BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Lecture 11: Molecular Design and Synthesis of Biomaterials Il: Inorganic Biomaterials Last time: hydrogel applications: molecular imprinting and responsive drug delivery Today biomineralization and biomimetic inorganic/organic composites Inorganic biomaterials Reading L A. Estroff and A D. Hamilton, ' At the interface of organic and inorganic chemistry bioinspired synthesis of composite materials, Chem. Mater. 13, 3227-3235 (2001) tephen Mann, 'Biomineralization Principles and Concepts in Bioinorganic Materials Chemistry, Ch 5 pp. 68-88, Oxford Univ Press(2001) 1 million orthopaedic surgeries involving bone grafting materials each year in US(R Langer et al. Tissue Eng, 1 o autografting is best but limited in the size of defects that can be corrected (J. South Orthop ssoc. 9, 91 (2000): Aust J Surg. 69, 7 allografting presents possibility of disease transmission(HIV, hep B)(S Mendenhall; Commetnary: the bone graft market in the United States, in Bone engineering J.e. Davis, ed, em squared incorporated 2000, Torotonto, Canada p 585-590) o synthetic solid hydroxyapatite resorbs very slowly(Clin Orthop. 157, 259(1981)) Bone Tissue Engineering JBRM 29, 359(1995)Ca phosphate JBMR 36, 17(1997) PLGA Biomaterials 19, 1405(1998)PLGA J Biomat Sci-polym ed, 7, 661(1996)PLGA-Ca phos composite Test Biomineralization and biomimetic inorganic crystals Structure and synthesis mechanisms of biomimetic inorganic crystals motivation for studying biomineralization o natural bone composite organization, with organic molecules (peptides and proteins) guiding inorganic crystal growth, allows shapes that defy the classical 230 space groups of crystalline materials o biomineralization processes vs laboratory methods Biological methods- benign synthesis methods 1. Precisely control upon morphologies and structures over several length scales 2. Occur at near neutral PH, ambient temperature and pressure 3. Customize and optimize properties of materials according to the environme Laboratory methods: 1. Rely on extreme pH conditions form specific morphologies or patterned structures 2. high temperature and/or high pressure synthesis 3. Simple structure o up to 3000X greater strength than pure inorganic crystal Lecture 11-Inorganic Biomaterials 1of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 1 of 12 Lecture 11: Molecular Design and Synthesis of Biomaterials III: Inorganic Biomaterials Last time: hydrogel applications: molecular imprinting and responsive drug delivery Today: biomineralization and biomimectic inorganic/organic composites Inorganic biomaterials Reading: L.A. Estroff and A.D. Hamilton, ‘At the interface of organic and inorganic chemistry: bioinspired synthesis of composite materials,’ Chem. Mater. 13, 3227-3235 (2001) Stephen Mann, ‘Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry,’ Ch. 5 pp. 68-88, Oxford Univ. Press (2001) • 1 million orthopaedic surgeries involving bone grafting materials each year in US (R. Langer et al. Tissue Eng., 1, 151 (1995)) o autografting is best but limited in the size of defects that can be corrected (J. South Orthop ssoc. 9, 91 (2000); Aust N Z J Surg. 69, 726 (1999)) o allografting presents possibility of disease transmission (HIV, hep B) (S. Mendenhall; Commetnary: the bone graft market in the United States, in Bone engineering J.e. Davis, ed., em squared incorporated 2000, Torotonto, Canada p. 585-590) o synthetic solid hydroxyapatite resorbs very slowly (Clin Orthop. 157, 259 (1981)) Bone Tissue Engineering: JBRM 29, 359 (1995) Ca phosphate JBMR 36, 17 (1997) PLGA Biomaterials 19, 1405 (1998) PLGA J Biomat Sci-polym ed, 7, 661 (1996) PLGA-Ca phos composite Test Biomineralization and biomimetic inorganic crystals1 Structure and synthesis mechanisms of biomimetic inorganic crystals2-4 • motivation for studying biomineralization o natural bone composite organization, with organic molecules (peptides and proteins) guiding inorganic crystal growth, allows shapes that defy the classical 230 space groups of crystalline materials o biomineralization processes vs. laboratory methods • Biological methods - benign synthesis methods 1. Precisely control upon morphologies and structures over several length scales 2. Occur at near neutral PH, ambient temperature and pressure 3. Customize and optimize properties of materials according to the environment • Laboratory methods: 1. Rely on extreme pH conditions form specific morphologies or patterned structures 2. high temperature and/or high pressure synthesis 3. Simple structure o o up to 3000X greater strength than pure inorganic crystal
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 o Applications biomaterials for bioengineering replication of trabecular bone structure and mechanical properties is still elusive low-cost, reproducible high-volume regenerative materials needed biomimetic structures are readily resorbed, promote vascularization and cellular biomaterials for other applications 8x8 Pieter Harting G original hand drawings of calcareous microstructures(1872) The complex morphology and microstructure of biological inorganic materials has long been appreciated o E.g. Harting s hand drawings from 1872 o natural organic/inorganic composites are used by nature as endo-and exo-sekeltons for their strong Lecture 11-Inorganic Biomaterials 2of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 2 of 12 o Applications: • biomaterials for bioengineering • replication of trabelcular bone structure and mechanical properties is still elusive • low-cost, reproducible high-volume regenerative materials needed • biomimetic structures are readily resorbed, promote vascularization and cellular differentiation • biomaterials for other applications Pieter HartingÕs original hand drawings of calcareous microstructures (1872) • The complex morphology and microstructure of biological inorganic materials has long been appreciated o E.g. Harting’s hand drawings from 1872 o natural organic/inorganic composites are used by nature as endo- and exo-sekeltons for their strong mechanical properties
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 unicellular organisms such as radiolarians and diatoms A hexagona: Microskeleton of 15KV 184419.0 BATHU Radiolarian: Microskeleton of amorphous silica Coccolith: CaCo, microskeleton central tenet of biomineralization o organic molecules regulate nucleation, growth, morphology, and assembly of inorganic crystals(Mann, o molecular recognition at organic-inorganic interface two mechanisms of templating complex natural crystals 1. interfacial crystal growth rystal nucleation at organized boundaries(Acc. Chem. Res. 30, 17( 1997); J Mater. Chem. 7 689(1997)) utilized by unicellular organisms such as radiolarians and diatoms, where lipid vesicles compartmentalize and control solution chemistry kinetically controlled crystal growth 2. epitaxial crystal growth from template proteins equilibrium crystal growth dictated by template interfacial crystal growth Utilization of two-phase systems for directing location of mineral crystallization three types used by nature and one(possibly)novel approach investigated by biomimetic chemists 1. Vesicular biomineralization 2. Microemulsion biomineralization 3. micellar biomineralization 4. Dendrimer biomineralization(novel?) Lecture 11-Inorganic Biomaterials 3of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 3 of 12 • unicellular organisms such as radiolarians and diatoms Radiolarian: Microskeleton of amorphous silica A. hexagona: Microskeleton of amorphous silica Coccolith: CaCO3 microskeleton • central tenet of biomineralization: o organic molecules regulate nucleation, growth, morphology, and assembly of inorganic crystals (Mann, 1993) o molecular recognition at organic-inorganic interfaces • two mechanisms of templating complex natural crystals: 1. interfacial crystal growth crystal nucleation at organized boundaries (Acc. Chem. Res. 30, 17 (!997); J. Mater. Chem. 7, 689 (1997)) utilized by unicellular organisms such as radiolarians and diatoms, where lipid vesicles compartmentalize and control solution chemistry kinetically controlled crystal growth 2. epitaxial crystal growth from template proteins equilibrium crystal growth dictated by template interfacial crystal growth5 • Utilization of two-phase systems for directing location of mineral crystallization • three types used by nature and one (possibly) novel approach investigated by biomimetic chemists: 1. Vesicular biomineralization 2. Microemulsion biomineralization 3. micellar biomineralization 4. Dendrimer biomineralization (novel?)
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Vesicular biomineralization Biological vesicular mineralization Use of phospholipids structures to compartmentalize inorganic deposition Micrometer-sized droplets of supersaturated inorganic ions stabilized in oil by surfactant Nucleation and growth of inorganic phase occurs at surfactant headgroups, grows into microdroplet Igroups don't match perfectly to crystal Lipid mesophases provide mulitiple organized micro-and nano-structures for crystal deposition Characteristics of biological vesicular mineralization 1. Construction of enclosed, organized reaction environment a. Often using lipid bilayer vesicles b. Mineralization can occur inside or outside a boundary layer 2. Control of physicochemical conditions inside reaction environment via transmembrane ion channels transporters, and selective permeability 3. Control of nucleation kinetics 4. Production of complex crystal shapes by varying lipid matrix during growth Most common mi inerals produced by this method in biology: o Silica(SiO2)(amorphous)from Si(oH)4 silicic acid -algae and bacteria o Calcium carbonate(CaCO3) from CaHCO3 -algae and bacteria o Hydroxyapatite(calcium phosphate, Cas(OH)(PO4)3)from Ca and P04 human bone Vesicle reactors used in biology: phospholipids bilayers o Lipids have 2 hydrocarbon tails, so they can't pack into spherical micelles(molecules must have a shape complementary to this organization- wedge-shaped with big heads and'small tails o Form ubiquitous bilayer structure instead 柴 OOC Fig-5.1 Phospholipids IR and R are long-chain moieties. illustration of mesophases formed by lipid self- (a)micelle, (b) reverse micelle (c)lamella bilayer vesicle, (e)hexagonal,(f)inverse Lecture 11-Inorganic biomaterials 4of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 4 of 12 Vesicular biomineralization6 Biological vesicular mineralization • Use of phospholipids structures to compartmentalize inorganic deposition • Micrometer-sized droplets of supersaturated inorganic ions stabilized in oil by surfactant • Nucleation and growth of inorganic phase occurs at surfactant headgroups, grows into microdroplet • Nonspecific (non-epitaxial) growth- headgroups don’t match perfectly to crystal structure • Lipid mesophases provide mulitiple organized micro- and nano-structures for crystal deposition • Characteristics of biological vesicular mineralization: 1. Construction of enclosed, organized reaction environment a. Often using lipid bilayer vesicles b. Mineralization can occur inside or outside a boundary layer 2. Control of physicochemical conditions inside reaction environment via transmembrane ion channels, transporters, and selective permeability 3. Control of nucleation kinetics 4. Production of complex crystal shapes by varying lipid matrix during growth • Most common minerals produced by this method in biology: o Silica (SiO2) (amorphous) from Si(OH)4 silicic acid – algae and bacteria o Calcium carbonate (CaCO3) from CaHCO3 – algae and bacteria o Hydroxyapatite (calcium phosphate, Ca5(OH)(PO4)3) from Ca++ and PO4- - human bone • Vesicle reactors used in biology: phospholipids bilayers o Lipids have 2 hydrocarbon tails, so they can’t pack into spherical micelles (molecules must have a shape complementary to this organization- wedge-shaped with ‘big heads’ and ‘small tails’ o Form ubiquitous bilayer structure instead •
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Control of crystal growth:' o Intracellular mineral deposition is controlled using microtubules and scaffolding proteins that allow the cell to pull on the vesicle, changing its shape and orientation during inorganic growth o Chemical deposition can also be controlled by nm-scale variation in spatial distribution or reactants E.g. sequentially-activated ion transporters lon flux Biosynthes Control of vesicle morphology SMT Scaffold Fig. 7.6 Pattern formation in intracellular biomineralization. B, biomineral; V, vesicle; MT, microtubule. Spatial control of chemical deposition Growth direction Lipid bilayer Growing mineral V equentially activated ion transporters (M Example: coccolith skeleton o Coccolithophorids: major group of calcifying algae o Production of lipid vesicles to confine calcite formation o Influx of calcium and carbonate ions o oriented nucleation of calcite crystals within enclosed vesicles o Control of crystal shape by active change in vesicle shape by cell's cytoskeleton Lecture 11-Inorganic Biomaterials 5of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 5 of 12 • Control of crystal growth:7 o Intracellular mineral deposition is controlled using microtubules and scaffolding proteins that allow the cell to pull on the vesicle, changing its shape and orientation during inorganic growth o Chemical deposition can also be controlled by nm-scale variation in spatial distribution or reactants E.g. sequentially-activated ion transporters (Mann, 2001) Growth direction Sequentially activated ion transporters Lipid bilayer Growing mineral Control of vesicle morphology: Spatial control of chemical deposition: • Example: coccolith skeleton o Coccolithophorids: major group of calcifying algae8 o Production of lipid vesicles to confine calcite formation o Influx of calcium and carbonate ions o oriented nucleation of calcite crystals within enclosed vesicles o Control of crystal shape by active change in vesicle shape by cell’s cytoskeleton
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Example: radiolarian skeleton o Silica-skeleton algae >1 mM Silic Fig- 2.18 The chemical nature of amorphous silica nucleated at exterior surface of icro-skeleton. Scale bar, 10 um. I rr ⊙ Stages in the morphogenesis of the diatom frustule, See text for details. SDV-silica deposition vesicles AV- aleolar vesicle PL-plasmalemma(lipid bilayer cell wall) ER-endoplasmic reticulum Example: human bone formation o Growth plate cartilage, tooth dentine(Clin. Orth. Re R 314, 266(1995)) 1. Matrix vesicles 100-200 nm diameter secreted. attach to matrix 2. Calcium and phosphate ions influx into vesicle nanoreactors 3. Calcium phophate supersaturate 4. Controlled precipitation in amorphous structure 5. Crystallizes into hydroxyapatite 6. Growing crystals burst vesicles 7. Growth continues, controlled by extracellular conditions Lecture 11-Inorganic biomaterials 6of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 6 of 12 • Example: radiolarian skeleton7 o Silica-skeleton algae Si(OH)4 Silicic acid > 1 mM Mineralization nucleated at exterior surface of vesicles Ôvesicle foamÕ SDV - silica deposition vesicles AV - aleolar vesicles PL - plasmalemma (lipid bilayer cell wall) ER - endoplasmic reticulum nanovesicles • Example: human bone formation o Growth plate cartilage, tooth dentine (Clin. Orth. Re. R 314, 266 (1995)) 1. Matrix vesicles 100-200 nm diameter secreted, attach to matrix 2. Calcium and phosphate ions influx into vesicle nanoreactors 3. Calcium phophate supersaturates 4. Controlled precipitation in amorphous structure 5. Crystallizes into hydroxyapatite 6. Growing crystals burst vesicles 7. Growth continues, controlled by extracellular conditions
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Hypertrophic chondrocytes/ odontoblasts Hydroxyapatite lon influx D CaHPO4 D crystallization nucleation Matrix vesicles Extracellular matrix lon channels Transport proteins synthetic( biomimetic) vesicular mineralization Vesicular mineralization nre 2, Porous vater on of vaterite microsph in carbon dioxide(A). The droplets become super- tanned water interface( B), which leaves an im- prrt as the emulsion is air dried (C) falbwiag eless af trpe (Green et al. 2002) chemistry of CaCO3 deposition in vesicles o Ca(aq)+ 2HCO3(aq)-> CaCO3(s)+ CO2 (aq)+ H2O [H2OJ[COzaallCaco3sI = constant [Caa I[HCO3 o e.g. algae: remove dissolved CO2 to induce more CaCO3 precipitation o supersaturation highest near bubbles of CO2, nucleation preferentially at surface of vesicle and grows Lecture 11-Inorganic biomaterials 7of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 7 of 12 Matrix vesicles Extracellular matrix Hypertrophic chondrocytes/ odontoblasts Ion channels Transport proteins - - - - - - - - - Acidic phospholipids Ca++ Ca++ PO4 - Ion influx CaHPO4 nucleation Hydroxyapatite crystallization synthetic (biomimetic) vesicular mineralization Vesicular mineralization (Green et al. 2002) • chemistry of CaCO3 deposition in vesicles: o Ca++(aq) + 2HCO3 - (aq) -> CaCO3(s) + CO2(aq) + H2O o Keq = [H2O][CO2(aq)][CaCO3(s)] [Ca(aq) ++ ][HCO3(aq ) ] 2 = constant o e.g. algae: remove dissolved CO2 to induce more CaCO3 precipitation o supersaturation highest near bubbles of CO2, nucleation preferentially at surface of vesicle and grows inward
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Vesicular mine H2o Alkylamine vesicles 的mi Synthetic vesicular growth does not yet exhibit the control seen in nature o Lack control over ion introduction into local spaces, ability to manipulate vesicle shape on the scale performed by microtubules and other cytoskeletal elements 31 Disk-shaped diatom Vesicular growth diatom facsimile Microemulsion biomineralization Aqueous solutions of biomineral emulsified by an organic surfactant in an organic phase Mineralization occurs from surface of droplet stabilizer and grows inward into aqueous droplet Lecture 11-Inorganic biomaterials 8of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 8 of 12 QuickTime™ and a Graphics decompressor are needed to see this picture. Vesicular mineralization Alkylamine vesicles -NH2 H2O H2O • Synthetic vesicular growth does not yet exhibit the control seen in nature: o Lack control over ion introduction into local spaces, ability to manipulate vesicle shape on the scale performed by microtubules and other cytoskeletal elements Disk-shaped diatom Vesicular growth diatom facsimile 30 µm Microemulsion biomineralization • Aqueous solutions of biomineral emulsified by an organic surfactant in an organic phase • Mineralization occurs from surface of droplet stabilizer and grows inward into aqueous droplet
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Organic(oil) phase Ae CaYCe: Figure 3. Vaterite spheroids formed from a microemulsion f octane: SDS: CaHCO3(71: 4: 25 wt %) Note the complex SDS surface patterning and uniform size of the spheres. Scale bar 10 um(Reproduced by permission from ref 14) Microemulsion mineralization OIr Figure 2. Porous vaterite microspheres waporatka feil attrated when the carbon dioxide slowly prec pate min fallowing release of tapped cOh Green et al. 2002) · Model of process 2. Loss of CO2 in solution increases the concentration of carbonate ions and thus the supersaturation r interface 1. Dissolved CO2 in the supersaturated solution separates into bubbles that are trapped at the oil-water interface 3. local supersaturation is highest next to CO2 bubbles, so first calcium carbonate precipitation occurs here Bubbles trapped at oil/water interface dictate foamed surface morphology of inorganic structure Vaterite formed in this process dictated by ostwald's rule of stages o Polymorph with highest solubility preferentially forms under kinetically-controlled Lecture 11-Inorganic biomaterials 9of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 9 of 12 Aq CaHCO3 SDS Organic (oil) phase Microemulsion mineralization (Green et al. 2002) • Model of process:6 1. Dissolved CO2 in the supersaturated solution separates into bubbles that are trapped at the oil-water interface 2. Loss of CO2 in solution increases the concentration of carbonate ions and thus the supersaturation 3. local supersaturation is highest next to CO2 bubbles, so first calcium carbonate precipitation occurs here • Bubbles trapped at oil/water interface dictate ‘foamed’ surface morphology of inorganic structure • Vaterite formed in this process dictated by Ostwald’s rule of stages: o Polymorph with highest solubility preferentially forms under kinetically-controlled conditions
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Bicontinuous microemulsions: interconnected microemulsion templates for mineralization Formed and then oil phase frozen to limit dynamic changes in liquid crystal nanochannel network during inorganic calcium phosphate crystallization Nanochannels formed with supersaturated solution; allowed to crystallize in surfactant encapsulated water channels Final structure composed of fibers with dimensions much larger than original nanochannels Growing calcium phosphate expands surfactant structure with time Frozen oil external Aqueo phase nanochannels Fig. 9.33 Micro-skeletal form of calcium phosphate prepared in bicontinuous microemulsions Scale bar, 1 um. Micellar biomineralization Growth of inorganic crystals from surface of micelles Model for microstructure development in one case: Ba CrO4-growth inside reverse micelles(water nanodroplets in oil phase) Cro4 6 nm Reverse micelle nanoreactors rmuoudasl menoyo her四u sulphosuccinate tld sufactant mmwe h. Irtetugeaton d the 如知可油出m间m出mm Lecture 11-Inorganic Biomaterials 10of12
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 10 of 12 Bicontinuous microemulsions: interconnected microemulsion templates for mineralization: • Formed and then oil phase frozen to limit dynamic changes in liquid crystal nanochannel network during inorganic calcium phosphate crystallization • Nanochannels formed with supersaturated solution; allowed to crystallize in surfactantencapsulated water channels. • Final structure composed of fibers with dimensions much larger than original nanochannels: • Growing calcium phosphate expands surfactant structure with time Aqueous nanochannels Frozen oil external phase Micellar biomineralization • Growth of inorganic crystals from surface of micelles9 • Model for microstructure development in one case: Ba++CrO4—growth inside reverse micelles (water nanodroplets in oil phase) sulphosuccinate surfactant Reverse micelle nanoreactors Organic (oil) phase Ba++ CrO42- 6 nm