Plasma Membrane Ca2+-ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization.

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+-Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1-4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4-8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Potassium Currents Activated by Depolarization in Odontoblasts.

Increased intracellular free Ca2+ concentrations elicit plasma membrane depolarization, which leads to the activation of K+ currents. However, the precise properties of K+ currents activated by depolarization in odontoblasts remain to be elucidated. The present study identified biophysical and pharmacological characteristics of time-dependent and voltage-activated K+ currents in freshly dissociated rat odontoblasts using patch-clamp recordings in a whole-cell configuration. Using a holding potential of -70 mV, outwardly rectifying time- and voltage-dependent currents were activated by depolarizing voltage. To record pure K+ conductance, we substituted Cl- in both the extracellular and intracellular solutions with gluconate-. Under these conditions, observation of K+ concentration changes in the extracellular solution showed that reversal potentials of tail currents shifted according to the K+ equilibrium potential. The activation kinetics of outward K+ currents were relatively slow and depended on the membrane potential. Kinetics of steady-state inactivation were fitted by a Boltzmann function. The half-maximal inactivation potential was -38 mV. Tetraethylammonium chloride, 4-aminopyridine, and α-dendrotoxin inhibited outward currents in odontoblasts in a concentration-dependent manner, suggesting that rat odontoblasts express the α-subunit of the time- and voltage-dependent K+ channel (Kv) subtypes Kv1.1, 1.2, and/or 1.6. We further examined the effects of Kv activity on mineralization by alizarin red and von Kossa staining. Continuous application of tetraethylammonium chloride to human odontoblasts grown in a mineralization medium over a 21-day period exhibited a dose-dependent decrease in mineralization efficiency compared to cells without tetraethylammonium chloride. This suggests that odontoblasts functionally express voltage-dependent K+ channels that play important roles in dentin formation.

Encapsulation of Either Hydrophilic or Hydrophobic Substances in Spongy Cellulose Particles.

We have reported cellulose particles with a spongy structure that we prepared by the solvent releasing method (SRM) from cellulose droplets composed of cellulose, 1-butyl-3-methylimidazoliumchrolide ([Bmim]Cl), and N,N-dimethylformamide (DMF). The spongy structure collapsed as the medium evaporated, resulting in dense cellulose particles. In this study, we encapsulated the hydrophilic and hydrophobic fluorescent substances in these particles to investigate the use of such particles in potential applications that require encapsulating of substances (e.g., drug delivery). Wet cellulose particles retained their spongy structure in both hydrophobic and hydrophilic media. When the spongy cellulose particles were dispersed in a solution containing nonvolatile solutes, these solutes were driven into the cellulose particles as media evaporated. Subsequently, the cellulose particles collapsed and encapsulated the nonvolatile solutes. Regardless of whether the solute was hydrophilic or hydrophobic, the encapsulation efficiency exceeds 80%. The maximum loading reflected the saturated solubility of solute in solution that filled the cellulose beads. Moreover, the encapsulated solute was released by dispersing the cellulose beads in the solvent, and the rate of release of the encapsulated solute could be controlled by coating the cellulose beads with a conventional polymer.

Preparation of cellulose particles using an ionic liquid.

Cellulose is a ubiquitous natural fiber used in various industrial materials and applications. We prepared micron-sized cellulose particles by the solvent releasing method (SRM) in which cellulose-[Bmim]Cl-N,N-dimethylformamide (DMF) droplets are dispersed in hexadecane (HD) containing dissolved surfactant. The dispersion is then poured into a large amount of 1-butanol. Since 1-butanol is miscible with HD, [Bmim]Cl, and DMF but not with cellulose, the cellulose particles precipitate out. FTIR and (1)H NMR analyses confirmed that this technique precipitated cellulose and completely removed [Bmim]Cl and DMF from the cellulose-[Bmim]Cl-DMF droplets. Interestingly, the obtained cellulose particles were almost the same size as the original droplets (cellulose, 7 wt%), indicating a microporous structure of the cellulose particles with a large medium content. Although the microporous structure collapsed as the medium evaporated, it was maintained by a freeze-drying technique.