title: Inhibition of osteoblast mineralization by phosphorylated phage-derived apatite-specific peptide.
abstract: Functionalization of biomaterials with material- and cell-specific peptide sequences allows for better control of their surface properties and communication with the surrounding environment. Using a combinatorial phage display approach, we previously identified the peptide VTKHLNQISQSY (VTK) with specific affinity to biomimetic apatite. Phosphorylation of the serine residues of the peptide (pVTK) caused a significant increase in binding to apatite, as well as a dose-dependent inhibition of osteoblast mineralization. In this study, we investigated the mechanisms behind pVTK mediated inhibition of mineralization using MC3T3 cells and testing the hypothesis that mineralization is inhibited via alteration of the Enpp1-TNAP-Ank axis. Inhibition of mineralization was not due to disruption of collagen deposition or calcium chelation by the negatively charged pVTK. The timing of peptide administration was important in inhibiting mineralization - pVTK had a greater effect at later stages of osteogenic differentiation (days 7-12 of culture corresponding to matrix maturation and mineralization), and could prevent progression of mineralization once it had started. pVTK treatment resulted in a significant decrease in ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) enzyme activity and gene expression. The expression of ankylosis protein (Ank), osteopontin (OPN) and Pit-1 genes was also significantly reduced with peptide treatment, while tissue non-specific alkaline phosphatase (TNAP), bone sialoprotein (BSP), and Runx2 gene expression was significantly higher. The ability of pVTK to inhibit mineralization can potentially be translated into therapeutics against pathological calcification seen in cardiovascular disease, osteoarthritis or craniosynostosis, or be used to prevent failure of biomaterials due to calcification, such as bioprosthetic heart valves.
authors.affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. Electronic address: jananir@umich.edu.
authors.forename: Janani
authors.initials: J
authors.lastname: Ramaswamy
authors.affiliation: Department of Orthodontics and Pediatric Dentistry, University of Michigan, Ann Arbor, MI, USA. Electronic address: hknam@umich.edu.
authors.forename: Hwa Kyung
authors.initials: HK
authors.lastname: Nam
authors.affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. Electronic address: sramara@umich.edu.
authors.forename: Harsha
authors.initials: H
authors.lastname: Ramaraju
authors.affiliation: Department of Orthodontics and Pediatric Dentistry, University of Michigan, Ann Arbor, MI, USA. Electronic address: nhatch@umich.edu.
authors.forename: Nan E
authors.initials: NE
authors.lastname: Hatch
authors.affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; Department of Biologic & Materials Sciences, University of Michigan, Ann Arbor, MI, USA. Electronic address: dhkohn@umich.edu.
authors.forename: David H
authors.initials: DH
authors.lastname: Kohn
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001031:Apatites / Q000737:chemistry*; D001672:Biocompatible Materials / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002454:Cell Differentiation; D002614:Chelating Agents / Q000737:chemistry; D003094:Collagen / Q000737:chemistry; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D058574:Integrin-Binding Sialoprotein / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D010012:Osteogenesis; D053495:Osteopontin / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D028061:Phosphate Transport Proteins / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011755:Pyrophosphatases / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013499:Surface Properties; D050817:Transcription Factor Pit-1 / Q000378:metabolism
chemical_list: D001031:Apatites; D001672:Biocompatible Materials; D002118:Calcium; D002614:Chelating Agents; D003094:Collagen; D050659:Core Binding Factor Alpha 1 Subunit; D058574:Integrin-Binding Sialoprotein; D053495:Osteopontin; D019151:Peptide Library; D010455:Peptides; D028061:Phosphate Transport Proteins; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D012694:Serine; D050817:Transcription Factor Pit-1; C494185:Pou1f1 protein, mouse; C493716:Runx2 protein, mouse; C506896:Spp1 protein, mouse; C410982:ank protein, mouse; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1
keywords: Biomineralisation; Osteoblast; Pathologic calcification; Peptide; Phage display