Calcium phosphate powder synthesis by out-of-phase pulsed sonoelectrochemistry.

High aspect ratio calcium phosphate (CaP) nanorods were achieved by out-of-phase pulsed sonoelectrodeposition from electrolytic aqueous bath composed of calcium nitrate, ammonium dihydrogenophosphate and surfactant at pH of 4.9. The nature of CaP phases was determined by powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR) and energy dispersive X-ray spectroscopy (EDX). The results reveal the predominantly presence of calcium deficient hydroxyapatite (CDHA). The transmission electron microscopy (TEM) analyzes highlighted that the nanorods are polycristalline and have an aspect ratio up to 30.

Phosphate-dependent stimulation of MGP and OPN expression in osteoblasts via the ERK1/2 pathway is modulated by calcium.

Inorganic phosphate (Pi) acts as a signaling molecule in bone-forming cells, affecting cell functions and gene expression. Particularly, Pi stimulates the expression of mineralization-associated genes such as matrix gla protein (MGP) and osteopontin (OPN) through the ERK1/2 pathway. With respect to the presence of elevated extracellular calcium and Pi levels during bone remodeling, we questioned whether calcium might play a role in the Pi-dependent effects in osteoblasts. We first showed by Western blot and real-time PCR that the concomitant presence of 10 mM Pi and 1.8 mM calcium is required to stimulate ERK1/2 phosphorylation and MGP/OPN genes expression. The mechanisms involved in the cellular effects of calcium in the presence of Pi were subsequently examined. Firstly, the use of the calcium-sensing receptor (CaSR) agonist gadolinium and the G-protein inhibitor pertussis toxin enabled us to determine that a CaSR mechanism is not involved in the Pi and calcium mediated cellular effects. By transmission electron microscopy, we next demonstrated that adding 10mM Pi to the culture medium containing 1.8mM calcium led to the formation calcium phosphate precipitates (CaPp). Moreover, treatment of osteoblasts with exogenous pre-synthesized CaPp stimulated ERK1/2 phosphorylation and MGP/OPN genes expression. In spite of high extracellular calcium and Pi concentrations, this stimulation was blunted in the presence of phosphocitrate, an inhibitor of crystal formation. Finally, we showed that despite that CaPp are not endocytosed, their effect on ERK1/2 phosphorylation and MGP/OPN genes expression were dependent on lipid rafts integrity. In summary, we showed that calcium is required for Pi-dependent ERK1/2 phosphorylation and regulation of mineralization-associated genes in osteoblasts and that its effect could originate from extracellular-related effects of CaPp that are dependent on the integrity of lipid rafts.

Inorganic phosphate regulates Glvr-1 and -2 expression: role of calcium and ERK1/2.

Sodium-dependent phosphate cotransporters are key regulators of phosphate homeostasis and play a major role in mineralized tissues remodelling. However, factors influencing their expression remain under consideration. In our study, modulation of type III sodium-dependent phosphate cotransporters expression by inorganic phosphate (Pi) was investigated in the murine odontoblast-like cell line MO6-G3. Experiments were designed to determine the effects of phosphate release on dental cells during tooth decay. By real-time RT-PCR we demonstrated that Glvr-1 and -2 expressions are up-regulated by Pi. The increase in Glvr-1 and -2 expressions was correlated with ERK1/2 phosphorylation and calcium/phosphate crystals formation in cultured wells. Using calcium-free culture conditions or the specific inhibitor of ERK phosphorylation (UO126), we demonstrated that Pi effects on Glvr-1 and -2 up-regulation require the presence of calcium and involve ERK signalling pathways. This study contributes to give new insights in the control of Pi transport during carious diseases.

Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apoptosis in the regulation of endochondral ossification.

UNLABELLED: Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification. INTRODUCTION: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. MATERIALS AND METHODS: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-(EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis). RESULTS: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces pi-induced mineralization. CONCLUSIONS: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.