Flowerlike Agglomerates of Calcium Carbonate Crystals Formed on an Eggshell Membrane Masakazu Takiguchi, Koichi Igarashi, Masayuki Azuma, and Hiroshi Ooshima* Graduate School of Engineering, Osaka City UniVersity, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 12 2754-2757
ReceiVed July 15, 2006; ReVised Manuscript ReceiVed September 27, 2006
ABSTRACT: Calcium carbonate was crystallized on an eggshell membrane using a specially designed crystallizer. The crystallizer used was a jacketed cylindrical glass vessel with two compartments divided by an eggshell membrane. The calcium chloride and sodium carbonate solutions were separately placed in the two compartments. Several kinds of peculiar agglomerates of calcium carbonate crystals formed on both surfaces of the eggshell membrane. Flowerlike agglomerates formed on the inner surface of the eggshell membrane that is the egg white side, but those were not formed on the outer surface of the membrane. The flowerlike agglomerate was composed of a hemispherical core and petal-like crystals growing around the core. We named it the CC (calcium carbonate)-flower. The different types of agglomerates formed on the outer surface of the membrane. For instance, when the outer surface of the membrane faced the calcium chloride solution, spherical agglomerates composed of long, needlelike crystals were obtained. We inferred that the surface structure of the eggshell membrane played an important role for the formation of such peculiar agglomerates. 1. Introduction There are many examples that inorganic crystals are the structural materials of the organisms. The eggshell is a typical example of such biominerals. The eggshell protects an inner embryo from external physical hazard and biological encroachment. The eggshell is mainly composed of calcium carbonate crystals (95% in weight).1 The other materials (3.5%) are organic matrices mainly consisting of glycoprotein and proteoglycans. The eggshell membrane is a thin film adhering inside the eggshell. The membrane plays an important role for the construction of the eggshell. The carbonate ions generated by respiration of the embryo pass through the eggshell membrane and react with calcium ions in the womb. The calcium carbonate crystals deposit on the surface of the eggshell membrane. The role of the eggshell membrane is to provide a nucleation point and affect structuring of the crystalline solid.2 In the previous work, we reported that the tubular structure of agglomerates of calcium carbonate crystals were formed on a cation-exchange membrane3 and that the cation-exchange membrane played an important role as a kind of template. In the present study, we attempted to crystallize calcium carbonate on the eggshell membrane. The method of the contact of calcium ions to carbonate ions was the same as that adopted in the previous work.3 The method was similar to that in the formation of the eggshell as described above. Namely, calcium chloride and sodium carbonate solutions were separately placed in the two vessels divided by an eggshell membrane. Calcium ions gradually transfer to the opposite side through the membrane to react with carbonate ions. The aim of the present work was to examine if the eggshell membrane might act as a template as functioned in nature. We also expected a novel morphology of agglomerates of calcium carbonate crystals. As a result, we found several structures of calcium carbonate agglomerates, including the flowerlike structure. 2. Materials and Methods The calcium chloride dihydrate and sodium carbonate used were of reagent grade (Wako Pure Chemicals Co. Ltd., Japan). The eggshell * To whom correspondence should be addressed. E-mail: ooshima@ bioa.eng.osaka-cu.ac.jp. Tel: 81-6-6605-2700. Fax: 81-6-6605-2701.
Figure 1. Schematic diagram of the crystallizer. membrane was prepared as follows. A hen egg was broken in half, and the contents were removed. The thin membrane adhering to the inside of the eggshell was carefully peeled off and washed in deionized water. It is known that the eggshell membrane is composed of three thin membranes, namely, the outer shell membrane, the inner shell membrane and the limiting membrane, from outside to inside.4 Therefore, the surface structure is different between both sides of an eggshell membrane. We used a whole eggshell membrane as a sheet of membrane. Here, we call the surface that makes contact with a shell the outer surface, and the opposite surface the inner surface. Figure 1 presents a schematic diagram of the crystallizer adopted in this study. The crystallizer was placed horizontally, as shown Figure 1. The crystallizer is composed of two jacketed cylindrical glass tubes, a membrane, silicon rubber sheets, and a horseshoe-shaped clamp. The inner diameter of the glass tube is 20 mm. The eggshell membrane was tightly fixed to the glass vessels by silicon rubber sheets and a horseshoe-shaped clamp. Calcium chloride and sodium carbonate were individually dissolved in deionized water and degassed to avoid the generation of bubbles during crystallization. The initial concentration of calcium chloride and sodium carbonate were 1.0 M; this concentration was arbitrarily adopted only to get a sufficient driving force for the penetration of calcium ions. The pH of sodium carbonate solution was adjusted to 12.0 with 5 M NaOH to allow the equilibrium between carbonate and bicarbonate to shift completely to the carbonate-ion side. The calcium chloride and the sodium carbonate solutions were placed in each compartment. The crystallization was carried out in two different modes; one in which the calcium chloride solution faced the inner surface of the membrane (Experiment A), and the other was the inverse (Experiment B). The crystallization was carried out at 15-50 °C for 1-10 days without stirring. Calcium ions and carbonate ions transferred to the opposite compartment through the eggshell membrane. As the result, calcium carbonate crystals grew on both surfaces of the membrane. After the crystallization for a given time, the membrane was recovered, gently rinsed with deionized water three times, and dried in air. The
10.1021/cg0604576 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/07/2006
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Figure 2. SEM images of the eggshell membrane. (A) Cross-section of the eggshell membrane. (B) Outer surface of the eggshell membrane. (C) Inner surface of the eggshell membrane. crystals on the membrane were coated with gold and observed by a scanning electron microscope (SEM; SHIMADZU EPM-810 and JEOL JSM-6500FE). The polymorphs of calcium carbonate crystals were identified by a powder X-ray diffraction meter (Rigaku, MiniFlex).
3. Results and Discussion Figure 2 shows the SEM images of the eggshell membrane used for this study. As mentioned above, the eggshell membrane is composed of three membranes, namely, the outer shell membrane, the inner shell membrane, and the limiting membrane.4 Panel A of Figure 2 shows a cross section of the membrane. The left upper side corresponds the outer surface of the eggshell membrane, which adheres to the eggshell. The inner shell membrane is sandwiched between the outer shell membrane and the limiting membrane. The total thickness of the eggshell membrane was about 70 µm. The outer surface of the eggshell membrane, namely, the surface of the outer shell membrane, is made of fibers, as shown in the panel B of Figure 2. Panel C of Figure 2 shows the inner surface of the membrane, namely, the surface of the limiting membrane. It was smoother than the outer surface. 3.1. Experiment A: The Inner Surface of the Eggshell Membrane Facing the Calcium Chloride Solution. Peculiar agglomerates of calcium carbonate crystals were obtained when the inner surface of the eggshell membrane faced the calcium chloride solution. Figure 3 shows the SEM images of the calcium carbonate crystals that grew on the inner surface of the membrane at 15 °C. Those are like flowers. Panel C of Figure 3 displays the flowerlike structure in more detail. We named the flowerlike agglomerate the CC (calcium carbonate)flower. Such a flowerlike structure of calcium carbonate crystals has not been reported. The size of the CC-flowers was about 30 µm in diameter. We found a hemispherical core at the center of the CC-flower, as presented in panel C of Figure 3. The size of the core presented in panel C is about 11 µm in diameter. Because the hemispheric core structure had not been observed on the inner surface of the eggshell membrane before crystallization as shown in panel C of Figure 2, the core must be made from calcium carbonate; it is composed of scalelike crystals. The hemispheric core is surrounded by many petal-like agglomerates. The length of the petal-like agglomerate was about 10 µm. A different type of petal was observed, as presented in panel D; the petals are composed of small platelike calcium carbonate crystals. The reproducibility of the CC-flowers was examined by repeating the same experiments. Panels E and F present the result. We could reproduce the CC-flowers, although the morphology of the CC-flowers was a little different from that presented in panel C. Those petals were short. The CCflowers with short petals were widely found on the inner surface of the membrane, as shown in panel E. It should be noted that in a natural egg, no crystal grows on the surface of the limiting membrane that faces egg white. This fact means that the formation of the CC-flowers is not natural, but artificial.
Figure 3. (A) SEM images of the calcium carbonate crystals formed on the inner surface of the eggshell membrane by crystallization at 15 °C for 10 days. (B) Enlarged view of the rectangular area in panel A. (C) Enlarged image of the flowerlike structure (the CC-flower). (D) Another type of the CC-flower differing in the morphology of petallike agglomerates. (E) CC-flowers obtained by repeating the same experiment. (F) Enlarged view of panel E.
However, it should also be true that the surface of the limiting membrane plays an important role for the formation of the CCflowers. The structure like the CC-flower has not been also observed in a natural egg. Figure 4 shows an X-ray powder diffraction pattern of the whole precipitate forming on the inner surface of eggshell membrane. The datum indicated that the crystals were a mixture of calcite and vaterite. The platelike crystals should be calcite and the scalelike crystals constructing the spherical cores should be vaterite. Calcium carbonate crystals also formed on the outer surface of the eggshell membrane that faced the sodium carbonate solution. Figure 5 presents the SEM image of crystals formed on the fibers constructing the outer surface of eggshell membrane. Cubic crystals that are calcite were observed all over the surface. We did not find any peculiar morphology. 3.2. Experiment B: The Outer Surface of the Eggshell Membrane is Facing to the Calcium Chloride Solution. Another experiment was carried out at 30 and 50 °C. Namely, the inner and outer surfaces were faced to the sodium carbonate
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Figure 7. SEM images of calcium carbonate crystals formed on the inner surface of the eggshell membrane.
Figure 4. XRD data of calcium carbonate crystals shown in Figure 3E; those were precipitated on the inner surface of the eggshell membrane. Closed squares and open circles indicate characteristic peaks of calcite and vaterite, respectively.
Figure 8. SEM images of calcium carbonate crystals formed on the outer surface of the eggshell membrane. (A) Spherical agglomerates. (B) Crack of the spherical agglomerate.
Figure 5. (A) SEM images of calcium carbonate crystals on the outer surface of the membrane. (B) Enlarged view of the rectangular area in panel A.
Figure 6. (A) SEM images of the early state of flowerlike structure. (B) Enlarged view of the rectangular area in panel A. (C) Enlarged view of the rectangular area in panel B.
and the calcium chloride solutions, respectively. This situation is same as that in the formation of natural eggshell, although the concentration of calcium ions and carbonate ions adopted here was much higher than that in nature. Calcium carbonate crystals were formed in both sides of the eggshell membrane in Experiment B as well as in Experiment A. Figures 6 and 7 show the agglomerates formed on the inner surface of the eggshell membrane. It is notable that the CC-flowers also formed on the inner surface even in Experiment B, as shown in panels A and B in Figure 6. The CC-flowers never formed on the outer surface in Experiment A either. This result makes us reconfirm that the structure of the inner surface of eggshell membrane is responsible for the growth of the CC-flower. The CC-flowers
shown in panel B may be in an early stage of the formation, because the petals are thin compared with those shown in Figure 3. We speculate the mechanism of the formation of the CCflower as follows. There might be spots on the surface of the limiting membrane, where calcium and carbonate ions gush. The hemispheric precipitate, which is composed of scalelike vaterite as shown in panel C of Figure 6, grows at the spot. Then crystals that become petals begin to grow around the hemispheric core. Even when one layer of petals are formed around the hemispherical core, calcium or carbonate ions would continue to spring through the gap between petals and the hemispheric core. Thus, petals would be formed layer by layer along the surface of the hemispheric core. If this speculation might be correct, the surface of the hemispheric core would be completely covered with petals at the end. Several products shown at the lower left in panel B of Figure 3 and also the CC-flower shown in panel D of Figure 3 may imply such an end. We infer from this speculation that the CC-flower is an intermediate product of an echinus-like agglomerate. Figure 7 presents the other precipitates formed on the inner surface of the eggshell membrane at 30 °C. Spherical agglomerates of calcite were observed in panel A. In a different view of the inner surface, candylike agglomerates were observed, as presented in panel B. Figure 8 shows SEM images of calcium carbonate crystals formed on the outer surface of the membrane. As can be seen in panel A, spherical agglomerates with rough surface were formed. The rough surface implies that the spherical agglomerates are composed of plenty of small particles with a uniform size. However, a crack site of the spherical agglomerate presented in panel B shows that the primary products constructing the agglomerates are not particles, but needlelike crystals. It is not clear if the needlelike crystals are single crystals. Figure 9 shows a different site of the outer surface of the eggshell membrane. Panel A presents the precipitates formed on the fiberlike structured surface of the outer shell membrane. Panel B presents those precipitates in large magnification. Panel C is an enlarged view of the rectangular area of panel B. The spherical agglomerates should be vaterite in appearance like the hemispheric cores of the CC-flowers. However, the crystals
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carbonate solutions and precipitates of calcium carbonate on the inner and outer surfaces of the membrane were observed. When the inner surface of the membrane faced the calcium chloride solution, the flowerlike agglomerate formed on the inner surface of the eggshell membrane. The flowerlike agglomerate was composed of a hemispheric core and the petal-like crystals growing around the core. We named the agglometare the CCflower. On the other hand, normal cubic crystals of calcite formed on the outer surface of the membrane. Any other structuralized precipitates were not observed.
Figure 9. (A) SEM image of the spherical core structure formed on the outer surface of the eggshell membrane. (B) SEM image of the spherical core in large magnification. (C) Magnified image of the surface of the spherical core.
formed around the spherical agglomerate were different from the petal-like agglomerates in the CC-flower. Many short rodlike crystals grew on the spherical agglomerates. And, as can be seen in Panel C, the short rodlike crystals were composed of thin platelike crystals that should be calcite. That short rodlike agglomerate may grow to the long needlelike crystal shown in panel B of Figure 8. Conclusions Calcium carbonate was crystallized on the eggshell membrane using a specially designed crystallizer. The eggshell membrane was placed between the calcium chloride and the sodium
When the outer surface of the membrane faced the calcium chloride solution, namely, in Experiment B, the spherical agglomerates formed on the outer surface of the membrane, as shown in Figures 8 and 9. Those spherical agglomerates were composed of the long, needlelike crystals. We also obtained the CC-flowers on the inner surface of the membrane at 50 °C that were the same as those when the inner surface was facing the calcium chloride solution. We conclude that the surface structure of the eggshell membrane played an important role for the formation of the peculiar agglomerates of calcium carbonate crystals as the CCflowers. References (1) Hincke, M. T.; Gautron, J.; Panhe´leux, M.; Garcia-Ruiz, J.; McKee, M. D.; Nys, Y. Matrix Biol. 2000, 19, 443-453. (2) Ajikumar, P. K.; Lakshminarayanan, R.; Valiyaveettil, S. Cryst. Growth Des. 2004, 4, 331-335. (3) Takiguchi, M.; Igarashi, K.; Azuma, M.; Ooshima, H. Cryst. Growth Des. 2006, 6, 1611-1614. (4) Bellairs, R.; Boyde, A. Z. Zellforsch. Mikros. Anat. 1969, 96, 237-249.
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