Mayumi Iijima

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

An animals 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.

Elongated Growth of Octacalcium Phosphate Crystals in Recombinant Amelogenin Gels under Controlled Ionic Flow

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.

Effects of F- on Apatite-Octacalcium Phosphate Intergrowth and Crystal Morphology in a Model System of Tooth Enamel Formation

SummaryIn order to study the effect of F- on tooth enamel-like apatite formation, crystal growth experiments were carried out in the presence of 0.1}2 ppm F- at 37°C and at pH 6.5 in a model system of enamel formation where octacalcium phosphate (OCP) was stable. Morphology changed from long and thin ribbons to small needle-like plates, and the product changed from OCP to apatite with an increase in F- concentration. In the presence of 0.1–1 ppm F-, apatite-OCP intergrowth took place, and crystals composed of apatite and OCP lamellas were formed. These crystals showed long and thin plate-like morphology and embedded an OCP lamella in the center of the crystal. The OCP lamella and its (100) planes were parallel to the (100) planes of apatite. The thickness of OCP decreased and that of apatite increased with an increase in F- concentration. Some apatite crystals obtained at 1 ppm F- embedded a central plane instead of the distinct OCP lamella. The result indicates that initially formed, thin, plate-like OCP acted as a template for the subsequent epitaxial overgrowth of apatite and, moreover, F- played an important role in regulating the apatite-OCP intergrowth.

Growth and structure of lamellar mixed crystals of octacalcium phosphate and apatite in a model system of enamel formation

Lamellar mixed crystals of octacalcium phosphate (OCP) and apatite were synthesized in a model system of enamel formation in the presence of 1 ppm F − at 37°C and at pH 6.5. The crystal has long and thin plate-like morphology and contained a distinct OCP lamella in the center of the apatite matrix. The thickness of the OCP lamella in the a -axis direction is one to several unit cells. Some apatite crystals embed a central layer instead of the distinct OCP lamella. The OCP lamella and the central layer are parallel to the (100) plane of the apatite, while the c -axis of the OCP is parallel to the c -axis of the apatite. Analysis suggests that (1) F − causes the growth of apatite on OCP and regulates the formation of the lamellar mixed crystals of OCP and apatite, (2) the OCP lamella acts as a template for the subsequent epitaxial growth of apatite, and (3) the lamellar mixed crystals grow mainly in the c-axis direction of both the OCP and apatite. These results strongly support the idea that enamel crystals take a thin and long ribbon-like morphology when the initially formed OCP acts as a template for the subsequent growth of apatite in the enamel formation.

Control of octacalcium phosphate and apatite crystal growth by amelogenin matrices

Tooth enamel, the hardest bioceramic composite in the vertebrate body, is the result of a cascade of intra- and extracellular events. Amelogenins, the principal extracellular matrix protein component of mineralizing enamel, have been considered to play substantial roles in controlling the growth and organization of enamel crystals. Considering octacalcium phosphate (OCP) as a precursor phase of enamel apatite crystallites, we have developed in vitro systems to grow OCP and apatite crystals in amelogenin matrices and therefore to investigate amelogenin–OCP and amelogenin–apatite interactions. This paper reviews our current findings on the effect of amelogenin on the morphology, size, phase and orientation of such crystals.

Transition of octacalcium phosphate to hydroxyapatite in solution at pH 7.4 and 37°C

Transition of octacalcium phosphate (OCP) to apatite was studied under the condition where Ca2+ ions were continuously supplied to the reacting solution at pH 7.4 and at 37°C. OCP crystals were grown and subsequently converted into apatite by discontinuing of Ca addition. The rectangular (100) blades of OCP crystals developed notches on their short edges in the early stage of transition. The depth of the notches increased along the c-axis direction with the progress of the reaction, giving rise to a slit-like texture. The direction of the slit formation seemed to relate to the spatial configuration of H2O molecules in the OCP lattice, which are released during the transition from OCP to apatite.

Interactions of Amelogenins with Octacalcium Phosphate Crystal Faces Are Dose Dependent

Amelogenins, the major protein components of the enamel extracellular matrix, are postulated to be involved in controlling the elongated and oriented growth of enamel carbonated apatite crystals. In order to clarify the functional role of amelogenin during the early stage of enamel biomineralization, octacalcium phosphate (OCP) crystals, known to be potent precursors of hydroxyapatite, were grown in 1–10% (w/w) native bovine and two recombinant murine amelogenins. Amelogenins were solution-like at 1% and formed gel at 10%, while 5% amelogenins became gel after reaction and it was inhomogeneous and porous. Morphological changes of OCP crystals were evaluated as the function of amelogenin concentration by analyzing the mean values of length, width, thickness, their reduction ratios (L/Lc, W/Wc, T/Tc) as well as L/W and W/T ratios. Length, width, and thickness decreased in a does-dependent manner. Length decreased almost linearly in 1%–10%, whereas width decreased drastically in 1%–5% while the decrease from 5% to 10% was small. As a result, elongated morphology of OCP crystal was most emphasized in 5% bovine amelogenins and rM166 and 2%–5% rM179. The size reduction was in the order of W/Wc < L/Lc < T/Tc. We therefore concluded that amelogenin interaction with crystal faces was in the order (010) > (001) > (100). At all concentrations, W/Wc was significantly the smallest. This indicated that the primary role of amelogenin was to decrease the width of OCP by blocking the hydrophobic (010) faces. We suggest that the drastic decrease of crystal width is the result of interaction of the densely packed nanospheres in 5%–10% amelogenin.

Fluoride Analysis of Apatite Crystals with a Central Planar OCP Inclusion: Concerning the Role of F− Ions on Apatite/OCP/Apatite Structure Formation

Abstract. To study the roles of F− ions in the formation of apatite crystals embedding octacalcium phosphate (OCP) lamella in the center of apatite (Ap), a range of the Ap/OCP/Ap lamellar-mixed crystals were synthesized under various concentrations of fluoride ion (F−) from 0.1–1.0 ppm at pH 6.5 and 37°C. The products were analyzed for the F− incorporation, F− distribution, and the amount of OCP and Ap by chemical analysis, X-ray diffraction (XRD), electron probe microanalysis (EPMA), and nuclear magnetic resonance (NMR) techniques. The F− content and the amount of apatite in the crystalline product increased with an increase in the F− concentration in solution, whereas the amount of OCP and the yield of total product decreased. EPMA indicated that F− ions are distributed in the crystals almost homogeneously. The combined analysis suggested that a low-substituted fluoridated hydroxyapatite (FHAp) grew on a small amount of F−-containing OCP or on a surface-reaction layer of OCP, which has accumulated a small amount of F−. The roles of F− ions were hypothesized as the reduction of the growth rate and/or the critical thickness in the a*-axis direction of OCP, the enhancement of hydrolysis of OCP, and the activation of the growth of FHAp, resulting in thinner OCP lamella and thicker apatite lamella in the a*-axis direction with an increase in F− concentration.

Oriented growth of octacalcium phosphate crystals on type I collagen fibrils under physiological conditions

Octacalcium phosphate (Ca8H2(PO4)6 · 5H2O, OCP) crystals grew on collagen fibrils withe preferred orientation in an experimental system at pH 6.5–7.0 and at 37°C. Directional and structural relations between the OCP crystals and the collagen fibrils were investigated by X-ray diffraction, and scanning and transmission electron microscopy. Three types of orientation were found: (1) parallel orientation of the c-axis of OCP to the fiber axis of collagen, (2) perpendicular orientation of the c-axis of OCP to the fiber axis, and the c-axis parallel to the collagen band, and (3) perpendicular orientation of the c-axis of OCP to the fibril itself. The X-ray diffraction and the TEM studies showed that a major part of OCP crystals grew along the collagen fibrils with the long axis (c-axis) parallel to the fiber axis. These observations demonstrate that OCP grew epitaxially on the collagen fibrils and strongly suggest that the collagen regulated the nucleation and crystal growth of OCP. The biological meaning of these phenomena is discussed.

Orientation of apatite and organic matrix in Lingula unguis shell

SummaryThe orientation relationship between apatite and organic matrix in shell ofLingula unguis (inarticulate brachiopod) was studied. The organic layers, mineralized layers, and decalcified mineralized layers were examined layer by layer using microbeam X-ray diffraction technique. Both organic layer and decalcified mineralized layer showed the diffraction pattern of β-chitin. The degree of orientation of apatite showed correlation to that of β-chitin: Well oriented diffraction patterns of apatite crystal and organic matrix were observed in the central part. In this part, the fiber axis of β-chitin was parallel to the c-axis of apatite. A close relationship of unit cell dimension between apatite and chitin was indicated. These strongly suggest that the fibrous structure of organic matrix assists the orientation of apatite crystals inLingula unguis shell.