Keywords

1 Introduction

Biomineralization is a widespread phenomenon in nature leading to the formation of a variety of solid inorganic structures by living organisms, such as intracellular crystals in prokaryotes; exoskeletons in protozoa, algae, and invertebrates; spicules and lenses; bone, teeth, statoliths, and otoliths; eggshells; plant mineral structures; and also pathological biominerals such as gall stones, kidney stones, and oyster pearls (Lowenstam and Weiner 1989; Mann et al. 1989; Simkiss and Wilbur 1989; Heuer et al. 1992; Arias and Fernandez 2008). By this process living organisms precipitate inorganic minerals on organic matrices. The resulting biominerals are deposited in elaborated shapes and hierarchical structures by interaction at the organic-inorganic interface, where the rate of crystal formation is regulated by the control of the microenvironment in which such mineralization events take place. One of the main biomineralization resulting mineral minerals is calcium carbonate (CaCO3), especially the more stable form, calcite. The avian eggshell is a good example of multifunctional biomineral where an organic scaffold (or matrix), the eggshell membrane, plays a crucial role in regulating mineral nucleation and growth by incorporating inorganic precursors, such as ions, ion clusters, and amorphous phases (Rao et al. 2017); other organic matrices complete the formation of a calcified layer (i.e., palisade) composed of calcite columns (Panheleux et al. 1999). Structurally, the eggshell is a multilayered calcitic bioceramic. As the egg migrates through the oviduct, the biomineral matures under the influence of biomolecular additives and an extracellular matrix (Nys et al. 1999; Fernandez et al. 2001). The matrix, primarily composed of collagen fibers, constitutes the eggshell membranes (ESM) (Arias et al. 1991). Each fiber exhibits a core surrounded by a glyco-proteinous material termed as the mantle. This serves as the first interface with the inorganic phase. During mineral growth, crystals nucleate on the outer side of the ESM at specialized sites called mammillae, mainly consisting of mammillan, a keratan sulfate proteoglycan together with the activity of carbonic anhydrase (CA). This metalloenzyme catalyzes the reversible hydration of carbonic dioxide to bicarbonate and a proton. In fact, it has been possible to mimic eggshell formation in vitro by adding the main organic components including CA, where an increase in calcium carbonate crystals growth and fusion was observed (Fernandez et al. 2004).

However, the use of enzymes in vitro is difficult, because of instability and the complexity for maintaining the catalytic function in chemical reactions (Lu et al. 2013). For that reason enzymatic immobilization on a solid substrate appears as a solution for this problem (Wanjari et al. 2013).

For in vitro biomineralization, a specific confined environment is needed, that means an inert scaffold which generates an almost two dimensional interface where the crystal nucleation takes place. Currently, many kinds of surfaces and/or supports are used for enzyme immobilization, and the ESM meets all the requirements to be used as a natural support for CA immobilization and in vitro biomineralization experiments.

In order to get insight into the mechanisms of chicken eggshell mineralization, ESM was used as a biotemplate for immobilizing carbonic anhydrase (CA).

2 Material and Methods

2.1 Carbonic Anhydrase (EC 4.2.1.1) Immobilization on ESM

ESM were obtained after 30 min incubation of an empty egg in 1% acetic acid to detach the membrane from de shell, and then ESM was incubated for another 48 h in 1% acetic acid to eliminate any remaining calcium carbonate crystals and then washed in deionized water three times (Arias et al. 2008).

For enzyme immobilization 1 mg of carbonic anhydrase (2500 units/mg, Sigma, St. Louis, MO, USA) in 1 mL deionized water was used; membranes were incubated for 1 h with 100 μL of this solution; then 20 μL of 2.5% glutaraldehyde for 30 min was used as cross-linking agent (Tembe et al. 2008). Then, membranes were washed with TRIS buffer solution pH 9 at 4 °C.

2.2 Crystallization Experiments

The crystallization assays were based on a variation of the sitting drop method developed elsewhere (Dominguez-Vera et al. 2000). Briefly, it consists of a chamber built with a 85 mm plastic petri dish having 18 mm in diameter central hole in its bottom, glued to a plastic cylindrical vessel (50 mm in diameter and 30 mm in height) (Fig. 4.1). The bottom of the petri dish was divided in 16 radii to assure an equidistant settling of odd number of polystyrene microbridges (Hampon Res., Laguna Niguel, CA). The microbridges were filled with 35 μL of 200 mM dihydrate calcium chloride solution in 200 mM Tris buffer, pH 9.0. The cylindrical vessel contained 3 ml of 25 mM ammonium carbonate. One strip of eggshell membrane with or without immobilized CA or with CA in solution was deposited on the bottom or on the top of each microbridge with the mammillary side facing up or upside down, respectively. Five replicates of each experiment were carried out inside the chamber at 20 °C for 24 h. After the experiments, eggshell strips were taken out of the microbridges, air-dried at room temperature, mounted on aluminum stubs with scotch double-sided tape, and coated with gold. Crystal morphology was observed and size estimated in an Hitachi TM 3000 scanning electron microscope.

Fig. 4.1
figure 1

Experimental setup used from growing in vitro CaCO3 crystals showing the location of eggshell membrane strips either on the bottom or the top of the microbridges containers

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3 Results

3.1 ESM on Bottom of the Microbridge 24 H Incubation

After 24 h incubation using ESM without treatment, rounded calcite crystals were observed deposited on the ESM (Fig. 4.2a), with an average size of 21.33 ± 1.97 μm (Table 4.1). By contrast, when ESM with immobilized CA was used, regular rhombohedral calcite crystals (Fig. 4.2b), 31 ± 2.08 μm in average size were obtained (Table 4.1). When intact ESM was used in combination with CA dissolved in the calcification medium, rounded calcite crystals (Fig. 4.2c) with an average size of 20 ± 3.68 μm (Table 4.1), similar to those observed in the first condition were obtained.

Fig. 4.2
figure 2

Eggshell membrane (ESM) on bottom of the microbridge after 24 h incubation: (a) ESM without treatment, 1000X; (b) ESM with immobilized CA, 1000X; (c) ESM without treatment but with CA dissolved in the calcification medium 1000X

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Table 4.1 Size of calcite crystals deposited on eggshell membrane (ESM) measured under different conditions
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3.2 ESM on Top of the Microbridge Facing the Calcification Medium After 24 H of Incubation

After 24 h incubation using ESM without treatment, polyhedral calcite crystals were obtained (Fig. 4.3a), with 22.1 ± 1.91 μm in average size (Table 4.1). But when ESM with immobilized CA was used, many polyhedral fused calcite crystals of 35.83 ± 3.13 μm in average size (Table 4.1) with symmetrical smooth edges (Fig. 4.3b) were observed. However, after using intact ESM with CA in solution, less polyhedral fused calcite crystals with curved edges (Fig. 4.3c) of 27.83 ± 2.47 μm in average size were observed (Table 4.1).

Fig. 4.3
figure 3

Eggshell membrane (ESM) on top of the microbridge facing calcification medium after 24 h incubation: (a) ESM without treatment, 1000X; (b) ESM with immobilized CA, 1000X; (c) ESM with CA in solution in the calcification medium 1000X

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4 Discussion

When in vitro CaCO3 crystallization experiments are done in a gas-liquid interface diffusion environment, the favorite place for crystal nucleation and growth must be considered. In fact, in this chamber-mediated experiment, CO2 comes from the gas phase, while carbonate ions are in aqueous solution. If the reaction is done without any additional scaffold, such as the eggshell membrane, it is expected that nucleation of CaCO3 crystals occurs close to the gas-liquid interface, and then, after a determined period of growth, crystals formed precipitate reaching the bottom of the reaction vessel (microbridge). However, here we showed that when an active heterogeneous nucleator scaffold, such as the eggshell membrane supplemented with immobilized or dissolved carbonic anhydrase, is located upside down at the gas-liquid interface for avoiding gravity effect, calcite nucleation and growth are sequestered by the scaffold from solution, thus affecting the morphology and size of the crystals formed. The morphology changes, including calcite aggregations occurs in a way that resembles the calcite column aggregation and fusion observed during natural eggshell formation.