Podosome-Driven Defect Development in Lamellar Bone under the Conditions of Senile Osteoporosis Observed at the Nanometer Scale.

The degradation mechanism of human trabecular bone harvested from the central part of the femoral head of a patient with a fragility fracture of the femoral neck under conditions of senile osteoporosis was investigated by high-resolution electron microscopy. As evidenced by light microscopy, there is a disturbance of bone metabolism leading to severe and irreparable damages to the bone structure. These defects are evoked by osteoclasts and thus podosome activity. Podosomes create typical pit marks and holes of about 300-400 nm in diameter on the bone surface. Detailed analysis of the stress field caused by the podosomes in the extracellular bone matrix was performed. The calculations yielded maximum stress in the range of few megapascals resulting in formation of microcracks around the podosomes. Disintegration of hydroxyapatite and free lying collagen fibrils were observed at the edges of the plywood structure of the bone lamella. At the ultimate state, the disintegration of the mineralized collagen fibrils to a gelatinous matrix comes along with a delamination of the apatite nanoplatelets resulting in a brittle, porous bone structure. The nanoplatelets aggregate to big hydroxyapatite plates with a size of up to 10 x 20 µm2. The enhanced plate growth can be explained by the interaction of two mechanisms in the ruffled border zone: the accumulation of delaminated hydroxyapatite nanoplatelets near clusters of podosomes and the accelerated nucleation and random growth of HAP nanoplatelets due to a nonsufficient concentration of process-directing carboxylated osteocalcin cOC.

First evidence of octacalcium phosphate@osteocalcin nanocomplex as skeletal bone component directing collagen triple-helix nanofibril mineralization.

Tibia trabeculae and vertebrae of rats as well as human femur were investigated by high-resolution TEM at the atomic scale in order to reveal snapshots of the morphogenetic processes of local bone ultrastructure formation. By taking into account reflections of hydroxyapatite for Fourier filtering the appearance of individual alpha-chains within the triple-helix clearly shows that bone bears the feature of an intergrowth composite structure extending from the atomic to the nanoscale, thus representing a molecular composite of collagen and apatite. Careful Fourier analysis reveals that the non-collagenous protein osteocalcin is present directly combined with octacalcium phosphate. Besides single spherical specimen of about 2 nm in diameter, osteocalcin is spread between and over collagen fibrils and is often observed as pearl necklace strings. In high-resolution TEM, the three binding sites of the γ-carboxylated glutamic acid groups of the mineralized osteocalcin were successfully imaged, which provide the chemical binding to octacalcium phosphate. Osteocalcin is attached to the collagen structure and interacts with the Ca-sites on the (100) dominated hydroxyapatite platelets with Ca-Ca distances of about 9.5 Å. Thus, osteocalcin takes on the functions of Ca-ion transport and suppression of hydroxyapatite expansion.

Magnesium as an intrinsic component of human otoconia.

OBJECTIVES: To investigate morphology changes of artificial otoconia (CGC) in the presence of magnesium during growth under in vitro conditions. METHODS: Investigating human otoconia by environmental scanning electron microscope and determining their magnesium content by energy-dispersive X-ray microanalysis (EDX). Comparing structural and morphological data of human and artificial otoconia (CGC, Ca1Mg0) without and with magnesium substitution (Ca1-xMgx). RESULTS: EDX- and X-ray data reveals that the inorganic component in human otoconia consists of calcite containing a minor amount of magnesium substitution (Ca1-xMgx). CGC containing magnesium (length 397.0 ± 146.4 µm, diameter 325.6 ± 100.1 µm) are slimmer and significantly smaller (p < .01) than pure CGC (length 548.6 ± 160 µm, diameter 373.0 ± 110.4 µm) and reveal a significant influence on the final morphology. The length/diameter ratio is significantly higher by incorporation of magnesium into CGC (1.84 ± 0.25 µm versus 1.48 ± 0.11 µm in pure CGC, p < .01), which brings the overall shape to a close relationship with human otoconia (1.98 ± 0.08 µm). CONCLUSIONS: Magnesium is an intrinsic component of human otoconia by partial substitution of calcium in the calcite crystal structure (Ca1-xMgx) and affects the development of the shape of artificial otoconia (calcite gelatin composites, CGC).

The sense of balance in humans: Structural features of otoconia and their response to linear acceleration.

We explored the functional role of individual otoconia within the otolith system of mammalians responsible for the detection of linear accelerations and head tilts in relation to the gravity vector. Details of the inner structure and the shape of intact human and artificial otoconia were studied using environmental scanning electron microscopy (ESEM), including decalcification by ethylenediaminetetraacetic acid (EDTA) to discriminate local calcium carbonate density. Considerable differences between the rhombohedral faces of human and artificial otoconia already indicate that the inner architecture of otoconia is not consistent with the point group -3m. This is clearly confirmed by decalcified otoconia specimen which are characterized by a non-centrosymmetric volume distribution of the compact 3+3 branches. This structural evidence for asymmetric mass distribution was further supported by light microscopy in combination with a high speed camera showing the movement of single otoconia specimen (artificial specimen) under gravitational influence within a viscous medium (artificial endolymph). Moreover, the response of otoconia to linear acceleration forces was investigated by particle dynamics simulations. Both, time-resolved microscopy and computer simulations of otoconia acceleration show that the dislocation of otoconia include significant rotational movement stemming from density asymmetry. Based on these findings, we suggest an otolith membrane expansion/stiffening mechanism for enhanced response to linear acceleration transmitted to the vestibular hair cells.

An NMR Study of Biomimetic Fluorapatite - Gelatine Mesocrystals.

The mesocrystal system fluoroapatite-gelatine grown by double-diffusion is characterized by hierarchical composite structure on a mesoscale. In the present work we apply solid state NMR to characterize its structure on the molecular level and provide a link between the structural organisation on the mesoscale and atomistic computer simulations. Thus, we find that the individual nanocrystals are composed of crystalline fluorapatite domains covered by a thin boundary apatite-like layer. The latter is in contact with an amorphous layer, which fills the interparticle space. The amorphous layer is comprised of the organic matrix impregnated by isolated phosphate groups, Ca3F motifs and water molecules. Our NMR data provide clear evidence for the existence of precursor complexes in the gelatine phase, which were not involved in the formation of apatite crystals, proving hence theoretical predictions on the structural pre-treatment of gelatine by ion impregnation. The interfacial interactions, which may be described as the glue holding the composite materials together, comprise hydrogen bond interactions with the apatite PO4(3-) groups. The reported results are in a good agreement with molecular dynamics simulations, which address the mechanisms of a growth control by collagen fibers, and with experimental observations of an amorphous cover layer in biominerals.

Polar Nature of Biomimetic Fluorapatite/Gelatin Composites: A Comparison of Bipolar Objects and the Polar State of Natural Tissue.

The correspondence of the state of alignment of macromolecules in biomimetic materials and natural tissues is demonstrated by investigating a mechanism of electrical polarity formation: An in vitro grown biomimetic FAp/gelatin composite is investigated for its polar properties by second harmonic (SHGM) and scanning pyroelectric microscopy (SPEM). Hexagonal prismatic seed crystals formed in gelatin gels represent a monodomain polar state, due to aligned mineralized gelatin molecules. Later growth stages, showing dumbbell morphologies, develop into a bipolar state because of surface recognition by gelatin functionality: A reversal of the polar alignment of macromolecules, thus, takes place close to that basal plane of the seed. In natural hard tissues (teeth and bone investigated by SPEM) and the biomimetic FAp/gelatin composite, we find a surprising analogy in view of growth-induced states of polarity: The development of polarity in vivo and in vitro can be explained by a Markov-type mechanism of molecular recognition during the attachment of macromolecules.

Synthesis and Characterization of Ba[CoSO]: Magnetic Complexity in the Presence of Chalcogen Ordering.

Barium thio-oxocobaltate(II), Ba[CoS2/2 O2/2 ], was synthesized by the reaction of equimolar amounts of BaO, Co, and S in closed silica ampoules. The title compound (Cmcm, a=3.98808(3), b=12.75518(9), c=6.10697(4) Å) is isostructural to Ba[ZnSO]. The use of soft X-ray absorption spectroscopy confirmed that cobalt is in the oxidation state +2 and tetrahedrally coordinated. Its coordination consists of two sulfur and two oxygen atoms in an ordered fashion. High-temperature magnetic susceptibility data indicate strong low-dimensional spin-spin interactions, which are suggested to be closely related to the layer-type crystal structure and perhaps the ordered distribution of sulfur and oxygen. Antiferromagnetic ordering below TN =222 K is observed as an anomaly in the specific heat, coinciding with a significant lowering of the magnetic susceptibility. Density functional theory calculations within a generalized-gradient approximation (GGA)+U approach identify an antiferromagnetic ground state within the square-like two-dimensional layers of Co, and antiferromagnetic correlations for nearest and next nearest neighbors along bonds mediated by oxygen or sulfur. However, this magnetic state is subject to frustration by relatively strong interlayer couplings.

Octacalcium phosphate - a metastable mineral phase controls the evolution of scaffold forming proteins.

The molecular structure of collagen type 1 can be understood as the result of evolutionary selection in the process of formation of calcium phosphate based biocomposites acting as load bearing components in living organisms. The evolutionary selection fulfills the principle of 'survival of the fittest' in a particular biological environment. Disk-like post-nucleation complexes of Ca2(HPO4)3 2- organized in ribbon-like assemblies in the metastable octacalcium phosphate (OCP) phase, and Ca3 triangles in the stable HAP phase had formed the crystallographic motifs in this selection process. The rotational as well as the translational symmetry of the major tropocollagen (TC) helix agree nearly perfectly with the corresponding symmetries of the OCP structure. The sequence of (Gly-X-Y) motifs of the three α chains constituting the TC molecule enables an optimized structural fit for the nucleation of Ca3 triangles, the directed growth of nanostructured OCP, and the subsequent formation of hydroxyapatite (HAP) in collagen macrofibrils by a topotaxial transition. The known connection between genetic defects of collagen type 1 and Osteogenesis imperfecta should motivate the search for similar dependences of other bone diseases on a disturbed molecular structure of collagen on the genetic scale.

Principles of calcite dissolution in human and artificial otoconia.

Human otoconia provide mechanical stimuli to deflect hair cells of the vestibular sensory epithelium for purposes of detecting linear acceleration and head tilts. During lifetime, the volume and number of otoconia are gradually reduced. In a process of degeneration morphological changes occur. Structural changes in human otoconia are assumed to cause vertigo and balance disorders such as benign paroxysmal positional vertigo (BPPV). The aim of this study was to investigate the main principles of morphological changes in human otoconia in dissolution experiments by exposure to hydrochloric acid, EDTA, demineralized water and completely purified water respectively. For comparison reasons artificial (biomimetic) otoconia (calcite gelatin nanocomposits) and natural calcite were used. Morphological changes were detected in time steps by the use of environmental scanning electron microscopy (ESEM). Under in vitro conditions three main dissolution mechanisms were identified as causing characteristic morphological changes of the specimen under consideration: pH drops in the acidic range, complex formation with calcium ions and changes of ion concentrations in the vicinity of otoconia. Shifts in pH cause a more uniform reduction of otoconia size (isotropic dissolution) whereas complexation reactions and changes of the ionic concentrations within the surrounding medium bring about preferred attacks at specific areas (anisotropic dissolution) of human and artificial otoconia. Owing to successive reduction of material, all the dissolution mechanisms finally produce fragments and remnants of otoconia. It can be assumed that the organic component of otoconia is not significantly attacked under the given conditions. Artificial otoconia serve as a suitable model system mimicking chemical attacks on biogenic specimens. The underlying principles of calcite dissolution under in vitro conditions may play a role in otoconia degeneration processes such as BPPV.

Interconnection of nanoparticles within 2D superlattices of PbS/oleic acid thin films.

Make it connected! 2D close-packed layers of inorganic nanoparticles are interconnected by organic fibrils of oleic acid as clearly visualized by electron holography. These fibrils can be mineralised by PbS to transform an organic-inorganic framework to a completely interconnected inorganic semiconducting 2D array.

The inner structure of human otoconia.

BACKGROUND: The architecture of human otoconia has been only poorly understood up to now. Currently, it is assumed that otoconia contain a central core surrounded by a shell. OBJECTIVES: To investigate the inner structure of human otoconia. METHODS: Human otoconia were investigated by environmental scanning electron microscopy (ESEM). The diffraction behavior was analyzed using X-ray techniques (XRD). Focused ion beam (FIB) slices of otoconia were investigated by transmission electron microscopy (TEM). The results were correlated with observations on degenerate human otoconia and decalcification experiments using ethylenediaminetetraacetic acid (EDTA). Artificial otoconia (calcite-gelatine and calcite-gelatine/agarose composites) were investigated in the same way and compared with human otoconia. RESULTS: Human otoconia represent highly mosaic-controlled calcite-based nanocomposites. The inner structure is composed of 3 + 3 branches with an ordered arrangement of nanocomposite particles and parallel orientation of fibrils. The surrounding belly is less ordered and appears more porous. Degenerate otoconia show a successive dissolution of the belly region exposing to the inner structure (branches) in later stages of degeneration. Artificial otoconia reveal identical chemical, crystallographic and morphologic patterns. They are, however, larger in size. CONCLUSION: Human otoconia show an inner architecture consisting of a less dense belly region and 3 + 3 more dense branches meeting at a central point (center of symmetry). The differences in volume densities and the resulting solubility may play a role in BPPV. Artificial otoconia may serve as a model for further investigations.

Detection of human utricular otoconia degeneration in vital specimen and implications for benign paroxysmal positional vertigo.

Otoconia are assumed to be involved in inner ear disorders such as benign paroxysmal positional vertigo (BPPV). Up to now, the distinct structure and morphology of intact and degenerate human utricular otoconia has been only poorly investigated on vital specimen. In this study, human otoconia were obtained from the utricle in five patients undergoing translabyrinthine vestibular schwannoma surgery. Specimens were examined by environmental scanning electron microscopy. Intact and degenerate otoconia as well as fracture particles of otoconia and bone were analyzed by energy dispersive X-ray microanalysis (EDX) and powder X-ray diffraction (XRD). Intact otoconia reveal a uniform size showing characteristic symmetry properties. Degenerative changes can be observed at several stages with gradual minor and major changes in their morphology including fragment formation. EDX analyses reveal the characteristic chemical composition also for otoconia remnants. XRD shows that intact and degenerate otoconia as well as remnants consist of the calcite modification. In conclusion, electron microscopy serves as a standard method for morphological investigations of otoconia. Human utricular otoconia show a uniform outer morphology corresponding to a calcite-based nanocomposite. Morphological changes provide further evidence for degeneration of utricular otoconia in humans, which might be a preconditioning factor causing BPPV. In case of uncertain origin, particles can be clearly assigned to otoconial origin using EDX and XRD analyses.

Gentamicin-induced structural damage of human and artificial (biomimetic) otoconia.

CONCLUSIONS: Gentamicin causes irreversible structural damage of human and artificial otoconia by progressive dissolution of calcite. The inner architecture of otoconia is strongly affected by degradation scenarios during gentamicin exposure. Artificial otoconia can be used as a model system mimicking the chemical attacks for detailed investigations. OBJECTIVES: To investigate the chemical interactions of gentamicin with natural calcite and human and artificial otoconia under in vivo conditions. METHODS: Pure calcite crystals and artificial and human otoconia were exposed to gentamicin injection solutions at various concentrations. Morphological changes were observed in time steps by the use of environmental scanning electron microscopy (ESEM). RESULTS: Dissolution of pure calcite crystals results in the formation of well oriented nanoshoots indicating an irreversible chemical reaction with gentamicin. Human and artificial otoconia reveal irreversible structural changes of their surface areas as well as of their inner structure, resulting in characteristic changes at different gentamicin concentrations. Minor changes are first observed by surface alterations and dissolution of calcite in the belly region. Major changes result in further reduction of the belly area reaching the center of symmetry. Finally, a complete dissolution of the branches takes place. Artificial otoconia provide detailed insight into surface alterations.

Intergrowth and interfacial structure of biomimetic fluorapatite-gelatin nanocomposite: a solid-state NMR study.

The model system fluorapatite-gelatin allows mimicking the formation conditions on a lower level of complexity compared to natural dental and bone tissues. Here, we report on solid-state NMR investigations to examine the structure of fluorapatite-gelatin nanocomposites on a molecular level with particular focus on organic-inorganic interactions. Using (31)P, (19)F, and (1)H MAS NMR and heteronuclear correlations, we found the nanocomposite to consist of crystalline apatite-like regions (fluorapatite and hydroxyfluorapatite) in close contact with a more dissolved (amorphous) layer containing first motifs of the apatite crystal structure as well as the organic component. A scheme of the intergrowth region in the fluorapatite-gelatin nanocomposite, where mineral domains interact with organic matrix, is presented.

Irregular shaped, assumably semi-crystalline calciumphosphate platelet deposition at the mineralization front of rabbit femur osteotomy: a HR-TEM study.

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high-resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow-up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex-shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix.

Mimicking the growth of a pathologic biomineral: shape development and structures of calcium oxalate dihydrate in the presence of polyacrylic acid.

The morphogenesis of calcium oxalate hydrates in aqueous solutions was investigated by varying the pH, oxalate concentration, and the concentration of the sodium salt of polyacrylate (PAA). With increasing amounts of PAA in solution, the shape of tetragonal calcium oxalate dihydrate (COD) changes from bipyramidal through elongated bipyramidal prisms to dumbbells and finally reverts to rodlike tetragonal bipyramidal prisms. PAA is incorporated into the prismatic zones of the growing COD crystals, thereby reducing the growth rate of the {100} faces along the <100> direction. Dumbbells start to develop through "non-crystallographic" branching from the prism faces and the formation of "multiple head" crystals. Adsorption of PAA on the rough surfaces of the splitting individuals supports the selection of new subindividuals and leads to the formation of core-shell patterns. The various shapes and structures of the biomimetic COD/PAA crystals and aggregates are closely related to the well-known "pathologic" individuals observed in the urine of patients with urinary disease (including urinary stones).

Exchange interactions through π-π stacking in the lamellar compound [{Cu(bipy)(en)}{Cu(bipy)(H2O)}{VO3}4]n.

Structural, magnetic, and powder and single-crystal electron paramagnetic resonance (EPR) studies were performed on [{Cu(bipy)(en)}{Cu(bipy)(H(2)O)}{VO(3)}(4)](n) (bipy = 2,2'-bipyridine, en = ethylenediamine), which is a new copper-vanadium hybrid organic-inorganic compound containing Cu(II) and V(V) centers. The oxovanadium units provide an anionic scaffolding to the structure, where two types of Cu(II) coordination modes, octahedral (Cu1) and square pyramidal (Cu2), contribute to the magnetic properties. The crystal structure contains layers including Cu1 and Cu2 ions, separated by stacked arrangements of 2,2'-bipyridine molecules. Each type of Cu(II) ion in these layers forms parallel spin chains described by exchange coupling parameters J(1) and J(2) for Cu1 and Cu2, respectively (exchange couplings defined as H(ex)(i,j) = -J(ij)S(i)S(j)), which, for necessity, are assumed to be equal to J. These chains are coupled by much weaker Cu1-Cu2 exchange interactions J(3) connecting neighbor Cu1 and Cu2 ions within a layer, through paths acting as rungs of a ladder chain structure. The average coupling J, which is antiferromagnetic (J < 0), according to the susceptibility data, is estimated with similar results with a mean field approximation (J = -1.4 cm(-1)), and with a uniform chain model (J = -1.7 cm(-1)). The EPR spectra of powdered samples and oriented single crystals are shown to be independent of J(1) and J(2), but are dependent on the weak coupling J(3), and the data allow a lower limit to be established: |J(3)| > 0.04 cm(-1). The spectra are also strongly sensitive to extremely weak coupling interactions with average magnitude J(4) between copper atoms in neighboring layers, separated by ∼10 Å, using the stacked 2,2'-bipyridine molecules, which produce a 2D-to-3D quantum phase transition. This is observed in single-crystal samples when the energy levels are changed with the orientation of the magnetic field. From the characteristics of these transitions, we estimate a value of |J(4)| = 0.0034 ± 0.0004 cm(-1) between Cu(II) ions in neighboring layers. This work emphasizes the important possibilities of EPR to evaluate extremely small exchange couplings between metal ions in a solid material, even in the presence of other much larger couplings.

Nanoporous titanium borophosphates with rigid gainesite-type framework structure.

Nanoporous titanium borophosphates have been synthesized which exhibit a rigid gainesite-type framework of polyhedra. The open-framework character is supported by the reversibility of de- and rehydration processes.