BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 176, No. 2, 1991
Pages 654-659
April 30, 1991
OBSERVATION OF THE PHASE TRANSITION IN THE GROWTH OF A BIOMINERALIZED CALCIUM CARBONATE L.J. Huang ",+ and H.D. Li D e p a r t m e n t of Materials Science and Engineering, Tsinghua University, Beijing, 100084, China *Chinese Center for Advanced Science and Technology (World Laboratory), P.O. Box 8730, Beijing, 100080, China Received March ii, 1991
SUMMARY: Transmission electron microscope studies of the cellular nucleation sites of a biomineralized calcium carbonate(aragonite) in the lamellar nacreous layer of the bivalvia Cristaria plicata (Leach) shell showed that the low density calcium particles were confined within some large vesicles of the bivalve mantle cells on which the crystalline aragonite phases were deposited. These vesicles served as the nucleation sites for the growth of the crystals. The crystalline phases at the first few lamellae were mostly imperfect while the whole nacreous layer acquitted itself into a highly oriented biomineralized aragonite. The structure of the vesicles is addressed in detail. ¢ 199~ Ao~demicP..... ~nc. The biomineralized crystalline phases are grown by deposition of biomineral particles secreted by organic cells in the organic matrix.
Efforts toward the understanding of the
biomineralized process have revealed the important role of the organic cells in the development of the biomineralized crystals and a variety of biomineral growth are hence termed as the "organic matrix mediated biomineralization". (1-3)
The mediator of the
biomineralization was thought to be mainly the organic constituents such as lipids, proteins and carbohydrates whereas the carriers of the biominerals were some small membranebound intra- and extracelluar vesicles.
These vesicles play the role to confining the
biomineraling zones within a localized volume without preferential alignment of the constituent biomineral particles and they were frequently observed being carrying toxic heavy metal biominerals during detoxification. (4,5) More sophisticated vesicles have been found as well in patterning some of the biomineralized crystals such as frustule (6), but their function during the nucleation of biominerals has not been identified. As the nucleation sites play a nontrivial part in the growth of the biominerals, it would be essential to have a + Present address: Surface Science Western, Natural Science Center, The University of Western
Ontario, London, Ontario, Canada N6A 5B7. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights' of reproduction in any form reserved.
654
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
clear knowledge of the nucleation sites within the cells. In this work, the nucleation sites in the cells of a bivalvia Cristaria plicata (Leach) were studied by transmission electron microscope. It was found that the calcium particles were confined within some large vesicles in the cell while the particles were transported by both the endoplasmic reticula, the Golgi body as well as some small vesicles. The growth of the biomineralized aragonite at the first few lamellae in the nacreous layers formed imperfect crystalline structures but the whole layer was a highly orientated aragonite. MATERIALS AND METHODS The bivalvia Cristaria plicata (Leach) were collected alive in a freshwater lake in the south of China. They were about 10 month old and hence were not fully developed. In this case, the biomineralization process within their cells were still under way. One could then expect that study of these cells at this stage could reveal some important features of biomineralization. Both cell and inner solid nacreous layer specimens for a Jeol JEM-200CX transmission electron microscope (TEM) examinations were prepared as follows. The cell specimens were selected from the front parts of the alive mantles of the Cristaria plicata. These parts were those which secreted nacreous layers and could be transferred as seeds into a mother shell for man-bred pearl shells. (7) The cell specimens were then immediately fixed by glutaric dialdehyde and osmium tetroxide in sequence. After dehydration, they were embedded into epoxy resin. The serially thin-sectioned specimens were fixed on T E M grids with support films and then stained by uranium iodide. The shells were air-dried and the solid inner nacreous layers abutted against the mantle cells were mechanically crushed and mounted on the T E M girds for measurement. The whole nacreous layers were separated from the other layers of the shells under an optical microscope and then subject to analysis by x-ray diffractometer. RESULTS AND DISCUSSION Figure l(a) is a T E M micrograph of the observed large vesicles within the cell specimens and Figure l(b) is the enlarged part of Figure l(a) showing its local structures.
These
vesicles are of a linear size about 3 lxm which is quite unusual with respect to normal cell constitution. (8) The dark particles inside the vesicles are of a linear size about 100 nm with a density of 24 particles/l~m z approximately. The selected area diffraction(SAD) approach was used at various areas to identify these particles and a typical diffraction pattern is shown in the inlet part of Figure l(b).
Indexing the SAD patterns indicated that most of the
diffraction spots were from the cubic calcium (Fm3m) with an expansion of the lattice spacing less than 3%. The partial indices are labelled on the inlet diffraction pattern of Figure l(b). The structure of the amorphous matrix where the particles situated could be deduced from the strong diffraction halos in the SAD patterns from which the scattering parameters, S=4~rsinO/2, of the matrix phase could be obtained. The ratios of these parameters are $1/$2=1.83 and Ss/$1=2.71, respectively. These parameters are much larger than those of amorphous inorganic solids (9) and hence imply a much dilute atomic package 655
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1. TEM micrographs of the nucleation sites of the biomineralized aragonite in the nacreous layer (Cristaria plicata). (a) The morphology of the vesicles confining low density calcium particles and (b) the enlarged part of (a) showing the local structures of the vesicles containing the mitochondrion, Golgi body and granules under transportation. The unlabelled diffraction spots are also from cubic calcium but of higher indices. 656
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the matrix. The expansion of the calcium lattice spacings could then be interpreted by the matrix effects. From Figure l(a) one sees that the calcium particles are all confined within these vesicles suggesting that these vesicles would be the nucleation sites for the future development of the calcium carbonate crystals. The whole vesicles shown in Figure l(a) is separated by the endoplasmic reticulum(Er) channels into some five subvesicles as pointed by the arrows.
These channels have a
structure basically the same as those surrounding the vesicles. Each of these subvesicles possessed a mitochondrion(m). From Figure l(b), it can be observed that the cristae in the mitochondrion are of a shape of a slender villus or blinding-ending tubule and they extend the full length of the mitochondrion. The density of the cristae is very high resulting in a very limited spacing between every two cristae.
This feature of the mitochondrion was
reported as a characteristics of an active and secreting cell. (8) The Golgi body(gb) abutted against the vesicle wall and the rich endoplasmic reticula around the vesicles presented the picture of the transportation process of the particles. Fine particles can be seen clustering at the outer side of the Golgi cisternae and the edges of the vesicles next to the endoplasmic reticula. These particles were then condensed by the Golgi body into larger particles as one sees from Figure l(b) that the small granule(gr) of a linear size 500 nm containing several particles is in between the Golgi body and the vesicle wall suggesting a further fusion of these two. This argument was also evidenced by the small granule which is nearly inside the vesicle as shown at the upper left corner of Figure l(b). To model the growth of the calcium carbonate phase from the nucleation sties, major efforts have been put on the matrix geometry (2,10) or thermodynamic approach. (11) It was reported (12)
that there existed a certain orientational correlation among the
mineralized aragonite and the protein of anti-parallel [~-sheet conformation and the chitin phase in the matrix. While it was difficult to identify the orientations of the present matrix at the nucleation sites from the SAD patterns, the inner surface layers of the shells were mechanically crushed and mounted onto the TEM grids for a measurement of the initial orientations of the crystals. Figure 2 shows both the TEM bright field morphology and the diffraction pattern of the layer. The present observation, however, showed that the structure of these layers are mostly imperfect. From the diffraction pattern (the inlet part of Figure 2), one sees that the layered crystals are not arrayed in a single orientation but rotated considerably forming a incomplete double diffraction pattern as indicated by the labelled triangle positions in the pattern. The diffraction spots of the single crystal aragonite also could be read out and are labelled by the large arrows which are identified to be the (111) aragonite with a spacing of d=0.341 nm whose intensity is however very weak. The strong diffraction spots indicated by both the triangles and small angles were identified to belong 657
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2. TEM morphology of the inner nacreous layers (Cristaria plicata). The inlet diffraction pattern indicates the imperfection of the crystals and the random rotated overlaid crystals but yet with a traceable orientation. The strong diffraction spots are from (001) planar group.
to (001) crystalline planar group and therefore the crystals were not along the c-axis as suggested to be the most common direction for biomineralized aragonite (10). To explore this point, the whole nacreous layer was measured by x-ray diffractometer. In order to make the obtained results comparable with the T E M diffraction data, both the solid layers and powder specimens milled from the nacreous layers were subject to x-ray analysis. It was found that the results obtained from these two kinds of specimens were qualitatively the same. The powder diffraction spectrum is presented in Figure 3. F r o m this figure, one sees that the crystals are indeed highly oriented and in the (100) orientation, or along the a-axis of the aragonite atomic cells. To reveal the possible epitaxial growth of the crystals will require the data of the protein structure of the present Cristaria plicata which is a topic currently under investigation. In conclusion, the nucleation sites for the biomineralized aragonite of the Cristaria plicata nacreous layers were constituted of some large vesicles confining low density calcium particles. These sites were included by rich endoplasmic reticula and contained a few mitochondria and Golgi bodies. The initial growth of the crystals abutted upon the 658
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
!
g I 15
I
50
i00
Figure 3. X-ray spectrum of the nacreous layer (Cristaria plicata) showing a strong orientation of (100) yet with one forbidden (312) plane. nucleation sites were mostly imperfect and the crystalline orientation of the nacreous layer of the Cristaria plicata was (100). A C K N O W L E D G M E N T S : The authors would like to express their sincere thanks to Dr. Y.Y. Liu of Institute of Zoology, Chinese Academy of Sciences, Dr. H,Q. Gong of Man-Bred Pearl Shell Plant of Wuxian and research staffs at the TEM Laboratory of Department of Biology, Peking University. The work is financially supported by the National Natural Science Foundation of China.
REFERENCES .
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
Lowenstam H.A. and Weiner S.(1989) On Biomineralization, Oxford University Press, New York. Mann S.(1983) in Structure and Bonding, (Clark M.J. et al EDS) Vol 54, 125-174 Springer-Verlag, Berlin. Simkiss IC and Wilbur K.M. (1989) Biomineralization: Cell Biology and Mineral Deposition, Academic Press, San Diego. Leadbeater B.S.C. (1979) Protoplasma, 98, 241. Blakemore R.P. and Frankel R.B. (1981) Sci. American, 245(6), 56-60. Volcani B.E. (1981) Silicon and siliceous Structures in Biological System, (Volcani B.E. and Simpson T.L. EDS), Springer-Verlag, Berlin. Lin C. (1974) Man-bred Pearl Shells, Science Press, Beijing.; Liu Y.Y., personal communication. Fawcett D.W.(1981) The Cells, p.446-448, W.B. Saunders Co. London. Chen H.S. (1980) Rep. Progr. Phys., 43, 353-379. Weiner S., Talmon Y. and Traub W.(1983) Int. J. Biol. Macromol., 5, 325-328. Wilbur ICM. and Saleuddin A.S.M. (1983) in The Mollusca,(A.S.M. Saleuddin and ICM. Wilbur Eds.) Vol.4 235-287, Academic Press, New York. Weiner S. and Traub W. (1980) FEBS Lett. 111, 311-315.
659
Vol. 176, No. 2, 1991
Pages 654-659
April 30, 1991
OBSERVATION OF THE PHASE TRANSITION IN THE GROWTH OF A BIOMINERALIZED CALCIUM CARBONATE L.J. Huang ",+ and H.D. Li D e p a r t m e n t of Materials Science and Engineering, Tsinghua University, Beijing, 100084, China *Chinese Center for Advanced Science and Technology (World Laboratory), P.O. Box 8730, Beijing, 100080, China Received March ii, 1991
SUMMARY: Transmission electron microscope studies of the cellular nucleation sites of a biomineralized calcium carbonate(aragonite) in the lamellar nacreous layer of the bivalvia Cristaria plicata (Leach) shell showed that the low density calcium particles were confined within some large vesicles of the bivalve mantle cells on which the crystalline aragonite phases were deposited. These vesicles served as the nucleation sites for the growth of the crystals. The crystalline phases at the first few lamellae were mostly imperfect while the whole nacreous layer acquitted itself into a highly oriented biomineralized aragonite. The structure of the vesicles is addressed in detail. ¢ 199~ Ao~demicP..... ~nc. The biomineralized crystalline phases are grown by deposition of biomineral particles secreted by organic cells in the organic matrix.
Efforts toward the understanding of the
biomineralized process have revealed the important role of the organic cells in the development of the biomineralized crystals and a variety of biomineral growth are hence termed as the "organic matrix mediated biomineralization". (1-3)
The mediator of the
biomineralization was thought to be mainly the organic constituents such as lipids, proteins and carbohydrates whereas the carriers of the biominerals were some small membranebound intra- and extracelluar vesicles.
These vesicles play the role to confining the
biomineraling zones within a localized volume without preferential alignment of the constituent biomineral particles and they were frequently observed being carrying toxic heavy metal biominerals during detoxification. (4,5) More sophisticated vesicles have been found as well in patterning some of the biomineralized crystals such as frustule (6), but their function during the nucleation of biominerals has not been identified. As the nucleation sites play a nontrivial part in the growth of the biominerals, it would be essential to have a + Present address: Surface Science Western, Natural Science Center, The University of Western
Ontario, London, Ontario, Canada N6A 5B7. 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights' of reproduction in any form reserved.
654
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
clear knowledge of the nucleation sites within the cells. In this work, the nucleation sites in the cells of a bivalvia Cristaria plicata (Leach) were studied by transmission electron microscope. It was found that the calcium particles were confined within some large vesicles in the cell while the particles were transported by both the endoplasmic reticula, the Golgi body as well as some small vesicles. The growth of the biomineralized aragonite at the first few lamellae in the nacreous layers formed imperfect crystalline structures but the whole layer was a highly orientated aragonite. MATERIALS AND METHODS The bivalvia Cristaria plicata (Leach) were collected alive in a freshwater lake in the south of China. They were about 10 month old and hence were not fully developed. In this case, the biomineralization process within their cells were still under way. One could then expect that study of these cells at this stage could reveal some important features of biomineralization. Both cell and inner solid nacreous layer specimens for a Jeol JEM-200CX transmission electron microscope (TEM) examinations were prepared as follows. The cell specimens were selected from the front parts of the alive mantles of the Cristaria plicata. These parts were those which secreted nacreous layers and could be transferred as seeds into a mother shell for man-bred pearl shells. (7) The cell specimens were then immediately fixed by glutaric dialdehyde and osmium tetroxide in sequence. After dehydration, they were embedded into epoxy resin. The serially thin-sectioned specimens were fixed on T E M grids with support films and then stained by uranium iodide. The shells were air-dried and the solid inner nacreous layers abutted against the mantle cells were mechanically crushed and mounted on the T E M girds for measurement. The whole nacreous layers were separated from the other layers of the shells under an optical microscope and then subject to analysis by x-ray diffractometer. RESULTS AND DISCUSSION Figure l(a) is a T E M micrograph of the observed large vesicles within the cell specimens and Figure l(b) is the enlarged part of Figure l(a) showing its local structures.
These
vesicles are of a linear size about 3 lxm which is quite unusual with respect to normal cell constitution. (8) The dark particles inside the vesicles are of a linear size about 100 nm with a density of 24 particles/l~m z approximately. The selected area diffraction(SAD) approach was used at various areas to identify these particles and a typical diffraction pattern is shown in the inlet part of Figure l(b).
Indexing the SAD patterns indicated that most of the
diffraction spots were from the cubic calcium (Fm3m) with an expansion of the lattice spacing less than 3%. The partial indices are labelled on the inlet diffraction pattern of Figure l(b). The structure of the amorphous matrix where the particles situated could be deduced from the strong diffraction halos in the SAD patterns from which the scattering parameters, S=4~rsinO/2, of the matrix phase could be obtained. The ratios of these parameters are $1/$2=1.83 and Ss/$1=2.71, respectively. These parameters are much larger than those of amorphous inorganic solids (9) and hence imply a much dilute atomic package 655
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1. TEM micrographs of the nucleation sites of the biomineralized aragonite in the nacreous layer (Cristaria plicata). (a) The morphology of the vesicles confining low density calcium particles and (b) the enlarged part of (a) showing the local structures of the vesicles containing the mitochondrion, Golgi body and granules under transportation. The unlabelled diffraction spots are also from cubic calcium but of higher indices. 656
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in the matrix. The expansion of the calcium lattice spacings could then be interpreted by the matrix effects. From Figure l(a) one sees that the calcium particles are all confined within these vesicles suggesting that these vesicles would be the nucleation sites for the future development of the calcium carbonate crystals. The whole vesicles shown in Figure l(a) is separated by the endoplasmic reticulum(Er) channels into some five subvesicles as pointed by the arrows.
These channels have a
structure basically the same as those surrounding the vesicles. Each of these subvesicles possessed a mitochondrion(m). From Figure l(b), it can be observed that the cristae in the mitochondrion are of a shape of a slender villus or blinding-ending tubule and they extend the full length of the mitochondrion. The density of the cristae is very high resulting in a very limited spacing between every two cristae.
This feature of the mitochondrion was
reported as a characteristics of an active and secreting cell. (8) The Golgi body(gb) abutted against the vesicle wall and the rich endoplasmic reticula around the vesicles presented the picture of the transportation process of the particles. Fine particles can be seen clustering at the outer side of the Golgi cisternae and the edges of the vesicles next to the endoplasmic reticula. These particles were then condensed by the Golgi body into larger particles as one sees from Figure l(b) that the small granule(gr) of a linear size 500 nm containing several particles is in between the Golgi body and the vesicle wall suggesting a further fusion of these two. This argument was also evidenced by the small granule which is nearly inside the vesicle as shown at the upper left corner of Figure l(b). To model the growth of the calcium carbonate phase from the nucleation sties, major efforts have been put on the matrix geometry (2,10) or thermodynamic approach. (11) It was reported (12)
that there existed a certain orientational correlation among the
mineralized aragonite and the protein of anti-parallel [~-sheet conformation and the chitin phase in the matrix. While it was difficult to identify the orientations of the present matrix at the nucleation sites from the SAD patterns, the inner surface layers of the shells were mechanically crushed and mounted onto the TEM grids for a measurement of the initial orientations of the crystals. Figure 2 shows both the TEM bright field morphology and the diffraction pattern of the layer. The present observation, however, showed that the structure of these layers are mostly imperfect. From the diffraction pattern (the inlet part of Figure 2), one sees that the layered crystals are not arrayed in a single orientation but rotated considerably forming a incomplete double diffraction pattern as indicated by the labelled triangle positions in the pattern. The diffraction spots of the single crystal aragonite also could be read out and are labelled by the large arrows which are identified to be the (111) aragonite with a spacing of d=0.341 nm whose intensity is however very weak. The strong diffraction spots indicated by both the triangles and small angles were identified to belong 657
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2. TEM morphology of the inner nacreous layers (Cristaria plicata). The inlet diffraction pattern indicates the imperfection of the crystals and the random rotated overlaid crystals but yet with a traceable orientation. The strong diffraction spots are from (001) planar group.
to (001) crystalline planar group and therefore the crystals were not along the c-axis as suggested to be the most common direction for biomineralized aragonite (10). To explore this point, the whole nacreous layer was measured by x-ray diffractometer. In order to make the obtained results comparable with the T E M diffraction data, both the solid layers and powder specimens milled from the nacreous layers were subject to x-ray analysis. It was found that the results obtained from these two kinds of specimens were qualitatively the same. The powder diffraction spectrum is presented in Figure 3. F r o m this figure, one sees that the crystals are indeed highly oriented and in the (100) orientation, or along the a-axis of the aragonite atomic cells. To reveal the possible epitaxial growth of the crystals will require the data of the protein structure of the present Cristaria plicata which is a topic currently under investigation. In conclusion, the nucleation sites for the biomineralized aragonite of the Cristaria plicata nacreous layers were constituted of some large vesicles confining low density calcium particles. These sites were included by rich endoplasmic reticula and contained a few mitochondria and Golgi bodies. The initial growth of the crystals abutted upon the 658
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
!
g I 15
I
50
i00
Figure 3. X-ray spectrum of the nacreous layer (Cristaria plicata) showing a strong orientation of (100) yet with one forbidden (312) plane. nucleation sites were mostly imperfect and the crystalline orientation of the nacreous layer of the Cristaria plicata was (100). A C K N O W L E D G M E N T S : The authors would like to express their sincere thanks to Dr. Y.Y. Liu of Institute of Zoology, Chinese Academy of Sciences, Dr. H,Q. Gong of Man-Bred Pearl Shell Plant of Wuxian and research staffs at the TEM Laboratory of Department of Biology, Peking University. The work is financially supported by the National Natural Science Foundation of China.
REFERENCES .
2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
Lowenstam H.A. and Weiner S.(1989) On Biomineralization, Oxford University Press, New York. Mann S.(1983) in Structure and Bonding, (Clark M.J. et al EDS) Vol 54, 125-174 Springer-Verlag, Berlin. Simkiss IC and Wilbur K.M. (1989) Biomineralization: Cell Biology and Mineral Deposition, Academic Press, San Diego. Leadbeater B.S.C. (1979) Protoplasma, 98, 241. Blakemore R.P. and Frankel R.B. (1981) Sci. American, 245(6), 56-60. Volcani B.E. (1981) Silicon and siliceous Structures in Biological System, (Volcani B.E. and Simpson T.L. EDS), Springer-Verlag, Berlin. Lin C. (1974) Man-bred Pearl Shells, Science Press, Beijing.; Liu Y.Y., personal communication. Fawcett D.W.(1981) The Cells, p.446-448, W.B. Saunders Co. London. Chen H.S. (1980) Rep. Progr. Phys., 43, 353-379. Weiner S., Talmon Y. and Traub W.(1983) Int. J. Biol. Macromol., 5, 325-328. Wilbur ICM. and Saleuddin A.S.M. (1983) in The Mollusca,(A.S.M. Saleuddin and ICM. Wilbur Eds.) Vol.4 235-287, Academic Press, New York. Weiner S. and Traub W. (1980) FEBS Lett. 111, 311-315.
659
