Pearl

Article pubs.acs.org/crystal

Primary Cell Culture of Fresh Water Hyriopsis cumingii Mantle/Pearl Sac Tissues and Its Effect on Calcium Carbonate Mineralization Dongni Ren,†,# Olga Albert,‡,# Minghui Sun,§ Werner E. G. Müller,‡ and Qingling Feng*,⊥ †

State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China ‡ ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, D-55128 Mainz, Germany § School of Economics and Management, Hebei North University, Zhangjiakou 075000, China ⊥ Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China ABSTRACT: Primary cell cultures of the fresh water Hyriopsis cumingii mantle and pearl sac tissues were produced in this study, and the influence of the tissue, cells, and secreted protein on calcium carbonate crystal nucleation and growth was studied. The study contributes to a further understanding of the influence of organic matrices on CaCO3 crystal formation. This research started from the protein level to the tissue/cell level, which is crucial for understanding the inorganic deposition process. The new data also add relevant theoretical approaches to an overall understanding of biomineralization processes. In the experimental groups with mantle or pearl sac tissue, the growth patterns of aragonite were similar: both started from a round disk-shaped amorphous calcium carbonate (ACC) and then turned to flowerlike aragonite aggregate. The whole crystal growing process was recorded by transmitted light microscopy. In the control group, without any tissue, there was no ACC found nor crystal phase transformation; it was pure calcite, and the crystal size enlarged as the culture time increased. stances, vaterite can be obtained by transformation of calcite,12 while the lackluster pearl is arranged by vaterite crystals, which changes its mechanical and optical properties and affects the pearl quality.13 The good solubility and biocompatibility of vaterite crystalline lackluster pearl enhance its potential use in biomedical materials. Formation of the mussel shell is related to the mantle tissue, which can secrete calcium that enters the extra-mantle matrix, where the final concentration of Ca2+ is about 10 mg/L.14 Calcium carbonate crystals nucleate in the calcium-rich environment and deposit alternatively with the organic matrix to form an inorganic crystal−organic matrix structure. In Mann’s review article,15 he concluded Weiner and Traub’s16,17 research results of the “Molecular correspondence at the inorganic−organic interface in the nacreous shell layer of Nautilus repertus”. In this paper, the schematic diagrams represent the organization of the inorganic−organic interface in shell nacre (Figure 1), which shows the importance of organic matrix effect on calcium carbonate mineralization in vivo.

1. INTRODUCTION Hyriopsis cumingii is a fresh water bivalve animal that produces pearls in southern China. Properties of the shell of this kind of mussel and pearls have been long and widely studied because they have special characteristics such as mechanical, optical and biological properties. Previous studies showed that the mineral phase in the bivalve mussel shell consists predominantly of two layers: the prismatic layer composed of long calcite sticks and the nacreous layer of hexagonal aragonite sheets.1,2 This peculiar but ordered structure in nacre is named as the “brick-mortar” structure; aragonite sheet is its “brick”, and organic matrix among these sheets is “mortar”, which can bond the inorganic crystal together to build a whole structure.1,3−6 Because of their optical property, the fresh water pearls can be divided into lustrous pearl and lackluster pearl. It is normally considered that the formation of lackluster pearl in mussel under natural conditions is an abnormal process.7,8 The study shows that the lustrous pearl is made out of pure aragonite crystals and possesses a similar composition and structure as shell nacre layer.9 The vaterite crystal is considered to be the most unstable crystalline structure of calcium carbonate, which can transform to calcite or aragonite easily.10 Pure vaterite mineral rarely exists in nature but can be synthesized by chemical methods under certain conditions.11 Also, under certain experimental circum© 2014 American Chemical Society

Received: November 6, 2013 Revised: January 23, 2014 Published: January 28, 2014 1149

dx.doi.org/10.1021/cg401657d | Cryst. Growth Des. 2014, 14, 1149−1157

Crystal Growth & Design

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In this study, tissue of H. cumingii mantle and pearl sac part was cultured under clean conditions for more than 30 days. The culture environment is similar to the living conditions of the mussel in nature. Effects of the tissue, cells, and their secreted protein on calcium carbonate crystal nucleation and growth were studied. The presented in vitro mineralization study presented here is a better way to approach and mimic the natural pearl as well as the mussel shell formation. Also this study contributes to a further in-depth understanding of our knowledge of the influence of organic matrices on CaCO3 crystal formation. In our approach, we started from the protein level to the tissue/cell level, a study direction that is crucial for an understanding of the inorganic deposition process. The new data should add also relevant theoretical approaches to an overall understanding of biomineralization processes in general.

2. MATERIALS AND METHODS 2.1. Materials and Solutions. 2.1.1. Hyriopsis Cumingii. For the experimental section, the fresh water mussel H. cumingii of less than one year old was chosen, as shown in Figure 2a (the length of the mussel is about 10 cm). Two tissue parts were selected: mantle and pearl sac tissues (Figure 2b).

Figure 1. Molecular correspondence at the inorganic−organic interface in the nacreous shell layer of Nautilus repertus. (a) Schematic representation of the structural relationship between protein sheets, aragonite crystals, and chitin fibers. There is a close geometric match between the periodicity of the β-sheet and the lattice-spacing in the ab face of aragonite. (b) Possible modes of molecular complementarity between Ca atoms in the aragonite ab face and aspartic acid resides organized in the sequence Asp-X-Asp (X, neutral residue) along the βsheet matrix interface. Ca2+ binding involves two or three ligands and is regulated along the interface to produce oriented crystal nuclei. (Reprinted with permission from ref 15. Copyright 1988 Nature Publishing Group.15−17

Lustrous pearl and shell nacre layers have a similar structure. There are two mainstream mechanisms that can explain their formation: the mineral bridge theory and the heterogeneous nucleation theory. The mineral bridge theory describes that there are holes in the organic matrix layer, which allow the aragonite crystals to grow continuously through different organic layers.18−21 The heterogeneous nucleation theory implies that the growth of each aragonite sheet is mediated by organic matrix separately.22 Both of the theories emphasize the connection between the calcium carbonate crystal and the organic matrix that is mainly composed of proteins, but these theories do not pay enough attention to the mussel tissue and cells that build the environment for crystal nucleation and growth.23,24 Nowadays, use of primary cell culture of bivalve animals to study the effect of tissue, cells, and secreted protein on calcium carbonate crystallization is concentrated on marine animals such as abalone, Pinctada f ucata, and Pinctada maxima.25−35 There are few studies of fresh water mussel tissue culture. There are mainly two obstacles for primary cell culture of fresh water mussel. First, with respect to marine mussels, there is a comprehensive gene bank that contains ample information about the genes and proteins that affect calcium carbonate crystallization during shell formation.36 In contrast, there is no gene bank built for fresh water mussel sequencing, since there are not enough biological studies being performed on those animals. Second, contamination in the fresh water mussel cell culture process is a very serious problem, and most of the contaminations come from the mussel itself and thus interfere with the crystalline process.37−39

Figure 2. Fresh water Hyriopsis cumingii. (a) Mussel shell construction; (b) anatomy of the mussel with earmarked mantle and pearl sac tissues.

2.1.2. Solution Preparation. For the solution preparation, 30 mg/L chloramphenicol solution (10% EtOH as solvent) and mix antibiotic solution were used as washing solutions to sterilize the mussel tissues. As nutrition medium, low-sugar Dulbecco’s modified Eagle medium (DMEM, GIBCO, USA) was used. A 1.6 mg/mL CaCl2 solution was added in the culture medium to increase the Ca2+ concentration. The antibiotic mix solution contains 100 units/mL penicillin (0242, Amresco, USA), 0.125 mg/mL gentamycin (0304, Amresco, USA), 0.1 mg/mL kanamycin (0408, Amresco, USA), 0.2 mg/mL streptomycin (0382, Amresco, USA), 0.1 mg/mL metronidazole (Yabao Medical, China), and 0.005 mg/mL amphotericin (E437, Amresco, USA). All solutions were prepared under sterile conditions and filtered with a paper filter with a pore size of 0.22 μm, then kept at 4 °C. 1150



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2.3.7. Polarized Light Microscopy. Medium and tissues in the culture plates were taken out, and the wells were washed with triple distilled water twice, 10 min each time. Samples were observed under a microscope with normal and polarized light.

2.2. Dissection Procedure. Mussels used for dissection were cultured in the laboratory for 15 days with a small amount of antibiotics in tap water for stabilization and sterilization. One mussel in good condition was chosenm and the shell was washed quickly with soap water, 75% alcohol, and tap water. The instruments used for the dissection (tweezers, scalpel, scissors) were sterilized with a UV light under laminar flow. After the treatment, the clean mussel was opened by cutting the adductor muscles, and the mantle and pearl sac tissues (Figure 2b) were cut off and placed in the culture dish, separately. Tissues were cut into about 1 mm2 pieces and washed with triple distilled water six times, then with chloramphenicol solution for 15 s, and with triple distilled water four more times. The two tissues were then washed with the mix antibiotic solution four times, each time for 30 min, then with triple distilled water six times, and dried under room temperature in the laminar flow. Sterilized glass slices (diameter of 13 mm) were placed in 24 cell culture well plates, rinsed with DMEM, and dried in air in the laminar flow. Mantle and pearl sac tissue pieces were placed onto these glass slices with clean needles. After 10 min, tissues were adhered well on glass slices, DMEM and CaCl2 solutions were added (the total amount in each well was 500 μL), and the final Ca2+ concentration in the experimental and control groups was set at 1.0 mg/mL. Cell culture well plates were placed in the incubator at 25 °C with 5% CO2, constantly. The mixed culture medium was changed every three days. 2.3. Characterization Method. 2.3.1. Transmitted Light Microscopy. The culture well plates were observed by microscope every day to see tissue, cells, and crystal changes. The microscope DM IL LED, equipped with a convert light-fluorescence microscope system (Leica, Germany) together with the camera DFC425C, was used. 2.3.2. Alizarin Red Staining. The medium in the culture plate was taken out, the wells were washed with phosphate buffered saline (PBS) twice, and the tissues and cells were then fixed in wells. The materials in the wells were stained with 10 g/L Alizarin Red solution, (pH = 4.2, Guoyao Chemical, China) for 30 min at 37 °C and then washed with PBS three times and observed under microscope. 2.3.3. DAPI Staining. The medium in the culture plate was taken out, the wells were washed with PBS twice, and 300 μL (2 μg/L) of 4′,6-diamidino-2-phenylindole (DAPI) staining solution was added to each well to cover all the tissues and cells. Culture plates were incubated at 25 °C for 5 min, the staining solution was taken out, and the wells were washed with PBS three times and observed under a fluorescence microscope. 2.3.4. Scanning Electron Microscopy (SEM). Medium and tissues in the culture plates were taken out, the wells were washed with PBS twice, and cells and crystals were fixed with 2.5% glutaraldehyde solution at 4 °C overnight. The fixation solution was taken out, and the wells were washed with PBS twice. The materials in the wells went through a dehydrate line by immersing the samples in ethanol and tbutyl alcohol (TBA). Subsequently, the samples were freeze-dried for 1 h to remove the liquid totally. The cells and crystals, attached on the glass plates, were coated with gold for 8 min, and then observed with SEM (JSM-7001F field emission SEM, Japan). 2.3.5. Transmission Electron Microscopy (TEM). The medium and tissues in the culture plates were taken out, and the wells were washed with triple distilled water and 75% ethanol solution, 10 min for each. The crystals on glass plates were scraped off with a knife and subjected to ultrasound in ethanol solution to disperse for 10 min. A total of 5− 10 drops were dropped on the microgrid for TEM observation. The TEM used in the experiment was JEM-200CX, with an acceleration voltage of 200 kV, point resolution 0.35 nm, and lattice resolution 0.14 nm, and the maximum tilt angle is X = ±25°, Y = ±25°. 2.3.6. Raman Spectrum. Medium and tissues in the culture plates were taken out, and the wells were washed with triple distilled water and 75% ethanol solution once, 10 min each time and dried at room temperature. Raman spectrum was carried out on the dry glass slides. The Raman spectrum equipment used was a RM2000 microscopic confocal Raman (Renishaw, Britain), with a resolution of 1 μm and an excitation wavelength of 514 nm. Raman shift range is 100−4000 cm−1, and spectral resolution is 1 wavenumber.

3. RESULTS AND DISCUSSION 3.1. Mantle and Pearl Sac Tissues and Cells. Figure 3 shows the variation trends of mantle and pearl sac tissues after

Figure 3. Cell secretion and proliferation of mantle and pearl sac tissues under transmission inverted light microscope. Left column: mantle tissue; right column: pearl sac tissue. Rows 1−5 represent tissues cultured after 12 h, 24 h, 4 days, 6 days, and 8 days (red arrows point to tissues).

culture. From this morphology, we can see that mantle tissue secretes cells at a very high speed. After 24 h of culture, there were a lot of cells in the mantle group (Figure 3c). The pearl sac tissue secreted cells at a lower speed; after 24 h in culture, there were only a few round-shaped cells around the tissue (Figure 3d). The cell secretion and proliferation of the mantle tissue accelerated after being cultured for a longer time. Figure 3g shows that most of the cells in the mantle group are long shaped, which are epithelial cells.40 The cell secretion and proliferation both in the mantle group and pearl sac group were favorable during the tissue culture process. After 6 days of culture, the cells still maintained their own characteristic shape (Figure 3j). 1151



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Figure 4. DAPI staining: (a) pearl sac tissue after 12 h; (b) mantle tissue after 12 h; (c) pearl sac tissue after 30 days; (d) mantle tissue after 30 days.

DAPI staining was carried out in the mantle and pearl sac groups after culturing for 12 h and 30 days. The cell number can be determined after DAPI staining on the basis of the DNA staining inside of the cells. After 12 h culture, there were no cells viewed in the pearl sac group (Figure 4a), and a few cells in the mantle group (Figure 4b). After 30 days of culture, the number of dissociative cells was much larger than that after 12 h of culture. In the mantle group (Figure 4d), the cell number was still larger than the pearl sac group (Figure 4c), but there was no significant difference. Under a higher observation ratio (400×), it can be seen that the long epithelial cells secreted from the mantle tissue is narrow and translucent at the two ends, but wide and opaque in the middle, and there is a close packed tubelike nucleus. There are also round-shaped cells in the view; some of those cells have large round nucleus, some of them have long tails, and some only show their nucleus (Figure 5Aa). The shape of cells in the pearl sac group is far different from that in the mantle group, most of the cells show a large round shape (Figure 5Ab) and

Figure 6. The initial and final morphologies of CaCO3 crystals in the control group (a, d); mantle group (b, e); and pearl sac group (c, f).

small round shape (Figure 5Ac). SEM figures also show the same type of cells (Figure 5B); the long epithelial cell is about 30 μm in length (Figure 5Ba), the large round cells are about 20 μm in diameter (Figure 5Bb), and small round cells are about 2 μm in diameter (Figure 5Bc). (The SEM size of the cells is different from the light microscopy observation due to the dehydration treatment.)

Figure 5. Observation of mantle and pearl sac tissue secreted cells. (A) Transmission inverted light microscope, (B) SEM figures, (C) Alizarin Red staining. (a) Epithelial cells secreted from mantle tissue, (b) large round cells secreted from pearl sac tissue, (c) small round cells secreted from pearl sac tissue. 1152



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Figure 7. Flowerlike aragonite crystals transformed from ACC and their growing process in experimental group with pearl sac tissue. (a) disorder linear deposition; (b) short stick deposition; (c) round or ellipse shaped disks; (d) large flat deposition; (e) water drop appeared in the middle of the deposition; (f) more water drops appeared; (g) growth of the water drops; (h) number of the “water-drop” granules increases; (i) flat deposition shrink; (j) “water-drop” granules assembled at two ends of the deposition; (k) the front and side looks of the flower-like crystals; (l) several aragonite flowerlike crystals.

Figure 8. Growing process of sticklike calcite crystal in control group without any tissue. (a) short stick deposition; (b) length of the stick deposition enlarged, simple aggregation of the sticks appeared; (c) further growth of the sticks in length; (d) large sticks appeared after 8 days of culture; (e) further growth of the large sticks in length; (f) simple aggregation of the large sticks; (g) several large stick depositions; (h) several aggregations of the large sticks; (i) a few more complex aggregations.

Alizarin Red staining was carried out on the three types of cells. The cell shape has a better look from the Ca staining inside the cells. Figure 4C shows the Alizarin Red staining result of the long epithelial cells secreted from mantle tissue, the large and small round shaped cells secreted from pearl sac

tissue, named as large pearl cell and small pearl cell, respectively.41 3.2. Calcium Carbonate Crystal Growth in Experimental Group and Control Group. The crystal formation that happened in pearl sac group was earlier than that in the 1153



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(Figure 6c). Their size (about 18.3 μm) is larger than those formed in control (about 6.8 μm) and mantle groups (about 6.4 μm). For the final morphology, the crystal form in the mantle group and pearl sac group are similar. They comprise as flowerlike crystal aggregates composed of “water-drop” shaped crystals connected to each other at the ends (Figure 6e,f). In the control group, the CaCO3 crystals formed a short stick shape (Figure 6d). An electrophoresis experiment (SDS-PAGE) was carried out to test the protein expression in the mantle and pearl tissues after culture (results not shown). In the pure culture medium of DMEM, there is no significant band, which means no biopolymer disturbance in the medium. Compare the fresh one with the one after culturing for 7 days and 21 days under the same treatment and in the same type of tissue, mantle, or pearl sac tissue, as the culture time increased. It appears that the influencing morphogenetic effect of the protein disappears. Staining of the proteins on the SDS-PAGE agar plates by silver shows that the tissues without disruption contain much fewer protein bands; there is barely any proteinaceous material visible Only a few protein bands in the mantle tissue with a size of 80, 65, 60, 58, 38, and 32 kDa can be distinguished. It can also be traced that the proteins come mainly from inside the cells. The protein assemblage in the fresh tissues after cell disruption is the richest one. The protein bands in the mantle and pearl sac tissues are similar, and the main difference lies in the abundance of the proteins. The major bands have a size of 140, 100, 85, 70, 60, 58, 52, 48, 38, 36, and 32 kDa. From the analyses outlined above, it can be deduced that there are a lot of protein species in the fresh water mussel tissues. During the tissue cultivation, along with CaCO3 crystal formation, the protein production varies. Proteins contained in the mantle tissue cells mainly are involved in tissue growth and cell proliferation, and those contained in the pearl sac tissue cells mainly are involved in calcium carbonate crystal mediation. 3.3. Growth Pattern of Flowerlike CaCO3 in the Experiment Group. In the primary cell culture of fresh water mussel, the Ca2+ concentration in the mix solution of DMEM and CaCl2 was set at 1.0 mg/mL. However, in the application of the calcium chelation method only a final Ca2+ concentration of about 0.3 mg/mL can be determined (the difference may be attributed to the testing method used and/or the Ca2+ lost during the preparation of the solutions). The carbon source during the CaCO3 formation originates from CO2 dissolving. Both the Ca2+ and the CO32‑concentrations are lower than those in the in vitro mineralization experiment.42 With the secreted proteins from tissue, the crystals grow at a slower rate and steady speed in the experimental group. Hence, this circumstance allows a more detailed observation of the crystal growth pattern. In the experimental groups with mantle and pearl sac tissues, a special flowerlike CaCO3 crystal appears, and the growth pattern was recorded by light microscopy (Figure 7). The crystal growth speed in the pearl group was higher than that in the mantle group. Figure 6a shows, after 4 days of culture, that there is a disorder linear deposition. We assume that this is a precursor for CaCO3 crystal formation: ACC. After 6 days of culture (Figure 7b), round- or ellipse-shaped disks appear, and their morphology is similar to the synthesized ACC.43 It is deduced that these deposits are composed of ACC. The deposits then grow with a split in the middle of the crystals. Moreover, they show a rotational symmetry pattern, with a

Figure 9. Amorphous calcium carbonate in the circle (A) coexistence of flowerlike aragonite crystal with round disklike ACC deposition in the early days of cell culture. (B) Microscopic analyses; (a) light microscopic image and (b) polarized light microscopy observation of round disklike ACC. (C) SEM and EDX figures of round disklike ACC. (D) TEM figure of round disklike ACC: (a) TEM observation; (b) broken ACC after high energy electron beam bombardment.

mantle group. After 6 days of culture, signs of early formed calcium carbonate crystals appeared in pearl sac group, whereas similar signs showed in the mantle group after 8 days of culture. The smaller amount of cells and earlier crystal formation in the pearl sac group, compared with the mantle group, indicate a stronger influencing effect on the crystal formation of the pearl sac tissue and cells as well as secreted proteins. Figure 6 shows the initial and final morphologies of the CaCO3 crystals in the experimental groups with mantle and pearl sac tissues, as well as the control group without any tissue. For the initial morphology, after 6 days of culture, the early sign of crystal formation in the control group appeared in a square shape (Figure 6a). In the mantle group, those signs came later; only until the eighth day, a sticklike deposition appeared (Figure 6b). In the pearl sac group, sign of the crystal appeared earlier (on the sixth day), and showed an ellipse shaped deposit 1154



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Figure 10. Raman spectra during transformation from ACC to flowerlike aragonite crystal. (a−f) Transformation from ACC to aragonite; (g) aragonite crystal in experimental group; (h) calcite crystal in the control group.

“peanut”-like morphology and reach a preliminary crystal characteristic. After 8 days of culture (Figure 7e), a “water-

drop”-like granule grows from the middle of the axis of symmetry. For a longer crystallization time, the number of 1155



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“water-drop” granules increases and their size expands. After 11 days, the “water-drop” granules grow to cone-shaped crystals and start to aggregate at two ends (Figure 7j). The crystal aggregate has a similar shape with the aragonite aggregate in the in vitro mineralization experiment.44,45 After 18 days, the crystals in the experimental group have a well-developed flowerlike morphology; Figure 6k shows the front and side of this special morphology. The growth pattern of sticklike CaCO3 crystal in the control group was also recorded (Figure 8). The growth process of this crystal is comparably simple. As crystallization time goes, the crystal shape does not change much, but the size gets larger, and there is a small amount of crystal aggregation. 3.4. Stabilization and Transformation of ACC in the Experiment Group. During the recording of crystal growth in the experimental groups with mantle and pearl sac tissues, showing a flowerlike CaCO3 crystal aggregate growth pattern, a round-shaped disk is observed (Figure 9A). Its morphology is similar to the ACC synthesized by chemical methods,46,47 but the size is larger than the “critical size” of ACC.48 Polarized light microscopy result shows that the optical property of this disk is different from the well-developed crystals (Figure 9Ba,b). SEM and energy dispersive X-ray (EDX) results show that its chemical composition is CaCO3 (Figure 9C). TEM tests on the disk find small particles (about 10 nm) in the disk (200−300 nm), which is much smaller than the SEM and microscopy results, which might come from the crystal water loss during the sample treatment, and the shape changes from round disk to a disordered shape (Figure 9Da). This stage might be the formation of nanosized prenucleation clusters. Selected area electron diffraction (SAED) shows that, after bombardment of the electron beam, this disk is smashed into pieces (Figure 9Da,b). It suggests that the ACC disk is fragile and unstable and can be easily destroyed under an electron beam with high energy. Normally, ACC is considered to be very unstable and can transform to other forms of CaCO3 crystals such as calcite or aragonite in a short time. It is very difficult to observe ACC in the in vitro mineralization experiments.49,50 In this primary cell culture experiment, this round-shaped ACC disk can exist in liquid for a much longer time, even for days, which might be the stabilization effect of secreted proteins from mantle or pearl tissues.51 Raman spectrum also proves that this disk is ACC (Figure 10). In the experimental group, it can finally transform to aragonite aggregate (Figure 10g). The factors for aragonite stabilization can be multiple, and among those, acidic proteins play an important role.52 Also, the existence of Mg2+ can suppress calcite rhombohedra formation and thus stabilize the aragonite phase. In the control group, without tissue, the polymorph of the crystal is calcite (Figure 10h). The morphology of calcite crystal is very different from those regular rhombohedra shape,44 and this might be attributed to the amino acids (glutamine and/or glycine) and/or Mg2+ that are coming from the culture medium (DMEM).52−54 The structure and morphology differences of aragonites in the experimental group and calcite in the control group can be caused by many reasons. Mann used a schematic diagram to illustrate how Asp-X-Asp groups in those acidic proteins could affect, stereochemically, aragonite crystallization.15 In Finnemore’s research on the biomimetic layer-by-layer structure of artificial nacre, he noted “the existence of Mg2+ can suppress calcite rhombohedra formation”.52 In addition, we assume that shell-forming tissue can secrete Mg2+ and by that further

suppress calcite formation in the experimental group. Ren et al studied the effect of functional groups, especially carboxyl in acidic proteins on CaCO3 crystallization.42 The influence factors on calcium carbonate crystallization is multiple and complex, further researches are needed for in-depth discussion.

4. CONCLUSIONS In the primary cell culture of mantle and pearl sac tissues from fresh water mussel, H. cumingii, the characteristics of the cells from tissues and the effect of tissue, cells, and secreted protein on CaCO3 crystal formation process are studied. By the observations during cell culture, it is found that, in the mantle group, most of the cells are long epithelial cells, 30 μm long (Figure 4Ba). The cell migration in the pearl sac group is slower than the mantle group. There are mainly two types of cells in the pearl sac group: large round cells with 20 μm in diameter and small round cells with 2 μm in diameter (Figure 4Bb,c). The CaCO3 mineralization in the pearl sac group is faster compared to the mantle with the control group. The CaCO3 crystallization process in the pearl sac and mantle groups are similar, and the growing process of a flowerlike aragonite aggregate was recorded. In the control group, without any tissue, the growth pattern of the sticklike calcite was also recorded. The calcite morphology is different from that in the in vitro mineralization experiment. This might be attributed to the influence of amino acids in the culture medium. At the very beginning of the flowerlike aragonite aggregation, a special round-shaped sediment is detected. Raman spectrum, polarized light microscopy, and TEM analyses all prove that it is ACC. It shows that, in this primary cell culture of fresh water mussel tissue, ACC can stably exist in the medium for a very long time and finally transform to flowerlike aragonite crystal under protein mediation.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 10 62782770. Fax: 86 10 62771160. E-mail: [email protected]. Web: http://www.mse. tsinghua.edu.cn/faculty/fengql/enindex.htm. Author Contributions #

D.R and O.A. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from National Natural Science Foundation of China (51361130032), Doctor Subject Foundation of the Ministry of Education of China (20120002130002). We thank Dr.Stéphanie Auzoux-Bordenave and her group's help, UMR BOREA 7208, Station de Biologie Marine du Muséum national d’Histoire naturelle (France) for the cell culture methodology transfer within the IRSES program (IRSES246987).

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