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In vitro growth of flat aragonite crystals between the layers of the insoluble organic matrix of the abalone Haliotis laevigata
Journal of Crystal Growth 358 (2012) 75–80
Contents lists available at SciVerse ScienceDirect
Journal of Crystal Growth journal homepage: www.elsevier.com/locate/jcrysgro
In vitro growth of flat aragonite crystals between the layers of the insoluble organic matrix of the abalone Haliotis laevigata Katharina I. Gries a,b,n, Fabian Heinemann a,1, Andreas Rosenauer b, Monika Fritz a a b
Institut f¨ ur Biophysik, Universit¨ at Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany Institut f¨ ur Festk¨ orperphysik, Universit¨ at Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany
a r t i c l e i n f o
abstract
Article history: Received 27 February 2012 Received in revised form 27 July 2012 Accepted 4 August 2012 Communicated by S.R. Qiu Available online 11 August 2012
Nacre of abalone shells consists of aragonite platelets and organic material, the so-called organic matrix. During the growth process of the shell the aragonite platelets grow into a scaffold formed by the organic matrix. In this work we tried to mimic this growth process by placing a piece of the insoluble organic matrix (which is a part of the organic matrix) of the abalone Haliotis laevigata in a crystallization device which was flowed through by CaCl2 and NaHCO3 solutions. Using this setup amongst others flat aragonite crystals grow on the insoluble organic matrix. When investigating these crystals in a transmission electron microscope it is possible to recognize similarities to the structure of nacre, like the formation of mineral bridges and growth between layers of the insoluble organic matrix. These similarities are presented in this paper. & 2012 Elsevier B.V. All rights reserved.
Keywords: A1. Biocrystallization A1. Transmission electron microscopy A2. In vitro crystal growth B1. Nacre B1. Insoluble organic matrix B1. Calcium carbonate
1. Introduction Nacre is a part of many mollusc shells and a very exceptional biomineral that exhibits a highly ordered structure consisting of vertically stacked aragonite platelets that are horizontally arranged in layers as well as organic matter. Aragonite is a CaCO3 polymorph with an orthorhombic unit cell (space group Pmcn No. 62). The diameter and the thickness of the aragonite platelets are approximately 5210 mm and 500 nm respectively. The c-axis of the aragonite unit cell is almost perpendicular to the face of the platelets and therefore to the surface of the shell. The organic matter is denominated as organic matrix. It just amounts to approximately 1.6% of the total mass of the nacreous part of the shell from abalone Haliotis laevigata [1] and surrounds the aragonite platelets. The organic matrix between vertically stacked aragonite platelets, the so-called interlamellar organic matrix, has a thickness of approximately 40 nm, whereas the thickness of the intertabular organic matrix, which is the organic matrix between horizontally arranged platelets, is much lower. Finally a brickand-mortar-like structure made of alternating layers of hard
n Corresponding author. Present address: Materials Science Center and Depart¨ Marburg, Hans-Meerwein-Straße 6, 35043 ment of Physics, Philipps Universitat Marburg, Germany. Tel.: þ49 6421 2822249; fax þ49 6421 28935. E-mail address: [email protected] (K.I. Gries). 1 Present address: Biophysical Institute, BIOTEC/TU Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
0022-0248/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcrysgro.2012.08.005
mineral and soft organic matter is formed causing the outstanding mechanical behaviour of nacre [2,3]. Apart from the subdivision of the organic matrix into the interlamellar and the intertabular organic matrix, a subdivision into the insoluble and the soluble organic matrix is common as well. These both components remain after demineralizing nacre using ethylenediaminetetraacetic acid (EDTA) or an acid such as diluted acetic acid. The soluble matrix contains all soluble proteins whereas the insoluble organic matrix contains all insoluble components like chitin and some proteins. The fraction of soluble proteins depends on the chemicals used for demineralization and on the concentration of these chemicals. The same applies to the fraction of the insoluble proteins. Thus, the composition of the soluble and insoluble organic matrix is depending on the method of demineralization. In abalone nacre the mineral and the organic phase do not grow in an alternating manner but the aragonite platelets grow in the preformed layers of the interlamellar organic matrix [4–8]. These preformed organic matrix layers are soaked by the extrapallial fluid containing ions as well as proteins that are incorporated into the shell during the growth process. In this process the newly formed aragonite platelets grow at first mainly in the c-direction until they reach a layer of the preformed interlamellar organic matrix and than spread out in the a- and b-direction until they hit upon neighbouring platelets. The proteins that are tracked between these platelets create the intertabular organic matrix. Before the aragonite platelets reach their final lateral magnitude, a new layer of platelets is generated above. This kind
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of growth process suggests that not only mineral bridges [9,10], which transfer information on the crystallographic orientation from one platelet to another trough the interlamellar organic matrix, play a significant role. Additionally the organic matrix acting as a substrate and maybe the inclosed proteins seem to be important components having a strong influence on the formation of flat and similarly oriented aragonite platelets in nacre. In a previously published paper we already documented the in vitro growth of flat aragonite crystals on the insoluble organic matrix of abalone Haliotis laevigata [8] using a crystallization device. This growth process took place in the presence of different concentrations of the soluble organic matrix. Under these circumstances lower concentrations slightly facilitated the growth of flat aragonite crystals, whereas higher concentrations caused an inhibition of any kind of crystal growth. In contrast to the aragonite platelets in nacre the in vitro grown flat aragonite crystals are not oriented with their caxis perpendicular to the interlamellar organic matrix, but exhibit completely different orientations that are constant in delimited regions but not all over the flat aragonite crystals. In the present paper we focus on the structure and composition of the in vitro grown flat aragonite crystals by using transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) spectroscopy. Additionally we describe the similarities between these in vitro grown crystals and the aragonite platelets in nacre.
2. Material and methods 2.1. Preparation of the insoluble organic matrix. To obtain the insoluble organic matrix from shells of the seasnail Haliotis laevigata (from Fred Glasbrenner, Abalone Exports, Laverton North, Victoria, Australia) the shells were first cleaned from coarse dirt with a steel brush. Afterwards the outer calcite layer was removed by blasting with corundum slurry (Al2O3, diameter 0.12–0.25 mm; slurry blaster WA70, Sigg, Jestetten, Germany). An incubation of the shell for 2 min in a solution consisting of equal parts of deionized water cleaned by a Milli-Q water purification device (Millipore, Academic Q-Gard2, Billerica, MA, U.S.A.) and sodium hypochloride (NaOCl, Roth, Karlsruhe, Germany) was performed to avoid organic impurities. After rinsing the shell thoroughly with deionized water, it was smashed with a hammer into pieces with diameters of 2–3 cm. To demineralize these nacre pieces they were dialysed against 100 mM EDTA (AppliChem, Darmstadt, Germany) at 4 1C and pH 5 under constant stirring (used dialysis tubes: Spectrum, Rancho Dominguez, CA, U.S.A.; molecular weight cutoff (MWCO) of 6– 8000 Da). To prevent bacterial growth, 0.02% of sodium azide (NaN3, Fluka, Sigma-Aldrich GmbH, Steinheim, Germany) was added to the dialysis solution. The total volume was exchanged 15 times at intervals of 2–3 days. Although acetic acid allows a much faster demineralization and is additionally easier to remove from the insoluble organic matrix, EDTA was used to avoid damage of the insoluble organic matrix by CO2 formation. 2.2. Crystallization device To grow CaCO3 crystals on the insoluble organic matrix a crystallization device was used. Fig. 1 shows a sketch of the cross section of the crystallization device. A photograph and a plane view sketch of the crystallization device were already presented in previous publications [8,11]. By placing a punched out piece of the insoluble organic matrix with a diameter of 14 mm and a neoprene ring (inner and outer diameter: 12 mm and 14 mm, height: 0.48 mm) as a spacer in the middle of the crystallization device, it was divided into two
Fig. 1. Sketch of a cross section of the used crystallization device in which the insoluble organic matrix was positioned. The device is divided into two compartments (I and II) with separate NaHCO3 and CaCl2 inflows. Both solutions mix at the position of the permeable insoluble organic matrix where a local super-saturation is generated.
compartments. Through separate supply tubes 10–20 mM CaCl2 (AppliChem, Darmstadt, Germany) and NaHCO3 (Sigma, St. Louis, MO, USA) solutions were pumped into the compartments. Due to the separation the salt solutions were not mixed until they reach the permeable insoluble organic matrix. The flow rate was 0.26–0.32 ml/min/side and the duration of the experiment was approximately 16 h. To obtain a constant temperature the crystallization device was placed in a water bath which was connected to a thermostat. After the treatment in the crystallization device the insoluble organic matrix was cut into two pieces and placed on aluminum foil where it was dried at room temperature ð 23 1CÞ. One of the pieces was placed in such a way on the foil that the side, which was oriented in the crystallization device towards the CaCl2 solution, points upwards. The second piece was placed the other way round. In some of the experiments different concentrations of the soluble organic matrix have been added to the CaCl2 and NaHCO3 solutions ( 0:02 and 1 mg=ml respectively). The influence of this addition on the probability of crystal growth was presented in a previous paper [8]. A direct influence on the results presented in the present paper was not observed. For that reason we refrained from explaining the preparation procedure of the soluble organic matrix at this point. If there is interest you may refer to the obove mentioned paper.
2.3. Transmission electron microscopy (TEM) For preparation of cross sections of the in vitro grown flat aragonite crystals a focused ion beam system (FIB; FEI, Nova 200, Eindhoven, The Netherlands) combined with the lift-out technique (using: manipulator, Kleindiek, Reutlingen, Germany; needle, Picoprobe, Naples, FL, U.S.A.) was used. A more detailed description and images of this procedure are provided in a previous publication [8]. The dual beam FIB system was combined with a scanning electron microscope (SEM), which was used to obtain images of the in vitro grown flat aragonite crystals. Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) investigations were performed on a TITAN 80/300 (FEI, Eindhoven, The Netherlands), which was equipped with a field emission gun and was operated at an acceleration voltage of 300 kV. With a mounted EDX-detector (SiLi (silicon lithium) detector, EDAX, Ametek, Meerbusch, Germany) energy dispersive X-ray (EDX) spectra were recorded to determine the elemental composition of the specimens.
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3. Results and discussion
3.1. Layered structure
In principle two different kinds of structures of the in vitro grown flat aragonite crystals appear: blocklike and layered structures. Flat aragonite crystals with these two structures cannot be distinguished just by their shape but only when their cross section is investigated using scanning electron microscopy (SEM) or (S)TEM. Fig. 2(a) and (b) shows SEM images of flat aragonite crystals with a blocklike structure whereas the aragonite crystals in (c) and (d) have a layered structure. From these images no obvious differences between the crystals are recognizable. The differences of the structures appear first in the cross sectional samples shown in the STEM images in Fig. 2(e) and (f). The blocklike structure (e) does not reveal any significant substructures. The bright layer consists of platinum, which was deposited during preparation in the FIB. In contrast the in vitro grown aragonite crystal shown in Fig. 2(f) reveals a layered structure where the crystal is interrupted by horizontal layers (f).
The flat aragonite crystals exhibiting a layered structure are of special interest because they remind of the vertically stacked aragonite platelets in nacre. The STEM image of the layered aragonite crystals in Fig. 2(f) shows that the layers between the crystals exhibit a lower intensity than the crystal. Therefore these layers contain less or lighter material than the surrounding crystal. This fact may rise the assumption that these layers between the crystals contain organic matter and consist of residues of the interlamellar organic matrix. This would imply that the in vitro growth process of the flat layered aragonite crystals in the crystallization device is comparable to the growth process in nacre where the aragonite platelets are built in the preformed layers of the organic matrix. First investigations to support this hypothesis have been performed using conventional TEM. An image of a cross sectional sample of an in vitro grown layered flat aragonite crystal is presented in Fig. 3(a). The dark layer on top consists of platinum deposited in the FIB. The bright
Fig. 2. (a)–(d) SEM images of in vitro grown aragonite crystals on the insoluble organic matrix of Haliotis laevigata. (a) and (b) Blocklike structure, (c) and (d) layered structure. (e) and (f) STEM images of the cross section of the in vitro grown aragonite crystals shown in (c) and (d). (e) Blocklike structure, (f) layered structure. Pt: platinum deposition, FC: flat aragonite crystal, M: insoluble organic matrix.
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Fig. 3. (a) TEM image of the cross section of a layered flat aragonite crystal. Pt: platinum, FC: flat aragonite crystal. (b) Magnified TEM image of the marked area in (a). (c)– (f) Diffractograms that have been created on the four regions marked in (b). The diffractogram in (c) refers to amorphous material, whereas the diffractograms in (d)– (f) refer to crystalline material. The arrowheads point on some identical reflections indicating a transfer of crystallographic orientation. As examples the arrows mark additional reflections that do not appear in the diffractogram in (d).
Fig. 4. STEM image of the cross section of a layered flat aragonite crystal. EDX spectrum 1 has been generated at a region within the aragonite crystal, whereas spectrum 2 has been generated at a region within the amorphous layer.
horizontal layer in the middle of the image between two crystalline regions is the object of interest: the layer that may consist of residues of the interlamellar organic matrix. The marked area is magnified in Fig. 3(b). A diffractogram (Fig. 3(c)) created within the bright layer reveals the amorphous structure of the material at that position. The diffractograms of the other areas marked in Fig. 3(b) are discussed later.
3.1.1. EDX investigations To determine the origin of the amorphous layers within the in vitro grown aragonite crystals EDX spectra of TEM samples have been recorded. Fig. 4 shows a STEM image of the cross section of a layered aragonite crystal and EDX spectra from regions within the aragonite crystal (spectrum 1) and the amorphous layer (spectrum 2), respectively. Both investigated regions exhibited the same size and
the same dwell time was used during the measurement. Spectrum 1, which was recorded from an area in the aragonite crystal, contains as expected C Ka1 , O Ka1 , Ca Ka1 and Ca Kb1 peaks. Additionally to these signals spectrum 2 exhibits a distinct silicon peak. At a first glance this signal is unexpected and does not prove that the layers are residues of the insoluble organic matrix. As none of the chemicals used during the experiment contain silicon, the origin of this peak should not be due to preparationrelated artefacts. Since no such a silicon peak was detected in spectrum 1, the used SiLi-EDX-detector could be excluded as a possible source as well. To find a plausible explanation for the appearance of the silicon peak a closer look on nacre is helpful. Fig. 5 shows a STEM image of a cross sectional nacre specimen with two vertically stacked aragonite platelets and the interlamellar organic matrix in between. The two rectangles mark the regions where the displayed EDX spectra have been recorded.
K.I. Gries et al. / Journal of Crystal Growth 358 (2012) 75–80
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Fig. 5. STEM image of a cross sectional nacre specimen. EDX spectrum 1 has been generated at a region within an aragonite platelet, whereas spectrum 2 has been generated at a region within the interlamellar organic matrix.
Spectrum 1 has been taken from an area within an aragonite platelet, whereas spectrum 2 has been detected from an area within the interlamellar organic matrix. Spectrum 1 contains signals that stem from CaCO3. Spectrum 2 contains a high carbon signal which is expected for an area including organic matter. Because of some surface irregularities the aragonite platelets protrude slightly into the scanned area. Therefore oxygen and calcium signals appear in that spectrum, too. Both of the spectra contain small but distinct silicon signals. The source of the silicon might be the seawater that include at a salinity of 35% a small amount of silicon of 2900 mg=l [12]. During the growth process the silicon may be incorporated into the crystalline and organic parts of the shell. The fact that silicon could be detected within the amorphous layer of the in vitro grown flat aragonite crystals is a hint that the layers are residues of the native insoluble organic matrix used as a template for crystal growth. Amorphous material developed during the experiment using purified components and thus in the absence of seawater would not contain silicon. The silicon signal detected in spectrum 2 in Fig. 4 is much more pronounced than the silicon signals detected in nacre (Fig. 5). Moreover it is conspicuous that it is higher than the calcium and carbon signals obtained from the same sample area. A possible explanation is that during the demineralization of the shell the part of the silicon, being originally located within the aragonite platelets, is concentrated in the layers of the insoluble organic matrix and therefore causes the increased silicon signal.
3.1.2. Appearance of mineral bridges Besides the fact that the amorphous layers are most likely residues of the insoluble organic matrix, which means that the aragonite crystals have grown within the scaffold of the insoluble organic matrix, another analogy with nacre appears. This analogy is shown in Fig. 3. The TEM image in Fig. 3(a) shows not only the residues of the insoluble organic matrix but also some kind of mineral bridges connecting the lower and the upper part of the aragonite crystal. Part of such a bridgelike structure is magnified in Fig. 3(b). In nacre mineral bridges transfer information on the crystallographic orientation from one aragonite platelet to the overlying one. This means that the orientation of the aragonite crystals above and below a mineral bridge is the same. To prove if this is also the case for in vitro grown flat aragonite crystals, diffractograms have been created within the bridgelike structure (e) and in regions within the aragonite crystals
below and above the bridgelike structure ((d) and (f)). The diffractograms contain mainly the same reflections. Some of these are marked with arrowheads. The existence of the identical reflections shows that there is no significant variation in the orientation of the aragonite crystal in the three regions. This implies that during the in vitro growth process the aragonite crystals not just grow within the scaffold of the insoluble organic matrix, but also through pores in the organic matrix forming some kind of mineral bridges like in nacre. Besides the identical reflections that appear in the three diffractograms ((d)–(f)) additional reflections are present in the diffractograms (e) and (f) and are marked with arrows. These additional reflections point out that the in vitro grown aragonite crystals are not perfect single crystals. This was already mentioned in [8] where a selected area diffraction aperture had to be used to obtain diffraction pattern which could be analyzed. Thus, the orientation of the aragonite crystals is just constant in delimited regions. The bridgelike structures transfer therefore information on the crystallographic orientation through the residues of the insoluble organic matrix only in locally limited areas. This is a significant difference to the structure of nacre and shows that the in vitro growth process still needs improvement. 3.2. Blocklike structure Besides the layered structure of the in vitro grown flat aragonite crystals, the existence of the blocklike structure should be considered, too. The appearance of these structures is actually not so surprising. A comparison with nacre shows that blocklike aragonite platelets appear that are not interrupted by the organic matrix [13,14]. These platelets are up to several micrometers in thickness. Additionally clear boundaries between horizontally adjacent platelets are neither in SEM nor in TEM images visible. Mostly these platelets build a layer on the inner surface of the shell which is oriented towards the body of the snail. Which processes control the formation of such a layer is still not known. But the same processes (induced for example by specific proteins that are located in some areas of the insoluble organic matrix) may influence the formation of flat blocklike aragonite crystals in our experiment.
4. Conclusions In this work we investigated flat aragonite crystals that have been grown on the insoluble organic matrix of the abalone Haliotis laevigata. To realize the formation of these crystals the insoluble
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organic matrix was placed in a crystallization device flowed through by CaCl2 and NaHCO3 solutions with concentrations of 10–20 mM. The investigation of the structure of the in vitro grown flat aragonite crystals was performed with a TEM. The flat aragonite crystals exhibited mainly two different structures: blocklike or layered. Because of the reminiscence of nacre structure, the layered structure of the in vitro grown flat aragonite crystals is of special interest. TEM investigations revealed that the layered structure is created by the intersection of the aragonite crystals with amorphous layers. EDX spectra recorded on these amorphous layers showed that they contain silicon. This is in contrast to the crystalline regions. Nacre specimens on the other hand contain silicon in the interlamellar organic matrix as well as in the aragonite platelets. One conclusion is that silicon is incorporated from the seawater into the shell during growth. Consequently all natural components of the in vitro grown samples contain silicon whereas the part of the sample created in the crystallization device (like the aragonite crystals) will not contain any silicon. In this light, it is most probable that the amorphous layers are residues of the insoluble organic matrix. Thus, the growth process is similar to that in nacre. An additional similarity to nacre is the appearance of bridgelike mineral structures connecting two vertically stacked parts of an in vitro grown aragonite crystal. As in nacre these mineral bridges transfer information on the crystallographic orientation through the layer of the organic matrix (respectively the residues of the insoluble organic matrix). In contrast to nacre the in vitro grown aragonite crystals maintain the orientation only in proximity to the bridgelike structures.
Acknowledgments K.I. Gries thanks the Physics International Postgraduate Programme (PIP) at Bremen University for sponsorship.
References [1] F. Heinemann, Investigation of Biopolymer–Mineral Interactions in the Natural Composite Material Nacre, Ph.D. Thesis, University of Bremen, 2008. [2] R. Wang, Z. Suo, A. Evans, N. Yao, I. Aksay, Deformation mechanisms in nacre, Journal of Materials Research 16 (9) (2001) 2485–2493. [3] K.S. Katti, D.R. Katti, Why is nacre so tough and strong? Materials Science and Engineering: C 26 (8) (2006) 1317–1324. [4] K.M. Towe, G.H. Hamilton, Ultrastructure and inferred calcification of the mature and developing nacre in bivalve mollusks, Calcified Tissue International 1 (1967) 306–318. [5] G. Bevelander, H. Nakahara, An electron microscope study of the formation of the nacreous layer in the shell of certain bivalve molluscs, Calcified Tissue Research 3 (1969) 84–92. [6] H. Nakahara, Calcification of gastropod nacre, in: P. Westbrock (Ed.), Biomineralization and biological metal accumulation, D. Reider Publishing Company, 1983, pp. 225–228. [7] H. Nakahara, Nacre formation in bivalve and gastropod molluscs, in: S. Suga, H. Nakahara (Eds.), Mechanisms and Phylogeny of Mineralization in Biological Systems, Springer-Verlag, 1991, pp. 343–350. [8] K. Gries, F. Heinemann, M. Gummich, A. Ziegler, A. Rosenauer, M. Fritz, Influence of the insoluble and soluble matrix of abalone nacre on the growth of calcium carbonate crystals, Crystal Growth and Design 11 (3) (2011) 729–734. ¨ ¨ [9] K. Gries, R. Kroger, C. Kubel, M. Schowalter, M. Fritz, A. Rosenauer, Correlation of the orientation of stacked aragonite platelets in nacre and their connection via mineral bridges, Ultramicroscopy 109 (3) (2009) 230–236. [10] F. Song, A.K. Soh, Y.L. Bai, Structural and mechanical properties of the organic matrix layers of nacre, Biomaterials 24 (2003) 3623–3631. [11] F. Heinemann, L. Treccani, M. Fritz, Abalone nacre insoluble matrix induces growth of flat and oriented aragonite crystals, Biochemical and Biophysical Research Communications 344 (1) (2006) 45–49. [12] K.K. Turekian, Oceans, Prentice-Hall, Inc., Englewood Cliffs, New Jersey, 1968. [13] X. Su, A.M. Belcher, C.M. Zaremba, D.E. Morse, G.D. Stucky, A.H. Heuer, Structural and microstructural characterization of the growth lines and prismatic microarchitecture in red abalone shell and the microstructures of abalone ‘flat pearls’, Chemistry of Materials 14 (7) (2002) 3106–3117. [14] M. Cusack, A. Freer, Biomineralization: elemental and organic influence in carbonate systems, Chemical Reviews 108 (11) (2008) 4433–4454.
Contents lists available at SciVerse ScienceDirect
Journal of Crystal Growth journal homepage: www.elsevier.com/locate/jcrysgro
In vitro growth of flat aragonite crystals between the layers of the insoluble organic matrix of the abalone Haliotis laevigata Katharina I. Gries a,b,n, Fabian Heinemann a,1, Andreas Rosenauer b, Monika Fritz a a b
Institut f¨ ur Biophysik, Universit¨ at Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany Institut f¨ ur Festk¨ orperphysik, Universit¨ at Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany
a r t i c l e i n f o
abstract
Article history: Received 27 February 2012 Received in revised form 27 July 2012 Accepted 4 August 2012 Communicated by S.R. Qiu Available online 11 August 2012
Nacre of abalone shells consists of aragonite platelets and organic material, the so-called organic matrix. During the growth process of the shell the aragonite platelets grow into a scaffold formed by the organic matrix. In this work we tried to mimic this growth process by placing a piece of the insoluble organic matrix (which is a part of the organic matrix) of the abalone Haliotis laevigata in a crystallization device which was flowed through by CaCl2 and NaHCO3 solutions. Using this setup amongst others flat aragonite crystals grow on the insoluble organic matrix. When investigating these crystals in a transmission electron microscope it is possible to recognize similarities to the structure of nacre, like the formation of mineral bridges and growth between layers of the insoluble organic matrix. These similarities are presented in this paper. & 2012 Elsevier B.V. All rights reserved.
Keywords: A1. Biocrystallization A1. Transmission electron microscopy A2. In vitro crystal growth B1. Nacre B1. Insoluble organic matrix B1. Calcium carbonate
1. Introduction Nacre is a part of many mollusc shells and a very exceptional biomineral that exhibits a highly ordered structure consisting of vertically stacked aragonite platelets that are horizontally arranged in layers as well as organic matter. Aragonite is a CaCO3 polymorph with an orthorhombic unit cell (space group Pmcn No. 62). The diameter and the thickness of the aragonite platelets are approximately 5210 mm and 500 nm respectively. The c-axis of the aragonite unit cell is almost perpendicular to the face of the platelets and therefore to the surface of the shell. The organic matter is denominated as organic matrix. It just amounts to approximately 1.6% of the total mass of the nacreous part of the shell from abalone Haliotis laevigata [1] and surrounds the aragonite platelets. The organic matrix between vertically stacked aragonite platelets, the so-called interlamellar organic matrix, has a thickness of approximately 40 nm, whereas the thickness of the intertabular organic matrix, which is the organic matrix between horizontally arranged platelets, is much lower. Finally a brickand-mortar-like structure made of alternating layers of hard
n Corresponding author. Present address: Materials Science Center and Depart¨ Marburg, Hans-Meerwein-Straße 6, 35043 ment of Physics, Philipps Universitat Marburg, Germany. Tel.: þ49 6421 2822249; fax þ49 6421 28935. E-mail address: [email protected] (K.I. Gries). 1 Present address: Biophysical Institute, BIOTEC/TU Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
0022-0248/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcrysgro.2012.08.005
mineral and soft organic matter is formed causing the outstanding mechanical behaviour of nacre [2,3]. Apart from the subdivision of the organic matrix into the interlamellar and the intertabular organic matrix, a subdivision into the insoluble and the soluble organic matrix is common as well. These both components remain after demineralizing nacre using ethylenediaminetetraacetic acid (EDTA) or an acid such as diluted acetic acid. The soluble matrix contains all soluble proteins whereas the insoluble organic matrix contains all insoluble components like chitin and some proteins. The fraction of soluble proteins depends on the chemicals used for demineralization and on the concentration of these chemicals. The same applies to the fraction of the insoluble proteins. Thus, the composition of the soluble and insoluble organic matrix is depending on the method of demineralization. In abalone nacre the mineral and the organic phase do not grow in an alternating manner but the aragonite platelets grow in the preformed layers of the interlamellar organic matrix [4–8]. These preformed organic matrix layers are soaked by the extrapallial fluid containing ions as well as proteins that are incorporated into the shell during the growth process. In this process the newly formed aragonite platelets grow at first mainly in the c-direction until they reach a layer of the preformed interlamellar organic matrix and than spread out in the a- and b-direction until they hit upon neighbouring platelets. The proteins that are tracked between these platelets create the intertabular organic matrix. Before the aragonite platelets reach their final lateral magnitude, a new layer of platelets is generated above. This kind
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of growth process suggests that not only mineral bridges [9,10], which transfer information on the crystallographic orientation from one platelet to another trough the interlamellar organic matrix, play a significant role. Additionally the organic matrix acting as a substrate and maybe the inclosed proteins seem to be important components having a strong influence on the formation of flat and similarly oriented aragonite platelets in nacre. In a previously published paper we already documented the in vitro growth of flat aragonite crystals on the insoluble organic matrix of abalone Haliotis laevigata [8] using a crystallization device. This growth process took place in the presence of different concentrations of the soluble organic matrix. Under these circumstances lower concentrations slightly facilitated the growth of flat aragonite crystals, whereas higher concentrations caused an inhibition of any kind of crystal growth. In contrast to the aragonite platelets in nacre the in vitro grown flat aragonite crystals are not oriented with their caxis perpendicular to the interlamellar organic matrix, but exhibit completely different orientations that are constant in delimited regions but not all over the flat aragonite crystals. In the present paper we focus on the structure and composition of the in vitro grown flat aragonite crystals by using transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) spectroscopy. Additionally we describe the similarities between these in vitro grown crystals and the aragonite platelets in nacre.
2. Material and methods 2.1. Preparation of the insoluble organic matrix. To obtain the insoluble organic matrix from shells of the seasnail Haliotis laevigata (from Fred Glasbrenner, Abalone Exports, Laverton North, Victoria, Australia) the shells were first cleaned from coarse dirt with a steel brush. Afterwards the outer calcite layer was removed by blasting with corundum slurry (Al2O3, diameter 0.12–0.25 mm; slurry blaster WA70, Sigg, Jestetten, Germany). An incubation of the shell for 2 min in a solution consisting of equal parts of deionized water cleaned by a Milli-Q water purification device (Millipore, Academic Q-Gard2, Billerica, MA, U.S.A.) and sodium hypochloride (NaOCl, Roth, Karlsruhe, Germany) was performed to avoid organic impurities. After rinsing the shell thoroughly with deionized water, it was smashed with a hammer into pieces with diameters of 2–3 cm. To demineralize these nacre pieces they were dialysed against 100 mM EDTA (AppliChem, Darmstadt, Germany) at 4 1C and pH 5 under constant stirring (used dialysis tubes: Spectrum, Rancho Dominguez, CA, U.S.A.; molecular weight cutoff (MWCO) of 6– 8000 Da). To prevent bacterial growth, 0.02% of sodium azide (NaN3, Fluka, Sigma-Aldrich GmbH, Steinheim, Germany) was added to the dialysis solution. The total volume was exchanged 15 times at intervals of 2–3 days. Although acetic acid allows a much faster demineralization and is additionally easier to remove from the insoluble organic matrix, EDTA was used to avoid damage of the insoluble organic matrix by CO2 formation. 2.2. Crystallization device To grow CaCO3 crystals on the insoluble organic matrix a crystallization device was used. Fig. 1 shows a sketch of the cross section of the crystallization device. A photograph and a plane view sketch of the crystallization device were already presented in previous publications [8,11]. By placing a punched out piece of the insoluble organic matrix with a diameter of 14 mm and a neoprene ring (inner and outer diameter: 12 mm and 14 mm, height: 0.48 mm) as a spacer in the middle of the crystallization device, it was divided into two
Fig. 1. Sketch of a cross section of the used crystallization device in which the insoluble organic matrix was positioned. The device is divided into two compartments (I and II) with separate NaHCO3 and CaCl2 inflows. Both solutions mix at the position of the permeable insoluble organic matrix where a local super-saturation is generated.
compartments. Through separate supply tubes 10–20 mM CaCl2 (AppliChem, Darmstadt, Germany) and NaHCO3 (Sigma, St. Louis, MO, USA) solutions were pumped into the compartments. Due to the separation the salt solutions were not mixed until they reach the permeable insoluble organic matrix. The flow rate was 0.26–0.32 ml/min/side and the duration of the experiment was approximately 16 h. To obtain a constant temperature the crystallization device was placed in a water bath which was connected to a thermostat. After the treatment in the crystallization device the insoluble organic matrix was cut into two pieces and placed on aluminum foil where it was dried at room temperature ð 23 1CÞ. One of the pieces was placed in such a way on the foil that the side, which was oriented in the crystallization device towards the CaCl2 solution, points upwards. The second piece was placed the other way round. In some of the experiments different concentrations of the soluble organic matrix have been added to the CaCl2 and NaHCO3 solutions ( 0:02 and 1 mg=ml respectively). The influence of this addition on the probability of crystal growth was presented in a previous paper [8]. A direct influence on the results presented in the present paper was not observed. For that reason we refrained from explaining the preparation procedure of the soluble organic matrix at this point. If there is interest you may refer to the obove mentioned paper.
2.3. Transmission electron microscopy (TEM) For preparation of cross sections of the in vitro grown flat aragonite crystals a focused ion beam system (FIB; FEI, Nova 200, Eindhoven, The Netherlands) combined with the lift-out technique (using: manipulator, Kleindiek, Reutlingen, Germany; needle, Picoprobe, Naples, FL, U.S.A.) was used. A more detailed description and images of this procedure are provided in a previous publication [8]. The dual beam FIB system was combined with a scanning electron microscope (SEM), which was used to obtain images of the in vitro grown flat aragonite crystals. Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) investigations were performed on a TITAN 80/300 (FEI, Eindhoven, The Netherlands), which was equipped with a field emission gun and was operated at an acceleration voltage of 300 kV. With a mounted EDX-detector (SiLi (silicon lithium) detector, EDAX, Ametek, Meerbusch, Germany) energy dispersive X-ray (EDX) spectra were recorded to determine the elemental composition of the specimens.
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3. Results and discussion
3.1. Layered structure
In principle two different kinds of structures of the in vitro grown flat aragonite crystals appear: blocklike and layered structures. Flat aragonite crystals with these two structures cannot be distinguished just by their shape but only when their cross section is investigated using scanning electron microscopy (SEM) or (S)TEM. Fig. 2(a) and (b) shows SEM images of flat aragonite crystals with a blocklike structure whereas the aragonite crystals in (c) and (d) have a layered structure. From these images no obvious differences between the crystals are recognizable. The differences of the structures appear first in the cross sectional samples shown in the STEM images in Fig. 2(e) and (f). The blocklike structure (e) does not reveal any significant substructures. The bright layer consists of platinum, which was deposited during preparation in the FIB. In contrast the in vitro grown aragonite crystal shown in Fig. 2(f) reveals a layered structure where the crystal is interrupted by horizontal layers (f).
The flat aragonite crystals exhibiting a layered structure are of special interest because they remind of the vertically stacked aragonite platelets in nacre. The STEM image of the layered aragonite crystals in Fig. 2(f) shows that the layers between the crystals exhibit a lower intensity than the crystal. Therefore these layers contain less or lighter material than the surrounding crystal. This fact may rise the assumption that these layers between the crystals contain organic matter and consist of residues of the interlamellar organic matrix. This would imply that the in vitro growth process of the flat layered aragonite crystals in the crystallization device is comparable to the growth process in nacre where the aragonite platelets are built in the preformed layers of the organic matrix. First investigations to support this hypothesis have been performed using conventional TEM. An image of a cross sectional sample of an in vitro grown layered flat aragonite crystal is presented in Fig. 3(a). The dark layer on top consists of platinum deposited in the FIB. The bright
Fig. 2. (a)–(d) SEM images of in vitro grown aragonite crystals on the insoluble organic matrix of Haliotis laevigata. (a) and (b) Blocklike structure, (c) and (d) layered structure. (e) and (f) STEM images of the cross section of the in vitro grown aragonite crystals shown in (c) and (d). (e) Blocklike structure, (f) layered structure. Pt: platinum deposition, FC: flat aragonite crystal, M: insoluble organic matrix.
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Fig. 3. (a) TEM image of the cross section of a layered flat aragonite crystal. Pt: platinum, FC: flat aragonite crystal. (b) Magnified TEM image of the marked area in (a). (c)– (f) Diffractograms that have been created on the four regions marked in (b). The diffractogram in (c) refers to amorphous material, whereas the diffractograms in (d)– (f) refer to crystalline material. The arrowheads point on some identical reflections indicating a transfer of crystallographic orientation. As examples the arrows mark additional reflections that do not appear in the diffractogram in (d).
Fig. 4. STEM image of the cross section of a layered flat aragonite crystal. EDX spectrum 1 has been generated at a region within the aragonite crystal, whereas spectrum 2 has been generated at a region within the amorphous layer.
horizontal layer in the middle of the image between two crystalline regions is the object of interest: the layer that may consist of residues of the interlamellar organic matrix. The marked area is magnified in Fig. 3(b). A diffractogram (Fig. 3(c)) created within the bright layer reveals the amorphous structure of the material at that position. The diffractograms of the other areas marked in Fig. 3(b) are discussed later.
3.1.1. EDX investigations To determine the origin of the amorphous layers within the in vitro grown aragonite crystals EDX spectra of TEM samples have been recorded. Fig. 4 shows a STEM image of the cross section of a layered aragonite crystal and EDX spectra from regions within the aragonite crystal (spectrum 1) and the amorphous layer (spectrum 2), respectively. Both investigated regions exhibited the same size and
the same dwell time was used during the measurement. Spectrum 1, which was recorded from an area in the aragonite crystal, contains as expected C Ka1 , O Ka1 , Ca Ka1 and Ca Kb1 peaks. Additionally to these signals spectrum 2 exhibits a distinct silicon peak. At a first glance this signal is unexpected and does not prove that the layers are residues of the insoluble organic matrix. As none of the chemicals used during the experiment contain silicon, the origin of this peak should not be due to preparationrelated artefacts. Since no such a silicon peak was detected in spectrum 1, the used SiLi-EDX-detector could be excluded as a possible source as well. To find a plausible explanation for the appearance of the silicon peak a closer look on nacre is helpful. Fig. 5 shows a STEM image of a cross sectional nacre specimen with two vertically stacked aragonite platelets and the interlamellar organic matrix in between. The two rectangles mark the regions where the displayed EDX spectra have been recorded.
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Fig. 5. STEM image of a cross sectional nacre specimen. EDX spectrum 1 has been generated at a region within an aragonite platelet, whereas spectrum 2 has been generated at a region within the interlamellar organic matrix.
Spectrum 1 has been taken from an area within an aragonite platelet, whereas spectrum 2 has been detected from an area within the interlamellar organic matrix. Spectrum 1 contains signals that stem from CaCO3. Spectrum 2 contains a high carbon signal which is expected for an area including organic matter. Because of some surface irregularities the aragonite platelets protrude slightly into the scanned area. Therefore oxygen and calcium signals appear in that spectrum, too. Both of the spectra contain small but distinct silicon signals. The source of the silicon might be the seawater that include at a salinity of 35% a small amount of silicon of 2900 mg=l [12]. During the growth process the silicon may be incorporated into the crystalline and organic parts of the shell. The fact that silicon could be detected within the amorphous layer of the in vitro grown flat aragonite crystals is a hint that the layers are residues of the native insoluble organic matrix used as a template for crystal growth. Amorphous material developed during the experiment using purified components and thus in the absence of seawater would not contain silicon. The silicon signal detected in spectrum 2 in Fig. 4 is much more pronounced than the silicon signals detected in nacre (Fig. 5). Moreover it is conspicuous that it is higher than the calcium and carbon signals obtained from the same sample area. A possible explanation is that during the demineralization of the shell the part of the silicon, being originally located within the aragonite platelets, is concentrated in the layers of the insoluble organic matrix and therefore causes the increased silicon signal.
3.1.2. Appearance of mineral bridges Besides the fact that the amorphous layers are most likely residues of the insoluble organic matrix, which means that the aragonite crystals have grown within the scaffold of the insoluble organic matrix, another analogy with nacre appears. This analogy is shown in Fig. 3. The TEM image in Fig. 3(a) shows not only the residues of the insoluble organic matrix but also some kind of mineral bridges connecting the lower and the upper part of the aragonite crystal. Part of such a bridgelike structure is magnified in Fig. 3(b). In nacre mineral bridges transfer information on the crystallographic orientation from one aragonite platelet to the overlying one. This means that the orientation of the aragonite crystals above and below a mineral bridge is the same. To prove if this is also the case for in vitro grown flat aragonite crystals, diffractograms have been created within the bridgelike structure (e) and in regions within the aragonite crystals
below and above the bridgelike structure ((d) and (f)). The diffractograms contain mainly the same reflections. Some of these are marked with arrowheads. The existence of the identical reflections shows that there is no significant variation in the orientation of the aragonite crystal in the three regions. This implies that during the in vitro growth process the aragonite crystals not just grow within the scaffold of the insoluble organic matrix, but also through pores in the organic matrix forming some kind of mineral bridges like in nacre. Besides the identical reflections that appear in the three diffractograms ((d)–(f)) additional reflections are present in the diffractograms (e) and (f) and are marked with arrows. These additional reflections point out that the in vitro grown aragonite crystals are not perfect single crystals. This was already mentioned in [8] where a selected area diffraction aperture had to be used to obtain diffraction pattern which could be analyzed. Thus, the orientation of the aragonite crystals is just constant in delimited regions. The bridgelike structures transfer therefore information on the crystallographic orientation through the residues of the insoluble organic matrix only in locally limited areas. This is a significant difference to the structure of nacre and shows that the in vitro growth process still needs improvement. 3.2. Blocklike structure Besides the layered structure of the in vitro grown flat aragonite crystals, the existence of the blocklike structure should be considered, too. The appearance of these structures is actually not so surprising. A comparison with nacre shows that blocklike aragonite platelets appear that are not interrupted by the organic matrix [13,14]. These platelets are up to several micrometers in thickness. Additionally clear boundaries between horizontally adjacent platelets are neither in SEM nor in TEM images visible. Mostly these platelets build a layer on the inner surface of the shell which is oriented towards the body of the snail. Which processes control the formation of such a layer is still not known. But the same processes (induced for example by specific proteins that are located in some areas of the insoluble organic matrix) may influence the formation of flat blocklike aragonite crystals in our experiment.
4. Conclusions In this work we investigated flat aragonite crystals that have been grown on the insoluble organic matrix of the abalone Haliotis laevigata. To realize the formation of these crystals the insoluble
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organic matrix was placed in a crystallization device flowed through by CaCl2 and NaHCO3 solutions with concentrations of 10–20 mM. The investigation of the structure of the in vitro grown flat aragonite crystals was performed with a TEM. The flat aragonite crystals exhibited mainly two different structures: blocklike or layered. Because of the reminiscence of nacre structure, the layered structure of the in vitro grown flat aragonite crystals is of special interest. TEM investigations revealed that the layered structure is created by the intersection of the aragonite crystals with amorphous layers. EDX spectra recorded on these amorphous layers showed that they contain silicon. This is in contrast to the crystalline regions. Nacre specimens on the other hand contain silicon in the interlamellar organic matrix as well as in the aragonite platelets. One conclusion is that silicon is incorporated from the seawater into the shell during growth. Consequently all natural components of the in vitro grown samples contain silicon whereas the part of the sample created in the crystallization device (like the aragonite crystals) will not contain any silicon. In this light, it is most probable that the amorphous layers are residues of the insoluble organic matrix. Thus, the growth process is similar to that in nacre. An additional similarity to nacre is the appearance of bridgelike mineral structures connecting two vertically stacked parts of an in vitro grown aragonite crystal. As in nacre these mineral bridges transfer information on the crystallographic orientation through the layer of the organic matrix (respectively the residues of the insoluble organic matrix). In contrast to nacre the in vitro grown aragonite crystals maintain the orientation only in proximity to the bridgelike structures.
Acknowledgments K.I. Gries thanks the Physics International Postgraduate Programme (PIP) at Bremen University for sponsorship.
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