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.
Highlights
Bone remodeling is a complex lifelong process that requires precise control of bone-resorbing osteoclasts and bone-forming osteoblasts for the maintenance of aging bone and repair of bone injuries
Primary osteoblasts attached well on both surfaces at 4 hours (Fig. 1A–B), cells seeded on samples pre-coated with enamel matrix derivative (EMD) began to form cell clusters after 24 hours (Fig. 1D) when compared to control (Fig. 1C)
Following cell clustering on EMD-coated samples, cells were immunolabeled for expression of cx43 and N-cad
Summary
Bone remodeling is a complex lifelong process that requires precise control of bone-resorbing osteoclasts and bone-forming osteoblasts for the maintenance of aging bone and repair of bone injuries. Osteoblasts are at the center of these processes by controlling matrix production and mineralization, receiving and processing mechanical and chemical signals to bone and most likely directing osteoclast function. This level of coordination demands sophisticated cell communication. Adherens junctions are intercellular structures that are formed through hemophilic, calcium-dependent cell-cell adhesion via cadherins. These molecules constitue a class of 30 single chain integral membrane glycoproteins composed of a long N-terminal extracellular domain, a single transmembrane domain, and a small intracellular C-terminal tail [3]. Cadherins play essential roles in fetal development of mesenchymal tissues including morphogenesis, osteogenesis and chondrocyte condensation [3,6,7]
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