Structural marvels of the eggshell
(appeared in April 2018)

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Shells of birds’ eggs take good care of the unborn chick, but let go when its time to hatch. , says S.Ananthanarayanan.

The eggshell’s first function is protection while the fertilized egg develops. During this time, the eggshell also dissolves from the inside, to be a source of calcium for the growing embryo. This thinning of the inner layers of the shell structure, in turn, helps weaken the shell, so that it cracks when the chick is ready to emerge.

Dimitra Athanasiadou, Wenge Jiang, Dina Goldbaum, Aroba Saleem, Kaustuv Basu, Michael S. Pacella, Corinna F. Böhm, Richard R. Chromik, Maxwell T. Hincke, Alejandro B. Rodríguez-Navarro, Hojatollah Vali, Stephan E. Wolf, Jeffrey J. Gray, Khanh Huy Bui and Marc D. McKee, from McGill University, Montreal, John Hopkins University, Baltimore, Friedrich-Alexander-University, Germany and the University of Ottawa, describe in the Journal, Science Advances, their study of how the nano-structure of the eggshell forms and how it facilitates its diverse functions during the incubation period. And then, the team shows that the protein that brings about the eggshell structure can create structure in non-biological material, just like it does in the eggshell.

The reptile and birds’ eggshell are remarkable containers that allow the embryo to develop in the rich nutrients of the egg, and in safety. The researchers discover that the eggshell is not the same at all parts of its thickness, and it is a protein called osteopontin (OPN), which was first found to help bone tissue to form, is the active agent that gives the eggshell its structure. Osteopontin is a chain molecule that has many points along its length where it shows a negative charge. Atoms of calcium and minerals bind to these points along the protein molecule. Osteopontin thus forms a scaffold and the calcium carbonate (the same thing as chalk), which forms 95% of the eggshell, grows into a matrix, to form a shell with the optimum structure that makes for strength.

"Eggshells are notoriously difficult to study by traditional means, because they easily break when we try to make a thin slice for imaging by electron microscopy," a press release reports Prof. McKee from McGill University to have said. "Thanks to a new focused-ion beam sectioning system recently obtained by McGill's Facility for Electron Microscopy Research, we were able to accurately and thinly cut the sample and image the interior of the shell."

Using these methods, the team finds that the eggshell material of the eggs of the domestic chicken (Galus gallus) consists of nano-granules, whose size varies as one passes from the outer surface of the eggshell to the inner surface. The paper in Science Advances explains that living things create a variety of biominerals, in the form of hardened structures, like human bones. In human bones, the material is mainly nano-crystals of calcium phosphate, which forms within a matrix of large organic molecules. In the case of other organisms, the material is mainly calcium carbonate (limestone or chalk) which form rigid structures, typically shells. In the case of shells of mollusk, coral skeletons, the granular structure has generally been found to be uniform, with the material behaving as if it were just one crystal. In the case of the eggshell, analysis has revealed a varying pattern in the grain size as one passes though the thickness of the shell.

It is found that in the outermost layer, called the vertical crystal layer (VCL), the grain size is 30 nanometers. The upper palisades layer (PL), where grain size is 33 nm, comes next. The grain size rises again to 59 nm in the middle PL, to 74 nm in the lower PL and 68 nm in the innermost mammillary layer (ML). This variation in the granular size was found to correspond to levels of OPN present – a large presence in the VCL resulted in fine grain size. While the gradient in grain size was noted, further study of the PL region, the largest in the section of the eggshell, revealed a pattern of clustering of domains from 5 to 7 nm in size. As a mechanism of how the pattern forms, the researchers suggest that it is similar to the formation of patterns in distribution of pigment, as in leopards’ spots or tigers’ stripes, and also related to the abundance of OPN during different stages of the eggshell formation.

The team also studied the progression of hardness and elasticity of the eggshell material. It was found that the outermost, VCL layer was the hardest and most elastic, with the properties reducing till the middle of the PL, and then rising towards the innermost of the ML. That the hardness was highest in the VCL, where the grain size was the smallest is in keeping with the known principles of composite materials, but the rise in hardness towards the ML is still not understood, the study says.

The first understanding from the study is the structure of the avian eggshell and its twin roles – in the inner layer being easily dissolved, to provide the embryo with calcium for bone development and in thereby weakening so that the chicken can hatch when it is ready. But an important finding is the finding that the shell strength can be varied by quantity of OPN that is present. It was shown by the team that even outside a biological setting, the mineralizing of calcium carbonate was just like in the eggshell when OPN was present. And then, more OPN led to finer grain size, as in the eggshell.

The paper says that a problem that the poultry industry faces is that when a hen has been laying for about a year, the shells of the eggs laid start getting brittle. This could allow salmonella infection and food safety. Knowledge about the eggshell structure and the role of OPN could suggest genetic engineering methods for stronger eggshells. Understanding the role of organic components in structure of composite materials would also help “inform design concepts for synthetic nanocomposites that have novel properties,” the study says.

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