title: Adsorption of enamel matrix proteins to a bovine-derived bone grafting material and its regulation of cell adhesion, proliferation, and differentiation.
abstract: BACKGROUND
  The use of various combinations of enamel matrix derivative (EMD) and grafting materials has been shown to promote periodontal wound healing/regeneration. However, the downstream cellular behavior of periodontal ligament (PDL) cells and osteoblasts has not yet been studied. Furthermore, it is unknown to what extent the bleeding during regenerative surgery may influence the adsorption of exogenous proteins to the surface of bone grafting materials and the subsequent cellular behavior. In the present study, the aim is to test EMD adsorption to the surface of natural bone mineral (NBM) particles in the presence of blood and determine the effect of EMD coating to NBM particles on downstream cellular pathways, such as adhesion, proliferation, and differentiation of primary human osteoblasts and PDL cells.
  
  
  METHODS
  NBM particles were precoated in various settings with EMD or human blood and analyzed for protein adsorption patterns via fluorescent imaging and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using fluorescent double-stranded DNA-binding dye. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding runt-related transcription factor 2, alkaline phosphatase (ALP), osteocalcin (OC), and collagen1α1 (COL1A1), and mineralization was assessed using red dye staining.
  
  
  RESULTS
  Analysis of cell attachment and cell proliferation revealed significantly higher osteoblast and PDL cell attachment on EMD-coated surfaces when compared with control and blood-coated surfaces. EMD also stimulated release of growth factors and cytokines, including bone morphogenetic protein 2 and transforming growth factor β1. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers, including COL1A1, ALP, and OC, in osteoblasts and PDL cells cultured on EMD-coated NBM particles.
  
  
  CONCLUSIONS
  The present results suggest that 1) EMD enhances osteoblast and PDL cell attachment, proliferation, and differentiation on NBM particles, and 2) blood contamination of the grafting material before mixing with EMD may inhibit EMD adsorption.
authors.affiliation: Department of Periodontology, School of Dental Medicine, University of Bern, Bern, Switzerland.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Miron
authors.forename: Dieter D
authors.initials: DD
authors.lastname: Bosshardt
authors.forename: Erik
authors.initials: E
authors.lastname: Hedbom
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Zhang
authors.forename: Beat
authors.initials: B
authors.lastname: Haenni
authors.forename: Daniel
authors.initials: D
authors.lastname: Buser
authors.forename: Anton
authors.initials: A
authors.lastname: Sculean
mesh_terms: D000327:Adsorption; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D000880:Anthraquinones; D001769:Blood; D055396:Bone Morphogenetic Protein 2 / Q000032:analysis; D018786:Bone Substitutes / Q000737:chemistry*; D002113:Calcification, Physiologic / Q000187:drug effects; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D020099:Coated Materials, Biocompatible / Q000737:chemistry* / Q000494:pharmacology; D024042:Collagen Type I / Q000032:analysis; D000090602:Collagen Type I, alpha 1 Chain; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D008855:Microscopy, Electron, Scanning; D008903:Minerals / Q000737:chemistry*; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology*; D015675:Osteocalcin / Q000032:analysis; D010513:Periodontal Ligament / Q000166:cytology* / Q000187:drug effects; D060888:Real-Time Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000032:analysis
chemical_list: D000880:Anthraquinones; C525718:BMP2 protein, human; C077540:Bio-Oss; D055396:Bone Morphogenetic Protein 2; D018786:Bone Substitutes; D020099:Coated Materials, Biocompatible; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit; D003746:Dental Enamel Proteins; D005456:Fluorescent Dyes; D008903:Minerals; C493709:RUNX2 protein, human; D053773:Transforming Growth Factor beta1; C109069:enamel matrix proteins; D015675:Osteocalcin; C010078:alizarin; D000469:Alkaline Phosphatase