General principles of biomineralization: Biologically induced mineralization 20 nm Biologically induced magnetite nanoparticles Ca2+ + 2HCO3-  CaCO3 + CO2 + H2O Biologically induced mineralization involves the adventitious precipitation of inorganic minerals by reaction of extraneous ions with metabolic products extruded across or into the cell wall. The mineral products are closely associated with the cell wall and crystallochemically heterogeneous.  

General principles: Biologically controlled mineralization uniform particle sizes well-defined structures and compositions high levels of spatial organization complex morphologies controlled aggregation and texture preferential crystallographic orientation higher-order assembly hierarchical structures Biologically controlled mineralization involves the specialized regulation of mineral deposition and results in functional materials with species-specific crystallochemical properties.

General principles: Site-directed biomineralization Intercellular – in the spaces between closely packed cells Intracellular – bilayer lipid vesicles enclosed compartments within the cell H2O 0.5 mm calcified coral Extracellular – on or within an insoluble macromolecular framework secreted outside the cell eggshell formation

General principles: Site requirements Spatial delineation – physical boundary for size and shape control Chemical regulation – for increasing ionic concentrations (ionic pumping) Diffusion limited ion flow – for controlling solution composition Organic surface – nucleation General principles: Control mechanisms Physiochemical Spatial Structural Morphological Constructional Gene pool Bioenergetics Biochemical

Boundary-organized biomineralization The delineation of biological environments is of key importance in boundary-organized biomineralization because it provides sites of controlled chemistry that are spatially defined. Control functions: spatial delineation – size, shape and organization of the mineral phase diffusion limited ion flow – ionic activities, solution composition, supersaturation mineral passivation – surface stabilization against dissolution and transformation ion accumulation and transport – supplying chemicals to remote intra- and extracellular mineralization sites mineral nucleation – regulating interfacial energies mineral transportation – moving mineralized structures to new construction sites.

Boundary-organized biomineralization; coccolith calcification uniport Ca2+ Ca2+ Ca2+ 2HCO3 - 2HCO3 - symport HCO3 - Coccoliths CaCO3 CA OH - CO2 H+ CO32- H2O CO2 Photosynthesis + Ca2+ Sugars SO42- SO42- Polysaccharides Golgi vesicle Medium Cell Light-dependent uptake: some CO2 fixed by photosynthesis  influence of light on calcification Some calcification occurs in dark using stored metabolic energy.

Chemical control of biomineralization Solubility The solubility (S) of an inorganic salt depends on the balance between lattice energy (L) and ion hydration (H) ion pairing (IP) and complexation (C) in aqueous solution. GS = GL - (GH + GIP + GC) Lattice substitutions are important in controlling the solubility of biological apatite Undersaturated [Ca ] 2+ /M HAP FAP Plaque fluid Saliva Supersaturated pH 100 10 1 0.1 5 6 7 Fluoride and tooth decay

Chemical control of biomineralization: Solubility product MnXm(solid)  nM+(aq) + mX-(aq)   Ksp = {M+}n . {X-}m The solubility product is a critical factor in determining the thermodynamic limit for the onset of inorganic precipitation. When the solubility product is less than the activity product (AP) of a solution then precipitation will occur until Ksp = AP. Problems Complexation with biological ligands (citrate/oxo/hydroxy) Covalent polymers (silica) Crystal size heterogeneity - Ostwald ripening Kinetic effects organic sheaths etc

Chemical control of biomineralization: Supersaturation Supersaturation is a measure to what extent a solution is out of equilibrium and represents the thermodynamic driving force for inorganic precipitation. Relative supersaturation, Absolute supersaturation   SR = AP/Ksp SA = (AP – Ksp)/Ksp The difference in chemical potential between the supersaturated solution and a solution at equilibrium with the solid is related to SR by  = kT lnSR Supersaturation is highly regulated in biology through the process of boundary-organized biomineralization.

Supersaturation control in spatial boundaries M n+ (n+1)+ MX Matrix X - E1 E2 H2O MC A + B H Direct mechanisms to increase S ion pumping ( + redox) ion complexation/decomplexation enzymic regulation of anions carbonic anhydrase (HCO3-) alkaline phosphatase (HPO42-) Indirect mechanisms ionic strength - Na+ and Cl- transport water extrusion – silica deposition? . proton pumping – pH changes

Chemical control of biomineralization: Nucleation G N * I B r r* Homogeneous nucleation The free energy of formation of a spherical nucleus, GN, is given by the difference between the surface (interfacial, I) and bulk (B) energies,   GN = GI - GB GI = + 4r2 where  = interfacial free energy per unit surface area, and GB = - 4r3 Gv 3 Vm where Gv = per mole solid-liquid phase change, and Vm () is the molar (molecular) volume. r* = critical size GN* = activation energy GN* = 1632 r* = 2Vm 3(kTlnSR)2 Gv   Rate; JN = Aexp(-GN*/kT)

Nucleation control in biomineralization JN SR*  SR increase  GN* decrease  JN increase GN*  (lnSR)-2 Need to control over catastrophic nucleation rate in pure solution at SR* GN*  3 small reductions in  can have a marked effect on JN and r* External substrate (dust etc) reduces  and hence increases JN at given value of SR. Heterogeneous nucleation occurs at lower SR and under control if SR* is not breached. GN* and JN determined by  and SR and biologically controlled by organic matrices and the membrane regulation of concentration gradients. JN A B C SR* SR Heterogeneous Homogeneous Extraneous particles with equal (A), variable (B) nucleation efficiencies. (C) without extraneous particles).

Oriented nucleation (epitaxy)   % mismatch CaCO3//KBr 3 CaCO3//NaI 1 CaCO3 //KCl -2 CaF2//NaBr 8 CaF2//NaCl 3 CaF2//KBr 21 A B C (A) non-oriented, (B) mosaic with crystals aligned only perpendicular to the substrate, (C) iso-oriented array with 3-D crystallographic alignment. Structural control in biomineralization involves the preferential nucleation of a specific crystal face or axis on the surface of an organic matrix. Key concepts: molecular recognition, lattice matching, electrostatic, stereochemical and structural complementarity.

Chemical control of biomineralization: Crystal growth Bulk diffusion Surface adsorption + dehydration 2D diffusion 1D KINK Crystal growth and termination are dependent on the level of supersaturation and occur through surface-controlled processes (active sites). STEP Rate of growth, JG = k (SA)x   k = rate constant, SA = absolute supersaturation Active sites - kinks, steps A B C mass transport (diffusion-limited) very high values of SA (x = 0) polynucleation (growth islands) high SA (x > 2) layer-by-layer growth moderate SA (x = 1) screw dislocation growth low SA (x = 2) screw dislocation

Chemical control of biomineralization: Crystal growth inhibitors apatite crystallization pH pH calcite crystallization + coccolith polysaccharides 7.4 8.0 t= 0 t = 4 min Mg2+ 7.2 7.5 control control 7.0 F- 7.0 1 2 5 10 15 Time / h Time / min sea urchin 50 m intercalation texture Soluble additives bind at steps/kinks, inhibit growth, change composition, structure and form.

Chemical control of biomineralization: Crystal morphology Crystal morphology (habit) is determined by the relative rates of growth of different crystal faces, with the slow growing surfaces dominating the final form. 010 110 100 Σ s{hkl}. A{hkl} = minimum CaCO3 control HPO42- Li+

Chemical control of biomineralization: Habit modification [001] [110] CaCO3 + [malonate]2- CaCO3 + polyaspartate Molecular-specific interactions: electrostatic, stereochemical and structural matching modify the surface energy or mechanism of growth, or both.

Chemical control of biomineralization: Polymorphism JN S (amorph) S (crystal) Amorphous Crystal Ksp (crystal) (amorph) X Activity product periodic order Ostwald-Lussac law of stages Crystallization proceeds along a series of structures with decreasing solubility and increasing thermodynamic stability. The structure of the critical nucleus is an important factor in controlling the crystallization pathway. disordered/hydrated

Polymorphism and phase transformations B G Solution (M+(aq) + X-(aq)) Amorphous GN(B) Gg(B) + GT1 GN(A) Gg(A) GT2 GT3 Final mineral (crystalline) CALCIUM CARBONATE CALCIUM PHOSPHATE Amorphous CaCO3 Amorphous CaP Accelerators and inhibitors Vaterite Brushite Aragonite Octacalcium phosphate Calcite Hydroxyapatite amorphous silica - biologically stable The chemical control of crystallization pathways that involve a sequence of kinetic inhibition and phase transformation can result in a high degree of selectivity in crystal structure and composition.

Phase transformations - examples CDL OE OCP HAP Calcium phosphate slow transformations of amorphous phase octacalcium phosphate to hydroxyapatite in situ solid state hydrolytic transformation x1 OCP (d100 = 1.868 nm)  x2 HAP (2d100 = 1.632 nm Calcium carbonate fast transformations of amorphous phase unless stabilized by organic sheaths high Mg2+ level in ACC  high Mg calcites (30 mol%) eg. sea urchin spicules Traces of OCP precursor are left as a central dark line in enamel HAP crystals. 1m Larval sea urchin spicule - early growth stage

Phase transformations - iron oxides Solid state transformation pH 7, 80 C Dissolution Reductive dissolution Rapid Slow -low Fe -low O2 -[H] agents -complexation -Fe2O3 FERRITIN Fe2O3·nH2O FeIII(aq) + FeII(aq) Magnetite Amorphous Ferrihydrite Goethite ferrihydrite  magnetite (bacteria, chitons) A1 A2 1.00 C Intensity B 0.95 0.90 0.85 57Fe Mössbauer -10.0 -5.0 0.0 5.0 10.0 Velocity (mm/sec) FeIII O OH + FeII OH+(aq) FeII + H+ + H2O Fe3O4 + H+ + H2O [FeIIIFeII(O)x(OH)y]n+