Norbert Cyran

The influence of scaffold architecture on chondrocyte distribution and behavior in matrix-associated chondrocyte transplantation grafts.

Scaffold architecture and composition are important parameters in cartilage tissue engineering. In this in vitro study, we compared the morphology of four different cell-graft systems applied in clinical cartilage regeneration and analyzed the cell distribution (DAPI nuclei staining) and cell-scaffold interaction (SEM, TEM). Our investigations revealed major differences in cell distribution related to scaffold density, pore size and architecture. Material composition influenced the quantity of autogenous matrix used for cellular adhesion. Cell bonding was further influenced by the geometry of the scaffold subunits. On scaffolds with widely spaced fibers and a thickness less than the cell diameter, chondrocytes surrounded the scaffold fibers with cell extensions. On those fibers, chondrocytes were spherical, suggesting a differentiated phenotype. Fiber sizes smaller than chondrocyte size, and widely spaced, are therefore beneficial in terms of improved adhesion by cell shape adaptation. They also support the differentiated stage of chondrocytes by preventing the fibroblast-like and polygonal cell shape, at least briefly.

Histochemical characterization of the adhesive organ of three Idiosepius spp. species

An adhesive organ is a prominent characteristic of the genus Idiosepius. Histological, histochemical and ultrastructural methods were applied to elucidate the nature of secretion of the epithelial cells of three Idiosepius species. The adhesive organs of Idiosepius biserialis and Idiosepius pygmaeus consist of five distinct cell types that can be distinguished morphologically and by the composition of their secretions. Histochemical analysis revealed that three cell types contain different sugar units and basic proteins, whereas the interstitial cells lacked secretory material. Acidic and sulfated substances were absent in Idiosepius secretions. The adhesive organ, but not the secretory material of the glandular cells, contained O-linked oligosaccharides. The histochemical analysis of the secretory products suggested that adhesion and release are not effected by a “duo-gland” adhesive system as in Euprymna scolopes. Idiosepius presumably uses a transitory adhesion, perhaps induced by secretion of a highly viscous gel. Release might be caused by contraction of the mantle musculature and/or chemical release mechanisms such as dilutors or enzymes.

Ultrastructural characterization of the adhesive organ of Idiosepius biserialis and Idiosepius pygmaeus (Mollusca: Cephalopoda)

Water drift and tidal rise make the use of bonding mechanisms beneficial for small benthopelagic or interstitial marine animals. Chemical adhesives for attachment are very common in molluscs; however, only a few cephalopods have glue producing organs. The family Idiosepiidae is characterized by an epithelial adhesive organ (AO) located on the posterior part of the dorsal mantle area. Previous morphological and histological studies described three non-glandular cell types (basal, interstitial and fusiform cells) and three glandular cell types (goblet, columnar and granular cells) containing protein and carbohydrate components. However, these studies provide different information about the nomenclature and characteristics of the cell types. The present ultrastructural analyses and a 3 D reconstruction of the AO of Idiosepius pygmaeus and Idiosepius biserialis therefore serve to investigate the cell distribution, the fine structure of the cells and possible interactions between the cells. We found that basal cells form a continuous cell layer along the basal membrane, overlapped by the other epithelial cells. Embedded in microvilli-covered interstitial cells the glandular cells are more or less evenly distributed within the AO. Goblet and granular cells are solitary glandular cells without conspicuous morphological characteristics, whereas the columnar cells are arranged in dense aggregations of 5―15 cells. Each columnar cell is enclosed by a narrow supporting interstitial cell which contains dense long i tudinal filament strands. The secretory process of the cells in the aggregation is synchronized. Each columnar cell aggregate bears approximately two ciliated sensory fusiform cells. The fusiform cells are connected to a neuronal network, aligned along the epithelium base. The results suggest that the bonding system is affected by two secretory cell types (granular and columnar cells). Both are similar in content, synthesis and secretory process but columnar cells are embedded in a particular cell environment. It is unclear in what way this arrangement is associated with the function of the AO. The neurons in several parts of the AO point to a neuronal control of the bonding mechanism. Comparisons with the AO cells of other cephalopods provide no indications for a morphological relationship between the adhesive systems.

Characterization of the Adhesive Systems in Cephalopods

Cephalopods are highly evolved invertebrates; since ancient times, they have been admired for their intelligence, their ability to change color within milliseconds and their flexible arms, equipped with suckers or hooks. The suckers are versatile, mainly used to attach mechanically (by a reduced-pressure systems with a low pressure up to 0.01 MPa) to hard or soft surfaces (Smith, 1996; Kier and Smith, 2002; Pennisi, 2002); its usage and force strength varies, from a “soft sensing” of unknown objects to a fast and forceful holding of resisting prey. The suckers also have a sensory function and are equipped with a large repertoire of numerous mechano- and chemoreceptors (Nixon and Dilly, 1977).

Morphological characterization of the glandular system in the salamander Plethodon shermani (Caudata, Plethodontidae)

Amphibians have evolved a wide variety of mechanisms that provide a certain degree of protection against predators, including camouflage, tail autonomy, encounter behavior and noxious or toxic skin secretions. In addition to these strategies, some amphibians release a glue-like secretion onto the surface of their skin when threatened. While some information regarding the origin and production of these adhesive secretions is available for frogs such as Notaden bennetti, these aspects are only partially understood in salamanders. We contribute to an earlier study and provide additional information regarding the origin, production, and characterization of the adhesive secretion in the red-legged salamander (Plethodon shermani) at a microanatomical level. When stressed, this salamander secretes a milky, viscous liquid from its dorsal and ventral skin. This secretion is extremely adhesive and hardens within seconds upon exposure to air. This study describes two cutaneous gland types (mucous and granular) in the dorsal and ventral epithelial tissue that differ considerably in their secretory content. While the smaller mucous glands contains flocculent to granular material, mostly acidic glycoproteins, the granular glands synthesize various granules of differing size and density that consist of basic proteinaceous material. The results strongly indicate that the secretions of both gland types from the dorsal as well as the ventral side form the adhesive mucus in Plethodon shermani, consisting of basic and acidic glycoproteins, glycoconjugates with mannose and α-L-fucose residues as well as lipid components.

Old and sticky—adhesive mechanisms in the living fossil Nautilus pompilius (Mollusca, Cephalopoda)

Nautiloidea is the oldest group within the cephalopoda, and modern Nautilus differs much in its outer morphology from all other recent species; its external shell and pinhole camera eye are the most prominent distinguishing characters. A further unique feature of Nautilus within the cephalopods is the lack of suckers or hooks on the tentacles. Instead, the animals use adhesive structures present on the digital tentacles. Earlier studies focused on the general tentacle morphology and put little attention on the adhesive gland system. Our results show that the epithelial parts on the oral adhesive ridge contain three secretory cell types (columnar, goblet, and cell type 1) that differ in shape and granule size. In the non-adhesive aboral epithelium, two glandular cell types (cell types 2 and 3) are present; these were not mentioned in any earlier study and differ from the cells in the adhesive area. The secretory material of all glandular cell types consists mainly of neutral mucopolysaccharide units, whereas one cell type in the non-adhesive epithelium also reacts positive for acidic mucopolysaccharides. The present data indicate that the glue in Nautilus consists mainly of neutral mucopolysaccharides. The glue seems to be a viscous carbohydrate gel, as known from another cephalopod species. De-attachment is apparently effectuated mechanically, i.e., by muscle contraction of the adhesive ridges and tentacle retraction.

Characterization of the adhesive areas in Sepia tuberculata (Mollusca, Cephalopoda)

Adhesion in cephalopods is either mechanical, involving a reduced‐pressure system of the arm and tentacle suckers, or is chemically mediated by special adhesive gland structures (as proposed for Euprymna, Idiosepius, and Nautilus). Four species of Sepia (S. typica, S. papillata, S. pulchra, and S. tuberculata) possess grooved structures on the ventral mantle surface and on the fourth arm pair, which are used to attach mechanically to the substratum. Because these areas are often partly covered with sand or debris, it has been hypothesized that chemical substances were involved in this attachment process. This study provides a histochemical and ultrastructural description of the glandular epithelium in the adhesive area of Sepia tuberculata. Two specific glandular cells (Type 1 and Type 2) are present in the epithelium, which differ clearly in their granule size and cellular structure. The aggregation of both cell types and their simultaneous secretion suggest that the secretions of both cell types work synergistically providing a two‐component adhesive system which supports the primarily mechanical sucker adhesion by making the arm surface sticky. J. Morphol. 2011.

Alterations in the mantle epithelium during transition from hatching gland to adhesive organ of Idiosepius pygmaeus (Mollusca, Cephalopoda)

Epithelial gland systems play an important role in marine molluscs in fabricating lubricants, repellents, fragrances, adhesives or enzymes. In cephalopods the typically single layered epithelium provides a highly dynamic variability and affords a rapid rebuilding of gland cells. While the digestive hatching gland (also named Hoyle organ) is obligatory for most cephalopods, only four genera (Nautilus, Sepia, Euprymna and Idiosepius) produce adhesive secretions by means of glandular cells in an adhesive area on the mantle or tentacles. In Idiosepius this adhesive organ is restricted to the posterior part of the fin region on the dorsal mantle side and well developed in the adult stage. Two gland cell types could be distinguished, which produce different contents of the adhesive. During the embryonic development the same body area is occupied by the temporary hatching gland. The question arises, in which way the hatching gland degrades and is replaced by the adhesive gland. Ultrastructural analyses as well as computer tomography scans were performed to monitor the successive post hatching transformation in the mantle epithelium from hatching gland degradation to the formation of the adhesive organ. According to our investigations the hatching gland cells degrade within about 1 day after hatching by a type of programmed cell death and leave behind a temporary cellular gap in this area. First glandular cells of the adhesive gland arise 7 days after hatching and proceed evenly over the posterior mantle epithelium. In contrast, the accompanying reduction of a part of the dorsal mantle musculature is already established before hatching. The results demonstrate a distinct independence between the two gland systems and illustrate the early development of the adhesive organ as well as the corresponding modifications within the mantle.

The short life of the Hoyle organ of Sepia officinalis: formation, differentiation and degradation by programmed cell death

Abstract Cephalopods encapsulate their eggs in protective egg envelopes. To hatch from this enclosure, most cephalopod embryos release egg shell-digesting choriolytic enzymes produced by the Hoyle organ (HO). After hatching, this gland becomes inactive and rapidly degrades by programmed cell death. We aim to characterize morphologically the development, maturation and degradation of the gland throughout embryonic and first juvenile stages in Sepia officinalis. Special focus is laid on cell death mechanisms and the presence of nitric oxide synthase during gland degradation. Hatching enzyme has been examined in view of metallic contents, commonly amplifying enzyme effectiveness. HO gland cells are first visualized at embryonic stage 23; secretion is observed from stage 27 onwards. Degradation of the HO occurs after hatching within two days by the rarely observed autophagic process, recognized for the first time in cephalopods. Nitric oxide synthase immunopositivity was not found in the HO cells after hatching, suggesting a possible NO role in cell death signalling. Although the HO ‘life course’ chronology in S. officinalis is similar to other cephalopods, gland degradation occurs by autophagy instead of necrosis. Eggs that combine a large perivitelline space and multi-layered integument seem to require a more complex and large gland system.

Characterization of the adhesive dermal secretion of Euprymna scolopes Berry, 1913 (Cephalopoda)

Bio-adhesion is a common and crucial process in nature and is used by several different species for camouflage, prey capture, hatching or to avoid drifting. Four genera of cephalopods belonging to four different families (Euprymna, Sepiolidae; Idiosepius, Idiosepiidae; Nautilus, Nautilidae; and Sepia, Sepiidae) produce glue for temporary attachment. Euprymna species live in near-shore benthic habitats of the Indo-Pacific Ocean, are nocturnal and bury into the seafloor during the day. The animals secrete adhesives through their epithelial glands to completely coat themselves with sand. In cases of danger, they instantaneously release the sandy coat as a sinking decoy to deflect predators. Earlier morphological investigations have shown that the adhesive gland cells of Euprymna scolopes are scattered on the dorsal epidermis. It has been proposed that neutral mucopolysaccharides, secreted by one gland type (goblet cells), are responsible for adhesion, whereas the release of the glue could be caused by acidic mucoproteins produced by ovate cells in the ventral epidermis. The ultrastructural re-investigation of the Euprymna epithelium in this study has indicated the presence of a new gland type (named flask cell), exclusively located in the dorsal epithelium and always neighboured to the known goblet cells. Based on our histochemical observations, the secretory material of the ovate cells does not display a strong reaction to tests for acidic groups, as had been previously assumed. Within the dermis, a large muscle network was found that was clearly distinctive from the normal mantle musculature. Based on our data, an antagonistic gland system, as previously proposed, seems to be unlikely for Euprymna scolopes. We hypothesize that the adhesive secretion is formed by two gland types (goblet and flask cells). The release of the sand coat may occur mechanically, i.e. by contraction of the dermal mantle muscle, and not chemically through the ovate cells.