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

Rho and rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation

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Gap junction-mediated cell-to-cell communication in bovine and human adrenal cells. A process whereby cells increase their responsiveness to physiological corticotropin concentrations.

We have studied the role of gap junction-mediated intercellular communication on the steroidogenic response of bovine (BAC) and human (HAC) adrenal fasciculo-reticularis cells in culture to corticotropin (ACTH). Indirect immunofluorescence analyses showed that intact human and bovine adreno-cortical tissue as well as HAC and BAC in culture expressed the gap junction protein connexin43 (also termed alpha 1 connexin). Both HAC and BAC were functionally coupled through gap junctions as demonstrated by microinjection of a low molecular mass fluorescent probe, Lucifer yellow. The cell-to-cell transfer of the probe was blocked by 18 alpha-glycyrrhetinic acid (GA), an inhibitor of gap junction-mediated intercellular communication. GA markedly decreased the steroidogenic response (cortisol production) of both HAC and BAC to low (10 pM) but not to high (5 nM) concentrations of ACTH. GA had no inhibitory effect on the steroidogenic response to 8 Br-cAMP (at either low or high concentrations) and did neither modify the binding of 125I-ACTH to its receptor nor the ACTH-induced cAMP production. BAC cultured at high or low cell densities (2.4 x 10(5) vs. 0.24 x 10(5) cells/cm2) exhibited distinct levels of intercellular communication and were differently responsive to sub-maximal ACTH concentrations. The ACTH ED50 values for cortisol production were 8.5 +/- 1.3 and 45 +/- 14 pM (P < 0.02) for BAC cultured at high and low density, respectively. In the presence of GA, there was a shift of the ACTH concentration-response curves in the two culture conditions. The ACTH ED50 of high density and low density cultured BAC increased 25- and 5-fold, respectively, and became similar (220 +/- 90 and 250 +/- 120 pM). These results demonstrate that gap junction-mediated communication between hormone-responsive and nonresponsive cells is one mechanism by which adrenal cells increase their responsiveness to low ACTH concentrations