Organic matrix-mediated biomineralization Functions: mechanical design – strength and toughness mineral passivation– stabilization from dissolution/phase transformation mineral nucleation– location and organization of nucleation sites – structure and crystallographic orientation boundary organization– partitioning with semi-permeable frameworks The organic matrix is a preformed insoluble macromolecular framework that is a key mediator of controlled biomineralization.

Organic matrices as mechanical frameworks Strain (  l/l) Stress/MPa Antler Femur Nacre Bone strength normalno matrix tension130 MPa 6 MPa compression150 MPa 40 MPa Young’s modulus 17 GPa 16 GPa (stress/strain = stiffness) Organic frameworks play an important role in the mechanical design of biomineralized tissues such as bones, shells and teeth. Many of the general functions of these biominerals – movement, protection, cutting and grinding – are dependent on mechanical properties, such as strength and toughness, which are specifically associated with inorganic-organic composites.

Macromolecules and the organic matrix - a general model Two-component model Nucleating Functional surface acidic macromolecules Hydrophobic Structural framework cross-linked macromolecules CaCO 3 Ca phosphate soluble/insoluble macromolecules silica HCl/EDTA HNO 3 /HF AspGlu  -Glu SerPSer Acidic macromolecules

SYSTEMFRAMEWORK ACIDIC Bone and dentineCollagenGlycoproteins (osteopontin, osteonectin) Proteoglycans (chondroitin sulfate) Gla-containing proteins Osteocalcin Tooth enamelAmelogeninGlycoproteins (enamelins) Mollusc nacre  -chitinGlycoproteins (nacrein, N66) Silk-like proteins (MSI 60) N16/N14 Lustrin A Crab cuticle  -chitinGlycoproteins Diatom shellsFrustulinsGlycoproteins (HEP200, silaffins) Silica SpongesSilicatein??? Plant silicaCelluloseProteins/carbohydrates Macromolecules and organic matrix-mediated biomineralization

Matrix macromolecules in bone collagen (90 wt%) + non-collagenous proteins and proteoglycans synthesis of helical polypeptide chains  enzymatic modification of amino acids (proline and lysine hydroxylation)  self-assembly of triple-stranded helix filaments  secretion into the extracellular space enzymatic removal of short peptides from filament ends  self-assembly of collagen fibrils  formation of cross-links  mature collagen fibrils  biomineralization Biosynthesis of collagen osteoblast extracellular space

Collagen – type I 1000 amino acids  30 % glycine (Gly) + 20% proline (Pro) + hydroxyproline (Hyp) [Gly-X-Y] 338 triplets often as [Gly-Pro-Hyp] Pro Gly ABC steric constraints  helical backbone small Gly  triple superhelix tropocollagen coiled-coil 3.3 residues /turn 280 nm 1.5 nm Tropocollagen interchain interactions: steric, H-bonding (NH-OC, OH) covalent crosslinks involving lysines

Tropocollagen – assembly of collagen fibrils N CC C C N N N Revised quarter-stagger model: five overlapping zones Mismatch due to crosslinks near C and N ends. Hole zones: 40 x 5 nm grooves [001] [110] - Top face Collagen Groove Direction Side face End face Collagen Fibril Axis HAP crystals aligned in hole zone/grooves

Non-collagenous proteins in bone MACROMOLECULESMOLECULAR COMPOSITION MASS (x 10 3 ) Acidic glycoproteins Osteonectin 44 (bovine)Asp/Glu Sialoprotein II 200Asp[Glu] 9 Phosphoprotein 40Asp/Glu/PSer Phosphophoryns (dentine) 100 (human)[Ser-Asp] n [PSer] 8 Proteoglycans (cartilage) Bone proteoglycans 350 chondroitin sulfate Cartilage proteoglycans1,000 chondroitin/keratin sulfate Gla proteins Osteocalcin 6  -carboxyGlu (x3) Matrix Gla protein 15  -carboxyGlu (x5)  -Glu Chondroitin 6-sulfateKeratan sulfate

Tooth enamel proteins Ameloblast Ca 2+ HPO nm nanosphere Amelogenin monomer c axis Hydroxyapatite crystal + enamelin sheath amelogenins 180 amino acids (hydrophobic, Pro, Leu..) 25k monomer  20 nm nanospheres (gel) spatial control of c axis growth Only 5 % organic macromolecules enamelins 60k highly acidic (Asp, Glu) sheath around HAP crystals

Matrix macromolecules from shell nacre Aragonite Acidic macromolecules Silk-fibroin-like hydrophobic proteins  -chitin a b c  - chitin; R = -NHCOMe Antiparallel  -pleated sheet MSI 60, N16; Ala, Gly-rich -CO, -NH hydrogen bonds Laminated hybrid structure nacrein [Asp-Glu-PSer]

Macromolecules from silica biomineralization Diatoms Frustulins HEP200 Silaffins (HF-extractable) high M r (75k) glycoproteins [Cys-Glu-Gly-Asp-Cys-Asp] + [Gly] n 25% Ser/Thrlow M r (4 to 17k) + 20% Asp/Glu polylysine repeats + oligo-N-methylpropylamino Sponge spiculesSilicatein x3 subunits; 20% Ser/Thr catalytic (hydrolytic) properties in vitro

Organic matrix-mediated nucleation The activation energy for nucleation is lowered by specific interfacial interactions between functional groups on the organic matrix and ions in supersaturated solution. no organic surface organic matrix G G * N(2) G * N(1) r r * (1) r * (2) – control nucleation rate and number of sites – organization of nucleation sites on organic surface – structural selectivity of mineral polymorphs – crystallographic alignment of nuclei on the organic surface.

Organic matrix-mediated nucleation – structural control A A A B B B G IIIIII 1no matrix 2matrix A, B polymorphs A kinetically favoured (no matrix) Outcomes I.promotion of non-specific nucleation - reduced activation energies for A and B, no change in the outcome of mineralization. II.promotion of structure-specific nucleation of polymorph B - crystallographic recognition at matrix surface; activation energy of state 2B < 2A III.promotion of a sequence of structurally non-specific to highly specific nucleation – variations in levels of recognition of nuclei A and B and reproducibility of matrix structure (genetic, metabolic, and environmental factors).

Interfacial molecular recognition Lattice geometry Charge Polarity Stereo- chemistry Space symmetry Topography Inorganic nucleus Organic matrix Lowering of the activation energy for nucleation can arise from matching of charge, polarity, structure and stereochemistry at the interface between an inorganic nucleus and organic macromolecular surface. The shape of the interface and the degree of chemical complementarity are important factors in this process.

Electrostatic accumulation – ionotropic model Anionic surface ligands accumulate metal cations by electrostatic binding (ionotropy) Site-directed ordering over nucleation scale by clustering – high spatial charge density High capacity binding  high localised supersaturation + Low affinity binding  migration of surface-bound ions to nucleus Or, charge matching of preformed nuclei in regions of high spatial charge density ABC

Fe III Fe II O2O2 ferroxidase centre 2Fe II + O 2 + 4H 2 O  2FeOOH + H 2 O 2 + 4H + nucleation groove Electrostatic accumulation – nucleation in ferritin

Surface topography concave convexplanar concave surfaces – high spatial charge density + good nucleation sites 3-D clustering of ions convex surfaces – dissipated charge density poor nucleation sites limit on number of nucleation sites planar substrates – localized charge distributions 2-D nucleation sites structural matching

Structural matching – the geometric model x y Nucleating crystal Organic matrix Distances between regularly spaced binding sites on the surface of the organic matrix are commensurate with lattice spacings in particular crystal faces. x nacre

Structural matching in nacre Tandem repeats of [Asp-X] explain the specific nucleation of the (001) face of aragonite on the surface of anti-parallel  -pleated sheet proteins in shell nacre. XRD and electron diffraction: a and b axes of  -sheet and lattice are co-aligned good matching along a directions; less so along b directions

Stereochemical matching in nacre Importance of side group stereochemistry coordination environments, multidentate binding, cooperativity charge balance ° 90° (001) face; values in nm Calcite ( ) vs aragonite ( ) (001) faces Similar lattice geometry but different Ca 2+ and CO 3 2- stereochemistry Calcite; CN = 6, planar CO 3 2- all coaligned Aragonite; CN = 9, planar CO 3 2- x2 types stereochemistry in crystal face

Oriented nucleation on soap films surfactant hydrophobic tail hydrophilic headgroup Langmuir monolayers air supersaturated CaHCO 3 (aq) Limiting area for single alkyl chain = 0.2 nm 2

Oriented nucleation of calcium carbonate under Langmuir monolayers CH 3 (CH 2 ) 16 COOHCH 3 (CH 2 ) 19 OSO 3 H unit cell a axis  to monolayer surface c axis  to monolayer surface NO oriented nucleation under CH 3 (CH 2 ) 17 OH Ca 2+ binding required !

A B carboxylate monolayer sulfated monolayer air supersaturated CaHCO 3 (aq) Ca 2+ binding 0.5 nm Matching of headgroup and orientation of CO 3 2- anions in nucleated crystal face Ca 2+ binding Matching of headgroup distance and Ca 2+ spacing in nucleated crystal face