Premature Osteoblast Clustering by Enamel Matrix Proteins Induces Osteoblast Differentiation through Up-Regulation of Connexin 43 and N-Cadherin

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

Tracking Endogenous Amelogenin and Ameloblastin In Vivo

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling

Ameloblastin Inhibits Cranial Suture Closure by Modulating Msx2 Expression and Proliferation

Deformities of cranial sutures such as craniosynostosis and enlarged parietal foramina greatly impact human development and quality of life. Here we have examined the role of the extracellular matrix protein ameloblastin (Ambn), a recent addition to the family of non-collagenous extracellular bone matrix proteins, in craniofacial bone development and suture formation. Using RT-PCR, western blot and immunohistochemistry, Ambn was localized in mouse calvarial bone and adjacent condensed mesenchyme. Five-fold Ambn overexpression in a K14-driven transgenic mouse model resulted in delayed posterior frontal suture fusion and incomplete suture closure. Moreover, Ambn overexpressor skulls weighed 13.2% less, their interfrontal bones were 35.3% thinner, and the width between frontal bones plus interfrontal suture was 14.3% wider. Ambn overexpressing mice also featured reduced cell proliferation in suture blastemas and in mesenchymal cells from posterior frontal sutures. There was a more than 2-fold reduction of Msx2 in Ambn overexpressing calvariae and suture mesenchymal cells, and this effect was inversely proportionate to the level of Ambn overexpression in different cell lines. The reduction of Msx2 expression as a result of Ambn overexpression was further enhanced in the presence of the MEK/ERK pathway inhibitor O126. Finally, Ambn overexpression significantly reduced Msx2 down-stream target gene expression levels, including osteogenic transcription factors Runx2 and Osx, the bone matrix proteins Ibsp, ColI, Ocn and Opn, and the cell cycle-related gene CcnD1. Together, these data suggest that Ambn plays a crucial role in the regulation of cranial bone growth and suture closure via Msx 2 suppression and proliferation inhibition