Mutable Collagenous Tissue: A Concept Generator for Biomimetic Materials and Devices.

Echinoderms (starfish, sea-urchins and their close relations) possess a unique type of collagenous tissue that is innervated by the motor nervous system and whose mechanical properties, such as tensile strength and elastic stiffness, can be altered in a time frame of seconds. Intensive research on echinoderm 'mutable collagenous tissue' (MCT) began over 50 years ago, and over 20 years ago, MCT first inspired a biomimetic design. MCT, and sea-cucumber dermis in particular, is now a major source of ideas for the development of new mechanically adaptable materials and devices with applications in diverse areas including biomedical science, chemical engineering and robotics. In this review, after an up-to-date account of present knowledge of the structural, physiological and molecular adaptations of MCT and the mechanisms responsible for its variable tensile properties, we focus on MCT as a concept generator surveying biomimetic systems inspired by MCT biology, showing that these include both bio-derived developments (same function, analogous operating principles) and technology-derived developments (same function, different operating principles), and suggest a strategy for the further exploitation of this promising biological resource.

Morphological and Physiological Aspects of Mutable Collagenous Tissue at the Autotomy Plane of the Starfish Asterias rubens L. (Echinodermata, Asteroidea): An Echinoderm Paradigm.

The mutable collagenous tissue (MCT) of echinoderms has the capacity to undergo changes in its tensile properties within a timescale of seconds under the control of the nervous system. All echinoderm autotomy (defensive self-detachment) mechanisms depend on the extreme destabilisation of mutable collagenous structures at the plane of separation. This review illustrates the role of MCT in autotomy by bringing together previously published and new information on the basal arm autotomy plane of the starfish Asterias rubens L. It focuses on the MCT components of breakage zones in the dorsolateral and ambulacral regions of the body wall, and details data on their structural organisation and physiology. Information is also provided on the extrinsic stomach retractor apparatus whose involvement in autotomy has not been previously recognised. We show that the arm autotomy plane of A. rubens is a tractable model system for addressing outstanding problems in MCT biology. It is amenable to in vitro pharmacological investigations using isolated preparations and provides an opportunity for the application of comparative proteomic analysis and other "-omics" methods which are aimed at the molecular profiling of different mechanical states and characterising effector cell functions.

From Food Waste to Innovative Biomaterial: Sea Urchin-Derived Collagen for Applications in Skin Regenerative Medicine.

Collagen-based skin-like scaffolds (CBSS) are promising alternatives to skin grafts to repair wounds and injuries. In this work, we propose that the common marine invertebrate sea urchin represents a promising and eco-friendly source of native collagen to develop innovative CBSS for skin injury treatment. Sea urchin food waste after gonad removal was here used to extract fibrillar glycosaminoglycan (GAG)-rich collagen to produce bilayer (2D + 3D) CBSS. Microstructure, mechanical stability, permeability to water and proteins, ability to exclude bacteria and act as scaffolding for fibroblasts were evaluated. Our data show that the thin and dense 2D collagen membrane strongly reduces water evaporation (less than 5% of water passes through the membrane after 7 days) and protein diffusion (less than 2% of BSA passes after 7 days), and acts as a barrier against bacterial infiltration (more than 99% of the different tested bacterial species is retained by the 2D collagen membrane up to 48 h), thus functionally mimicking the epidermal layer. The thick sponge-like 3D collagen scaffold, structurally and functionally resembling the dermal layer, is mechanically stable in wet conditions, biocompatible in vitro (seeded fibroblasts are viable and proliferate), and efficiently acts as a scaffold for fibroblast infiltration. Thus, thanks to their chemical and biological properties, CBSS derived from sea urchins might represent a promising, eco-friendly, and economically sustainable biomaterial for tissue regenerative medicine.

Studying Echinodermata Arm Explant Regeneration Using Echinaster sepositus.

Echinoderms are marine invertebrate deuterostomes known for their amazing regenerative abilities throughout all life stages. Though some species can undergo whole-body regeneration (WBR), others exhibit more restricted regenerative capabilities. Asteroidea (starfish) comprise one of the few echinoderm taxa capable of undergoing WBR. Indeed, some starfish species can restore all tissues and organs not only during larval stages, but also from arm fragments as adults. Arm explants have been used to study cells, tissues and genes involved in starfish regeneration. Here, we describe methods for obtaining and studying regeneration of arm explants in starfish, in particular animal collection and husbandry, preparation of arm explants, regeneration tests, microscopic anatomy techniques (including transmission electron microscopy, TEM) used to analyze the regenerating explant tissues and cells plus a downstream RNA extraction protocol needed for subsequent molecular investigations.

Echinoderm regenerative response as a sensitive ecotoxicological test for the exposure to endocrine disrupters: effects of p,p'DDE and CPA on crinoid arm regeneration.

Echinoderms are valuable test species in marine ecotoxicology and offer a wide range of biological processes appropriate for this approach. Regenerating echinoderms can be regarded as amenable experimental models for testing the effects of exposure to contaminants, particularly endocrine disrupter compounds (EDCs). As regeneration is a typical developmental process, physiologically regulated by humoral mechanisms, it is highly susceptible to the action of pseudo-hormonal contaminants which appear to be obvious candidates for exerting deleterious actions. In our laboratory experiments, selected EDCs suspected for their antiandrogenic action (p,p'-DDE and cyproterone acetate) were tested at low concentrations on regenerating specimens of the crinoid Antedon mediterranea. An integrated approach which combines exposure experiments and different morphological analyses was employed; the obtained results suggest an overall pattern of plausible endocrine disruption in the exposed samples, showing that processes such as regenerative growth, histogenesis, and differentiation are affected by the exposure to the selected compounds. These results confirm that (1) regenerative phenomena of echinoderms can be considered valuable alternative models to assess the effects of exposure to exogenous substances such as EDCs, and (2) these compounds significantly interfere with fundamental processes of developmental physiology (proliferation, differentiation, etc...) plausibly via endocrine alterations. In terms of future prospects, taking into account the increasing need to propose animal models different from vertebrates, echinoderms represent a group on which ecotoxicological studies should be encouraged and specifically addressed.

Marine-derived collagen biomaterials from echinoderm connective tissues.

The use of marine collagens is a hot topic in the field of tissue engineering. Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCTs) which can represent an innovative source of collagen to develop collagen barrier-membranes for Guided Tissue Regeneration (GTR). In the present work we used MCTs from different echinoderm models (sea urchin, starfish and sea cucumber) to produce echinoderm-derived collagen membranes (EDCMs). Commercial membranes for GTR or soluble/reassembled (fibrillar) bovine collagen substrates were used as controls. The three EDCMs were similar among each other in terms of structure and mechanical performances and were much thinner and mechanically more resistant than the commercial membranes. Number of fibroblasts seeded on sea-urchin membranes were comparable to the bovine collagen substrates. Cell morphology on all EDCMs was similar to that of structurally comparable (reassembled) bovine collagen substrates. Overall, echinoderms, and sea urchins particularly, are alternative collagen sources to produce efficient GTR membranes. Sea urchins display a further advantage in terms of eco-sustainability by recycling tissues from food wastes.

Triphenyltin alters androgen metabolism in the sea urchin Paracentrotus lividus.

Androgen metabolism (androstenedione and testosterone) has been assessed in the digestive tube and gonads of the echinoderm Paracentrotus lividus exposed to different concentrations of the biocide triphenyltin (TPT) in a semi-static water regime for 4 weeks. Key enzymatic activities involved in both synthesis and metabolism of androgens, namely 17beta-hydroxysteroid dehydrogenases (17beta-HSDs), 3beta-HSDs, 5alpha-reductases, P450-aromatase, palmitoyl-CoA:testosterone acyltransferases (ATAT) and testosterone sulfotransferases (SULT), were investigated in digestive tube and/or gonads of control and TPT-exposed specimens in an attempt to see whether androgen metabolism was altered by exposure. In agreement with previous data for vertebrates, exposure to TPT led to a concentration dependent decrease of P450-aromatase that was statistically significant at the highest TPT concentration tested (225ng/L). Additionally, increased metabolism of testosterone to form dihydrotestosterone (DHT) and 5alpha-androstane-3beta,17beta-diol was observed, suggesting increased 5alpha-reductase activity in the gonads of TPT-exposed individuals. Interestingly, exposure to TPT induced testosterone conjugating activities in organisms exposed to medium (SULT) and high (ATAT and SULT) TPT concentrations. Despite the changes of androgen metabolizing enzymes, testosterone levels in gonads remained rather stable. In contrast, an increase in testosterone and a concomitant decrease in estradiol were observed in the coelomic fluid of TPT-exposed organisms. Overall, the data indicate the ability of TPT to modulate androgen metabolism and circulating steroid levels in P. lividus and suggest the existence of regulatory mechanisms to maintain stable endogenous levels of testosterone in gonads. This study also contributes to a better knowledge of echinoderm endocrinology.

Mechanical properties of the compass depressors of the sea-urchin Paracentrotus lividus (Echinodermata, Echinoidea) and the effects of enzymes, neurotransmitters and synthetic tensilin-like protein.

The compass depressors (CDs) of the sea-urchin lantern are ligaments consisting mainly of discontinuous collagen fibrils associated with a small population of myocytes. They are mutable collagenous structures, which can change their mechanical properties rapidly and reversibly under nervous control. The aims of this investigation were to characterise the baseline (i.e. unmanipulated) static mechanical properties of the CDs of Paracentrotus lividus by means of creep tests and incremental force-extension tests, and to determine the effects on their mechanical behaviour of a range of agents. Under constant load the CDs exhibited a three-phase creep curve, the mean coefficient of viscosity being 561±365 MPa.s. The stress-strain curve showed toe, linear and yield regions; the mean strain at the toe-linear inflection was 0.86±0.61; the mean Young's modulus was 18.62±10.30 MPa; and the mean tensile strength was 8.14±5.73 MPa. Hyaluronidase from Streptomyces hyalurolyticus had no effect on creep behaviour, whilst chondroitinase ABC prolonged primary creep but had no effect on secondary creep or on any force-extension parameters; it thus appears that neither hyaluronic acid nor sulphated glycosaminoglycans have an interfibrillar load transfer function in the CD. Acetylcholine, the muscarinic agonists arecoline and methacholine, and the nicotinic agonists nicotine and 1-[1-(3,4-dimethyl-phenyl)-ethyl]-piperazine produced an abrupt increase in CD viscosity; the CDs were not differentially sensitive to muscarinic or nicotinic agonists. CDs showed either no, or no consistent, response to adrenaline, L-glutamic acid, 5-hydroxytryptamine and γ-aminobutyric acid. Synthetic echinoid tensilin-like protein had a weak and inconsistent stiffening effect, indicating that, in contrast to holothurian tensilins, the echinoid molecule may not be involved in the regulation of collagenous tissue tensility. We compare in detail the mechanical behaviour of the CD with that of mammalian tendon and highlight its potential as a model system for investigating poorly understood aspects of the ontogeny and phylogeny of vertebrate collagenous tissues.

Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus.

The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes within physiological timescales, the only major exception being the reversible destiffening of the mammalian uterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (sea urchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions in timescales of around a second to minutes. Elucidation of the molecular mechanism underlying such mutability has implications for the zoological, ecological and evolutionary field. Important information could also arise for veterinary and biomedical sciences, particularly regarding the pathological plasticization or stiffening of connective tissue structures. In the present investigation we analyzed aspects of the ultrastructure and biochemistry in two representative models, the compass depressor ligament and the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three different mechanical states. The results provide further evidence that the mechanical adaptability of echinoderm connective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher glycosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compass depressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues. The possible involvement of GAG in the mutability phenomenon will need further clarification. During the shift from a compliant to a standard condition, significant changes in GAG content were detected only in the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling) and biochemistry (two alpha chains) were found between the two models and mammalian collagen. Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLAST alignment highlighted the uniqueness of sea urchin collagen with respect to mammalian collagen.

Primary cell cultures from sea urchin ovaries: a new experimental tool.

In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

The reaction of the sponge Chondrosia reniformis to mechanical stimulation is mediated by the outer epithelium and the release of stiffening factor(s).

Although sponges are still often considered to be simple, inactive animals, both larvae and adults of different species show clear coordination phenomena triggered by extrinsic and intrinsic stimuli. Chondrosia reniformis, a common Mediterranean demosponge, lacks both endogenous siliceous spicules and reinforcing spongin fibers and has a very conspicuous collagenous mesohyl. Although this species can stiffen its body in response to mechanical stimulation when handled, almost no quantitative data are available in the literature on this phenomenon. The present work was intended to quantify the dynamic response to mechanical stimulation both of intact animals and isolated tissue samples in order to evaluate: (i) the magnitude of stiffening; (ii) the relationship between the amount of stimulation and the magnitude of the stiffening response; (iii) the ability of the whole body to react to localized stimulation; (iv) the possible occurrence of a conduction mechanism and the role of the exopinacoderm (outer epithelium). Data on mesohyl tensility obtained with mechanical tests confirmed the difference between stimulated and non-stimulated isolated tissue samples, showing a significant relationship between ectosome stiffness and the amount of mechanical stimulation. Our experiments revealed a significant difference in tensility between undisturbed and maximally stiffened sponges and evidence of signal transmission that requires a continuous exopinacoderm. We also provide further evidence for the presence of a chemical factor that alters the interaction between collagen fibrils, thereby changing the mechanical properties of the mesohyl.