Preliminary Research Report, January 30, 2015

Engineering  Tough  Materials:  Biomimetic  Eggshell   Preliminary  Research  Report,  January  30,  2015   Dr.  Michelle  L.  Oyen  (with  PhD  stude...
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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

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  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

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