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Crystal orientation preference and formation mechanism of nacreous layer in mussel
ARTICLE IN PRESS
Journal of Crystal Growth 258 (2003) 402–408
Crystal orientation preference and formation mechanism of nacreous layer in mussel W.T. Hou*, Q.L. Feng Department of Materials Science and Engineering, Laboratory of Advanced Materials Science, Tsinghua University, Beijing 100084, People’s Republic of China Received 23 June 2003; accepted 2 July 2003 Communicated by M. Schieber
Abstract Using a new and simple etching method to investigate the tablets of nacre, it is possible to observe a great deal of tablets in a large range. We found that most tablets have the same formal orientations after citric acid treatment. This phenomenon occurred not only in the same lamina but also along the direction perpendicular to nacreous plane. Based on the additional selected area diffraction patterns of transmission electron microscope, it is proposed that the formal orientations could indicate the crystal orientations of aragonite tablets. Statistical analyze shows that there is a main (type I tablets) and a secondary (type II tablets) preferred orientation with an angle of around 60 between them. Two possible modes of structural correspondence between protein sheets and aragonite lattice are assumed to interpret the orientation preference. On the other hand, the cluster character of type II tablets strongly supports that mineral bridges of the organic matrix have significant function in the crystal growth. At last, the mechanisms of structural correspondence and mineral bridges are combined to explain the formation process of nacreous layer in mussel. r 2003 Elsevier B.V. All rights reserved. PACS: 81.10.Aj; 81.65.Cf; 87.64.Dz Keywords: A1. Biomineralization; A1. Crystal orientations; A1. Etching; A3. Mineral bridges; A3. Structural correspondence; B1. Aragonite
1. Introduction Nacre of mollusk shell is a composite of aragonite crystals and protein-polysaccharide matrix. Its fracture toughness is estimated to be 1000 times greater than that of aragonite minerals, *Corresponding author. Tel.: +86-10-62782770; fax: +8610-62771160. E-mail addresses: [email protected] (W.T. Hou), [email protected] (Q.L. Feng).
although the inorganic platelets have approximately 99% of the mass and 95% of the volume [1,2]. It is hoped that the mechanism of biologic controlling to form this microstructure could give us inspiration in developing advanced materials [3,4]. The aragonite tablets in nacre is basically a single crystal with their c-axis perpendicular to the tablet plane [5,6], yet we have known little about aand b-axes of them. Some works have shown that adjacent tablets may have same a- and b-axes and
0022-0248/$ - see front matter r 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-0248(03)01551-3
ARTICLE IN PRESS W.T. Hou, Q.L. Feng / Journal of Crystal Growth 258 (2003) 402–408
these works supported the hypothesis of mineral bridges [7–9]. But until now no report has been seen to investigate large numbers of tablets mainly because of the limitation in quantity of transmission electron microscope (TEM) studies. We recently use a simple etching method to accomplish this investigation. The simplified two-component mode for the organization of the matrix shows that hydrophobic biopolymers (chitin and silk-fibroin proteins) form a framework on which the hydrophilic acidic macromolecules are aligned to provide a structurally well-defined charged interface for nucleation [10]. It has been found that EDTA-soluble proteins extracted from aragonitic or calcitic mollusk shell layers have stretches of (Asp-Y) n-type sequence with a predominantly b-sheet conformation and they are sufficient to control the phase of deposited calcium carbonate [11–15]. Several theories have been developed to explain the biofabrication of nacre and in these heterepitaxial is widely accepted as the interlamellar organic sheet that serves as template of inorganic crystal nucleation in the aspects of electrostatic accumulation, structural correspondence and stereochemical requirements [10]. While another hypothesis of mineral bridges based on the observation of gastropod shell suggests continuous growth of aragonite crystals [16], it accords very well with the stacked arrangement of growth front of abalone nacre [9]. Our research results of aragonite tablets in mussel show both features of heterepitaxial and that of mineral bridges. They may concur in the formation process of bivalve nacre.
2. Materials and methods Sample prepared from the 2-year-old bivalve mollusk shells, Mytilus edulis, were grown in the Bohai Sea at Qinhuangdao in North China. It was cleaned in 5% NaOH solution for 10 min to eliminate the organic proportion on the surface. Then we used 10 wt% ethylenediaminetetraacetic acid (EDTA) solution or 0.018 M citric acid solution to etch on the aragonite tablets of nacreous layer. The concentration of the acid
403
solutions need not be invariable and it continually declined as the reaction was proceeding. The etching time was set from 5 min to 24 h for citric acid and 12 or 24 h for EDTA. Then the surface was washed by deionized water and deposited with gold sputtering. After that, observation was performed using a Hitachi S-450 scanning electron microscope (SEM). Sample of TEM was prepared from a part of shell that had been abraded into a slice as thin as possible with only nacreous layer. Then, ion-mill method was used to make the sample thin enough to observe in a Hitachi-800 TEM.
3. Results and discussion Fig. 1a shows the nacre tablets after NaOH treatment, the proteins on/between the tablets are almost dissolved and washed off. Figs. 1b and c show the morphology after EDTA treatment. It can be seen that EDTA first yields some etch-holes into the tablets, most possibly from dislocation points on the surface. Then the hole becomes larger and larger until this tablet disappears (black arrow in Fig. 1b). This kind of etching mode induces the flower-like morphology. And with the increase of etching time, this morphology remains unchanged, except that the etched depth increased (Fig. 1c). On the other hand, the morphology of the nacre surface after citric acid treatment is unlike that after EDTA treatment. First dissolved part in a tablet is not on the top face but on the side face. Most aragonite tablets exhibit same rectangular form and a preferred formal orientation (Fig. 2). This formal orientation preference is not only in a range of several tablets but are also in a much larger area as shown in Fig. 2c (at least 100 mm). This phenomenon can be seen in different tablets either along the tablet plane or along the direction perpendicular to nacreous plane. For convenience, we call them type I tablets. Whereas, there still exist tablets in some domains that have different formal orientation with type I tablets (black circles in Figs. 2a and b), these tablets are normally in some clusters and also have same gradient. We call them type II tablets.
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Fig. 1. SEM morphology of the nacre tablets after treatment. (a) After NaOH treatment, the proteins on/between the tablets are almost dissolved and washed off. The forms of the tablets are various. (b) After NaOH and EDTA treatment successively, many etch holes can be seen on the top face of the tablets. The etching time is 12 h. (c) Similar flower-like morphology after etching for 24 h. Scale: a, 13.8 mm; b, 7.3 mm; c, 7.4 mm.
Fig. 2. After citric acid treatment, aragonite tablets exhibited similar rectangular form and preferred formal orientation. The length proportion of the adjacent formal edges is approximately 5:8. (a), (b) The etching time is 10 min. (c) The etching time is 24 h. Scale: a, 11.6 mm; b, 9.3 mm; c, 22 mm; d, 7.7 mm.
Does the formal orientation indicate the crystal orientation of aragonite tablets? We think it does. As the surface proteins had been eliminated, it could not adsorb on the aragonite to affect the etching process. When the citric acid is added, it prefers reacting with the edge atoms to the normal surface atoms. With the dissolution going on, the form of the tablets changes from various to similar probably because of the anisotropy of the aragonite crystals. In order to ascertain the crystal orientation of the individual tablets, we used selected area diffraction (SAD) method of TEM. The patterns are shown in Fig. 3a. Every tablet is
selected in separate visual fields to make certain that they are not at a distance of several tablets. It is obvious from Fig. 3c that their crystal orientations incline to be normally distributed, although they are not completely in one direction. This investigation confirmed the SEM results. Statistical chart of directions in Fig. 2a reveals that 63% of the orientations of aragonite tablets are concentrated within thirty degrees from –20 to 10 (Fig. 4). Type I tablets have high proportion (the main peak in Fig. 4) and type II tablets have a lower proportion (the two secondary peaks in Fig. 4).
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Fig. 3. (a) SAD patterns of some tablets selected from different visual fields of TEM. (b) The crystal indexes and a-axis direction of a5 are labeled out as an instantiation. (c) The defined directions of 90 , 0 , 90 and eight a-axis (a1–a8) of tablets are shown in (a). These a-axes decline to be normally distributed around 30 except a6.
As we demonstrated the formal orientation indicated the crystal orientation, its preference could be interpreted by structural correspondence. There are two kinds of organic matrix in the mollusk shell, EDTA-soluble proteins and EDTAinsoluble proteins. Soluble proteins have a common amino-acid sequence of Asp-X-Asp (X is a neutral residue). Sequence of this type in antiparallel b-pleated sheet of silk-fibroin-like proteins
could present optimum binding (and hence nucleation) configuration for Ca2+ or CaCO3 microcrystals at the matrix interface [17]. Lavi et al. found that the synthesized polypeptide of (Asp-Leu)n is capable of specifically inducing aragonite formation [15]. Poly-L-Asp is also believed to control the oriented nucleation of aragonite in collagenous matrices [19]. Recently works of Orme et al. has shown that Asp can
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Fig. 4. Two hundred tablets in Fig. 2a are selected and measured. The defined 90 , 0 and 90 direction is same with that in Fig. 3c. The Savitzky-Golay smoothing curve in the chart reveals that there is a main peak around 7 and two secondary peaks around 59 and 53 . 63% of the degrees are in the range from –20 to 10 .
adsorbed on the (1 0 4) steps of calcite to change the step-edge energy [20]. All these works indicated that Asp plays a leading role of biominerialization in vivo. Weiner et al. had found in mollusk shell that the antiparallel b-pleated sheet of the matrix and the overlaying aragonite crystal lattice are matched in orientation at the interface [18]. We also observed type II tablets in their SEM morphology of the bivalve, Neotrigonia margaritacea. It implied that the phenomenon is ubiquitous in mollusk shell of bivalve. Based on the matching assumption, we attempted to find the reason of orientation preference. As the c-axis of the aragonite is perpendicular to the tablet surface, we only consider the ab-face matching to the proteins. Fig. 5 is the schematic representation of the possible geometrical matching, of which Mode 1 is the most optimum. The matching errors (Table 1) and number of matching points of the three modes are discussed below. Mode 1 is the most possible matching. Its matching error is very low except that of [0 1 0] direction, 13.3%. But it is sufficient to firmly bind a microcrystal of three or four lattice size, as the protein sheets are more likely to bind ionic regiment or CaCO3 microcrystal than Ca2+ atoms
Fig. 5. Schematic model of the structural relationships between protein sheets and ab-face of aragonite crystals. The basal rectangle lattice with crosses represents the main structure of antiparallel b-pleated sheets, which have many possible Asp positions. The rectangles with spheres (Ca atoms) represent the ab-face of aragonite. There are three possible matching modes. Their matching errors are listed in Table 1.
to produce oriented crystal nuclei [17]. There are four matching points as seen in Fig. 5. Mode 2 has three perfect matching points in a line of [1 1 0] direction. If they are bound, other Ca2+ atoms in the microcrystal will search for matching and stabilize the binding. Mode 3 is comparatively difficult to be stabilized, as it has less matching point and higher matching errors. Geometric analysis indicates that mode 2 and 3 matching have a gradient of 58 and 34.5 with mode 1 matching (Table 1). The angles between peaks in Fig. 4 (52 and 60 ) are very close to the gradient of mode 2. This angle coherence implies that types I and II tablets may be related with modes 1 and 2 matching. When numerous microcrystals of aragonite compete to be nucleus, these two most stable matching modes have high probability to happen in statistics. So most tablets (type I) have same orientation because their nuclei are mode 1 matching, and some tablets (type II) have a gradient of around 60 with type I tablets because their nuclei are mode 2 matching. From the black circles in Figs. 2a and b, we can see that type II tablets are not individual with each other but in some clusters. If every tablet of nacreous layer is grown from an aragonite nucleus
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Table 1 Matching errors of the three modes between protein sheets and Ca atoms Mode
Matching errors of a [1 0 0] length (%)
1 2 3
4.3 12.6 18.3
Gradient with mode 1
b [0 1 0] length (%) 13.3 0.6 5.3
[1 1 0] length (%)
[1 0 0] direction
[0 1 0] direction
[1 1 0] direction
— 1.3 —
0 2.5 1.5
0 6.2 0
— 0 —
stabilized by protein sheets, tablets of mode 2 matching would equably distributed in three dimensions of nacreous layer. The cluster character can be interpreted only through the growth mechanism of mineral bridges. A lower lamina aragonite tablet can grow through the pores in the organic interlamellar sheets (mineral bridges) to form a new tablet. This new tablet preserves the crystal orientation of the old one and hence may transfer the character to the upper lamina. Since type I tablets has high proportion, this process in them is not as obvious as that in type II tablets. These two mechanisms of structural correspondence and mineral bridges are dissimilar but not incompatible with each other. There are two possible relationships between them in vivo: successive or competitive. Successive relationship can be described as follows. Formation of the primary tablets of nacreous layer is controlled by structural correspondence. The later formed tablets are all grown from the primary ones through mineral bridges (Fig. 6a). So type II tablets’ orientation may be inherited and observed. Competitive relationship can be described as all tablets of nacreous layer are formed either by structural correspondence or through mineral bridges. If there are several pores in the organic sheets in the growth area, calcium carbonate may grow on the base of under lamina minerals to release supersaturation. If there are no pores in the growth area, heteroepitaxial nucleation may happen on the protein sheets surface to allow calcium carbonate growing up. We propose that the difference lies in the density and size of pores in the matrix sheets. In gastropod shell such as abalone, organic sheets have pores of 20–100 nm and same space size between them [7,9]. The large size and high density of pores allow
0 758 734.5
Fig. 6. Two-dimension paradigm of structural correspondence and mineral bridges induced nacreous layer. The grey lines represent matrix sheets and the white and black zones represent type I tablets and cluster of type II tablets, respectively. Notice the mineral bridges along the growth direction. (a) Successive relationship. A primary type II tablet grows to the outer surface of nacreous layer through mineral bridges. (b) Competitive relationship. Construction correspondence induced type II tablets equably distributed in three dimensions. And they may or may not grow to the next lamina.
aragonite to grow vertically without much difficulty before a tablet shapes up laterally. The result is that the growth surface of gastropod exhibits stacked arrangement of aragonite crystals. Whereas in bivalve shell, pores of organic sheets are likely to have smaller size and growth into these pores would additionally increase the surface area, which is unstable in thermodynamics for the growing crystal. So the crystals first choose to grow laterally to form a complete tablet until they have to grow to the next lamina. This exhibits brick-wall type arrangement. If the density of pores in bivalve shell
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protein sheets is not very low, aragonite crystals may keep on growing through them as described in Fig. 6a. If the density is too low that not every tablet can snatch at least one pore in its territory, Fig. 6b may happen. It depends on the pore density. Further research should be focused on these. 4. Conclusion There are two types of tablets in mollusk shell of bivalve, Mytilus edulis. Type I tablets have almost same orientations of a- or b-axes. Type II tablets have lower proportion, and their crystal orientation has a gradient around 60 with that of type I. They exhibit a kind of cluster character. Two modes of structure correspondence are developed to interpret the orientation preference. (0 0 1) face of aragonite microcrystals decline to match on the b-pleated sheet of proteins and the stabilized microcrystals may become nucleus to grow into aragonite platelets. Mineral bridges have significant function in the growth of nacreous layer. Crystals may grow through them and become the nucleus of another tablet. That is why type II tablets assemble into some clusters. The mechanism of structure correspondence and mineral bridges may concur in the formation of nacreous layer of bivalve. The possible successive and competitive relationships can explain the observation results very well. Acknowledgements This work is supported by National Natural Science Foundation of china, Grant 50272035.
References [1] A. Jackson, J. Vincent, R. Tuener, Proc. R. Soc. Lond. B 234 (1988) 415. [2] F.Z. Cui, Q.L. Feng, Biomaterials, Science China Press, Beijing, China, 1996, p.78. [3] G.M. Bond, R.H. Richman, W.P. McNaughton, J. Mater. Eng. Performance 4 (3) (1995) 334. [4] S. Mann, D.D.A. Rchibald, J.M. Didymus, T. Douglas, B.R. Heywood, F.C. Meldrum, N.J. Reeves, Science 261 (1993) 1286. [5] M.E. Marsh, R.L. Sass, Science 208 (1980) 1262. [6] S. Mann, in: S. Mann, W. Williams, R.J.P. Williams (Eds.), Biomineralization: Chemical and Biological Perspectives, VCH, Weinheim, 1989, p. 146. [7] S. Manne, C.M. Zaremba, R. Giles, L. Huggins, D.A. Walters, A. Belcher, D.E. Morse, G.D. Stucky, J.M. Didymus, S. Mann, P.K. Hansma, Proc. Roy. Soc. Lond. B 256 (1994) 17. [8] Q.L. Feng, F.Z. Cui, G. Pu, R.Z. Wang, H.D. Li, Mater. Sci. Eng. C 11 (2000) 19. [9] T.E. Schaffer, C.I. Zanetti, R. Proksch, M. Fritz, D.A. Walters, N. Almqvist, C.M. Zaremba, A.M. Belcher, B.L. Smith, G.D. Stucky, D.E. Morse, P.K. Hansma, Chem. Mater. 9 (1997) 1731. [10] S. Mann, Nature 332 (10) (1988) 119. [11] S. Weiner, L. Hood, Science 190 (1975) 987. [12] G. Falini, S. Albeck, S. Weiner, L. Addadi, Science 271 (1996) 67. [13] A.M. Belcher, X.H. Wu, R.J. Christensen, P.K. Hansma, G.D. Stucky, D.E. Morse, Nature 381 (2) (1996) 56. [14] Q.L. Feng, G. Pu, Y. Pei, F.Z. Cui, H.D. Li, T.N. Kim, J. Crystal Growth 216 (2000) 459. [15] Y. Lavi, S. Albeck, A. Brack, S. Weiner, L. Addadi, Chem.-Eur. J. 3 (1998) 389. [16] N. Watabe, Prog. Cryst. Growth Charact. 4 (1981) 99. [17] A.P. Wheeler, K.W. Rusenko, J.W. George, Compounds Biochem. Physiol. B 87 (4) (1987) 952. [18] S. Weiner, W. Traub, Phil. Trans. R. Soc. Lond. B 304 (1984) 425. [19] G. Falini, Int. J. Inorg. Mater. 2 (2000) 455. [20] C.A. Orme, A. Noy, A. Wierzbicki, M.T. McBride, M. Grantham, H.H. Teng, P.M. Dove, J.J. DeYoreo, Nature 411 (2001) 775.
Journal of Crystal Growth 258 (2003) 402–408
Crystal orientation preference and formation mechanism of nacreous layer in mussel W.T. Hou*, Q.L. Feng Department of Materials Science and Engineering, Laboratory of Advanced Materials Science, Tsinghua University, Beijing 100084, People’s Republic of China Received 23 June 2003; accepted 2 July 2003 Communicated by M. Schieber
Abstract Using a new and simple etching method to investigate the tablets of nacre, it is possible to observe a great deal of tablets in a large range. We found that most tablets have the same formal orientations after citric acid treatment. This phenomenon occurred not only in the same lamina but also along the direction perpendicular to nacreous plane. Based on the additional selected area diffraction patterns of transmission electron microscope, it is proposed that the formal orientations could indicate the crystal orientations of aragonite tablets. Statistical analyze shows that there is a main (type I tablets) and a secondary (type II tablets) preferred orientation with an angle of around 60 between them. Two possible modes of structural correspondence between protein sheets and aragonite lattice are assumed to interpret the orientation preference. On the other hand, the cluster character of type II tablets strongly supports that mineral bridges of the organic matrix have significant function in the crystal growth. At last, the mechanisms of structural correspondence and mineral bridges are combined to explain the formation process of nacreous layer in mussel. r 2003 Elsevier B.V. All rights reserved. PACS: 81.10.Aj; 81.65.Cf; 87.64.Dz Keywords: A1. Biomineralization; A1. Crystal orientations; A1. Etching; A3. Mineral bridges; A3. Structural correspondence; B1. Aragonite
1. Introduction Nacre of mollusk shell is a composite of aragonite crystals and protein-polysaccharide matrix. Its fracture toughness is estimated to be 1000 times greater than that of aragonite minerals, *Corresponding author. Tel.: +86-10-62782770; fax: +8610-62771160. E-mail addresses: [email protected] (W.T. Hou), [email protected] (Q.L. Feng).
although the inorganic platelets have approximately 99% of the mass and 95% of the volume [1,2]. It is hoped that the mechanism of biologic controlling to form this microstructure could give us inspiration in developing advanced materials [3,4]. The aragonite tablets in nacre is basically a single crystal with their c-axis perpendicular to the tablet plane [5,6], yet we have known little about aand b-axes of them. Some works have shown that adjacent tablets may have same a- and b-axes and
0022-0248/$ - see front matter r 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-0248(03)01551-3
ARTICLE IN PRESS W.T. Hou, Q.L. Feng / Journal of Crystal Growth 258 (2003) 402–408
these works supported the hypothesis of mineral bridges [7–9]. But until now no report has been seen to investigate large numbers of tablets mainly because of the limitation in quantity of transmission electron microscope (TEM) studies. We recently use a simple etching method to accomplish this investigation. The simplified two-component mode for the organization of the matrix shows that hydrophobic biopolymers (chitin and silk-fibroin proteins) form a framework on which the hydrophilic acidic macromolecules are aligned to provide a structurally well-defined charged interface for nucleation [10]. It has been found that EDTA-soluble proteins extracted from aragonitic or calcitic mollusk shell layers have stretches of (Asp-Y) n-type sequence with a predominantly b-sheet conformation and they are sufficient to control the phase of deposited calcium carbonate [11–15]. Several theories have been developed to explain the biofabrication of nacre and in these heterepitaxial is widely accepted as the interlamellar organic sheet that serves as template of inorganic crystal nucleation in the aspects of electrostatic accumulation, structural correspondence and stereochemical requirements [10]. While another hypothesis of mineral bridges based on the observation of gastropod shell suggests continuous growth of aragonite crystals [16], it accords very well with the stacked arrangement of growth front of abalone nacre [9]. Our research results of aragonite tablets in mussel show both features of heterepitaxial and that of mineral bridges. They may concur in the formation process of bivalve nacre.
2. Materials and methods Sample prepared from the 2-year-old bivalve mollusk shells, Mytilus edulis, were grown in the Bohai Sea at Qinhuangdao in North China. It was cleaned in 5% NaOH solution for 10 min to eliminate the organic proportion on the surface. Then we used 10 wt% ethylenediaminetetraacetic acid (EDTA) solution or 0.018 M citric acid solution to etch on the aragonite tablets of nacreous layer. The concentration of the acid
403
solutions need not be invariable and it continually declined as the reaction was proceeding. The etching time was set from 5 min to 24 h for citric acid and 12 or 24 h for EDTA. Then the surface was washed by deionized water and deposited with gold sputtering. After that, observation was performed using a Hitachi S-450 scanning electron microscope (SEM). Sample of TEM was prepared from a part of shell that had been abraded into a slice as thin as possible with only nacreous layer. Then, ion-mill method was used to make the sample thin enough to observe in a Hitachi-800 TEM.
3. Results and discussion Fig. 1a shows the nacre tablets after NaOH treatment, the proteins on/between the tablets are almost dissolved and washed off. Figs. 1b and c show the morphology after EDTA treatment. It can be seen that EDTA first yields some etch-holes into the tablets, most possibly from dislocation points on the surface. Then the hole becomes larger and larger until this tablet disappears (black arrow in Fig. 1b). This kind of etching mode induces the flower-like morphology. And with the increase of etching time, this morphology remains unchanged, except that the etched depth increased (Fig. 1c). On the other hand, the morphology of the nacre surface after citric acid treatment is unlike that after EDTA treatment. First dissolved part in a tablet is not on the top face but on the side face. Most aragonite tablets exhibit same rectangular form and a preferred formal orientation (Fig. 2). This formal orientation preference is not only in a range of several tablets but are also in a much larger area as shown in Fig. 2c (at least 100 mm). This phenomenon can be seen in different tablets either along the tablet plane or along the direction perpendicular to nacreous plane. For convenience, we call them type I tablets. Whereas, there still exist tablets in some domains that have different formal orientation with type I tablets (black circles in Figs. 2a and b), these tablets are normally in some clusters and also have same gradient. We call them type II tablets.
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Fig. 1. SEM morphology of the nacre tablets after treatment. (a) After NaOH treatment, the proteins on/between the tablets are almost dissolved and washed off. The forms of the tablets are various. (b) After NaOH and EDTA treatment successively, many etch holes can be seen on the top face of the tablets. The etching time is 12 h. (c) Similar flower-like morphology after etching for 24 h. Scale: a, 13.8 mm; b, 7.3 mm; c, 7.4 mm.
Fig. 2. After citric acid treatment, aragonite tablets exhibited similar rectangular form and preferred formal orientation. The length proportion of the adjacent formal edges is approximately 5:8. (a), (b) The etching time is 10 min. (c) The etching time is 24 h. Scale: a, 11.6 mm; b, 9.3 mm; c, 22 mm; d, 7.7 mm.
Does the formal orientation indicate the crystal orientation of aragonite tablets? We think it does. As the surface proteins had been eliminated, it could not adsorb on the aragonite to affect the etching process. When the citric acid is added, it prefers reacting with the edge atoms to the normal surface atoms. With the dissolution going on, the form of the tablets changes from various to similar probably because of the anisotropy of the aragonite crystals. In order to ascertain the crystal orientation of the individual tablets, we used selected area diffraction (SAD) method of TEM. The patterns are shown in Fig. 3a. Every tablet is
selected in separate visual fields to make certain that they are not at a distance of several tablets. It is obvious from Fig. 3c that their crystal orientations incline to be normally distributed, although they are not completely in one direction. This investigation confirmed the SEM results. Statistical chart of directions in Fig. 2a reveals that 63% of the orientations of aragonite tablets are concentrated within thirty degrees from –20 to 10 (Fig. 4). Type I tablets have high proportion (the main peak in Fig. 4) and type II tablets have a lower proportion (the two secondary peaks in Fig. 4).
ARTICLE IN PRESS W.T. Hou, Q.L. Feng / Journal of Crystal Growth 258 (2003) 402–408
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Fig. 3. (a) SAD patterns of some tablets selected from different visual fields of TEM. (b) The crystal indexes and a-axis direction of a5 are labeled out as an instantiation. (c) The defined directions of 90 , 0 , 90 and eight a-axis (a1–a8) of tablets are shown in (a). These a-axes decline to be normally distributed around 30 except a6.
As we demonstrated the formal orientation indicated the crystal orientation, its preference could be interpreted by structural correspondence. There are two kinds of organic matrix in the mollusk shell, EDTA-soluble proteins and EDTAinsoluble proteins. Soluble proteins have a common amino-acid sequence of Asp-X-Asp (X is a neutral residue). Sequence of this type in antiparallel b-pleated sheet of silk-fibroin-like proteins
could present optimum binding (and hence nucleation) configuration for Ca2+ or CaCO3 microcrystals at the matrix interface [17]. Lavi et al. found that the synthesized polypeptide of (Asp-Leu)n is capable of specifically inducing aragonite formation [15]. Poly-L-Asp is also believed to control the oriented nucleation of aragonite in collagenous matrices [19]. Recently works of Orme et al. has shown that Asp can
ARTICLE IN PRESS 406
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Fig. 4. Two hundred tablets in Fig. 2a are selected and measured. The defined 90 , 0 and 90 direction is same with that in Fig. 3c. The Savitzky-Golay smoothing curve in the chart reveals that there is a main peak around 7 and two secondary peaks around 59 and 53 . 63% of the degrees are in the range from –20 to 10 .
adsorbed on the (1 0 4) steps of calcite to change the step-edge energy [20]. All these works indicated that Asp plays a leading role of biominerialization in vivo. Weiner et al. had found in mollusk shell that the antiparallel b-pleated sheet of the matrix and the overlaying aragonite crystal lattice are matched in orientation at the interface [18]. We also observed type II tablets in their SEM morphology of the bivalve, Neotrigonia margaritacea. It implied that the phenomenon is ubiquitous in mollusk shell of bivalve. Based on the matching assumption, we attempted to find the reason of orientation preference. As the c-axis of the aragonite is perpendicular to the tablet surface, we only consider the ab-face matching to the proteins. Fig. 5 is the schematic representation of the possible geometrical matching, of which Mode 1 is the most optimum. The matching errors (Table 1) and number of matching points of the three modes are discussed below. Mode 1 is the most possible matching. Its matching error is very low except that of [0 1 0] direction, 13.3%. But it is sufficient to firmly bind a microcrystal of three or four lattice size, as the protein sheets are more likely to bind ionic regiment or CaCO3 microcrystal than Ca2+ atoms
Fig. 5. Schematic model of the structural relationships between protein sheets and ab-face of aragonite crystals. The basal rectangle lattice with crosses represents the main structure of antiparallel b-pleated sheets, which have many possible Asp positions. The rectangles with spheres (Ca atoms) represent the ab-face of aragonite. There are three possible matching modes. Their matching errors are listed in Table 1.
to produce oriented crystal nuclei [17]. There are four matching points as seen in Fig. 5. Mode 2 has three perfect matching points in a line of [1 1 0] direction. If they are bound, other Ca2+ atoms in the microcrystal will search for matching and stabilize the binding. Mode 3 is comparatively difficult to be stabilized, as it has less matching point and higher matching errors. Geometric analysis indicates that mode 2 and 3 matching have a gradient of 58 and 34.5 with mode 1 matching (Table 1). The angles between peaks in Fig. 4 (52 and 60 ) are very close to the gradient of mode 2. This angle coherence implies that types I and II tablets may be related with modes 1 and 2 matching. When numerous microcrystals of aragonite compete to be nucleus, these two most stable matching modes have high probability to happen in statistics. So most tablets (type I) have same orientation because their nuclei are mode 1 matching, and some tablets (type II) have a gradient of around 60 with type I tablets because their nuclei are mode 2 matching. From the black circles in Figs. 2a and b, we can see that type II tablets are not individual with each other but in some clusters. If every tablet of nacreous layer is grown from an aragonite nucleus
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407
Table 1 Matching errors of the three modes between protein sheets and Ca atoms Mode
Matching errors of a [1 0 0] length (%)
1 2 3
4.3 12.6 18.3
Gradient with mode 1
b [0 1 0] length (%) 13.3 0.6 5.3
[1 1 0] length (%)
[1 0 0] direction
[0 1 0] direction
[1 1 0] direction
— 1.3 —
0 2.5 1.5
0 6.2 0
— 0 —
stabilized by protein sheets, tablets of mode 2 matching would equably distributed in three dimensions of nacreous layer. The cluster character can be interpreted only through the growth mechanism of mineral bridges. A lower lamina aragonite tablet can grow through the pores in the organic interlamellar sheets (mineral bridges) to form a new tablet. This new tablet preserves the crystal orientation of the old one and hence may transfer the character to the upper lamina. Since type I tablets has high proportion, this process in them is not as obvious as that in type II tablets. These two mechanisms of structural correspondence and mineral bridges are dissimilar but not incompatible with each other. There are two possible relationships between them in vivo: successive or competitive. Successive relationship can be described as follows. Formation of the primary tablets of nacreous layer is controlled by structural correspondence. The later formed tablets are all grown from the primary ones through mineral bridges (Fig. 6a). So type II tablets’ orientation may be inherited and observed. Competitive relationship can be described as all tablets of nacreous layer are formed either by structural correspondence or through mineral bridges. If there are several pores in the organic sheets in the growth area, calcium carbonate may grow on the base of under lamina minerals to release supersaturation. If there are no pores in the growth area, heteroepitaxial nucleation may happen on the protein sheets surface to allow calcium carbonate growing up. We propose that the difference lies in the density and size of pores in the matrix sheets. In gastropod shell such as abalone, organic sheets have pores of 20–100 nm and same space size between them [7,9]. The large size and high density of pores allow
0 758 734.5
Fig. 6. Two-dimension paradigm of structural correspondence and mineral bridges induced nacreous layer. The grey lines represent matrix sheets and the white and black zones represent type I tablets and cluster of type II tablets, respectively. Notice the mineral bridges along the growth direction. (a) Successive relationship. A primary type II tablet grows to the outer surface of nacreous layer through mineral bridges. (b) Competitive relationship. Construction correspondence induced type II tablets equably distributed in three dimensions. And they may or may not grow to the next lamina.
aragonite to grow vertically without much difficulty before a tablet shapes up laterally. The result is that the growth surface of gastropod exhibits stacked arrangement of aragonite crystals. Whereas in bivalve shell, pores of organic sheets are likely to have smaller size and growth into these pores would additionally increase the surface area, which is unstable in thermodynamics for the growing crystal. So the crystals first choose to grow laterally to form a complete tablet until they have to grow to the next lamina. This exhibits brick-wall type arrangement. If the density of pores in bivalve shell
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protein sheets is not very low, aragonite crystals may keep on growing through them as described in Fig. 6a. If the density is too low that not every tablet can snatch at least one pore in its territory, Fig. 6b may happen. It depends on the pore density. Further research should be focused on these. 4. Conclusion There are two types of tablets in mollusk shell of bivalve, Mytilus edulis. Type I tablets have almost same orientations of a- or b-axes. Type II tablets have lower proportion, and their crystal orientation has a gradient around 60 with that of type I. They exhibit a kind of cluster character. Two modes of structure correspondence are developed to interpret the orientation preference. (0 0 1) face of aragonite microcrystals decline to match on the b-pleated sheet of proteins and the stabilized microcrystals may become nucleus to grow into aragonite platelets. Mineral bridges have significant function in the growth of nacreous layer. Crystals may grow through them and become the nucleus of another tablet. That is why type II tablets assemble into some clusters. The mechanism of structure correspondence and mineral bridges may concur in the formation of nacreous layer of bivalve. The possible successive and competitive relationships can explain the observation results very well. Acknowledgements This work is supported by National Natural Science Foundation of china, Grant 50272035.
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