Jun Zhang1,2, Peter Eisenhauer3, Ozҫan Kaya4, Alexander R Vaccaro1,3, Carol Diallo1, Andrzej Fertala1, Theresa A Freeman5. 1. Sidney Kimmel Medical College Department of Orthopaedic Surgery, Thomas Jefferson University, 501 Curtis Bldg., 1015 Walnut St., Philadelphia, PA, 19107, USA. 2. Orthopaedic Medical Center, 2nd Hospital of Jilin University, 218 Ziqiang Street, Changchun, 130041, People's Republic of China. 3. Rothman Institute at Jefferson, 925 Chestnut Street, Philadelphia, PA, 19107, USA. 4. Department of Orthopedics and Traumatology, Istanbul University, Istanbul, 34093, Turkey. 5. Sidney Kimmel Medical College Department of Orthopaedic Surgery, Thomas Jefferson University, 501 Curtis Bldg., 1015 Walnut St., Philadelphia, PA, 19107, USA. Theresa.freeman@jefferson.edu.
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.
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.
Authors: Kristin M Hennessy; Beth E Pollot; William C Clem; Matthew C Phipps; Amber A Sawyer; Bonnie K Culpepper; Susan L Bellis Journal: Biomaterials Date: 2009-01-20 Impact factor: 12.479
Authors: G J Eaton; Q-S Zhang; C Diallo; A Matsuzawa; H Ichijo; M J Steinbeck; T A Freeman Journal: Cell Death Dis Date: 2014-11-13 Impact factor: 8.469