Multiscale formation of mineralized tissues: Insight from the 3D structure of the reptilian eggshell
Joseph Deering* 1 , Valentin Nelea1 , Marc D. McKee1,2 * Presenting author, Postdoctoral Fellow (since 2022)
1 Faculty of Dental Medicine and Oral Health Sciences, UU直播, Montreal, Quebec 2 Department of Anatomy and Cell Biology, UU直播, Montreal, Quebec
Fiber-based extracellular matrices are found in many mineralized tissues across the animal kingdom. Bones and teeth have calcium-phosphate mineral embedded in their fibrous extracellular matrix, whereas mineralized eggshells have the mineral calcium carbonate, like that found in inner ear otoconia. Mineralized eggshells oviposited by birds and reptiles provide a rigid, durable, and protective exterior to eggs that resists pathogen ingress and allows for water and gaseous exchange for a growing embryo. While avian eggshells are highly organized into a columnar mineral structure originating from a fibrous organic membrane, reptilian eggshells vary considerably across species in the continuity, thickness, and structure of the mineral layer. Similar biomineralization principles are thought to operate in all of these mineralized tissues, despite their diversity. Here we present the first multiscale 3D structural analysis of the organic and mineral components of the Eublepharis macularius (common leopard gecko) eggshell, including the first use of cryogenic samplepreparation methods for these shells. Light microscopy, polarized light microscopy, atomic force microscopy, and transmission electron microscopy were used to view broad and fine structure. Submicron X-ray micro-computed tomography (X-ray microscopy) was used for assessing mid-range order of the calcareous assembly in 3D. Focused ion beam (FIB) serial sectioning coupled with scanning electron microscopy (SEM) was used to generate 3D volumes of eggshell with nanometre-scale resolution. The calcitic mineral phase of the gecko eggshell takes on three microstructural motifs at the shell surface: nodular scutes (~40 渭m), intermittent blunt or star-shaped mineral rosettes (~1 mm), and smaller mineral corollas (~10 渭m). Individual scutes have two hierarchical mineral structures 鈥� mineral nanospheres (~500 nm) and mineral nanodomains (~40 nm). Scute mineral was observed to dissolve after short-term exposure to aqueous media, highlighting the lability of biogenic calcium carbonate in these shells, and the need for cryo-preservation methods. Such structural information forms a baseline for observing eggshell response to environmental changes, for assessing the stability and architecture of synthetic and biological calcium carbonate systems, and for characterization of other mineralized tissues.
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