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.

Exposure to polystyrene nanoplastics induced physiological and behavioral effects on the brittle star Ophiactis virens.

Nanoplastic contamination has become an issue of environmental concern but the information on the potential adverse effects of nanoplastics on marine ecosystems is still limited. Therefore, the aim of this work was to investigate the effects of the exposure to polystyrene nanoplastics (PS-NPs; 0.05, 0.5 and 5 µg/mL) on the brittles star Ophiactis virens. Diverse endpoints at different levels of biological organization were considered, including behavior, arm regeneration capacity and oxidative stress. PS-NPs were observed on the brittle star body surface but not in inner tissues. Accumulation of PS-NPs was observed in the pre-buccal cavity of animals exposed to 5 µg/mL PS-NPs which also displayed delayed righting activity and an oxidative stress condition. Nevertheless, no effect was observed on arm regeneration efficiency at any tested PS-NPs concentration. Overall, our results highlighted that prolonged exposure to high amounts of PS-NPs could interfere at least partially with the physiology of O. virens.

Discovery and functional characterization of neuropeptides in crinoid echinoderms.

Neuropeptides are one of the largest and most diverse families of signaling molecules in animals and, accordingly, they regulate many physiological processes and behaviors. Genome and transcriptome sequencing has enabled the identification of genes encoding neuropeptide precursor proteins in species from a growing variety of taxa, including bilaterian and non-bilaterian animals. Of particular interest are deuterostome invertebrates such as the phylum Echinodermata, which occupies a phylogenetic position that has facilitated reconstruction of the evolution of neuropeptide signaling systems in Bilateria. However, our knowledge of neuropeptide signaling in echinoderms is largely based on bioinformatic and experimental analysis of eleutherozoans-Asterozoa (starfish and brittle stars) and Echinozoa (sea urchins and sea cucumbers). Little is known about neuropeptide signaling in crinoids (feather stars and sea lilies), which are a sister clade to the Eleutherozoa. Therefore, we have analyzed transcriptome/genome sequence data from three feather star species, Anneissia japonica, Antedon mediterranea, and Florometra serratissima, to produce the first comprehensive identification of neuropeptide precursors in crinoids. These include representatives of bilaterian neuropeptide precursor families and several predicted crinoid neuropeptide precursors. Using A. mediterranea as an experimental model, we have investigated the expression of selected neuropeptides in larvae (doliolaria), post-metamorphic pentacrinoids and adults, providing new insights into the cellular architecture of crinoid nervous systems. Thus, using mRNA in situ hybridization F-type SALMFamide precursor transcripts were revealed in a previously undescribed population of peptidergic cells located dorso-laterally in doliolaria. Furthermore, using immunohistochemistry a calcitonin-type neuropeptide was revealed in the aboral nerve center, circumoral nerve ring and oral tube feet in pentacrinoids and in the ectoneural and entoneural compartments of the nervous system in adults. Moreover, functional analysis of a vasopressin/oxytocin-type neuropeptide (crinotocin), which is expressed in the brachial nerve of the arms in A. mediterranea, revealed that this peptide causes a dose-dependent change in the mechanical behavior of arm preparations in vitro-the first reported biological action of a neuropeptide in a crinoid. In conclusion, our findings provide new perspectives on neuropeptide signaling in echinoderms and the foundations for further exploration of neuropeptide expression/function in crinoids as a sister clade to eleutherozoan echinoderms.

More than a simple epithelial layer: multifunctional role of echinoderm coelomic epithelium.

In echinoderms, the coelomic epithelium (CE) is reportedly the source of new circulating cells (coelomocytes) as well as the provider of molecular factors such as immunity-related molecules. However, its overall functions have been scarcely studied in detail. In this work, we used an integrated approach based on both microscopy (light and electron) and proteomic analyses to investigate the arm CE in the starfish Marthasterias glacialis during different physiological conditions (i.e., non-regenerating and/or regenerating). Our results show that CE cells share both ultrastructural and proteomic features with circulating coelomocytes (echinoderm immune cells). Additionally, microscopy and proteomic analyses indicate that CE cells are actively involved in protein synthesis and processing, and membrane trafficking processes such as phagocytosis (particularly of myocytes) and massive secretion phenomena. The latter might provide molecules (e.g., immune factors) and fluids for proper arm growth/regrowth. No stem cell marker was identified and no pre-existing stem cell was observed within the CE. Rather, during regeneration, CE cells undergo dedifferentiation and epithelial-mesenchymal transition to deliver progenitor cells for tissue replacement. Overall, our work underlines that echinoderm CE is not a "simple epithelial lining" and that instead it plays multiple functions which span from immunity-related roles as well as being a source of regeneration-competent cells for arm growth/regrowth.

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.

Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach.

Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.

Diverse and Productive Source of Biopolymer Inspiration: Marine Collagens.

Marine biodiversity is expressed through the huge variety of vertebrate and invertebrate species inhabiting intertidal to deep-sea environments. The extraordinary variety of "forms and functions" exhibited by marine animals suggests they are a promising source of bioactive molecules and provides potential inspiration for different biomimetic approaches. This diversity is familiar to biologists and has led to intensive investigation of metabolites, polysaccharides, and other compounds. However, marine collagens are less well-known. This review will provide detailed insight into the diversity of collagens present in marine species in terms of their genetics, structure, properties, and physiology. In the last part of the review the focus will be on the most common marine collagen sources and on the latest advances in the development of innovative materials exploiting, or inspired by, marine collagens.

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.

Interactive effects between sinking polyethylene terephthalate (PET) microplastics deriving from water bottles and a benthic grazer.

The information concerning the toxicity of sinking microplastics (MPs) on benthic marine animals, particularly benthic grazers, is still scant. No study focused on biological weathering of sinked MPs operated by benthic organisms. This study aims at investigating the ingestion and the effects induced by 7-days dietary exposure to environmentally relevant amount (8, 80 and 800 particles/g of food) of irregular shaped and sized (diameter 12.6-1,065 µm; mean diameter 316 ± 12 µm) polyethylene terephthalate microplastics (PET-MPs) on a common marine benthic grazer, the sea urchin Paracentrotus lividus. Adverse effects were investigated on digestive tract at biochemical (oxidative stress biomarkers) and tissue level (histopathological analyses). Potential alteration of MP structure/surface and PET macromolecules due to the ingestion of PET-MPs within the sea urchin digestive tract were investigated. Results showed that PET-MPs were efficiently egested by sea urchins without producing histological alterations on digestive tract tissues, only inducing a slight modulation of oxidative status. Sea urchin grazing activity and the related transit of PET-MPs within animal digestive tract slightly affected MP structure and PET composition. These findings suggest that PET-MPs might represent an hazard for benthic grazer organisms, which can partially contribute to the degradation of PET in marine ecosystems.

Plastics and biodegradable plastics: ecotoxicity comparison between polyvinylchloride and Mater-Bi® micro-debris in a freshwater biological model.

The improper release of plastic items and wastes is nowadays one of the main environmental and social problems, whose solution or mitigation represents a great challenge worldwide. In this context, the growing use of the so-called biodegradable plastics could represent a possible solution in the short to medium term. The few information known about the ecological impact of these materials on freshwater organisms, especially the ones relative to the micro-debris derived from their aging, prompted us to study the comparison of the sub-lethal effects eventually caused by plastic and biodegradable plastic micro-debris on the mussel Dreissena polymorpha, which represents an excellent biological model for the freshwater ecosystems. We selected two powders of polyvinylchloride (PVC) and Mater-Bi® administered at 1 mg/L to D. polymorpha specimens in semi-static conditions for 14 days. The presence of micro-debris was evaluated on mussel tissues and pseudo-faeces using advanced microscopy techniques. The sub-lethal effects were investigated on exposed mussels at 6 and 14 days using a suite of biomarkers of cellular stress, oxidative damage, and genotoxicity. Lastly, we compared the ecotoxicity of these two materials integrating each endpoint in the Biomarker Response Index. Microscopy observations highlighted the surprising absence of micro-debris in the gut lumen and tissues of exposed mussels, but the presence of both PVC and Mater-Bi® micro-debris in the pseudo-faeces, suggesting a possible efficient elimination mechanism adopted by mussels to avoid the micro-debris gulping. Consequently, we did not observe significant sub-lethal effects, except for the glutathione-S-transferase activity modulation after 6 days of exposure.

Regeneration in Stellate Echinoderms: Crinoidea, Asteroidea and Ophiuroidea.

Reparative regeneration is defined as the replacement of lost adult body parts and is a phenomenon widespread yet highly variable among animals. This raises the question of which key cellular and molecular mechanisms have to be implemented in order to efficiently and correctly replace entire body parts in any animal. To address this question, different studies using an integrated cellular and functional genomic approach to study regeneration in stellate echinoderms (crinoids, asteroids and ophiuroids) had been carried out over the last few years. The phylum Echinodermata is recognized for the striking regeneration potential shown by the members of its different clades. Indeed, stellate echinoderms are considered among the most useful and tractable experimental models for carrying comprehensive studies focused on ecological, developmental and evolutionary aspects. Moreover, most of them are tractable in the laboratory and, thus, should allow us to understand the underlying mechanisms, cellular and molecular, which are involved. Here, a comprehensive analysis of the cellular/histological components of the regenerative process in crinoids, asteroids and ophiuroids is described and compared. However, though this knowledge provided us with some clear insights into the global distribution of cell types at different times, it did not explain us how the recruited cells are specified (and from which precursors) over time and where are they located in the animal. The precise answer to these queries needs the incorporation of molecular approaches, both descriptive and functional. Yet, the molecular studies in stellate echinoderms are still limited to characterization of some gene families and protein factors involved in arm regeneration but, at present, have not shed light on most of the basic mechanisms. In this context, further studies are needed specifically to understand the role of regulatory factors and their spatio-temporal deployment in the growing arms. A focus on developing functional tools over the next few years should be of fundamental importance.

Evaluation of uptake and chronic toxicity of virgin polystyrene microbeads in freshwater zebra mussel Dreissena polymorpha (Mollusca: Bivalvia).

Microplastics (MPs), plastic debris smaller than 5mm, are widely found in both marine and freshwater ecosystems. However, few studies regarding their hazardous effects on inland water organisms, have been conducted. For this reason, the aim of our research was the evaluation of uptake and chronic toxicity of two mixtures (MIXs) of virgin polystyrene microbeads (PMs) of 10µm and 1µm in size (MIX 1, with 5×105 of 1µmsizePMs/L and 5×105 of 10µmsizePMs/L, and MIX 2 with 2×106 of 1µmsizePMs/L and 2×106 of 10µmsizePMs/L) on freshwater zebra mussel Dreissena polymorpha (Mollusca: Bivalvia) during 6 exposure days. The PM uptake in the mussel body and hemolymph was assessed using confocal microscopy, while the chronic toxicity of PMs was evaluated on exposed mussels using a comprehensive battery of biomarkers of cellular stress, oxidative damage and neuro- genotoxicity. Confocal microscopy analyses showed that MPs concentrated in the gut lumen of exposed mussels, absorbed and transferred firstly in the tissues and then in the hemolymph. The results revealed that PMs do not produce oxidative stress and genetic damage, with the exception of a significant modulation of catalase and glutathione peroxidase activities in mussels exposed to MIX 1. Regarding neurotoxicity, we observed only a significant increase of dopamine concentration in mussels exposed to both MIXs, suggesting a possible implication of this neurotransmitter in an elimination process of accumulated PMs. This research represents a first study about the evaluation of virgin MP toxicity in zebra mussel and more research is warranted concerning the long term neurological effects of virgin MPs.

Fundamental aspects of arm repair phase in two echinoderm models.

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored. Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools. Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models. Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage.

An integrated view of asteroid regeneration: tissues, cells and molecules.

The potential for repairing and replacing cells, tissues, organs and body parts is considered a primitive attribute of life shared by all the organisms, even though it may be expressed to a different extent and which is essential for the survival of both individual and whole species. The ability to regenerate is particularly evident and widespread within invertebrates. In spite of the wide availability of experimental models, regeneration has been comprehensively explored in only a few animal systems (i.e., hydrozoans, planarians, urodeles) leaving many other animal groups unexplored. The regenerative potential finds its maximum expression in echinoderms. Among echinoderm classes, asteroids offer an impressive range of experimental models in which to study arm regeneration at different levels. Many studies have been recently carried out in order to understand the regenerative mechanisms in asteroids and the overall morphological processes have been well documented in different starfish species, such as Asterias rubens, Leptasterias hexactis and Echinaster sepositus. In contrast, very little is known about the molecular mechanisms that control regeneration development and patterning in these models. The origin and the fate of cells involved in the regenerative process remain a matter of debate and clear insights will require the use of complementary molecular and proteomic approaches to study this problem. Here, we review the current knowledge regarding the cellular, proteomic and molecular aspects of asteroid regeneration.

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.

Wound repair during arm regeneration in the red starfish Echinaster sepositus.

Starfish can regenerate entire arms following their loss by both autotomic and traumatic amputation. Although the overall regenerative process has been studied several times in different asteroid species, there is still a considerable gap of knowledge as far as the detailed aspects of the repair phase at tissue and cellular level are concerned, particularly in post-traumatic regeneration. The present work is focused on the arm regeneration model in the Mediterranean red starfish Echinaster sepositus; to describe the early cellular mechanisms of arm regeneration following traumatic amputation, different microscopy techniques were employed. In E. sepositus, the repair phase was characterized by prompt wound healing by a syncytial network of phagocytes and re-epithelialisation followed by a localized subepidermal oedematous area formation. Scattered and apparently undifferentiated cells, intermixed with numerous phagocytes, were frequently found in the wound area during these first stages of regeneration and extensive dedifferentiation phenomena were seen at the level of the stump, particularly in the muscle bundles. A true localized blastema did not form. Our results confirm that regeneration in asteroids mainly relies on morphallactic processes, consisting in extensive rearrangement of the existing tissues which contribute to the new tissues through cell dedifferentiation, redifferentiation, and/or migration.

Re-growth, morphogenesis, and differentiation during starfish arm regeneration.

The red starfish Echinaster sepositus is an excellent model for studying arm regeneration processes following traumatic amputation. The initial repair phase was described in a previous paper in terms of the early cicatrisation phenomena, and tissue and cell involvement. In this work, we attempt to provide a further comprehensive description of the later regenerative stages in this species. Here, we present the results of a detailed microscopic and submicroscopic investigation of the long regenerative phase, which can be subdivided into two subphases: early and advanced regenerative phases. The early regenerative phase (1-6 weeks p.a.) is characterized by tissue rearrangement, morphogenetic processes and initial differentiation events (mainly neurogenesis and skeletogenesis). The advanced regenerative phase (after 6 weeks p.a.) is characterized by further differentiation processes (early myogenesis), and obvious morphogenesis and re-growth of the regenerate. As in other starfish, the regenerative process in E. sepositus is relatively slow in comparison with that of crinoids and many ophiuroids, which is usually interpreted as resulting mainly from size-related aspects and of the more conspicuous involvement of morphallactic processes. Light and electron microscopy analyses suggest that some of the amputated structures, such as muscles, are not able to replace their missing parts by directly re-growing them from the remaining tissues, whereas others tissues, such as the skeleton and the radial nerve cord, appear to undergo direct re-growth. The overall process is in agreement with the distalization-intercalation model proposed by Agata and co-workers. Further experiments are needed to confirm this hypothesis.

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.

Production, characterization and biocompatibility of marine collagen matrices from an alternative and sustainable source: the sea urchin Paracentrotus lividus.

Collagen has become a key-molecule in cell culture studies and in the tissue engineering field. Industrially, the principal sources of collagen are calf skin and bones which, however, could be associated to risks of serious disease transmission. In fact, collagen derived from alternative and riskless sources is required, and marine organisms are among the safest and recently exploited ones. Sea urchins possess a circular area of soft tissue surrounding the mouth, the peristomial membrane (PM), mainly composed by mammalian-like collagen. The PM of the edible sea urchin Paracentrotus lividus therefore represents a potential unexploited collagen source, easily obtainable as a food industry waste product. Our results demonstrate that it is possible to extract native collagen fibrils from the PM and produce suitable substrates for in vitro system. The obtained matrices appear as a homogeneous fibrillar network (mean fibril diameter 30-400 nm and mesh < 2 µm) and display remarkable mechanical properties in term of stiffness (146 ± 48 MPa) and viscosity (60.98 ± 52.07 GPa·s). In vitro tests with horse pbMSC show a good biocompatibility in terms of overall cell growth. The obtained results indicate that the sea urchin P. lividus can be a valuable low-cost collagen source for mechanically resistant biomedical devices.

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.