P15 Peptide Stimulates Chondrogenic Commitment and Endochondral Ossification

Zhang J, et al., 41(7):1413-1422, Int Orthop., 2017

PP15, a synthetic 15 amino acid biomimetic peptide sequence derived from the alpha (α)-1 chain of collagen I has been known to enhance osteogenic differentiation and bone formation via an assumed interaction with α2β1 integrin. Reported herein is a study of the putative direct binding mechanism of P15 peptide to α2β1 integrin and the effect of P15 on bone formation. Surface Plasmon Resonance (SPR) was used to study the binding interaction between P15 and α2β1 integrin. All SPR experiments were performed using a Pall ForteBio Pioneer instrument equipped with COOH1 sensor chip. The sensor chip surface was activated using a 1:1 mixture of EDC/NHS. Subsequently, neutrAvidin was allowed to bind to the activated surface until a response plateau was reached. The residual active sites were blocked by using Tris-HCl (pH 8.5). The biotinylated P15 was injected over the neutrAvidin-coated sensor chip until all available biotin-binding sites were saturated. Bovine serum albumin (BSA) was used to block any non-specific binding sites. The kinetics of P15-α2β1 integrin binding was analyzed by injecting an active recombinant variant of α2β1 integrin in increasing concentrations over the P15 immobilized sensor chip surface. After each assay, the sensor chip surface was regenerated by injecting HCl. SPR data obtained were globally fitted. The association rate constants (kon), dissociation rate constants (koff) , and the equilibrium dissociation constant (KD) values were determined. Overall results of this study demonstrate that though there is no direct binding interaction between P15 and α2β1 integrin, P15 enhance integrin signaling, thereby increasing the differentiation of both osteoblasts and chondrocytes.

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