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 crystals 1 Structure and synthesis mechanisms of biomimetic inorganic crystals 2-4 ? 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 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 Harting?s 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 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: CaCO 3 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 growth 5 ? 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 Lecture 11 – Inorganic Biomaterials 4 of 12 Vesicular biomineralization 6 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 (SiO 2 ) (amorphous) from Si(OH) 4 silicic acid – algae and bacteria o Calcium carbonate (CaCO 3 ) from CaHCO 3 – algae and bacteria o Hydroxyapatite (calcium phosphate, Ca 5 (OH)(PO 4 ) 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 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 algae 8 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 Lecture 11 – Inorganic Biomaterials 6 of 12 ? Example: radiolarian skeleton 7 o Silica-skeleton algae Si(OH) 4 Silicic acid > 1 mM Mineralization nucleated at exterior surface of vesicles ?vesicle foam? 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 Lecture 11 – Inorganic Biomaterials 7 of 12 Matrix vesicles Extracellular matrix Hypertrophic chondrocytes/ odontoblasts Ion channels Transport proteins - - - - - - - - - Acidic phospholipids Ca ++ Ca ++ PO 4 - Ion influx CaHPO 4 nucleation Hydroxyapatite crystallization synthetic (biomimetic) vesicular mineralization Vesicular mineralization (Green et al. 2002) ? chemistry of CaCO 3 deposition in vesicles: o Ca ++ (aq) + 2HCO 3 - (aq) -> CaCO 3 (s) + CO 2 (aq) + H 2 O o K eq = [H 2 O][CO 2(aq) ][CaCO 3(s) ] [Ca (aq) ++ ][HCO 3(aq) ] 2 =constant o e.g. algae: remove dissolved CO 2 to induce more CaCO 3 precipitation o supersaturation highest near bubbles of CO 2 , nucleation preferentially at surface of vesicle and grows inward 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 -NH 2 H 2 O H 2 O ? 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 Lecture 11 – Inorganic Biomaterials 9 of 12 Aq CaHCO 3 SDS Organic (oil) phase Microemulsion mineralization (Green et al. 2002) ? Model of process: 6 1. Dissolved CO 2 in the supersaturated solution separates into bubbles that are trapped at the oil-water interface 2. Loss of CO 2 in solution increases the concentration of carbonate ions and thus the supersaturation 3. local supersaturation is highest next to CO 2 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 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 surfactant- encapsulated 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 micelles 9 ? Model for microstructure development in one case: Ba ++ CrO 4 —growth inside reverse micelles (water nanodroplets in oil phase) sulphosuccinate surfactant Reverse micelle nanoreactors Organic (oil) phase Ba ++ CrO4 2- 6 nm BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 11 of 12 (M. Li et al. 1999) ? Changing the ratio of cations to anions in the crystal alters its interaction with the organic surfactant and thus the assembly/growth properties in the microemulsion o With excess barium, cationic faces form on the crystal ? Strong ionic binding between headgroups and crystal prevents controlled growth and precipitation of growing crystals leads to eventual nanofilament formation o With excess chromate, anionic faces form that don’t bind surfactant ? No longer assembled by surfactant-surfactant interactions, leading to spherical nanoparticles 5:1 Ba ++ : CrO 4 2- cationic faces 1:5 Ba ++ : CrO 4 2- anionic faces ? Dendrimer biomineralization 10 o Approach: ? Hydrophobic surfactant tails self-assembled on the surface of dendrimer particles BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 11 – Inorganic Biomaterials 12 of 12 ? Surface-modified dendrimers mixed with aqueous biomineral solutions to drive crystallization ? After 4 days, calcite rhombohedrons grown around amorphous calcium carbonate spheres were observed o Coexistence of these 2 phases previously only observed in biological systems o Significant role of organic component in both nucleating and stabilizing amorphous phase? References 1. Estroff, L. A. & Hamilton, A. D. At the interface of organic and inorganic chemistry: Bioinspired synthesis of composite materials. Chemistry of Materials 13, 3227-3235 (2001). 2. Ozin, G. A. Morphogenesis of biomineral and morphosynthesis of biomimetic forms. Accounts of Chemical Research 30, 17-27 (1997). 3. Green, D., Walsh, D., Mann, S. & Oreffo, R. O. C. The potential of biomimesis in bone tissue engineering: Lessons from the design and synthesis of invertebrate skeletons. Bone 30, 810-815 (2002). 4. Almqvist, N. et al. Methods for fabricating and characterizing a new generation of biomimetic materials. Materials Science & Engineering C-Biomimetic and Supramolecular Systems 7, 37-43 (1999). 5. Walsh, D., Hopwood, J. D. & Mann, S. Crystal Tectonics - Construction of Reticulated Calcium-Phosphate Frameworks in Bicontinuous Reverse Microemulsions. Science 264, 1576-1578 (1994). 6. Walsh, D., Lebeau, B. & Mann, S. Morphosynthesis of calcium carbonate (vaterite) microsponges. Advanced Materials 11, 324-328 (1999). 7. Mann, S. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry (Oxford Univ. Press, New York, 2001). 8. Young, J. R., Davis, S. A., Bown, P. R. & Mann, S. Coccolith ultrastructure and biomineralisation. J Struct Biol 126, 195-215 (1999). 9. Li, M., Schnablegger, H. & Mann, S. Coupled synthesis and self-assembly of nanoparticles to give structures with controlled organization. Nature 402, 393-395 (1999). 10. Donners, J. J. J. M. et al. Amorphous calcium carbonate stabilised by poly(propylene imine) dendrimers. Chemical Communications, 1937-1938 (2000).