it is of great importance in both production of hard 
 tissues and in mediating the interaction of biota with various terrestrial , 
 aquatic , and marine environments . moreover , biomineralization offers 
 potential approaches to the formation of complex synthetic materials . otoconia are small biominerals found in the saccule and utricle of 
 the ear . as integral components of sense organs of linear acceleration 
 and gravity , otoconia 
 consist of an organic matrix , surrounded by a mineral shell composed 
 of crystalline calcium carbonate . the 
 crystalline phase is one of three polymorphs : calcite ( mammals and 
 birds ) , aragonite ( amphibians and fish ) , and vaterite ( primitive jawfish ) . it has been suggested by pote and ross that the 
 selection of polymorphs is directed by specific major matrix proteins 
  unique to that polymorph  , though proof of that hypothesis is currently lacking . otoconia in 
 mammals are unique in that they are the only functional mammalian 
 biomineral that consists of calcite instead of hydroxyapatite ( calcium 
 phosphate ) , which is the mineral phase of bone and teeth . in humans , calcitic otoconia are about 510 m in length and exhibit 
 triplanar ( 104 ) end facets . the otoconia are formed during embryogenesis 
 with maturation occurring during a brief perinatal period after which 
 they are believed to be static until demineralization begins with 
 aging . decalcification occurs with increasing 
 age and degrades the mineral resulting in loss of balance . benign 
 positional vertigo ( bpv ) often takes place as a result of displacement 
 of otoconia into the semicircular canal due to head trauma or age - related 
 otoconial fragmentation . in spite of the important role otoconia play 
 in human balance and movement , little is known about their development , 
 maintenance , and pathological processes at a molecular level . previous studies suggest that the organic 
 matrix of otoconia serves 
 as a framework on which the calcite crystals deposit and grow . in mammals and birds , the insoluble scaffold 
 protein otolin-1 was identified to be the core protein of the matrix . otolin-1 , belonging to the c1q / tnf and 
 collagen superfamilies , is similar to the network forming type x collagen . on the basis of the established role that collagens 
 play in biomineralization and the recent demonstration that otolin-1 
 forms self - assembled networks in vitro , otolin-1 is expected to self - assemble in vivo into an extracellular 
 scaffold structure essential for the directed nucleation and growth 
 of the calcite crystal . otolin-1 appears to be recruited 
 into the otoconial matrix by otoconin 90 ( oc90 ) , which is the principal otoconial protein and accounts for 
 more than 90% of the soluble phase . oc90 
 is essential for the formation of this matrix and the deposition of 
 otolin-1 . otolin-1 has been reported 
 to interact with oc90 when they are coexpressed . in the absence of oc90 , the organic matrix was reported 
 to be nearly absent , and the efficiency of crystal formation was reduced 
 by 50% . in addition , oc90 was found 
 to facilitate nucleation and control crystal size and morphology . in vitro coadministration of oc90 with otolin-1 to crystal growth 
 solution produced synergistic effects on crystal morphology that resulted 
 in calcite crystals resembling otoconia . many other proteins in the otoconia matrix have also been 
 identified , 
 including fetuin a , osteopontin ( opn ) , laminin alpha 3 , sparc - like 
 protein 1 , and myosin regulatory light polypeptide 9 , all of which 
 are calcium - binding proteins . fetuin a is abundant 
 in serum and was first identified in 
 1944 as a major protein component of bovine fetal serum . it is a known inhibitor in the formation of 
 calcium compounds , such as hydroxyapatite , due to its strong ca - binding property and accounts for about 50% 
 of the capacity of serum to inhibit precipitation of ca - salts , like 
 calcium phosphate . opn also serves as 
 an inhibitor of other ca minerals including calcium oxalate , calcite , and hydroxyapatite . rounded and poorly formed facets of both the 
 ( 010 ) and ( 101 ) faces of calcium oxalate and ( 104 ) faces of calcite 
 indicate that opn interacts with steps on the crystal faces . phosphorylation of opn was determined to be an important factor 
 in the inhibitory effects . however , zhao et al . argued that opn was not required for otoconia 
 formation since no change of calcium concentration was observed in 
 the ultrastructure in the absence of opn . we report results 
 from atomic force microscopy ( afm ) , scanning 
 electronic microscopy ( sem ) , optical microscopy , and electrophoretic 
 light scattering ( els ) investigations into the role of three key otoconial 
 proteins fetuin a , opn , and oc90in the formation of 
 calcite . in situ afm studies of the effects of these proteins on the 
 growth of atomic steps on calcite surfaces revealed interactions with 
 the steps and the resulting control on growth kinetics and morphology . sem images illustrated the relationship of changes in step kinetics 
 to alterations in growth morphology . optical microscopy enabled quantification 
 of the kinetics of calcite nucleation on films of these proteins formed 
 through deposition on mica surfaces . interfacial energies controlling 
 the thermodynamic barriers to nucleation were determined from measurements 
 of calcite nucleation rate as a function of supersaturation . these 
 interfacial energies were correlated with the charge density of the 
 individual proteins as determined by zeta potential measurements obtained 
 from els analysis . commercial sources were used for fetuin 
 a ( alpha 2 hs - glycoprotein from american research product , inc . ) and 
 opn ( recombinant mouse osteopontin from r&d systems , inc . ) . the recombinant opn was also phosphorylated , but details about the 
 extent of phosphorylation were not available . recombinant histidine 
 tagged mouse oc90 protein was produced utilizing the freestyle max 
 293 expression system ( invitrogen ) to transfect nonadherent human 
 embryonic kidney ( hek293-f ) cells with the expression plasmid pcdna3.1-oc90-his 
 per the manufacturer s directions . the transfected cells were 
 incubated at 37 c and 8% co2 for 7 days in freestyle 
 293 medium ( invitrogen ) . the medium containing the secreted protein 
 was extracted from the cell culture , and the protein was purified 
 chromatographically by affinity on an akta purifier with a 1 ml histrap 
 column ( ge healthcare ) . total protein concentration was determined 
 by the bradford assay ( biorad ) . purity and identity of the proteins were determined by coomassie staining and western blot using a 1:1000 
 dilution of polyclonal mouse anti - his c - terminal antibody ( abcam ) 
 following sds - polyacrylamide gel electrophoresis on an anykd ( biorad ) 
 gel . in situ measurements of the effects 
 of all proteins on calcite monolayer step growth were carried out 
 by afm ( digital instruments j scanner , nanoscope iiia and v controllers , 
 bruker corporation , billerica , ma ) . all measurements took place at 
 1 atm and room temperature ( 25 c ) . freshly cleaved calcite chips 
 of optical - quality iceland spar ( ward s scientific , chihuahua , 
 mexico ) of approximately 2 mm  2 mm  1 mm in dimension 
 were mounted in the fluid cell . supersaturated calcite solutions were 
 made by dissolving reagent sodium bicarbonate ( nahco3 , 
 aldrich ) and dehydrate calcium chloride ( cacl2 , aldrich ) 
 into deionized water ( 18.2 m ) . the supersaturation  
 was defined asand calculated by the commercial 
 software 
 phreeqc to be 0.9 and 2.3 . ph was adjusted 
 by adding hcl to the solutions to maintain it at 7.44 , which 
 is close to the human physiological value . the ionic strength of all 
 solutions was fixed at 0.2 m. reactant solutions were continuously 
 pumped through the fluid cell by a syringe pump at a flow rate of 
 60 ml / h , and all images were collected with silicon tips on silicon 
 nitride cantilevers ( appnano inc . ) . the crystal surfaces were allowed 
 to grow for an hour before taking images to ensure quality and stability . step velocities were calculated by measuring the change in apparent 
 step orientations between continuous upward and downward afm images 
 collected in contact mode . for each solution composition , the growth experiments were repeated at least three times , 
 and step speed data were typically collected on two to three growth 
 spirals in each experiment . the data points presented here represent 
 an average of the measured values for each growth spiral . in addition , 
 calcite crystals were grown by the carbonate diffusion method through 
 the slow evaporation method of nh4hco3 into 
 a cacl2 solution as reported . briefly , they were grown on clean 12 mm glass coverslips , placed 
 into wells of a cell culture dish containing 7.5 mm cacl2 solutions to which aliquots of the proteins had been added . the 
 culture dish was covered with aluminum foil containing a few pinholes 
 and placed into a sealed glass chamber containing nh4hco3 . crystals were grown for 48 h and then examined with a jeol 
 jsm 6320f field emission scanning electron microscope at 5 kv after 
 sputter coating with gold and palladium to increase the conductivity . freshly cleaved mica surfaces 
 were incubated in solution for 8 h. after incubation , they were removed 
 from solution , rinsed , and dried with compressed air . afm tapping 
 mode was used to examine the morphology of proteins adsorbed on the 
 mica surfaces . nucleation of 
 calcite on all three 
 proteins films prepared as described above were investigated in situ 
 using an inverted optical microscopy . cacl2 and nahco3 solutions at equal concentrations 
 from 20 mm to 45 mm were mixed and pumped through the reaction cell 
 at a flow rate of 120 ml / h . the location of nucleation events was 
 recorded as the number density of nuclei vs time . 50 nm of each protein 
 was dissolved in a 1 mm cacl2 solution at ph 6.8 . zeta 
 potential measurements of each protein were done by sz-100 nanoparticle 
 analyzer ( horiba , japan ) at 1 atm and room temperature ( 25 c ) . growth hillocks on calcite ( 104 ) faces 
 exhibit two crystallographically distinct types of steps , commonly 
 referred to as  obtuse  and  acute  . the 
 difference in the kinetics of growth for the two types of steps is 
 evident from the difference in the interstep spacing , which is a direct 
 measure of the relative step speeds . previous work at approximately ph 8.4 showed that calcite growth hillocks consisted of straight steps 
 with sharp corners , as seen in figure 1a . the 
 results presented here ( figure 1a , b ) demonstrate 
 that both types of steps become rounded when ph is decreased from 
 8.4 to 7.4 , independent of the addition of proteins . the addition of protein at sufficiently 
 high concentration can lead to further rounding , step pinning , or 
 obscuring of the steps , presumably due to coverage of the surface 
 by a protein film . afm images of calcite growth spirals in ( a , b ) protein - free 
 solutions 
 at  = 2.3 for ( a ) ph = 8.4 and ( b ) ph = 7.4 ; ( c ) 50 nm fetuin 
 at ph = 7.4 , ( d ) 80 nm opn at ph = 7.4 and ( e ) 40 nm oc90 at ph = 
 7.4 . all images were captured with obtuse steps up and acute sites 
 at the bottom , as marked in ( a ) . panels ( a ) and ( b ) demonstrate the 
 effect of ph on hillock morphology . steps are partially covered or 
 pinned by fetuin a , opn , or oc90 in panels ( c  e ) respectively 
 and the step velocity drops to about 50% of the original value . image 
 sizes are ( a ) and ( b ) 3  3 m ; ( c ) to ( e ) 4  4 m . ( note that the differences in step spacing seen in ( c  e ) reflect 
 the differences in step speed obtained with each protein . ) from sequences of images like those in figure 1 , we measured the dependence of step speed on protein 
 concentration 
 over the range required to see significant inhibition for all of the 
 proteins ( figure 2 ) . at the lowest protein 
 concentrations used here , steps could be easily resolved and their 
 speeds measured . as the protein concentration increased and steps 
 became difficult to resolve ( figure 1c  e ) , 
 step speeds could no longer be measured . afm measurements of step 
 propagation rate vs concentration along 
 both obtuse and acute directions for solutions containing ( a ) fetuin , 
 ( b ) opn , and ( c ) oc90 . normalized step speeds are given to emphasize 
 the relative enhancement and/or inhibition and minimize the effects 
 of inherent run - to - run variations in the step speed values . solid lines :  = 0.9 ; dashed lines in 
 ( a ) :  = 2.3 . error bars represent 10% of the measured value 
 and are based upon error analyses in our previous investigations . decreases in the step speed were 
 seen for both step - types as oc90 
 and opn concentrations were increased . at an oc90 concentration of 
 only 40 nm , step speeds dropped to < 50% of the values in protein - free 
 solution ( figure 2c ) , showing that oc90 is 
 a potent inhibitor of calcite growth . the slightly stronger inhibition 
 of the acute step over that of the obtuse steps is consistent with 
 the change in crystal morphology seen in sem images of crystals grown 
 in the presence of the protein ( figure 3a , b ) 
 and is also consistent with the results of lu et al . and moreland et al . ( a ) 0.5 m oc90 ; ( b ) 1 m oc90 ; ( c ) 1.59 m recombinant 
 mouse opn ; ( d ) 3.82 m recombinant mouse opn ; ( e ) 1.02 m 
 human fetuin a ; ( f ) 2.04 m human fetuin a. scale bar : a  c 
 20 m ; d  f 10 m . opn had less impact on step speeds ( figure 2b ) and crystal morphology ( figure 3c , d ) than 
 oc90 but still acted as an inhibitor of growth . opn in concentrations 
 from 1.59 to 3.82 m caused a marked modification of crystal 
 shape with increasing concentration ( figure 3c , d ) . modification became evident at 1.59 m with the ( 104 ) 
 face becoming rounded and exhibiting a narrow ring - like band along 
 the c axis ( figure 3c ) . these 
 modifications significantly increased at 3.82 m with strong 
 rounding of the ( 104 ) faces becoming apparent . in contrast to 
 oc90 and opn , fetuin a exhibited either enhancement 
 or inhibition depending on protein concentration and solution supersaturation . 
 at sufficiently high supersaturations ( 2.3 ) and low fetuin a concentration 
 ( < 40 nm ) , an obvious increase in step speed was observed , while 
 the protein exerted an inhibitory effect at concentrations greater 
 than 40 nm ( figure 2a ) . as the protein concentration as shown in figure 3d , e , the modification of 
 crystal shape seen with increasing fetuin a concentration was similar 
 to that observed with the other two proteins . at a concentration of 
 1.02 m , a series of rounded macrosteps ( figure 3e ) were observed on one side of the crystal while at concentrations 
 of 2.04 m ( figure 3f ) , significant rounding 
 of acute steps and a broad ring - like band comprised of the ( hk0 ) faces 
 ( i.e. , parallel to the c - axis ) were obtained , though 
 the effects on morphology were less pronounced than with either opn 
 or oc90 . figure 4 shows 
 the morphology of adsorbed fetuin , opn , and oc90 on mica . after the 
 same incubation time in the same electrolyte solution , all three formed 
 isolated irregular islands on the mica surface . in addition , comparison 
 with bare mica ( figure s1 , supporting information ) indicates that the surface was covered with protein , though the 
 exact thickness is unknown . this is consistent with our expectation 
 that these are soluble proteins and are not likely to form cross - linked 
 filaments or three - dimensional networks as we observed previously 
 for the otoconial protein otolin-1 . ex situ 
 afm height images ( 1.0  1.0 m ) following absorption 
 of ( a ) 50 nm fetuin a ( b ) 50 nm opn , and ( c ) 50 nm oc90 on freshly 
 cleaved mica . image sizes are ( a ) to ( c ) 1  1 m . using 
 an in situ optical assay described 
 previously , we measured the rate of calcite 
 nucleation on adsorbed films of all of the proteins over a range of 
 supersaturations . figure 5a shows a typical 
 dependence of nucleation rate j0 on time t for a given  ( see also figures 
 s2 and s3 , supporting information ) . taking the linear portion 
 of the curve as the quasi - steady state nucleation rate , we derived 
 the dependence of j0 on supersaturation . the results are plotted in figure 5b as ln(j0 ) vs the inverse supersaturation squared . in 
 all cases , the rate increases with increasing  and exhibits 
 a roughly linear dependence of ln(j0 ) 
 on 1/ . ( a ) 
 an example of number of calcite nuclei vs time on a film of oc90 at 
  = 4.90 . the slope of the linear portion of the curve for each 
 supersaturation corresponds to the steady - state nucleation rate , j0 . ( b ) natural logarithm of the steady - state 
 nucleation rate vs the inverse of the supersaturation squared exhibiting 
 the linear relationship predicted by eq 3 for 
 all substrates . the slope of the line b is proportional 
 to the substrate - specific free energy barrier . ( c ) the zeta potential exhibiting 
 an inverse linear relationship between protein surface charge density 
 and interfacial energy . we found that all three 
 proteins had negative values of surface charge , which in the order 
 of maximum to minimum were ( absolute charge value ) : fetuin a ( 30.1 
  1.8 mv ) > oc90 ( 17.1  2.1 mv ) > opn ( 0.53 
  0.23 mv ) . the propagation of 
 steps on single crystal surfaces of calcite and other biomineral phases 
 and the impact of peptides and proteins on that process have been 
 explored in great detail over the past 15 years . the salient point of those studies with respect to the 
 current work is that soluble peptides and proteins often produce differential 
 inhibition of calcite steps due to step - specific binding affinity 
 that results in shape modification similar to that seen in figure 3 . the acute steps are often more susceptible to 
 inhibition and induce an elongation of the crystals along the crystallographic 
 ( 001 ) direction with a rounding of the ( hk0 ) faces . 
 in the case of aspartic acid - rich peptides from abalone nacre protein , 
 fu et al . related the effect on the morphology of the calcite atomic 
 steps to the overall changes in crystal morphology . given the fact that fetuin a , opn , and oc90 have strong affinity 
 to calcium , the results presented here indicate that each protein 
 adsorbs to calcite steps and blocks calcium or carbonate ions from 
 attaching to the kink sites . step speed is thereby 
 reduced at low protein concentrations , as seen in figure 2 . the morphologies seen in figure 3 are indicative of rounding of the acute steps , which is usually 
 associated with preferential binding to those steps . though preferential 
 inhibition is not discernible in the speeds of the steps at the much 
 lower concentrations where the data must be collected , the preferential 
 rounding is apparent in the hillock morphologies of figure 1 . observation of overall inhibition and step rounding 
 provides a rationale for the evolution in crystal shape and may provide 
 insights to the source of the characteristic shape of otoconia . the growth - enhancing regime observed with fetuin a ( figure 2a ) has been reported in a number of protein  mineral 
 systems and was explained 
 as a catalytic process in which the activation barrier for solute 
 addition to the crystal is reduced in the presence of the proteins 
 at low concentration . in the growth - inhibiting 
 regime , the critical concentration of fetuin a required to fully stop 
 step propagation increases with solution supersaturation . this is 
 due to the inverse relationship between calcite critical length and 
 supersaturation , so less protein is needed to pin the edges and stop 
 the steps at low supersaturation . classical nucleation theory 
 ( cnt ) quantifies the steady state rate of nucleation j0(m s ) through1where a is a kinetic 
 prefactor , kb is boltzmann constant , and t is temperature . g * is the free 
 energy barrier 
 to the formation of a critical size nucleus given by2here f is the shape - dependent 
 factor for crystal nuclei ,  is the molecular volume of the 
 crystal , which equals 6.13  10 m per molecule for calcite , and  
 is the net interfacial energy . combining eqs 1 and 2 gives3 in which the slope , b , is 
 given by4 thus , for any value of  , g * can be obtained directly from b through5the shape factor f depends 
 on the orientation of the nucleus . however , it does not change significantly 
 from one to another . for all protein 
 films , we observed calcite nucleation with random orientations . using a shape factor of 17 , which is the average for calcite rhombs 
 nucleating on the ( 012 ) and ( 104 ) faces , we estimate from the slopes 
 in figure 5b that the values of  range 
 from a low of 77  0.5 mj / m for opn to a high of 
 117  1.4 mj / m for fetuin a , with the oc90 film falling 
 at an intermediate values of 96  1.1 mj / m ( table 1 ) . the values for heterogeneous nucleation on opn 
 and oc90 are substantially less than that for homogeneous nucleation 
 in the bulk solution where  = 109 mj / m . this reduction arises from the interaction between the nucleating 
 crystal and the substrate as well as the interaction between the solution 
 and substrate . for heterogeneous nucleation , 
 the net interfacial energy is determined by three independent interfacial 
 energies for the crystal  liquid ( cl ) , substrate these are related by the expression:6where h is a constant that 
 depends on nucleus shape ( e.g. , h = 0.525 for a calcite 
 rhomb nucleating on the ( 012 ) face and 
 0.276 for the ( 104 ) face ) . cl can be treated as 
 constant in our cases so the differences in interfacial energy are 
 controlled by the relative values of sc and ls . the 
 value of sc is determined by the strength 
 of crystal binding to the substrate , while 
 ls is largely controlled by surface charge density , 
 surface hydrophilicity , or hydrophobicity . from eq 6 we infer that ( sc  ls)fetuin > ( sc ls)oc90 > ( sc  ls)opn . in the case 
 of polysaccharides 
 reported by giuffre et al . , higher negative 
 charge density resulted in lower ls and thus larger 
  and higher  nucleation barrier . hamm et al . found that larger values of the binding energy 
 between the substrate and the crystal resulted in lower values of 
 sc and thus smaller . the zeta potential 
 measurements indicate that the proteins exhibit a similar control 
 to that seen for the polysaccharides . as figure 5c shows , the zeta potential exhibits an inverse linear relationship 
 between the surface charge density and the interfacial energy , with 
 the large negative zeta potential of fetuin a leading to the lowest 
 value of  and the small zeta potential of opn giving the largest 
 value of . ( it is worth noting that analysis of net charge 
 at ph 7.44 based on sequence alone gives a relative ranking that is 
 exactly reversed from that determined by zeta potential , showing that 
 structural factors impact the relative number of charged residues 
 exposed at the surfaces of the proteins and highlighting the utility 
 of the zeta potential data . ) the magnitude of the changes in 
 the free energy barrier is even 
 more significant than the changes in  , because the barrier 
 contains  ( eq 1 ) . moreover , 
 because the barrier enters into the rate j0 as the argument of an exponential , the increase in nucleation rates 
 due the observed differences in  can be dramatic . for example , 
 at a supersaturation equal to 5.37 , taking all other terms in eqs 1 and 2 to be equal , a drop 
 in  from 109 to 77 mj / m would result in an increase 
 in nucleation rates by a factor of 3.75  10 , showing 
 that opn and oc90 may serve as potent promoters of nucleation . the results reported here demonstrate that fetuin a , opn , and oc90 
 are inhibitors of calcite growth through step pinning , presumably 
 due to their high calcium binding affinity . the dependence of nucleation 
 rate on supersaturation shows that the interfacial free energy of 
 calcite on fetuin a ( 117 mj / m ) is similar to that for 
 calcite in bulk solution , while opn and oc90 reduce it to 72.46 and 
 91.21 mj / m , respectively . zeta potential measurements 
 reveal that fetuin a carries more negative charge than oc90 and opn . given the previously demonstrated relationship between the surface 
 charge density and solution  substrate interfacial energy , these 
 results indicate that the variation in interfacial energy observed 
 here is due to the differences in protein surface charge density , 
 which impacts the affinity of the substrates for the aqueous solution 
 ( i.e. , the hydrophilicity ) . the consequence is that while fetuin a 
 presents a higher free energy barrier to nucleation than the bulk 
 solution , opn and oc90 present much lower free energy barriers .
we 
 investigated the roles of three proteins associated with the 
 formation of otoconia including fetuin a , osteopontin ( opn ) , and otoconin 
 90 ( oc90 ) . 
 in situ atomic force microscopy ( afm ) studies of the effects 
 of these proteins on the growth of atomic steps on calcite surfaces 
 were performed to obtain insight into their effects on the growth 
 kinetics . 
 we also used scanning electron microscopy to examine the 
 effects of these proteins on crystal morphology . 
 all three proteins 
 were found to be potent inhibitors of calcite growth , although fetuin 
 a promoted growth at concentrations below about 40 nm and only became 
 an inhibitor at higher concentrations . 
 we then used in situ optical 
 microscopy to observe calcite nucleation on films of these proteins 
 adsorbed onto mica surfaces . by measuring the calcite nucleation rate 
 as a function of supersaturation , the value of the interfacial energy 
 that controls the free energy barrier to heterogeneous nucleation 
 was determined for each protein . 
 opn and oc90 films led to significantly 
 reduced interfacial energies as compared to the value for homogeneous 
 calcite nucleation in bulk solution . 
 the value for fetuin a was equal 
 to that for bulk solution within experimental error . 
 zeta potential 
 measurements showed all of the proteins possessed negative surface 
 charge and varied in magnitude according to sequence fetuin a > 
 oc90 
 > opn . 
 in addition , the interfacial energies exhibited an inverse 
 scaling with the zeta potential . 
 in analogy to previous measurements 
 on polysaccharide films , this scaling indicates the differences between 
 the proteins arise from the effect of protein surface charge on the 
 solution  substrate interfacial energy .