## Kazuyoshi Endo · Toshihiro Kogure Hiromichi Nagasawa *Editors*

# Biomineralization

From Molecular and Nano-structural Analyses to Environmental Science

Biomineralization

The 14th International Symposium on Biomineralization was held during October 9–13, 2017, at Tsukuba International Congress Center in Tsukuba, Japan

Kazuyoshi Endo • Toshihiro Kogure Hiromichi Nagasawa Editors

## Biomineralization

From Molecular and Nano-structural Analyses to Environmental Science

*Editors* Kazuyoshi Endo Department of Earth and Planetary Science The University of Tokyo Graduate School of Science Tokyo, Japan

Hiromichi Nagasawa Department of Applied Biological Chemistry Graduate School of Agricultural and Life Sciences The University of Tokyo Tokyo, Japan

Toshihiro Kogure Department of Earth and Planetary Science The University of Tokyo Graduate School of Science Tokyo, Japan

ISBN 978-981-13-1001-0 ISBN 978-981-13-1002-7 (eBook) https://doi.org/10.1007/978-981-13-1002-7

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© The Editor(s) (if applicable) and The Author(s) 2018, corrected publication 2018. This book is an open access publication.

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### **Preface**

Biomineralization is a process by which organisms form mineral-like inorganic substances inside or outside their bodies. Biominerals are diverse in terms of structure, composition, morphology and role, depending on organisms. The research area of biomineralization is really interdisciplinary in methodology, ranging from microscopic observation to molecular biology. It includes not only basic researches mainly focusing on the mechanisms and evolutionary processes of biomineral formation but also applied researches including medical, dental, agricultural, environmental and materials sciences. Therefore, the society of biomineralization research constitutes a group of researchers with a variety of background. It is a good stimulation for us to get together and exchange ideas for new developments.

The International Symposium on Biomineralization began in 1970 and was held irregularly up to the 10th symposium but thereafter regularly every 2 years. This volume was planned as a record for the proceedings of the 14th International Symposium on Biomineralization (BIOMIN XIV): From Molecular and Nanostructural Analyses to Environmental Science. The symposium was held during October 9–13, 2017, at Tsukuba International Congress Center in Tsukuba, Japan. A total of 210 participants, including four accompanying persons, from 18 countries and areas attended this symposium. The participants included 157 regular researchers and 49 students, who contributed to the 106 oral presentations, including 10 keynote lectures, and 84 poster presentations. The presentations were classified into the following 8 topics:

This volume consists of 42 articles which are arranged in the order of the above topics. Most of them are original articles, and a few are reviews. The contributors were those who chose to submit their manuscript. All articles were peer-reviewed. Although the volume does not necessarily represent the whole contents of the symposium, it contains articles from all 8 topics. Thus, we are able to understand the present status of the cutting-edge of various aspects in biomineralization research from this volume. The volume has an appendix, which comprises valuable SEM and TEM images taken and left unpublished by late Dr. Hiroshi Nakahara, a distinguished researcher majoring in electron microscopy of biominerals. This appendix was arranged by Dr. Mitsuo Kakei, who selected the photographs from about 160 slides, which were shown on the screen during the lunch times in the symposium.

The symposium and the publication of this volume were supported by a number of Japanese scientific societies, to which we extend our gratitude. We express our sincere thanks to the Naito Foundation, Tokyo Ouka Foundation for the Promotion of Science and Technology, Kato Memorial Bioscience Foundation, Inoue Foundation for Science, Suntory Foundation for Life Sciences, Life Science Foundation of Japan, Tsukuba Tourism and Convention Association, and Tsukuba City for their financial support. Thanks are also due to Japanese Fossil Museum, Kouchiken Nihonkei Hozonkai, K. MIKIMOTO & Co. Ltd., Mikimoto Pharmaceutical Co., Ltd., Kao Corporation, LOTTE Co., Ltd., and Kotegawa Sangyo Co., Ltd., for their kind donations.

April, 2018

Tokyo Japan Kazuyoshi Endo Toshihiro Kogure Hiromichi Nagasawa

### **Contents**

#### **Part I Structure and Analysis of Biominerals**

viii

#### Contents

## **Part I Structure and Analysis of Biominerals**

## **Chapter 1 On the Transition Temperature to Calcite and Cell Lengths for Various Biogenic Aragonites**

#### **Taiga Okumura, Masahiro Yoshimura, and Toshihiro Kogure**

**Abstract** In order to understand the mineralogical difference between biogenic aragonites and their geological or synthetic ones, the transition temperature from aragonite to calcite by heating and the cell lengths of a number of biogenic aragonites have been measured using conventional and high-temperature XRD, as well as those of abiotic ones. Among 21 specimens, most biogenic aragonites showed a transition temperature 60–100 °C lower than that for abiotic ones. However, the shells of land snails showed almost similar transition temperatures. The temperature range from the beginning to the completion of the transition was also varied among the biogenic aragonites. On the other hand, the axial ratios (*a/b* and *c/b*) of aragonites in marine molluscan species were considerably larger than those of abiotic ones. However, aragonites in freshwater molluscan species and land snails showed axial ratios similar to abiotic ones. X-ray microanalysis suggested that the origin of such abnormal cell lengths was sodium incorporated in the aragonite crystals, not due to lattice distortion induced by the intracrystalline organic molecules proposed in previous researches.

**Keywords** Aragonite · Calcite · Transition temperature · Cell lengths · Axial ratio · Sodium

#### **1.1 Introduction**

Aragonite is one of the polymorphs of anhydrous calcium carbonate (CaCO3) and thermodynamically slightly less stable than calcite at the ambient temperature and pressure in which organisms are alive. However, aragonite commonly occurs by biomineralization processes. It has been often reported that biominerals possess distinct characteristics and properties which are not observed in their geological or

T. Okumura · M. Yoshimura · T. Kogure (\*)

Department of Earth and Planetary Science, The University of Tokyo, Tokyo, Japan e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS><EMAIL_ADDRESS>
synthetic counterparts. Biogenic aragonite is not the exception. For instance, Koga and Nishikawa (2014) investigated the transition from coral aragonite to calcite by heating in a thermogravimetric (TG)-differential thermal analysis (DTA) apparatus. They found that when the temperature was increased at a certain rate, the temperature at which the aragonite-calcite phase transition occurred was around 100 °C lower for the coral aragonite than the geological ones. Please note that this temperature is not corresponding to that at which the thermodynamic stability between aragonite and calcite is reversed. They ascribed such a low temperature for the transition to the existence of interstitial water between aragonite crystals, which was released during the transition.

On the other hand, the crystallographic parameters of biominerals have been also reported to be specific, compared to abiotic minerals. Pokroy et al. (2004, 2007) found that the cell lengths of aragonite in the shells of three molluscan species were slightly different from those of abiotic minerals; the *a-* and *c*-lengths are longer, and the *b*-length is shorter. They suggested that this "distortion" was induced by intracrystalline organic molecules, namely, a biological effect.

These two examples as the specific characters of biogenic aragonite are interesting to consider, for instance, the diagenetic effect on biominerals to form fossils. However, it is not certain whether such characters can be observed ubiquitously in biogenic aragonite formed by other species, families, etc. The present study investigated more than 15 specimens for biogenic aragonite as well as geological and synthetic ones, with respect to their temperatures for aragonite-calcite transition by heating and cell lengths.

#### **1.2 Materials and Methods**

The aragonite specimens investigated are listed in Table 1.1. In general, all specimens except the synthetic ones were powdered using an agate mortar and pestle for X-ray diffraction (XRD). The two synthetic aragonite specimens (Syn-PVA and Syn-Mg) were prepared according to Kim et al. (2005) and Kitano (1962) using polyvinyl alcohol (PVA) and MgCl2, respectively. In case of multilayered shells with a calcite layer, the calcite layer was removed by grinding with a micro drill, and then the remaining aragonite layer(s) was crushed into powder using an agate mortar and pestle. They were washed with distilled water and ethanol and then dried in an oven.

In the present study, we used an X-ray diffractometer (Rigaku SmartLab) with a high-temperature specimen holder (Rigaku DHS 900), to measure the temperature for the transition from aragonite to calcite. A copper X-ray tube was used and CuKα was selected by Ni filter. X-ray was detected using a silicon strip detector (Rigaku D/teX Ultra 2). The powdered specimens were processed into a disk of 3 mm in diameter and 0.5 mm thick by a press and placed on the high-temperature specimen holder. The temperature was raised at a rate of 5 °C, and 2*θ* was scanned repeatedly

**Table 1.1** Aragonite specimens investigated in this study

a Collection of The University Museum, The University of Tokyo

from 25.5° to 30.5° at a rate of 5°(2*θ*)/min. One hundred eleven reflection of aragonite and 104 reflection of calcite were recorded in the 2*θ* range.

The XRD measurement to refine the cell lengths of aragonite was conducted using a Rint-Ultima+ diffractometer (Rigaku) with CuKα radiation monochromated with Ni filter and a silicon strip detector (Rigaku D/teX Ultra 2). The 2*θ* range was 10°–90° with continuous scan and a rate of 1° (2*θ*)/min. Data was collected at every 0.02° (2*θ*). The powder specimens were mounted in a shallow dimple on a nonreflective plate made of silicon. The cell lengths (*a*, *b*, and *c*) of aragonite were calculated from the 2*θ* values of around 48 reflections, using PDXL software (Rigaku).

Finally, the chemical composition, particularly the concentration of sodium and chlorine, in aragonite was analyzed using an electron-probe microanalyzer (EPMA, JEOL JXA-8530F). The specimens for EPMA were prepared by embedding fragments of the minerals or shells in epoxy resin, polishing with diamond paste and colloidal silica. Finally amorphous carbon was coated by vacuum deposition for electron conductivity.

#### **1.3 Results and Discussion**

Figure 1.1 represents an example of the high-temperature XRD measurement. The temperature at the right of each pattern indicates the temperature at the beginning of the scanning. Please note that it took only 1 min for the 2*θ* scan, and the rate to increase temperature was 5 °C/min. The peaks from aragonite were gradually decreased, and conversely 104 peak of calcite was increased, indicating the progress of the phase transition. We estimated by extrapolation the beginning temperature at which 104 peak of calcite had the integrated intensity and the ending temperature at

**Fig. 1.1** An example of in situ high-temperature XRD pattern to determine the temperature for the aragonite-calcite transition

**Fig. 1.2** The beginning and ending temperatures of the transition for aragonite specimens of various origins. PT indicates phase transition

which 111 peak of aragonite completely lost the intensity. The two temperatures for all biogenic aragonites of several geological and synthetic aragonites are shown in Fig. 1.2. As reported in the previous work (Koga and Nishikawa 2014), biogenic aragonite showed lower beginning and ending temperatures for the transition than geological and synthetic aragonites. Particularly, the beginning temperature for the coral was extremely low. However, the transition temperatures for terrestrial molluscan shells, or land snails, were similar to those for abiotic aragonite, suggesting that biogenic aragonites are not always less stable than abiogenic ones. The origin of the difference of the temperature for the transition is not clear at present. Koga and Nishikawa (2014) proposed that the origin is intercrystalline water in the coral aragonite because water molecules were detected by mass spectroscopy at the transition. However, this is not convincing because the release of water may not be the origin of the transition but the accompanied phenomenon of the transition. It should be revealed by further investigations in the future.

The cell lengths of most of the samples are shown in Fig. 1.3. In the figure, the cell lengths are expressed with the ratio to those of geological aragonite from Sefrou, Morocco (*a* = 4.9629 (10) Å, *b* = 7.9690 (15) Å, *c* = 5.7430 (11) Å), according to Pokroy et al. (2007). As Pokroy et al. (2007) reported, *a*- and *c*-lengths are considerably larger, and *b*-length is shorter for some aragonites in molluscan shells,

**Fig. 1.3** The differences of the cell lengths of various aragonites from that of a geological sample (Sefrou, Morocco). The specimens surrounded with the rectangle are marine molluscan shells and coral

but this trend is not true for all biogenic aragonites. Among all samples investigated, the trend was distinctly observed only for marine molluscan shells. From this result, we supposed that salinity or incorporation of sodium and/or chlorine in the aragonite structure may have resulted in such a systematic change of the cell lengths. Hence, we conducted quantitative analysis of sodium/chlorine concentrations in the samples using EPMA. It was revealed that the content of chlorine was extremely low for all samples and not related to the cell lengths. On the other hand, the concentration of sodium was considerably varied, probably depending on the environments where the aragonites were formed. Considering the origin of the samples, the relationship between the axial ratio (*a*/*b* and *c*/*b*) and the sodium concentration was summarized as shown in Fig. 1.4. From the figure, it is apparent that distinct cell lengths or anisotropic *lattice distortions* observed in some biogenic aragonite compared to geological and synthetic ones are originated from the incorporation of sodium in the crystal structure of aragonite. We insist that the abnormality is not owing to intracrystalline organic molecules as proposed in the previous works (Pokroy et al. 2004, 2007).

Pokroy et al. (2004) considered Na-substitution in biogenic aragonite to change the cell lengths, but they denied its possibility because the ionic radii of Na+ and Ca2+ are so close that a small substitution of Na+ in aragonite cannot change its cell lengths significantly. It is probably true if we suspect only the isomorphic substitution, but the charges of the two cations are different, and some substitution for anions must be accompanied. As stated above, chlorine was not sufficiently detected

**Fig. 1.4** The axial ratios (*a*/*b* in the upper and *c*/*b* in the lower figure) vs. concentration of Na in the aragonites of various origins. The concentration of Na is expressed as wt.% of Na2CO3

in the sodium-bearing aragonites. One possibility is the incorporation of proton or hydroxyl (OH−) which substitutes O2− or HCO3 − instead of CO3 2−. Probably Na+ and OH− reside closely in the aragonite structure, and they locally modify the atomic arrangement around the substitution, which may affect the cell lengths. Of course it is not clear why the *a*- and *c*-lengths were elongated and *b*-length was shrunk by such substitutions, but the idea of intracrystalline organic molecules also cannot explain the anisotropic change. Pokroy et al. (2007) reported that when the biogenic aragonites were heated to 350 °C in air, the cell lengths became identical to those of geological aragonite, which they proposed the evidence for organic molecules as the origin of the lattice distortion. However, our preliminary experiment revealed that when a marine molluscan shell (*N. pompilius*) was annealed at around 350 °C, the concentration of sodium was significantly decreased, probably by diffusing away from the aragonite structure. Accordingly, the Na-substitution can also explain the annihilation of abnormality of the cell lengths by heating.

**Acknowledgments** We are grateful to Prof. A. Checa (Univ. Granada) and Prof. T. Sasaki (Univ. Tokyo) for donating valuable shell samples, Prof. M. Suzuki (Univ. Tokyo) for instructing the preparation of the synthetic aragonite, Mr. K. Fukawa (Univ. Tokyo) for assisting XRD measurement, and Mr. K. Ichimura (Univ. Tokyo) for assisting EPMA analysis.

#### **References**

Kim W, Robertson RE, Zand R (2005) Effects of some nonionic polymeric additives on the crystallization of calcium carbonate. Cryst Growth Des 5:513–522

Kitano Y (1962) The behavior of various inorganic ions in the separation of calcium carbonate from a bicarbonate solution. Chem Soc Jpn 35:1973–1980

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Chapter 2 TEM Study of the Radular Teeth of the Chiton** *Acanthopleura japonica*

**Mitsuo Kakei, Masayoshi Yoshikawa, and Hiroyuki Mishima**

**Abstract** The radula chiton teeth, *Acanthopleura japonica*, were examined using transmission electron microscopy (TEM). After cutting into segments corresponding roughly to three developmental stages from the onset of tooth development, the middle and the fully matured stages, toluidine blue staining has given the posterior side three different color patterns, colorless, reddish-brown, and black colors, respectively. At the colorless stage, the microvilli attached along the surface of the tooth cusp appeared to be dissembled and convert into the lamellar structure in the tooth interior. At the reddish-brown stage, the electron density between fibrous layers increased. A complex of tiny clusters of grains appeared along the fibrous layers. They seemed to aggregate each other to become larger. At the black stage, multiple layers consisting of irregular-shaped and various size of iron minerals were formed. After treating with an aqua regia solution, organic substances have remained between iron minerals, suggesting the abrasion-resistant role at the posterior side of chiton teeth during feeding. In addition, these minerals were randomly arranged. The lattice intervals of the ion minerals varied at an approximate range from 4.8 to 10.2 Å. Also, we have confirmed clearly the lattice fringe of apatite crystal in the core region.

**Keywords** Chiton · Transmission electron microscopy · Iron minerals · Apatite crystal

M. Kakei (\*) Tokyo Nishinomori Dental Hygienist College, Tokyo, Japan e-mail<EMAIL_ADDRESS>
M. Yoshikawa

H. Mishima Department of Dental Engineering, Tsurumi University School of Dental Medicine, Yokohama, Japan e-mail<EMAIL_ADDRESS>
The original version of this chapter was revised. A correction to this chapter is available at https://doi.org/10.1007/978-981-13-1002-7\_44

Division of Orthodontics, Meikai University School of Dentistry, Sakado, Japan e-mail<EMAIL_ADDRESS>
#### **2.1 Introduction**

The lateral teeth of the radula exhibit a sequential series of tooth developments along with the production of iron biominerals from the organic stage to the fully mineralized stage. Regarding the iron biominerals in the radula teeth of the chiton, the teeth were composed of multilayers of iron oxide, predominately in the form of magnetite, but also in other forms of ferrihydrite and lepidocrocite (Lowenstam 1967). Therefore, many studies have focused mainly on these iron biominerals in the tooth cusp (Lowenstam 1967; Kim et al. 1986; Weaver et al. 2010; van der Wal 1989; Martin et al. 2009; Han et al. 2011). The tooth cusps of posterior side are mainly reinforced by the mineralization of magnetite at the matured stage. Due to its hardness, TEM study conducted by making thin sections has not been available in particular for the fully matured chiton teeth so far. On the other hand, the presence of a calcium phosphate mineral in the tooth core region has been examined using various techniques for a considerable time (Lowenstam and Weiner 1985; Kim et al. 1986; Evans et al. 1992; Evans and Alvarez 1999; Lee et al. 2000). Although a study using electron microscopy has been tried to demonstrate the presence of apatite crystal (Evans et al. 1992), the precise structure of apatite crystal has not yet fully elucidated. In this study, we have conducted the present study to demonstrate the unique iron mineral deposits and verify the detailed structure of apatite crystal mineral using an electron microscope.

#### **2.2 Materials and Methods**

Samples of the chiton *Acanthopleura japonica* were collected at Hachijojima's coastal area, Tokyo, Japan. Radulae were extracted from the chiton. After the removal of the soft tissue, radulae were cut into segments corresponding roughly to the three developmental stages of radula teeth from the onset of tooth development, the middle, and the fully matured stages, by using a razor blade. Then samples were subjected to examine using transmission electron microscope. They were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 1 h at 5 °C, post-fixed with 1% osmium tetroxide in the same buffer for 1 h at 5 °C, dehydrated by passage through a series of ascending ethanol concentrations, and then embedded in Araldite 502. Thick sections were stained with toluidine blue solution. Based on different color patterns at the posterior side of ralular teeth, these developmental stages tentatively called the initial stage with colorless, the middle stage with reddish-brown, and the fully matured stage with black color were examined. Thin sections were obtained with a Porter Blum MT2 ultra-microtome (SORVALL) equipped with a diamond knife. Sections were stained with saturated uranyl acetate and lead citrate, and some were left unstained. Also, some treated with an aqua regia solution were subjected to study. Then, they were examined under a JEM 100CX electron microscope (JEOL) at an accelerating voltage of 80 kV.

**Fig. 2.1** Toluidine blue staining of the radula chiton teeth at three different stages. The change of color patterns at the posterior side reflects the degree of iron mineralization process. (**a**) The initial stage, (**b**) the middle stage, and (**c**) the fully matured stage. Toluidine blue-stained sections. Bar = 150 μm

#### **2.3 Results and Discussion**

Toluidine blue staining showed three different color patterns at the posterior side of three developmental stages from the initial stage with colorless, the middle stage with reddish-brown, and the fully matured stage with black color, respectively (Fig. 2.1).

At the colorless stage of the onset of tooth development, the internal structure of tooth cusps was filled with fibrous layers arranging in relatively parallel to the tooth surface at the anterior side (Fig. 2.2a). On the other hand, at the posterior side, fibrous layers bent upward adjacent to the inside of tooth cusp and run through toward the tooth tip (Fig. 2.2b).

At the reddish-brown stage of horizontal section, it was noted that higher magnifications of unstained sections have demonstrated that the deposits of ion minerals appeared immediately and grew more quickly at the posterior side than at the anterior side. So, it is suitable to examine the mineral deposits at the anterior side of the tooth cusp. A comparison between stained and unstained sections showed that the electron-dense zones were sandwiched by electron-lucent fibrous layers (Fig. 2.3). This suggests that the electron-dense zones might store mineral ions. At this stage, fibrous layers were altered to be the plume structure (Fig. 2.4a). Double-stained section showed the small and discrete particles were developed along fibrous layers. Also, it have been observed that a complex of tiny clusters of fine grains showing different electron density, which looked like a bunch of grapes, developed associating with the plume structure (Fig. 2.4b). The developed fine grains showing different electron densities seemed to aggregate each other and increase its size to create larger nonuniform grains.

**Fig. 2.2** TEM observations of the initial stage of chiton tooth. The arrangement of fibrous layer in the anterior side is different from posterior side of tooth cup. Double-stained sections. Bar = 0.5 μm

**Fig. 2.3** TEM observations of the middle stage. By comparing stained with unstained sections, the space between organic layers shows a relatively high electron density. Double-stained (**a**) and unstained (**b**) sections. Bar = 1.0 μm

**Fig. 2.4** High magnification of the plume structure (**a**) and the development of iron mineral grains (**b**) at the middle stage. The fine grains of iron minerals develop along the plume structure (**b**). Double stain. Bars = 100 nm (**a**), 2.0 μm (**b**)

At the black color stage of longitudinal sections, TEM study has demonstrated that the multilayers of iron minerals at the posterior side showed a brick-like or veneer-like wall structure (Weaver et al. 2010) (Fig. 2.5a). Each iron layer seemed to be comprised of relatively large minerals (Fig. 2.5b). Also, it was noted that small minerals were scattering in distribution. After treating with an aqua regia solution, organic substances remained between iron minerals (Fig. 2.6a, b). Although the role of organic matrix is not fully elucidated, it has been considered that the organic matrix may control the mineralization processes (van der Wal et al. 2000; Nemoto et al. 2012) and contribute to resist crack and increase the tensile strength and flexibility during the feeding by the teeth (Evans et al. 1990; van der Wal et al. 2000).

Iron minerals observed near the core (Fig. 2.7a, b) and in the magnetite (Fig. 2.7c, d) regions are shown in Fig. 2.7. Regarding the lattice fringes of iron minerals, the estimate of lattice intervals of these iron minerals was ranging from 4.8 to 10.2 Å, approximately. On the basis of the observation at a high magnification of Fig. 2.5b, it has been considered that small iron minerals might gather together to create a lump of iron minerals. It is also considered that random arrangement of iron minerals could prevent the radula teeth from becoming magnetic and attracting ion sand which comes from the sandy beach.

TEM observation has clearly demonstrated the crystal fringe of apatite crystal in the core region (Fig. 2.8). Whether the crystals are fluorapatite have been discussed previously (Lowenstam 1967; Kim et al. 1986; Evans et al. 1992; Evans and Alvarez 1999; Lee et al. 2000). To our knowledge, the biologically induced apatite crystals are divided into central dark line (CDL)-free and CDL-bearing types (Kakei et al. 2016). Viewing from the CDL-free type of crystal structure in the core region of the chiton teeth, we assumed that fluorapatite was formed.

**Fig. 2.5** TEM observations of the posterior region at the fully matured stage. The mineral layers consist of a brick-like wall or so-called veneer structure at the posterior side (**a**). Each mineral layer consists of both large angular and small minerals (**b**). No stain. Bars = 1.0 μm (**a**), 200 nm (**b**)

**Fig. 2.6** TEM observations of aqua regia solution-treated sections. Treating with an aqua regia solution shows organic substances remained between iron minerals and suggesting a possible role of glue. (**a**) 5 min treatment, (**b**) 3 min treatment. Arrows indicate organic substances. Doublestained sections. Bar = 100 nm (**a**), 20 nm (**b**)

**Fig. 2.7** TEM observations of iron minerals in the posterior region. Lattice fringes of iron minerals are recognized (**a**–**d**). Iron minerals are randomly arranged (**d**). No stain. Bar = 10 nm

**Fig. 2.8** TEM observations of calcium phosphate mineral in the core region. Two apatite crystals are confirmed. No stain. Bar = 10 nm

#### **References**

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Chapter 3 Experimental Cremation of Bone: Crystallite Size and Lattice Parameter Evolution**

**Martina Greiner, Balazs Kocsis, Mario F. Heinig, Katrin Mayer, Anita Toncala, Gisela Grupe, and Wolfgang W. Schmahl**

**Abstract** In this study we investigate pristine and experimentally incinerated bovine bone material with differing annealing times and temperatures from 100 to 1000 °C to analyse the crystallographic change of natural bone mineral during cremation. We used X-ray powder diffraction (XRPD) and Fourier transform infrared (FTIR) spectroscopy as complementary methods. We observe a structural change of bone mineral during cremation. Our study highlights that there are only few or even no hydroxyl ions in pristine bone mineral (bioapatite), which is a carbonate-hydroapatite rather than a hydroxyapatite. A significant recrystallization reaction from bioapatite to hydroxyapatite takes place at elevated temperatures from 700 °C (after 30 min cremation time). This process is associated with a significant increase of crystallite size, and it involves an increase of hydroxyl in the apatite lattice that goes along with a depletion of water and carbonate contents during cremation. Our first results highlight the importance of both time and temperature on the recrystallization reaction during cremation.

**Keywords** Carbonated apatite · Calcium phosphate · Bioapatite · Bone · X-ray diffraction · FTIR · Rietveld refinement

K. Mayer · A. Toncala · G. Grupe

e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
M. Greiner (\*) · B. Kocsis · M. F. Heinig · W. W. Schmahl

Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität, Munich, Germany

e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
Fakultät für Biologie, Anthropologie und Humangenetik, Ludwig-Maximilians-Universität, Martinsried, Germany

#### **3.1 Introduction**

The Forschergruppe FOR 1670 project on human transalpine mobility in the Late Bronze Age to Early Roman times performs isotope studies on archaeological bone finds. During that age, cremating the deceased was the primary burial custom (Grupe et al. 2015). To understand bone alteration by cremation, we study the evolution of bone crystallography and crystallite size as a function of cremation temperature and annealing time for bovine bone by FTIR and X-ray diffraction. Mammal bone mineral is a nanocrystalline material consisting of an apatite mineral that is chemically far more complex than hydroxyapatite and can be approximated as (Ca,Mg,Na)10-x((PO4)6-x(CO3)x)(OH1-y-z, (CO3)y, (H2O)z)2 (Elliott 2002; Rey et al. 2007). It comprises between 5 and 8 wt% carbonate, which substitutes in the [OH]− site (A-type substitution) as well as the [PO]4 3- site (B-type substitution) of the apatite structure (LeGeros et al. 1969; Wopenka and Pasteris 2005; Pasteris et al. 2012; Yi et al. 2013). Previous studies showed that recrystallization of the bioapatite and crystallite growth mainly sets in from about 600 °C (Piga et al. 2009; Schmahl et al. 2017), but small changes are already apparent at low temperatures of 100 °C (Harbeck et al. 2011). In this study, we want to define more precisely the cremation temperature and annealing time where hydroxyapatite crystallization sets in.

#### **3.2 Materials and Methods**

Pieces of compact bone were cut from the tubular part of bovine femur; the endosteal and periosteal surfaces were mechanically removed. Samples were ultrasonically washed in deionized H2O. After air-drying, the bones were defatted for 5 days with diethyl ether in a Soxhlet and air-dried. Finally, the samples were homogenized to a fine powder and passed through a 100 μm sieve.

Cremation experiments were carried out between 100 and 1000 °C in air (oxidizing conditions) in steps of 100 °C with 150 min annealing time. After data analysis of the initial results, we focussed on the temperatures of 650 and 700 °C and used shorter exposure times between 10 and 60 min in intervals of 10 min.

X-ray diffractograms were collected on a General Electric 3003 powder diffractometer in Bragg-Brentano reflection geometry. Cu-Kα1 radiation was selected with an exposure time of 1000s.

All samples were mixed with NIST 660b LaB6 as internal standard (2–5 wt%). The FULLPROF code (Rodríguez-Carvajal 1993; Rodríguez-Carvajal and Roisnel 2004) was applied for data evaluation and Rietveld refinement (Rietveld 1969). The Thompson-Cox-Hastings method for convolution of instrumental resolution with size and isotropic microstrain broadening (Thompson et al. 1987) was applied. For refinement, a hexagonal symmetry model for carbonated apatite was used (Wilson et al. 2004).

Infrared spectra were measured on a Bruker Equinox FTIR instrument with a resolution of 4 cm−1 with 128 scans.

#### **3.3 Results**

#### *3.3.1 X-Ray Diffraction: 100 °C Intervals*

Figure 3.1a shows X-ray powder diffraction patterns of untreated and cremated bones up to 1000 °C. The untreated bone mineral displays extremely broadened peaks. The broadening decreases only little with 150 min heat treatment up until 600 °C, whereas the diffraction pattern sharpens considerably from 700 °C upwards.

Exemplary Rietveld refinements of samples cremated at 600 °C and 700 °C for 150 min annealing time are shown in Fig. 3.1b, c. Enlarged images of the 31–35° 2θ section show overlapping apatite diffraction peaks in Fig. 3.1b, whereas peaks in Fig. 3.1c can clearly be distinguished from each other. The lattice parameters and crystallite sizes obtained by Rietveld refinement of diffractograms measured at room temperature for the 150 min heat-treated samples are shown in Fig. 3.1d, e. Note the sharp rise of the crystallite size setting in at 700 °C. The lattice parameters show an initial increase with annealing treatment and then sharply drop at temperatures where the grain size sharply increases.

#### *3.3.2 X-Ray Diffraction: 10 Min Intervals*

The X-ray diffractograms of the bovine bone cremated at 650 °C from 10 to 60 min depict a steady narrowing of the diffraction peaks with increasing annealing time. Nevertheless, a broad peak shape remains after 60 min annealing (Fig. 3.2a). The unit cell parameters shrink (Fig. 3.2b, c) already after 20 min cremation. The crystallite size increases more or less steadily with elapsed annealing time, whereas the biggest change is between 30 and 40 min with an increase of the crystallite size of 99.4(2) Å to 126.6(3) Å (Fig. 3.2c).

X-ray diffractograms of bovine bone cremated at 700 °C from 10 to 60 min show a broad diffraction peak shape from 10 to 30 min annealing time. Samples annealed for 40 min or longer depict sharper peaks. Bovine bone incinerated at 700 °C shows a steady increase of the crystallite size up to 30 min experimental cremation. From 30 to 40 min elapsing time, we observe a significant jump from 117.85(2) Å to 294.8(3) Å (2.5 times larger) (Fig. 3.2f). Moreover, we observe a decrease of lattice parameters a and c until 40 min annealing times. This trend is reversed after 50 min annealing (Fig. 3.2e, f).

**Fig. 3.1** (**a**) Comparison of the 10–60° 2θ range of X-ray diffractograms (Cu-Kα1) of cremated bones from 100 to 1000 °C; (**b**) Rietveld refinement of bovine bone cremated at 600 °C, 150 min annealing. *Red dots*, observed data points; *black line*, calculated XRD profile; *bottom blue line*, difference of observed and calculated data; *green vertical bars*, positions of diffraction peaks; *top row*, bone apatite; *bottom row*, LaB6 standard. Enlarged region shows 31–35° 2θ range with overlapping 121, 112, 030 and 022 peaks; (**c**) Rietveld refinement of bovine bone cremated at 700 °C after 150 min annealing. Enlarged region shows 30–35° 2θ range with clearly distinct 121, 112, 030 and 022 peaks; (**d**) Lattice parameters a (=b) and c of untreated and annealed bone material at temperatures from 100 to 1000 °C after 150 min annealing; (**e**) Unit cell volume and crystallite size of untreated and cremated bones from 100 to 1000 °C after 150 min annealing

**Fig. 3.2** (**a**) Comparison of the 10–80° 2θ range of X-ray diffractograms (Cu-Kα1) of untreated and cremated bones at 650 °C, 10–60 min annealing; (**b**) Lattice parameters a (=b) and c of untreated and cremated bones at 650 °C, 10–60 min annealing; (**c**) Unit cell volume and crystallite size of untreated and cremated bones at 650 °C, 10–60 min annealing; (**d**) 10–80° 2θ range of X-ray diffractograms of untreated and cremated bones at 700 °C, 10–60 min annealing; (**e**) Lattice parameters a(=b) and c of untreated and cremated bones at 700 °C, 10–60 min annealing; (**f**) Unit cell volume and crystallite size of untreated and cremated bones at 700 °C, 10–60 min annealing

#### *3.3.3 FTIR*

Figure 3.3a shows a comparison of IR spectra for different annealing temperatures. Characteristic phosphate group absorption bands at 470–480 cm−1 (ν2PO4 3−), 500– 750 cm−1 (ν4PO4 3−), ~962 cm−1 (ν1PO4 3−) and 980–1120 cm−1 (ν3PO4 3−) were identified according to Destainville et al. (2003) and Raynaud et al. (2002). Annealed bone at 1000 °C shows a well-differentiated hydroxyl libration peak at ~632 cm−1 which is absent in the FTIR spectra of untreated bovine bone (Fig. 3.3b).

Absorption bands at 873–879 cm−1 and 1400–1458 cm−1 were attributed to ν2CO3 2− and ν3CO3 2− (Fleet 2009; Grunenwald et al. 2014; Rey et al. 1989). The intensity of carbonate bands begins to decrease from 400 °C with increasing annealing temperatures and can barely be observed for the sample cremated at 1000 °C (see Fig. 3.3a). For untreated bovine bone, we observe broad H2O absorption bands from ~3000 to 3600 cm−1 (Brubach et al. 2005). These peaks lose intensity with heat treatment and disappear for the 700 °C and higher heat treatments (Fig. 3.3c).

**Fig. 3.3** (**a**) Comparison of the 400–1800 cm−1 FTIR spectra of untreated and cremated bovine bone at 400, 500, 600, 700 and 1000 °C (annealing time 150 min); (**b**) Enlargement of the 450– 750 cm−1 region of Fig. 3.2a) with ν2PO4 3− and ν4PO4 3− vibration bands and emerging OH− libration band with increasing cremation temperature; (**c**) 2600–3800 cm−1 region of untreated and cremated bone (400, 500, 600, 700 and 1000 °C) indicating decreasing H2O absorption bands and increasing OH− stretching mode with increasing cremation temperature

The OH stretch vibration gives a sharp peak at 3570 cm−1 (González-Díaz and Hidalgo 1976; González-Díaz and Santos 1977; Vandecandelaere et al. 2012). The band at ~632 cm−1 was assigned to the OH− libration mode (Destainville et al. 2003). In the untreated bone spectra and for low annealing temperatures, neither the OH− libration peak near 630 cm−1 nor the sharp and distinct OH− stretching vibration peak at 3570 cm−1 is present. However, these OH− signals emerge with annealing at 400 °C and significantly increase and become well-differentiated for samples annealed at temperatures of 700 °C or higher (see Fig. 3.3b, c).

#### **3.4 Discussion**

In the original bone, we observe broad diffraction peaks (Figs. 3.1a and 3.2a, b) due to the nanoscale dimension of the bone apatite crystallites. Further, the high intensity of the carbonate and H2O vibration bands and absence of OH− bands quite clearly indicate that bone apatite is a carbonate-hydro-apatite rather than a hydroxyapatite (Fig. 3.3a–c). Loong et al. (2000) using inelastic neutron scattering and Pasteris et al. (2004) using Raman spectroscopy came to similar conclusions.

With extended annealing treatment, the diffraction peaks of the bone mineral get sharper; the (CO3) 2− infrared signals decrease, while the OH− infrared peaks increase. Within the broad water band, one can clearly see the rising of the OH− stretching is a function of increasing annealing temperature (Fig. 3.3c). This indicates a reaction from bioapatite (carbonate-hydro-apatite) to hydroxyapatite with heat treatment, as the material approaches stoichiometric chemistry. This reaction is associated with growth of the crystallites; the growth becomes more rapid at temperatures from 700 °C and higher (Figs. 3.1a and 3.2d). At the same time, loss of carbonate and water and their replacement by OH− in the structure lead to a decrease of unit cell parameters (Figs. 3.1d, e and 3.2b–f).

While observing these structural changes occurring, it cannot be simply concluded that the process occurs homogeneously within the apatite lattice. It is just as likely that the bioapatite decomposes and that hydroxyapatite is formed in a heterogeneous reaction at the expense of the decomposing bioapatite. It must be borne in mind that our techniques integrate over the whole volume of the sample, and the diffraction peak positions as well as IR frequencies of both mineral phases (bioapatite and hydroxyapatite) are very close and initially severely broadened. Thus our techniques might well record a superposition of the signals of both phases coexisting. Here it is important to note that the distinct increase of the crystallite size for temperatures of and above 700 °C sets in after 30 min of annealing time (Fig. 3.2e, f). This may be indicative of a heterogeneous rather than a homogeneous reaction from bioapatite to hydroxyapatite. For an increase of crystallite size of only one homogeneous apatite phase by Ostwald ripening, the evolution of crystallite size with time (Fig. 3.2f) should have the opposite curvature than observed.

The time dependence (Fig. 3.2) of the reaction process becomes important when concluding from bone crystallinity to cremation temperature. Up to 40 min cremation time, we see the same trend for lattice parameters when comparing 650 and 700 °C: a decrease of lattice parameters a and c and therefore a shrinkage of the unit cell coupled with an increase of the crystallite size (Fig. 3.2b–f). After more than 30 min annealing at 700 °C, we see more rapidly increasing lattice parameters, an effect which is not observable in the 650 °C temperature experiments. Moreover, at

700 °C after 30 min annealing, we observe also a significant change in crystallite size which we attribute to a progressive recrystallization reaction of bioapatite to hydroxyapatite. At that time, the decomposition products (essentially carbon) of organics such as collagen, which comprise about 35 wt% of the original bone material (Rogers and Zioupos 1999), are almost completely gone, and the hydroxyapatite crystallites can grow without being impeded by films of organics or their residues which separate the crystallites.

Note that bone annealed at 400 °C for 150 min displays essentially the same crystallite sizes as bone treated for 20 min at 700 °C (~97 Å), and bone annealed at 600 °C for 150 min displays similar crystallite sizes as bone treated at 650 °C for 50 min (~133 Å). Our experiments reveal that it is important to consider the influence of time when concluding from material state on cremation conditions.

**Acknowledgement** We thank the Deutsche Forschungsgemeinschaft (DFG) for funding the project in Forschergruppe FOR1670 under Schm930/12-1 and Gr 959/20-1,2.

#### **References**

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Chapter 4 Effect of Carbonic Anhydrase Immobilized on Eggshell Membranes on Calcium Carbonate Crystallization In Vitro**

**M. Soledad Fernández, Betzabe Montt, Liliana Ortiz, Andrónico Neira-Carrillo, and José Luis Arias**

**Abstract** The eggshell membranes (ESM) serve as the first interface with the inorganic phase during eggshell formation. During mineral growth, crystals nucleate on the outer side of the ESM at specialized sites called mammillae, mainly consisting of mammillan, a keratan sulfate proteoglycan together with the activity of carbonic anhydrase (CA).

In order to get insight into the mechanisms of chicken eggshell mineralization, ESM was used as a biotemplate for immobilizing carbonic anhydrase (CA) and study in vitro calcite crystallization. Here, we showed that when the eggshell membrane supplemented with immobilized or dissolved carbonic anhydrase is located at the gas-liquid interface, calcite nucleation and growth are sequestered by the ESM scaffold from solution, thus affecting the morphology and size of the crystals formed.

**Keywords** Eggshell membrane · Carbonic anhydrase · Biomineralization

#### **4.1 Introduction**

Biomineralization is a widespread phenomenon in nature leading to the formation of a variety of solid inorganic structures by living organisms, such as intracellular crystals in prokaryotes; exoskeletons in protozoa, algae, and invertebrates; spicules and lenses; bone, teeth, statoliths, and otoliths; eggshells; plant mineral structures; and also pathological biominerals such as gall stones, kidney stones, and oyster pearls (Lowenstam and Weiner 1989; Mann et al. 1989; Simkiss and Wilbur 1989; Heuer et al. 1992; Arias and Fernandez 2008). By this process living organisms precipitate inorganic minerals on organic matrices. The resulting biominerals are deposited in elaborated shapes and hierarchical structures by interaction at the organic-inorganic interface, where the rate of crystal formation is regulated by the

M. S. Fernández (\*) · B. Montt · L. Ortiz · A. Neira-Carrillo · J. L. Arias Faculty of Veterinary Sciences, University of Chile, Santiago, Chile

e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS><EMAIL_ADDRESS><EMAIL_ADDRESS>
control of the microenvironment in which such mineralization events take place. One of the main biomineralization resulting mineral minerals is calcium carbonate (CaCO3), especially the more stable form, calcite. The avian eggshell is a good example of multifunctional biomineral where an organic scaffold (or matrix), the eggshell membrane, plays a crucial role in regulating mineral nucleation and growth by incorporating inorganic precursors, such as ions, ion clusters, and amorphous phases (Rao et al. 2017); other organic matrices complete the formation of a calcified layer (i.e., palisade) composed of calcite columns (Panheleux et al. 1999). Structurally, the eggshell is a multilayered calcitic bioceramic. As the egg migrates through the oviduct, the biomineral matures under the influence of biomolecular additives and an extracellular matrix (Nys et al. 1999; Fernandez et al. 2001). The matrix, primarily composed of collagen fibers, constitutes the eggshell membranes (ESM) (Arias et al. 1991). Each fiber exhibits a core surrounded by a glycoproteinous material termed as the mantle. This serves as the first interface with the inorganic phase. During mineral growth, crystals nucleate on the outer side of the ESM at specialized sites called mammillae, mainly consisting of mammillan, a keratan sulfate proteoglycan together with the activity of carbonic anhydrase (CA). This metalloenzyme catalyzes the reversible hydration of carbonic dioxide to bicarbonate and a proton. In fact, it has been possible to mimic eggshell formation in vitro by adding the main organic components including CA, where an increase in calcium carbonate crystals growth and fusion was observed (Fernandez et al. 2004).

However, the use of enzymes in vitro is difficult, because of instability and the complexity for maintaining the catalytic function in chemical reactions (Lu et al. 2013). For that reason enzymatic immobilization on a solid substrate appears as a solution for this problem (Wanjari et al. 2013).

For in vitro biomineralization, a specific confined environment is needed, that means an inert scaffold which generates an almost two dimensional interface where the crystal nucleation takes place. Currently, many kinds of surfaces and/or supports are used for enzyme immobilization, and the ESM meets all the requirements to be used as a natural support for CA immobilization and in vitro biomineralization experiments.

In order to get insight into the mechanisms of chicken eggshell mineralization, ESM was used as a biotemplate for immobilizing carbonic anhydrase (CA).

#### **4.2 Material and Methods**

#### *4.2.1 Carbonic Anhydrase (EC <IP_ADDRESS>) Immobilization on ESM*

ESM were obtained after 30 min incubation of an empty egg in 1% acetic acid to detach the membrane from de shell, and then ESM was incubated for another 48 h in 1% acetic acid to eliminate any remaining calcium carbonate crystals and then washed in deionized water three times (Arias et al. 2008).

**Fig. 4.1** Experimental setup used from growing in vitro CaCO3 crystals showing the location of eggshell membrane strips either on the bottom or the top of the microbridges containers

For enzyme immobilization 1 mg of carbonic anhydrase (2500 units/mg, Sigma, St. Louis, MO, USA) in 1 mL deionized water was used; membranes were incubated for 1 h with 100 μL of this solution; then 20 μL of 2.5% glutaraldehyde for 30 min was used as cross-linking agent (Tembe et al. 2008). Then, membranes were washed with TRIS buffer solution pH 9 at 4 °C.

#### *4.2.2 Crystallization Experiments*

The crystallization assays were based on a variation of the sitting drop method developed elsewhere (Dominguez-Vera et al. 2000). Briefly, it consists of a chamber built with a 85 mm plastic petri dish having 18 mm in diameter central hole in its bottom, glued to a plastic cylindrical vessel (50 mm in diameter and 30 mm in height) (Fig. 4.1). The bottom of the petri dish was divided in 16 radii to assure an equidistant settling of odd number of polystyrene microbridges (Hampon Res., Laguna Niguel, CA). The microbridges were filled with 35 μL of 200 mM dihydrate calcium chloride solution in 200 mM Tris buffer, pH 9.0. The cylindrical vessel contained 3 ml of 25 mM ammonium carbonate. One strip of eggshell membrane with or without immobilized CA or with CA in solution was deposited on the bottom or on the top of each microbridge with the mammillary side facing up or upside down, respectively. Five replicates of each experiment were carried out inside the chamber at 20 °C for 24 h. After the experiments, eggshell strips were taken out of the microbridges, air-dried at room temperature, mounted on aluminum stubs with scotch double-sided tape, and coated with gold. Crystal morphology was observed and size estimated in an Hitachi TM 3000 scanning electron microscope.

#### **4.3 Results**

#### *4.3.1 ESM on Bottom of the Microbridge 24 H Incubation*

After 24 h incubation using ESM without treatment, rounded calcite crystals were observed deposited on the ESM (Fig. 4.2a), with an average size of 21.33 ± 1.97 μm (Table 4.1). By contrast, when ESM with immobilized CA was used, regular

**Fig. 4.2** Eggshell membrane (ESM) on bottom of the microbridge after 24 h incubation: (**a**) ESM without treatment, 1000X; (**b**) ESM with immobilized CA, 1000X; (**c**) ESM without treatment but with CA dissolved in the calcification medium 1000X

**Table 4.1** Size of calcite crystals deposited on eggshell membrane (ESM) measured under different conditions

**Fig. 4.3** Eggshell membrane (ESM) on top of the microbridge facing calcification medium after 24 h incubation: (**a**) ESM without treatment, 1000X; (**b**) ESM with immobilized CA, 1000X; (**c**) ESM with CA in solution in the calcification medium 1000X

rhombohedral calcite crystals (Fig. 4.2b), 31 ± 2.08 μm in average size were obtained (Table 4.1). When intact ESM was used in combination with CA dissolved in the calcification medium, rounded calcite crystals (Fig. 4.2c) with an average size of 20 ± 3.68 μm (Table 4.1), similar to those observed in the first condition were obtained.

#### *4.3.2 ESM on Top of the Microbridge Facing the Calcification Medium After 24 H of Incubation*

After 24 h incubation using ESM without treatment, polyhedral calcite crystals were obtained (Fig. 4.3a), with 22.1 ± 1.91 μm in average size (Table 4.1). But when ESM with immobilized CA was used, many polyhedral fused calcite crystals of 35.83 ± 3.13 μm in average size (Table 4.1) with symmetrical smooth edges (Fig. 4.3b) were observed. However, after using intact ESM with CA in solution, less polyhedral fused calcite crystals with curved edges (Fig. 4.3c) of 27.83 ± 2.47 μm in average size were observed (Table 4.1).

#### **4.4 Discussion**

When in vitro CaCO3 crystallization experiments are done in a gas-liquid interface diffusion environment, the favorite place for crystal nucleation and growth must be considered. In fact, in this chamber-mediated experiment, CO2 comes from the gas phase, while carbonate ions are in aqueous solution. If the reaction is done without any additional scaffold, such as the eggshell membrane, it is expected that nucleation of CaCO3 crystals occurs close to the gas-liquid interface, and then, after a determined period of growth, crystals formed precipitate reaching the bottom of the reaction vessel (microbridge). However, here we showed that when an active heterogeneous nucleator scaffold, such as the eggshell membrane supplemented with immobilized or dissolved carbonic anhydrase, is located upside down at the gasliquid interface for avoiding gravity effect, calcite nucleation and growth are sequestered by the scaffold from solution, thus affecting the morphology and size of the crystals formed. The morphology changes, including calcite aggregations occurs in a way that resembles the calcite column aggregation and fusion observed during natural eggshell formation.

**Acknowledgment** Work supported by Fondecyt project 1150681 from CONICYT.

#### **References**

Simkiss K, Wilbur KM (1989) Biomineralization. Academic, San Diego

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Chapter 5 Proteomic Analysis of Venomous Fang Matrix Proteins of** *Protobothrops flavoviridis* **(Habu) Snake**

**Tomohisa Ogawa, Asa Sekikawa, Hajime Sato, Koji Muramoto, Hiroki Shibata, and Shosaku Hattori**

**Abstract** Venomous animals have specialized venom delivery apparatus such as nematocysts, stings, and fangs in addition to the poisonous organs consisting venom gland or sac, which produce and stock the venom. Snake is one of the major venomous animals, of which fangs are connected to the venom gland to inject the venom into prey. Snake's venomous fangs showed the unique characteristics including mechanical strength and chemical stability. Especially, *Protobothrops flavoviridis* (habu) snake fangs showed the resistance against its venom digestive proteases, whereas the bones and teeth of mouse were completely digested in the gastrointestinal tract, although habu fangs were also drawn into the body with the prey. These observations suggest that structural differences exist between venomous fangs and mammalian bones and teeth.

In this study, to reveal the molecular properties of venomous snake fangs, the matrix proteins of *P. flavoviridis* (habu) snake venom fang were analyzed by using proteomics experiments using 2D-PAGE and TOF MS/MS analyses. As a result, several biomineralization-related proteins such as vimentin, tectorin, adaptin, and collagen were identified in the venomous fang matrix proteins. Interestingly, the inhibitory proteins against venomous proteins such as metalloproteinase and PLA2 were also identified in fang's matrix proteins.

T. Ogawa (\*) · A. Sekikawa · H. Sato · K. Muramoto

Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan

e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>koji.muramoto.d5@ tohoku.ac.jp

H. Shibata Division of Genomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan e-mail<EMAIL_ADDRESS>
S. Hattori Amami Laboratory of Injurious Animals, The Institute of Medical Science, The University of Tokyo, Kagoshima, Japan e-mail<EMAIL_ADDRESS>
**Keywords** Biomineralization · Matrix protein · Proteome · Snake · Venomous fang

#### **5.1 Introduction**

Venomous animals such as sea anemone, jellyfish, lizards, scorpion, fish, arachnids, bees, and snakes produce chemical weapon, toxic proteins, and peptides cocktail to kill and capture pray. They deliver the toxins as venom into prey through the sophisticated venom delivery systems consisting of an exocrine gland, a lumen, venom duct, and also injector such as nematocyst, sting, fangs, harpoon-like sting, and spine. These venomous apparatuses are thought to have evolved from the general biological organs, namely, an ovipositor, a tooth, radula, and dorsal fin, respectively. Snake is one of the major venomous animals, of which fangs are connected to the venom gland to inject the venom into prey. Venomous snakes can be classified into two groups according to the fang systems, front fanged (elapid and vipers) and rear fanged (grass snakes), and frontal fangs are further divided into two types, grooves and tubes (Kardong 1979; Savitzky 1980; Jackson 2002; Kuch et al. 2006). Vonk et al. (2008) reported the evolutionary origin and development of snake fangs, showing that front fangs develop from the posterior end of the upper jaw and are strikingly similar in morphogenesis to rear fangs. In the anterior part of the maxilla of front-fanged snakes, gene expression of sonic hedgehog, which is responsible among other things for the formation of the teeth, is suppressed. Despite such extensive studies and the recent genome sequence analyses for two venomous snakes, the king cobra (*Ophiophagus hannah*) (Vonk et al. 2013) and the five-pacer viper (*Deinagkistrodon acutus*) (Yin et al. 2016), the matrix proteins of venomous fangs, their evolutionary origins, and the biomineralization mechanisms of venomous fangs are still poorly understood.

*Protobothrops flavoviridis* (habu) snake that inhabits Ryukyu (Okinawa, Tokunoshima, and Amami) Islands are dangerous snakes having various toxic peptides and proteins (multiple protein families) as venom. Their venomous fangs are frequently lost and drawn into their own body with the prey after injection of the venom. Interestingly, venomous fangs are excreted with no change and no digestion, whereas the bones and teeth of the mouse (prey) are completely digested. These observations suggest that structural differences between venomous fangs and mammalian bones and teeth exist. In addition, it is conceivable that the adaptive evolution of the venomous organ and venomous fang bestowed them to have resistance to digestive juices. Thus, the snake fangs show the unique characteristics including mechanical strength and chemical stability.

In this study, to reveal the characteristics of habu snake fangs such as chemical stability, and their molecular evolution, proteomic analyses of fang matrix proteins were conducted by using 2D-PAGE and MALDI-TOF MS/MS.

#### **5.2 Materials and Methods**

#### *5.2.1 Materials*

The crude venomous fangs of *Protobothrops flavoviridis* (habu) snakes captured in Amami Island, Kagoshima Prefecture, Japan, were collected by dissection of the head from sacrifice. Subsequently, fangs and tissues were separately rinsed with phosphate-buffered saline and stored at −80 °C until use. Immobiline DryStrip for two-dimensional electrophoresis and the IPG buffer (pH 3–11) were obtained from GE Healthcare UK Ltd. (Buckinghamshire, England). Silver Stain MS kit was purchased from Wako Pure Chemical Industry, Ltd. (Osaka, Japan). *Achromobacter* protease I and *Staphylococcus aureus* V8 protease were obtained from Wako Pure Chemicals (Osaka, Japan) and Sigma-Aldrich Co. (St. Louis, MO, USA), respectively. ZipTip C18 was purchased from Millipore (Massachusetts, USA). All other reagents were of the best commercially available grade from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).

#### *5.2.2 Isolation and Characterization of the Matrix Proteins from the Venomous Fang*

Venomous fangs of habu snakes were decalcified with 50% formic acid at room temperature for 2 days. Then, the decalcified matrix proteins were dissolved in 6 M guanidinium hydrochloride in 50 mM Tris-HCl buffer (pH 8.8) containing 200 mM NaCl at 60 °C. After TCA-acetone precipitation, the pellet was dissolved in 8 M urea in 100 mM Tris-HCl buffer (pH 8.2) at 60 °C. For two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), fang matrix proteins were directly dissolved in 400 μl of rehydration buffer (8 M urea, 4% CHAPS, 2% immobilized pH gradient (IPG) buffer (pH 3-11NL), DeStreak reagent (15 mg/ml), and 0.002% bromophenol blue) and were loaded onto IPG strips. After rehydration for 12 h, isoelectric focusing (IEF) was performed at 20 °C using the running conditions of the following focusing program: 500 V for 1 h, a gradient to 1000 V for 1 h, a gradient to 8000 V for 3 h, and 8000 V for 1.5 h (3225 V, 50 μA, 19,742 Vhs). After running IEF, IPG strips were equilibrated in a reducing equilibration buffer for 15 min and subsequently alkylated with iodoacetamide. Then, IPG strips were transferred onto 15% polyacrylamide gel (18×16 cm) and embedded with 0.5% agarose and electrophoresed. Gels were stained using Silver Stain MS kit or Coomassie Brilliant Blue.

#### *5.2.3 Proteome Analysis*

The spots on 2D gel were cut into pieces and washed with Milli-Q water. After the gels were dehydrated by acetonitrile with gentle agitation and completely dried in vacuo, gel samples were reduced by 10 mM DTT for 1 h at 56 °C. After cooling and washing by 25 mM ammonium bicarbonate buffer for 10 min, the gel samples were treated with 55 mM iodoacetamide in 25 mM ammonium bicarbonate solution in the dark. After removal of the solvent to be completely dried, gel particles were digested by *Achromobacter* protease I (Lys-C) or V8 protease at 37 °C for one night. After concentrating the digest in speed vacuum, samples were desalted on ZipTip C18 (Millipore). Samples were separated by using a DiNa Nano LC system equipped with a DiNa MALDI spotting device (KYA Technologies Co., Tokyo, Japan) and applied to MALDI-TOF MS and tandem MS/MS analysis using TOF/TOF™ 5800 Analyzer (AB SCIEX). Enzyme-digested matrix proteins without 2D-PAGE were also analyzed by nanoLC-MALDI-TOF MS/MS. Molecular masses were calibrated using the Sequazyme Peptide Mass Standards Kit (Applied Biosystems). Protein identification was performed by searching of each MS/MS spectrum against the protein sequence databases derived from the RNA-seq data of *P. flavoviridis* snake fang-forming tissues by using ProteinPilot software (version 3.0; AB Sciex) with the Paragon method.

#### **5.3 Results and Discussion**

#### *5.3.1 Isolation and Characterization of the Matrix Proteins from* **P. flavoviridis** *Venomous Fangs*

First, the decalcification conditions of venomous fangs were investigated by using hydrochloric acid and formic acid, respectively. The complete decalcification of the venomous fang without protein degradation was achieved by 50% formic acid at 30 °C for 2 days, resulting typical yield of 5.6 mg from 1.0 g of *P. flavoviridis* fangs, while the decalcification of fang by 10% HCl treatment caused the degradation of proteins (Fig. 5.1a). Then, the matrix proteins were subjected to the proteome analysis, *in-gel* enzymatic digestions for mass spectrometry characterization with 2D-PAGE and the shotgun proteomics of enzymatic digestions of total matrix proteins by nanoLC-MS/MS, respectively, after dissolved in 6 M guanidinium at 60 °C and concentrated by TCA-acetone precipitation.

**Fig. 5.1** Proteomic analysis of *P. flavoviridis* fang matrix proteins

Brief explanation of procedure through decalcification and mass spectrometry analysis (**a**) and typical profiles of digested matrix proteins on nanoLC-MALDI-TOF MS/MS (**b**)

**Fig. 5.2** 2D-PAGE profile of *P. flavoviridis* venomous fang matrix proteins Samples #1 to #5 were analyzed by nanoLC-MALDI-TOF MS/MS analysis after *in-gel* digestion, respectively

#### *5.3.2 Proteome Analysis of the Fang Matrix Proteins*

To identify the array of proteins in *P. flavoviridis* venomous fangs, the extracted matrix proteins were subjected to the 2D-PAGE (pH 3–11), resulting in identification in acidic region of around 20 appreciable major spots, of which pI values ranging from 4 to 6 (Fig. 5.2). These fang matrix proteins were roughly divided into five groups based on the molecular mass numbers: 55 kDa (sample #1), 40 kDa (#2), 35 kDa (#3), 30 kDa (#4), and 25 kDa (#5) proteins. Preliminary proteomic analyses of these protein spots allowed the identification of major components of fang matrix proteins including type I collagens alpha-1 and alpha-2 and UV excision repair protein RAD23-like protein (Table 5.1). Interestingly, antihemorrhagic factor HSF, which is a proteinaceous serum inhibitor against own venom metalloproteinases, was also detected as a matrix protein. However, these proteomic data from 2D-PAGE could not provide satisfactory results.

To improve the proteomic data of *P. flavoviridis* fang matrix proteins, a direct shotgun proteomic analysis was conducted. As a result of 4 independent experiments of shotgun proteomics, 36 proteins were identified as fang matrix proteins (Table 5.2). In addition to the type I collagen alpha1 (isoform X1 and X2) and alpha2 chains, the collagens type VI alpha2 and alpha3 chains and type XI alpha1 and alpha2 chains were identified. Because type I collagen has been reported to be related to the formation of dentin and enamel, contributing to the nanoscale architecture in the teeth (Wallace et al. 2010), type I collagen seems to be an important

aSample numbers #2 to #4 correspond to the number of 2D-PAGE spots in Fig. 5.2

**Table 5.2***Protobothrops flavoviridis* fang matrix proteins identified by shotgun proteomic analyses

Trial/protein

Peptides

**Table 5.2** (continued)

Trial/protein

Peptides

(continued)

**Table 5.2** (continued)

Trial/protein

Peptides

(continued)

**Table 5.2** (continued) component of venomous snake fang. Furthermore, the type VI collagen, which forms microfibrils and is primarily associated with the extracellular matrix of skeletal muscle and bone marrow, and type XI collagen, which is found in the cartilage of the nose and external ears in human, were also identified as matrix proteins in venomous fang, suggesting the unique distribution of type VI and type XI collagens as part of the fang matrix. On the other hand, noncollagenous dentin matrix proteins including proteoglycans (PGs), glycoproteins, serum proteins, enzymes, and growth factors are deemed to play structural, metabolic, and functional roles as key components in the mineralization process of dentin (Orsini et al. 2009). Shotgun proteomic analysis showed the fang noncollagenous dentin matrix proteins include proteoglycan such as decorin (1\_2, 3\_4, 4\_13 in Table 5.2) and biglycan (1\_8, 4\_4), glycoproteins such as osteonectin (secreted protein acidic and rich in cysteine: SPARC) (1\_5, 4\_8), the SIBLING proteins such as dentin matrix acidic phosphoprotein 1 (4\_12), and serum proteins such as albumin (1\_4, 3\_5, 4\_1), phospholipase A2 inhibitor (1\_14) and antihemorrhagic factors, HSF (1\_9, 2\_2, 3\_2, 4\_2), and HSFlike protein (1\_7, 3\_6). The coexistence of these serum inhibitors as fang matrix proteins explains why venomous fang is stable against own venom enzymes compared with mouse-derived teeth and bones. Compared with the homologous proteins in mouse, several fang matrix proteins such as dentin matrix acidic phosphoprotein 1 (36%), titin-like protein (31%), transferrin-like protein (26%), and serum inhibitors including albumin (32%) and PLA2 inhibitor (29%) showed lower sequence similarities, suggesting that these differences in matrix proteins might be related to the functional differences and distinctive properties between venomous fang and mouse's teeth.

In this study, we identified 36 matrix proteins from *P. flavoviridis* snake fangs by proteomics analyses. They include proteinaceous inhibitor against own venom enzymes in addition to several types of collagens (types I, VI, and XI) and noncollagenous dentin matrix proteins. More recently, we have decoded the whole genome sequence of *P. flavoviridis* snakes (Shibata et al. 2018, in press). Further investigations are needed to elucidate the biomineralization mechanisms of venomous fang and their biological functions.

**Acknowledgments** The authors thank Prof. Noriyuki Satoh, Drs. Shinichi Yamasaki and Kanako Hisata, Okinawa Institute of Science and Technology Graduate University (OIST), Onna, Okinawa, for providing RNA-seq data from *P. flavoviridis* fang-forming tissues.

This study was partly supported by Grants-in-Aid of MEXT, Japan (#24651130 and #23107505 to TO). This study was also partly performed in the collaborative Research Project Program of the Medical Institute of Bioregulation, Kyushu University.

#### **References**

Jackson K (2002) How tubular venom-conducting fangs are formed. J Morphol 252:291–297 Kardong KV (1979) Protovipers and the evolution of snake fangs. Evolution 33:433–443 Kuch U, Müller J, Mödden C, Mebs D (2006) Snake fangs from the Lower Miocene of Germany:

evolutionary stability of perfect weapons. Naturwissenschaften 93:84–87

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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## **Chapter 6 Characterization of Goldfish Scales by Vibrational Spectroscopic Analyses**

**Masayuki Nara, Yusuke Maruyama, and Atsuhiko Hattori**

**Abstract** Scales of bony fishes are calcified tissues that contain osteoblasts, osteoclasts, and bone matrix, all of which are similar to those found in mammalian bone. The scales are composed of hydroxylapatite (HAP) and extracellular matrix which is mainly type I collagen fibers. We investigated the scales from goldfish by Fourier transform infrared (FTIR) and Raman spectroscopic analyses to characterize the components in the scales. The attenuated total reflection (ATR)-FTIR spectrum obtained from the surface coat of the normal goldfish scale was quite different from that of the backside coat of the same scale. The former showed a strong band at 1013 cm−1 , which was assignable to HAP, and weak bands at about 1643, 1415, and 870 cm−1 , whereas the latter showed a typical protein profile: strong bands at about 1631, 1550, and 1240 cm−1 , which were assignable to amide-I, amide-II, and amide-III of collagen, respectively. We also investigated the local structure of goldfish scales by using micro-Raman spectroscopy. The Raman spectrum of the normal scale showed the amide-I band at about 1640 cm−1 from the collagen and the PO4 3− symmetric stretching band at about 961 cm−1 from the HAP. We discuss the implication of Raman and FTIR profiles for normal and regenerating goldfish scales.

**Keywords** Vibrational spectroscopy · Goldfish scale · Regeneration · Hydroxylapatite · Carbonate apatite

#### **6.1 Introduction**

Goldfish scale is a calcified tissue that contains osteoblasts, osteoclasts, and bone matrix, all of which are similar to those found in mammalian bone (Azuma et al. 2007). The scale is composed of hydroxyapatite (HAP) and extracellular matrix which is mainly type I collagen fibers, forming a highly ordered three-dimensional structure. It has been known that scales regenerate after being removed. Therefore,

College of Liberal Arts and Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan

M. Nara (\*) · Y. Maruyama · A. Hattori

e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
the goldfish scale is interesting from the viewpoint of the model system for the regeneration of mammalian bone.

Vibrational spectroscopy is useful for characterizing developmental changes in bone and other mineralized tissues. Infrared spectroscopy is used for examining the mineral properties and collagen maturity of bones (Boskey and Mendelsohn 2005; Paschalis et al. 2011) as well as protein structures such as collagen and osteocalcin (Jackson et al. 1995; Barth 2007; Mizuguchi et al. 2001). Raman spectroscopy is also available for bones and fish scales to provide information about protein and mineral components (Ikoma et al. 2003a, b).

In the present study, FTIR and Raman spectroscopic analyses were applied to scales from goldfish to characterize the components of the normal and regenerating ones. In particular, we focused on the pocket side of them, since the epidermal side of scales is more complicated because of the contribution of chromatophore.

#### **6.2 Materials and Methods**

We prepared normal scales and regenerating scales after 1 week and 2 weeks, extracted from goldfishes (*Carassius auratus*). As can be seen in Fig. 6.1, the picture of the regenerating scale after 2 weeks was morphologically different from that of the normal scale. There were about ten circular ridges for regenerated scales after 2 weeks, although a lot of circular ridges were observed for normal scales.

Attenuated total reflection (ATR)-Fourier transform infrared (FTIR) measurements were performed at 25 °C on a PerkinElmer Spectrum-One FTIR spectrometer equipped with a universal ATR unit and a liquid nitrogen-cooled MCT detector at resolution 2 cm−1 . A scale sample was placed on a diamond/ZnSe 1-reflection topplate (PerkinElmer). The sampling depth of the ATR method was approximately 1–2 μm over the range of 2000–1000 cm−1 . We obtained the ATR-FTIR spectrum for the spot (about 1.5 mm in diameter) with the accumulations of 200 scans.

**Fig. 6.1** Pictures of a normal scales (left) and a regenerating scale after 2 weeks (right) of goldfish (*Carassius auratus*)

Raman spectra were collected at room temperature (25 °C) with a Raman microscope (Kaiser HoloLab 5000 of Kaiser Optical System Inc.) using 532 nm Nd-YAG laser (2–5 mW at the sample surface), holographic transmission grating, and chargecoupled device (CCD). The spectral resolution in the present system is approximately 4.8 cm−1 . We obtained the Raman spectrum for a spot (about 2 μm in diameter) with two accumulations of 30 s each. The sampling depth of the Raman measurement was about 100 μm. A scale was set on a slide glass covered with aluminum foil, and the pocket sides of the surface were measured by Raman spectroscopy.

#### **6.3 Results and Discussion**

#### *6.3.1 Characterization of a Normal Scale from Goldfish by FTIR Spectra*

Figure 6.2 shows ATR-FTIR spectra of the surface and backside coats of a normal goldfish scale in the pocket side. Figure 6.2a showed a strong band at 1013 cm−1 , which was assignable to HAP, and weak bands at 1647, 1546, 1415, and 871 cm−1 . The bands at 1647 and 1546 cm−1 were probably assignable to proteins. The bands

**Fig. 6.2** ATR-FTIR spectra from (**a**) the surface coat and (**b**) the backside coat of the normal goldfish scale

**Fig. 6.3** ATR-FTIR second-derivative spectra of (**a**–**c**) the surface coat and (**d**–**f**) the back coat in the pocket side of three type of scales from goldfish: (**a**, **d**) a normal scale, (**b**, **e**) a regenerating scale after 1 week, and (**c**, **f**) a regenerating scale after 2 weeks. The second derivatives were multiplied by −1

at 1415 and 871 cm−1 were assigned to carbonate (CO3 2−). Figure 6.2b showed a protein profile of collagen: strong bands at 1631, 1550, and 1240 cm−1 , which are undoubtedly assignable to amide-I, amide-II, and amide-III, respectively.

Figure 6.3a, d shows the ATR-FTIR second-derivative spectra of the corresponding spectra in Fig. 6.2. The second-derivative spectrum of surface coat showed the bands at 1654, 1015, 981, and 781 cm−1 in Fig. 6.3a, whereas that of the backside coat showed three bands at 1696, 1652, and 1625 cm−1 in the region of amide-I, one band at 1550 cm−1 in the region of amide-II, and one band at 1240 cm−1 . Obviously, the spectral profile for the surface coat reflected the HAP as the main component of the calcified layer, while the spectral profile for the back coat reflected the collagen as the main component of the fibrillary layer. The amide-I band observed from the surface coat may be due to proteins other than collagen, since the profile was different from that of collagen. The bands at 1415 and 871 cm−1 are probably originated from CO3 2− (Ikoma et al. 2003b).

#### *6.3.2 Characterization of Regenerating Scales from Goldfish by FTIR Spectra*

Figure 6.3b, e shows the second-derivative spectra of the surface and back coats of the regenerating scale after 1 week in the pocket side, respectively. The band at 1017 cm−1 caused by HAP in Fig. 6.3b was quite sharper than the corresponding band of normal scale in Fig. 6.3a. The spectral profile of the amide-I region was similar to that of the back coat, which suggested that the ATR spectra reflected the fibrillary layer as well as the calcified layer, since the thickness of the calcified one was less than 2 μm. The spectral profile of the back coat of regenerating scales after 1 week (Fig. 6.3e) was almost the same as that of the back coat of a normal scale (Fig. 6.3d).

Figure 6.3c, f shows the second-derivative spectra of the surface and back coats of the regenerating scale after 2 weeks in the pocket side, respectively. The absorbance of amide-I band was weaker than that of Fig. 6.3b, which suggested that the calcified layer became thicker. The spectral profile of the back coat was almost the same as that of normal scale (Fig. 6.3d).

It was difficult to obtain an infrared spectrum of fish scale by using transmission mode, because the thickness of the normal scale is >100 μm and the absorbance of most interesting bands was saturated. ATR measurements have an advantage over transmission measurement, because ATR technique makes it possible to obtain not only the spectral profiles of fish scales but also the information about the surface and backside coats of the scales separately. Therefore, ATR-FTIR spectroscopy is promising for monitoring HAP and collagen components during the regeneration process of goldfish scale.

#### *6.3.3 Raman Spectra from Normal and Regenerating Scales of Goldfish*

Figure 6.4a, b shows Raman spectrum from the area between adjacent circular ridges in a normal scale. The bands at about 1669 cm−1 and 1242 cm−1 were assignable to collagen amide-I and amide-III, respectively, and the intense band at about 961 cm−1 was originated from the PO4 3− symmetric stretching band for HAP (Penel et al. 1998). The intensity at 961 cm−1 was quite weak in the most outer part of the circular ridges, suggesting that HAP layer is not formed or thin on the collagen layers. The band at 1070 cm−1 was observed at the center of the scale, which was caused by type-B carbonate apatite (Awonusi et al. 2007). This interpretation was also confirmed by the FTIR bands at 1415 and 871 cm−1 (Fig. 6.2a). Furthermore, we found that the HAP layer became slightly thicker along the ridge line than the area between adjacent circular ridges (data not shown).

**Fig. 6.4** Raman spectra from (**a**, **b**) the normal goldfish scale and from (**c**–**e**) the regenerating scale after 2 weeks. The spot position for (**a**, **c**) were on the outer part of the scale in the pocket side, and the spot positions for (**b**, **d**, **e**) were on the inner part of the scale in the pocket side. The spot position on (**d**) was in the middle of (**c**, **e**)

Figure 6.4c–e shows the Raman spectra from the area between adjacent circular ridges in regenerating scales after 2 weeks. The spectral profiles of the regenerating scales were similar to those of the normal ones except for the band at 961 cm−1 , which were weaker than that of normal scale (Fig. 6.4a, b). The intensities of the band at 961 cm−1 were the order (c) < (d) < (e). This result demonstrated that the HAP layers were formed from the center part to the outer part during the regeneration process of the scale.

Micro-Raman spectroscopy is a useful method for understanding the local components of goldfish scale because Raman spectroscopic method has higher resolution in the area compared with FTIR spectroscopic method. ATR-FTIR provides information regarding the surface component, because the sampling depth of Raman measurements was 100 μm. Therefore, Raman spectroscopy in combination with ATR-FTIR spectroscopy is powerful for analyzing HAP and carbonate apatite of the regenerating scales.

#### **References**

Barth A (2007) Infrared spectroscopy of proteins. Biochim Biophys Acta 1767:1073–1101

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Chapter 7 Relationship Between Bone Morphology and Bone Quality in Female Femurs: Implication for Additive Risk of Alternative Forced Molting**

**Natsuko Ishikawa, Chihiro Nishii, Koh-en Yamauchi, Hiroyuki Mishima, and Yoshiki Matsumoto**

**Abstract** Calcium (Ca) storage in bone has a relationship with eggshell production. Forced molting by feeding restriction for older hens improves eggshell quality but leads to a decline in the bone quality. Dietary minerals improve the eggshell and bone quality; however, the effect on bone quality post-molting has not been clarified. This study evaluated the effects of dietary Ca and minerals on the eggshell and bone quality during both pre- and post-molting periods. The bone quality was evaluated by measurement of bone density and Fourier transform infrared spectroscopy (FT-IR) analysis. The eggshell quality was evaluated by morphological observation of the mammillary cores by scanning electron microscope and FT-IR analysis. The high Ca concentration feed group showed a low bone density post-molting and a high carbonate/phosphate ratio pre-molting. In high mineral concentration feed group, the eggshell strength, the thickness, and the proportion of large mammillary core areas were significantly higher (*p* < 0.05) than control group post-molting. These results suggest that the eggshell strength increases as the proportion of mammillary core areas increases. Furthermore, the high carbonate/phosphate ratio promoted a decrease in bone density. Therefore, the concentration of dietary mineral is strongly related with the maintenance of eggshells and bone quality post-molting.

 Yamauchi · Y. Matsumoto (\*) Faculty of Agriculture, Kagawa University, Miki, Japan e-mail<EMAIL_ADDRESS>
Department of Dental Engineering, Tsurumi University School of Dental Medicine, Yokohama, Japan

#### **7.1 Introduction**

A high egg laying ratio of laying hens is maintained by the metabolic mechanism of calcium (Ca) which is the main ingredient of eggshell. Approximately 70% of Ca derived from feed is accumulated in the marrow bones, a characteristic feature of birds, and approximately 68% of Ca in the bones is used for eggshell formation (Itoh 1971). Therefore, Ca storage in bones has a direct relationship with eggshell production. Consequently, the decline in Ca metabolism of the aging hens which leads to an increase in the number of broken eggs is a key issue in the poultry industry. In Japan, forced molting by feeding restriction of hens is applied in the latter stages of the laying period to improve the hen-day egg production and the eggshell quality and to extend the laying period (Roland and Bushong 1978). However a decrease in bone quality and bone density is caused by forced molting (Mazzuco and Hester 2005). Therefore, it is necessary to address the decline of bone quality in post-molting. Several studies have reported that dietary minerals and the particle size of Ca can lead to an improvement in the bone and eggshell quality (Calvo et al. 1982; Guinotte et al. 1991; Mekada et al. 1976). However, the effect on bone quality in post-molting has not yet been clarified. This study evaluated the effects of the particle size of Ca and the mineral components contained in feed on the eggshell and bone quality in fasting hens during pre- and post-molting periods.

#### **7.2 Material and Method**

#### *7.2.1 Breeding Test*

Sixteen egg-laying hens (Julia) were equally divided into four feeding groups, a control group (Group C) and three experimental groups (Group D, E, and F): Ca concentration (CaCO3) was 3.8% CaCO3 for Group C and 4.6% CaCO3 for each experimental group by weight. CaCO3 particle size was 40% CaCO3 of 1–1.4 mm diameter for Groups C, D, and F. Group E was fed with powdered CaCO3 less than 0.3 mm diameter. All groups, except Group F, received the same concentration of mineral additives, a mineral premix containing manganese sulfate, iron (III) sulfate, zinc sulfate, copper (II) sulfate, calcium iodate, and cobalt (II) sulfate. Group F received a richer mineral concentration.1 The hens were fed from the age of 160 days old. Feed was given every day and there was no restriction on water intake by the hens. Forced molting by feeding restriction was applied for 8 days from the age of 503 days old to 510 days old. After forced molting, hens were refeed according to their group's respective feeding specification. All experiments were conducted in

1The amount of minerals added to feed is proprietary information and subject to a confidentiality agreement.

accordance with the regulations of the Kagawa University Animal Care and Use Committee.

#### *7.2.2 Sample Collection*

When the sample hens were 458 days old, two hens from each groups (a total of eight hens) were slaughtered, and femur samples were collected. The samples were regarded as pre-molting samples (PRE). When the hens were 512 days old, two hens from each groups (a total of eight hens) were slaughtered after 24 h of refeeding, and their femur samples were collected. These samples were regarded as postmolting samples (POST). The femur heads were fixed in 4% paraformaldehyde phosphate buffer solution (pH 7.4) and demineralized with 0.5 M EDTA (pH 8.0) for 3 weeks. Femur heads were embedded in paraffin and sectioned to obtain 10 μm sections.

#### *7.2.3 Bone Density*

The sections were stained by the Azan-Mallory method. The trabecular and bone marrow areas in femoral cancellous bone were observed at 10× magnification under a fluorescence microscope (Keyence Corp., Japan), and their central parts, 5500 μm away from periosteum, were analyzed using the software WinROOF version 7.4 (Mitani Corp., Japan). The area ratio of the trabecular to total area was determined as bone density.

#### *7.2.4 Fourier Transform Infrared Analysis of the Femoral Cortex*

The qualitative characteristics of the femoral cortex were determined using Fourier transform infrared spectroscopy (FT-IR) (Jasco Corp., Japan) analysis. The femoral cortex was powdered and mixed with potassium bromide (KBr). Spectroscopic analysis was based on two parameters: the calcification and the carbonate/phosphate ratio (C/P ratio) of the samples, both in PRE and POST. The calcification was calculated as the ratio of the area of the phosphate bands (900–1200 cm−1 ) to the area of the amide I bands (1585–1720 cm−1 ). The C/P ratio was calculated as the ratio of the area of the carbonate bands (850–890 cm−1 ) to that of the phosphate bands (900–1200 cm−1 ) (Boskey and Camacho 2007).

#### *7.2.5 Eggshell Quality Test*

The eggshell quality tests were performed twice in both PRE and POST. Eggshell thickness was determined three times on the eggshell equator for eight eggs per group. Eggshell strength was determined by an eggshell strength meter (Fujihira Industry Co., Ltd., Japan). The eggshells of 501 and 680 days old hens were used as morphological samples. The eggshells were cut into about 5 mm squares, and the eggshell membrane was dissolved with a mixture of 6% sodium hypochlorite, 4.12% sodium chloride, and 0.15% sodium hydroxide (Radwan et al. 2010). The mammillary cores were observed at 200× magnification under a scanning electron microscope (SEM) (JCM-6000: JEOL Ltd., Japan). The areas of approximately 100 mammillary cores were analyzed per piece, and 4 pieces per group were examined. Thereafter, the histogram of the areas of the mammillary cores was produced.

In the FT-IR analysis, the eggshells, including their membranes, were powdered and mixed with KBr. Spectroscopic analysis was calculated using the ratio of the peak at 873 cm−1 to the peak at 713 cm−1 , a parameter to evaluate the carbonate purity of the eggshells (Rodriguez-Navarro et al. 2015).

#### *7.2.6 Statistics*

Statistical processing was done using the IBM SPSS version 19 software (IBM Corp., USA). The significant differences among the groups were analyzed by the Tukey's HSD and Games-Howell post hoc tests. The level of significance was set at 5%, and the trend level was set at 10%.

#### **7.3 Results**

#### *7.3.1 Bone Density*

The trabecular areas in POST from all groups were significantly lower than those in PRE (*p* < 0.05). The area ratio of the trabecular to total area in PRE did not differ between any groups. However, the area ratio of the trabecular to total area in POST was significantly lower in Groups D and E than those in Groups C and F (*p* < 0.05) (Fig. 7.1a, b).

7 Relationship Between Bone Morphology and Bone Quality in Female Femurs…

**Fig. 7.1** (**a**) The trabecular and bone marrow areas in PRE and POST (black area, trabecular area; gray area, bone marrow area; white area, background). (**b**) The area ratio of the trabecular to total area in PRE and POST (a, b bars with different superscripts within a group are different at *p* < 0.05, *n* = 4)

#### *7.3.2 Fourier Transform Infrared Analysis of Femoral Cortex*

The calcification of the femoral cortex in Group F significantly decreased in POST (*p* < 0.05), whereas that in the other groups showed a decreasing trend (*p* < 0.1) (Fig. 7.2a). The C/P ratio of Groups D and E were significantly higher than Group F in PRE (*p* < 0.05) (Fig. 7.2b).

#### *7.3.3 Egg Quality Test*

Eggshell strength in Group F was significantly higher than those in Group C in POST (*p* < 0.05) (Fig. 7.3a). Eggshell thickness in Group F was significantly higher than those in Groups C and E in POST (*p* < 0.05) (Fig. 7.3b). The mammillary cores were observed as shown in Fig. 7.4a, and the proportions of the area of the eggshell mammillary cores did not differ in any group in PRE (Fig. 7.4b). There was a significant difference between Group C and Group F in POST with respect to their mammillary core areas larger than 6000 μm2 (*p* < 0.05) (Fig. 7.4b). The ratio of the peak at 873 cm−1 to the peak at 713 cm−1 did not differ in any groups in PRE and POST (Fig. 7.5).

**Fig. 7.2** (**a**) The calcification of the femoral cortex (a, b bars with different superscripts within a group are different at *p* < 0.05, *n* = 6). (**b**) The C/P ratio of the femoral cortex (a, b bars with different superscripts within a group are different at *p* < 0.05, *n* = 6)

**Fig. 7.3** (**a**) Eggshell strength in PRE and POST (a, b bars with different superscripts within a group are different at *p* < 0.05, *n* = 16). (**b**) Eggshell thickness in PRE and POST (a, b bars with different superscripts within a group are different at *p* < 0.05, *n* = 16)

**Fig. 7.4** (**a**) SEM microphotographs of eggshell mammillary core in PRE and POST. (**b**) Histogram of the areas of mammillary cores in PRE and POST (*n* = 4)

#### **7.4 Discussion**

In this study, we focused on the effects of the mineral components and the particle size of CaCO3 contained in feed and we investigated the morphology and quality of bone and eggshell in PRE and POST. The area ratio of the trabecular to total area is an index for evaluating bone density (Hagino, 2005). It was thought that a decrease in bone density of the femoral cancellous bone was due to the influence of forced molting because of the area ratio of the trabecular to total area diminished in all groups in POST (Fig. 7.1b). Thus, three experimental groups' feeds did not suppress the decline of the bone density.

Calcification is an index for evaluating bone strength, and high calcification indicates that the storage function of mineral in bone is high (Boskey and Camacho 2007). The decline of bone strength of the femoral cortex was also caused by the impact of forced molting because the calcification decreased in all groups in POST

(Fig. 7.2a). Furthermore, when the hardness of the part of the cortex of the femoral diaphysis was determined by Vickers hardness tester, the result of bone hardness and bone calcification were linked (data not shown), and further analysis is necessary about bone strength.

The C/P ratio is used as an index of the purity of bone minerals, and a low C/P ratio indicates a high degree of crystal purity (Malluche et al. 2012). The finding of this study suggested that crystal purity was low in PRE in Groups D and E (Fig. 7.2b). The main constituent of bone minerals are present in the bone as calcium phosphate and undergo a phase change to convert to stable apatite due to maturation. Apatite in bones replaces ions, such as carbonate ions(CO3 2−) and phosphate ions, in the crystal lattice. Apatite containing a lot of CO3 2− is reported to have high solubility and increase bone dissolution (Suetsugu 1996; Gourion-Arsiquaud et al. 2012). This suggested that the solubility of apatite was high in Groups D and E in PRE because a ratio of carbonate in Groups D and E were higher than Group F. Therefore, we think that the reduction of bone density was promoted further in Groups D and E in POST. These differences were thought to be due to the difference in the ratio of Ca and other mineral concentration in feed.

Eggshell strength and thickness in Group F were significantly higher than those in Group C in POST (Fig. 7.3a, b). Since zinc and manganese contained in mineral additives in feed have a relationship in eggshell formation, it was conclusive that the high mineral concentration feed improves eggshell quality and supports the previous research which mineral addition in feed improve eggshell quality (Mekada et al. 1976). Eggshell is formed by mammillary core formation on the eggshell membrane and deposition of Ca as a starting point from the mammillary cores. It was reported that as the number of large mammillary cores where several mammillary cores fused increases, the eggshell strength increases (Solomon 2010; Stefanello et al. 2014). As a result of the large number of mammillary core areas larger than 6000 μm2 in POST, it was thought that the eggshell strength was linked the size of mammillary cores (Fig. 7.4b). Therefore, in addition to evaluating conventional eggshell strength, the morphological observation of the mammillary cores which is the microstructure of eggshell suggested that it is a new means to evaluate eggshell quality. The mechanism of action which mineral components in feed change the form of eggshell structure requires further investigation.

In the FT-IR analysis of eggshells, it was reported that the purity of carbonate changes during eggshell formation (Rodriguez-Navarro et al. 2015). We examined if the eggshell crystal quality could be evaluated after laying, however, the influence of feed on eggshell crystal quality was not detected.

The present result suggested that high mineral concentration feed improved eggshell quality, and high Ca concentration feed promoted the reduction of bone density in POST. In conclusion, the present study has demonstrated that the ratio of the minerals except Ca and the Ca concentration in the feed is important for maintenance of eggshell quality and bone quality in pre- and post-molting. It is necessary to analyze the bone metabolism in order to investigate the effect of feed in the future.

**Acknowledgments** This study was supported by Ise Foods, Inc., Japan, and Nichiwa Sangyo Co., Ltd., Japan, by providing hens and feeds.

#### **References**

Suetsugu Y (1996) Carbonate ions in apatite structure. Inorg Mater 3:48–54

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Chapter 8 Spectroscopic Investigation of Shell Pigments from the Family Neritidae (Mollusca: Gastropoda)**

**Toshiyuki Komura, Hiroyuki Kagi, Makiko Ishikawa, Mana Yasui, and Takenori Sasaki**

**Abstract** Molluscan shells display a wide variety of pigmentation patterns. The diversity in molluscan shell color reflects the variety of different chemical species in the shell surface. Chemical characteristics of molluscan shell pigments have been extensively investigated, and compounds including porphyrins, polyenes, and melanins were identified as shell pigments. Here, we investigated shell pigments in 24 species in the family Neritidae using Raman spectroscopy. An excitation wavelength of 514.5 nm revealed two types of Raman spectra. One was characterized by two peaks ranging in wavenumber from 1100–1200 to 1500–1600 cm−1 , which indicate the presence of polyenes. Another type remained unassigned, implying the presence of other pigments such as porphyrins or melanins. The Raman spectra indicated a different distribution of the two types of pigments in shells. The patterns of the Raman spectra had no obvious relationship with taxonomical classification lower than the genus level and typical habitats. Measurement of the Raman spectrum at an excitation wavelength of 442 nm suggested that the wavelength can distinguish polyenes from other types of pigments.

M. Yasui

#### T. Sasaki

T. Komura (\*) · H. Kagi

Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
M. Ishikawa

Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Faculty of Animal Health Technology, Yamazaki Gakuen University, Hachioji, Tokyo, Japan e-mail<EMAIL_ADDRESS>
Department of Resources and Environmental Engineering, School of Creative Science and Engineering, Waseda University, Shinjuku-ku, Tokyo, Japan e-mail<EMAIL_ADDRESS>
The University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, Japan e-mail<EMAIL_ADDRESS>
**Keywords** Mollusc · Neritidae · Shell pigment · Polyene · Porphyrin · Raman spectroscopy

#### **8.1 Introduction**

The phylum Mollusca is the second largest taxon in the animal kingdom. It is very diverse in size, shape, color, habitats, and other traits. The diverse pigmentation patterns on shells are a key trait. Diversity in molluscan shell color reflects the wide variety of chemical species included in the shell. Investigating the origin of shell pigmentation is very important to understand the evolutionary history of Mollusca (Williams 2017). Furthermore, chemical speciation of shell pigment molecules could provide insight into the interaction of pigment molecules with other shell components (Hedegaard et al. 2006), which is important for the better understanding of biomineralization during shell formation.

Shell pigments have been extensively investigated (Williams 2017; Ishikawa et al. 2013 and references therein). Comfort (1949a, b) reported the presence of porphyrin compounds in several groups of gastropods including the family Neritidae (described later) using ultraviolet fluorescence and absorption chromatography. Subsequently, the presence of polyene compounds was reported using Raman spectroscopy. For example, Hedegaard et al. (2006) estimated the chain length of conjugated polyenes and discussed the chemical modification of polyenes contained in the shell of the gastropod *Cypraea moneta* (Cypraeidae). Polyene pigments were observed in various taxa. However, the target samples used in previous studies were limited to common shells with strong colors. Here, we comprehensively studied the family Neritidae and conducted a preliminary study to detect porphyrin pigments using Raman spectroscopy. The family Neritidae comprises small- or medium-sized snails that inhabit a wide variety of shallow-water environments such as brackish areas and intertidal rocky zones. The family is characterized by clear and vivid color bands (Fig. 8.1), which make spectroscopic analysis easy to perform. Their various habitats are expected to have an effect on pigmentation because shell colors are affected by environmental factors (e.g., Sokolova and Berger 2000).

#### **8.2 Materials and Methods**

We investigated 24 species from 5 genera of the family Neritidae by using Raman spectroscopy (Table 8.1). Raman spectra were obtained from each differently colored area using an exposure time of 10 s for each measurement. Ten spectra were obtained for each area. Estimated energy resolution ranged from 1 to 2 cm−1 . Two Raman spectrometers with different excitation wavelengths (514.5 and 442 nm) were used (Table 8.2). The excitation wavelength of 514.5 nm has been previously demonstrated to detect polyenes. The excitation wavelength of 442 nm is near the Soret maxima of porphyrins and thus is expected to excite porphyrins that are

**Fig. 8.1** *Neritina waigiensis* in the family Neritidae. A section of the shell denoted by the white square was used for Raman analysis (see Fig. 8.3)

**Table 8.1** Collection numbers, species, locality, and typical habitats of the investigated samples

(continued)

**Table 8.1** (continued)

Scientific names and typical habitats follow the designation schemes of Tsuchiya and Kano (2017) and Eichhorst (2016)

**Table 8.2** Excitation wavelength, laser source, laser power, calibration standard, and objective lens magnification data of the two Raman spectrometers

Laser power could be attenuated through the optical paths for both instruments

present in the mollusc shell (Comfort 1949a). We performed intact shell surface analysis without chemical treatments. The samples examined were registered in The University Museum, The University of Tokyo.

#### **8.3 Results and Discussion**

#### *8.3.1 Raman Spectra at 514.5 nm*

Figure 8.2 shows representative Raman spectra obtained from yellow, red, and black color bands of *N. waigiensis*. The Raman spectra of the black and red parts were similar, while the spectrum of the yellow part was markedly different from both. The spectrum obtained from the yellow part was characterized by two peaks with wavenumbers ranging from 1100–1200 cm−1 (ν5) to 1500–1600 cm−1 (ν1). These two peaks are assigned to polyene pigments contained in the shell (e.g., Bergamonti et al. 2013). These polyene-type spectra were obtained from the species listed in Table 8.3. While Raman shifts of ν5 and ν1 varied in all species, the observed values were within a certain range.

The spectra obtained from red and black parts of *N. waigiensis* had no obvious peaks that could be assigned to polyenes. This type of spectrum was also obtained from the species listed in Table 8.4. Similar spectra from the gastropod *Clunculus pharaonius* (Trochidae) were reported by Merlin and Delé-Dubois (1986) and Williams et al. (2016). The red and black pigments of *C. pharaonius* were identified as uroporphyrin and eumelanin, respectively, using high-performance liquid chromatography (Williams et al. 2016). However, the authors argued that the Raman spectra did not convey any definitive information concerning shell pigments, and it

**Fig. 8.2** Raman spectra obtained from each color band of *N. waigiensis* using 514.5 nm excitation. White circles in the figure indicate measurement points for Raman analysis

**Table 8.3** Colors and wavenumbers of the ν5 and ν1 peaks using the 514.5 nm excitation from species containing polyenes

was quite unclear whether the obtained spectra were assignable to uroporphyrin or eumelanin included in the shell. Presently, we also could not conclusively identify shell pigment from the Raman spectra. However, the difference in chemical origins of the black and red pigmentations of *N. waigiensis* from that of yellow pigmentation is a novel finding. It is worth noting that the Raman peaks obtained from red and black regions of *N. waigiensis* appeared at a similar frequency to those in the spectrum of eumelanin reported by Mbonyiryivuze et al. (2015). It is conceivable that the peaks we observed were due to background fluorescence.

Shell pigmentation may be relevant to taxonomical classification (Comfort 1949a, Williams 2017). However, the patterns and peak wavenumber of the Raman spectra (Tables 8.1, 8.2, and 8.3) had no obvious relationship with the taxonomical classification at the genus or lower level or with typical habitats. Different types of spectra could be observed even in the same individuals.

We fabricated the shell section perpendicular to the apertural margin of *N. waigiensis* (Fig. 8.3). The thickness of red- and black-colored layers was approximately 30 μm. The Raman spectra obtained from five points are shown in Fig. 8.4. No obvious peaks assignable to polyenes were observed from points 1 to 2. Two weak peaks assignable to polyenes were detected at point 3. The obtained spectra

**Table 8.4** Colors and Raman shifts of representative peaks obtained at 514.5 nm excitation wavelength from species displaying other spectra

**Fig. 8.3** (**a**) A section perpendicular to the apertural margin of *N. waigiensis*. The black and yellow patterns on the shell exterior are visible on the upper side of the figure. The numbers 1–5 indicate measured points. (**b**) Scanning electron microscopy image of the section of *N. waigiensis*. The multilayered structure of the shell is evident. The outermost region is a prismatic layer (P) that corresponds to the colored layer in (**a**). The inner layer has a crossed-lamellar structure (CL). These two figures were obtained from different individuals of *N. waigiensis*

raise the possibility that the concentration of polyenes increases continuously from the interior of the shell to the exterior surface. In contrast, black and red pigmentation appeared only in the narrow range on the surface, indicating that their distributions are quite different from the distribution of polyenes.

**Fig. 8.4** Raman spectrum obtained from each measured point in Fig. 8.3

**Fig. 8.5** Raman spectrum obtained from each color band of *N. waigiensis* using the 442 nm excitation wavelength. The white circles in the figure indicate the measurement points for Raman analysis

#### *8.3.2 Raman Spectra at 442 nm Excitation*

Figure 8.5 shows Raman spectra obtained from *N. waigiensis* using the 442 nm excitation wavelength. Clear peaks assignable to polyenes were observed from the yellow and red parts of the shell surface, but not from the black part. Polyene-type

spectrum from the red part may be attributed to the polyenes of the yellow part existing just under the red color band on the surface. Williams et al. (2016) argued that it is difficult to distinguish polyenes from porphyrins only by the Raman spectra. However, our results suggest that polyenes can be distinguished from other pigments because the background fluorescence was relatively low compared to that for the peak heights at ν5 and ν1.

#### **8.4 Conclusions**

Analysis using the 514.5 nm excitation wavelength demonstrated that the pigmentation in the family Neritidae has at least two types of origins: polyenes and other unknown pigments such as porphyrins or melanins. The data from a shell section of *N. waigiensis* suggest that these two types of pigments are distributed in a different manner in the section. The patterns of the Raman spectra did not display obvious relationships with taxonomical classification and habitats. The Raman spectra obtained using the 442 nm excitation wavelength suggest that polyenes can be distinguished from other pigments by the presence of strong peaks of ν5 and ν1. However, definitive information on shell pigments was difficult using only Raman spectroscopy. Other analytical techniques such as high-performance liquid chromatography could be needed and will be studied.

**Acknowledgment** We thank Dr. Natsuhiko Sugimura from the Research Support Center at Waseda University for providing support for the measurement of the Raman spectra.

#### **References**

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

## **Chapter 9 3D Visualization of Calcified and Non-calcified Molluscan Tissues Using Computed Tomography**

**Takenori Sasaki, Yu Maekawa, Yusuke Takeda, Maki Atsushiba, Chong Chen, Koji Noshita, Kentaro Uesugi, and Masato Hoshino**

**Abstract** Three-dimensional (3D) reconstruction is an essential approach in morphological studies in biology and paleontology. Seeking an optimized protocol for nondestructive observations, we attempted 3D visualization of various molluscan shells and animals with X-ray micro-computed tomography (micro-CT). Calcified parts of molluscs were easily visualized except for cases with marked differences in thickness heterogeneity. 3D imaging of shell microstructure was difficult. Visualization of soft tissue requires staining to enhance the image contrast. Especially for soft tissues, synchrotron X-ray microtomography is the most advanced method to generate clear 3D images. 3D data facilitates morphological quantification, enabling calculations of length and volume even for very complex forms. X-ray micro-CT is extremely useful in the morphologic examination of

Y. Takeda The University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

Department of Earth and Planetary Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido, Japan e-mail<EMAIL_ADDRESS>
C. Chen

Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa, Japan e-mail<EMAIL_ADDRESS>
K. Noshita

K. Uesugi · M. Hoshino

T. Sasaki (\*) · Y. Maekawa · M. Atsushiba

The University Museum, The University of Tokyo, Bunkyo-ku, Tokyo, Japan e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS><EMAIL_ADDRESS>
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan e-mail<EMAIL_ADDRESS>
Japan Synchrotron Radiation Research Institute, Sayo-gun, Hyogo, Japan e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
mineralized and soft tissues, although microstructural and histological details should be supplemented by other microscopic techniques.

**Keywords** Micro-CT · Synchrotron CT · Reconstruction · Anatomy

#### **9.1 Introduction**

The importance of computed tomography (CT) has been established in biology and paleontology. CT has also been used in examinations of vertebrate morphology (Gignac et al. 2016; du Plessis et al. 2017), and micro-CT has been used in invertebrate biology, mainly in entomology studies (Friedrich et al. 2014; Wipfler et al. 2016). The approach has been infrequently used for other animal groups. In malacology, the use of micro-CT is restricted to a small number of case studies involving anatomy (Golding and Jones 2007; Golding et al. 2009), ontogeny (Kerbl et al. 2013), paleontology (Takeda et al. 2016), and shell morphology (Monnet et al. 2009; Liew and Schilthuizen 2016; Noshita et al. 2016).

The application of CT is still in the testing and development stage. Having an optimized protocol for the nondestructive micro-CT analysis of various morphological characteristics would help expand the scope of CT applications and was the focus of the present study.

#### **9.2 Material and Methods**

The molluscan specimens used in this study are all registered and deposited in The University Museum, The University of Tokyo (UMUT). The abbreviations RM and CM in the registration numbers indicate recent and Cenozoic molluscs, respectively. Animals were fixed in 5% formaldehyde solution for several days, washed in tap water, and then preserved in 70% ethanol. Intact animals were extracted from shells by soaking the specimens in approximately 10% HCl to completely dissolve the shells. Most specimens were first digitally imaged (Fig. 9.1a–e) using a ScanXmate B100TSS110 industrial micro-CT device (Comscantecno Co., Ltd.) at UMUT. Scan parameters were adjusted according to samples and included source voltage (70– 100 kV), source current (approximately 40–150 μA), and exposure time per frame (0.4–1.0 s). The total number of frames was 1200, the number of pixels of the detector was 1024 × 1012, and the highest resolution was approximately 2 μm. Samples larger than 3 cm were scanned in the laboratory of Comscantecno Co. Ltd. For scanning, embedding the sample in a heat-generated hole in Styrofoam on a rotating stage was the most effective method of sample fixation. For soft portions of samples, high-resolution X-ray CT was also performed (Fig. 9.1f–h) at the hutch 3 of the beamline BL20B2 in SPring-8 (Hyogo, Japan) with a resolution of 13.16 μm per pixel and an X-ray energy of 25 keV. Three-dimensional (3D) visualization was conducted using Molcer Plus (White Rabbit Corporation), OsiriX (OsiriX Foundation), and Amira 3.5.5 (Visage Imaging, Inc.) software.

**Fig. 9.1** CT facilities used in this study. (**a–e**) Industrial CT (UMUT). (**b**) X-ray tube. (**c**) Sample stage. (**d**) X-ray detector. (**e**) Sample holder. (**f–h**) Synchrotron CT (SPring-8 BL20B2). (**g**) Magnified view of sample stage. Arrowhead indicates sample. (**h**) X-ray detector

#### **9.3 Results**

#### *9.3.1 Shells*

Most samples of calcified shells were scanned without difficulty using conventional industrial micro-CT (Fig. 9.2). The internal structure of shells was also perfectly reconstructed from scan data. Exceptions included shells of specific groups of gastropods, with shells from different regions having extremely disparate thicknesses. For example, members of the families Ellobiidae (Figs. 9.2c, d), Olividae, and Conidae have shells in which the internal whorls are much thinner than the outer wall owing to the secondary resorption that occurs during growth. In such cases, the thinner inner whorls disappear or became patchy in distribution when the settings were optimized for the outer shell surface. Conversely, if contrast were enhanced to reveal the inner structures, outer surfaces were rendered unusable by the increased noise levels (the overexposure effect). So far, no viable solution to this problem has been identified in our current system.

Digitization using micro-CT is an excellent approach to display the overall 3D morphology and the internal structure of molluscan shells. Examples of specimens of small Cenozoic fossil gastropods are presented in Fig. 9.2e–j. Virtual slicing of type specimens along an arbitrary plane is possible only using micro-CT. Prominent columellar folds (Fig. 9.2f, h) or denticles in the outer lip (Fig. 9.2h) were important features to morphologically diagnose species. Construction of images from apical (Fig. 9.2i) and basal (Fig. 9.2j) views for fragile and minute specimens is easily acquired. However, imaging of the same specimens by photography with a digital camera and binocular microscope carries the risk of specimen loss or damage. Micro-CT allows the detailed nondestructive observation of the delicate structures of shells.

**Fig. 9.2** Example of 3D reconstruction of calcified shells from industrial micro-CT data. (**a**, **b**) *Volutoconus bednalli* (Brazier, 1878) (Volutidae). UMUT RM32762. (**c**, **d**) *Ellobium aurismidae* (Linnaeus, 1758) (Ellobiidae). UMUT RM32763. Arrowhead indicates missing inner wall as artifact. (**e**, **f**) *Cerithiella trisulcata* (Yokoyama, 1922) (Newtoniellidae). UMUT CM20781. Holotype. (**g**–**i**) *Tiberia pseudopulchella* (Yokoyama, 1920) (Pyramidellidae). UMUT CM20242. Holotype. (**i**) Apical view. (**j**) Adapical view. Shell height: **a**, **b** = 90.6 mm; **c**, **d** 88.8 mm; **e**, **f** = 5.2 mm; **g**, **h** = 7.0 mm. Software: Molcer Plus

However, the use of micro-CT for shells is not entirely versatile. Most importantly, growth lines, shell layers, and shell microstructure cannot be observed. We were unable to detect any structure on sliced shell planes in the CT-derived 3D data. The observation of these fine structures still necessitates mechanical destruction or cutting of the actual shell samples.

Another issue is that high-resolution scanning of a specific part of a large shell is not possible. To achieve high-resolution scan data, it is necessary to place a sample

as close to the X-ray source as possible (for any given detector image size, the realized resolution depends on the field of view and, thus, the size of the specimen). In conventional micro-CT, it is necessary to rotate the specimen. Thus, if the sample is large, it must be placed further away from the X-ray source, which will decrease the resolution. This dilemma cannot be overcome at the present time.

#### *9.3.2 Soft Tissues*

Simultaneous visualization of both the shell and the soft regions from a single specimen could not be achieved with satisfactory results. If various parameters are optimized for shells, the settings are far from optimal for the soft regions, and vice versa. The only way to achieve high-quality scans for both features from a single individual is to obtain the shell scan first and to decalcify it to scan the soft regions. This destroys the shell. Therefore, development of a new algorithm or methodology is desired to enable the nondestructive scans of precious specimens (such as intact types) to reveal the anatomy of soft portions.

Applying CT to soft parts is more challenging than applying it to calcified parts. Animal tissues usually do not display any contrast in CT images without the aid of contrast-enhancing substances. Figure 9.3 shows an example of Patellogastropoda. In this organism, only iron-mineralized radular teeth are visible if the specimen is not treated with a contrast agent (e.g., iodine).

Installation of samples on the rotating stage is also an extremely sensitive part of the scanning process. If a wet sample is scanned while it is exposed to air, it quickly becomes dehydrated and deformed, which will result in a very blurred image that is

**Fig. 9.4** Soft part of *Conus ebraeus* Linnaeus, 1758 (Conidae). UMUT RM32765. (**a**) Formalinfixed animal with shell decalcified. (**b**–**d**) 3D reconstruction with software Molcer Plus from scan data of industrial CT. Body length = 19 mm

**Fig. 9.5** Soft part of *Lottia dorsuosa* (Gould, 1859) (Lottiidae). UMUT RM32764. Comparison of scanning results between conventional industrial micro-CT (**a**) and synchrotron CT (**b** SPring-8 BL20B2). *dg* digestive gland, *es* esophagus, *f* foot, *i* intestine, *mm* mantle margin, *pb* pedal blood sinus, *pc* pallial cavity, *rds* radular sac, *st* stomach. Body length = 22 mm

unusable. Therefore, samples should be contained in a liquid medium such as ethanol, water, or physiological saline water in a (relatively) X-ray transparent container. Wrapping a sample with X-ray transparent film in a Styrofoam container can be an effective solution. Expelling air bubbles from the medium is essential because the bubbles can cause sample movement when they expand as they are subjected to heat generated during prolonged scanning.