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J Dent Res 81(1): 69-73, 2002
© 2002 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

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

M. Iijima*,1, Y. Moriwaki1, H.B. Wen2,3, A.G. Fincham2, and J. Moradian-Oldak2

1 Asahi University School of Dentistry, Dental Materials and Technology, 1851-1 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-0296, Japan;
2 University of Southern California, School of Dentistry, Center for Craniofacial Molecular Biology, Los Angeles, CA, USA; anad
3 present address, DePuy, a Johnson & Johnson company, PO Box 988, 700 Orthopaedic Drive, Warsaw, IN 46581-0988, USA;

*corresponding author, iijima{at}dent.asahi-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

KEY WORDS: amelogenins • enamel crystal • OCP • crystal elongation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the process of enamel crystal formation in mammalian amelogenesis, thin ribbon-like crystals initially deposit in the enamel matrix and eventually grow into flat-hexagonalprisms (Rohnholm, 1962; Nylen et al., 1963). The nature of the mineral phase of the initial deposits is equivocal, while it is widely accepted that mature mammalian enamel crystals are a prototype of carbonate-containing fluoridated hydroxyapatite. Octacalcium phosphate [OCP: Ca8H2(PO4)6 5H2O], which is a metastable phase of hydroxyapatite and has a structure similar to that of apatite, can grow into ribbon-like morphology under physiological condition by controlling the direction of Ca ion supply (Iijima, 2001) and transforms to apatite in solution (Brown et al., 1962). Therefore, mechanisms of enamel apatite formation that involve OCP as a precursor of enamel apatite have been proposed (Brown, 1965; Nelson et al., 1989; Iijima et al., 1992; Miake et al., 1993). In the present study, we selected OCP crystals as a primary model for investigating the interactions of amelogenin proteins with calcium phosphate minerals.

Since amelogenins occupy more than 90% of the matrix proteins (Eastoe, 1965; Termine et al., 1980), enamel crystals grow under the influence of amelogenins. Although little was known about the effects of amelogenins on the growth of OCP, some recent studies revealed that amelogenins affect the morphology of OCP in a manner that provides some clues about the growth of tooth enamel crystals. From 1 to 2% porcine amelogenins incorporated into a gelatin gel resulted in elongation of OCP crystals (Wen et al., 2000). In 10% bovine amelogenin gel, enclosed in a micro-space where ionic diffusion was controlled by a cation-selective membrane and a dialysis membrane, the morphology of OCP crystals changed from ribbon-like to prismatic and cylindrical fibers (Iijima et al., 2001). In both systems, however, heterogeneous amelogenin fractions from in vivo sources were used; therefore, the majority of the components were the molecules without the hydrophilic C-terminal region.

For a better understanding of the mechanisms of modulation of OCP morphology, we focused, in the present study, on the effects of highly purified and homogenous amelogenins free of non-amelogenin proteins. For this purpose, two types of recombinant murine amelogenins were used: (a) rM179, which has the hydrophilic C-terminal residues and lacks an amino terminal methionine and a phosphorylated serine residue; and (b) rM166, which lacks the hydrophilic C-terminal residues present in rM179. Furthermore, the microstructure of 10% rM179 gel used for OCP crystallization was analyzed, and the effects of amelogenins on OCP growth were compared with those of other gel systems, such as polyacrylamide and agarose.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Amelogenin Preparation
Recombinant murine amelogenins, rM179 (M = 20 kDa) and rM166 (M = 18.6 kDa), were expressed in E. coli, and isolated and purified by high-performance liquid chromatography (HPLC) as previously reported (Moradian-Oldak et al., 1994; Simmer et al., 1994). Analysis of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC revealed their purity to be higher than 95%.

Sample Preparation for Atomic Force Microscopy (AFM)
The 10% rM179 amelogenin gel-like matrix was prepared under conditions resembling those of OCP crystallization. Approximately 1.1 mg of lyophilized protein was dissolved in 12 µL of cold ammonium phosphate (10 mM, pH 6.5). The procedure was performed directly on a freshly cleaved mica surface. The thin film of amelogenin gel-like matrix (1 mm thick and 10 mm in diameter) was fixed for 2 hrs at 37°C by means of Karnovsky fixative. This procedure has been shown to preserve the microstructural features of the nanosphere containing gel matrix (Wen et al., 1999, 2001). The fixed gel was then equilibrated in ascending series of ethanol, air-dried, and immediately taken for AFM observation via a Nanoscope IIIa Scanning Probe Microscope System (Digital Instruments, Inc., Santa Barbara, CA, USA). We obtained the images by operating a Tapping mode Etched Silicon Probe (model OTESPA-10) in air at a scanning rate of 0.5-1.0 Hz.

Crystal Growth Experiment
All chemicals used were reagent grade, and all solutions were prepared with de-ionized and double-distilled water (DDW). Reaction was carried out at 37°C for 3 days in a dual-membrane system, where a cation-selective membrane (CMVTM, Asahi Glass Co., Tokyo, Japan) and a dialysis membrane (Visking Cellulose Tubing; Union Carbide Co.) were used to enclose about 15 µL of reaction solution containing the proteins (Iijima et al., 2001). Ca2+ and PO43- ions were allowed to diffuse into the reaction space from mutually opposite sides through the cation-selective membrane and the dialysis membrane, respectively. A 100-mL quantity of NH4H2PO4 + (NH4)2HPO4 (10 mM, 1:1 molar ratio) was used as a PO4 solution reservoir and 1.8 mL of 10 mM Ca(CH3COO)2 H2O as a Ca solution reservoir, which were adjusted to pH 6.5 at 37°C by dilute HCl solution. About 1.4 mg of amelogenin weighed by a microbalance was mixed with 13 µL of cold DDW to make a 10% solution on a cold dish floated on ice-water. Some parallel reactions were carried out in 10% polyacrylamide (PAA) gel, containing 0.5% cross-linking reagent (N,N'-methylenebisacrylamide), and 0.5% agarose (Sigma) and 10% bovine albumin (Sigma) for comparison. In the present study, % wt was used to unify the total amount of protein in solution. In the case of agarose, since 10% agarose gel was much harder than other gels, 0.5% was used, which showed hardness close to that of other gels.

After the reaction, the gel still fixed on the membrane was rinsed superficially with distilled water, frozen quickly at -80°C, and lyophilized. Crystals were identified by an x-ray diffractometer (XRD) (Rigaku, RINT 2500). The morphology of crystals was observed by means of a scanning electron microscope (Hitachi, S4500). Crystal sizes were measured on the SEM photographs, in which crystal faces were properly situated for dimension measurement. The number of data points was in the range of 10 to 78 for each dimension. Their mean values and standard deviations (SD) were calculated. Mean values were compared by Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fig. 1Go shows a Tapping mode AFM image in air of a 10% rM179 amelogenin gel. To investigate the microstructure of amelogenin gel used for OCP crystallization, we constructed the gel under conditions resembling those of crystallization and examined it by AFM. The resulting gel was composed of spherules with diameters of about 10-20 nm (Fig. 1Go). The size distribution of these nanospheres was uniform.



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  		Figure 1. Direct visualization of amelogenin nanospheres (10-20 nm in diameter) in 10% rM179 amelogenin gel by a Tapping mode AFM in air. The gel was formed in 10 mM phosphate buffer at pH 6.5, with conditions resembling those of OCP crystallization, and fixed at 37°C (see MATERIALS & METHODS).

 
SEM images of crystals grown in 10% (wt/vol) rM179 and rM166 gels are shown in Figs. 2b and 2cGo, along with the crystals grown in other materials (Figs. 2d,2e,2fGo). As can be seen from the scale bars in Fig. 2Go and the TableGo, these crystals were significantly smaller than crystals grown without proteins (control crystal in Fig. 2aGo). XRD patterns of these products were those of OCP with characteristic 100, 200, and 110 reflections; thus, they were identified asOCP. Control crystals are the largest and have a ribbon-like morphology (Fig. 2aGo). Both rM179 and rM166 caused a unique morphological change: The majority of the OCP crystals were prismatic and cylindrical fibers (Figs. 2b, 2cGo), which are unusual to the morphology of OCP crystals. A few crystals were ribbon-like. In contrast, crystals grown in 10% polyacrylamide (Fig. 2dGo) and 0.5% agarose gels (Fig. 2eGo) and 10% albumin (Fig. 2fGo) had a ribbon-like morphology which is similar to that of the control crystals.



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  		Figure 2. SEM images of crystals grown (a) without protein, in (b) 10% rM179, (c) 10%r M166, (d) 10% polyacrylamide gel, (e) 0.5% agarose gel, and (f) 10% albumin. Note that OCP crystals in a, d, e, and f show typical ribbon-like morphology, while the cylindrical and prismatic fibers grew only in amelogenin gels, regardless of the existence of the hydrophilic C-terminal of amelogenin.

 

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  		Table. Crystal Size and the Size Reductiona of Crystals Grown in Various Materialsb
 
The length, width, and thickness of crystals grown in rM179, rM166, PAA, albumin, and agarose are listed in the TableGo, and data obtained in our previous study (Iijima et al., 2001 ) are included for comparison. Among these materials, amelogenins, PAA, and agarose formed gel, whereas gelatin (Type B, 75 Bloom) and albumin did not under the present conditions. Since all measured crystals did not necessarily start growing simultaneously, variances of each dimension, and hence SD, were large. The mean values of width and thickness of crystals grown in 10% rM179 gel and those in 10% rM166 gel were almost the same (P = 0.076 for width; P = 0.42 for thickness), while length in the rM179 gel was significantly (P < 0.0001) smaller than that in the rM166 gel. The PAA and agarose gels showed similar size reduction modes (L/Lo, W/Wo, and T/To in the TableGo). In contrast, the size reduction modes of rM179 and rM166 were different from those of the former gels: The lengths of crystals grown in rM179 and rM166 were significantly (P < 0.0001) shorter than those of crystals grown in polyacrylamide and agarose gels, but longer than those of crystals grown in albumin. Both amelogenin gels showed a large decrease in width and a small decrease in thickness. The widths of crystals grown in rM179 and rM166 were significantly smaller (P < 0.0001) than those of crystals grown in polyacrylamide and agarose gels, smaller (rM166 < gelatin, P = 0.09) than that of gelatin, but larger than that of albumin (rM179 > albumin, P = 0.0018). The thickness of crystals grown in rM179, rM166, polyacrylamide, and agarose gels was almost half that of control crystals and about 2.5 times that of crystals grown in 10% albumin and gelatin.

The length-to-width ratio (L/W) (Fig. 3aGo) and the width-to-thickness ratio (W/T) (Fig. 3bGo) show unique changes caused by amelogenins: Both rM179 and rM166 resulted in crystals with L/W ratios larger than those of any other crystals, except gelatin, and W/T ratios smaller than those of any other crystals. Although the L/W ratio of crystals grown in gelatin was similar to that of crystals grown in amelogenins, the W/T ratio was 3 times larger than that in amelogenins.



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  		Figure 3. Effects of 10% gels of bovine amelogenins, rM166, rM179, polyacrylamide, and 10% albumin and gelatin on the morphology of OCP crystal, which are represented by (a) the length-to-width ratio (L/W) and (b) the width-to-thickness ratio (W/T) of crystals. Aspect ratios and their standard deviations (SD) were calculated based on the mean values and SD values of each dimension in the TableGo. Error bars represent SD values. (c) Schema ofOCP crystal, showing length, width, thickness, crystal faces, and crystallographic axes.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the model system, recombinant amelogenins rM179 and rM166 in a 10% gel state uniquely altered the morphology of OCP crystals. AFM imaging of the 10% rM179 gel revealed that, under the conditions used for crystallization, the full-length amelogenin molecules assemble into nanospheres. The amelogenin nanospheres have been shown to be the basic building blocks of 30% (w/w) rM179 gels (Wen et al., 2001), as well as gels of extracted porcine amelogenin (Wen et al., 1999). In the latter study, amelogenins were extracted from developing enamel matrix and were a mixture of 25 kDa, 23 kDa, 20 kDa and smaller fractions. The porcine amelogenins assembled into 8- to 20-nm nanospheres that further assembled into 40- to 300-nm spherules in a 35% gel. The presence of nanospheres in recombinant human amelogenins has been independently confirmed by cryo-transmission electron-microscopy-vitrified ice (Leytin et al., 1998). Highly conserved sequences of amelogenins across species suggest that the bovine amelogenins which were used previously (Iijima et al., 2001) also assemble into nanospheres in the 10% gel. AFM and dynamic light-scattering analysis of rM166 (Moradian-Oldak, 2001) have revealed the presence of the nanospheres formed by this engineered amelogenin. Thus, in this system, OCP crystals grew in gels densely packed with amelogenin nanospheres, analogous to enamel crystals growing in the enamel matrix composed with amelogenin nanospheres (Fincham et al., 1994). Therefore, we speculate that the effect of amelogeninon OCP morphology is related to the quaternary structure of the amelogenin gel. Polyacrylamide gel has the closed-cellular structure with the pore size of a few microns (Ruchel and Brager, 1975). Due to the cell walls, the mechanical interference of the polyacrylamide gel toward the growing crystals is supposed to be larger than that of the amelogenin gel, which is packed with nanospheres. Nevertheless, the decrease in length and width in amelogenin gels was larger than that in the polyacrylamide gel, while the decrease in thickness in amelogenin gels was almost the same as that in the polyacrylamide gel. This could be partly ascribed to interaction between amelogenin nanospheres and OCP crystals. An additional factor that could influence the crystal growth process is the mode of ionic diffusion that can be affected by the structure and composition of the gels.

The degree of crystal size reduction (L/Lo, W/Wo, and T/To in the TableGo) indicates that rM179 and rM166 interacted strongly with the (010), then with the (001), and weakly with the (100) face of the OCP crystal (for crystal faces, see Fig. 3cGo). This tendency was also observed in 10% bovine amelogenins (Iijima et al., 2001). Thus, the mode of the interaction was common among rM179, rM166, and bovine amelogenins, regardless of the existence of the hydrophilic C-terminal and the homogeneity of molecular weight. This suggests that some common factors among these amelogenins regulated the interaction in 10% gels. The most plausible factor is hydrophobicity. Since the reduction in thickness caused by rM179 (T/To = 0.41) was almost the same as those caused by bovine amelogenins (T/To = 0.45) and rM166 (T/To = 0.37), the distribution of the hydrophilic C-terminal in nanospheres in 10% gel might differ from that in diluted solution, thus giving rise to the hydrophobic property. Hydrophobic nanospheres would not react strongly with the (100) face, which exposes water molecules, preferring to react with the (010) face, which does not expose water molecules (Iijima et al., 2001). Indeed, in gelatin, albumin, polyacrylamide gel, and agarose gel, which are rather hydrophilic, prismatic and cylindrical crystals were not obtained (Fig. 2Go).

Furthermore, the L/Lo value indicates that the interaction of rM166 and rM179 with the (001) face was weaker than that of bovine amelogenins. In other words, the inhibitory effects of rM166 and rM179 on the growth in the c-axis direction of OCP were smaller than those of bovine amelogenins. This may well be connected to the initial elongated growth of enamel crystals. In the secretory stage, the full-length amelogenin molecules, which correspond to rM179, are secreted, the hydrophilic C-terminal is soon removed, and amelogenin becomes shorter, like rM166. In the enamel matrix, when it is full of those amelogenins, enamel crystals grow drastically in length. Although it is well-accepted that enamel crystals grow as carbonate-containing hydroxyapatite, it is proposed that they initially deposit as OCP-like precursors (Brown, 1965; Nelson et al., 1989; Iijima et al., 1992; Miake et al., 1993).

Regarding the interaction mechanism with amelogenin gel and crystal faces, one of the problems that remain but should be clarified relates to molecular conformation and/or the arrangement of a functional group, such as Ser residue, near the surfaces of nanospheres, because such a group could determine the property of the nanosphere surface and hence the interactionwith crystal faces.

Although this system does not perfectly simulate the in vivo condition, such as nucleation points and the directions of Ca and PO4 ion flow, it can deal with growth under one directional ionic supply, and this is its peculiarity. As has been established, the one-directional Ca ion flow enhances the elongation of OCP crystals, when growth was compared with that in calcium phosphate solutions (Iijima, 2001).

In summary, the morphological change of OCP in 10% rM179 and rM166 gels was unique, regardless of the existence of the hydrophilic C-terminal, when compared with that in gelatin, albumin, PAA gel, and agarose gel. The inhibitory effects of both rM179 and rM166 in the directions of length and width of OCP crystals were less effective than that of a heterogeneous mixture of degraded bovine amelogenins. The present experimental evidence supports our hypothesis that, in addition to other enamel extracellular matrix proteins (such as enamelins; Doi et al., 1984), amelogenin nanospheres are directly involved in controlling the morphology of crystals during enamel biomineralization (Fincham et al., 1995).


   ACKNOWLEDGMENTS
 
The authors acknowledge the technical assistance of Mr. Narbeh Gharakhanian in protein expression and purification, and the continuous support and encouragement of Dr. Chuck Shuler in the Center for Craniofacial Molecular Biology. This work was partly supported by NIH-NIDCR research grant DE12350 (J.M.O), and by the Miyata Grant from Asahi University (M.I.).

Received February 19, 2001; Last revision November 9, 2001; Accepted November 28, 2001


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Brown WE (1965). A mechanism for growth of apatite crystals. In: Tooth enamel II. Stack MV, Fearnhead RW, editors. Bristol: John Wright Ltd., pp. 11-14.

Brown WE, Smith JP, Lehr JR, Frazier AW (1962). Octacalcium phosphate and hydroxyapatite. Nature 196:1048–1055.

Doi Y, Eanes ED, Shimokawa H, Termine JD (1984). Inhibition of seeded growth of enamel apatite crystals by amelogenins and enamelin proteins in vitro. J Dent Res 63:98–105.[Abstract/Free Full Text]

Eastoe JE (1965). The chemical composition of bone and tooth. Adv Fluorine Res Dent Caries Prev 3:5–16.

Fincham AG, Moradian-Oldak J, Sarte PE (1994). Mass-spectrographic analysis of a porcine amelogenin identifies a single phosphorylated locus. Calcif Tissue Int 55:398–400.[Medline]

Fincham AG, Moradian-Oldak J, Diekwisch TGH, Lyaruu DM, Wright JT, Bringas P, et al. (1995). Evidence for amelogenin "nanospheres" as functional components of secretory-stage enamel matrix. J Struct Biol 115:50–59[Medline]

Iijima M (2001). Formation of octacalcium phosphate in vitro. In: Monographs in oral science. Vol. 18. Octacalcium phosphate. Basel, Switzerland: Karger AG, pp. 17-49.

Iijima M, Tohda H, Suzuki H, Yanagisawa T, Moriwaki Y (1992). Effect of F- on apatite-octacalcium phosphate intergrowth and crystal morphology in a model system of tooth enamel formation. Calcif Tissue Int 50:357–361.[Medline]

Iijima M, Moriwaki Y, Takagi T, Moradian-Oldak J (2001). Effects of bovine amelogenins on the crystal growth of octacalcium phosphate in a model system of tooth enamel formation. J Cryst Growth 222:615–626.

Leytin V, Arad T, Matlis S, Daphni L, Mao Z, Shay B, et al. (1998). Cryo-transmission electron microscopy and atomic force microscopy of the recombinant human amelogenin protein, self-assembled into nanospheres. In: Workshop on the structure and development of dental enamel. Oslo, Norway: University of Oslo.

Miake Y, Shimoda S, Fukae M, Aoba T (1993). Epitaxial overgrowth of apatite crystals on the thin-ribbon precursor at early stages of porcine enamel mineralization. Calcif Tissue Int 53:249–256.[Medline]

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Moradian-Oldak J, Simmer JP, Lau EC, Sarte PE, Slavkin HC, Fincham AG (1994). Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering. Biopolymers 34:1339–1347.[Medline]

Nelson DGA, Barry JC, Shields SCP, Glena R, Featherstone JDB (1989). Crystal morphology, composition, and dissolution behavior of carbonated apatites prepared at controlled pH and temperature. J Colloid Interf Sci 130:467–479.

Nylen MU, Eanes ED, Omnell KÅ (1963). Crystal growth in rat enamel. J Cell Biol 18:109–123.[Abstract/Free Full Text]

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Wen HB, Moradian-Oldak J, Leung W, Bringas P Jr, Fincham AG (1999). Microstructure of an amelogenin gel matrix. J Struct Biol 126:42–51.[Medline]

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