Engineering Tough Materials: Biomimetic Eggshell Preliminary Research Report, January 30, 2015 Dr. Michelle L. Oyen (with PhD student H. Burak Caliskan) Cambridge University Engineering Dept. Trumpington Street Cambridge, CB2 1PZ UK Approved for public release;distribution unlimited
Abstract
This preliminary report has two parts. First, the literature on biomimetic calcite synthesis is reviewed, with an aim of establishing gold-‐standard techniques for making biomimetic eggshell in large quantities. The literature is found to be surprisingly uniform in that most controlled studies of calcite biomineralization have utilized a vapor diffusion technique, where calcium is in solution and carbon and oxygen atoms or ions are introduced as a gaseous phase. Although well controlled, this process is slow and produces small quantities of material. As such, the evidence supports our continuing with solution-‐based calcite synthesis with a mind towards scale-‐up of material synthesis for eggshell-‐like material in large quantities. Second, the results of a series of preliminary experiments on this project are presented, comparing calcitic materials we produce with natural eggshell. Spectroscopy is utilized for examining the mineralization of calcite and the presence of amorphous calcium carbonate in the calcite matrix. Thermal analysis is used to establish the presence of organic materials within calcium carbonate. Next steps on this project will add a focus on how little is known about the quantitative thermodynamics of calcite formation in the presence of organic molecules, such as eggshell proteins, in order to facilitate larger-‐scale production of eggshell-‐like organic-‐inorganic composite materials.
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Engineering Tough Materials: Biomimetic Eggshell
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This preliminary report has two parts. First the literature on biomimetic calcite synthesis is reviewed with an aim of establishing gold-?????standard techniques for making biomimetic eggshell in large quantities. The literature is found to be surprisingly uniform in that most controlled studies of calcite biomineralization have utilized a vapor diffusion technique where calcium is in solution and carbon and oxygen atoms or ions are introduced as a gaseous phase. Although well controlled this process is slow and produces small quantities of material. As such the evidence supports our continuing with solution-?????based calcite synthesis with a mind towards scale-?????up of material synthesis for eggshell-?????like material in large quantities. Second the results of a series of preliminary experiments on this project are presented comparing calcitic materials we produce with natural eggshell. Spectroscopy is utilized for examining the mineralization of calcite and the presence of amorphous calcium carbonate in the calcite matrix. Thermal analysis is used to establish the presence of organic materials within calcium carbonate. Next steps on this project will add a focus on how little is known about the quantitative thermodynamics of calcite formation in the presence of organic molecules such as eggshell proteins in order to facilitate larger-?????scale production of eggshell-?????like organic-?????inorganic composite materials. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: a. REPORT
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Introduction The physical structure of the eggshell is the basis of its strength. An eggshell is made up of ~95% calcium carbonate in the form of calcite, one of the most widespread mineral compounds in nature. Biomaterials are almost always the outcome of an organic-‐inorganic collaboration. Eggshells contain a few percent of organic material, largely protein, which contribute to the physical properties of the final material. Natural minerals are in fact formed through an organic-‐ inorganic interplay; biomineralization is itself regulated by protein (Figure 1). The mechanisms by which the organic content regulates biomineralization are poorly understood. There is very little information, for instance, on the thermodynamics of biomineralization [Navrotsky 2004]. Although, state-‐of-‐the-‐ art techniques for calcium carbonate synthesis in the presence of various organic and inorganic molecules provide an opportunity for studying the formation of biominerals at the molecular level, almost all of the information that the current literature offers is only qualitative. Unravelling these mechanisms quantitatively could pave the way to engineering bio-‐inspired materials, and is absolutely critical in scale-‐up of materials synthesis from small-‐scale laboratory experiments to military-‐scale materials applications.
Figure 1. Schematic representation of the functional mechanism of ovocleiden-‐ Figure 7: 17, Schematic of ithe functioning mechanism of (Protein a protein trepresentation hought to be crucial n eggshell calcite biomineralization. OC-17. (Protein was downloaded fromCommons Wikimedia Commons structure structure was downloaded from Wikimedia [http://commons.wikimedia.org/wiki/Category:Images]) [http://commons.wikimedia.org/wiki/Category:Images]) Calcite synthesis C-type lectin-like proteins are mineralization mediators in many different avian species. A unique C-type lectin-like protein purified forout some avian species, Calcium carbonate forms easily bwas y precipitation of solution, and is typically found igoose, n one oturkey, f three duck, crystalline polymorphs, calcite, vand aterite and aragonite. It such as for chicken, guinea fowl, pheasant[21], ostrich[41], can be synthesized by simply ixing solutions f calcium of chloride CaCl2・2H2O) rhea and emu[42]. There are differences inmthe amino acid osequences these (difand sodium carbonate (Na CO3) or sodium bicarbonate (NaHCO3). Our ferent C-type lectin-like proteins. Goose2 C-type lectin-like protein ansolcalcin[43], laboratory typically uses both solutions at 200 mmol, mixing to combine directly for instance, only shares a 36% similarity withinfuse OC-17. ostrich or using syringe pumps to slowly each Importantly, solution into athe vessel for mixing. We two have also developed an alternate soaking that (AS) are, technique for forming calcium eggshell contains different C-type lectin-like proteins, struthiocalcin-1 carbonate in the difference presence oin f othe rganic molecules, mixing with organics (in our and struthiocalcin-2[41]. This ostrich may beby related the enhanced mechanical properties of the eggshell because as previously described, intracrystalline proteins counter-balance the mechanical shortcomings of the brittle calcium carbonate. In addition, the ostrich eggshell was shown to possess an extraordinary preservation ability for its intra-crystalline content[7]. There is little knowledge
(HA–GEL).20 ural formation hell, where the agenous memmineral micromaterial—2% ralized layer. ited using the nd demineralaction of the between the of the sample om a series of nts. Once the osite has been be controlled ymorphism of e either calcite scope (SEM) morphology is ch the mineral als that calcite mber of ASP of the natural EL composite neously nucle-
preparation
d; J Sainsbury to release the % by weight ution, pH 7.4, ellets (Sigmae of the memh contains the crystals of the
te and gelatin –20% GEL by bloom gelatin um carbonate distilled water subsequently transferred to e conical tube de from parts roup, Billund,
Conical tubes were then rolled for a further 2 days with the lid removed to allow evaporation of water from the sample, at which point the slurry had formed a solid. After forming a solid, samples were transferred to an autodesiccator for 7 days to dry completely. After the reference composite samples hadcbeen they were weighed to confirm that stock solutions and then particular ase, tdried, he protein gelatin) in with these same all water ahad evaporated and ohence thatfor thea weight dipping substrate in each f four check beakers prescribed number of cycles percentage in the ufinal solid is thesynthesis have included the (Figure 2). ofOgelatin ther studies sing homogeneous solution-‐based calcite same was added tocrystals the drywpowder mixture; forsurfaces all use oas f natural calcite ith freshly cleaved to aid in crystal samples, the [total mass solid Owas withinin 61% of the nucleation Elhadj et aofl. the 2006]. ur focus the last few years has always original mass of the two powders. involved solution-‐based formation of calcium carbonate (and calcium phosphate Calcite powder was purchased Sigma-Aldrich [Strange and Oyen 2011] in earlier from work), but for the first Aim of this project, the (U.K.). Vaterite was by omixing literature was rpowder eviewed to eproduced stablish the ptions 20 for mL calcite synthesis and the 21 of a 200 mmol Ca ion solution with 20 mL potential for large-‐scale production of eggshell-‐like bof iomimetic material. (NB athat 200this mmol CO2! ion solution, followed by immediate report’s r eferences a re n ot i nclusive o f t he l iterature surveyed.) 3 additionThe of 20vast mLmofajority 100% oethanol (Sigma-Aldrich, U.K.) f research into biomimetic mineralization [Gomez-‐ 24–26 toMorales arrest vaterite calcite. mixed, the [Addadi et al. 1987] et al. 2conversion 010] has uto tilized a vapor Once diffusion process resulting solution was centrifuged, the remaining fluid for calcium carbonate formation. In this process, calcium chloride is in solution, drained and the aprecipitate but the off, carbonate toms are dried. in gas form, often from ammonium carbonate powder, which vaporizes easily. The ammonium carbonate dissociates, and the C. Automated carbon dioxide vASP apor interacts with the calcium in solution to precipitate calcium carbonate. A vused ariation on this approach ubbles carbon dioxide gas The ASP, Fig. 1, was to coprecipitate CaCO3b –GEL directly i nto a c alcium s olution t o a chieve t he s ame effect [Lakshminarayanan et composites on both glass coverslips and demineralized al. 2006]. eggshell membrane substrates. The solutions for this were 21been modified to incorporate a wide The v apor d eposition rocess Ca has produced as follows: a 200 pmmol ion solution range of otorganic molecules [Meldrum 2003], b5.88 oth ngatural stabilized pH 7.4 was prepared by dissolving of and synthetic, to examine h ow t hese o rganic m olecules i nfluence t he t ypes (polymorphs) of calcium chloride dihydrate (CaCl2"2H2O; Sigma-Aldrich calcium cLtd., arbonate resent, and shape o(Sigmaf crystals, and their orientation. Company U.K.)pin 20 mLthe ofs1ize mol Tris-HCl The i nfluence o f i norganic s ubstitutions, o f m agnesium Aldrich Company Ltd., U.K.) diluted with 180 mL of for calcium in particular, has aAlso been widely examined [Gower 2008]. The vapor deposition process has DW. 200 mmol CO2! 3 solution was prepared by diluting been u sed s pecifically in the study of biomimetic eggshell in at least two research 80 mL of 0.5 mol Na2CO 3 solution (Sigma-Aldrich Comgroups t al. mL 2004; Lakshminarayanan et al. 2006]. Small pany Ltd.,[Fernandez U.K.) with e120 of DW. Gelatin was included modifications t o t he v apor d iffusion m ethod i nclude the use of a polymer-‐ in both of these solutions by heating the solutions to 80 °C induced precursor PILP) Odom, 2000] or a modification to and addingliquid between 2.5 and(15 g of[Gower dry 180and bloom gelatin the p recise s tructural s et-‐up o f t he v apor d iffusion c hamber [Gomez-‐Morales et extracted from porcine skin (Sigma-Aldrich Company Ltd., al. 2010] but cycle these of are clearly tweaks n the overall same process. U.K.). For one theall ASP, glasssmall coverslips or oeggshell
FIG. 1. The from Ref. 16). Both thefCaCl Figure 2. TASP he fwith our gelatin step a(adapted lternate soaking process or forming calcium carbonate 2 and Na CO solutions are 200 mmol, with between 2.5 and 15 g of 3 with 2gelatin [Armitage et al. 2012]. Both the CaCl2 and Na2CO3 solutions are 200 gelatin in w each mL of solution. mmol, ith 200 between 2.5 and 15 g of powdered gelatin added to each 200 mL
beaker of solution.
Mater. Res., Vol. 27, No. 24, Dec 28, 2012
wnloaded: 18 Dec 2012
IP address: 129.169.141.149
The challenge with a vapor diffusion process from a scale-‐up perspective is that it is slow, taking as long as several days to form calcium carbonate [Addadi et al. 1987]. This makes it an acceptable vehicle for studying basic aspects of mineralization in an extremely controlled manner, such as the influence of the presence of various organic molecules, but for the manufacture of large scale materials it is impractical. The AS process is relatively faster, but the process is overall not very well controlled. The act of forming calcium carbonate by just mixing solutions is also not well controlled, and there is insufficient time for organic molecules to influence the process, and become incorporated into the crystals, making it not very biomimetic and unlikely to yield material with the desired robust mechanical properties, and fracture toughness in particular. In addition to challenges with reaction rate, in all of these cases a major limitation to the calcite formation is in the size of the crystals formed. While natural eggshell is up to millimeters thick with nearly through-‐ thickness calcite crystals, the crystals reported in biomimetic biomineralization works are micrometers in size. After 100 cycles of the AS process, the crystals were approximately 4 μm in diameter, compared with sub-‐1 μm after the first cycle [Armitage et al. 2012]. Limiting the literature on optimized large-‐scale biomimetic calcite synthesis with the incorporation of organic molecules is the lack of quantitative thermodynamic data about the process of eggshell biomineralization. Overall eggshell has been studied dramatically less than other calcium carbonate materials, such as nacre and various marine organisms. The production of a complete chicken eggshell takes only 18 hours. Research on eggshell has largely been confined to the food (poultry) industry, and interest in mechanical properties of egg was mostly concerning the transport of eggs to supermarkets. It is largely suspected that, as with many other biomineralization processes, there is a multi-‐step formation process with calcite as an end result (Figure 3) and intermediate phases of amorphous calcium carbonate, vaterite and aragonite [Gower 2008]. This hypothesis is consistent with the co-‐existence of all three crystalline phases in nature, since the vaterite and aragonite are meta-‐ stable and calcite is the final stable product. An important role of this mechanism is to allow the organism to mold the amorphous form in any desired shape to produce complex architectures. This mechanism was shown to be possible in the context of reprecipitation of quail eggshell [Lakshminarayanan et al. 2006]. The role of proteins in the amorphous precursor phase is proposed to be crucial because of their ability to keep the amorphous form stable until the crystal transformation is needed [Gower 2008]. A thorough understanding of how this mechanism is used by living organisms is desirable to enable production of biomimetic inorganic materials in large quantities.
Figure 3. Reaction coordinate diagrams of classical nucleation single reaction (A) and likely biomineralization multiple-‐reaction pathways. (Adapted from Figure 6: Reaction coordinate diagrams of classical(B) nucleation (A) and biominerali[Gower 2008].) sation (B) pathways. (Adapted from Refs. 4 & 23) Preliminary Experiments starts with the deposition of an amorphous precursor phase which then transforms Biological mineral formation requires the precipitation inorganic crystals to a crystal passing through meta-stable forms[30]. Thisof pathway offers from many ada solution. Although it is now widely accepted that the first phase in an uthis nstable amorphous fis orm, hich then transform to to mould vantages. An biomineralization important roleis of mechanism towallow the organism meta-‐stable and finally to a stable crystal, precipitation from a solution is still the the amorphouskey form desired shape toprocesses produce such as step fin or aany lmost all mineralization in ncomplex ature. It is tarchitectures hen reasonable to analyze mineralization only after the common solid inorganic material formation, but seashells[4]. This mechanism wasnot shown to be in many different biominerals also during the crystallization of the mineral. Spectroscopy is one of the most such as zebra convenient fish bones[32] or tquail Calcium carbonate for methods o study eggshells[33]. mineralization during crystal formation. Tstructures, his technique allows monitoring mineral formation in real-‐time, thereby providing a instance, is known to transform from amorphous to meta-stable vaterite or aragomethod to study the kinetics of trans-‐ formation of the amorphous precursor phase. nite and finally end up with stable calcite (Figure 6) [4]. The role of the proteins We therefore use Fourier Transform InfraRed spectroscopy (FTIR) as a in amorphous useful precursor is cproposed to bevia crucial because of their tool for phase examining alcite formation, a Perkin-‐Elmer Spotlight 100 ability to Attenuated Total Reflection Fourier Transform InfraRed (ATR-‐FTIR) keep the amorphous form stable until the crystal transformation is needed[4]. In adspectrometer. Figure 4 shows the spectrum for commercially available (Sigma Aldrich) calcite do powder as a reference. The sstable pectra for three types of natural dition, some biominerals accomplish to form transient phases such as the eggshell show a calcite-‐dominance with small residual “bumps” at the location aragonite crystals in nacre by possibly harnessing specialiazed proteins. A thorough indicating amorphous calcium carbonate (Figure 5). A time sequence of FTIR from this immediately after CaCl nd NaHCO (both Sigma Aisldrich) solution understandingscans of how mechanism is aused by 3organisms required to produce mixing to 30 minutes later demonstrates the development of the calcite peaks biomimetic inorganic materials. and the diminution, but not complete removal, of the amorphous signal (Figure 6).
The avian eggshell is epitome of organic-inorganic interplay. As is the case for almost all biological minerals, the organic content of the eggshell matrix is responsible for the regulation of mineral deposition. Although, there are more than 400 proteins
Figure 17: 4The Absorption at 1414 Figure . The FTIR FTIR spectrum spectrum of of ccommercial ommercial CaCO CaCO33 c. alcite powder peaks (Sigma 1 1 1 -‐1 -‐1 -‐1 cm Aldrich). , 872 cmAbsorption and 712 p cm eaks are at 1specific 414 cmpeaks , 872 for cmcalcite and 712 cm are specific to calcite. 4.3 Fourier Transform InfraRed Spectroscopy Anal-
ysis of Minerals Biological mineralisation was once thought to follow the classical nucleation theory. As mentioned in the previous chapters, however, natural organisms use a different pathway to produce inorganic materials. It is now widely accepted that, the mineral formation in nature proceeds through an amorphous precursor phase which is then followed by meta-stable phases. The end product of biomineralisation is thermodynamically most stable crystal form, such as, calcite. In this study, FTIR is used to monitor amorphous to crystal transition in real-time. The possibility to observe phase transformation allow to study the effect of different proteins during mineral formation. Eggshell mineralisation specific protein OC-17, for instance, has been proposed to trigger calcium carbonate clusters to transform into calcite at the very beginning of mineralisation. FTIR experiments showed that without any organic content the mineralisation of CaCO proceeds through an amorphous then transforms to crystalline 318: Figure The FTIR spectra of ostrich, phase quail which and hen eggshells. Rectangle shows Figure 5. The FTIR spectra of ostrich, 1quail and hen eggshells. The rectangle amorphous absorption peaks at 1086 cm calcite. Figure 17, shows the FTIR spectrum of commercial 3 . cm-‐1. Small shows amorphous calcium carbonate absorption peaks aCaCO t 1086 Although it is cknown avian eggshells organic peaks an be othat bserved at 1 648 cm-‐1are . composed of crystalline calcite, complex in whichcalcium eggshellcarbonate. are produced. oviduct ofa aresult female they alsoenvironment contain amorphous ThisThe is probably of bird the contains thousands of different chemicals and eggshell is produced through many
different interactions between proteins and 40 inorganic molecules. This environment possess a non-equilibrium condition in which crystal transition is hardly possible. On the other hand, the function of proteins is to ensure that the calcium carbonate clusters are transformed into calcite. As can be seen in Figure 18, eggshell still contain amorphous material.
Figure 20: 6The spectra of oCaCO forfor 303min upon mixing CaCl Figure . The spectra f CaCO mineralization 0 min upon mixing CaCl and and 3 3mineralisation NaHCO show Sthe amorphous to acrystal transformation from the beginning NaHCO pectra show the morphous to crystal transformation from 3 solutions. 3 . Spectra (bottom) to the end of mineralisation (top) Rectangles 1-2 and 3-4 indicate the beginning (bottom) to the end of mineralization (top) in time sequence. the amorphous and 2calcite peaks, respectively. Rectangles (1086 specific cm-‐1) and 1-‐3-‐4 (1414 cm-‐1, 872 cm-‐1 and 712 cm-‐1) indicate the amorphous and calcite specific peaks, respectively. 4.4 Purification of Avian Eggshell Protein OC -17 Protein purification is a key step to study the function of these crucial molecules. Because biological minerals contain organic molecules, the interplay of Although, molecular dynamics studies in order to understand proteins with inorganic crystals is awere key pconducted rocess, which requires protein the functioning mechanism of OC-17, there is still experimental evidence to confirm purification and protein analysis, both of wno hich are ongoing. Moreover, the occlusion o f o rganic c ontent i nto c alcite c rystals i s a c rucial p art o f t his s tudy and molecular simulations. One of the aims of this study is to purify OC-17 from different can be observed using nalysis (TGA, erkin-‐Elmer) in order avian species’ eggshells andthermogravimetric study the effect of athis protein onPthe mineralisation of to understand the nature of protein preservation within calcite crystals. The CaCO addition, ofto pure OC-17 energetics 3 . In loss weight of the thermodynamic crystals before cproperties alcite starts melt gives tand he athe pproximate of calcite-OC-17 will ibe studied tolattice. shed some on the percentage of interactions organic matter n the crystal This light is shown for thermodynatural chicken eggshell in Figure 7, sand howing a fnature ew percent weight loss at proteins. a mid-‐range namics of eggshell mineralisation on the of intra-crystalline For temperature f ar l ower t han t he d issociation t emperature o f c alcite i tself ( around these reasons, OC-17 was purified from powdered chicken eggshell by dissolving the 800° C). This is consistent with the couple of percent of organic material known calcified of shells in HCl solution. Next, insoluble discarded and8) to be layer present in natural eggshell. The same general fraction result is was observed (Figure remaining proteins separated using on the sizeaof proteins. when w e make were calcite in solution in ultrafiltration the presence obased f bovine serum lbumin commercially awere vailable (Sigma using Aldrich) model protein we are using as a Two(BSA), sets ofaultrafiltrations conducted filtration tubes with different pore the eggshell proteins we are currently working to The purify. Calcite sizes.stand-‐in Finally,for the purified fraction was analysed by SDS-PAGE. image of the made using the same solutions and process but no protein present does not have electrophoresis gel is w shown Figure 21. any appreciable eight in loss across the same temperature range (Figure 9), In Figure 21,the theidea bands atthis the w left of the image show the protein marker that is a supporting that eight loss is uniquely associated with the protein or other organic proteins material included the calcite in either natural shell synthesis mixture of different with knownin molecular weights. The sample’s molecular (Figure 7) or biomimetic calcite formation (Figure 8). 43
Figure 26: TGA result for the calcite crystals grown in the absence of any protein in the mineralisation solution
o FigureFigure 27: TGA result forfor the chicken eggshell crystals. Weight lossC at 350 7. TGA result chicken eggshell. The weight loss at 350° can be C indicates the intra-crystalline protein degradation attributed to intra-‐crystalline protein degradation. 49
Figure . TGA result for calcite crystals grown in solution in the presence f Figure 825: TGA result for the calcite crystals grown in the presence of BSA.oWeight bovine serum o albumin protein (a well-‐characterized model protein available loss at 300 C indicates the intra-crystalline protein degradation commercially, Sigma Aldrich). The overall picture is similar to that for natural eggshell with integrated protein (Figure 7). conditions. Figure 4.6, shows the TGA analysis of pure calcite crystals that were synthesized using the same parameters with that of BSA-entrapped calcites. Comparison of TGA analysis of eggshell crystals and biomimetic synthesis of BSA entrapped calcite may give some hints to understand intra-crystalline content of biominerals. For this reason, chicken eggshell was ground into fine powder and analysed using TGA under the same experimental conditions. The result of the TGA analysis of eggshell powder is shown in Figure 4.6. Similar to the BSA-entrapped calcite crystals, the intra-crystalline organic content of chicken eggshell did exhibit a weight loss during heat treatment. The tem-
Figure 26: TGA result for the calcite crystals grown in the absence of any protein Figure 9. TGA result for calcite grown in solution in the absence of any added in the mineralisation solution protein. In contrast to both natural eggshell (Figure 7) or lab-‐synthesized calcite with BSA protein included (Figure 8) there is approximately zero weight loss at temperatures less than 600° C, above which dissociation of the calcite itself is expected. Conclusion and Outlook Despite attracting little attention, an important aspect of mineralization is its thermodynamic nature. As is the case for all such studies, the most reliable method for extracting thermodynamic information is to use calorimetry. This will be a critical component of the next phase of this project. The aim is to gain quantitative understanding of the mineralization pathway proposed in Figure 3, and to establish whether this pathway is universally true (i.e. does the material always “stop” at each metastable phase in the figure, or perhaps does some calcite “jump” from amorphous calcium carbonate directly to calcite?). We previously observed that in solution-‐based calcium carbonate synthesis, we tended to obtain vaterite when there was no organic material (protein) present and calcite when organic material was present [Armitage et al. 2012]. This result will be replicated with quantitative measurements using calorimetry. We will further continue with the 2nd and 3rd Aims as laid out in the original proposal, examining the influence of texture and organic molecules isolated from natural eggshell on the formation of calcite. o Figure 27: TGA result for the chicken eggshell crystals. Weight loss at 350 C indicates the intra-crystalline protein degradation
49
References Addadi L, Moradian J, Shay E, Maroudas NG, Weiner S, A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation: Relevance to biomineralization. Proc. Natl. Acad. Sci. USA 84 (1987) 2732-‐6. Armitage OE, Strange DGT, Oyen ML, Biomimetic Calcium Carbonate-‐Gelatin Composites as a Model System for Eggshell Mineralization. Journal of Materials Research 27 (2012) 3157 − 64. Elhadj S, Salter EA, Wierzbicki A, De Yoreo JJ, Han N, and Dove PM: Peptide controls on calcite mineralization: Polyaspartate chain length affects growth kinetics and acts as a stereochemical switch on morphology. Cryst. Growth Des. 6 (2006) 197–201. Fernandez MS, Passalacqua K, Arias JI, Arias JL, Partial biomimetic reconstitution of avian eggshell formation. J. Structural Biol. 148 (2004) 1-‐10. Gomez-‐Morales J, Hernandez-‐Hernandez A, Sazaki G, Garcia-‐Ruiz JM, Nucleation and polymorphism of calcium carbonate by a vapour diffusion sitting drop crystallisation technique. Cryst. Growth Des. 10 (2010) 963-‐9. Gower, L. B. Biomimetic Model Systems for Investigating the Amorphous Pre-‐ cursor Pathway and Its Role in Biomineralization. Chemical Reviews 108 (2008) 4551– 4627. Gower LB, Odom DJ, Deposition of calcium carbonate films by a polymer-‐induced liquid-‐precursor (PILP) process. J. Crystal Growth 210 (2000) 719-‐34. Lakshminarayanan R, Loh XJ, Gayathri S, Sindhu S, Banerjee Y, Kini RM, Valiyaveettil S. Formation of transient amorphous calcium carbonate precursor in quail eggshell mineralization: an in vitro study. Biomacromolecules. 7 (2006) 3202-‐9. Meldrum FC, Calcium carbonate in biomineralisation and biomimetic chemistry. Intl. Mater. Rev. 48 (2003) 187-‐224. Navrotsky, A. Energetic clues to pathways to biomineralization: Precursors, clusters, and nanoparticles. Proc. Natl. Acad Sci. USA 101 (2004) 12096–12101. Strange DGT and Oyen ML, Biomimetic Bone-‐Like Composites Fabricated Through an Automated Alternate Soaking Process, Acta Biomaterialia 7 (2011) 3586 − 94.