An investigation of the nanostructural features of avian eggshell

The avian eggshell is a highly ordered bioceramic with both inorganic and organic constituents. The eggshell takes approximately 18 hours to form in the shell gland region of the hen's oviduct, a process which is repeated every 24 hours in modern hybrid laying hens, which are capable of laying in excess of 300 eggs per annum. Although the formation of the eggshell is rapid, it still results in a structure that is highly organised and which displays unique functional properties that depend on the interplay between its mineral and protein phases. The interactions between the inorganic and organic components during the formation of the eggshell however are poorly understood but it is likely that they occur at the nanometer level. Thus, it is hypothesised that structural variation at the nanostructural level will impinge on the overall structural integrity and mechanical performance of the eggshell. In this thesis. X-ray diffraction (XRD), small angle X-ray scattering (SAXS) and microfocus small angle X-ray scattering (? SAXS) were applied to investigate the nanostructure of the eggshell. A description of these different techniques is given in Chapter 2 along with theoretical considerations as to how the resulting data presented in subsequent chapters was analysed. In Chapter 3, thin sections of normal and abnormal eggshells, where the layers were structurally intact, were analysed using ? SAXS. The results of this experiment suggest that there are nanostructural features within the different layers of normal eggshells, especially in the mammillary layer. The size dimensions of these nanopores/nanovoids were subsequently estimated to be between 3 - 5 nm in both visually normal and abnormal eggshells. However, despite being of similar size, the distribution of these nanopores or voids was found to be disrupted in the abnormal eggshell samples. In Chapter 4, XRD and SAXS were used to analyse powdered eggshell samples which represented different stages of gestation. In this case, the diffraction and scattering data produced were used to calculate an average size measurement of the structural features within bulk eggshell samples. The X-ray diffraction data indicated that the crystallite sizes were large, between 54-232 nm. However, size dimensions of approximately 5.7 - 7.2 nm were observed from the analysis of the SAXS data confirming the hypothesis that SAXS was measuring nanovoids within the crystallites arising from the presence of embedded protein. A further study using SAXS to compare the nanostructural features of eggshells of varying mechanical strengths is presented in Chapter 5. Here, powdered eggshells from young and aged hens were compared and found to contain an average nanovoid size value of approximately 5.9 nm and 5.8 nm, respectively. These size dimensions were confirmed by a parallel study in which mercury intrusion porosimetry was used to investigate pore size distribution. It was concluded that the average size of nanovoids is comparable in eggs of different mechanical strength. Chapter 6 describes the results of an investigation in which SAXS was used to monitor the nucleation events which take place when calcium carbonate is grown in vitro with and without the presence of eggshell proteins. The results of this study although preliminary suggest that the initial nucleation event is extremely rapid and provides a unique insight into the size of the calcium carbonate crystals which initially form. The main conclusions from this thesis are summarised in Chapter 7, in relation to the effect that changes in eggshell nanostructure could have on the overall structure and function of this complex biomineralised structure. Potential further studies are also discussed.