Phosphate and Carbonate in the Biomineralization of Chicken Eggshells and the Increase in Eggshell Thickness Through Nanodroplet Addition

The presence of hydroxyapatite (HAp) in the Cuticle of laying-hen eggshells was investigated through a comprehensive characterization of its morphology, composition, crystallographic structure, and thermal behavior. Comparison with bone-derived hydroxyapatite confirmed the similarity of the mineral phase. Examination of the same region at different imaging contrasts revealed unidirectional (nanofibrous) calcite growth within the Vertical Layer (VL) and Palisade Layer (PL). Shell thickening in these layers appears to occur through an additive mechanism involving the successive deposition of nanodroplets containing, according to our hypothesis, the mineral phase, water, and organic components. This multiphasic system generates lamellae that progressively increase in thickness through the continuous incorporation of new nanodroplets onto the pre-existing surface. Such a nanodroplet-mediated growth mechanism also provides a plausible explanation for the formation of the micropores observed in the VL and PL. The proposed additive mechanism is further supported by the presence of nanohemispheres attached to growing lamellae and the similar diameters of cuticle nanospheres and Vertical Layer nanohemispheres. Fractures observed in the Vertical Layer indicate structural continuity between the Cuticle and the VL, suggesting that additive growth involves a continuous supply of HAp, possibly across the entire uterine surface, followed by a previously undescribed process in which calcium phosphate nanospheres dissolve and/or transform into calcium carbonate nanofibers.