P15 peptide stimulates chondrogenic commitment and endochondral ossification

Int Orthop. 2017 Jul;41(7):1413-1422. doi: 10.1007/s00264-017-3464-8. Epub 2017 Apr 8.

Abstract

Purpose: The synthetic 15 amino acid biomimetic peptide sequence (P15) derived from a region of the alpha (α)-1 chain of collagen I, has been shown to promote α2 integrin activation and enhance intramembranous ossification. In this study, we ask if the P15 peptide also enhances bone formation through endochondral ossification, and determine if direct binding of α2 integrin with P15 mediates integrin activation.

Methods: Mesenchymal cells (C3H10T1/2) were cultured in chondrogenic media and the expression of chondrogenic markers and integrin activation was determined by Western blot and fluorescent immunohistochemistry. A biosensor assay was used to determine if binding occurred between P15 and α2 β1 integrin. Finally, an in vivo model of endochondral ossification was used to determine the effect of P15 on bone formation.

Results: In the presence of P15, chondrogenesis and activation of α5 integrin were enhanced, as observed by both Western blot analysis and immunoflourescent staining. A biosensor assay investigating the specificity of the interaction between P15 with α2β1 integrin determined direct binding does not occur. When P15 was added to Matrigel implanted in a murine endochondral ossification model, in the presence of bone morphogenic protein-2 (BMP-2), a significant increase in chondrocyte differentiation and mineralization was observed.

Conclusion: P15 does not directly activate integrins by binding, but does upregulate integrin signaling to enhance differentiation of both osteoblasts and chondrocytes to increase both intramembranous and endochondral bone formation.

Keywords: Bone density; Chondrogenesis; Collagen; Endochondral ossification; Integrin; MicroCT analysis; P15 peptide.

MeSH terms

  • Animals
  • Biosensing Techniques
  • Blotting, Western
  • Bone Morphogenetic Protein 2 / metabolism
  • Cell Culture Techniques
  • Cell Differentiation / drug effects
  • Chondrocytes / drug effects*
  • Chondrocytes / metabolism
  • Chondrogenesis / drug effects*
  • Collagen / pharmacology*
  • Drug Combinations
  • Fluorescent Antibody Technique
  • Integrins / metabolism*
  • Laminin
  • Mesenchymal Stem Cells / cytology
  • Mice
  • Osteoblasts / drug effects
  • Osteogenesis / drug effects*
  • Peptide Fragments / pharmacology*
  • Proteoglycans
  • Signal Transduction

Substances

  • BMP2 protein, human
  • Bone Morphogenetic Protein 2
  • Drug Combinations
  • Integrins
  • Laminin
  • Peptide Fragments
  • Proteoglycans
  • cell-binding peptide P-15
  • matrigel
  • Collagen