Fibrillary Plate Research Articles

Simple preparation of hydroxyapatite nanostructures derived from fish scales

The nanostructured hydroxyapatite (HAp) formation in fish scales were investigated. Calcium and phosphate ions were effectively accumulated in the scales by the immersion processes. The HAp nanoparticles arranged in one direction in the internal fibrillary plate layers were observed, revealing the crystal growth along with the collagen fibers to resultantly form the porous structures. Thus, the arrangement of collagen fibers inside the scales effectively form the HAp nanostructures.

Hierarchical structure and cytocompatibility of fish scales from Carassius auratus

To study the structure and the cytocompatibility of fish scales from Carassius auratus, scanning electron microscopy (SEM) was used to observe the morphology of fish scales treated with different processing methods. Based on varying morphologies and components, the fish scales can be divided into three regions on the surface and three layers in vertical. The functions of these three individual layers were analyzed. SEM results show that the primary inorganic components are spherical or cubic hydroxyapatite (HA) nanoparticles. The fish scales have an ~60° overlapped plywood structure of lamellas in the fibrillary plate. The plywood structure consists of co-aligned type I collagen fibers, which are parallel to the HA lamellas. X-ray diffraction (XRD), thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) and Fourier transform infrared (FTIR) analysis indicate that the main components are HA and type I collagen fibers. MC3T3-E1 cell culture results show a high cytocompatibility and the ability to guide cell proliferation and migration along the scale ridge channels of the fish scales. This plywood structure provides inspiration for a structure-enhanced composite material.

High Resolution Scanning Electron Microscope Examination of the Fish Scale: Inspiration for Novel Biomaterials

A Scanning Electron Microscopic study on the fish scale of Cyprinus carpio communis (freshwater carp), depicts remarkable structural and compositional characteristics which may be used as inspiration for novel biomaterial design. The fracture surface structure and sintered fish scale reveals an osseous layer on its dorsal side. It has a compact heterogeneous crystalloid-like structure of assorted shapes and sizes. The ventral side is made of orthogonally arranged mineralized needle like crystalloids template embedded in a fibrillary plate. Frozen scales revealed that this dorsal layer may contain high atomic number elements as it appeared bright with back scattered electron signals. The ventral side consists of collagen plates and a matrix, which are arranged orthogonally in a double-twisted, plywood-like structure. There are alternate crystalloid and matrix which are arranged orthogonally and forming 15-17 layers in between these two sides. This provides useful information of scale composition. Design features from the structure of the fish scale may be useful in the development of functional biomaterials, in various different fields including nano-composites, biopolymers and natural source of hydroxyapatite, used in applied therapeutic, pharmaceutical industries and semiconductor technology. The tolerance of the fish scale to high temperature and very low temperature cooling revealed unique characteristics for biomaterial fabrication. The orthogonally arranged ventral side plates of collagen embedded in proteoglycans may also prove to be a good source of scaffold material for cell culturing for tissue engineering.

Structure and composition of teleost scales from snakehead Channa argus (Cantor) (Perciformes: Channidae)

The structure of teleost scales from snakehead Channa argus was investigated using thermogravimetric analysis (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy‐dispersive analysis of X‐rays (EDAX), Fourier transform infra‐red spectroscopy (FTIR) and X‐ray diffraction (XRD). Thermal treatment of fish scales indicates that the fibrillary plate is partially calcified. SEM shows two kinds of scale denticles, arranged along the circuli in the anterior field and the lateral fields, respectively. TEM indicates the stratum laxum with abundant fibrils, chromatophores and capillary blood vessels within the scale covering, and shows the fibrillary plate as an ‘orthogonal plywood structure’ of stratified lamellae, consisting of 80–100 nm diameter collagen fibres co‐aligned in individual lamellae and alternated by c. 90° of the fibre alignment between adjacent lamellae. EDAX, FTIR and XRD show that the mineral phase of the scales is a carbonated hydroxyapatite with a Ca:P molar ratio of 1·85.

Microstructure, mechanical, and biomimetic properties of fish scales from Pagrus major

The fish scale of Pagrus major has an orthogonal plywood structure of stratified lamellae, 1–2 μm in thickness, consisting of closely packed 70- to 80-nm-diameter collagen fibers. X-ray diffraction, energy-dispersive X-ray analysis, and infrared spectroscopy indicate that the mineral phase in the scale is calcium-deficient hydroxyapatite containing a small amount of sodium and magnesium ions, as well as carbonate anions in phosphate sites of the apatite lattice. The tensile strength of the scale is high (∼90 MPa) because of the hierarchically ordered structure of mineralized collagen fibers. Mechanical failure occurs by sliding of the lamellae and associated pulling out and fracture of the collagen fibers. In contrast, demineralized scales have significantly lower tensile strength (36 MPa), indicating that interactions between the apatite crystals and collagen fibers are of fundamental importance in determining the mechanical properties. Thermal treatment of fish scales to remove the organic components produces remarkable inorganic replicas of the native orthogonal plywood structure of the fibrillary plate. The biomimetic replica produced by heating to 873 K consists of stratified porous lamellae of c-axis-aligned apatite crystals that are long, narrow plates, 0.5–0.6 μm in length and 0.1–0.2 μm in width. The textured inorganic material remains intact when heated to 1473 K, although the size of the constituent crystals increases as a result of thermal sintering.

Focal Scale Damage Among Chum Salmon (Oncorhynchus keta) of Hokkaido, Japan

Fifty percent of chum salmon (Oncorhynchus keta) sampled from seven hatcheries in Hokkaido, Japan, were found to have either damage to the osseous scale layer where a hole resulted or intrusion of the osseous layer into the underlying fibrillary plate. Damage resulting in a hole is not related to age, body size, sex, or hatchery stock size. Intrusion of the osseous layer is similarly not related to body size, sex, or stock size; however, this phenomenon may be related to age. All damage was confined to the first, and rarely extended into the second, annual growth zone, suggesting occurrence during that period and retention of the mark throughout the life cycle. No evidence was found to support the hypothesis that either type of focal scale damage is a result of reduced diet. Examination of historical records from one hatchery revealed that the phenomenon of a hole has occurred for at least the past 10 yr at that location. It is not presently possible to identify hatchery of origin using these traits; however, such permanent, uniformly occurring traits may be of use in the identification of chum salmon originating from Hokkaido.

Supracellular microtubule alignments in cell layers associated with the secretion of certain fish scales.

Intercellularly aligned microtubule arrays are present in cell layers associated with the growth and secretion of scales in the zebra fish Brachydanio rerio and the neon tetra fish Hyphessobrycon innesi. The layers in question are: the osteoblast layer that covers the ossified outer surface of a scale, and the layer of fibroblasts that is situated immediately underneath the inner collagenous surface of a scale's fibrillary plate. In certain osteoblasts, the proximal portions of microtubules (with respect to centrosomes) run closely alongside the anterior margin of each cell where it flanks one of a scale's ridge-shaped circuli. These osteoblasts and microtubule portions are arranged in aligned rows that are parallel to circuli. However, the distal portions of the microtubules curve into an orientation that is approximately at right angles to circuli and they are aligned with each other and similar microtubule portions in adjacent osteoblasts. Such microtubule alignments only occur in osteoblasts that are associated with circuli. In Hyphessobrycon osteoblasts situated elsewhere on a scale's surface, microtubules radiate from cell centres but their distal portions curve into alignment with each other and are oriented alongside cell margins. The proximal portions of fibroblast microtubules radiate from centrally positioned centrosomes but the distal portions curve into alignment with each other and distal microtubule portions in neighbouring fibroblasts. The overall pattern of microtubule alignment is similar to that of collagen fibres, which these fibroblasts are secreting onto the fibrillary plate. The immunofluorescence protocol that was used to demonstrate the microtubule alignments described above did not reveal such alignments in the osteoblast and fibroblast layers associated with scales of the brown trout Salmo trutta fario. These findings are assessed in terms of intra-and inter-cellular control of microtubule alignment, and decentralized reorientation of microtubules at distances of several micrometres from centrosomal microtubule-organizing centres. The functional significance of the relationships between microtubule alignment and supracellular patterns of alignment that take place as collagen deposition and ossification proceed during scale formation is also considered.

Fine Structure of the Developing Scale in the Cichlid Hemichromis bimaculatus (Pisces, Teleostei, Perciformes)

AbstractThe study of the formation and structure of the early teleost scale and its associated cells has been carried out on Hemichromis bimaculatus fry using in toto staining with alizarin and transmission electron microscopy techniques. Results of the study show very rapid scale formation in Hemichromis. The papilla of the scale differentiates a little in advance of the bone scale formation. No epidermal cells are involved in the constitution of the scale pocket made up of scleroblasts. In Hemichromis, as in other teleost scales, the osseous layer is the first one to be secreted by, presumably, only the scleroblasts. Then the scleroblasts specialize in their functions. Superficial ones are involved in the formation of osseous circuli; marginal scleroblasts are responsible for growth in diameter of the scale; while deep scleroblasts allow the scales to thicken owing to the progressive addition of collagen fibrils organized in a “plywood‐like” structure which constitutes the fibrillary plate of the scale. Mineralization occurs very rapidly within the osseous layer in the form of hydroxyapatite‐like crystal deposits. The fibrillary plate is not yet mineralized in Hemichromis at the stages studied here, but presumably is later. Results obtained in Hemichromis are discussed against similar data available in the literature on teleost scale formation.

Comparative aspects of calcium dynamics in calcified tissues in the goldfish Carassius auratus.

The comparative nature of calcium physiology in bone, scales and otoliths was studied in young goldfish, carassius auratus. Net calcium uptake by the fish was estimated to be 143 μg/gbody weigyt/day. Of that, 79% was distributed in bone, 13% in scales, 3.5% in otoliths and 4.5% in soft tissues. Scales showed the highest incorporation of 45Ca per mg-tissue weight after 1 or 2 days in 45Ca-containing water; bone came second and otoliths last. However, 35 days after transfer to non-radioactive water, the other of descending radioactivity had changed to otoliths, bone and scales, reflecting defferent rates of calcium turnover. In bone, prelabeled 45Ca activity increased for the fist 2 days after transfer and then decreased gradually (biological half-life, T0.5=94 days). In otoliths, prelabeled radioactivity consistently increased for 35 days. Scales showed two phases of calcium turnover. They lost about 33% of theri prelabeled radioactivity during the first 7 days (T0.5=10.5 days) in ono-radioactive water, but thereafter the rate of decrease slowed down greatly (T0.5=210 days). These two phases of calcium turnover were found in the osseous layer (including calcium crystals in the fibrillary plate) of scales, indicating the presence of physiologically labile as well as stable forms of calcium in the layer.

Ultrastructure of scales in a teleost (Carassius auratus L.) after use of rapid freeze-fixation and freeze-substitution.

New data on the ultrastructural features of the elasmoid scales of Carassius auratus have been obtained by use of rapid freezing with subsequent freeze-substitution in anhydrous solvents. These are compared with the results obtained using conventional aqueous fixatives. The external layer of the scales is composed of randomly oriented collagen fibres. In the first stages of mineralization, mineral deposits are located in the interfibrillary substance where dense granules appear to be active sites of mineralization. Spheritic mineralization occurs in this layer. The fibrillary plate is composed of two kinds of collagen fibres. Most of them are organized in lamellae forming the "plywood-like structure". They are thicker than the so-called "TC fibres", which are oriented from the basal part towards the superficial layer. These TC fibres are involved in the first stages of mineral deposition in the fibrillary plate where inotropic mineralization occurs. The mineral phase is almost always located in the interfibrillary matrix in both layers of the elasmoid scale. In this respect, teleost scales differ from those described so far in other lower vertebrates.

The fine structure of calcified Mandl's corpuscles in teleost fish scales

Calcified Mandl's corpuscles present in the internal layer (or fibrillary plate) of the teleost fish scale were studied by transmission and scanning electron microscopy for a better understanding of this special type of mineralization process. The corpuscles show a great variability in their structure, form and surface features depending on the arrangement of the collagen fibrils in the internal layer of the different fish species studied, on the localization of the corpuscles in the scale and on the technical treatment to which the scale is subjected.

Visualization of microtubules of cells in situ by indirect immunofluorescence.

Microtubule staining patterns can be visualized within cells in situ on the surface of fish scales from the squirrel fish, Holocentrus ascensionis, and the common goldfish, Carassius auratus, after incubation with antibodies to sea urchin tubulin and fluorescein-labeled goat antibodies to rabbit immunoglobulin G. Chromatophores in situ from both species reveal a radial microtubule framework that orients the alignment of pigment granules. Innervating fibers of erythrophores on the H. ascensionis scale can also be observed. In situ, pseudo-epithelial cells called scleroblasts show microtubule patterns with a remarkable degree of similarity within a selected region. Over 90% of the cells have a microtubule framework that is nearly superimposable from cell to adjacent cell. The microtubules in scleroblasts are few and form a simple radial framework with a localized microtubule organizing center (MTOC). Microtubules in scleroblasts in vitro emanate from localized MTOCs but are much less radially organized than in situ. Scleroblasts in situ on the scale of C. auratus show microtubules that curve abruptly into coalignment with phase striations on the fibrillary plate. The phase striations arise from the orthogonal plies of collagen in intimate association with the scleroblasts. The role of microtubules in scleroblasts may thus be to provide orientation for collagen fibrillogenesis, analogous to their role in orientation of cellulose fibers in plants. That cells in situ exhibit highly related and coordinated microtubule staining patterns reaffirms that the cytoskeleton plays an important role in the organization of differentiated tissues.

Studies on formation and resorption of fish scales. IV. Ultrastructure of developing scales in newly hatched fry of the sheepshead minnow, Cyprinodon variegatus (Atheriniformes: Cyprinodontidae).

Scale formation in Cyprinodon variegatus was found to be initiated at about 26 to 30 days after hatching. Ultrastructural investigation revealed that within 4 to 6 h in the first-formed scales the marginal cells begin to flatten and differentiate into osteogenic cells, which later change to osteoblasts and fibroblasts. These cells are separated from the surrounding epithelial cells by a basal lamina. The osteoid is formed by the marginal and osteogenic cells; the osseous layer by the osteoblasts; and the fibrillary plate by the fibroblasts. The osteoid is formed within 2 to 3 h after the initiation of the scale, and within 20 to 24 h the osseous layer is formed. Hydroxyapatite crystals are deposited in the matrix of the osseous layer without apparent association with collagen fibers. No matrix vesicles or dense bodies are evident at the sites of calcification. The fibrillary plate arises 18 to 20 h after the initiation of the scale. It is also partially calcified, but not before the third week of scale formation. The crystals develop almost exclusively between the collagen fibers at the extreme edge of the calcifying front, but solid calcification of the fibers results with futher growth of the crystals. The fibroblasts appear to participate in calcification of the fibrillary plate.

Studies on fish scale formation and resorption. III. Fine structure and calcification of the fibrillary plates of the scales in Carassius auratus (Cypriniformes: Cyprinidae).

Electron microscopic investigation of scales of the goldfish Carassius auratus revealed that the lamellae of fibrillary plates contain sheet-like structures composed of vertically oriented collagen fibers embedded in an organic matrix. The fibers (TC fibers) are smaller in diameter (35-45 nm) than those of the lamellae and the matrix is stained intensely with lead citrate. The sheet-like structures as well as the lamellae are formed by fibroblasts located beneath the lamellae. The orientation of collagen fibers of the sheets and the lamellae seems to be controlled by the orientation of the ridges and invaginations of the surface of the fibroblasts. The fibrillary plate of C. auratus was found to be partially calcified. Calcification was initiated by the deposition of needle-like or flaky crystals of hydroxyapatite in the organic matrix of the sheet-like structure and proceeded into the TC fibers and the matric region of the lamellae. The potassium pyroantimonate-osmium tetroxide method showed a heavy concentration of calcium in the osteoblasts, fibroblasts, and in the matrix regions of the fibrillary plate. Calcium-containing precipitates were also present in the "hole zone" of the collagen fibers in the lamellae, but the significance of this location in calcification remains to be elucidated.

Alkaline phosphatase activity in the skin of the Common goby, Pomatoschistus microps (Krøyer), in relation to cycles in scale and body growth

A histochemical study of the distribution of alkaline phosphatase in the skin of the Common goby, Pomatoschistus microps, showed that the enzyme is present in the dermis, not in the epithelium, and is more pronounced around the growing edge of the scale and below the fibrillary plate. The intensity of the enzyme reaction is higher in regenerated scales. In conjunction with the phenology of P. microps, a method was adopted to measure the enzyme activity in the skin, including scales, for successive months over a period of two years. Enzyme activity displayed seasonal fluctuations which were closely related to both body and scale growth. Enzyme activity also reflected other phenological cycles during the life‐span of P. microps.

Studies on fish scale formation and resorption. I. Fine structure and calcification of the scales in Fundulus heteroclitus (Atheriniformes: Cyprinodontidae).

Fine structure of the scales of Fundulus heteroclitus was examined by scanning and transmission electron microscopy. The concentric ridges of the scale surface were characterized by the presence of minute, highly calcified, denticles or tooth-like processes. Needle-shaped crystals of hydrox-yapatite were precipitated not only in the osseous layer but in the intimate lamellae of the fibrillary plate except in portions just below the grooves. The calcification of the osseous layer was observed to proceed by filling the matrix with patches of crystals. The fibrillary plate appeared to calcify by invasion of crystals from the upper calcified zone into spaces between collagen fibers.

サクラマス雄魚の早熟残留型にみられる鱗吸収と関連細胞について

Scale resorption was found to occur in the precocious male parr of the masu salmon (Oncorhynchus masou) during autumn and winter. A histological study was made on those resorbing scales and the associated cells. Resorption commences at the posterior scale margin and advances gradually to the anterior. In a cross section, both the osseous layer and the fibrillary plate seem to be affected successively by the activity of cells attached around the resorbing margin. In the early phase of reorption, these cells show a morphological resemblance to those of the growing scale margin. After considerable destruction of the margin, however, there appear multinucleated giant cells which are similar to the osteoclasts of bone tissues. It is presumed that the cells involved in the resorption of the scale may be derived from ancestor cells in the connective tissue of the scale pocket, or from a functional conversion of the scale forming cells.

サケ鱗発生初期の微細構造

Early phases of scale development in the chum salmon (Oncorhynchus keta) were studied by electron microscopy. The osseous matrix, composed of fine collagenous fibrils and dense ground substance, is secreted from cells of the scale papilla aggregated in the subepidermal connective tissue. The presence of dilated cisternae of the granular endoplasmic reticulum, rather numerous mitochondria, and abundant free ribosomes characterizes these osteogenic cells. Calcification of the matrix begins with the appearance of clusters of needle-shaped hydroxyapatite crystallites being limited within small dense particles scattered in the matrix. As the calcification progresses, they increase in number as well as in size. Thick collagen fibrils are added onto the lower surface of the calcified osseous layer by another kind of cells to form the fibrillary plate. These cells, rectangular in form, are characterized by a parallel array of highly extensive granular endoplasmic reticulum and well-developed Golgi zones with lysosomal bodies. The scale ridge arises as a partial thickening of the osseous layer. The ridge in cross section appears to be formed when an upper osteogenic cell is overlaid obliquely by the adjacent outer cell in the process of flattening of cells of the scale margin.

Fine structure of scale development in the teleost, Brachydanio rerio.

AbstractMature and embryonic scales of the zebrafish, Brachydanio rerio, were examined by light and electron microscopy. Each scale consists of a mineralized “osseous layer” superficially and a deeper, non‐mineralized, “fibrillary plate.” The mineralizing matrix contains randomly oriented filaments in decalcified sections, whereas the fibrillary plate is composed of orthogonally arranged lamellae of banded collagen fibrils embedded in electron dense material.Scale papillae and small scales first appear in the midbody region of fry between 0.95 and 1.14 cm long. The matrix of the osseous layer is produced prior to the fibrils of the fibrillary plate. Foci of mineral deposition appear in this matrix soon after its production, and increase gradually in number and extent. Cells surrounding the periphery of the scale are continuous with two layers of cells beneath the inner surface and with two layers extending a variable distance over the superficial surface. These “scale‐associated” cells are separated from the dermal collagen by other investing cell processes. The probable roles of these cells in scale formation are discussed and the need for further investigation of the fish scale as a mineralizing system is stressed.

Mucopolysaccharides and the Calcification of the Scale of the Roach (Leuciscus rutilus)

ABSTRACT The scale of the roach (Leuciscus rutilus L.) was examined for state of calcification, metachromasy, and reaction to the periodic acid / Schiff (PAS) test. Metachromasy and a positive PAS-reaction imply acid mucopolysaccharides in the bony layer. There is a great increase in these reactions and in reactions for bone salts in the transition zone between the uncalcified and calcified part of the bony layer. These reactions imply that the bond between the osseoid and the inorganic fraction of the bony layer is through SO2-and PO4-groupings. The fibrillary plate lacks metachromasy, but shows a positive PAS-reaction. Before calcification a strong orthochromasy points to acid groups in connexion with the collagen. Under the radii there is no orthochromasy, and the PAS-reaction is negative. When calcifying, the fibrillary plate loses its orthochromasy and the mineral matter is deposited as large crystal-complexes. In regenerating scales the reactions are weaker than in normal scales.