Arg‐Gly‐Asp Sequence/Integrin α5β1‐Mediated Signal Involvement in Embryonic Enchondral Ossification by Exo Utero Development System

Enchondral ossification is a fundamental mechanism for longitudinal bone growth during vertebrate development. In vitro studies suggested that functional blockade with RGD peptides or with an antibody that interferes with integrin α5β1–ligand interactions inhibited pre‐hypertrophic chondrocyte differentiation. The purpose of this study is to elucidate in vivo the roles of the integrin α5β1‐mediated signal through the Arg‐Gly‐Asp (RGD) sequence in the cell–extracellular matrix (ECM) interaction in embryonic enchondral ossification by an exo utero development system. We injected Arg‐Gly‐Asp‐Ser (RGDS) peptides and anti‐integrin α5β1 antibody (α5β1 ab) in the upper limbs of mouse embryos at embryonic day (E) 15.5 (RGDS‐injected limbs, α5β1 ab‐injected limbs), and compared the effects on enchondral ossification with those found in the control limbs (Arg‐Gly‐Glu‐Ser peptide‐, mouse IgG‐, or vehicle‐injected, and no surgery) at E16.5. In the RGDS‐injected limbs, the humeri were shorter and there were fewer BrdU‐positive cells than in the control limbs. The ratios of cartilage length and area to those of the humerus were higher in the RGDS‐injected limbs. The ratios of type X collagen to type 2 collagen mRNA and protein (Coll X/Coll 2) were significantly lower in the RGDS‐injected limbs. In those limbs, TUNEL‐positive cells were hardly observed, and the ratios of fractin to the Coll X/Coll 2 ratio were lower than in the control limbs. Furthermore, the α5β1 ab‐injected limbs showed results similar to those of RGDS‐injected limbs. The present in vivo study by exo utero development system showed that RGDS and α5β1 ab injection decreased chondrocyte proliferation, differentiation, and apoptosis in enchondral ossification, and suggested that the integrin α5β1‐mediated ECM signal through the RGD sequence is involved in embryonic enchondral ossification. © 2014 American Society for Bone and Mineral Research.

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