In Vivo Analysis of Arg-Gly-Asp Sequence/Integrin [alpha]5[beta]1-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 [alpha]5[beta]1-ligand interactions inhibited pre-hypertrophic chondrocyte differentiation. The purpose of this study is to elucidate in vivo the roles of the integrin [alpha]5[beta]1-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 [alpha]5[beta]1 antibody ([alpha]5[beta]1 ab) in the upper limbs of mouse embryos at embryonic day (E) 15.5 (RGDS-injected limbs, [alpha]5[beta]1 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 [alpha]5[beta]1 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 [alpha]5[beta]1 ab injection decreased chondrocyte proliferation, differentiation, and apoptosis in enchondral ossification, and suggested that the integrin [alpha]5[beta]1-mediated ECM signal through the RGD sequence is involved in embryonic enchondral ossification. © 2014 American Society for Bone and Mineral Research. [PUBLICATION ABSTRACT]