Bioinspiring Chondrosia reniformis (Nardo, 1847) collagen-based hydrogel: a new extraction method to obtain a sticky and self-healing collagenous material

Collagen is a natural and abundant polymer that serves multiple functions in both invertebrates and vertebrates. As collagen is the natural scaffolding for cells, collagen-based hydrogels are regarded as ideal materials for tissue engineering applications since they can mimic the natural cellular microenvironment. Chondrosia reniformis is a marine demosponge particularly rich in collagen, characterized by the presence of labile interfibrillar crosslinks similarly to those described in the mutable collagenous tissues (MCTs) of echinoderms. As a result single fibrils can be isolated using calcium-chelating and disulphide-reducing chemicals. In the present work we firstly describe a new extraction method that directly produces a highly hydrated hydrogel with interesting self-healing properties. The materials obtained were then biochemically and rheologically characterized. Our investigation has shown that the developed extraction procedure is able to extract collagen as well as other proteins and Glycosaminoglycans (GAG)-like molecules that give the collagenous hydrogel interesting and new rheological properties when compared to other described collagenous materials. The present work motivates further in-depth investigations towards the development of a new class of injectable collagenous hydrogels with tailored specifications.The authors gratefully acknowledge the financial support from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement ERC-2012-ADG 20120216-321266 (ERC Advanced Grant project ComplexiTE), as well as from the European Regional Development Fund (ERDF) under the projects “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechonologies, marine-derived biomaterials and stem cells” (NORTE-01-0145-FEDER-000021), supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and 0687_NOVOMAR_1_P, co-financed by transborder cooperation programme POCTEP. The authors are also thankful to the Area Marina Protetta Portofino (Italy) for permission to collect sponge specimens and to Daniela Candia (University of Milan, Italy) and Marco Giovine (University of Genoa, Italy) for the logistical support on the sponge sampling and immediate processing. We are grateful to the two anonymous referees for improving the quality of the present article.info:eu-repo/semantics/publishedVersio

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.(undefined)info:eu-repo/semantics/publishedVersio

Ecophysiology of mesohyl creep in the demosponge Chondrosia reniformis (Porifera: Chondrosida)

Chondrosia reniformis is a common marine demosponge that shows striking tissue plasticity and unusual body deformability. This sponge can develop long and slender outgrowths extending from the parental body. According to some authors, this phenomenon, called \u201ccreeping\u201d, can be related to asexual reproduction, atypical mechanisms of \uablocalized\ubb locomotion or passive response to environmental stress. Here we address this phenomenon by means of an interdisciplinary approach consisting of field survey, experimental field studies and experimental laboratory studies. During field survey and field experimental survey we observed that the instability of substratum is an important factor that trigs the beginning of creeping. The sponge size does not seem to be directly involved in the occurrence of the phenomenon. Specimens of Bergeggi (Ligurian Sea, northern Italy) show a high correlation between the creeping phenomenon and the sea temperature; this seems to support the hypothesis that the phenomenon is related to asexual reproduction, which is in its turn seasonally regulated by environmental temperature. In addition, experimental laboratory studies performed in different mechanical conditions on isolated samples of both ectosome and choanosome showed that temperature affects mesohyl mechanical properties: the lower is the temperature the stiffer is the mesohyl. The different physiological states recorded by the laboratory experiments are expressions of the mechanical adaptability of the collagenous mesohyl of C. reniformis and suggest that stiffness variability is under cellular control. On the basis of present results we can infer that C. reniformis can exert some control on the creeping phenomenon and that the primary factors implied in inducing creeping phenomena are the instability of substratum and the temperature. Interestingly the capability to modulate the mechanical properties of the collagenous matrix is an uncommon feature that C. reniformis shares with the mutable collagenous tissue (MCT) of Echinoderms. This close analogy, which is supported by morphological and physiological evidence, is an intriguing point that opens a wide range of evolutionary and functional questions

Sea urchins and mechanically adaptable connective tissues: alternative sources for biomaterial design

Sea urchins, as all echinoderms (starfish, sea cucumber, etc.), possess connective tissues that undergo drastic changes in their mechanical properties (Mutable Collagenous Tissues: MCTs). Mammalian connective tissues rarely undergo significant changes within a physiological timescale, the only major exception being the destiffening then restiffening of the mammalian uterine cervix at the end of pregnancy. In contrast, MCT can switch reversibly between stiff and compliant conditions in timescales of seconds to minutes following nervous stimulation. Considering this, MCT could be an inspiration for new matrices capable of changing their molecular and structural conformation in response to external stimuli. Furthermore, elucidating the molecular mechanism underlying MCT mutability could have implications for veterinary and biomedical science, particularly regarding the pathological plasticization or stiffening of connective tissue structures. The MIMESIS (Marine Invertebrate Models & Engineered Substrates for Innovative bio-Scaffolds) project has being developed within this scientific context. This contribution presents a review of the distinctive features of MCT together with the first results aimed to the production of MCT-derived matrices as cell culture /tissue regeneration substrates

Extraction of collagen/gelatin from the marine demosponge Chondrosia reniformis (Nardo, 1847) using water acidified with carbon dioxide: process optimization

Marine sponges are a rich source of natural bioactive compounds. One of the most abundant valuable products is collagen/gelatin, and therefore, presents an interesting alternative source for pharmaceutical and biomedical applications. We have previously proposed an innovative green technology for the extraction of collagen/gelatin from marine sponges based in water acidified with carbon dioxide. In this work, we have optimized the process operating conditions towards high yields and collagen quality as well as to reduce extraction procedure duration and energy consumption. The process extraction efficiency is higher than 50%, corresponding to a yield of approximately 10% of the sponge dry mass, obtained for mild operating conditions 10 bars and 3h. The extracted material was characterized by Scanning Electron Microscopy (SEM), Rheology, Fourier Transformed Infrared Spectroscopy (FTIR), Circular Dichroism (CD), Aminoacid Analysis and SDS-PAGE. The extracts were found to be composed of highly pure mixtures of collagen and gelatin with similar properties of collagen isolated from other marine sources. The cytotoxicity evaluation, performed with L929 cells, has proven the biocompatibility of the material extracted. Overall, the results obtained demonstrate the efficiency of this approach and the high industrial potential of this technology to obtain marine collagen/gelatin with properties suitable for biomedical applications.FCT through Grant EXP/QEQ-EPS/0745/2012, SWIMS (Subcritical Water Isolation of compounds from Marine Sponges)European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement numbers REGPOT-CT2012-316331-POLARIS and KBBE-2010-266033 (project SPECIAL)European Regional Development Fund (ERDF) under the project “Novel smart and biomimetic materials for innovative regenerative medicine approaches” RL1-ABMR-NORTE-01- 0124-FEDER-000016), cofinanced by North Portugal Regional Operational Programme (ON.2, O Novo Norte), under the National Strategic Reference Framework (NSRF)European Research Council grant agreement ERC-2012- ADG 20120216-321266 for project ComplexiT

The smart connective tissue of echinoderms: a materializing promise for biotech applications

Echinoderm Mutable Collagenous Tissues (MCTs) undergo nervously mediated, drastic and reversible changes in their passive mechanical properties. MCT mutability is involved in autotomy,posture maintenance and motility, and, as a consequence, it influences all aspects of echinoderm biology (nutrition, reproduction, habitat selection, self-defense and predatory behavior) representing a key-factor for the ecological success of the phylum. Besides this, MCT performance represents a topic of remarkable interest for many different applied fields. A biomimetic research route looks at MCTs as a source of inspiration for the development of smart and innovative biomaterials with great potential for in vitro and in vivo applications when controlled and reversible plasticization and/or stiffening of the extracellular matrix is required. The MIMESIS (Marine Invertebrates Models & Engineered Substrates for Innovative bio-Scaffolds) project has been developed within this scientific context. The selected echinoderm model is the common sea urchin Paracentrotus lividus. This project is based on a multidisciplinary approach combining functional biology with biomaterial engineering. A brief review of recent morphological, biomolecular, biomechanical and biochemical results on P.lividus MCTs are here presented in a biotechnological perspective, taking into account also a promising application regarding the use of MCT-derived substrata for cell culture studies

Effects of enzymes on the creep behaviour of <i>P. lividus</i> CDs.

<p>The numbers of values in the primary and secondary creep groups are shown respectively in parentheses. Means sharing the same superscript letter are significantly different (^a<i>P</i> = 0.001; ^b<i>P</i> = 0.005; Mann-Whitney tests). There were no other statistically significant differences between enzyme-treated CDs and corresponding control (PBS- or Tris-ASW-treated) CDs.</p><p>Effects of enzymes on the creep behaviour of <i>P. lividus</i> CDs.</p

Effects of pharmacological agents on the relative viscosity of <i>P</i>. <i>lividus</i> CDs.

<p>Effects of pharmacological agents on the relative viscosity of <i>P</i>. <i>lividus</i> CDs.</p

Diagrammatic representation of the CD of <i>P</i>. <i>lividus</i>.

<p>This depicts part of a longitudinal section; not to scale. The CD consists of parallel discontinuous collagen fibrils (cf), which are connected by interfibrillar crossbridges containing chondroitin sulphate/dermatan sulphate proteoglycans (ic). Hyaluronic acid (hy) is present but of unknown function (its disposition on the surface of the collagen fibrils is conjectural). Bundles of beaded microfibrils (mf) occur between the collagen fibrils. The main cellular components are the somata and processes of electron-dense granule-containing juxtaligamental cells (jlc). Agranular cell processes, which may be cholinergic axons, are in close contact with the juxtaligamental cells and are shown as transverse sections (ax).</p

Effects of chondroitinase ABC on the force-extension behaviour of <i>P. lividus</i> CDs.

<p>Effects of chondroitinase ABC on the force-extension behaviour of <i>P. lividus</i> CDs.</p