The inner ear of vertebrates contains a complex arrangement of enclosed sacs and channels which are the sites of the senses of balance and hearing . Parts of these sensory systems are called maculae, acting as gravity receptor organs responding to linear accelerations . The sensory transduction depends on the inertial mass of a calcium carbonate biomineral (so-called statoliths) which, in case of fish and amphibians consists of aragonite or in rare cases even vaterite (so-called otoliths) [3, 4], while reptiles, birds and mammals produce the calcite modification (so-called otoconia) . A major difference between otoliths (“ear stones”; mm sized)  and otoconia (“ear dust”; μm sized)  is that otoliths display a daily growth pattern, whereas adult otoconia undergo only minor changes with life time which, however, may be connected with degeneration effects [7, 8]. Otoconia are present as an array of thousands of tiny biomineral specimens in each of the maculae (utricle and saccule), and are surrounded by a liquid called endolymph . Each otoconium is characterized by a barrel-shaped habit with triplanar faceted ends (rhombohedral truncation) [10, 11]. Scanning electron microscopy (SEM) images of human otoconia are presented in Fig. 1.
Inspired by nature’s skill to take over control of the formation and shape development of functional materials (biominerals) in living systems, big activities have been started since decades, in order to mimic these growth scenarios in the laboratory. Several attempts have been made with respect to calcite-based biominerals, especially by modifying the basic rhombohedral shape of calcite crystals in the presence of various organic (biogenic) additives. Elongated, rhombohedrally truncated calcite specimens with a habit more or less similar to the shape of otoconia (see Fig. 1) are reported in a few publications, only. These include spindle-shaped specimens obtained in the presence of malonic acid , the development of additional (sometimes even structured) faces extending nearly parallel to the c-axis direction of calcite in the presence of γ-carboxyglutamate or α-aminosuccinate , glycoproteins extracted from sea-urchin spines , and nacre proteins .
The potential of organic additives to act as growth modifiers is mainly ascribed to the binding activities of the molecules to specific planes of the respective crystal structure, thereby inhibiting the growth of the plane. With regard to calcite, carboxylate groups of small dicarboxylic acid molecules (such as malonic acid) have been reported to inhibit crystal growth upon binding to the (110) planes . Sulfonate groups, on the other hand, are reported to stabilize the (001) planes [17, 18]. From these observations it was assumed that polymers containing the two different growth modifiers would have a special effect on the shape development of calcite crystals. By use of the commercial polyelectrolyte, poly(4-styrenesulfonate-co-malonic acid) (PSS-co-MA) random copolymer, isometric rhombohedrally truncated calcite specimens were obtained with planar rhombohedral faces (104) interconnected by six curved and rough faces arranged around the central –3 axis of the trigonal individuals [19, 20], an overall shape which reminds of pseudo-dodecahedra. TEM images of ultrathin slices suggest the presence of aggregated nanoparticles and incorporated PSS-co-MA between the nanocrystal domains. Because of the common orientation of the nanoparticles the individuals are representative for the so-called mesocrystalline state . A polymer content of approximately 2.2 wt.-% was determined by thermogravimetric (TG) analysis .
Biominerals can be classified as inorganic/organic nanocomposite systems. Even though the organic part is only involved as the minority component, the molecules take over control of the nucleation szenarios and the shape development. The outer shape of a biomineral (and this also holds for biomimetically grown materials) does not give any information on the inner structure which may even be developed to a hierarchical pattern. This conclusion is of great significance in order to avoid any doubtful interpretation of biomimetically grown individuals indicating structural relations which, in fact, may not be present at all. While the significance of organic molecules acting as growth modifiers by specific adsorption on special crystal planes remains undisputable, this situation is just part of a more complex story. The real challenge for deeper understanding of the nucleation and shape development of a biomineral (or a biomimetic material) is to clarify the structural relations between the inorganic and the organic components from the atomic level to the meso/macro-scale. This strategy was successfully followed in recent investigations on fluorapatite-gelatine composites, and it was shown that even perfectly developed hexagonal prismatic individuals (the Bragg diffraction pattern corresponds to a single crystal) are not just “simple” single crystals, but actually represent a nano-composite superstructure with an additionally integrated (mesoscopic) hierarchy of gelatine microfibrils [22, 23].
Our research on otoconia and their biomimetic growth by double-diffusion in gelatine-gel matrices was initiated just by accident. In order to bring the fluorapatite-gelatine system [22, 23] more into line with biogenic conditions, we added carbonate for the formation of carbonate-apatite . Under these modified conditions we sometimes observed the appearance of otoconia-shaped calcite-gelatine composite specimens . After having a more detailed look at the medical literature it became clear that the peculiar shape of biogenic (human) otoconia, their shape development and the resulting biofunctionality had been only poorly investigated. Significant discrepancies in the interpretation of the inner architecture of otoconia were also obvious. With this in mind, and after having learned about the structural complexity of fluorapatite-gelatine nanocomposites [22, 23], we started to extensively investigate biomimetic and human otoconia in order to get deeper insight into their structural relations and funcionality.
Biomimetic morphogenesis and bulk characterization in relation to human otoconia
Biomimetic otoconia were grown by double-diffusion in gelatin-gel matrices [25, 26]. The horizontal tubes of the U-shaped diffusion cells were filled with gelatine-gel while the vertical tubes contained aqueous solutions (pH = 7.4) of CaCl2, and Na2CO3, respectively. The formation of the otoconia-shaped calcite-gelatine composite is mainly observed close to the calcium source. The experiments were run between 1 and 10 days. To isolate the composite particles at various growth stages, the gelatin plugs were pressed out of the central tubes, cut into slices, washed with hot water and centrifuged. More detailed information on the growth conditions are given in .
The complex morphogenesis of biomimetic otoconia is shown in Fig. 2. Within days, the shape development evolves from stage 1 (an arrangement characterized by six “trumpet-like” branches) via various intermediate states that are dominated by the six branches (stages 2–3). The branches grow fast and develop their basal faces to the final state (stage 4) where they meet at both ends of the individual, thereby each forming straight common edges and rounded boundaries in direction of the belly region. The belly region grows with temporal delay and appears to be structured on a small particle scale but with a preferred (common) orientation of the subunits which are dominated by rhombohedral contours (see stage 3). The spiky structures on the basal planes of the branches which are clearly seen in early states of the shape development (see stage 2) are assumed to indicate the positions of gelatine molecules, which will be discussed in the chapters INNER ARCHITECTURE and ATOMISTIC SIMULATIONS in more detail.
Apart from the more pronounced and rounded belly region, the biomimetically grown calcite-gelatine composite specimens in their final state of development reveal morphological characteristics similar to those for adult otoconia of mammals, with an overall symmetry close to –3m (see Fig. 1 and Fig. 2 bottom right (4)). As concerns to their size, the artificial specimens have to be classified as “more than giant”: The mean length of “normal” biogenic otoconia is about 10 μm , but “giant” biogenic otoconia up to 80 μm in length are also observed . It can be assumed that the shape development of the biomimetic specimens to the “closed otoconial habit” may, in general, be finished at a composite size of about 100 μm. This assumption is supported by the observation that biomimetic samples after a growth period of five days consist of composite particles in their final state of shape development with a size distribution between 100 μm and 400 μm . At the same time, this means that biomimetic otoconia keep growing until the end of the diffusion experiment. Finally, it should be pointed out, that the “ideal” symmetry –3m of biomimetic and biogenic otoconia is only given, in case that all the basal faces of an otoconium are of the same size.
The inorganic component of human otoconia consists of the calcite modification of calcium carbonate, and at least parts of the individuals have already been reported to behave like single crystals [29, 30]. The X-ray powder pattern of biomimetically grown otoconia also confirms the presence of calcite as the inorganic component . Completely developed specimens of biomimetic and biogenic (human) otoconia were glued on glass capillaries for investigation by single crystal X-ray methods [25, 26]. Optical micrographs of the individuals together with the respective Bragg diffraction patterns are shown in Fig. 3. The X-ray patterns are representative for single crystals and the crystal structure of calcite was refined from both specimens (Biomimetic/Biogenic: R-3c, ZH = 6, aH = 4.9880(4)/4.9836(7) Å, c = 17.096(2)/17.071(4) Å, R1 = 0.025/0.068). The plane faces of the bigger biomimetic otoconia were indexed as the rhombohedral planes (104). The same indexing can be assumed for the plane faces of the biogenic specimens. The curved (ellipsoidal) shape of the belly area indicates the presence of small calcite subunits with common crystallographic orientation which generate the habit of otoconia. As can be derived from Fig. 2 (stage 3), the subunits are characterized by a rhombohedral shape. A simplified model of an otoconium generated by small rhombohedra with 3D-periodic arrangement is shown in Fig. 4, and reflects the main characteristics of the otoconial habit (plane rhombohedral faces together with a curved (ellipsoidal) belly surface) . The “ideal” symmetry corresponds to –3m.
To preliminarily conclude the bulk characterization of biomimetic otoconia in relation to biogenic (human) specimens, we also have to consider their density and the amount and nature of the organic components within the composites. For biogenic otoconia the presence of foreign (inorganic) components is of additional interest. The density of biomimetic otoconia (2.563 g cm–3) as determined by the He-gas pressure technique , is similar to the values determined for rat otoconia (2.47–2.65 g cm–3) . The density measurements of biogenic otoconia are affected by their small size and the fact that the organic components attached to the surface cannot be completely removed without destruction. Density values for human otoconia are not available up to now, but a close relation to biomimetic otoconia can be expected. The biomimetically grown otoconia contain an amount of gelatine between 1.9 wt-% and 2.6 wt-% as determined by TG measurements and chemical analyses . For human otoconia a maximum value of 3.2 wt-% for the organic components (mainly glycoproteins and glycosaminoglycans) [11, 33, 34] was determined by spectroscopic investigations . This value (3.2 wt-%) may be even lower because of the surface organics which could not be completely removed from the tiny individuals. At first glance, the nature of the proteins incorporated in biomimetic (gelatine ≙ denatured collagen) and biogenic (glycolysated collagen) otoconia seems to be contradictory. This situation will be discussed and clarified in the chapter ATOMISTIC SIMULATIONS. Microprobe analyses of human otoconia revealed the presence of trace amounts of Na, Mg, P, S, Cl, and K . The reason for the presence of these non-Ca elements is not clear at present: either their detection is caused by remnants of the endolymph, and/or the minority elements (or some of them) intrinsically belong to the inorganic part of otoconia (e.g., substitution effects). The role of Mg is just under investigation .
Inner architecture and composite structure of otoconia
The peculiar shape development of otoconia which is characterized by fast growing rhombohedral faces (see Fig. 2) gives rise for the assumption that the inner architecture also reflects this kind of peculiar morphogenesis. Step by step decalcification of biomimetic and human otoconia was performed by treatment with ethylenediaminetetraacetate (EDTA) in order to gradually observe differences in the dissolution behavior and to get information on the inner structure of the composite [25, 26, 31, 37]. The first observations made on biomimetic otoconia are presented in Fig. 5. The optical micrographs taken during successive decalcification reveal that the belly parts are more easily dissolved than the branches. After complete decalcification the organic component (gelatine) keeps the shape of the former composite (images 5b and 5c taken in solution). A more detailed picture of the inner architecture of biomimetic otoconia is presented in Fig. 6. The SEM image of a partially dissolved fracture area reveals that the rhombohedral branches seem to develop from a common “point” close to the center of the specimen. The belly area appears to be more porous and less ordered compared with the branches. Partially dissolved human otoconia are shown in Fig. 7. The image taken by environmental scanning electron microscopy (ESEM) clearly shows that the belly area is completely dissolved while the branches remain nearly unaffected. A net or organic fibrils within the former belly area of the composite interconnects the two rhombohedral branch areas which appear to be already completely separated. In principle, the significant anisotropy of the decalcification behavior of otoconia fully agrees with the rough surface area and the more porous structure of the belly volume (Fig. 4 and Fig. 6) which allows for easier attack of the complexing agent (EDTA) compared with the more ordered and more dense branches. For further discussion, a 3D-model of a single otoconium is shown in Fig. 8, together with a human specimen and a partially decalcified individual. From these images it becomes evident that deviations from ideal symmetry –3m caused by differences in size of the rhombohedral planes lead to considerable consequences in the distribution of dense and more porous regions within the volume of an otoconium.
After having gained first insight into the basic (mesoscopic) principles of the inner architecture of otoconia, the next step of investigation was directed on higher resolution techniques, such as transition electron microscopy (TEM) [25, 26, 37]. Sample preparations (thin slices) were performed by the focused ion beam (FIB) method in various (selected) directions through representative otoconial specimens at different growth stages. The ion scanning image of a biomimetic otoconium in an early stage of morphogenesis (see Fig. 2) is shown in Fig. 9a. The bar indicates the area from which the TEM lamella was cut by the FIB technique. The cut leads through one of the branches and parts of the surrounding belly area. TEM images of branch and belly (overview in Fig. 9b) clearly reveal the presence of structural differences within the composite volume. The belly region (Fig. 9c) is only poorly crystalline and consists of nanodomains in a mosaic arrangement as well as of pores. Compared with the branch area which exhibits a perfect periodic pattern (Fig. 9d and inset (FFT) top left) the FFT of the belly region (inset Fig. 9c, top left) displays only a small number of weak spots due to reduced crystallinity. The overall crystallographic orientations of branch and belly areas are identical which fully agrees with the X-ray Bragg diffraction properties of the completely developed biomimetic specimens that are representative for single crystals (Fig. 3). An enlarged TEM image of the branch area (Fig. 9b) is shown in Fig. 10a which reveals parallel traces of about 10 nm in thickness corresponding to calcified microfibrils stretching out along the branch direction (104) and being perfectly integrated into the periodic calcite pattern of the dense branch. The corresponding fast Fourier Transform (FFT) processed image (Fig. 10b) gives rise to superstructure lattice spots which are caused by parallel ordering of the fibrils with a spacing of about 25 nm. In case of human otoconia, the same arrangement of branches and fibrils is observed. Figure 11a represents a longitudiual FiB cut of a human otoconium with the corresponding electron diffraction pattern of the whole cut (Fig. 11b) which, again, is representative for a single crystal in the sense of a mesocrystal . The branches from the rhombohedral end faces contain the fibrils in parallel arrangement running normal to the rhombohedral planes, while the belly part appears less ordered (Fig. 11c) which is consistent with the random net of fibrils remaining after decalcification (Fig. 7). The packing sequence of fibrils within the branches corresponds to a periodicity of about 20 nm (Fig. 11d) which is similar to the periodicity of 25 nm in biomimetic specimens (see Fig. 10).
Further detailed investigations on thin slices of biomimetic and human otoconia [25–27] reveal that the six branches nearly meet at a common “point” close to the center of the specimen. A model of the resulting dumbbell-shaped entity is displayed in Fig. 12a. The orientation of the fibrils within the branches as indicated by the white arrows (Fig. 12b) reflects their parallel arrangement within each branch and their orientation perpendicular to the rhombohedral end faces. The spiky structures on top of the rhombohedral planes (as already mentioned for early growth states of the biomimetic individuals; see Fig. 2) are assumed to represent the outgrowth pattern of the fibrils (see also Fig. 6).
Atomistic simulations: nucleation of calcium carbonate at fiber proteins
A recently presented molecular dynamics simulation procedure mimicking ion-by-ion association and self-organization during apatite-collagen nucleation  was transferred to the formation of calcium carbonate-collagen composites . To allow direct comparability to the previous study of apatite-collagen composite nucleation, the simple (Gly-Pro-Hyp)n collagen model was adopted. The first aggregation steps were found in full analogy for both systems: Ca2+ ions are incorporated into the helical center and stiffen the triple-helical structure, while phosphate and carbonate ions are attached laterally. The simulations related to apatite reveal that during aggregate growth the phosphate ions remain at lateral positions whereas carbonate ions also enter the triple helices which leads to increased unfolding. As a consequence, calcium carbonate aggregates are formed within the collagen backbone, thus pushing the peptide strands apart. This situation is presented in Fig. 13 and shows the destructive force of calcium carbonate during attachment at/in collagen.
The major organic component in otoconia of mammals (and birds) is a highly glycosylated protein, named otonin 90, coming to more than 90% of the soluble organic matrix [40, 41]. The principal insoluble scaffold protein, otolin-1, contributes to the extracellular matrix of the inner ear, and is assumed to be responsible for the growth and anchoring of otoconia [42–44]. Otolin-1 is known as a glycosylated collagen-like protein specific for calcite-based biominerals. The aggregate growth simulations were thus repeated , in order to investigate the interplay of otolin-1 with calcium and carbonate ion association. Instead, for the glycolysated protein otolin-1, the picture is substantially different from Fig. 13: Fig. 14 shows that the nucleation of calcium carbonate aggregates occurs at the contact regions of disaccharide side chains and the aqueous solution. Upon formation of larger ion agglomerates, this scenario leads to an intergrowth of (largely disordered) calcium carbonate with the side chains of otolin-1. At the same time, the triple-helical structure (backbone) remains intact. The collagen-like protein otolin-1 which contains a particularly high degree of glycolysated amino acids provides association sites more lateral to the backbone and avoids destructive calcium – carbonate contacts within the triple-helix. The calculated occurrence profile for Ca···O distances within the largely disordered ion clusters associated to otolin-1 shows that the corresponding distances are characteristic for the calcite and the aragonite crystal sructures. By taking into account Ca2+ contacts to oxygen atoms of carbonate groups, saccharide groups and water molecules, the averaged coordination number is 6.97 . In the crystal structures of calcite and aragonite the coordination numbers for calcium by oxygen are 6 and 7, respectively.
Biomimetic otoconia have been grown by double-diffusion in gelatine-gel matrices [25, 26] which, at first glance, seems to be contradictory to the calculations showing the destructive force of calcium carbonate during attachment at/in nonglycolysated collagen . However, commercial gelatine still contains 0.5–1.0 wt.-% of covalently bound saccharides  which are not detached during gelatine processing (degeneration) from collagen and which help for calcium carbonate nucleation without disruption of the triple-helical structure. At the same time, the triple-helices are stiffened to form the parallel alignment within the branches as presented in Figs. 10 and 12.
The very first nucleation steps during the morphogenesis of otoconia can be described as the formation of areas of largely disordered calcium carbonate, stabilized by the organic substrate and water (H2O, H+, OH–). This metastable state is often called “amorphous calcium carbonate (ACC)” acting as a precursor phase for the formation of calcium carbonate based biominerals [46–48]. The term “ACC”, however, should be avoided and is misleading in the sense of thermodynamics: The metastable phase always contains chemical components which do not belong to the chemical formula CaCO3. Regardless of this general comment, the actual formation of calcite is expected at much later stages of aggregate growth and is not observed on the basis of atomistic simulations which are limited to the infancy of nucleation. However, it was possible to obtain TEM images showing the appearance of calcite nanocrystals in gelatine-gel matrices at very early stages of the biomimetic morphogenesis of otoconia (see Fig. 15) which remind of pre-states in the course of mesocrystal formation . The nanocrystals are characterized by their rhombohedral shape which is consistent with the shape of the subunits of the belly area of otoconia as shown in Fig. 2 (stage 3) and which is also consistent with the sketch presented in Fig. 4. The area marked by the red frame in Fig. 15 seems to indicate a tendency for controlled agglomeration of the nanocrystals. A directing role of the organic component (gelatine), however, cannot be deduced from the TEM image.
Basic assumptions for the function of otoconia
From the medical point of view, it is generally accepted that otoconica couple mechanic forces to sensory hair cells, in order to detect linear acceleration and gravity for the purpose of maintaining bodily balance . Human (mammalian) otoconia are formed at late embryonic stages and normally undergo only minor changes with life time, although some maintenance after formation may be required . Head tilts and linear head motion cause displacement of the otoconial array (tens of thousands of individuals), producing a shearing force which deflects the underlying sensoring hair cells in the sensory epithelium (macula). Anchoring of otoconia is essential for optimal vestibular function and balance .
Bringing together the medical knowledge of otoconia with the more detailed basic observations of the present investigation, and taking into account nature’s strategy to develop biomaterials in direction of optimal functionality, we are now able to take the next step in deeper understanding of the mechanics of the otoconial system. The central point in this connection concerns the conclusion that the composite nature and the complex inner architecture of otoconia are of great significance going far beyond simple particles characteristics with a well-defined density, only (gravity receptor).
Otoconia are not only anchored to the sensory epithelium but are also interconnected by a net of protein fibrils as shown in Fig. 16. On the one hand, the protein fibrils represent the organic component of otoconia which is integrated into the composite system. On the other hand, the fibrils extend over the volume of otoconia into the surrounding endolymph, thereby forming the interconnecting net (linking filaments) . It can be assumed that especially the highly ordered fibrils spreading out of the rhombohedral faces of otoconia contribute to interotoconial linking. As the otoconial array on the sensory epithelium responds to mechanical forces (acceleration) with a collective answer, the question arises whether the anisotropic density distribution within the volume of otoconia (dumbbell-shaped, more dense branch region (see Fig. 12) and more porous belly area (see Fig. 6) has an advantage over a more homogeneous (isotropic) structure. In this connection it has to be pointed out that the first model for the inner architecture of otoconia was based on a core-shell scenario as derived from SEM investigations on accidentally freeze-fractured samples . The respective SEM image leading to the assumption of a core-shell structure is presented in Fig. 17 (left). The branch-belly model in Fig. 17 (right), however, clearly shows that a cut parallel to the trigonal axis affecting small parts of the branches and a big part of the belly area leads to an image faking the presence of a core-shell arrangement. Coming back to the branch-belly architecture and the habit of otoconia, the “ideal” symmetry –3m is only given in case that all the rhombohedral faces of an otoconium are of the same size (see chapters BIOMIMETIC MORPHOGENESIS and INNER ARCHITECTURE). This criterion immediately leads to an arrangement of branches which is also consistent with the symmetry –3m, and the center of symmetry coincides with the center of the volume of an otoconium. With respect to the density distribution within otoconia, the center of gravity, in the centrosymmetric case, coincides with the center of symmetry. However, careful inspection of the shape of otoconia (biomimetic as well as biogenic/human) with special focus on the size of the rhombohedral faces and their distribution over the surface of the specimens seems to indicate that the majority of otoconia does not meet the criterion for “ideal” symmetry, but rather indicates the presence of non-centrosymmetric density distribution. Representative examples are shown in Fig. 18.
The chemical composition of the liquid medium (endolymph) surrounding the otoconia has mainly been investigated for guinea pigs [53–55]. Although consistent quantitative data are not available, the qualitative presence of the following components is much certain: proteins, sodium, potassium, calcium, chloride and (hydrogen)carbonate. The pH-value under vivo conditions is around the neutral point (7.0 ± 0.3). A significant viscosity of the endolymph is assumed to be caused by the presence of specific (glyco)proteins . A more recent investigation of human endolymph reveals that the chemical composition is not substantially different from compositions found for guinea pigs .
By summarizing the knowledge on otoconia, their inner architecture, their anchoring and interconnections as well as the surrounding endolymph, the following (preliminary) picture regarding functionality can be deduced: Otoconia are floating within the endolymph and keep their positions in case of immobility of the system. These resting positions are caused and stabilized by a number of facts, such as (i) the porous belly area of otoconia acting as buoyancy ring, (ii) anchoring of otoconia at the gelatineous membrane  and interconnection by a net of fibrils, and (iii) the viscous endolymph moistening the otoconia and penetrating into their porous belly areas. A simplified and schematic sketch of the sensory system is shown in Fig. 19. In case of linear acceleration, otoconia (as a whole) start to move not only because of their inertial mass but they also start to rattle within the net of fibrils (because of their non-centrosymmetric density distribution) thereby affecting neighboring regions. Displacements and movements within the otoconial array produce shearing forces within the gelatineous membrane which then deflect the sensory hair cells (nerve fibers in Fig. 19) in the sensory epithelium. Although the given picture of the functionality of the system has to be taken as a very first approximation, it already reflects the actual complexity of this sensory arrangement.
Degeneration of otoconia
Otoconia related balance disorders are prevalent. Human saccular and utricular otoconia show gradual changes in morphology (degeneration) during life time . Degeneration is associated with structural damage, a successive loss of otoconial material and related mass reduction. It has also been shown that structural changes of human otoconia in the utricle, such as fragment formation, take place gradually and are assumed to cause otoconia dislodgment into the endolymph leading to benign paroxsysmal positional vertigo (BBPV) . Furthermore, it has been proposed that otoconia dislodgment plays a role in peripheral vestibulopathies such as Ménièr’s disease (MD) and vestibular drops attacks [63, 64]. Otoconia stay intact under in vivo conditions as long as the surrounding endolymph is saturated with regard to chemical components in solution which keep the equilibrium conditions for long-time stability constant. Changes in pH, ionic shifts and complexing agents have been identified as the underlying principles causing structural changes in the sense of degeneration of otoconia . The low stability of calcite against chemical attacks in aqueous solution, together with the fact that otoconia are already formed during embryonic stages and normally do not undergo significant growth- and repair-scenarios with life time, clearly explain their sensitivity against any change in chemical composition of the surrounding liquids in the inner ear. Especially nowadays, this situation is of significant importance because of the various pharmaceutical products we take in the course of our life, and which reach our inner ear.
Age related degenerate otoconia are shown in Fig. 16. Although the destructive agents are not known in this special case, the image is characteristic for chemical attacks which, in general, start in the belly regions leading to extensive holes and fissures . The dissolution scenarios proceed in analogy to the developments shown in Figs. 5–7 which are finally characterized by complete dissolution of the belly areas and formation of fragments which mainly represent parts of the branches. In any case, otoconia may lose their contacts to the interconnecting fibril net and, as a consequence, dislodgement into the endolymph is possible (see above: BBPV and MD).
Although classified as othotoxic, aminoglycosides (such as gentamicin) are widely used as antibiotics. The othotoxicity of aminoglycosides is mainly discussed in connection with blocking mechanisms in, and death of sensory hair cells [65, 66]. The structure of gentamicin indicates that the molecule may act as a complexing agent for calcium ions. In fact, gentamicin causes irreversible structural damage of otoconia by progressive dissolution of calcite . The sequence of ESEM-images presented in Fig. 20 impressively verifies the complete dissolution of the calcite component of human otoconia within minutes. Again, the chemical attack starts in the belly area and extends over the branches until the otoconium is completely dissolved. The images clearly reveal that otoconia as solid composite specimens are threatened by chemical attacks, in general.
Conclusion and outlook
The successful growth of artificial (biomimetic) otoconia led to a fundamental breakthrough in the fields of biomineralisation as well as inner-ear research with the focus on balance and sensing of linear accelerations. From the point of view of biomineralisation, artificial otoconia represent the first example of mimicking a solid functional biomaterial not only in outer shape but also in composite structure and hierarchical inner architecture. From the medical point of view, the possible use of biomimetic otoconia for fast and immediate investigation of structural and chemical changes as well as for observations of their movement behavior under external (mechanical) influences are of great significance. Finally, even the bigger size of biomimetic otoconia is advantageous in several respects.
The interdisciplinary approach with the aim of deeper understanding of the growth, structure and function of otoconia required a close cooperation between the disciplines chemistry and medicine. After having extensively investigated the composite nature and the hierarchical architecture of otoconia, a significant step in direction of their function and general principles of degeneration could be taken. The sum of facts known up to now, allows an immediate outline of suitable strategies for future work which has to be done on this subject. To shortly name some of the most pressing topics, these will be directed on the question of symmetry (density distribution), the development of repair scenarios, and the arrangement and size distribution of otoconia as a whole.
The author gratefully acknowledges the close and fruitful co-operation with all the authors which contributed to the common publications [22–26, 31, 36–40]. Particular thanks is given to Jana Buder, Horst Borrmann, Raul Cardoso-Gil, Wilder Carrillo-Cabrera and Paul Simon (all: MPI for Chemical Physics of Solids, Dresden, Germany) as well as to Leif Erik Walther (University Medicine Mannheim, Germany) for constructive support in recent years.
W. Kahle, M. Frotscher. Color Atlas and Textbook of Human Anatomy, Vol. 3, Nervous System and Sensory Organs (5thed.). Thieme Medical Publishers, Stuttgart (2003).Google Scholar
K. Simkiss, K. M. Wilbur. Biomineralisation: Cell Biology and Mineral Deposition. Academic Press, San Diego (1989).Google Scholar
E. Bäuerlein. Biomineralisation: Progress in Biology, Molecular Biology and Application. Wiley-VCH, Weinheim (2004).Google Scholar
E. Bäuerlein. Handbook of Biomineralisation: Biological Aspects and Structure Formation. Wiley-VCH, Weinheim (2007).Google Scholar
P. Westbroek, E. W. de Jong. Biomineralisation and Biological Metal Accumulation. D. Reidel Publ. Comp., Dordrecht (1983).Google Scholar
M. D. Ross, D. Peacor, L. G. Johsson, L. F. Allard. Ann. Otol. Rhinol. Laryngol.85, 310 (1976).Google Scholar
Y. S. Jang, C. H. Hwang, J. Y. Shin. Laryngoscope116, 996 (2006).Google Scholar
D. Berggren, E. Klein, R. Wroblewski, M. Anniko. Acta Otolaryngol. 112, 779 (1992).Google Scholar
M. D. Ross, K. G. Pote. Philos. Trans. R. Soc. London Ser. B.304, 445 (1984).Google Scholar
U. Lins, M. Farina, M. Kurc, G. Riordan, R. Thalmann, J. Thalmann, B. Kachar. J. Struct. Biol.131, 67 (2000).Google Scholar
S. Mann, J. Webb, R. J. P. Williams. Biomineralization: Chemical and Biochemical Perspectives. VCH, Weinheim (1989).Google Scholar
S. Mann. Biomineralization: Principles and Concepts in Bioinorganic Materials Chemistry. Oxford University Press, Oxford (2001).Google Scholar
G. Fu, S. R. Qiu, C. A. Orme, D. E. Morse, J. J. De Yoreo. Adv. Mater.17, 2678 (2005).Google Scholar
S. Mann, D. D. Archibald, J. M. Didymus, T. Douglas, B. R. Heywood, F. C. Meldrum, N. J. Reeves. Science261, 1286 (1993).Google Scholar
A.-W. Xu, M. Antonietti, S. H. Yu, H. Cölfen. Adv. Mater.20, 1333 (2008).Google Scholar
R.-Q. Song, H. Cölfen, A.-W. Xu, J. Hartmann, M. Antonietti. Amer. Chem. Soc. Nano3, 1966 (2009).Google Scholar
H. Cölfen, M. Antonietti. Mesocrystals and Nonclassical Crystallization. Wiley, Hoboken (2008).Google Scholar
Y.-X. Huang, J. Buder, R. Cardoso-Gil, Y. Prots, W. Carrillo-Cabrera, P. Simon, R. Kniep. Angew. Chem. Int. Ed.47, 8250 (2008).Google Scholar
L. A. Everett, I. A. Beleyantseva, K. Noben-Trauth, R. Cantos, A. Chen, S. I. Thakkar, S. L. Hoogstraten-Miller, B. Kachar, D. K. Wu, E. D. Green. Hum. Mol. Genet.10, 153 (2001).Google Scholar
D. Carlstrom, H. Engstrom. Acta Oto-Laryngol.45, 14 (1955).Google Scholar
D. Carlstrom. Biol. Bull.125, 441 (1963).Google Scholar
L. E. Walther, A. Blödow, J. Buder, R. Kniep. PLOS ONE9, 1 (2014).Google Scholar
H. Li, L. A. Estroff. J. Amer. Chem. Soc.129, 5480 (2007).Google Scholar
C. D. Fermin. Microsc. Res. Techn.25, 297 (1993).Google Scholar
R. Kniep, J. Buder, L. Walther. In preparation (2015).Google Scholar
Y. Wang, P. E. Kowalski, I. Thalmann, D. M. Ornitz, D. L. Mager, R. Thalmann. Proc. Natl. Acad. Sci. USA95, 15345 (1998).Google Scholar
W. Lu, D. Zhou, J. J. Freeman, I. Thalmann, D. M. Ornitz, R. Thalmann. Hearing Res.268, 172 (2010).Google Scholar
Y. W. Lundberg, X. Zhao, E. N. Yamoah. Brain Res.1091, 47 (2006).Google Scholar
E. Murayama, P. Herbomel, A. Kawakami, H. Takeda, H. Nayasawa. Mechanisms Development122, 791 (2005).Google Scholar
M. R. Deans, J. M. Peterson, G. W. Wong. PLOS ONE5, 12765 (2010).Google Scholar
T. Koide, K. Nataya. In: Top. Curr. Chem.: Collagen Primer in Structure, Processing and Assembly. J. Brinkmann, H. Notbohm, P. K. Müller (Eds.). Springer, Berlin, 247 (2005).Google Scholar
J. L. Arias, M. S. Fernandez. Chem. Rev.108, 4475 (2008).Google Scholar
J. J. M. Lenders, A. Dey, P. H. H. Bomans, J. Spielmann, M. R. Hendrix, G. deWith, F. C. Meldrum, S. Harder, N. A. Sommerdijk. J. Am. Chem. Soc.134, 1367 (2012).Google Scholar
R. Thalmann, E. Ignatova, B. Kachar, D. M. Ornitz, I. Thalmann. Ann. N. Y. Acad. Sci.942, 162 (2001).Google Scholar
X. Zhao, S. M. Jones, E. M. Yamoah, Y. W. Lundberg. Neurosci.153, 289 (2008).Google Scholar
L. Walther, A. Wenzel, J. Buder, M. B. Bloching, R. Kniep, A. Blödow. Eur. Arch. Otorhinolaryngol.271, 3133 (2014).Google Scholar
C. Morgenstern, H. Miyamoto, W. Arnold, K.-H. Vosteen. Acta Otolayryngol.93, 187 (1982).Google Scholar
N. Mori, N. Uozumi, H. Furuta, H. Hoshikawa. Acta Otolaryngol. Suppl. 533, 12 (1998).Google Scholar
V. Couloigner, M. Teixeira, O. Sterkers, E. Ferrary. Acta Otolaryngol.119, 200 (1999).Google Scholar
H. Rask-Andersen, J. E. DeMott, D. Bagger-Sjöbäck, A. N. Salt. Hear. Res.138, 81 (1999).Google Scholar
V. Couloigner, A. B. Grayeli, O. Sterkers, E. Ferrary. Ann. Otol. Rhinol. Laryngol.117, 123 (2008).Google Scholar
A. Campos, P. V. Crespo, J. M. Garcia, M. C. Sanchez-Quevedo, M. Ciges. Acta Otolaryngol.119, 203 (1999).Google Scholar
D. J. Lim. Scanning Electron Microsc.3, 929 (1979).Google Scholar
M. D. Ross, T. E. Komorowski, K. M. Donoran, K. G. Pote. Acta Otolaryngol.103, 56 (1987).Google Scholar
C. D. Fermin, D. Lychakov, A. Campos. Histol. Histopath.113, 1103 (1998).Google Scholar
L. G. Johnson, J. E. Hawkins. Ann. Otol. Rhinol. Laryngol.81, 179 (1972).Google Scholar
S. Takano, H. Iguchi, H. Sakamoto, H. Yamane, M. Anniko. Acta Otolaryngol.133, 692 (2013).Google Scholar
A. P. Calzada, I. A. Lopez, G. Ishiyama, A. Ishiyama. Otol. Neurotol.33, 1593 (2013).Google Scholar
L. P. Rybak, C. A. Whitworth. Drug Discov. Today10, 1313 (2005).Google Scholar
E. M. Priuska, J. Schacht. Biochem. Pharmacol.50, 1749 (1995).Google Scholar
L. Walther, A. Wenzel, J. Buder, A. Blödow, R. Kniep. Acta Oto-Laryngol.134, 111 (2014).Google Scholar