title: Premature osteoblast clustering by enamel matrix proteins induces osteoblast differentiation through up-regulation of connexin 43 and N-cadherin.
abstract: In recent years, enamel matrix derivative (EMD) has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43) and N-cadherin (N-cad) as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.
authors.affiliation: Department of Periodontology, School of Dental Medicine, University of Bern, Bern, Switzerland. richard.miron@zmk.unibe.ch
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Miron
authors.forename: Erik
authors.initials: E
authors.lastname: Hedbom
authors.forename: Sabrina
authors.initials: S
authors.lastname: Ruggiero
authors.forename: Dieter D
authors.initials: DD
authors.lastname: Bosshardt
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Zhang
authors.forename: Corinna
authors.initials: C
authors.lastname: Mauth
authors.forename: Anja C
authors.initials: AC
authors.lastname: Gemperli
authors.forename: Tateyuki
authors.initials: T
authors.lastname: Iizuka
authors.forename: Daniel
authors.initials: D
authors.lastname: Buser
authors.forename: Anton
authors.initials: A
authors.lastname: Sculean
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D015153:Blotting, Western; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002449:Cell Aggregation / Q000187:drug effects; D002450:Cell Communication / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000494:pharmacology; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D003746:Dental Enamel Proteins / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D058574:Integrin-Binding Sialoprotein / Q000235:genetics / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D015820:Cadherins; D020099:Coated Materials, Biocompatible; D018031:Connexin 43; D003746:Dental Enamel Proteins; D058574:Integrin-Binding Sialoprotein; C109069:enamel matrix proteins; D015675:Osteocalcin; D000469:Alkaline Phosphatase