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Elaborate Architecture of the Hierarchical Hen’s Eggshell

Jie Zhou1,2, Shutao Wang1, Fuqiang Nie1, Lin Feng3 ( ), Guangshan Zhu4, and Lei Jiang1 ( ) 1 Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Organic Solids, Institute of Chemistry, Chinese

Academy of Sciences, Beijing 100190, China 2 Graduate School of the Chinese Academy of Sciences, Beijing 100049, China 3 Department of Chemistry, Tsinghua University, Beijing 100084, China 4 State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Changchun 130023, China Received: 15 September 2010 / Revised: 23 October 2010 / Accepted: 27 October 2010 © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2010

ABSTRACT Eggshells are one of the most common and well-studied biomaterials in nature and exhibit unique properties of gas conduction. However, the morphologies of eggshells at the submicro-/nano-scale and their impact on eggshell functions remain unclear. In this work, the architecture of hen’s eggshell at different length scales has been systematically investigated by scanning electron microscopy (SEM) and environmental SEM (ESEM). It is found that the skeleton of calcium carbonate (CaCO3) has hierarchical structures at nano- to micro-scales: primary nano-particles of ~10 nm loosely congregate giving a porous and rough texture, and compose the upper-level morphologies including submicro spheres, nano-rods, rhombohedral-cleavage pattern and slices, which are elaborately arranged in a surface layer, palisade layer and mammillary layer along the radial direction. Accordingly, the pore system exhibits a three-level hierarchy, namely nano-scale pores (between nano-rods and primary nano-particles), submicro-scale pores (“bubble pores”) and micro-scale pores (opening of “gas pores”). Further investigation shows that hen’s eggshell regulates gas conduction through adjusting the sizes and numbers of submicro-scale “bubble pores”. Based on our observations, a new description of hen’s eggshell is presented, which amends the conventional view of micro-scale, straight and permeating “gas pores”, and reveals the role of hierarchical pores in gas conduction and contamination resistance. KEYWORDS Eggshell, hierarchy, morphology, porous calcium carbonate, gas conduction

1. Introduction

Eggshell has long attracted the attention of resear- chers because of its unique properties of gas conduction. Eggshell is one the most common and well-studied biomaterials in nature, and its ultrastructure and porosity have been carefully investigated. Tyler and co-workers reported a pioneering series of work in

the 1950s. They developed some basic experiment methods [1, 2] and analyzed the resulting data to determine the pore distribution [3], and revealed some important structural features of eggshell by plastic models, including the honeycomb structure of the inner surface and the presence of three layers along the radial direction [4]. In addition, eggshell membranes have been investigated by electron microscopy using

Nano Res. 2011, 4(2): 171–179 ISSN 1998-0124DOI 10.1007/s12274-010-0067-8 CN 11-5974/O4Research Article

Address correspondence to Lei Jiang, jianglei@iccas.ac.cn; Lin Feng, fl@mail.tsinghua.edu.cn

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stained ultrathin slices, and membrane thickness, fiber density and diameter were measured [5, 6]. Although early researchers tried hard to obtain a clear picture of the eggshell structure, they probably obtained some inaccurate information by preparing specimens via violent treatment, as well as the limitations of microscopes available in those days. From the 1970s, with the aid of modern scanning electron microscopes (SEM), relatively accurate observations of eggshells were gradually obtained and SEM images at the micro-scale have been utilized [7–12]. However, the morphologies of hen’s eggshell at the submicro-/nano-scale remains unclear, and the structural hierarchy of hen’s eggshell and its impact on eggshell function have not yet been reported.

On the other hand, gas exchange across the eggshell and shell-membrane has also aroused considerable interest. Permeability to O2, CO2 and water vapor have been directly measured or calculated [13–22], and the effects of many internal and environmental factors have been considered, including the incubation time [13, 16], metabolic rate [17, 18], colloid osmotic pressure of egg white [13, 16], egg species [18], humidity [16], anisotropic gas conductance [19] and regional gas tension [20–22]. Although so many factors have been investigated, the structural features of the eggshell itself, especially the morphology at the submicro-/nano-scale, have not yet been described.

Here, we present a full-scale observation of hen’s eggshell from the micro-scale to submicro-/nano-scale which revealed the complex hierarchical structure of skeletal calcite and the pore system, and the ultrastructure of the shell-membrane. Further inves- tigation indicated that hen’s eggshell regulates gas conduction through adjusting the sizes and numbers of submicro-scale “bubble pores” and implies the importance of cooperation of hierarchical pores in gas conduction and contamination resistance.

2. Experimental

All the hen’s eggs were picked from a local chicken farm in Beijing, China. After the yolk and egg white were removed, the eggshell with its shell membrane was split into pieces of ~1 cm2 area by a fine file, and divided into four groups. The first group was kept raw

in order to observe the cuticular colloid membrane. The second group was washed with deionized water and dried by nitrogen flow. The third group was immersed in sodium hypochlorite for 2 min to remove organic components in the shell, and then treated with a graded series of ethanol for dehydration. The keratin-fiber membrane was also removed from eggshell by peeling and burnishing. In order to observe the shell-membrane, the fourth fraction was immersed in dilute acetic acid (5%, v/v) for 15 h to dissolve the calcium carbonate. Eventually, the samples from each group were cut into smaller pieces, and then observed by SEM (JEOL JSM-6700F, Japan) at 3.0 kV, or ESEM (FEI Quanta 200 FEG, Holland). For gas conduction experiments, samples from the blunt pole or the middle region of the eggshell were carefully washed with deionized water and dried by nitrogen flow, and then half were immersed in sodium hypochlorite for 2 min to remove the keratin-fiber membrane. Then all the samples were glued to the penetration cell by sealant, and used as the blocking membrane. Gas flux of O2 or CO2 reaching the membrane was adjusted by a controlled mass flow meter and kept constant; gas pressure in front of the membrane was adjusted by a leak valve and also kept constant. Gas flux after the membrane was measured by a soap-film flow meter.

3. Results and discussion

3.1 Outer face of hen’s eggshell

The outer face of hen’s eggshell was observed by SEM and ESEM (Figs. 1 and 2). In the raw samples, there is a cuticular colloid layer over the shell, but it is difficult to observe under SEM (Fig. 1(a)) since the layer tends to be destroyed by electron beam. In the ESEM images, an amorphous colloid layer with a little roughness of beads and pits can be clearly seen, however (Figs. 1(b) and 1(c)). In the washed samples, there are some gaps and pores existing in certain areas and connecting with each other; most gaps have irregular widths of up to several micrometers, while most pores have diameters of 10–20 μm (Fig. 2(a)). From the enlarged image of a gap, it is clear that there are many submicro spheres at gap edges, and that the gap is actually a row of missing spheres (Fig. 2(b)). Similarly,

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Figure 1 SEM and ESEM images of the cuticular colloid layer in raw eggshells. (a) SEM image: the cuticular colloid layer is destroyed. (b) and (c) ESEM images of the intact cuticular colloid layer show a little roughness of beads and pits

Figure 2 SEM images of the outer face of the shell. (a) Gaps and pores are clear in the low-magnification SEM image. Enlarged images of gaps and pores show that a gap is actually a row of missing submicro spheres (b), while a pore is a bucket-shaped pit where submicro spheres are absent (c). (d) Image of a randomly picked “gas pore” focused on its inside reveals it is blocked beneath a thin surface layer. After the organic components are removed, spheres of submicro-scale are very clear (e), and ordered nano-rods emerge where the spheres have fallen off (f)

a pore is a bucket-shaped pit where submicro spheres are absent (Fig. 2(c)). Those pores are likely to be described as entrances to the so-called “gas pore” in the conventional model, whereas our further observations revealed that most of them are only shallow openings through the surface layer and are blocked beneath this thin layer (Fig. 2(d)). This is inconsistent with the conventional picture, where pores are said to permeate through the whole eggshell and provide a channel connecting the exterior with the interior of the eggshell. Figure S-1 in the Electronic Supplementary Material (ESM) provides more detailed SEM images of a series of “gas pores” over a large area surface. When the fragments of eggshell were treated to completely remove the cuticular colloid, numerous spheres of diameter 100–300 nm were clearly seen: these are assemblies of nano-particles of diameter ~10 nm (Fig. 2(e)). Such spheres can also be clearly seen at the brinks of pores and gaps. Beneath the cuticular spheres, a highly oriented nano-rod array (width: ~10 nm, length: ~1 μm)—which is also an assembly of ~10 nm nano-particles—is exposed where the spheres have fallen off (Fig. 2(f)).

3.2 Radial section and inner face of hen’s eggshell

SEM images of the radial section of hen’s eggshell are shown in Fig. 3. From the exterior to the egg white, the eggshell has anisotropic structures in the radial direction and can be mainly subdivided into the surface layer, palisade layer and mammillary layer (Fig. 3(a)). The surface layer (~10 μm) accounts for a small pro- portion of the thickness of the whole shell (300–350 μm), and is made up of numerous oriented nano-rods and submicro spheres, composed of nano-particles, just like the outer face (Figs. 3(b) and 3(c)). The palisade and mammillary layers make up the majority of the shell. In the palisade layer, CaCO3 crystals having a rhombohedral-cleavage pattern with straight edges predominate and some “bubble pores” are distributed throughout (Fig. 3(d)). When the edges and pores were probed at the nano-scale, a very porous and loose texture was observed (Fig. 3(e)). The main morphology of the mammillary layer involves slices of CaCO3 crystals with distributed “bubble pores” (Fig. 3(f)), and each slice has obvious nano-scale roughness (Fig. 3(g)).

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Figure 3 SEM images of side-view of the shell. (a) A low magnification side-view of the eggshell where the membrane has been removed. The surface layer is composed of an oriented layer of nano-rods covered with a layer of submicro spheres on top (b), and these submicro spheres consist of nano-particles assembled together and adhering to the lower layer through organic com- ponents (c). In the palisade layer, CaCO3 crystals with rhombohedral- cleavage pattern and straight edges are dominant, and pores of ∼250 nm are distributed (d). A zoomed image (e) shows a section of the edge and a pore in detail. Slices of CaCO3 crystals with pores of ∼250 nm are the main morphology in the mammillary layer (f), and exhibit a porous and rough texture (g)

These “bubble pores” should be an important structural element of the shell for gas exchange, and so a further analysis of pore diameter distribution in the palisade and mammillary layers was carried out.

Figure 4 shows images of the inner face of the eggshell. When the shell-membrane was peeled by hand, numerous mammillaries could be faintly seen through a layer of residual membrane fibers (Fig. 4(a)). When the inner face was burnished using a piece of

sandpaper, the texture of interlaced inorganic CaCO3 crystals and organic keratin fibers was obvious (Fig. 4(b)), and the loose structure of calcite particles could be seen at higher magnification (Fig. 4(c)). After the keratin fibers were completely removed by oxidation and dehydration, thousands of mammillaries emerged (Fig. 4(d)). From the enlarged view of one mammillary, places where keratin fibers were removed can be easily recognized (Fig. 4(e)), and the loose and rough texture of crystals is exposed in a further enlarged image (Fig. 4(f)).

3.3 The fiber-membrane of hen’s eggshell

The fiber-membrane beneath the shell was observed by SEM as shown in Fig. 5. In order to clearly observe the fiber-membrane, the shell made of CaCO3 was removed by being immersed in dilute acetic acid for 15 h. On the outer face (the face adhering to the shell) of the fiber-membrane, many knobs formed by assembly of fibers are distributed (Figs. 5(a) and 5(b)) and may act as anchors to increase the strength of linking between the membrane and mammillaries on the inner face of the shell. A comparatively smooth layer of amorphous organic colloid instead of fibers can be seen on the inner face (contacting the egg white) of the fiber-membrane (Fig. 5(c)), and this so-called “limiting film” is believed to have the function of preventing egg white from leaking. However, this “limiting film” is not perfectly seamless, and breaches of different size are distributed throughout. From a side view (Fig. 5(d)), the fiber-membrane can be divided into two parts: an outer part (Fig. 5(e)) and an inner part (Fig. 5(f)). The outer membrane, composed of thicker fibers, is clearly looser than the inner membrane with its thinner fibers. Some of thick fibers in the outer mem- brane have a core–shell structure and internal holes (as shown in the insert in Fig. 5(d)). Figure 6 shows face view images of the fiber-membrane from the inside to the outside and corresponding statistical analyses of the fiber diameter, which indicate that the fiber diameters gradually change from 1.5–2.5 μm at the innermost surface to 2.5–5.0 μm at the outermost surface.

3.4 Distribution of “bubble pores” and gas conduction

For analysis of the distribution of “bubble pores”,

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samples were selected from either the middle region or the blunt pole of the eggshell, where the air cell usually exists. Ten samples were selected for each position. For each sample, ten SEM images of the palisade layer and mammillary layer at 10 000 times magnification were taken. Then the diameters of all the “bubble pores” in these images were measured

and recorded, and the numbers of “bubble pores” with diameter within a selected range were counted. In both regions of the eggshell, pores with diameter of ~250 nm are in the majority. The middle region contains a considerable number of pores with diameter less than 250 nm (Fig. 7(a)), whereas the blunt pole of the eggshell contains more “bubble pores” having

Figure 4 SEM images of the inner face of the shell. (a) When the shell-membrane was peeled by hand, the mammillaries can befaintly seen through a residual fiber layer. When the inner face of the eggshell was burnished by a piece of sandpaper, the texture ofinterlaced inorganic CaCO3 crystals and organic keratin fibers is obvious (b), and the loose structure of calcite particles can be seen (c).(d)–(f) After the organic components were oxidized by being immersed in sodium hypochlorite solution for 2 min, keratin fibers werecompletely removed, and rough morphology appeared

Figure 5 SEM images of the fiber-membrane beneath the shell. The shell has been removed by being immersed in dilute acetic acid (5%,v/v) for 15 h to dissolve the calcium carbonate. (a) and (b) Protuberances formed by assembled fibers are distributed all over the outsideface (adhering to the eggshell) of the shell-membrane. (c) The inside face (contacting the egg white) of the shell-membrane. Few fibers canbe seen, likely formed by amorphous organic colloids, and it is smoother than the outside face. Side views of the shell-membrane: (d) isthe cross-section of both outer and inner shell-membranes. The insert is the section of a single fiber, exhibiting a core–shell structurewith holes inside; (e) and (f) are magnified images of the outer and inner shell-membrane, respectively

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Figure 6 Diameter of the fiber-membrane from the inside to the outside: (a) inner surface of the inner membrane; (b) outer surface of the inner membrane; (c) inner surface of the outer membrane; (d) outer suface of the outer membrane. The scale bar is 10 μm. The inner membrane having fibers of diameter 1.5–2.5 μm is more compact than the outer membrane with fibers having diameters of 2.5–5 μm

diameters within the range 250–400 nm (Fig. 7(b)). The higher number of larger size pores at the blunt pole result in a more porous and looser texture, which is favorable for gas exchange.

To confirm that hen’s eggshell regulates gas con- duction by adjusting the sizes and numbers of these “bubble pores”, the gas permeability of O2 (Figs. 7(c) and 7(d)) and CO2 (Figs. 7(e) and 7(f)) across the eggshell was measured. Samples were from either the middle region or the blunt pole of the eggshell, with (Figs. 7(c) and 7(e)) or without (Figs. 7(d) and 7(f)) membrane. In particular, as the two-layered shell-membrane separates to form the air cell at the blunt pole and only the outer membrane remains tightly attached to the shell, the blunt pole sample with membrane actually only contains the outer shell– membrane. Although there is a great variation between different samples and there are a small number of exceptions, the permeability of the blunt pole samples (red dots) is clearly better than the middle region ones (black squares). This is consistent with the speculation that blunt pole of the eggshell contains more and larger

“bubble pores”, resulting in a more porous and loose texture which is favorable for gas exchange.

3.5 Discussion

Based on the above observations, a novel description of hen’s eggshell is presented in Fig. 8. The shell can be mainly divided into the surface layer, palisade layer and mammillary layer, with a cuticular colloid layer over the shell and a fiber-membrane beneath the shell. In spite of different morphologies of CaCO3 samples in the different layers—namely, nano-rods and submicro spheres in the surface layer, rhombohedral-cleavage pattern in the palisade layer, and slices in mammillary layer–they all consist of primary nano-particles of diameter ~10 nm. Moreover, these nano-particles do not pack tightly, so that all of the above structures show a rough and porous texture.

On the other hand, the pore system of the shell contains at least three hierarchical levels of pores: first, micro- or meso-pores, with size less than 10 nm, ascribed to the interstitial voids between the packed primary nano-particles; second, “bubble pores” of ~250 nm, distributed throughout the palisade and mammillary layers; third, pores of several micrometers, i.e., the opening of conventional “gas pores”. The structural hierarchy of the pore system is believed to play an important role in exchanging gas and resisting contamination. In a typical description widely adopted by early researchers, micro-scale pores permeate the shell, directly connecting with the exterior [14]. Although some different pore types were identified [4, 10], all of them have been considered to have micrometer-scale accesses directly connecting to the outside. However, our observation of “gas pores” reveals that most of the so-called “funnel-like” pores are only shallow openings through the surface submicro-sphere layer, and “blocked” by compactly arrayed nano-rods beneath the sphere layer (Fig. 2(d) and Fig. S-1). This nano-rod layer should be an important barrier to exterior contaminants. As the layer (<10 μm) is very thin and accounts for only a small proportion in the thickness of the whole shell (300–350 μm), gases can easily permeate through voids between nano-rods and primary nano-particles and reach the palisade and mammillary layers that mainly

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Figure 7 Diameter distribution of “bubble pores” in the palisade and mammillary layers. (a) Middle region of the eggshell. Pores withdiameters less than 250 nm account for the majority, while those larger than 300 nm are relatively rare. (b) Blunt pole of the eggshell,where the air cell is usually located. Pores smaller than 200 nm are rare and most pores are within the range 200–400 nm. The modaldiameter of the pores is ∼250 nm in both cases. Gas penetration experiments: O2 (c) and (d) and CO2 (e) and (f) are used as test gases.Samples are from either the middle region or the blunt pole of eggshell, with (c, e) or without (d, f) membrane. In either case, penetrabilityof the blunt pole samples (red dots) is clearly better than the ones from the middle region (black squares)

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make up of the shell, with their numerous “bubble pores” of size ~250 nm. Our statistical calculation of “bubble pore” sizes and gas conduction experiments suggest a close relationship between “bubble pores” and gas conduction, and we believe that the “bubble pores” in the palisade and mammillary layers are main gas pathway. Thus, the paradox that gases can be exchanged whilst exterior contaminants are still obstructed can be resolved: micro-scale pores (openings of “gas pores”) and submicro-scale pores (“bubble pores”) construct the main pathway of gas conduction for embryo respiration during incubation, while the compact nano-rod layer with its nano-scale pores (between nano-rods and primary nano-particles) insulate the interior from the exterior and provide the necessary protection and resistance, without resisting gas conduction.

4. Conclusions

Hen’s eggshell has the ability to exchange gases while obstructing environmental contamination because of its elaborate architecture involving structural hierarchy. Numerous “bubble pores” in the palisade and mammillary layers provide the main gas pathway, and hen’s eggshell can regulate gas conduction by adjusting the sizes and numbers of these submicro- scale pores. In addition, the structural hierarchy of the pore system and cooperativity between pores of

different scales is believed to be the key to the com- bined gas conduction and contamination resistance. The natural elaborate architecture of eggshell may give us inspiration in designing biomimetic gas-exchange systems, such as the film to extend the shelf life of foods and life-support cabins for use in spacecraft.

Acknowledgements

The authors are grateful for financial support from the National Research Fund for Fundamental Key Projects (Nos. 2010CB934700, 2009CB930404, and 2007CB936403), and the National Natural Science Foundation of China (Nos. 20974113, 20601005). The Chinese Academy of Sciences is gratefully acknowledged.

Electronic Supplementary Material: Figure S-1, showing more detailed SEM images of a series of “gas pores” which are higher magnifications of a large area SEM image of the eggshell surface, is available in the online version of this article at http://dx.doi.org/10.1007/s12274-010-0067-8.

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Figure 8 A description of hen’s eggshell. The shell can be mainly divided into the surface layer, palisade layer and mammillary layerfrom the exterior to the egg white, with a cuticular colloid layer over the shell and a keratin-fiber membrane beneath the shell. Themorphologies of CaCO3 involve nano-rods covered by submicro spheres in the surface layer, rhombohedral-cleavage pattern in thepalisade layer, and slices in the mammillary layer. There are openings of conventional “gas pores” at the surface layer, and “bubblepores” distributed throughout the palisade and mammillary layers

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