An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation

To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca2+](i)) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca2+](i) signal can be propagated to the neighboring cells. No [Ca2+](i) response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks. Published by Elsevier Ltd.

Intercellular calcium wave propagation in linear and circuit-like bone cell networks

In the present study, the mechanism of intercellular calcium wave propagation in bone cell networks was identified. By using micro-contact printing and self-assembled monolayer technologies, two types of in vitro bone cell networks were constructed: open-ended linear chains and looped hexagonal networks with precisely controlled intercellular distances. Intracellular calcium responses of the cells were recorded and analysed when a single cell in the network was mechanically stimulated by nano-indentation. The looped cell network was shown to be more efficient than the linear pattern in transferring calcium signals from cell to cell. This phenomenon was further examined by pathway-inhibition studies. Intercellular calcium wave propagation was significantly impeded when extracellular adenosine triphosphate (ATP) in the medium was hydrolysed. Chemical uncoupling of gap junctions, however, did not significantly decrease the transferred distance of the calcium wave in the cell networks. Thus, it is extracellular ATP diffusion, rather than molecular transport through gap junctions, that dominantly mediates the transmission of mechanically elicited intercellular calcium waves in bone cells. The inhibition studies also demonstrated that the mechanical stimulation-induced calcium responses required extracellular calcium influx, whereas the ATP-elicited calcium wave relied on calcium release from the calcium store of the endoplasmic reticulum.

Preparation of uniform calcium alginate gel beads by membrane emulsification coupled with internal gelation

With the objective of making calcium alginate gel beads with small and uniform size, membrane emulsification coupled with internal gelation was proposed. Spherical gel beads with mean size of about 50 mum, and even smaller ones in water, and with narrow size distribution were successfully obtained. Experimental studies focusing mainly on the effect of process parameters on bead properties were performed. The size of the beads was mainly dependent on the diameter of the membrane pores. High transmembrane pressure made for large gel beads with wide size distribution. Low sodium alginate concentration produced nonspherical beads, whereas a high concentration was unsuitable for the production of small beads with narrow distribution. Thus 1.5% w/v was enough. A high surfactant concentration favored the formation of small beads, but the adverse effect on mass transfer should be considered in this novel process. (C) 2002 Wiley Periodicals, Inc.

Characterization of structure and diffusion behaviour of Ca-alginate beads prepared with external or internal calcium sources

Ca-alginate beads were prepared with either external or internal calcium sources. The structures of both beads were investigated with the aid of scanning electron microscopy (SEM) and confocal microscopy. It was shown that the beads with internal calcium source had a looser structure and bigger pore size than those with external calcium source. The attempts to interpret the difference were carried out by determining the Ca content within the beads at various times, which indicated that it was the different gelation mechanisms that caused the difference of structures of both beads. Furthermore, it was also found that the diffusion rate of haemoglobin (Hb) within the beads with an internal calcium source was faster than that of the beads with an external one, which was consistent with the observation of their structures.

Specific Adsorption of Trace Amounts of Calcium and Strontium by Hydrous Oxides of Iron and Aluminum1

Freshly prepared Fe and Al hydrous oxide gels and the amorphous product of heating gibbsite selectively adsorbed traces of Ca and Sr from solutions containing a large excess (∼1M) of NaNO3. The fraction of the added Ca (Sr) adsorbed depended principally on the suspension pH, the amount of solid present, and to a lesser extent on the NaNO3 concentration. Significant Ca and Sr adsorption occurred on the Fe and Al gels, and heated gibbsite, at pH values below the points of zero charge (8.1, 9.4, and 8.3±0.1, respectively), indicating specific adsorption. The pH (± 0.10) at which 50% of the Ca was adsorbed (pH50) occurred at pH 7.15 for the Fe gel (0.093M Fe), 8.35 for the Al gel (0.093M Al), and 6.70 for the heated gibbsite (0.181M Al); for Sr, the pH50 values were 7.10, 9.00, and 6.45, respectively. For the Fe gel and heated gibbsite, an empirical model based on the law of mass action described the pH dependence of adsorption reasonably well and suggested that for each Ca or Sr fraction adsorbed, approximately one proton was released. Failure of the Al gel to fit this model may have resulted from its rapid aging.

Biomimetic crystallization of unusual macroporous calcium carbonate spherules in the presence of phosphatidylglycerol vesicles

We report the interesting finding that crystallization of calcium carbonate (CaCO3) in the presence of dimyristoylphosphatidylglycerol (DMPG) vesicles by a simple gas diffusion method results in the formation of unusual microscopic CaCO3 spherules. The experimental results indicate that the as-prepared CaCO3 spherules, which have a complex macroporous structure, are predominantly vaterite. It is believed that DMPG vesicles play an important role in the process of crystallization, and the possible formation mechanism is proposed.

Direct electrochemistry of horseradish peroxidase immobilized in calcium carbonate microsphere doped with phospholipids

Protein electrochemistry affords a direct method to study the biological electron transfer processes. However, supplying a biocompatible environment to maintain the native state of protein is all-important and challengeable. Here, we chose vaterite, one of the crystalline polymorphs of calcium carbonate, with highly porous nature and large specific surface area, which was doped with phospholipids, as the matrix to immobilize horseradish peroxidase (HRP). The integrity of HRP was kept during the simple immobilization procedure. By virtue of this organic/inorganic complex matrix, the direct electrochemistry of HRP was realized, and the activity of HRP for catalyzing reduction of O-2 and H2O2 was preserved.

Use of atomic force microscopy for imaging the initial stage of the nucleation of calcium phosphate in Langmuir-Blodgett films of stearic acid

The nucleation of calcium phosphate on the substrate of steatic acid Langmuir-blodgett film at the initial stage was investigated by atomic force microscopy. Nano-dots, nano-wires and nano-islands were observed in sequence for the first time, reflecting the nucleation of calcium phosphate and the molecular arrangement of carboxylic layer. The nucleation rates perpendicular and parallel to the carboxylic terminal group were estimated from the height and diameter of the calcium phosphate crystals, respectively. And this stage was distinct from the late explosive grown stage, in which the change of the morphology was not obvious. The approaches based on this discovery would lead to the development of new strategies in the controlled synthesis of inorganic nano-phases and the assembly of organized composite and ceramic materials.