Matrix Heterogeneity in the Radular Teeth of the Chiton Acanthopleura hirtosa

The structure and organization of the organic matrix of the cusps of the major lateral teeth of the chiton Acanthopleura hirtosahave been examined using conventional light and transmission electron microscopy techniques and by using the protein ferritin as an ultrastructural probe. The results show major structural differences in the organic matrix between the surface layers of the anterior (calcified) region and the posterior (magnetite-mineralized) region and their respective underlying regions. In addition, the central (lepidocrocite-mineralized) region of the tooth has been examined and shown to consist of bundles of fibres arranged such that they display a tightly interwoven pattern. It is suggested that while the structural organization of surface fibres readily permits the passage of ions required for mineralization, the architecturally discrete distribution of biominerals found in mature chiton teeth is due mostly to spatial delineation of the tooth by matrix macromolecules in the central region of the tooth.     

红条毛肤石鳖齿舌主侧齿中铁还原酶分布及与生物矿化的关系

以红条毛肤石鳖Acanthochiton rubrolineatus(Lischke)齿舌为材料,通过切片和酶组织化学技术,在光镜和电镜下对齿舌主侧齿的微结构及高铁还原酶的存在进行观察,从微观角度了解齿舌主侧齿齿尖的矿化机理。结果显示,成熟主侧齿由齿尖和齿基组成。齿尖结构由外至内分为三层,最外层为磁铁矿层,前后齿面磁铁矿层的厚度不等,后齿面约50μm,前齿面约5-10μm。向内依次为棕红色的纤铁矿层,厚约10μm,及略显黄色的有机基质层,有机基质层占据着齿尖内部的大部分结构。高分辨透射电镜下显示磁铁矿由条状四氧化三铁颗粒组成,长约2-3μm,宽约100-150nm。齿舌的矿化是一个连续过程,不同部段处于不同的矿化阶段,齿舌囊上皮细胞沿囊腔分布,并形成齿片。未矿化的新生主侧齿齿尖中存在由有机基质构成的网状结构。随矿化的进行,有机基质内出现矿物颗粒。初始矿化的齿尖外表面有一个细胞微突层,微突的另一端为囊上皮细胞,矿物质经由微突层达齿尖并沉积于有机基质中,齿尖随之矿化并成熟。初始矿化齿尖的外围有大量的三价铁化物颗粒,稍成熟的齿尖外围同时还出现二价铁化物。新生或初始矿化主侧齿齿尖外围的囊上皮细胞中有大量球形类似于铁蛋白聚集体的内容物,直径0.6-0.8μm,球体由膜包围。齿舌囊上皮组织中存在三价高铁还原酶,此酶分布于上皮细胞的膜表面,可能与齿尖表面磁铁矿的生成有一定的关系。       

A new biomineral identified in the cores of teeth from the chiton <Emphasis Type="Italic"> Plaxiphora albida</Emphasis>

The hydrated iron(III) oxide limonite is reported for the first time as a biomineral. In situ laser Raman spectra of the tooth cores from major lateral teeth of the chiton Plaxiphora albida are compared with those of synthetic and mineral iron phosphates and iron oxides. Raman spectra measured on iron phosphate and iron oxide standard materials are shown to be easily distinguishable from one another. The central tooth cores of mature P. albida teeth do not show any evidence for the presence of a separate iron phosphate mineral. Rather, in each tooth a narrow band of the hydrated iron(III) oxide limonite is shown to separate the magnetite of the tooth surface from a central core region comprising both lepidocrocite and limonite. The high concentration of phosphorus in P. albida tooth cores, previously observed by energy dispersive spectroscopy, is not associated with a separate iron phosphate mineral, indicating that this element may be adsorbed onto the surface of the iron oxide minerals present. The failure to detect a separate iron(III) phosphate is discussed with reference to other chiton species that display high levels of iron and phosphorus in the cores of their mature major lateral teeth.     

The chiton stylus canal: An element delivery pathway for tooth cusp biomineralization

A detailed investigation of the stylus canal situated within the iron mineralized major lateral teeth of the chiton Acanthopleura hirtosa was undertaken in conjunction with a row‐by‐row examination of cusp mineralization. The canal is shown to contain columnar epithelial tissue similar to that surrounding the mineralized cusps, including the presence of iron rich particles characteristic of the iron storage protein ferritin. Within the tooth core, a previously undescribed internal pathway or plume is evident above the stylus canal, between the junction zone and mineralizing posterior face of the cusp. Plume formation coincides with the appearance of iron in the superior epithelium and the onset of mineralization at tooth row 13. The plume persists during the delivery of phosphorous and calcium into the tooth core, and is the final region of the cusp to become mineralized. The presence of the stylus canal was confirmed in a further 18 chiton species, revealing that the canal is common to polyplacophoran molluscs. These new data strongly support the growing body of evidence highlighting the importance of the junction zone for tooth mineralization in chiton teeth, and indicate that the chemical and structural environment within the tooth cusp is under far greater biological control than previously considered. J. Morphol. 2009. © 2008 Wiley‐Liss, Inc.     

The junction zone: Initial site of mineralization in radula teeth of the chiton Cryptoplax striata (Mollusca: Polyplacophora)

Elemental composition and distribution in individual teeth of the whole radula of the chiton Cryptoplax striata were analyzed using energy-dispersive spectroscopy. Both the element deposited and its position within the tooth vary according to the stage of mineralization. The initial site of mineralization is the junction zone, the region between the tooth cusp and base. In this region, the first element to be deposited is iron, followed by phosphorus and then calcium. Iron deposition next commences in the tooth cusp cap, where it proceeds rapidly, being virtually complete within 12 tooth rows. By contrast, mineralization in the core of the tooth cusp does not commence until well down the radula and consists initially of iron and phosphorus with the addition of a small amount of calcium 6 rows later. While mineralization in the tooth base commences early in radula development, it continues right through to the fully mature end of the radula. A number of minor elements are also found at various stages of mineralization. The data obtained have been used to construct a schematic of the progression of mineralization along the length of the radula. © 1996 Wiley-Liss, Inc.     

Atomic force microscopy study of tooth surfaces

Atomic force microscopy (AFM) was used to study tooth surfaces in order to compare the pattern of particle distribution in the outermost layer of the tooth surfaces. Human teeth and teeth from a rodent (Golden hamster), from a fish (piranha), and from a grazing mollusk (chiton) with distinct feeding habits were analyzed in terms of particle arrangement, packing, and size distribution. Scanning electron microscopy and transmission electron microscopy were used for comparison. It was found that AFM gives high-contrast, high-resolution images and is an important tool as a source of complementary and/or new structural information. All teeth were cleaned and some were etched with acidic solutions before analysis. It was observed that human enamel (permanent teeth) presents particles tightly packed in the outer surface, whereas enamel from the hamster (continuously growing teeth) shows particles of less dense packing. The piranha teeth have a thin cuticle covering the long apatite crystals of the underlying enameloid. This cuticle has a rough surface of particles that have a globular appearance after the brief acidic treatment. The similar appearance of the in vivo naturally etched tooth surface suggests that the pattern of globule distribution may be due to the presence of an organic material. Elemental analysis of this cuticle indicated that calcium, phosphorus, and iron are the main components of the structure while electron microdiffraction of pulverized cuticle particles showed a pattern consistent with hydroxyapatite. The chiton mineralized tooth cusp had a smooth surface in an unabraded region and a very rough structure with the magnetite crystals (already known to make part of the structure) protruding from the surface. It was concluded that the structures analyzed are optimized for efficiency in feeding mechanism and life span of the teeth.     

Fine structure of the mineralized teeth of the chiton Acanthopleura echinata (Mollusca: Polyplacophora)

The major lateral teeth of the chiton Acanthopleura echinata are composite structures composed of three distinct mineral zones: a posterior layer of magnetite; a thin band of lepidocrocite just anterior to this; and apatite throughout the core and anterior regions of the cusp. Biomineralization in these teeth is a matrix-mediated process, in which the minerals are deposited around fibers, with the different biominerals described as occupying architecturally discrete compartments. In this study, a range of scanning electron microscopes was utilized to undertake a detailed in situ investigation of the fine structure of the major lateral teeth. The arrangement of the organic and biomineral components of the tooth is similar throughout the three zones, having no discrete borders between them, and with crystallites of each mineral phase extending into the adjacent mineral zone. Along the posterior surface of the tooth, the organic fibers are arranged in a series of fine parallel lines, but just within the periphery their appearance takes on a "fish scale"-like pattern, reflective of the cross section of a series of units that are overlaid, and offset from each other, in adjacent rows. The units are approximately 2 microm wide and 0.6 microm thick and comprise biomineral plates separated by organic fibers. Two types of subunits make up each "fish scale": one is elongate and curved and forms a trough, in which the other, rod-like unit, is nestled. Adjacent rod and trough units are aligned into large sheets that define the fracture plane of the tooth. The alignment of the plates of rod-trough units is complex and exhibits extreme spatial variation within the tooth cusp. Close to the posterior surface the plates are essentially horizontal and lie in a lateromedial plane, while anteriorly they are almost vertical and lie in the posteroanterior plane. An understanding of the fine structure of the mineralized teeth of chitons, and of the relationship between the organic and mineral components, provides a new insight into biomineralization mechanisms and controls.     

Contribution of Raman spectroscopy to identification of biominerals present in teeth of Acanthopleura rehderi,Acanthopleura curtisiana,and Onithochiton quercinus

Raman spectroscopic investigations of the major lateral teeth of the chitons Acanthopleura rehderi and Acanthopleura curtisiana indicate that, in addition to the magnetite of the cutting surface and a carbonated hydroxyapatite in the central tooth core, these species deposit limonite in place of the lepidocrocite reported for other members of the genus Acanthopleura. A comparison of the spectra from these species with those of Onithochiton quercinus, which also deposits limonite, indicates that the current assignment of these species to Acanthopleura may not be appropriate. Biomineralization of the major lateral teeth may be a useful parameter to include in the taxonomic classification of chiton species.     

Characterization of the calcium biomineral in the radular teeth of Chiton pelliserpentis

The radula in a group of molluscan invertebrates, the chitons (Polyplacophora), is a ribbon-like apparatus used for feeding and which bears a series of distinctive mineralized teeth called the major lateral teeth. While some chiton species deposit only iron biominerals in these teeth, many others deposit both iron and calcium. In this study, the calcium biomineral in the teeth of one of the latter types of species, the Australian east-coast chiton, Chiton pelliserpentis, has been isolated and examined for the first time. Spectroscopic and crystallographic techniques have identified the biomineral as a carbonate-substituted apatite with significant fluoride substitution also likely. Fourier-transform infrared and laser Raman spectroscopy indicated that the carbonate content was less than that of either bovine tibia cortical bone or human tooth enamel. X-ray diffraction analysis showed the biomineral to be poorly crystalline due to small crystal size and appreciable anionic substitution. The lattice parameters were calculated to be a=9.382?Å and c=6.883?Å, which are suggestive of a fluorapatite material. It is postulated that structural and biochemical differences in the tooth organic matrix of different chiton species will ultimately determine if the teeth become partly calcified or iron mineralized only.     

In situ Raman spectroscopic studies of the teeth of the chiton Acanthopleura hirtosa

 In situ Raman spectroscopy, in combination with energy dispersive spectroscopy, has been used for the first time to determine the identities and locations, at the micron level, of mineral phases present in single chiton teeth that have been extensively mineralized. At the later stages of development the major lateral teeth of the chiton Acanthopleura hirtosa show characteristic spectroscopic evidence for the presence of lepidocrocite (γ-FeOOH), magnetite (Fe₃O₄), and an apatitic calcium phosphate. Goethite (α-FeOOH) and ferrihydrite (5 Fe₂O₃·9 H₂O), which have been detected previously in teeth at the early stages of mineralization, were not detected in this mature tooth. The spatial distribution of these phases was determined, providing evidence for the presence of a discrete layer of lepidocrocite between the magnetite and apatite regions, illustrating the complexity of the biomineralization process. The technique of laser Raman microscopy is shown to be ideal for the examination of small biomineralized structures in situ, such as chiton teeth. Received: 6 July 1998 / Accepted: 19 August 1998     

Magnetic and structural properties of magnetite in radular teeth of chiton Acanthochiton rubrolinestus

The teeth of the Polyplacophora Chiton Acanthochiton Rubrolinestus contain biomineralized magnetite crystallites whose biological functions in relation to structure and magnetic properties are not well understood. Here, using superconducting quantum interference device (SQUID) magnetometry, we find that the saturation magnetization (σ(s)) and the Verwey transition temperature (T(v)) of tooth particles are 78.4 emu/g and 105 K, respectively. These values are below those of the stoichiometric magnetite. An in situ examination of the structure of the magnetite-bearing region within an individual tooth using high-resolution transmission electron microscopy indicates magnetite microcrystals form electron dense polycrystalline sheets with typical lengths of about 800 nm and widths of about 150 nm. These polycrystalline sheets are arranged regularly along the longitudinal direction of the tooth cutting surface. In addition, the crystallites in polycrystalline sheets take on generally good crystallinity. The magnetic microstructures of in situ magnetic force microscopy demonstrate that the [111] easy direction of magnetite microcrystals are aligned along the length of the tooth, whereas the [111] direction is parallel to the thickness of the tooth. Both M?ssbauer spectra and magnetization versus temperature measurements under field cooled and zero-field cooled conditions do not detect superparamagnetic magnetite crystallites in the mature major lateral tooth particles of this chiton.     

海洋软体动物齿舌中磁铁物的研究

在扫描电镜下对红条毛肤石鳖（ Ａｃａｎｔｈｏｃｈｉｔｏｎ ｒｕｂｒｏｌｉｎｅａｔｕｓ Ｌｉｓｃｈｋｅ） 齿舌进行了观察,用原子力和磁力显微镜及超导量子干涉器（ＳＱＵＩＤ） 式磁强计对齿舌中的磁铁物Ｆｅ３ Ｏ４ 进行了分析和测量,实验证明齿舌中含有磁铁物Ｆｅ３ Ｏ４ ,每个齿舌约含Ｆｅ３ Ｏ４ ０．２ ｍｇ ,占齿舌重量的１５ ％ ,矿物重量的４０％ ,磁化强度约为０．０２×１０ － ３ Ａｍ２／ 个,相当于１４Ａｍ２／ｋｇ ,并且磁铁物主要存在于第一侧齿的齿尖上,同时Ｆｅ３ Ｏ４ 晶体在齿片表面上的排列及磁畴结构具有方向性。     

Proteomic analysis from the mineralized radular teeth of the giant Pacific chiton, Cryptochiton stelleri (Mollusca)

The biomineralized radular teeth of chitons are known to consist of iron-based magnetic crystals, associated with the maximum hardness and stiffness of any biomineral. Based on our transmission electron microscopy analysis of partially mineralized teeth, we suggest that the organic matrix within the teeth controls the iron oxide nucleation. Thus, we used Nano-LC-MS to perform a proteomic analysis of the organic matrix in radular teeth of the chiton Cryptochiton stelleri in order to identify the proteins involved in the biomineralization process. Since the genome sequence of C. stelleri is not available, cross-species similarity searching and de novo peptide sequencing were used to screen the proteins. Our results indicate that several proteins were dominant in the mineralized part of the radular teeth, amongst which, myoglobin and a highly acidic peptide were identified as possibly involved in the biomineralization process.     

Silicification of the medial tooth in the blue crab Callinectes sapidus

Observations of cuticular structures mineralized with silica within the Crustacea have been limited to the opal teeth of copepods, mandibles of amphipods, and recently the teeth of the gastric mill in the blue crab Callinectes sapidus. Copepod teeth are deposited during premolt, with sequential elaboration of organic materials followed by secretion of silica into the tooth mold. The timing of mineralization is in stark contrast to that of the general integument of crustaceans in which calcification is completely restricted to the postmolt period. To determine the timing of molt‐related deposition and silicification of the teeth of the gastric mill, the medial tooth of the blue crab C. sapidus was examined histologically and ultrastructurally across the molt cycle. Histological data revealed deposition of the organic matrix of the epicuticle and exocuticle during premolt. No evidence of postmolt changes in the thickness of the epicuticle and exocuticle, or any deposition of endocuticle, was observed. Scanning electron microscopy revealed degradation of the outer surface of the old tooth during premolt. During premolt, epithelial structures resembling papilla appeared to secrete a fibrous web that coalesces to become the matrix of the new tooth. Semi‐quantitative elemental analyses indicated simultaneous deposition of silica and organic matrix, and demonstrated a homogeneous distribution of silicon throughout the epicuticle of the tooth at all stages. However, there is evidence of deposition (presumably silicification) during postmolt as spaces between the papillae become filled in. Thus, the pattern and timing of deposition and silicification of the tooth are different from both teeth of copepods and the general exoskeleton of decapods, and may facilitate rapid resumption of feeding and consumption of the exuvia in early postmolt. J. Morphol. 277:1648–1660, 2016. © 2016 Wiley Periodicals, Inc.     

The mineralization and hardness of the radular teeth of the limpet Patella vulgata L.

Summary A study of the Patella vulgata radula has been made using: the scanning electron microscope in its normal and compositional contrast modes of operation, the electron microprobe analyser, ion etching with argon ions and microhardness testing.Only iron, silicon and small amounts of sulphur were detected in the radula. The teeth can be subdivided into a cusp, a junctional area where the cusp is joined to the base, and the base which is embedded in the radular membrane. From a study of longitudinal vertical and transverse sections of the mature teeth it was found that the cusp could be subdivided into a posterior iron-rich area (44–51% Fe, 1–6% Si) and an anterior silicon-rich area (22–30% Fe, 27–32% Si). The junctional zone consisted of a poorly mineralised layer at its border with the cusp and an iron-rich layer where it joined the base. The upper part of the base (5% Fe, 16% Si) could be clearly differentiated from the silicon-rich anterior and lower parts of the base (3–4% Fe, 28–35% Si). No minerals were detected in the membrane. The changes in the mineral content of the teeth cusps along the length of the radula were studied. Iron appeared in the cusps at the 25th row and the concentration increased to 28% at the 50th row. The iron was here evenly distributed throughout the cusp. Silicon appeared in the anterior part of the cusp at the 50th row and as it increased in concentration so the iron was displaced, and at the same time the concentration of iron increased in the posterior part of the cusp. Mineralization appeared to be complete by the 150th row.The teeth cusps appear to consist of 800 Å fibres grouped into 1 thick bundles and the tooth appears to be covered by a thin enamel-like layer. It is suggested that the fibres contain the silicon-rich phase and the matrix the iron-rich phase.The significance of the arrangement of the fibres and the distribution of the minerals are discussed with relation to the function of the teeth.We wish to thank Mr. A. Rees and Mr. A. Davies for their technical assistance; Prof. Lewis and Dr. James for the use of the Electron Microprobe; and the S.R.C. for their financial support.     

湖北郧西黄龙洞更新世晚期人类牙齿磨耗与使用痕迹

对2004—2006年在黄龙洞发现的7枚人类牙齿磨耗与使用痕迹的观察显示: 除具有正常牙齿相互接触造成的磨耗外, 黄龙洞人类牙齿还呈现出一些特殊的使用痕迹, 包括明显的前部牙齿釉质破损与崩裂、上颌侧门齿齿冠唇面釉质破损、上颌前部牙齿齿间邻接面沟等。根据这些牙齿使用痕迹集中在前部牙齿, 釉质破损与崩裂主要分布在靠近切缘的上颌门齿唇面及下颌门齿舌面的情况, 推测生活在黄龙洞的更新世晚期人类经常使用前部牙齿从事啃咬、叼衔、或剥离等动作, 并可能将前部牙齿作为工具使用。分布在前部牙齿的齿间邻接面沟提示当时人类经常从事剔牙活动。黄龙洞人类前部牙齿的使用痕迹与当时人类获取、处理及食用附着在骨骼上的筋或肉的动作密切相关, 当时人类的食物构成中可能包含有较多的肉类及粗纤维植物。     

Macrofibres in the cuticle of the crab Callinectes gladiator (Benedict)

The cuticle of Callinectes gladiator resembles that of Carcinus maenas (Dennell, 1973) in containing large sinuous unbranched fibres lying between the laminae of the calcified zone. The overlying pigmented and calcified zone also contains large fibres, but these are more densely packed, branched, and tubular. In addition, much finer parallel fibres have been observed in horizontal sections.     

Biomineralization of limpet teeth: a cryo-TEM study of the organic matrix and the onset of mineral deposition

The continuously growing limpet radula contains teeth at various stages of maturity and thus provides an excellent opportunity for studying the processes and mechanisms of their mineralization. We report here on our structural investigations of the pre-formed chitin matrix and the initial deposition and growth of goethite (α-FeOOH) crystals within the matrix. By using cryo-techniques, in which unstained sections of the teeth are examined in a frozen-hydrated state in a transmission electron microscope (TEM), we were able to characterize the process without introducing artifacts normally associated with the staining, dehydration, and embedding required for conventional TEM. The unmineralized matrix consists of relatively well ordered, densely packed arrays of chitin fibers, with only a few nanometers between adjacent fibers. There are clearly no pre-formed compartments that control goethite crystal size and shape; rather, crystals must push aside or engulf the fibers as they grow. By examining teeth nearly row-by-row around the onset of mineralization, we were able to image the first-formed mineral within the chitin matrix. These linear deposits of goethite appear to nucleate on the chitin fibers, which thus control the orientation of the crystals. Crystal growth, on the other hand, is apparently not influenced by the matrix, in contrast to many other biomineralization systems.     

Ontogeny and phylogeny of tooth attachment modes in actinopterygian fishes

There are four major tooth attachment modes in actinopterygians. Type 1 mode is characterized by complete ankylosis of the tooth to the attachment bone; it is the primitive attachment mode for actinopterygians. In Type 2 mode there is a ring of collagen between the tooth base and the bone. In Type 3 mode mineralization extends near or to the bone at the anterior tooth border, and there is a relatively large collagen area on the posterior surface of the tooth; Type 3 teeth are hinged with an anterior axis of rotation. Type 4 teeth also have a relatively large posterior collagen area, but there is no collagenous connection between the anterior basal tooth border and the attachment bone; Type 4 teeth are hinged, with a posterior axis of rotation. Types 2, 3, and 4 attachment modes appear to result from retardation of mineralization and resemble, with some modifications, ontogenetic stages in the development of Type 1 mode; they are considered to be paedomorphic features. Attachment modes 2, 3, and 4 are each associated with a major evolutionary lineage within the Teleostei. The degree to which paedomorphosis has been a factor in teleostean evolution is discussed.     

S.E.M. and polarization microscopy of nemertean stylets

The ultrastructure of the stylets produced by nine species of nemerteans has been examined by scanning electron microscopy (S.E.M.) and polarized light microscopy. Stylets are solid, nail-shaped structures that typically reach lengths of 50–200 μm. Each stylet is composed of a centrally located organic matrix surrounded by an inorganic cortex that contains calcium and phosphorus. When viewed at high magnifications, fine granules can be seen throughout the organic matrix, and the cortex appears to be composed of densely packed homo-geneous material. Fractured specimens and whole matrices isolated from decalcified stylets reveal a close correspondence between the shape of the organic matrix and that of the surrounding cortex. This similarity in morphology suggests that the organic matrix serves as a template during calcification of the stylet. The fact that abundant material can be seen in the core of incinerated stylets, and in the central region of stylets that had been soaked for several hours in sodium hypochlorite, supports the hypothesis that the organic matrix is also highly calcified. Polarization microscopy of nemertean stylets indicates that they are composed of a crystalline, rather than amorphous, form of calcium phosphate. The probable organization of the calcium phosphate crystals is discussed.