Effect of phosphoproteins on intracellular calcification of bacteria.

This study aimed to evaluate the effects of phosphoproteins on bacterial mineralization. Dental calculus formation is attributed to bacterial mineralization in the oral cavity; however, the influence of phosphoproteins (which are abundant in saliva) is not clear. The model bacterium Escherichia coli was suspended in a calcification solution containing casein as a model phosphoprotein. To evaluate mineralization independent of bacterial metabolism, bacteria killed by heat treatment at 70°C were compared with viable bacteria. After incubation at 37°C for 24 h, the mode of calcification was observed using electron microscopy and energy dispersive x-ray spectroscopy. Solutions without casein produced precipitation in solution, which was identical to that in experiments without bacteria. In contrast, calcification solutions with 200 ppm casein only produced calcium phosphate deposition intracellularly. Without heat treatment, intracellular calcification rarely occurred, even when casein was added. Thus, phosphoproteins promoted intracellular calcification of dead bacteria; this is similar to the calcification of insoluble matrices, such as collagen fibrils, promoted by acidic polymers. We concluded that intracellular calcification is caused by the collagen fibril-like behavior of dead bacteria. The promotion of intracellular calcification of dead bacteria by phosphoproteins suggested a basic principle of dental calculus formation.

Microstructure and mineral components of the outer dentin of Chimaera phantasma tooth plates.

Tooth plates are a unique dental organ found in holocephalan fishes and lungfish. The chimaeroid tooth plates are atypical in terms of biomineralization, due to the hard tissue composition of whitlockite and apatite, while those of lungfish and other vertebrates are composed of apatite. The tooth plates are overlaid by a thin veneer-outer dentin-whose composition and role are not known. We aimed to test whether the outer dentin is composed of whitlockite or apatite, and whether it protects the osteodentin from abrasion and supports its overall strength. For this purpose, the mineral components and microstructure of outer dentin were studied. Our analyses of the outer dentin from the anterior (vomerine) tooth plates of Chimaera phantasma revealed that the mineral component is magnesium- and carbonate-containing calcium-deficient apatite and that the outer dentin has a three-zone structure. The main body is sandwiched between thin zones, which are less mineralized than the main body. Furthermore, in the outer zone and the main body, a higher-order structure was formed in accordance with the organization of wide and narrow fibers. Mineralization made the main body a composite of bundles of fibers and apatite. Transmission electron microscopy showed a structural relationship between apatite and the fibrous component on which the apatite was formed. Such a structure of the main body could be highly effective as a framework to resist abrasion and support the overall strength of the tooth plate.

Coherent surface structure induces unique epitaxial overgrowth of metastable octacalcium phosphate on stable hydroxyapatite at critical fluoride concentration.

The phase transformation from soluble calcium phosphates to less-soluble hydroxyapatite (HAP) is a thermodynamically natural route. This process is irreversible, and effective use of poorly reactive HAP to repair teeth that have no cellular metabolism remains challenging. However, this thermodynamically controlled transformation may apparently be reversed through the fast nucleation and growth of metastable phases, leading to a reactive HAP surface. Here, the assembled HAP-nanorod phase is demonstrated to change into the metastable octacalcium phosphate (OCP) phase in a calcium phosphate solution containing 0.8 ppm fluoride. Grown OCPs display parallel surface streaks and their 11¯0 and 00l (l: odd) electron-diffraction spots are often not visible. The streaked, elongated OCP gradually grows into large plates with flat surfaces that exhibit an intense11¯0 spot. Crystal-structure models reveal that the unique epitaxial overgrowth of OCP on HAP occurs since both materials share coherent {100} faces, resulting in the distinctive disappearance of 11¯0 and 00l OCP spots. A polysynthetic twin model that reliably explains this disappearance is proposed for the growth of OCP. This apparent reverse phase transformation produces hybrid calcium phosphates consisting of HAP cores and highly reactive outer OCP layers that are promising for the repair of dentin caries. STATEMENT OF SIGNIFICANCE: This paper demonstrates important and interesting finding regarding formation of calcium phosphates in relation to their crystal structures. We first show that hydroxyapatite (HAP), the major constituent of human teeth and bone, can reversely change to its precursor, octacalcium phosphate (OCP), contrary to thermodynamic-stability rule. This apparent reverse phase transformation occurs through sharing the coherent {100} faces of both materials under controlled fluoride concentration. Nanoscale similarity of two crystal surfaces enables structurally shared epitaxial overgrowth of OCP on HAP aided by faster growth rate of OCP than that of HAP. This reaction produces hybrid crystal consisting of outer OCP and core HAP, that has not been known before and is able to be applied to dentin caries repair.

A unique mineralization mode of hypermineralized pleromin in the tooth plate of Chimaera phantasma contributes to its microhardness.

Tooth plates of the chimaeroids, holocephalian fishes, are unique dental hard tissues. Unlike the teeth of other animals, the tooth plates are located on the roof of the mouth and in the lower jaw. Their tooth plates consist, to a large extent, of lightly mineralized tissue (osteodentin) and hypermineralized tissue (pleromin). Notably, the mineral phase of pleromin is whitlockite, while that of other animals is apatite. Dietary habits of chimaeroids and wearing features of their tooth plates suggest an extreme hardness of pleromin, but this has never been investigated. We examined the microhardness of the tooth plate of Chimaera phantasma and found that pleromin in the biting region was extremely hard, comparable with the hardness of mature tooth enamel, whereas the hardness of immature pleromin was lower than that of bovine dentin. The hardness of osteodentin, on the other hand, was equivalent to that of bovine dentin and almost the same throughout the tooth plate. Immature pleromin was sparsely packed with oval crystals of whitlockite and, as pleromin matures, the space between crystals was filled with small intercrystalline materials. The maturing process of pleromin could partly contribute to its remarkable hardness and have some implications for designing novel biomaterials.

Artificial enamel induced by phase transformation of amorphous nanoparticles.

Human tooth enamel has tightly packed c-axis-oriented hydroxyapatite (HAP: Ca10(PO4)6(OH)2) nanorods with high elastic modulus. Fabrication of an enamel architecture in vitro supports the repair of teeth using HAP; however, existing methods require complex and laborious steps to form an enamel-like structure. Here we present a very simple and effective technique for forming artificial enamel in near-physiological solution using a substrate composed of amorphous calcium phosphate (ACP) nanoparticles. Without any functionalized modification of the substrate surface, faint dissolution and successive phase transformation automatically induce formation of an intermediate layer of low-crystalline HAP nanoparticles, on which highly oriented HAP nanorods grow by geometrical selection. We also show that an enamel structure forms on a substrate of amorphous calcium carbonate when the surface nanoparticles react so as to form an intermediate layer similar to that in ACP. Our results demonstrate that there is a wide range of substrate choices for nanorod array formation. Contrary to current understanding, a stable surface designed in nanoscale is not essential for the growth of arranged guest crystals. Reactive amorphous nanoparticles and their transformation efficiently induce a nanorod array structure.

The cooperation of enamelin and amelogenin in controlling octacalcium phosphate crystal morphology.

Enamel matrix proteins, including the most abundant amelogenin and lesser amounts of enamelin, ameloblastin, and proteinases, play vital roles in controlling crystal nucleation and growth during enamel formation. The cooperative action between amelogenin and the 32-kDa enamelin is critical to regulating the growth morphology of octacalcium phosphate crystals. Using biophysical methods, we investigated the interaction between the 32-kDa enamelin and recombinant pig amelogenin 148 (rP148) at pH 6.5 in phosphate-buffered saline (PBS). Dynamic light scattering results showed a trend of increasing particle size in the mixture with the addition of enamelin to amelogenin. Upon addition of the 32-kDa enamelin, the shift and intensity decrease in the ellipticity minima of rP148 in the circular dichroism spectra of rP148 illustrated a direct interaction between the 2 proteins. In the fluorescence spectra, the maximum emission of rP148 was blue shifted from 335 to 333 nm in the presence of enamelin as a result of complexation of the 2 proteins. Our results demonstrate that the 32-kDa enamelin has a close association with amelogenin at pH 6.5 in PBS buffer. Our present study provides novel insights into the possible cooperation between enamelin and amelogenin in macromolecular coassembly and in controlling enamel mineral formation.

Tooth enamel proteins enamelin and amelogenin cooperate to regulate the growth morphology of octacalcium phosphate crystals.

To examine the hypothetical cooperative role of enamelin and amelogenin in controlling the growth morphology of enamel crystals in the post-secretory stage, we applied a cation selective membrane system for the growth of octacalcium phosphate (OCP) in the truncated recombinant porcine amelogenin (rP148) with and without the 32kDa enamelin fragment. Enamelin alone inhibited the growth in the c-axis direction more than rP148, yielding OCP crystals with the smallest aspect ratio of all conditions tested. When enamelin was added to the amelogenin "gel-like matrix", the inhibitory action of the protein mixture on the growth of OCP in the c-axis direction was diminished, while that in the b-axis direction was increased. As a result, the length to width ratio (aspect ratio) of OCP crystal was markedly increased. Addition of enamelin to amelogenin enhanced the potential of amelogenin to stabilize the amorphous calcium phosphate (ACP) transient phase. The ratio of enamelin and amelogenin was crucial for stabilization of ACP and the growth of OCP crystals with larger aspect ratio. The cooperative regulatory action of enamelin and amelogenin was attained, presumably, through co-assembling of enamelin and amelogenin. These results have important implications in understanding the growth mechanism of enamel crystals with large aspect ratio.

Direct transformation from amorphous to crystalline calcium phosphate facilitated by motif-programmed artificial proteins.

An animal's hard tissue is mainly composed of crystalline calcium phosphate. In vitro, small changes in the reaction conditions affect the species of calcium phosphate formed, whereas, in vivo, distinct types of crystalline calcium phosphate are formed in a well-controlled spatiotemporal-dependent manner. A variety of proteins are involved in hard-tissue formation; however, the mechanisms by which they regulate crystal growth are not yet fully understood. Clarification of these mechanisms will not only lead to the development of new therapeutic regimens but will also provide guidance for the application of biomineralization in bionanotechnology. Here, we focused on the peptide motifs present in dentin matrix protein 1 (DMP1), which was previously shown to enhance hydroxylapatite (HAP) formation when immobilized on a glass substrate. We synthesized a set of artificial proteins composed of combinatorial arrangements of these motifs and successfully obtained clones that accelerated formation of HAP without immobilization. Time-resolved static light-scattering analyses revealed that, in the presence of the protein, amorphous calcium phosphate (ACP) particles increased their fractal dimension and molecular mass without increasing their gyration radii during a short period before precipitation. The protein thus facilitated reorganization of the internal structure of amorphous particles into ordered crystalline states, i.e., the direct transformation of ACP to HAP, thereby acting as a nucleus for precipitation of crystalline calcium phosphate. Without the protein, the fractal dimension, molecular mass, and gyration radii of ACP particles increased concurrently, indicating heterogeneous growth transformation.

The development of carbonate-containing apatite/collagen composite for osteoconductive apical barrier material.

The current report describes the properties of a new apical barrier material formulated from carbonate-containing apatite (CAp) and collagen. CAp particles of around 50 nm were deposited on reconstituted collagen fibers. CAp/col with about 60 wt % CAp (corresponding to apatite content of bone) was obtained after 1 day of calcification. CAp content increased up to about 80 wt % in a 15-day calcification reaction. CAp/col was composed of fine calcified collagen fibers. The crystallinity and Ca/PO(4) ratio of CAp were comparable to those of bone apatite. The mixture of CAp/col and saline reached a pH of about 9. The optimum powder-to-liquid ratio (P/L) to set into a root canal was determined to be 1.2. Furthermore, the mixture (P/L = 1.2) condensed in a root canal was liquid permeable. Thus, the CAp/col was expected as an apical barrier material with osteoconductivity.

Control of apatite crystal growth by the co-operative effect of a recombinant porcine amelogenin and fluoride.

Recently, we used native amelogenins extracted from developing pig enamel to examine the combined effect of fluoride and amelogenins on the growth of octacalcium phosphate (OCP) and apatite crystals. The purpose of the present study was to investigate this combined effect using a highly purified recombinant amelogenin. We applied porcine amelogenin (rP172) and fluoride in a dual-membrane system as a model for tooth enamel formation. The combination of rP172 and fluoride in this system resulted in the formation of rod-like apatite crystals. On the other hand, without fluoride, rod-like OCP crystals of a comparable size were formed, and rather large hexagonal prisms of mixed crystals of OCP and apatite grew without amelogenins. Thus, highly purified and homogeneous recombinant amelogenin, in co-operation with F, regulated the mineral phase, habit, and size of crystals in the same manner as the extracted heterogeneous porcine amelogenins. We suggest that in both cases the control over the crystal phase and morphology was a direct effect of amelogenin protein serving as a scaffold for apatite mineralization.

Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).

We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.

Formation of Giant Liposomes from Crystalline Complexes of Monoalkylammonium Surfactants and 4-Hydroxybiphenyl.

A parallel rather than a perpendicular alignment of aromatic compounds with respect to surfactant molecules (see schematic representation) is preferred for the formation of a liposome structure, because the perpendicular alignment would reduce the hydrophilicity of the aggregate. This is the result of studies on crystalline complexes of monoalkylammonium halides and various aromatic compounds.