Polycarboxylated Eggshell Membrane Scaffold As Template for Calcium

crystals

Article Polycarboxylated Eggshell Membrane Scaﬀold as Template for Calcium Carbonate Mineralization

José L. Arias *, Karla Silva, Andrónico Neira-Carrillo , Liliana Ortiz, José Ignacio Arias, Nicole Butto and María Soledad Fernández Faculty of Veterinary and Animal Sciences, University of Chile, Santiago 8820808, Chile; [email protected] (K.S.); [email protected] (A.N.-C.); [email protected] (L.O.); [email protected] (J.I.A.); [email protected] (N.B.); [email protected] (M.S.F.) * Correspondence: [email protected]

Received: 8 August 2020; Accepted: 5 September 2020; Published: 9 September 2020

Abstract: Biomineralization is a process in which specialized cells secrete and deliver inorganic ions into confined spaces limited by organic matrices or scaffolds. Chicken eggshell is the fastest biomineralization system on earth, and therefore, it is a good experimental model for the study of biomineralization. Eggshell mineralization starts on specialized dispersed sites of the soft fibrillar eggshell membranes referred to as negatively charged keratan sulfate mammillae. However, the rest of the fibrillar eggshell membranes never mineralizes, although 21% of their amino acids are acidic. We hypothesized that, relative to the mammillae, the negatively charged amino acids of the fibrillar eggshell membranes are not competitive enough to promote calcite nucleation and growth. To test this hypothesis, we experimentally increased the number of negatively charged carboxylate groups on the eggshell membrane fibers and compared it with in vitro calcite deposition of isolated intact eggshell membranes. We conclude that the addition of poly-carboxylated groups onto eggshell membranes increases the number of surface nucleation sites but not the crystal size.

Keywords: eggshell mineralization; poly-glutamic acid

1. Introduction Biomineralization is a widespread phenomenon in nature through which living organisms fabricate a large variety of solid organic-inorganic composite structures, such as intracellular crystals in prokaryotes; exoskeletons in protozoa, algae, and invertebrates; spicules; lenses; bone; teeth; statoliths; otoliths; eggshells; and plant mineral structures, as well as pathological biominerals such as gall stones, kidney stones, and oyster pearls [1–8]. In this process, specialized cells secrete and deliver inorganic ions into confined spaces limited by organic matrices or scaffolds. The resulting biominerals are formed in elaborate shapes and hierarchical structures across several length scales 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 [9]. Although there are several types of biominerals, the main mineral resulting from biomineralization is calcium carbonate (CaCO3), specifically the more stable form, calcite. Considering its size, the chicken eggshell is the fastest biomineralization system on earth, where 5 g of calcite are deposited every day on each egg. Therefore, the avian eggshell is a good experimental model for the study of biomineralization. Structurally, the eggshell is a multilayered calcitic bioceramic built on different organic scaffolds produced by different cells located along defined regions of the avian oviduct in the form of an assembly line [10,11]. The first and innermost scaffolds are the fibrillar eggshell membranes (ESM), which on one hand surround the egg albumen (colloquial: egg white), and on the other hand support discrete accumulations of a different organic composition referred to as mammillae [12]. It has been well

Crystals 2020, 10, 797; doi:10.3390/cryst10090797 www.mdpi.com/journal/crystals Crystals 2020, 10, 797 2 of 10 established that the eggshell membranes and mammillae play a crucial role in regulating mineral nucleation and growth by incorporating inorganic precursors, such as ions, ion clusters, and amorphous phases [13–15]; other organic matrices complete the formation of a calcified layer (i.e., palisade) composed of polycrystalline calcite columns that are normal to the eggshell surface [16–18]. Eggshell membrane fibers are composed of mainly type X collagen and other proteins [19–21], and physiologically, they never mineralize in spite of the fact that 21% of their amino acids are acidic (glutamic and aspartic acids) [22]. A review of biomineralization discusses that in other biomineralized systems, such as bone and sea shells, one of the roles of these acidic amino acids is in structural matching for binding ions in a regular array at the surface of an organic matrix [5]. However, there is no differential explanation for additives incorporated in solution or, forming part of a co-polymer or a hydrogel, adsorbed or chemically coupled to a solid scaffold in an inorganic precipitation solution at equivalent saturation concentrations. In particular, eggshell calcite crystal mineralization starts and grows on negatively charged keratan sulfate-rich structures, referred as to mammillae, but not on the surface of the eggshell membrane fibers [23]. We hypothesized that the negatively charged amino acids of the eggshell membrane fibers are not competitive enough to promote calcite nucleation and growth relative to the mammillary sites. To test this hypothesis, we experimentally increased the number of negatively charged carboxylate groups on the eggshell membrane fibers and compared this to in vitro calcite deposition on isolated intact eggshell membranes.

2. Materials and Methods

2.1. Reagents and Materials Chemicals: Poly-l-glutamic acid (sodium salt) (15,000–50,000 mol wt), anhydrous sodium sulfate, methanol, tetraﬂuoroboric acid etherate, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and lithium hydroxide were from Merck KGaA, (formerly Sigma-Aldrich), Darmstadt, Germany. Plasticware: Falcon®polypropylene conical centrifuge tubes (Corning, Glendale, AZ, USA) and micro-bridges (Hampton Research, Laguna Niguel, CA, USA) were used.

2.2. Eggshell Membrane (ESM) Functionalization Protocols for functionalization were adapted from Greene and Wuts [24]. ESM were obtained from commercial White Leghorn chicken eggs. Empty eggs were ﬁlled with 1% acetic acid solution for 30 min to detach the membrane from the shell, and then ESM were incubated for another 48 hours in 1% acetic acid solution to eliminate any remaining calcium carbonate crystals, washed three times in deionized water, and manually detached from the shell [25]. Strips, 3 mm wide 20 mm long were × trimmed oﬀ the isolated ESM. Functionalization of ESM with poly-glutamic acid is diagrammed in Figure1, and the reaction was followed by Fourier Transform Infrared spectroscopy (FTIR) using a FTIR/FTNIR bench-top spectrometer (INTERSPEC 200-X, Interspectrum OÜ, Tartumaa, Estonia).

2.2.1. Protection of the Carboxyl Groups of Poly-glutamic Acid Briefly, 15 mg of poly-l-glutamic acid sodium salt (P-Glu) was mixed with 7 mg of sodium sulfate and suspended in 10 mL of methanol in a 50-mL Falcon tube. Then, 2 mL of tetrafluoroboric acid etherate was added with a micropipette. The mixture was stirred at room temperature for 15 h. The resulting liquid was carefully removed by pipette suction, washed with 5 mL of methanol, centrifuged at 2500 g for 5 min, filtered with a Millipore filter, dried at room temperature, and saved × properly [26]. This resulting product, where carboxyl groups from poly-glutamic acid were protected with methyl groups, was named P-Glu-CH3. Crystals 2020Crystals, 10, 797 2020, 10, x FOR PEER REVIEW 3 of 10 3 of 10

Figure 1. Diagrammatic representation of poly-glutamic acid coupling to eggshell membrane scaﬀold. Figure 1. Diagrammatic representation of poly-glutamic acid coupling to eggshell membrane scaffold. Top to bottom: poly-glutamic acid carboxyl group protection, crosslinking of methyl carboxylate Top to bottom: poly-glutamic acid carboxyl group protection, crosslinking of methyl carboxylate poly-glutamate to eggshell membrane, and cleavage of methyl groups to restore carboxylate groups to poly-glutamate to eggshell membrane, and cleavage of methyl groups to restore carboxylate groups the poly-glutamic acid coupled to the eggshell membrane scaﬀold. to the poly-glutamic acid coupled to the eggshell membrane scaffold.

2.2.2. Crosslinking of P-Glu-CH3 to Eggshell Membranes 2.2.1. Protection of the Carboxyl Groups of Poly-glutamic Acid ESM strips totaling 300 mg were introduced into a 5-mL Falcon tube and suspended in crosslinking solution consistingBriefly, of: 15 10 mg mL of 0.1poly M-L MES-glutamic (2-N-morpholine-ethane acid sodium salt (P sulfonic-Glu) was acid), mixed 0.5 M withNaCl, 7 and mg pHof sodium 6.0 activationsulfate bu andffer, suspend whereed 4 mgin 10 of mL EDC of (1-ethyl-(3,3-dimethyl-aminopropyl)-carbodiimide)methanol in a 50-mL Falcon tube. Then, 2 mL of tetrafluoroboric and 6 mg of NHSacid etherate (N-hydroxysuccinimid) was added with a were micropipette. added, stirred, The mixture and allowed was stirred to react at forroom 15 temperature min at room for 15 h. The resulting liquid was carefully removed by pipette suction, washed with 5 mL of methanol, temperature. P-Glu-CH3 was dissolved in 1 mL phosphate-buffered saline (PBS) pH 7.5 and added to the crosslinkingcentrifuged solution. at 2500 The × g for reaction 5 min, was filtered allowed with toa Millipore proceed forfilter, 2 h dried at room at room temperature temperature, [27,28 and]. saved The ESMproper stripsly were [26]. filtered This resulting and washed product, twice where with carboxyl 3:1 methanol:water. groups from poly This-glutamic product wasacid namedwere protected with methyl groups, was named P-Glu-CH3. P-Glu-CH3 ESM strips.

2.2.3. Cleavage2.2.2. Crosslinking of Methyl Groups of P-Glu to- RestoreCH3 to E Carboxylateggshell Membranes Groups

P-Glu-CHESM3 ESM strips strips totaling were suspended 300 mg inwere 10 mL introduced of 3:1 methanol:water, into a 5-mL 240 Falcon mg of lithium tube and hydroxide suspended in were added,crosslinking and the reactionsolution was consisting allowed of: to 10 proceed mL of at0.1 4 M◦C MES for 15(2- hN [-29morpholine]. Then the-ethane strips were sulfonic filtered acid), 0.5 M with ethanolNaCl, on and a Millipore pH 6.0 filter. activation This active buffer, polycarboxylated where 4 mg ofEMS EDC was (1 named-ethyl- P-Glu(3,3-dimethyl ESM. -aminopropyl)- carbodiimide) and 6 mg of NHS (N-hydroxysuccinimid) were added, stirred, and allowed to react 2.3. Calciumfor 15 Carbonate min at room Crystallization temperature. Experiments P-Glu-CH3 was dissolved in 1 mL phosphate-buffered saline (PBS) ThepH crystallization 7.5 and added assay to the was crosslinking based on solution. a variation The ofreaction the sitting was allowed drop method to proceed developed for 2 h at room elsewheretemperature [30]. Brieﬂy, [27,28] the. The assay ESM consists strips ofwere a chamberfiltered and built wash withed antwice 85-mm with diameter3:1 methanol:water plastic . This Petri dishproduct having was a central named hole P-Glu of-CH 18 mm3 ESM in strips. diameter at the bottom, with the chamber glued to a plastic cylindrical vessel (50 mm in diameter and 30 mm in height) (Figure2a). Calcium carbonate 2.2.3. Cleavage of Methyl Groups to Restore Carboxylate Groups crystallization was done on micro-bridges located inside the chamber. The micro-bridge is a small

P-Glu-CH3 ESM strips were suspended in 10 mL of 3:1 methanol:water, 240 mg of lithium hydroxide were added, and the reaction was allowed to proceed at 4 °C for 15 h [29]. Then the strips

Crystals 2020, 10, x FOR PEER REVIEW 4 of 10

were filtered with ethanol on a Millipore filter. This active polycarboxylated EMS was named P-Glu ESM.

2.3. Calcium Carbonate Crystallization Experiments The crystallization assay was based on a variation of the sitting drop method developed elsewhere [30]. Briefly, the assay consists of a chamber built with an 85-mm diameter plastic Petri dish having a central hole of 18 mm in diameter at the bottom, with the chamber glued to a plastic cylindrical vessel (50 mm in diameter and 30 mm in height) (Figure 2a). Calcium carbonate Crystals 2020, 10, 797 4 of 10 crystallization was done on micro-bridges located inside the chamber. The micro-bridge is a small bridge (inverted U) manufactured from clear polystyrene, which contains a smooth, 35-µL capacity bridgeconcave (inverted depression U) manufactured able to hold a from volume clear of polystyrene, liquid, essentially which containsa hemispherical a smooth, micro 35-µ-well,L capacity in the concavecenter of depression the top region able to of hold the abridge. volume Micro of liquid,-bridges essentially were fill aed hemispherical with 35 µL of micro-well, 200 mM calcium in the centerchloride of the dihydrate top region solution of the bridge.in 200 mM Micro-bridges Tris buffer, were pH filled9.0. The with cylindrical 35 µL of 200vessel mM contained calcium chloride 3 mL of dihydrate25 mM ammonium solution in 200bicarbonate. mM Tris buOneffer, 3- pHmm 9.0. wide The X cylindrical 20-mm long vessel ESM contained or P-Glu 3 ESM mL of strip 25 mM was ammoniumdeposited on bicarbonate. the top of Oneeach 3-mmmicro- widebridge X 20-mmwith the long mammillary ESM or P-Glu side ESMfacing strip down was in deposited contact with on the the topcalcium of each chloride micro-bridge solution, with leaving the mammillary enough space side between facing down the contour in contact of the with strip the and calcium the perimeter chloride solution,of the micro leaving-well enough to allow space free between diffusion the from contour the ofchamber the strip to and the theCaCl perimeter2 solution of (Figure the micro-well 2b). Strips to allowends free were di ff attachedusion from to the chamber micro-well to the edges CaCl with2 solution by double (Figure-sided2b). Strips tape. Fiveends replicateswere attached of each to theexperiment micro-well were edges carried with out by double-sidedinside the chamber tape. Five at 20 replicates °C for 24 ofh. eachAfter experiment the experiments, were carried and before out insidethe ESM the strips chamber were at taken 20 ◦C out for of 24 the h. Aftermicro- thebridges, experiments, the solut andion inside before the the micro ESM- stripswell was were removed taken outwith of a the micropipette micro-bridges, and replaced the solution twice inside with thedistilled micro-well water, wasand removedthen it was with dehydrated a micropipette with a and 50% replacedto 100 % twice ethanol with gradient distilled solution water, andseries. then Then it was the dehydratedESM strips were with removed, a 50% to 100 mounted % ethanol on aluminum gradient solutionstubs with series. double Then-side thed ESMtape, strips air-dried were for removed, 24 h at room mounted temperature, on aluminum and coated stubs with gold. double-sided After the tape,crystallization air-dried for assays, 24 h at the room ESM temperature, strips mounted and coated on with aluminum gold. After stubs the were crystallization analyzed. assays, Crystal themorphology ESM strips was mounted observed on aluminumand size was stubs estimated were analyzed. on a Jeol Crystal JSM-IT300LV morphology (Jeol USA, was observedInc., Peabody, and sizeMA, was USA) estimated scanning on electron a Jeol JSM-IT300LV microscope (Jeol (SEM) USA, operated Inc., Peabody, at 20 kV MA, with USA) an EDX scanning AZTec electron Oxford microscopedetector. Ten (SEM) randomized operated selected at 20 kV fields with anof EDXthe SEM AZTec micropgraphs Oxford detector. of five Ten replicates randomized were selectedused for fieldscalculating of the SEMcrystal micropgraphs number and ofsize. five Statistical replicates analyses were used of forcrystal calculating size and crystal density number were done and size.with StatisticalInfoStat 2014 analyses (Córdoba, of crystal Argentina) size and software.density were For done determining with InfoStat the type 2014 of (C ó datardoba, distribution, Argentina) a software.Shapiro–Wilk For determining test was used. the The type p of> 0.05 data statistical distribution, significance a Shapiro–Wilk level between test was the used. control The (ESM)p > 0.05 and statisticalpolycarboxylated significance eggshell level membrane between the (P- controlGlu ESM) (ESM) samples and polycarboxylatedwas analyzed by Student eggshell t membranetest. Results (P-Gluare expressed ESM) samples as mean was ± SD. analyzed by Student t test. Results are expressed as mean SD. ±

Figure 2. Diagram of the experimental crystallization chamber. (a) Gas diﬀusion chamber design, (b) location of the eggshell membrane strips on the top of the micro-bridges.

Figure 2. Diagram of the experimental crystallization chamber. (a) Gas diffusion chamber design, (b) location of the eggshell membrane strips on the top of the micro-bridges.3. Results and Discussion.

3. Results and Discussion As expected, the main components of the ESM are carbon, hydrogen, nitrogen, oxygen, and sulfur. As has been shown elsewhere [31,32], the Fourier Transform Infrared (FTIR) spectrum of ESM (Figure 3) shows, in the region of higher wavenumbers, an intensive peak at 3271 cm−1, which corresponds to the stretching mode of O–H and N–H groups. Peaks at 2923 and 2102 cm−1 correspond Crystals 2020, 10, 797 5 of 10 to the asymmetric stretching vibrations of the C–H bonds present in =C–H and =C–H2 groups, while in the region with lower wavenumbers, the peaks at 1628 cm−1, 1531 cm−1, and 1446 cm−1 represent the 1 1 1 lowerC=O wavenumbers, stretching, N the–H peaks bending at 1628 amide cm − absorption, 1531 cm− bonds, and, 1446 and cm CH− 2 representscissoring the bonds, C=O stretching, respectively. N–HEggshell bending membrane amide absorptions coupled bonds, to methyl and esterified CH2 scissoring poly-glutamate bonds, respectively. (P-Glu-CH3 Eggshell ESM) showed membranes a similar coupledspectrum to methyl to the esterified intact eggshell poly-glutamate membranes. (P-Glu-CH However,3 ESM) when showed the methyl a similar groups spectrum were cleaved to the intact from P- −1 eggshellGlu-CH membranes.3 ESM restoring However, the carboxylate when the methyl groups, groups the FTIR were spectrum cleaved from showed P-Glu-CH a new3 peakESM restoringat 1441 cm − 1 theindicative carboxylate of groups, absorption the FTIR of COO spectrum symmetric showed stretch a newing peak as atdescribed 1441 cm− forindicative pure poly of- absorptionγ-glutamic ofacid COO[33,− 34].symmetric After calculation stretching asof describedthe absorbance for pure integra poly-tedγ-glutamic area of th acide P [-33Glu,34 ESM]. After FTIR calculation spectrum of (Figure the −1 1 absorbance1) in the integratedrange of 1 area400 to of 1800 the P-Glu cm ESMusing FTIR a Gaussian spectrum model (Figure and1) ina polynomial the range of baseline 1400 to 1800 of order cm − 5, it usingwas a Gaussianpossible to model theoretical and aly polynomial estimate the baseline unblocked of order carboxylic 5, it was possiblegroups that to theoretically were able to estimate affect the theCaCO unblocked3 crystallization. carboxylic In groups accordance that were with able the empirical to affect the formula CaCO of3 crystallization.poly-glutamic acid, In accordance considering a withtheoretical the empirical polydispersity formula of index poly-glutamic of 1, it contains acid, considering a proportion a theoreticalof 4 carboxylic polydispersity groups per index 7 carbony of l 1, itgroups, contains that a proportionis a quotient of of 4 0.57. carboxylic A Gaussian groups integrated per 7 carbonyl area of – groups,564.5346 that was is obtained a quotient, representing of 0.57. A Gaussianthe carbon integratedyl groups area indistinctively of –564.5346 wasderived obtained, from representingcarboxylic or the other carbonyl sources groups. However, indistinctively we found −1 1 derivedthat the from peak carboxylic of 1441 cm or other showed sources. an integrate However,d area we of found −230 that.7645 the, representing peak of 1441 the cm fraction− showed of ionized an integratedcarboxylate area of groups.230.7645, Thus, representing the quotient the fraction of the ofionized ionized carboxylate carboxylate groups groups. divided Thus, the by quotient the total − of thecarbonyl ionized groups carboxylate is 0.41. groups By quotients divided comparison, by the total carbonyl the LiOH groups cleavage is 0.41. had By an quotients effectiveness comparison, of 71.9%. the LiOH cleavage had an effectiveness of 71.9%.

Figure 3. FTIR spectra of intact eggshell membrane (ESM), carboxyl group-protected poly-glutamic

acidFigure coupled 3. FTIR to eggshell spectra membrane of intact eggshell (P-Glu-CH membrane3 ESM), (ESM), and poly-glutamic carboxyl group acid-protected coupled topoly eggshell-glutamic 1 membraneacid coupled (P-Glu to ESM). eggshell Ionized membrane symmetric (P-Glu carboxylate-CH3 ESM), groups and arepoly shown-glutamic at 1441 acid cm coupled− . to eggshell membrane (P-Glu ESM). Ionized symmetric carboxylate groups are shown at 1441 cm−1. In our assay system, when calcium carbonate crystallization assays are done with a variation of the sittingIn our drop assay method system, using when micro-bridges calcium carbonate inside a crystallization closed mini-chamber assays are (Figure done2 with), but a withoutvariation of anythe additive, sitting COdrop2 producedmethod using from micro (NH4-)HCObridges3 interacts inside a firstclose withd mini the-chamber surface of(Figure the hemispherical 2), but without micro-wellany additive, containing CO2 produced the CaCl2 fromsolution. (NH Thus,4)HCO under3 interacts this condition,first with the nucleation surface ofof CaCOthe hemispherical3 crystals occursmicro first-well atthis containing interface, the and CaCl after2 solution. reaching Thus a certain, under size, this these condition, crystals nucleation leave the surface of CaCO and3 crystals sink in the CaCl2 solution, reaching the micro-bridge bottom and forming typical rhombohedral calcite crystals of different sizes probably due to a diffusion phenomenon of CO2 in the calcium chloride solution (Figure4). Hence, under this experimental condition, extreme caution must be taken when the eggshell membrane scaffold is used as an additive for driving heterogeneous nucleation on its surface. In fact, when an eggshell membrane strip is deposited on the bottom of the micro-bridge, it becomes very difficult to differentiate the crystals that are deposited by gravity on the eggshell membrane scaffold from those that grow bottom-up because of the nucleation and growth effect of the eggshell membrane proper. This, therefore, is the reason why ESM or P-Glu ESM strips were Crystals 2020, 10, x FOR PEER REVIEW 6 of 10

occurs first at this interface, and after reaching a certain size, these crystals leave the surface and sink in the CaCl2 solution, reaching the micro-bridge bottom and forming typical rhombohedral calcite crystals of different sizes probably due to a diffusion phenomenon of CO2 in the calcium chloride solution (Figure 4). Hence, under this experimental condition, extreme caution must be taken when the eggshell membrane scaffold is used as an additive for driving heterogeneous nucleation on its surface. In fact, when an eggshell membrane strip is deposited on the bottom of the micro-bridge, it Crystalsbecomes2020 very, 10, 797 difficult to differentiate the crystals that are deposited by gravity on the eggshell6 of 10 membrane scaffold from those that grow bottom-up because of the nucleation and growth effect of the eggshell membrane proper. This, therefore, is the reason why ESM or P-Glu ESM strips were placedplaced on the top ofof eacheach micro-bridgemicro-bridge withwith thethe mammillarymammillary side facingfacing down in contact with the calcium chloride solution. solution. Under Under this this condition condition,, the the effect effect of of crystal crystal deposition deposition because because of ofgravity gravity is isavoided avoided,, and and the the crystallization crystallization occurring occurring on on the the eggshell eggshell membrane membrane surface surface reflect reflectss the proper proper influenceinfluence ofof thethe template.template. As shown in Figure5 5,, crystalline crystalline calciumcalcium carbonatecarbonate aggregatesaggregates occurredoccurred preferentially onon mammillarymammillary sitessites ofof intact eggshell membranes (Figure(Figure5 5a,a,c).c). ThisThis featurefeature resemblesresembles veryvery well what occurs during natural eggshell formation, where calcium carbonate depositiondeposition begins on the outer outer mammillary mammillary side of the the eggshell eggshell membranes, membranes, closely associated with the negatively negatively charged keratan-sulfatekeratan-sulfate proteoglycan proteoglycan-rich-rich mammillae, and and then then progresses progresses in in an an upward direction. The finalfinal complex architecture ofof thethe eggshelleggshell calcifiedcalcified layerlayer isis the result of the interaction of calcium carbonate crystals crystals with with organic organic matrix matrix molecules molecules consisting consisting of proteins of and proteins proteoglycans and proteoglycans [11,15,35,36]. In[11,15,35,36]. contrast, when In contra a poly-glutamicst, when a poly acid-functionalized-glutamic acid-functionalized eggshell membrane eggshell membrane is used, large is used, amounts large ofamou calciticnts of aggregates calcitic aggregates grow not onlygrow on not mammillary only on mammillary sites but directly sites but on directly the eggshell on the membrane eggshell fibersmembrane (Figure fibers5b,d). (Figure In fact, 5b, ad). threefold In fact, a greater threefold amount greater of amount crystals of was crystals grown was on grown the poly-glutamic on the poly- acid-functionalized eggshell membrane compared with the intact eggshell membrane: 2334 677 vs. glutamic acid-functionalized eggshell membrane compared with the intact eggshell membrane:± 2334 674 213 crystals per square millimeter, respectively (Figure6a). However, there is no significant ± 677± vs. 674 ± 213 crystals per square millimeter, respectively (Figure 6a). However, there is no difference between the size of the crystals formed on both scaffolds: 10.49 2.9 vs. 10.66 4.32 µm, significant difference between the size of the crystals formed on both scaffolds:± 10.49 ± 2.9 ±vs. 10.66 ± respectively4.32 µm, respectivel (Figure6yb). (Figure Therefore, 6b). itTherefore, is reasonable it is toreasonable conclude to that conclude the addition that ofthe poly-carboxylated addition of poly- groupscarboxylated onto eggshell groups on membranesto eggshell increases membranes the numberincreases of the surface number nucleation of surface sites nucleation but, because sites of bu thet, inorganicbecause of ion the concentration, inorganic ion theconcentration, crystal size the reached crystal at size 24 h r iseached not aff atected. 24 h is not affected.

Figure 4. Representative scanning electron microscopy (SEM) observation of normal rhombohedral Figure 4. Representative scanning electron microscopy (SEM) observation of normal rhombohedral calcite crystals grown on the bottom of the micro-bridge under the experimental conditions but in the calcite crystals grown on the bottom of the micro-bridge under the experimental conditions but in the absence of any additive. absence of any additive.

Crystals 2020, 10, 797 7 of 10 Crystals 2020, 10, x FOR PEER REVIEW 7 of 10

Figure 5. Representative SEM observation of calcite crystals grown on intact eggshell membrane (a,c) Figure 5. Representative SEM observation of calcite crystals grown on intact eggshell membrane vs(a., c growing) vs. growing on poly on poly-glutamic-glutamic acid acid-coupled-coupled eggshell eggshell membrane membrane (b, (db).,d Different). Diﬀerent kind kindss of of crystal crystal aggregationsaggregations are are shown. shown. Arrows Arrows illustrate illustrate some some mammillary mammillary sites. sites.

Our results apparently disagree with other findings in which poly-glutamic acid associated with eggshell membranes induced an aragonite phase [37]. However, these authors did the experiments under different and not directly comparable conditions such as preadsorbing rather than chemically coupling the poly-glutamic acid to the eggshell membranes, and also using a different calcification protocol. Additionally, different effects on calcium carbonate crystallization have been described when poly-glutamic acid is not supported on a solid scaffold but instead is incorporated as an additive in solution [38]. Under these conditions, the addition of poly-glutamic acid to the CaCO3 precipitation solution substantially inhibits both nucleation and crystal growth of stable calcite compared with the unstable vaterite polymorph, probably as a consequence of kinetic constraints through a stronger binding of acidic polypeptide by the calcite surfaces than by the vaterite surfaces [38]. Therefore, care must be taken when the effects of additives are compared with additives incorporated in solution or,

Crystals 2020, 10, 797 8 of 10 forming part of a copolymer or hydrogel, adsorbed or chemically coupled to a solid scaﬀold in an Crystalsinorganic 2020, precipitation 10, x FOR PEER solution REVIEW at equivalent saturation concentrations. 8 of 10

Figure 6. Statistical analysis (mean SD) of the density (a) and size (b) of calcite crystals grown on Figure 6. Statistical analysis (mean ±± SD) of the density (a) and size (b) of calcite crystals grown on intact eggshell membrane or on poly-glutamic acid-coupled eggshell membrane. intact eggshell membrane or on poly-glutamic acid-coupled eggshell membrane. 4. Conclusions Our results apparently disagree with other findings in which poly-glutamic acid associated with Based on the results obtained in this study, it seems that widespread poly-glutamic negatively eggshell membranes induced an aragonite phase [37]. However, these authors did the experiments undercharged different sites attached and not to directly the eggshell comparable membrane conditions ﬁbers compete such as preadsorbing with the natural rather mammillary than chemically keratan sulfate negatively charged sites for calcite nucleation and growth under equivalent experimental coupling the poly-glutamic acid to the eggshell membranes, and also using a different calcification protocol.conditions. Additionally, This study also different oﬀers effects a possible on explanationcalcium carbonate for the mechanismcrystallization of nucleation have been and described crystal whengrowth poly of calcium-glutamic carbonate acid is andnot supporteda possible role on ofa solid negatively scaffold charged but instead chemical is groups incorpo coupledrated as on an a solid substrate on calcium carbonate biomineralization. additive in solution [38]. Under these conditions, the addition of poly-glutamic acid to the CaCO3 precipitationAuthor Contributions: solutionConceptualization, substantially inhibits J.L.A.; bothmethodology, nucleation A.N.-C., and N.B.; crystal investigation, growth of K.S., stable L.O., calcite M.S.F., comparedJ.I.A.; writing—original with the unstable draft, review vaterite and editing,polymorph, J.L.A., M.S.F.,probably J.I.A.; as funding a consequence acquisition, of J.L.A. kinetic All authorsconstraints have throughread and a agreed stronger to the binding published of acidic version polypeptide of the manuscript. by the calcite surfaces than by the vaterite surfaces [38]. Therefore, care must be taken when the effects of additives are compared with additives incorporated in solution or, forming part of a copolymer or hydrogel, adsorbed or chemically coupled to a solid scaffold in an inorganic precipitation solution at equivalent saturation concentrations.

Crystals 2020, 10, 797 9 of 10

Funding: The research described in this paper was financially supported by the Chilean Government National Agency of Research and Development (ANID), grant Fondecyt N◦ 1180734. Acknowledgments: The authors would like to thank Rocío Orellana of the Scanning Electron Microscopy Laboratory, Faculty of Dentistry, University of Chile. The authors would also like to thank David A. Carrino (Cleveland, OH) for providing insightful comments on the manuscript language style, spelling and scientific expertise suggestions. Conflicts of Interest: The authors declare no conflict of interest.

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