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.

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.

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.

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.

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.

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.