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Microstructure and mechanical design in the lantern ossicles of the regular sea‐urchin Paracentrotus lividusi A scanning electron microscope study M. Daniela Candia Carnevali , Francesco Bonasoro & Giulio Melone To cite this article: M. Daniela Candia Carnevali , Francesco Bonasoro & Giulio Melone (1991) Microstructure and mechanical design in the lantern ossicles of the regular sea‐urchin Paracentrotus lividusi A scanning electron microscope study, Bolletino di zoologia, 58:1, 1-42, DOI: 10.1080/11250009109355726 To link to this article: http://dx.doi.org/10.1080/11250009109355726

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Date: 25 January 2017, At: 02:02

Boll. Zool. 58: 1-42 (1991)

Microstructure and mechanical design in the lantern ossicles of the regular sea-urchin Paracentrotus lividusi A scanning electron microscope study

inorganic and organic components co-operate to achieve a structure which is remarkably hard and resistant to different types of mechanical stress. The significance of these two different structural solutions and the functional implications of their numerous variations are discussed in the light of current knowledge of lantern mechanics. KEY WORDS: Sea-urchins - Dental ossicles - Microstructure Mechanical design.

M. DANIELA CANDIA CARNEVALI FRANCESCO BONASORO GIULIO MELONE Dipartimento di Biologia, Universita di Milano, via Celoria 26, 1-20133 Milano (Italy)

AKNOWLEDGEMENTS We are very grateful to Prof. G. Lanzavecchia for his valuable advices and encouragements during the praparation of this paper. We are also indebted to Dr. I. C. Wilkie for his precious and careful critical review of the manuscript and to Dr. E. D'Auria for her help during the work. This research has been supported by a grant of Consiglio Nazionale delle Ricerche (CNR), Roma.

INTRODUCTION

ABSTRACT Echinoids are the echinoderm group which has explored and exploited most efficiently the potential of the endoskeleton in a range of extremely advanced and sophisticated adaptive solutions. The most ingenious of these adaptations are employed in the dental apparatus, whose different elements represent a striking example of the versatility of the skeletal tissue. The dental ossicles (jaws, rotulae, compasses and teeth), though having the same basic organization, show a wide range of structural and functional solutions: inorganic phase and organic stroma are variously combined and integrated in a very plastic and adaptable tissue, which is able to fulfil very specific mechanical requirements. On the whole, all the different arrangements shown by the dental elements can be considered as differentiations of the two limit-models of skeletal microarchitecture, represented respectively by classical porous stereom and a composite lamellar structure. The first structural model is by far the most common: all the lantern ossicles, except the tooth, show a rather complete range of variations on the theme of the threedimensional stereom, including either the usual labyrinthic pattern or other types of more specialized stereoms (laminar, galleried, fascicular, microperforate, imperforate), whose presence is closely related to the association with interacting elements (ligaments, muscles, other skeletal parts) and to specific functional requirements. In any case, remarkable differences can be observed easily between the superficial and the internal stereom microstructure, in parallel with as many conspicuous variations in the distribution and the organization of the associated stroma. The second model, on the contrary, can be detected only in the tooth and consists of a unique composite structure (primary and secondary elements), showing a number of specific differentiations in different zones: the primary structure consists of unique mineral elements (lamellar plates and prisms), which form the main framework of the tooth; the secondary structure, on the contrary, consists of stereom plates of more or less modified structure, which cement and reinforce the primary structure. The tooth is a true masterpiece of constructional design, where (Received 15 September 1990 - Accepted 5 November 1990)

The mesodermal calcareous endoskeleton is one of the most significant features of the echinoderms. The skeletal elements differ remarkably from each other in their general morphology, both in the different classes and in the individual animal, depending on their anatomical and functional role. On the other hand, they all consist of the same structural components and, except in some peculiar situations, show a common general organization. As is well known, each skeletal plate is composed of an inorganic component, usually represented by a tridimensional mesh of calcite trabeculae, termed stereom, and of an organic component, termed stroma, represented by the connective tissue filling the pores of the stereom. This terminology, recently criticized as inadequate by Markel et al. (1989) on the basis of detailed histological considerations, seems nevertheless at present to be the simpler and the more suitable to describe the two phases of echinoderm skeletal tissue. The characteristic porous structure of the stereom (average porosity usually about 50%) shows a number of advantages, represented first of all by the lightness and the quick and economical growth of the overall structure. Moreover such a tridimensional framework offers a firm anchorage for muscles and ligaments: the different solutions adopted for the attachment of muscles and ligaments were recently described in detail respectively by Stauber & Markel (1988) and Smith et al. (1990). The inorganic component of each skeletal element, either plate or spicule, behaves optically as if it were a monocrystal of calcite (Nichols & Currey, 1968; Wainwright et al., 1976; Inoue' & Okazaki, 1977). However, according to some authors it probably consists of a polycrystalline microstructure whose optical behaviour is due to many crystals oriented in the same direction (Towe, 1967; Pearse & Pearse, 1975; O'Neill, 1981; Emlet, 1982). A polycrystalline arrangement has been described in some elements at the level of the external layers corresponding to the articular surfaces, and seems

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M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

to be correlated with the peculiar mechanical requirements of these areas (Markel et al., 1971; Markel, 1976b; 1979). For similar functional reasons, a variable amount of MgCO3 is often found in the mineral phase of the skeleton in association with the usual main CaCO3 component (Schroeder et at., 1969; Weber, 1969; Weber et al., 1969). In addition the mineral phase contains a small amount (from 0.05 to 0.1%) of organic matrix, which is intimately bound to the calcite itself and helps to increase its mechanical resistance (Emlet, 1982; Burkhardtefa/., 1983; Weiner, 1985; Markel etal., 1986). The stroma consists of the usual components of connective tissue, i.e., different types of cells, fibrils and extracellular matrix. The sclerocytes are the only characterizing elements of the skeleton: they are associated closely in a functional syncytium and are responsible for depositing calcite crystals inside wide vacuolar system included in the syncytium itself (Markel et al., 1986). The other types of cells (fibroblasts, phagocytes, morula cells) are all elements usually found in echinoderm connective tissue. The organic matrix coat in the intrasyncytial cavity, which consists mainly of proteoglycans, constitutes the necessary substratum for the biomineralization process (Dubois & Chen, 1989; Markel et al., 1989). The fibrous elements, represented by fibrils of different types, including collagen, increase remarkably the mechanical resistance of the fragile skeletal tissue to fractures. A conspicuous collagen component, on the other hand, is limited specifically to the articular areas, where it is responsible for the firm connection between the different plates (Moss & Mehan, 1967) or is involved in the interactions with muscles and ligaments. The structural organization and, most of all, the relationship and the ratio between stereom and stroma seem to vary in the different situations. The presence of the organic component is also correlated with the potential for growth and repair of the skeletal tissue.

Markel and coworkers in a long series of papers (for a review see Markel & Gorny, 1973; Markel et al., 1977). Nevertheless all the elements of the dental apparatus merit morphofunctional analysis and are very suitable for studying the structural organization of the skeletal tissue in relation to the different mechanical roles of the parts. The lantern of the regular sea-urchins, as is well known, is a very complex biomechanical system consisting of variously articulated skeletal pieces, muscles and ligaments, all involved in the different motor activities of the apparatus during feeding, scraping and digging (Candia Camevali et al., 1988; Lanzavecchia et at., 1988; Andrietti et al., 1990; Candia Camevali & Andrietti, 1990). These multiple roles impose on the apparatus precise constructional and mechanical requirements which have to be respected both in the overall arrangement of its components and in the architecture of the single pieces. Therefore, an analysis of the mechanical design of the skeletal parts seems to be the indispensable key for a complete functional interpretation of their adaptive features. This paper takes into account these aspects of macro- and micro-structural organization and mechanical design of the endoskeleton in the different ossicles of the lantern of the regular seaurchin Paracentrotus lividus. This species is a representative of the camarodonts, a group which has evolved the most advanced and mobile lantern type in the echinoids (Smith, 1984). Our aim is to provide a comprehensive view of the possible functional adaptations of both stereom and stroma in the skeletal pieces which, although assembled to form a single apparatus and engaging in common general activities, also fulfil at the same time specific mechanical requirements. Preliminary results of this research were published in abstract form (Candia Camevali & Melone, 1987).

Echinoids are perhaps the echinoderm group whose evolutionary success and adaptive capabilities owe most to the characteristic features of the skeleton. In fact the skeleton provides these animals with protection, support and movement and consists of three functionally different parts: the test, the accessory appendages and the masticatory apparatus, or lantern. The general architecture of these skeletal parts and the respective macro- and micro-structure of the single pieces are related closely to their specialized functions and can be compared in order to correlate the possible structural modulations with specific mechanical roles. The test plates and the accessory articulating appendages are by far the best studied skeletal parts from both the structural/ morphofunctional and comparative points of view (Raup, 1966; Nissen, 1969; Jensen, 1972; Re'gis, 1977; Markel & Roser, 1983; Re'gis & Thomassin, 1985, to quote only a few). The masticatory apparatus, on the contrary, has until now not been investigated so broadly or comparatively in terms of microarchitecture, apart from the tooth, whose structure was extensively described by

MATERIALS AND METHODS Specimens of Paracentrotus lividus, collected near Naples, were kept for months in artificial sea water (16-18 °C) and fed with lettuce. Lantern preparations were isolated from the tests, dissected and prepared according to the following methods: Scanning electron microscopy Different specimens were employed: a) completely digested specimens, treated with IN NaOH (for 2-3 weeks) which removes completely the organic component and gives perfectly clean disassembled ossicles. These samples, intact or suitably fractured, were air-dried and then processed immediately according to standard SEM methods; b) semi-digested specimens, treated with 0.1 N NaOH (for 1 week) which partially removes the soft tissues while emphasizing the ligaments, and then prepared according to a); c) intact specimens, fixed with 2% glutaraldehyde- 4% paraformaldehyde mixture in 0.1 M cacodylate buffer and post-fixed in 1 % osmic acid in 0.1 M cacodylate buffer. The different ossicles were then isolated and suitably fractured, critical point dried and processed for observation in the SEM (Cambridge Stereoscan 250 MK2, at 5 or 10 kV).

MICROSTRUCTURE AND MECHANICAL DESIGN

3

Light microscopy

Stereom microstructural organization

Whole specimens as well as isolated skeletal parts were prepared according to standard histological methods (Bouin fixative or neutral buffered formalin, and paraffin embedding). The sections were stained by means of different techniques (Haematoxylin-eosin, Mallory trichrome stain, Milligan trichrome stain, PicroindigocarminePAS method) and then observed under a Jenaval light microscope.

In the different species the anatomical structure of the individual ossicles of the lantern show remarkable differences which represent specific functional adaptations of the masticatory apparatus to different feeding behaviours, and are usually considered important phylogenetic characters. A similar range of diversity can be detected in the ossicle microstructure, not only in the different groups but also in a single animal, depending on the mechanical requirements. In other words, the skeletal pieces can vary a lot in their macro- and microstructure according to their functional roles, due to the remarkable plasticity of the skeletal tissue. The possible architectural modulations in relation to specific needs are well known for the skeletal plates of the sea-urchin test (Raup, 1966; Nissen, 1969; Jensen, 1972; Re'gis, 1977; Smith, 1980; Telford, 1985a, b). In particular, Smith (1980) described no fewer than ten different structural models of stereom in the echinoid test (extant and extinct) variously combined in the plates according to the needs and to the species (Fig. 2). The ossicles of the dental apparatus, where the morphofunctional conditions seem to be even more diversified than in the test plates, merit analysis according to the same criteria.

RESULTS

Gross anatomy of the lantern framework The dental apparatus of camarodont sea-urchins consists of two anatomically and functionally distinct parts: the perignathic girdle and the lantern itself (Fig. 1). The

Jaw

Fig. 1 - Schematic reconstruction of the lantern system in Paracentrotus: the arrangement of the main muscles and ligaments is shown on the right, a = auricle; c = compass; cd = compass depressor ligaments; e = elevator muscle; i = interpyramidal muscle; j = jaw; p = protractor muscle; pm = peristomial membrane; r = retractor muscle; ro = rotula; t = tooth.

first is part of the internal test and forms five regular and prominent ambulacral ridges (the auricles), which are considered a distinctive advanced feature of the most movable and evolved lanterns; the second constitutes an independent complex, connected to the test by means of muscle bundles (protractors and retractors) and ligamental structure (peristomial membrane and compass depressors), which are inserted on the perignathic girdle. The lantern consists of forty different ossicles connected by a variety of joints and arranged to form a pentamerous framework. The main skeletal components are the five jaws, each one formed by four complementary pieces. Each jaw forms an alveolus for a long tooth, which is inserted rigidly inside the jaw itself and protrudes from it as a sharp tip. Very specialized hinge-joints connect together the adjacent jaws at the lantern base by means of interposed articular ossicles known as rotulae. The system is completed by five other ossicles, the compasses, which are located above the rotulae but are not involved directly in the basic mechanics of the lantern.

The jaw complex (two complementary demi-pyramids plus two complementary demi-epiphyses acting as an integrated functional unit) shows by far the most diverse macro- and micro-structure of the lantern components. Due to the variety and the complexity of its interactions and articular relationships with the other elements of the lantern, the jaw offers in its surface structure in particular a wide range of microstructural features over and above various individual modulations due to size, age, etc. Each jaw is shaped like a hollow triangular pyramid consisting, on the whole, of an outer face, two latero-radial sides and an aboral base (Fig. 1). The outer face. The outer face (Plates la and 2a) opens in a wide triangular window, the foramen magnum, which reveals the tooth lodged inside and whose lower border is raised to form dental brackets. It is subdivided longitudinally by the medial zig-zag suture line between the two complementary demi-pyramids (Plate la, e, f)> which is ideally in continuity with the joint between the upper arches of the complementary demi-epiphyses (Plate 2a, d); this is a distinctive feature of the camarodont lantern. The intrapyramidal suture looks very tight for its whole length, apart from the oral region, where a thin gap filled by bundles of ligaments can be always detected (Plate li). On the upper part of the jaw two symmetrical oblique indentations mark laterally the articulations between demi-pyramids and demiepiphyses (Plate 2a, b, c): in this case the joint never looks very tight and is provided everywhere with clearly visible ligaments (Plate 2c, f). The labyrinthic stereom

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M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

LABYRINTHIC

GALLERIED

LAMINAR

FASCICULAR

MICROPERFORATE

IMPERFORATE

Fig. 2 - Diagram of the main stereom fabrics employed in the ossicles of the dental apparatus of Paracentrotus, tooth excluded (redrawn and modified from Smith, 1980).

(Fig. 2) seems to be the dominant microstructural pattern in this outer face. On the other hand, the trabecular network can vary a lot according to the zones, going from the dense and superficially smooth labyrinthic stereom, extending over all the non-specialized surfaces (Plate lb), to the finer type, present at the specialized attachment areas of protractor and retractor muscles, represented by the fossae of the epiphysal arch (Plate 2c, e) and the subapical fossae of the pyramid (Plate lg). In this last case, the superficial pattern is sensibly modified in a fine, open and irregular construction (Smith, 1980), characterized by irregularly shaped pores and many free-ended trabeculae, which provide suitable firm anchorage to the muscle fibers. Moreover, the wide attachment areas of retractor muscles are regularly perforated by a series of

small holes, which interrupt the uniform structure of the superficial stereom. These holes presumably represent the access for the nerve branchings (Plate lg, h) to the deeper ossicle. This interpretation could explain the frequent occurrence, in histological sections, of tracts of nerve fibres running inside the skeletal tissue of the jaw (Plate 21a). Other structural specializations include zones of galleried stereom (Fig. 2), characterizing the areas close to the intrapyramidal suture (Plate le, f): it is, however, interesting to remark the different orientation of the galleries, which run transversely in correspondence with the narrow borders of the suture itself, whereas are longitudinally directed in the remaining broad areas. Furthermore, zones of fascicular and even microperforate stereom (Fig. 2) are found in the «doorposts» of the foramen (Plate lc, d).

MICROSTRUCTURE AND MECHANICAL DESIGN

The latero-radial sides. The two symmetrical lateroradial sides complete the lateral surface of the jaw (Plate 3a). The morphology of these sides reveals very clearly the relationships between the adjacent jaws. A marked articular line separates the upper epiphyseal portion from the pyramidal face below (Plate 4a). The whole area, apart from its respective oral and aboral ends, is used mostly as attachment area for the interpyramidal muscles and is transversely «striated» by a uniform sequence of raised strips separated by furrows and ending in the prominent teeth of the comb-like inner border (Plate 3b). There is a striking macro- and micro-structural correspondence between these ribbon-like attachment ridges and the respective interpyramidal muscle bundles (Lanzavecchia et al., 1988): in each strip the skeletal organization itself follows the parallel arrangement of the flattened muscle fibers by employing a rather modified form of very fine and dense galleried stereom which is superficially characterized by parallel, longitudinally aligned and thin protruding trabeculae (Plate 3c). The precise longitudinal direction of galleries and trabeculae tends, then, to vanish in the irregular meshes of a typical more or less dense labyrinthic stereom in the extreme edges of this wide articular area (Plate 3d). These results can be conveniently compared with the corresponding histological sections of the jaw complex, which show in detail the relationships and the connections between muscle and ossicle (Plates 8d and 21b), achieved by means of typical connective "composite tendons» coiling around the skeletal trabeculae (Stauber & Markel, 1988). The remaining oral end of the lateral surface (Plate 3e) loses completely any type of peculiar superficial pattern and is homogeneously arranged with more compact types of stereom (very dense labyrinthic and microperforate). On the other hand, the aboral terminal area (epiphyseal base), which is engaged in the most important movable joint of the lantern (Plate 4a), shows only very limited areas of unspecialized stereom (Plate 4b, c): in fact, whenever the articular surfaces of epiphysis and rotula directly interact in the complex rotular joint, wide zones of microperforate, and even imperforate, stereom occur mostly in correspondence with the main tubercles and ledges (crista and epicrista, according to Markel, 1976a, 1979) (Plate 4d, e, f). The ab'gral base. The external view of the jaw-complex is finaljy completed by its aboral base (Plate 5a). The limited surface of this side, belonging completely to the epiphysis, is mostly exposed outside and, therefore, shows a preferential microstructural arrangement of unspecialjzed labyrinthic stereom (Plate 5b, d), which even at the level of the intraepiphyseal joint does not seem to change significantly its random pattern (Plate 5d). The suture itself does not look very tight, the firm connection between the complementary parts consisting of strong bundles of ligaments (Plate 5g) even externally running across the joint. Since the rotular joint involves not only the jaw lateral surfaces but also part of its aboral base, we

5

find articular specializations analogous to those described previously, i.e., well defined areas of microperforate or imperforate stereom related to the grooves and prominences (Plate 5c, e), which involve also the lateral surface (see above) and are complementary to those shown by the rotular surface. Moreover, there are some limited areas for the insertion of the strong ligaments anchoring the rotula to the epiphysis (Plates 5e, f and 2Id): these areas show the specialized galleried pattern characterizing the attachment of many other articular ligaments (see below). The sutures. Beside these microstructural differences shown by the whole jaw-complex, a detailed analysis of the articular relationships between the four complementary elements reveals unexpected and striking functional modulations. Taking into account the broad longitudinal suture between the demi-pyramids, and in particular the opposing articular areas of the complementary ossicles (Plate 6a, c, e), it is possible to recognize two longitudinal bands, which correspond to a clear sequence of distinct structural patterns in relation to the complexity of the articulation involved. The more external band is characterized by a regular arrangement of densely packed pegs (Plate 6b, d), typical of sutural galleried stereom (Smith, 1980).These pegs are generally roundish in shape and geometrically ordered in a square lattice, but can often fuse together to give oblong prominences, always regularly oriented, according to the position. The inner band, on the contrary, consists of coarser and less specialized galleried stereom (Plate 6b), with larger pores and thinner trabeculae, characterized superficially by a rather regular distribution of small thorns and free-ended trabeculae. This band seems in its turn subdivided into distinct sub-bands, where the superficial organization of the stereom varies more gradually. The inner border of the band, for instance, shows often remarkable roughness and coarseness not shared at all by the other sub-bands (Plate 6f). By comparing these results with the histological sections of the jaw, it is possible to check that the outer band coincides with the true suture area, where the complementary skeletal surfaces firmly fit in, connected by only a limited amount of short sutural collagen fibres (Plate 8a, c). These fibres find suitable insertions in the galleries of the stereom, which run perpendicular to the articular surface. The inner band, on the other hand, corresponds to a specialized part of the same articulation, where the true suture seem to be replaced by a less tight articular relationship (Plate 8a, b). Well-developed ligamental bundles are inserted on the respective articular parts: since the limited extent of the galleried stereom, which runs perpendicularly to the articular surface, allows these ligaments to penetrate not very deeply the skeletal structure, most of the fibres realize a very effective anchorage by looping around the skeletal trabeculae (Plate 8b). The massive presence of ligaments and the not very tight connection between the two complementary parts provide evidence for a dif-

6

ferent type of joint, which can be considered a semimovable articulation (Lanzavecchia et al., 1988). This condition of articular bivalence is generally shared by the other joints involved in the jaw complex, even if the situations seem to be much more diverse. For instance, with regard to the joint between the two demiepiphyses, the articular area, strictly limited to the epiphyseal arch, shows an organization closely comparable to that described for the intrapyramidal joint (Plate 7f, g). With regard to the demi-pyramid - demiepiphysis joint, on the other hand, the unevenness of the articular surfaces often makes it very difficult to recognize a regular sequence of stereom patterns and to define their borders (Plate 7a). In this case the areas corresponding to the semi-movable joint tend to be generally more widespread, whereas the true suture areas are limited to only some external zones (Plate 7b, c, d, e). In any case there are many local modifications of structure and organization in relation to specific parts: the presence of a wide cavity system inside the pyramid (Plate 7a, b), and, to a lesser extent, inside the epiphysis (Plate 7d), modifies and interrupts the articular surface of these ossicles, by interposing, at the level of the internal cavities, discontinuous lamellae or layers of labyrinthic, and even laminar, stereom (Plate 7a, b, d). The histological sections (Plate 8d, e) confirm the articular pattern implied by the microstructural analysis. The inner surface. A number of minor modifications can be detected in the stereom microstructure of the inner surfaces of the jaw (Plate 9a), the most relevant being shown by the longitudinal dental guide. The internal side of the frontal part of the pyramid is marked by two symmetrical, parallel strips of dense galleried stereom (Plate 9b, c), which continue into the two dental brackets and provide attachment areas for the ligamental bundles connecting pyramid and tooth. Due to its limited interactions with other elements of the lantern, the remaining internal side shows a widespread presence of dense labyrinthic stereom, only occasionally substituted by morespecialized patterns. Thus fascicular or perforate stereom can be found in all the areas more exposed to mechanical stresses, for instance at the level of the inner adaxial borders of the latero-radial side (Plate 9d, e). The internal architecture. The deeper skeletal architecture of the jaw is also not homogeneous, but, as happens in the test plates (Smith, 1980), shows many differences in microstructure which are revealed when the ossicle is fractured at random or cut along known planes. By transecting a demi-pyramid at different levels, we can appreciate conspicuous macro- and micro-structural changes through the thickness of the ossicle. A cut at an intermediate level (Fig. 3b and Plate 10a) shows a layered organization which can be locally modified and consists of different types of stereom. In particular, we can distinguish the microstructure of the wide lateral expan-

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

sions from that of the central body of the pyramid. In the first case (Plate lOe) the arrangement tends to be rather compact and consists of, from outside to inside, a thin layer of galleried stereom, a thicker fascicular layer, and, not always present, a thick microperforate layer which is so well-developed in some areas that it completely replaces all other stereom types (see the doorposts of the foramen). In the central body, on the other hand, a porous microstructure prevails (Plate 10c) and the first two layers are followed by a conspicuous component of labyrinthic stereom, which gives way to some sheets of laminar stereom before opening into the wide central multicavity system. These cavities (Plate 10c, d) are very irregularly shaped and mostly interconnected; they vary remarkably in number and distribution according to the level in the ossicle and to its size. They tend to be reinforced by septa and strands of retiform or laminar stereom passing through and partially filling the holes, which end by being completely obliterated towards the oral end of the pyramid. These internal channels probably allow the nerves to pass through. When the pyramid is cut closer to the top (Fig. 3c and Plate 10b), its internal architecture shows an overall compactness and uniformity: its multistratified structure consists prevalently of microperforate, fascicular and very dense galleried stereom (sometimes so regular and aligned as to become rectilinear: see Plate lOf), often distributed in this order from outside to inside. A compact microperforate pattern tends to prevail completely in the most exposed apical parts (Plate lOh), even though it can be covered by thin layers of more specific stereom types (Plate lOg) in correspondence with the specialized superficial areas (for instance, near the intrapyramidal suture). This type of distribution pattern in cross section is also seen when the ossicle is cut longitudinally (Fig. 4b and Plate 1 la). In particular, if the pyramid is cut through its central body, both the stratification of stereom types within the ossicle and their progressive transition in longitudinal direction become very clear (Plate lib, c, d). Analogous results can be obtained in the demiepiphysis. This ossicle shows on the whole a rather compact microstructure. As can be seen by cutting it transversely or longitudinally (Fig. 4d and Plate lie, f), the' dominant stereom is the very dense galleried type, oriented preferentially according to the main longitudinal axis of the jaw itself. This internal frame is then covered by other thinner layers of superficial stereom (labyrinthic or microperforate depending on the specific areas). The internal cavity in this ossicle (Plate 7d) is so negligible that it must have a minimal effect on the deeper microstructure. Rotula Five small articular ossicles, the rotulae, are radially inserted in the aboral base of the lantern (Fig. 1): as is well known, they perform a prominent role in the movable

7

MICROSTRUCTURE AND MECHANICAL DESIGN

II II

II

It 11 II 1! II 1

_ =

MICROPERFORATE

.

=

LAMINAR

GALLERIED

LABYRINTHIC

FASCICULAR

Fig. 3 - Internal architecture of the jaw. a) Schematic frontal view of a demi-pyramid cut along two possible transverse planes, b and c) Schematic cross-sectional plans corresponding to the surface fractures shown in a) and illustrating the distribution of stereom fabrics. Intermediate cross section (b); apical cross section (c).

interpyramidal hinge-joints. Each rotula fits perfectly into the respective epiphyses (Plate 12a) and interacts with them in a sequence of different articular relationships: in particular, where the main joint is located, the abaxial rotular head is embedded tightly between the epiphyses, while at the level of the minor joints the adaxial rotular part lies directly upon the epiphyses. In spite of its small size, a number of macro- and micro-structural specializations can be found in the external architecture of the rotula.

The aboral face. The aboral face is smooth and rather homogeneous (Plate 12b): it interacts only with the compass, which sits on the top of the rotula but is linked to it only at the level of the adaxial (inner) rotular head, where there is a typical movable joint (Plate 13a, d). Consequently, in correspondence with this small specialized area, the very dense and compact labyrinthic stereom characterizing the whole aboral surface is replaced by a pit of even more compact stereom (Plate 12d). The greater porosity of the surrounding zones allows the at-

8

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

Fig. 4 - Internal architecture of the jaw. a) Schematic side view of a demi-pyramid cut along a possible longitudinal plane, b) Schematic longitudinal plan corresponding to the fracture of a) and showing the arrangement of different stereom types, c) Schematic side view of a demiepiphysis cut along a possible longitudinal plane, d) Schematic cross-sectional plan corresponding to the fracture of c) and showing the distribution of stereom fabrics. Hatching as in Figure 3-

tachment of the ligamental bundles connecting the compass end to the rotular adaxial head (Plate 12g). The oral face. Both the macro- and the microstructure of the oral face are much more varied (Plate 12c): due to its complex articular involvement with the adjacent epiphyses, this surface shows a symmetrical arrangement of articular specializations, which complement precisely those of the epiphyses. These specializations are par-

ticularly obvious with regard to the prominent condyle of the abaxial head, which contributes mostly to the hinge-joint with the epiphysis (Plate 12f) and to a lesser extent to the lateral borders of the oral side, where grooves and small tubercles are developed in sequence (Plate 12e, h). The stereom pattern is the usual smooth microperforate, suitable for reducing friction between the contact surfaces in the movable joints. These areas are intercalated with others where labyrinthic or

9

MICROSTRUCTURE AND MECHANICAL DESIGN

galleried stereom occur offering convenient attachment sites for the ligaments that interconnect the ossicles. Finally, some areas of transversely or obliquely oriented fascicular stereom are restricted to the middle portion of both the abaxial and adaxial heads (Plate 12e, f).

The internal architecture. The cross fractures of the compass show a uniform internal architecture. The microstructure is very compact and, as in the rotula, consists mostly of dense fascicular stereom regularly aligned along the longitudinal axis of the ossicle (Plate 13i).

The internal architecture. Despite its varied external morphology, the rotula shows, on the whole, a very homogeneous and compact internal structure. At any level the microstructural organization seems always to consist mostly of a very dense fascicular stereom (Plate 12i), oriented along the longitudinal axis of the ossicle itself: this compact pattern is then covered by thin superficial layers of stereom organized according to the patterns described above.

Tooth

Compass The aboral face of the lantern framework is completed by five small and slender compasses which lie upon the rotulae (Plate 13a). In profile each compass is slightly bent in an oral direction and consists of two complementary pieces, the hook and the fork, joined by means of a suture. We can distinguish an aboral and an oral face; and an abaxial biforked end and an adaxial hooked end. The aboral face. The convex aboral surface is dominated by rather dense labyrinthic stereom (Plate 13c), whose arrangement changes according to the specialization of the particular area. Specific attachment areas for ligaments are found at the level of the forked abaxial end (Plate 13e, g) and of the tapered adaxial end (Plate 13d), where the long compass depressor bundles and the short compass-rotula ligaments are inserted respectively. Moreover, a modified form of the same stereom can be recognized in the lateral compass «flaps» (Plate 13b), where the elevator muscle bundles are inserted in a regular ribbon-like arrangement reflected also in the corrugated ossicle structure. This arrangement can be replaced locally by more aligned types of stereom, particularly by galleried stereom in the zones immediately ( adjacent to the suture (Plate 13d). The oral face. The oral compass face is very similar in microstructure to the aboral one (Plate 13h). Here the dense labyrinthic stereom is more widespread, only occasionally interrupted by very limited areas where more specialized fabrics occur (galleried stereom, within the suture, or other minor modifications). The suture. Concerning the complementary surfaces involved in the suture (Plate 130. their features are quite similar to those described for the jaw sutures, and a similar articular significance can be attributed to the two identifiable stereom patterns: a band of densely packed pegs and a band of thorny labyrinthic stereom.

The tooth is the only lantern ossicle previously studied in detail: only a restricted description will be given here of its elaborate and unique structure which is well known due to the extensive work of Markel and coworkers (Markel, 1969, 1970a, b, c, 1973, 1974, 1986b; Markel & Gorny, 1973; Markel & Titschack, 1969; Markel et al., 1971, 1977). On the other hand we will take into account more extensively some structural aspects, never described before, which provide the mechanical analysis with significant morphofunctional details. The general architecture. The variety of mechanical problems faced by the tooth during its manifold activities are resolved by means of advanced structural solutions. The tooth of camarodont sea-urchins is strongly subjected to high bending moments, i. e., to simultaneous stresses of both compression and tension, which arise more from impact with the substratum during digging and scraping than from actual chewing activity. The morphofunctional adaptations for these activities are reflected in both its macroscopic shape (that of a slightly bent T-girder (Fig. 5, Plates 14a and 15e), and its internal microstructure. The remarkable structural diversity shown by the lateral surface of the tooth in different zones (abaxial face, lateral sides and adaxial keel) depends on and is closely related to its peculiar internal microarchitecture which displays a unique structural pattern. It consists of different primary and secondary elements assembled in a variety of combinations (Jensen, 1979). The primary elements are monocrystalline lamellar plates (Fig. 5) which are approximately conical in shape and consist of laminar parts (primary and side plates) and fibrous parts (prisms). They are superimposed on each other and ordered in two rows which are partly interlocked in the medial plane. The secondary elements are polycrystalline plates, deposited at a different time inside and upon the framework of primary elements, which are connected and embedded firmly in a sort of solid cemented structure. The varying distribution of these secondary elements is mainly responsible for the remarkable structural differences characterizing the various regions of the tooth: a) the plumula, i.e., the aboral growing tip (Plate 14b, e), where the primary structure is easily recognizable because of the lack of superimposed secondary plates; b) the shaft, i.e., the intermediate parts (Plate 14c, f), where the presence of the secondary elements increases progressively making the tooth surface increasingly compact towards the oral end; c) the sharp and hard chewing tip (Plate I4d, g), where the secondary structure forms conspicuous and con-

10

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

Fig. 5 - Primary structure of the tooth (redrawn and modified from Markel et a!., 1977). a) Schematic side view of the tooth cut along a transverse plane, b) Reciprocal arrangement of some primary elements, c) Side view of a primary element seen from the medial plane, d) Adaxial view of a primary element, e) Cross section of the tooth, p = prisms; pip = primary lamellar plate; psp = primary side plate.

tinuous scales surrounding the primary elements. This zone is subjected to continuous wear because of impact with the substratum: flaking occurs in a direction coinciding with the organization of the primary elements, whose continual detachment maintains the sharpness of the tooth tip, like a self-whetting chisel (Markel et aL, 1977; Smith, 1980). The primary structure. The complex composite organization of the tooth is confirmed by observations of various types of fractures, which show unequivocally the correspondence between the differences in internal structure and the peculiar arrangement of both the different elements and their constituent parts. We shall avoid here a unnecessary re-description of the

remarkable, but well known, differentiation of the tooth's internal microarchitecture. Nevertheless, there are some new details, which are necessary to provide an overall view of the morphofunctional adaptations of the echinoid skeletal microstructure. These aspects concern particularly the deeper organization of the primary elements of the tooth which changes markedly from the plumula to the oral tip. Both the laminar parts (primary and side plates) and the fibrous components (prisms) of the primary elements seem to undergo a considerable and progressive increase in compactness, which is revealed only by analysing and comparing their internal structure when the tooth is fractured at different levels. At the growing part (Plate 15a, f), all these elements are never compact and solid throughout their thickness, but

MICROSTRUCTURE AND MECHANICAL DESIGN

show wide internal cavities (only partially filled by stroma in the intact samples - see below). The resulting structure of each laminar plate resembles that of a bistratified «wafer» (Plate 15a, b), while the prisms look like bundles of thin bamboos with irregular and multiple cavities inside (Plate 15f, h). Starting from the intermediate tooth region, however, all these cavity systems tend to be progressively filled due to a conspicuous thickening of the calcareous wall, and, from the shaft to the chewing tip, both lamellae (Plate 15c, d) and prisms (Plate 15e, g) become increasingly compact and solid. The secondary structure. With regard to the secondary elements, there is an even more marked structural differentiation in the different tooth regions. The secondary plates, as described above, reinforce and cement to different degrees the tooth primary structure, apart from the plumula, and consist of connecting elements which differ much in structure and in functional significance from the primary elements. In fact, they cannot be regarded as another microstructural device unique to the tooth, as is the case with the primary elements, but seem to represent further examples of already known microstructural specializations of the usual stereom. Detailed analysis of both the superficial and the internal microarchitecture of the tooth can provide us with evidences for this. Starting from the aboral end of the shaft, for instance, we can follow the first appearance of the secondary plates, which are deposited and distributed only at certain areas on a largely uncovered primary structure. These areas are represented especially by two broad lateral bands, where the secondary elements are already well developed and consist of a monolayer of longitudinally oriented and interconnected trabeculae (Plates 14b and 16b), arranged like superficial galleried stereom. These trabeculae are directly connected, without structural interruption, to the edges of the primary side plates (Plate l6i), where characteristic thorny processes begin to develop very early (Plate 16a) and provide the trabeculae themselves with suitable support and attachment points. On the other hand, in other surface areas (see, for instance, the tooth frontal band, where the primary plates overlap one another - Plate l6e - or the lateral, very indented borders of the side plates - Plate l6g, h) the deposition of secondary plates is limited to a thin layer of compact stereom, which begins to surround and cover both lamellae and prisms in a sort of coherent amalgam. In any case, at this level, the scarce secondary component seems to be limited to the abaxial part of the tooth, while in the adaxial keel it is missing. It is even more difficult to detect the presence of secondary plates in the deep tooth structure: fractures show a largely discontinuous internal organization consisting mostly of primary elements whose reciprocal arrangement is discernible only when the organic stroma is subjected to at most mild digestion. It is very difficult to distinguish the presence of a hypothetical secondary structure which must be very reduced, because the ex-

11

tensive fibrillar network of the stroma tends to conceal everything. At the level of the intermediate shaft the secondary structure becomes more conspicuous: it is well-developed throughout the whole tooth and consists of both superficial and deeper components. On the tooth surface it is possible to follow the progressive enhancement of this secondary component, which tends to cover uniformly the primary skeleton with a layer of microperforate and then of imperforate stereom. This condition is shared by the frontal part (Plates I4f and I6f), the lateral sides and the keel (Plate 14c), the only exception being the two lateral strips, where the trabecular structure previously described is still recognizable, although it is more compact (Plate 16c). It is worthwhile pointing out how the microstructure of these strips, which represent the articular areas between the tooth and pyramid, fits neatly into that of the corresponding bands observed on the internal surface of the pyramid (see above). On the other hand, the articular relationship involving these specialized regions of tooth and pyramid is clearly shown in histological sections, which emphasize the ligament bundles and their attachment areas on the respective skeletal pieces (Plate 21e, f). The remarkable presence of the secondary elements is also evident in the internal tooth microstructure. In particular, at the level of the lamellar zone (Plate l6j), this component is represented by wide flattened plates of microperforate stereom alternating with the primary lamellae, while at the level of the fibrous zone it consists of small, partly overlapping, imperforate and irregular plates (calcareous disks, according to Markel etal., 1977), which are packed together tightly and surround the prisms in a sort of reinforced concrete (Plate 16k). Since in the intermediate shaft both lamellae and prisms have already completed their development and consist of a quite solid microstructure, fractures of the tooth at this level generally show a very compact internal arrangement (Plate 15e). Finally, at the level of the sharp oral tip which juts out from the pyramidal alveolus and where, as is well known, the tooth structure reaches its maximal hardness and strength, the secondary component dominates the primary one in the form of a homogeneous cement of imperforate stereom which covers and binds together everything (Plate 161). The resulting superficial and internal structure is therefore very uniform: even the two lateral strips lose their peculiar trabecular arrangement which is progressively «scraped off» as soon as the tooth emerges from the pyramid and is replaced by the usual homogeneous layers of compact stereom (Plate l6d). Stroma organization The structure of the complete endoskeleton, including its organic component, can be examined by avoiding any pre-treatment of the dental pieces. The distribution of this organic component varies according to the different patterns of the associated mineral phase, but seems in

12

general to be organized much more uniformly in all the ossicles, with the usual exception of the tooth. Therefore, regarding the jaw-complex, the rotula and the compass, we can refer to a common condition which shows only very insignificant modifications.

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

(Plates 8a, d, and 21b, f). In general, of course, the distribution of the stroma depends strictly on the organization of the mineral phase, and its amount tends to be inversely proportional to the compactness of the stereom. Stroma of the tooth

Stroma of the dental ossicles Firstly, in all these skeletal elements the connective tissue forms a thin, fibrous sheath which covers the external surfaces of the ossicle like a periosteum (Plate 17b, d). This tight membrane follows closely the network of the inorganic component and creeps into it, upholstering completely the outermost galleries and pores (Plate 17c). Whenever the skeletal surface is exposed and is not engaged in interactions with other elements (skeletal pieces, muscles or ligaments), the fibrous connective sheath is completed by an outer epithelium, represented by the typical coelomic lining with its flagellated collarcells (Plate 17a). In all the other cases the connective tissue itself is directly involved and connected with the related elements, often giving rise to local thickenings and to peculiar specializations (see, for instance, the insertions of muscles and ligaments, or the articular relationships between the ossicles etc.). The arrangement and the possible modifications of this fibrous sheath can be well appreciated by observing the histological sections stained by the Milligan trichrome method which emphasizes very clearly the connective components (Plate 20a). In particular, it is possible to distinguish a conspicuous cellular component, probably represented by fibroblasts and other types of cells (phagocytes etc.) and to point out an unequivocal collagenous component, rather scant in the exposed areas but well-developed in the previously mentioned specialized zones (Plate 21a, b, c, d). Towards the deeper ossicle (Plates 17e, f, g and 20b) this compact tissue is then progressively replaced by a connective network, which tends to become thinner as the inner part is reached (Plate 17h). This weft penetrates the whole stereom porosity, fitting tightly into its structure, whatever that may be, and consists mainly of bundles of fibrils, interlaced in various directions. On the basis of SEM studies alone it is difficult to identify these fibrils: as in the extracellular stroma of the skeletal plates in other echinoderms, (Markel & Roser, 1985), they probably consist mostly of very thin unstriated microfibrils, whereas the presence of definite collagen fibrils seems to be limited only to some areas. In any case, throughout this reticular stroma, it is possible to distinguish also a cellular component, represented by typical sclerocytes and by usual connective tissue elements like fibroblasts, phagocytes or morula cells, which are recognizable in the sections by their different morphology and, sometimes, by their staining properties. The number of cells is higher, on the whole, in the peripheral parts than in the deeper ossicle (Plate 20a, b), and increases remarkably at specialized zones, where a peculiar type of interaction with other tissues takes place

In the tooth, the stromal organization varies strikingly in parallel with the structural differences in the inorganic component described above. In particular, the aboral growing end is the zone with by far the richest organic component: it is completely enveloped by the dental sac, i.e., that peculiar compartment of the peripharyngel coelom where the elements of the tooth are renewed continually. By removing this adherent sac, it is possible to expose the plumula (Plate 18a), which is covered by its own tight and fibrous membrane. In the extreme end of the plumula, just below this fibrous sheath, a great number of irregularly arranged proliferating cells, the future odontoblasts, are associated with a lot of fibrillar material (Plate 18b). Like the plumula, the rest of the tooth is covered by a very thin, uniform sheath (Plate 18c), which is continuous with the internal stroma (Plate 20c) and gives rise to the two well defined bands of ligaments connecting the abaxial tooth face to its alveolus (Plate 21e, f). These ligaments provide the tooth with a firm anchorage to the jaw but also allow it to progress slowly lengthwise during its continuous growth. The adaxial part of the tooth is provided with an additional covering layer represented by the lining of the inner surface of the jaw, which extends to cover the tooth keel. Obviously all these external covernings are lost in the protruding chewing tip. In the inner structure of the tooth itself we have to consider separately the usual three functional regions. In the aboral end, thick layers of stroma alternate with the laminar plates and the prisms constituting the primary structure (Plate 18d). These layers consist of a number of odontoblasts (Plate 18e) that are rather regular in size, shape and distribution, and of a conspicuous felt of interlaced fibrils. The close network formed by these fibrils not only covers and connects together all the individual inorganic elements, but even penetrates inside the primary elements whose inner cavities are filled, at least partially, by an irregular weft of fibrils (Plate 19a, b, c). At the shaft level, the stroma tends to be replaced progressively by the mineral phase, represented by the secondary plates described previously, and shrinks to form a progressively sparse interlamellar network (Plate 19d). Due to the increased compactness developed in both lamellae and prisms, the intralamellar stroma in particular regresses totally (Plate 19e). The odontoblasts which are responsible for the pattern of biomineralization, are imprisoned inside the inorganic component, interconnections being possible only through fine gaps left in the secondary component (Plate 18f). In the oral end, finally, the inorganic component is so compact that it fills all possible gaps (Plate 19f): the interlamellar organic component is virtually lacking and

MICROSTRUCTURE AND MECHANICAL DESIGN

the cells are definitely encased inside the composite structure of primary and secondary elements cemented together. The histological sections cut at different tooth levels show very clearly how the stroma is organized in a very complicated and varied pattern, which follows strictly the arrangements of the mineral phase (Plate 20c, d, e).

DISCUSSION Detailed analysis of the microstructure of the dental ossicles in Paracentrotus leads us to a series of considerations concerning the skeletal structures of echinoids in particular and of echinoderms, in general. General considerations As stated above, echinoids are the echinoderm group which have explored and exploited most successfully the potential of the dermaskeletal structure and in which, for this reason, the endoskeleton itself seems to play its most significant role. The lantern, in particular, seems to represent the most striking example of the surprising versatility of the skeletal tissue: the plasticity pointed out in the microstructural organization of its different ossicles shows very well how versatile the endoskeleton is and confirms the breadth of its functional performance. In the one apparatus it is possible to discern wide range of morphofunctional solutions which cannot be found in less functionally specialized situations. On the whole, the different arrangements shown by the dental elements can be regarded as differentiations of only two limit-models of skeletal microarchitecture, represented respectively by the classical porous stereom and by the composite lamellar structure. The first structural solution is applied most extensively throughout the lantern ossicles, where there is a complete range of variations on the theme of the three-dimensional stereom. The usual labyrinthic stereom, which can be regarded as the basic constructional design adopted by echinoids in their skeletal pieces (Smith, 1980), is employed widely in the lantern ossicles, but is often associated with other more specialized stereom types (Fig. 2). The distribution of these specialized microstructural arrangements is related to very localized mechanical requirements and justified by constructional criteria closely comparable with those followed, for instance, in vertebrate endoskeleton. Mechanical

interpretation

Taking into account what is already known about the overall movements of the lantern and the mechanical relationships between its different skeletal pieces (Lanzavecchia et ah, 1988; Andrietti et al., 1990; Candia Carnevali & Andrietti, 1990), and the basic principles of mechanical design, it is possible to interpret more thoroughly the structural solutions adopted by the lantern. Such structural analysis is, of course, particularly complicated for integrated systems like the dental ap-

13

paratus, which consists of many structurally and functionally different elements. The structural design of the individual elements, and of the system as a whole, is much more complex than that of engineering structures where the roles of each component can be very precisely defined, because the ossicles are subjected to very complicated stress patterns, and are able to move reciprocally and to transmit rather considerable force. Moreover, there is a need to find the optimal design of an element which will both resist forces applied in fixed directions and provide them with insertions for muscles and ligaments that will offer the maximum mechanical advantages (Wainwright et al., 1976). It should be remembered that the dental apparatus is not only used during feeding to perform chewing movements, but also plays an important role in other activities like scraping or burrowing, particularly in the case of Paracentrotus. Because of these different functions, it is very difficult to reduce to a schematic form the different patterns and wide ranges of stresses to which each jaw is subjected. Since they represent the main supports of the lantern framework and its main movable component, the jaws have to bear and transmit forces of different types, the only exception being weight, which can be completely disregarded due to the effects of buoyancy (Candia Carnevali et al., 1988; Andrietti et al., 1990). Due to the activities of protractor, retractor and interpyramidal muscles, and to the resistence of the substratum.during movements, the jaw complex has to withstand a wide range of stresses such as tension, compression, bending and torsion. The skeleton resists easily these different forces by means of a number of both macro- and micro-structural adaptations. One device, well known in constructional design, is for the cross-sectional area of a given element to be altered according to mechanical requirements (Wainwright et al., 1976): in fact, the maximum stress allowed is a function of the moment of inertia of the section. This can provide us with a good intuitive explanation for the variable shape and size of the jaw in cross section (Figs 3, 4). Although it is not appropriate to provide a detailed structural analysis here, it is worthwhile stressing how the hollow internal structure of the jaw itself seems to match the general principle according to which (as long as the compressive forces are transmitted over some distance and the other forces are kept low enough), for equal cross-sectional area, a hollow structure is more effective than a solid one. Another way to respond to the different functional requirements is represented by the employment of specialized stereom types, whenever the specific mechanical stresses exceed locally those imposed on unspecialized labyrinthic stereom. Thus, in the zones most stressed by the tensile action exerted by muscles and ligaments, the microstructural pattern tends to be aligned with the direction of the forces, the most compatible design being more or less dense, suitably oriented galleried stereom. On the other hand, in the attachment zones of muscles and ligaments themselves this regular pattern can be modified to provide the two types

14

of soft tissues with the specific skeletal insertions required (Stauber & Markel, 1988; Smith et al., 1990). When, the total load is particularly high (especially with regard to compressive stresses), the skeletal material tends to show an increase in density through a drastic reduction in internal porosity: in these cases, the most compact stereom fabrics are employed, such as the fascicular, microperforate or imperforate types, which occur in highly stressed parts, including some portions of the jaw (see, for instance, the oral end, which is loaded by most of the stress borne directly by the tooth) or some whole ossicles (see the rotula, subjected to a strong compression by the adjacent epiphyses because of its articular position). Finally, besides a number of other types of functional adaptations of the skeletal structure, it is worthwhile highlighting a device that occurs frequently in the lantern ossicles (see the four complementary parts of the jaw or the two pieces of the compass), i.e., the division of an element into sub-components. The usual mechanical advantage represented by the internal subdivision of an overloaded structure into different elements (Gordon, 1980) is probably more effective because of the peculiar way the parts are connected together. In fact the widespread employment of complex bivalent articulations between two complementary elements enables the structure to be resistant and deformable at the same time. Moreover, if we assume that the ligaments involved in the semi-movable part of these joints may consist of mutable collagenous tissue (MCT), the range of possible performances could broaden considerably: the presence of this type of tissues whose tensile properties can be rapidly and reversibly modified (Wilkie, 1984; Wilkie & Emson, 1988) and which is employed widely in the dental apparatus in other articular situations (Lanzavecchia et al., 1988; Candia Carnevali et al., 1990; Wilkie,, Andrietti & Candia Carnevali, unpublished results) could represent a key-solution with great adaptive significance. In any case the porous structure of the stereom, which is well aligned with the direction of the ligaments, allows the collagen bundles to insert very deeply and firmly inside the ossicle, thus reinforcing the articular region. The most advanced example of mechanical adaptation of the skeletal material is undoubtedly represented by the tooth. The tooth structure of camarodont sea-urchins has been studied and described more extensively than that of other sea-urchins since it is ideal for a complete functional and mechanical analysis, although we have not considered this in detail here. The tooth is the most stressed lantern element due to its direct impact with the substratum during chewing, scraping and burrowing. Since during lantern activity the teeth are always dragged adaxially (towards the centre of the lantern) by their respective jaws, they are exposed to specific directional forces. The functional strategies adopted in order to minimize the conspicuous stresses set up by bending, torsion or buckling, entail both adaptation of the general shape (with particular reference to the cross sectional

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

area) and use of stronger materials. The tooth of Paracentrotus, in fact, is slightly bent and keeled, macroscopically shaped like a T-girder, where the abaxial part is mostly subjected to compressive forces and the adaxial part is subjected to tensile forces. The mechanical adaptation of the tooth is even more evident in its microarchitecture and is correlated with the choice of a constructional material which meets the concurrent requirements of hardness and resistance. The teeth of sea-urchins show a unique architecture found in no other echinoderm skeletal parts and the cost of their construction and maintenance must have a significant impact on the metabolism of echinoids, which is in general very low (Emson, 1985). This is perfectly analogous to the situation in vertebrates, where the strongest and stiffest materials are employed in the teeth. These materials, however, have a definite disadvantage in being very "expensive" in a metabolic sense: in both cases, therefore, the animals tend to use them sparingly and only where they are indispensable (Wainwright et al., 1976). We have already quoted the exhaustive work of Markel and coworkers (Markel, 1969, 1970a, b, c, 1973, 1974; Markel & Titshack, 1969; Markel et al., 1971, 1977; Markel & Gorny, 1973) on the tooth structure of different echinoids. It is sufficient to remember that the tooth employs a typical composite material. Composite materials, i.e., materials consisting of two phases, a lamellar or fibrous component and a homogeneous matrix, are widely used in engineering (e.g., plastics reinforced by glass fibres or reinforced concrete). There is a surprising analogy between the artificial types of composite materials and those employed in nature, although the echinoid tooth is original in utilizing the same material, calcite, for both phases, which consist of primary and secondary elements decribed previously. The arrangement and distribution of this material in the tooth follow precise mechanical criteria and the resulting structure shows an extraordinary and varied mechanical design, which can be regarded as an extreme example of specialization of the echinoderm skeletal material. We have mentioned above the different types of stress normally acting on the tooth, but which are not distributed uniformly along the whole tooth. The internal structure of the tooth is adapted perfectly to respond to these stresses, due to the differential distribution of its elements. Thus, the lamellar components (primary plates) are distributed mainly in the zones of maximum compressive stress (abaxial part), whereas the fibrous elements (prisms) are the main component of the zones of maximum tensile stress (adaxial part). Even although the mechanical requisites of the tooth are determined mostly by the unique primary structure, they also depend on the secondary structure, which cements together all the different primary elements in a sort of reinforced concrete and contributes significantly to the morphofunctional differentiation of the different regions of the tooth. The secondary plates, which can be interpreted as simply plates of the usual three-

MICROSTRUCTURE AND MECHANICAL DESIGN

dimensional stereom, are indispensable for improving the mechanical resistance of the whole structure and providing it with suitable insertions for the ligaments. The overlapping of primary and secondary structures reaches a state of maximum solidity and compactness at the oral end: here, due to the effect of shearing forces, whole tooth elements split off one by one, allowing the tooth to renew continuously its sharp end (Markel et al., 1977). A maximum hardness, on the other hand, is limited to the inner tooth core (the stony part: Markel et al., 1977), where there is an optimal combination of primary lamellae and prisms with secondary elements, and the MgCO, content of the secondary component reaches its greatest value (Schroeder et al., 1969). The tooth structure, due to its continuous growth, illustrates well how morphofunctionally versatile the echinoderm endoskeleton can be and how many structural modifications it can undergo owing to its remarkable structural and functional adaptability. It is worthwhile stressing how this plasticity is related closely to the capacities of the organic component to control and direct in detail the progressive development of the inorganic skeleton from both outside and inside. In particular one class of cells, the odontoblasts, united functionally in a syncytium, is responsible for the two subsequent processes of mineralization which give rise to primary and secondary elements (Markel et al., 1986): the determinant factor for these different activities seems to be represented by a different organic matrix coat inside the vacuolar cavity where the calcite deposition takes place. The organic stroma (including in this term organic matrix, fibrils and cells, i.e., syncytial sclerocytes and other types of cells) undoubtedly plays a significant role in the echinoderm endoskeleton. Leaving aside its specific involvement in the development and growth of the skeletal tissue (discussed extensively by Markel and coworkers: Markel et al., 1986, 1989), we have to take into account some possible effects of the interaction between inorganic and organic component on the mechanical properties of the dental ossicles. Firstly it should be noted that in the echinoderm endoskeleton the fibrous component of the internal stroma, which is quantitatively not very significant, is never oriented along preferential directions. This seems to limit considerably any possible mechanical contribution from the organic conponent. Even so, the presence of the stroma generally increases the intrinsic resistance of the mineral phase of the skeleton to compression, tension and torsion stresses (the inorganic calcite is much less resistant to mechanical tests: Weber, 1969), by introducing remarkable advantages. The most frequent cause of breakage of materials, including calcite, is the spread of small fractures and superficial cracks which frequently af-

15

fect the material itself (Wainwright et al., 1976; Gordon, 1980). Discontinuity in the internal structure, due to the presence of pores (as in the common stereoms) or to the sub-division into non-homogeneous elements (as in the tooth) reduces appreciably and inhibits this effect by arresting crack propagation (Gordon, 1980), while the suitable filling provided by the organic component, which is much more elastic and deformable, contributes massively to the absorbance of deformation energy (Currey, 1962). Whatever the microstructure of the inorganic component (with the exception of some zones of maximal compactness), it is always completely penetrated by the stroma, whose functional significance, be it in the jaw or tooth, is the same. The presence of the living stroma also offers many other advantages: it facilitates the maintenance of the skeletal structure, by supplying convenient means to repair possible damage and permitting physiological rearrangements, by means of which a significant metabolic function of the skeletal tissue can be performed. In absence of true vascularization of the tissue itself, the close connection with the coelomic lining can provide it with suitable access to circulating elements like coelomocytes. Furthermore, the organic stroma facilitates relationships with the muscular and ligamental components, via the well known specialized insertions (Stauber & Markel, 1988; Smith et al., 1990), which are directly continuous with the stroma itself.

CONCLUSIONS

The echinoderms, and echinoids in particular, have devised at the endoskeleton level a series of extremely advanced and sophisticated structural solutions, the most effective and «ingenious» of which are shown in the dental apparatus. Through the analysis of the different solutions applied in this apparatus, it is clear that echinoids have profited advantageously by all the intrinsic potentialities of their skeletal material, which turns out to be structurally and functionally an extremely versatile tissue, capable of fulfilling different requirements: in this tissue calcareous material is employed in a unique way to build an inorganic framework, whose different microstructural patterns have to be considered as many mechanical adaptations and whose «open» structure is perfectly integrated and permeated by an organic stroma. Thus, due to the plastic interaction of these two components, the limited properties of the inorganic calcite are remarkably improved and transcended in a skeletal structure where remarkable size and strength are coupled with lightness, economy of material and adaptability, as well as great mobility. Only in the vertebrate endoskeleton can such a comparable range of properties be found.

16

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

Plate 1 -Jaw: outer face, a) Comprehensive frontal view showing the general structure of an isolated jaw. The four complementary pieces and the most characteristic aspects of the jaw are well recognizable. The wide foramen magnum and the sutured epiphyseal arch are distinctive features of the camarodont lantern, de = demi-epiphysis; dp = demi-pyramid; sf = subapical fossa; fm = foramen magnum; arrows = sutures. Bar = 1 mm. b) Demi-pyramid: dense labyrinthic stereom of an unspecialized superficial area. Bar = 5 um. c) Demi-pyramid: lower portion of a doorpost of the foramen showing a typical fascicular stereom microstructure. Bar = 200 um. d) Demi-pyramid: microperforate stereom of the upper portion of a doorpost of the foramen. Bar = 80 um. e) Detail of the intrapyramidal suture. The galleried stereom is the prevailing microstructural pattern in the areas close to the suture. The galleries of the stereom run mainly parallel to the suture: however, their direction becomes perpendicular in correspondence with the narrow borders of the suture itself. Bar = 200 urn. f) Enlargement showing the tight zig-zag intrapyramidal suture. In the borders of the suture the galleries of the galleried stereom run perpendicular to the suture plane. Bar = 30 um. g) Demi-pyramid: detail of the subapical fossa showing the wide insertion area of the retractor muscle. The uniform superficial layer of fine labyrinthic stereom is interrupted by the presence of regularly arranged holes. Bar = 400 um. h) Enlargement of one of the holes shown in Figure g. Bar = 40 um. i) Enlargement of the oral tract of the intrapyramidal suture. The complementary pieces are separated by a thin interspace filled by parallel bundles of ligaments, which look well preserved by the mild digestion. The dominant microstructural pattern of this limited area is the labyrinthic stereom. Bar = 30 um. Plate 2 -Jaw. outer face, a) Comprehensive frontal view of the epiphyseal arch of a jaw, seen in the whole context of the lantern. The complex relationships with the other skeletal components are shown, c = compass; db = dental bracket; ef = epiphyseal fossa; r = rotula; t = tooth; arrows = sutures. Bar = 400 um. b) Detail of a demi-epiphysis. The side view emphasizes the oblique demi-epiphysis/ demi-pyramid suture. The movable rotular joint is shown as well. Bar = 300 um. c) Demi-epiphysis: the dominant microstructural pattern is the labyrinthic stereom. In particular the detail shows the wide attachment fossa for the protractor muscle and the demi-epiphysis/ demi-pyramid suture. The connection between the complementary skeletal parts does not look very tight. Bar = 400 um. d) Detail of the intraepiphyseal suture. The labyrintic stereom is the dominant pattern in the areas adjacent to the suture. Bar =100 um. e) Epiphyseal fossa: in correspondence with the wide insertion area of the protractor muscle the labyrinthic stereom acquires a finer and more open texture, providing the muscle fibers with suitable anchorage. Bar = 200 um. f) Detail of the demi-epiphysis/ demi-pyramid articulation showing a wide ligamental band connecting the articular parts. Notice the deep insertion of the ligaments, which look well preserved by the mild digestion process employed. Bar = 100 um. Plate 3 -Jaw: latero-radial side, a) Comprehensive lateral view of a whole jaw. A marked articular line (arrows) separates the epiphyseal from the pyramidal part. The adaxial inner border shows prominent comb-like indentations, de = demi-epiphysis; dp = demi-pyramid; Bar = 1 mm. b) Detail of the attachment area of the interpyramidal muscle. The whole area looks transversely striated by regular ridges which continue in the teeth of the indented border. Bar = 400 urn. c) Enlargement of Figure b showing the specialized type of galleried stereom employed for the attachment area of the interpyramidal muscles. The pattern is characterized by longitudinally aligned and parallel trabeculae which follow the arrangement of the muscle fibers. Bar = 60 um. d, e) Details of the oral end of the latero-radial side of the jaw. d) Close to the borders of the attachment area of the interpyramidal muscle, the regular transversely striated pattern is replaced by a more uniform superficial layer of dense labyrinthic stereom. e) Very dense labyrinthic (to the top left) and microperforate stereom (on the right) are the dominant superficial patterns of the conical terminal region. Bars of Figures d and e = 400 um. Plate 4 -Jaw. Latero-radial side, a) Lateral view of the overall epiphyseal portion. Since this part is mostly engaged in the movable rotular joint, its articular surface shows a number of structural specializations, abc = abaxial crista; it = intermediate turbercle; ads = adaxial saddle; arrows = demi-epiphysis/ demi-pyramid suture. Bar = 1 mm. b) Detail of the adaxial inner border of the epiphyseal region. The dense labyrinthic stereom seems to be the dominant microstructural pattern of this unspecialized area. Notice the abrupt transition in microstructure from the pyramidal to the epiphyseal part. Bar = 200 um. c) Enlargement of Figure b emphasizing the network of the labyrinthic stereom. Fascicular stereom of the pyramidal region to the bottom left. Bar = 50 um. d, e, f) Rotular joint: details of some articular specializations. The micropeforate and the imperforate stereom are the characteristic patterns of the intermediate tubercle (d), the abaxial crista (e) and the adaxial saddle (f), i.e., the areas most subjected to the friction with the correspondent rotular surfaces. A narrow lateral strip contributes to the attachment area of the interpyramidal muscle (f) by employing a fine, open and oriented labyrinthic stereom. Bars of Figures d and f = 200 urn; bar of Figure e = 100 um. Plate 5 -Jaw: aboral base, a, b) Comprehensive aboral views of the jaw seen in an isolated sample (a) or in the context of the whole lantern (b). The very limited aboral surface, completely belonging to the epiphysis, shows a number of articular specializations, abc = abaxial crista; ads = adaxial saddle; it = intermediate tubercle; rli = insertion area of the rotular ligament; arrows = intraepiphyseal suture. Bar of Figure a = 800 (im; bar of Figure b = 400 um. c, d, e, f) Enlargements of Figure a. The dense labyrinthic stereom prevails in all the unspecialized area (c, d). When the surface interacts with the rotula, giving rise to conspicuous grooves and relieves (c,e), the patterns employed are the microperforate and the imperforate stereom. A specialized type of galleried stereom is then specifically developed at the level of the insertion areas of the strong rotular ligament (f, on the right). Bar of Figure c = 600 um; bar of Figure d = 50 um; bar of Figure e = 400 um; bar of Figure f = 80 um. g) Detail of the intraepiphyseal suture. The strong ligaments connecting together the articular parts is well preserved thanks to the mild semi-digestion of the samples. Bar = 200 um. Plate 6 -Jaw: sutural surfaces, a, c, e) Intrapyramidal suture. Details of the articular surface: a, aboral tract; b, intermediate tract; c, oral tract. The articular surface always consists of two well defined zones, greatly different in microstructure.The spatial ratio between the two zones varies from one tract to the other. Bars of Figures a and c = 200 um; bar of Figure e = 400 um. b) Enlargement of Figure a. The outer band of the articular surface (to the bottom) is characterized by a regular arrangement of densely packed pegs, distinctive of the sutural galleried stereom. This band corresponds to the true suture zone. Also the inner band (to the top) consists of galleried stereom, which, however, is superficially arranged in a coarser pattern with larger pores and free-ended trabeculae. This band seems to be subdivided into well defined subbands. At this level the suture is not very tight and the connection between the two articular parts cannot be very rigid. Bar =100 um. d) Enlargement of Figure c showing the pegged regular arrangement distinctive of the outer band of the suture. Bar = 50 urn. f) Enlargement of Figure e showing a detail of the inner sutural band, which provides the ligaments with suitable attachments. A remarkable roughness characterizes the internal zone of this band. Bar = 50 um. Plate 7 - Jaw: sutural surfaces, a) Demi-epiphysis/ demi-pyramid suture. Comprehensive view of the whole articular surface of a demipyramid. The low magnification gives a general idea of the very different articular specializations involved. Notice the wide intrapyramidal cavity, ic = intrapyramidal cavity; is = inner surface; dg = dental guide; arrows = intrapyramidal suture. Bar = 1 mm. b) Enlargement of

MICROSTRUCTURE AND MECHANICAL DESIGN

17

Figure a showing well recognizable articular areas greatly different in microstructure. In this case the true suture is restricted to the outer areas (on the left) whereas the attachment area of the interarticular ligaments involves all the remaining surface. Bar = 400 um. c) Enlargement of Figure b showing a detail of the different stereom microstructure in the two articular areas. The pegged arrangement of the sutural galleried stereom (on the left) is abruptly replaced by the more open and coarse pattern of the attachment area of the interarticular ligaments (on the right). Bar = 40 um. d) Demi-epiphysis-demi-pyramid suture. Detail of the articular surface of the demi-epiphysis. Two different articular zones are well recognizable. Notice the not very deep intraepiphyseal cavity. Bar = 200 um. e) Enlargement of Figure d showing the different microstructural pattern of the two articular areas involved in the same joint. Sutural area (to the bottom) and attachment area of the interarticular ligaments (to the top). Bar = 100 (im. f) Intraepiphyseal suture. Comprehensive view of the whole articular surface. Bar = 400 Jim. g) Enlargement of Figure f showing the distinctive patterns of the two articular areas involved. The sutural area is characterized by regularly arranged pegs (to the top) whereas the attachment area of the interarticular ligament shows the typical porous network with free-ended trabeculae (to the bottom). Bar = 100 um. Plate 8 -Jaw: histological organization of the sutures, a) Cross section of a demi-pyramid. The detail shows the structure of the intrapyramidal suture, which looks clearly subdivided into two differently arranged parts. In the true suture the complementary parts tightly fit in and are connected by a limited amount of short collagen fibres. In the other half articulation the connection is not so tight, but well developed bundles of ligaments cross the articular gap penetrating for a certain depth into the skeletal thickness. Mallory trichrome staining, sa = sutural part of the articulation; la = ligamentous part of the articulation . Bar = 50 um. b, c) Semi-tangential sections of an intrapyramidal suture, b) Detail of the ligamentous part of the articulation showing the terminal loops of the collagen bundles around the skeletal trabeculae. c) Detail of the sutural part of the articulation showing the perfect matching of the two demi-pyramids and the limited amount of collagen fibres involved. Milligan trichrome staining. Bars of Figure b and c = 30 um. d) Longitudinal section of a jaw, involving the demi-epiphysis/ demi-pyramid suture. The bivalent structure of the joint is shown. In this case the sutural part of the articulation is restricted to a very small area (sa) whereas the ligamentous part is much more developed (la). The terminal loops of the collagen fibres are clearly discernible. The Milligan trichrome stain evidentiates very clearly the collagen component (in green). Muscular component in red. im = interpyramidal muscles. Bar = 50 um. e) Enlargement of Figure d showing a detail of the arrangement of the interarticular ligaments and, in particular, their terminal loops around the skeletal trabeculae. In correspondence with these articular zones, the skeletal stroma is very abundant and rich in cells. Milligan trichrome staining. Bar = 30 um. Plate 9 -Jaw: inner surface, a) Comprehensive view of a half jaw seen from its internal side. A marked articular line separates the complementary demi-epiphysis and demi-pyramid (arrows), ps = articular surface of the intrapyramidal suture; es = articular surface of the intraepiphyseal suture; dg = dental guide. Bar = 200 |im. b) Detail of the dental guide marked by a clearly recognizable strip of dense galleried stereom longitudinally oriented with respect to the pyramid (to the top). The labyrinthic stereom is widely diffused in the other less specialized areas (to the bottom). Bar =100 urn. c) Enlargement of Figure b showing the regular, longitudinally aligned pattern of the galleried stereom. Bar = 30 um. d) Detail of the inner adaxial border. The fascicular (on the left) and the microperforate stereom (on the right) are the distinctive patterns of this limited aera. Bar = 200 um. e) Enlargement of Figure d showing a detail of the fascicular stereom. At the level of the indented border this pattern is replaced by a dense labyrinthic stereom. Bar = 80 um. Plate 10 - Jaw: internal architecture, a) Comprehensive view of an intermediate transverse fracture of a demi-pyramid showing the internal architecture of the ossicle, which consists of a triangular central body (cb) and a wide lateral wing (lw). ic = intrapyramidal cavity. Bar = 500 fim. b) Comprehensive view of a transverse fracture of a demi-pyramid: the cut is closer to the oral end. The internal microarchitecture looks much more uniform and compact, cb = central body; lw = lateral wing. Bar = 400 um. c, d, e) Enlargements of Figure a showing some aspects of the internal architecture of the demi-pyramid. c) Central body: different stereom layers are well recognizable. Two bands of galleried and fascicular stereom are developed, in this sequence, near the outer surface (to the bottom), whereas labyrinthic stereom predominates centrally. d) Detail of the central cavity, which looks internally covered and subdivided by sheets of laminar stereom. e) lateral wing of the demi-pyramid: the stereom arrangement is rather compact. The outer layers of galleried and fascicular stereom (to the top) are followed by an inner thick layer of microperforate stereom (to the bottom). Bars of Figure c and d = 100 ^m; bar of Figure e = 50 um. f, g, h) Enlargements of Figure b showing some microsctructural aspects distinctive of this type of apical fracture, f) Detail of the central body of the demi-pyramid: alternating internal layers of galleried (to the top) and fascicular stereom (to the bottom) are shown. A well defined area of rectilinear stereom is recognizable as well (to the top right), g) Superficial layers of galleried stereom in correspondence with the sutural articular surface (to the top left), h) Lateral wing of the demi-pyramid: microperforate and imperforate stereom are the dominant patterns of the most exposed external regions. Bar of Figure f = 100 um; bar of Figure g = 40 um; bar of Figure h = 50 um. Plate 11 -Jaw: internal architecture, a) Aboral part of a demi-pyramid cut along a longitudinal plane, crossing its central body. The fracture shows a very complex internal architecture of the ossicle, dg = dental guide; arrows = demi-epiphysis/demi-pyramid articular surface; ic = intrapyramidal cavity; Bar = 600 um. b, and c) Enlargements of Figure a. b) Typical laminar arrangements of the stereom microstructure in correspondence with the internal cavity, c) Different stereom layers of the ossicle thickness. Galleried stereom is developed near the outer surface (to the bottom), whereas labyrinthic stereom predominates centrally (to the top). Bar of Figure b = 20 um; bar of Figure c = 200 um. d) Detail of the same fracture of figure a, but seen in its oral part. The compactness of the microstructural pattern is very enhanced: the main internal layers consist of microperforate and fascicular stereom externally delimited by a thin layer of dense labyrinthic stereom. Bar = 1 0 0 um. e) Detail of a longitudinal fracture of a demi-epiphysis crossing its central body. The dominant internal pattern is represented by a regularly aligned and dense galleried stereom. The stereom is oriented along to the main longitudinal axis of the whole jaw. Bar = 20 um. f) Enlargement of Figure e showing the regular arrangement of pores and trabeculae of the galleried stereom. Bar = 10 um. Plate 12 - Rotula. a) Top view of a rotula seen in the context of the lantern. The compass is partly removed to emphasize the main articular features of the rotular joint, c = compass; de = demi-epiphysis. Bar = 500 um. b) Rotula seen from its aboral side. Very dense and compact labyrinthic stereom characterizes this smooth and homogeneous external face, abh = abaxial head; adh = adaxial head. Bar = lmm. c) Rotula seen from its oral side. This internal face shows a symmetrical arrangement of articular specializations in correspondence with the areas directly interacting with the epiphyses. abc = abaxial condyle; adg = adaxial groove; if = intermediate fossa. Bar = 1 mm. d) Detail of the adaxial rotular head showing, in particular, the articular fossa for the compass. Bar = 400 urn. e, f, h) Enlargements of Figure c showing some specialized articular areas, which are characterized by a smooth layer of microperforate and imperforate stereom: e, Adaxial groove; f, Condyle of the abaxial head; h, Intermediate fossa. Some areas of fascicular stereom are limited to the central regions (e, f, on the right), whereas the lateral sides offer wide insertion areas to the rotular ligaments (e, on the left). Bar of Figure e = 400 um; bar of Figure f = 200 um; bar of Figure

18

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

h = 100 um. g) Rotula-compass joint: the ligamental bundles connecting the compass to the adaxial rotular head are firmly inserted in a fine and porous network of labyrinthic stereom. The ligaments are well preserved by the mild semi-digestion of the sample. Bar =100 urn. i) Cross fracture showing the very homogeneous and compact internal structure, which mainly consists of very dense fascicular stereom, regularly oriented along the longitudinal axis of the ossicle. Bar = 40 um. Plate 13 - Compass, a) Top view of a compass-rotula complex. The structure of the compass is shown in its more general aboral aspects, abf = abaxial fork; adh = adaxial hook; If = iateral flap. Bar = 1 mm. b, c and e) Enlargements of Figure a showing some details of the stereom microstructure of the aboral face, b) Fine, open and irregular labyrinthic stereom of a lateral flap. This area, used for the insertion of the elevator muscles, shows a corrugated superficial structure, c) Dense labyrinthic stereom of an unspecialized superficial area, e) Dense and smooth labyrinthic stereom of the abaxial forked end. Bars of Figures b and e= 200 um; bar of Figure c = 50 um. d) Detail of the adaxial hooked end of the compass including its connection with the rotula. The areas close to the suture are typically characterized by galleried stereom whose galleries are perpendicular to the suture itself. Bar = 200 urn. f) Sutural surface. The whole articular area shows the typical bivalent pattern of the other sutures consisting of a pegged sutural zone (to the bottom) and of an attachment area for the interarticular ligament (to the top). Bar = 100 um. g) Detail of the abaxial forked end showing the specialized and deep insertion of the compass depressor ligaments on the porous and trabecular skeletal network. Bar = 200 fim. h) Detail of the abaxial end seen from its oral side. The dense labyrinthic stereom is widely diffused and only occasionally replaced by other more specialized stereom types. Bar = 500 um. i) Cross fracture to show the very compact and homogeneous internal architecture. The deeper microstructure consists of dense fascicular stereom oriented along the longitudinal axis of the ossicle. Bar = 50 um. Plate 14 - Tooth: overall structure, a) Comprehensive lateral view of a whole tooth showing the general features of its structure. The plumula is partly removed. Bar = 400 um. b, e) Details of the aboral growing end seen in lateral (b) and frontal view (e): the primary lamellar structure is widely uncovered and well recognizable, since the exiguous secondary component is only represented by a thin trabecular layer limited to the lateral band, pip = primary lamellar plates; psp = primary side plates; st = secondary trabeculae. Bar of Figure b = 50 um; bar of Figure e = 100 um. c, f) Details of the intermediate part seen in lateral (c) and frontal view (f): at this level the primary structure is covered and concealed by the conspicuous secondary component, which form a compact and homogeneous amalgam of microperforate and imperforate stereom. k = keel; pip = primary lamellar plates; sc = secondary component; st = secondary trabeculae. Bar of Figure c = 100 um; bar of Figure f = 200 um. d, g) Details of the oral chewing tip seen in lateral (d) and frontal view (g): the secondary component prevails on the primary one forming a very solid and hard cemented structure. Bar of Figure d = 400 um; bar of Figure g = 200 um. Plate 15 - Tooth: primary structure, a) Detail of an isolated primary plate of the aboral growing end. The lateral border clearly shows the hollow internal structure of the lamellar plate. Bar = 1 0 um. b) Enlargement of Figure a showing the wide hollow internal interspace of the lamellar plate. Bar = 5 ^im. c) Intermediate tooth part: detail of an oblique fracture showing the compact internal structure of the primary plates, pp = primary plates; arrows = secondary component. Bar = 40 um. d) Enlargement of Figure c. The lamellar plate looks rather compact through its whole thickness and its internal cavity seems to be completely obliterated. Bar = 5 um. e) Intermediate tooth part: comprehensive view of a cross fracture. The whole structure is very compact and solid, both in its primary (lamellar plates and prisms) and secondary components, p = prisms; pp = primary plates. Bar = 200 um. f) Aboral growing part: detail of an oblique fracture involving the prism zone. The hollow structure of the prisms is clearly recognizable. Bar = 10 um. g) Intermediate tooth part: detail of a cross fracture through the prism zone. At this level the prisms show a remarkably increased thickness and a very homogeneous and compact structure. Bar = 40 um. h) Aboral growing part: detail of the wide internal cavity of a prism. Bar = 2 um. Plate 16 - Tooth: secondary structure, a) Lateral surface of the aboral part of the tooth: early deposition of secondary elements in form of thorny processes on the edges of the primary plates. Bar = 40 um. b) Lateral surface of the aboral-intermediate part. At this more advanced level the secondary elements form a wide longitudinal band characterized by a regular monolayer of interconnected trabeculae. This band is employed as insertion area by the ligaments connecting the tooth to the jaw. Bar = 30 um. c) Lateral surface of the intermediate-oral part. At this level the tooth justs out from its jaw: the secondary trabecular arrangement of the lateral strips is progressively replaced by a more and more homogeneous and compact layer of «pegged» stereom. Bar = 20 urn. d) Lateral surface of the oral end of the tooth. The secondary component forms a very compact and thick layer of imperforate stereom. Notice the residual pegs of the lateral band. Bar = 20 um. e, f) Frontal surface of the aboral-intermediate part. The progressive deposition of secondary elements on the primary structure is shown. At a more aboral level, the presence of secondary elements is limited to a thin and very discontinuous layer of compact stereom (e), which subsequently tends to become a continuous and dense layer and to cover the primary plates more uniformly. Bars of Figures e and f = 50 um. g, h) Lateral surface of the intermediate part. The progressive deposition of the secondary elements is shown at keel level (g). The lateral indented borders of the side plates are welded together by a more and more compact layer of secondary elements. Bar of Figure g = 200 um; bar of Figure h = 80 um. i) Lateral surface of the intermediate part of the tooth: detail of an oblique superficial fracture. The connection and the welding between primary lamellar plates and secondary trabeculae is shown. Bar = 20 um. j and k) Details of a cross intermediate fracture. The internal microarchitecture of the tooth consists of closely connected primary and secondary elements, j) Lamellar zone: the secondary plates follow the arrangement of the primary plates and very well show their porous structure of microperforate stereom. k) Fibrous zone: the secondary plates are here represented by small, partly overlapped compact plates, which strictly cover and conceal the prisms. Bar of Figure j = 20 um; bar of Figure k = 100 um. 1) Oral end of the tooth; detail of an oblique superficial fracture. The secondary component forms an homogeneous cement of imperforate stereom, which covers and amalgamates the primary structure. Bar = 20 um. Plate 17 - Stroma organization, a) Jaw. Detail of the outer coelomic epithelium lining the external exposed surfaces of the ossicle. The typical flagellated collar-cells are clearly recognizable. Bar = 4 urn. b) Jaw. Detail of the external surface. A strip of the connective sheath covering the ossicle has been raised and turned inside out. Bar =100 um. c) Jaw. Detail of the external surface. The tight periosteum-like connective sheath is partly removed to show its intimate connection with the underlying skeletal structure: the external pores and galleries seem to be penetrated by the periosteum itself. Bar = 20 um. d) Jaw. Detail of the connective sheath covering the ossicle, which seems to consist of a thick fibrous structure. Bar = 4 um. e)Jaw. Detail of a superficial fracture showing the fibrous and close network of the organic stroma. Bar = 4 um. f and g) Jaw. Details of deeper fractures to show the distribution and the organization of the stroma in comparison with the inorganic component. The connective network is now very thin and mainly consists of bundles of fibrils variously interlaced. Some scattered cell elements are recognizable, f) Oblique fracture; g) Cross fracture. Bar of Figure f = 10 um, Bar of Figure g = 5 um. h) Rotula. Detail of a cross fracture. The organic component is very exiguous in relation to the compactness of the stereom microstructure, but is continuous throughout the whole thickness of the ossicle. Bar = 40 um.

MICROSTRUCTURE AND MECHANICAL DESIGN

19

Plate 18 - Stroma organization, a) Tooth. Adaxial view of the plumula showing its continuous and tight fibrous covering. Bar = 100 \im. b) Tooth. Terminal end of the plumula: the detail shows a large amount of proliferating cells associated to fibrillar material. Bar = 5 |xm. c) Tooth. Detail of the lateral surface of the aboral- intermediate part: a uniform continuous fibrous sheath covers the whole skeletal structure, underlining its lamellar outlines. Bar = 10 |xm. d) Tooth. Detail of the lateral surface of the aboral- intermediate part after removing the outer connective sheath. The laminar plates are alternated with layers of stroma consisting of a number of odontoblasts tightly associated to a conspicuous felt of interlaced fibrils. Bar = 10 iim. e) Enlargement of Figure d showing a typical odontoblast and the fine structure of the surrounding stroma. Bar = 2 ^m. 0 Tooth. Detail of the lateral surface of the intermediate part: the stroma is progressively replaced by inorganic component. In particular the intralamellar stroma tends to regress completely, while the odontoblasts remain imprisoned inside the calcareous walls. Bar = 20 \im. Plate 19 - Stroma organization, a, b and c) Tooth. Details of fractured primary plates (a, c) and prisms (b) from the aboral part. The network of the fibrillar stroma penetrates even inside the hollow structure of the primary elements, partially filling their internal cavities (a, b). The resulting primary structure is therefore completely interconnected, both externally and internally, by the organic stroma (c). Bar of Figure a = 5 urn; bar of Figure b = 8 tun; Bar of Figure c = 10 \im. d, e and f) Tooth. Detail of fractured primary plates from the intermediate (d), intermediate-oral (e) and oral part (f) showing the progressive withdrawing of the organic stroma from the deep structure. Due to the increased compactness and thickness of the primary plates (d, e) and to the remarkable presence of secondary elements (f), the overall amount of stroma is progressively reduced and is replaced by inorganic component. Some odontoblasts partly or completely cemented in the calcareous phase are shown (e and f, to the bottom right). Bar of Figure d = 1 tim; bar of Figure e = 8 ^m; bar of Figure f = 20 |xm. Plate 20 - Stroma histological organization, a) Jaw. Detail of a cross section showing the distribution and the structure of the stroma in a superficial zone of the ossicle. The outer periosteum-like sheath and the internal network are emphasized by the Milligan trichrome staining, which marks the collagen component in green. Bar = 30 (im. b) Jaw. Detail of an oblique section showing the internal organization of the stroma. The transition to different arrangements is well recognizable. Dense labyrinthic stereom to the left bottom; galleried stereom to the top right. Picroindigocarmine-PAS staining. Bar = 30 urn. c) Tooth. Detail of a longitudinal sagittal section of the plumula. The amount of stroma is very conspicuous and shows a complex arrangement in the different zones, varying according to the organization of the inorganic component, abl = abaxial lamellar part; adf = adaxial fibrous part. The connective sheath, very rich in collagen fibres, is stained in green. Milligan trichrome staining. Bar = 30 \im. d) Tooth. Detail of a longitudinal sagittal section of the intermediate part showing the distribution of the organic stroma. The overall amount of stroma is very reduced and is strictly related to the arrangement and the distribution of the inorganic component, abl = abaxial lamellar part; adf = adaxial fibrous part. Milligan trichrome staining. Bar = 30 \im. e) Tooth. Detail of a cross section of the intermediate part showing the different arrangement of the stroma in relation to the primary and secondary structure of the tooth. Abaxial part to the bottom; keel to the top. Mallory trichrome staining. Bar = 50 nm. Plate 21 - Stroma and related soft tissues: histological organization, a) Jaw. Detail of a cross intermediate section. The overall organization of the organic stroma network and its relationships with the related soft tissues are clearly shown. In particular, the Milligan trichrome staining marks, the specialized attachment of the interpyramidal muscles (im). Notice the wide intrapyramidal cavity and the nerve branchings running inside (n). Bar = 1 0 0 nm. b and c) Jaw. Details of the insertions of the interpyramidal (b) and the retractor muscles (c) in longitudinal section. The "composite tendons* and in particular their typical terminal loops around the skeletal trabeculae are clearly shown. The Milligan trichrome stain allows to distinguish very well the muscle from the connective tissue, im = interpyramidal muscle; rm = retractor muscle. Bar of Figure b = 30 |im; bar of Figure c = 50 \im. d) Jaw. Detail of a longitudinal frontal section showing the attachment of the rotular ligaments. The ligamental bundles penetrate the skeletal structure, by fraying in sub-bundles and firmly anchoring to the trabeculae. Milligan trichrome staining. Bar = 30 urn. e) Jaw. Detail of a cross section showing the ligamental band connecting the jaw to the tooth. Milligan trichrome staining, j = jaw; t = tooth. Bar = 50 um. f) Enlargement of Figure e to show in detail the structure of the ligament and its connection with the related skeletal parts. Bar = 30 (im.

M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

MICROSTRUCTURE AND MECHANICAL DESIGN

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M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

MICROSTRUCTURE AND MECHANICAL DESIGN

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MICROSTRUCTURE AND MECHANICAL DESIGN

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M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE

MICROSTRUCTURE AND MECHANICAL DESIGN

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Märkel K., 1974 - Morphologie der Seeigelzahne V. Die Zahne der Clypeastroida. Z. Morph. Tiere, 78: 221-256. Märkel K., 1976a - Das Wachstum der «Lanteme des Aristoteles» und seine Anpassung an die Funktion der Lanterne (Echinodermata, Echinoidea). Zoomorphologie, 86: 25-40. Märkel K., 1976b - On the teeth of sea urchins. Thalassiajugoslavica, 72:207-211. Märkel K., 1979a - The lantern of Aristotle. In: M. Jangoux (ed.), Echinoderms: Present and Past. Balkema, Rotterdam, pp. 91-92. Märkel K., 1979b - Structure and growth of the cidaroid socket-joint lantern of Aristotle compared to the hinge-joint of non-cidaroid regular echinoids (Echinodermata, Echinoidea). Zoomorphology, 94: 1-32. Märkel K., Gorny P., 1973 - Zur funktionellen Anatomie der Seeigelzahne. Z. Morph. Tiere, 75: 223-242. Märkel K., Gorny P., Abraham K., 1977 - Microarchitecture of sea urchin teeth. Fortschr. Zool., 24: 103-114. Märkel K., Kubanek F., Willgallis A., 1971 - Polykristalliner Calcit bei Seeigeln. Z. Zellforsch. Mikrosk. Anat., 119: 355-377. Märkel K., Röser U., 1983 - The spine tissues in the echinoid Eucidaris tubuloides. Zoomorphology, 103: 25-41. Märkel K., Röser U., 1985 - Comparative morphology of echinoderm calcified tissues: Histology and ultrastructure of ophiuroid scales. Zoomorphology, 105: 197-207. Märkel K., Röser U., Mackenstedt U., Klostermann M., 1986 Ultrastructural investigation of matrix-mediated biomineralization in echinoids (Echinodermata, Echinoidea). Zoomorphology, 106: 232-243. Märkel K., Röser U., Stauber M., 1989 - On the ultrastructure and the supposed function of the mineralizing matrix coat of sea urchins (Echinodermata, Echinoida). Zoomorphology, 109: 79-87. Märkel K., Titschack H., 1969 - Morphologie der Seeigelzahne. I. Der Zahn von Stylocidaris affinis. Z. Morph. Tiere, 64: 179-200. Moss M. L., Meehan M., 1967 - Sutural connective tissues in the test of an echinoid Arbacia punctulata. Acta anat., 66: 279-304. Nichols D., Currey J. D., 1968 - The secretion, structure, and strength of echinoderm calcite. In: S. M. McGee-Russel & K. F. A. Ross (eds), Cell structure and its interpretation. Arnold, London, pp. 251-261. Nissen H. U., 1969 - Crystal orientation and plate structure in echinoid skeletal units. Science, 166: 1150-1152. O'Neill P. L., 1981 - Polycrystalline echinoderm calcite and its fracture mechanics. Science, 213: 646-648. Pearse J. S., Pearse V. B., 1975 - Growth zones in the echinoid skeleton. Am. Zool., 15, 731-753. Raup D. M., 1966 - The endoskeleton. In: R. A. Boolootian (ed.), Physiology of Echinodermata. J. Wiley and Sons, New York, Ch. 16. Regis M. B., 1977 - Organisation microstructurale du stereome de l'Echinoide Paracentrotus lividus (Lamarck) et ses eventuelles incidences physiologiques. C. R. Acad. Sei. Paris, 285 D: 189-192. Regis M. B., Thomassin B. A., 1985 - Macro- and microstructure of the primary spines in Asthenosoma varium Grube (Echinothuridae: Echinoidea): Affinities with the Diadematidae and Toxopneustidae. In: B. F. Keegan & B.D.S. O'Connor (eds), Echinodermata. Balkema, Rotterdam, pp. 321-332. Schroeder J. H., Dwornik E. J., Papike J. J., 1969 - Primary protodolomite in echinoid skeletons. Geol. Soc. Amer. Bull., SO: 1613-1616. Smith A. B., 1980 - Stereom microstructure of the echinoid test. Special papers in Palaeontology n. 25. The Palaeontological Association, London pp. 81. Smith A. B., 1984 - Echinoid paleobiology. George Allen & Unwin, London pp. 190. Smith D. S., Del Castillo J., Morales M., Luke B., 1990 - The attachment of collagenous ligament to stereom in primary spines of the sea-urchin, Eucidaris tribuloides. Tissue & Cell., 22: 157-176. Stauber M., Märkel K., 1988 - Comparative morphology of muscleskeleton attachments in the Echinodermata. Zoomorphology, 108: 137-148.

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M. D. CANDIA CARNEVALI, F. BONASORO, G. MELONE Weber J. N., Greer R., Voight B., White E., 1969 - Unusual strenght properties of echinoderm calcite. J. Ultrastruct. Res., 26: 355-366. Weiner S., 1985 - Organic matrixlike macromolecules associated with the mineral phase of sea urchin skeletal plates and teeth. J. exp. Zool., 234: 7-15. Wilkie I. C., 1984 - Variable tensility in echinoderm collagenous tissues: a review. Mar. Behav. Physiol., 11: 1-34. Wilkie I.C., Emson R. H., 1988 - Mutable collagenous tissues and their significance for echinoderm paleontology and phylogeny. In: C.R.C. Paul & A. B. Smith (eds), Echinoderm phylogeny and evolutionary biology. Oxford Science Publications, Liverpool Geological Society, pp. 311-330.