title: Enamel matrix derivative: protein components and osteoinductive properties.
abstract: BACKGROUND
  Although enamel matrix derivative (EMD) has demonstrated the ability to promote angiogenesis and osteogenesis both in vitro and in vivo, the specific elements within the EMD compound responsible for these effects remain unknown.
  
  
  METHODS
  Nine different protein pools from a commercially produced EMD were collected based on molecular weight. Six of these pools, along with the complete EMD unfractionated compound and positive and negative controls, were tested for their ability to induce bone formation in a calvarial induction assay. Immunocytochemistry of phosphorylated SMAD1/5/8 (phospho-SMAD), osterix, and vascular endothelial growth factor A (VEGF-A) was carried out at selected time points. Finally, proteomic analysis was completed to determine the specific protein-peptide content of the various osteoinductive pools.
  
  
  RESULTS
  One of the lower-molecular-weight pools tested, pool 7, showed bone induction responses significantly greater than those of the other pools and the complete EMD compound and was concentration dependent. Dynamic bone formation rate analysis demonstrated that pool 7 was optimally active at the 5- to 10-μg concentration. It was demonstrated that EMD and pool 7 induced phospho-SMAD, osterix, and VEGF-A, which is indicative of increased bone morphogenetic protein (BMP) signaling. Proteomic composition analysis demonstrated that pool 7 had the highest concentration of the biologically active amelogenin-leucine-rich amelogenin peptide and ameloblastin 17-kDa peptides.
  
  
  CONCLUSIONS
  These studies demonstrate that the low-molecular-weight protein pools (7 to 17 kDa) within EMD have greater osteoinductive potential than the commercially available complete EMD compound and that the mechanism of action, in part, is through increased BMP signaling and increased osterix and VEGF-A. With this information, selected components of EMD can now be formulated for optimal osteo- and angio-genesis.
authors.affiliation: Department of Periodontics, Wilford Hall Medical Center, Lackland Air Force Base, San Antonio, TX.
authors.forename: Bradshaw M
authors.initials: BM
authors.lastname: Stout
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Alent
authors.forename: Peter
authors.initials: P
authors.lastname: Pedalino
authors.forename: Ryan
authors.initials: R
authors.lastname: Holbrook
authors.forename: Jelica
authors.initials: J
authors.lastname: Gluhak-Heinrich
authors.forename: Yong
authors.initials: Y
authors.lastname: Cui
authors.forename: Marie A
authors.initials: MA
authors.lastname: Harris
authors.forename: Anja C
authors.initials: AC
authors.lastname: Gemperli
authors.forename: David L
authors.initials: DL
authors.lastname: Cochran
authors.forename: David E
authors.initials: DE
authors.lastname: Deas
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Harris
mesh_terms: D053523:Amelogenin / Q000032:analysis; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003746:Dental Enamel Proteins / Q000032:analysis* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D051379:Mice; D023421:Models, Animal; D008970:Molecular Weight; D010012:Osteogenesis / Q000187:drug effects; D010294:Parietal Bone / Q000187:drug effects; D010521:Periosteum / Q000187:drug effects; D020543:Proteome / Q000032:analysis; D051898:Smad1 Protein / Q000032:analysis / Q000494:pharmacology; D051902:Smad5 Protein / Q000032:analysis / Q000494:pharmacology; D051904:Smad8 Protein / Q000032:analysis / Q000494:pharmacology; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000032:analysis / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000494:pharmacology
chemical_list: C490890:Ambn protein, mouse; D053523:Amelogenin; D019485:Bone Morphogenetic Proteins; D003746:Dental Enamel Proteins; D020543:Proteome; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D051902:Smad5 Protein; C497798:Smad5 protein, mouse; D051904:Smad8 Protein; C491621:Smad9 protein, mouse; D000076182:Sp7 Transcription Factor; C479466:Sp7 protein, mouse; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C109069:enamel matrix proteins; C067432:leucine-rich amelogenin peptide; C467480:vascular endothelial growth factor A, mouse