Electrolysis as a Universal Approach for Isolation of Diverse Chitin Scaffolds from Selected Marine Demosponges.

Three-dimensional chitinous scaffolds often used in regenerative medicine, tissue engineering, biomimetics and technology are mostly isolated from marine organisms, such as marine sponges (Porifera). In this work, we report the results of the electrochemical isolation of the ready to use chitinous matrices from three species of verongiid demosponges (Aplysina archeri, Ianthella basta and Suberea clavata) as a perfect example of possible morphological and chemical dimorphism in the case of the marine chitin sources. The electrolysis of concentrated Na2SO4 aqueous solution showed its superiority over the chemical chitin isolation method in terms of the treatment time reduction: only 5.5 h for A. archeri, 16.5 h for I. basta and 20 h for the S. clavata sample. Further investigation of the isolated scaffolds by digital microscopy and SEM showed that the electrolysis-supported isolation process obtains chitinous scaffolds with well-preserved spatial structure and it can be competitive to other alternative chitin isolation techniques that use external accelerating factors such as microwave irradiation or atmospheric plasma. Moreover, the infrared spectroscopy (ATR-FTIR) proved that with the applied electrochemical conditions, the transformation into chitosan does not take place.

Adsorption of Cationic Dyes on a Magnetic 3D Spongin Scaffold with Nano-Sized Fe3O4 Cores.

The renewable, proteinaceous, marine biopolymer spongin is yet the focus of modern research. The preparation of a magnetic three-dimensional (3D) spongin scaffold with nano-sized Fe3O4 cores is reported here for the first time. The formation of this magnetic spongin-Fe3O4 composite was characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential thermal analysis (DTA) (TGA-DTA), vibrating sample magnetometer (VSM), Fourier-transform infrared spectroscopy (FTIR), and zeta potential analyses. Field emission scanning electron microscopy (FE-SEM) confirmed the formation of well-dispersed spherical nanoparticles tightly bound to the spongin scaffold. The magnetic spongin-Fe3O4 composite showed significant removal efficiency for two cationic dyes (i.e., crystal violet (CV) and methylene blue (MB)). Adsorption experiments revealed that the prepared material is a fast, high-capacity (77 mg/g), yet selective adsorbent for MB. This behavior was attributed to the creation of strong electrostatic interactions between the spongin-Fe3O4 and MB or CV, which was reflected by adsorption mechanism evaluations. The adsorption of MB and CV was found to be a function of pH, with maximum removal performance being observed over a wide pH range (pH = 5.5-11). In this work, we combined Fe3O4 nanoparticles and spongin scaffold properties into one unique composite, named magnetic spongin scaffold, in our attempt to create a sustainable absorbent for organic wastewater treatment. The appropriative mechanism of adsorption of the cationic dyes on a magnetic 3D spongin scaffold is proposed. Removal of organic dyes and other contaminants is essential to ensure healthy water and prevent various diseases. On the other hand, in many cases, dyes are used as models to demonstrate the adsorption properties of nanostructures. Due to the good absorption properties of magnetic spongin, it can be proposed as a green and uncomplicated adsorbent for the removal of different organic contaminants and, furthermore, as a carrier in drug delivery applications.

Potential Biomedical Applications of Collagen Filaments derived from the Marine Demosponges Ircinia oros (Schmidt, 1864) and Sarcotragus foetidus (Schmidt, 1862).

Collagen filaments derived from the two marine demosponges Ircinia oros and Sarcotragus foetidus were for the first time isolated, biochemically characterised and tested for their potential use in regenerative medicine. SDS-PAGE of isolated filaments revealed a main collagen subunit band of 130 kDa in both of the samples under study. DSC analysis on 2D membranes produced with collagenous sponge filaments showed higher thermal stability than commercial mammalian-derived collagen membranes. Dynamic mechanical and thermal analysis attested that the membranes obtained from filaments of S. foetidus were more resistant and stable at the rising temperature, compared to the ones derived from filaments of I. oros. Moreover, the former has higher stability in saline and in collagenase solutions and evident antioxidant activity. Conversely, their water binding capacity results were lower than that of membranes obtained from I. oros. Adhesion and proliferation tests using L929 fibroblasts and HaCaT keratinocytes resulted in a remarkable biocompatibility of both developed membrane models, and gene expression analysis showed an evident up-regulation of ECM-related genes. Finally, membranes from I. oros significantly increased type I collagen gene expression and its release in the culture medium. The findings here reported strongly suggest the biotechnological potential of these collagenous structures of poriferan origin as scaffolds for wound healing.

Calcite Nanotuned Chitinous Skeletons of Giant Ianthella basta Marine Demosponge.

Marine sponges were among the first multicellular organisms on our planet and have survived to this day thanks to their unique mechanisms of chemical defense and the specific design of their skeletons, which have been optimized over millions of years of evolution to effectively inhabit the aquatic environment. In this work, we carried out studies to elucidate the nature and nanostructural organization of three-dimensional skeletal microfibers of the giant marine demosponge Ianthella basta, the body of which is a micro-reticular, durable structure that determines the ideal filtration function of this organism. For the first time, using the battery of analytical tools including three-dimensional micro-X-ray Fluorescence (3D-µXRF), X-ray diffraction (XRD), infra-red (FTIR), Raman and Near Edge X-ray Fine Structure (NEXAFS) spectroscopy, we have shown that biomineral calcite is responsible for nano-tuning the skeletal fibers of this sponge species. This is the first report on the presence of a calcitic mineral phase in representatives of verongiid sponges which belong to the class Demospongiae. Our experimental data suggest a possible role for structural amino polysaccharide chitin as a template for calcification. Our study suggests further experiments to elucidate both the origin of calcium carbonate inside the skeleton of this sponge and the mechanisms of biomineralization in the surface layers of chitin microfibers saturated with bromotyrosines, which have effective antimicrobial properties and are responsible for the chemical defense of this organism. The discovery of the calcified phase in the chitinous template of I. basta skeleton is expected to broaden the knowledge in biomineralization science where the calcium carbonate is regarded as a valuable material for applications in biomedicine, environmental science, and even in civil engineering.

Progress in Modern Marine Biomaterials Research.

The growing demand for new, sophisticated, multifunctional materials has brought natural structural composites into focus, since they underwent a substantial optimization during long evolutionary selection pressure and adaptation processes. Marine biological materials are the most important sources of both inspiration for biomimetics and of raw materials for practical applications in technology and biomedicine. The use of marine natural products as multifunctional biomaterials is currently undergoing a renaissance in the modern materials science. The diversity of marine biomaterials, their forms and fields of application are highlighted in this review. We will discuss the challenges, solutions, and future directions of modern marine biomaterialogy using a thorough analysis of scientific sources over the past ten years.

Electrochemical Approach for Isolation of Chitin from the Skeleton of the Black Coral Cirrhipathes sp. (Antipatharia).

The development of novel and effective methods for the isolation of chitin, which remains one of the fundamental aminopolysaccharides within skeletal structures of diverse marine invertebrates, is still relevant. In contrast to numerous studies on chitin extraction from crustaceans, mollusks and sponges, there are only a few reports concerning its isolation from corals, and especially black corals (Antipatharia). In this work, we report the stepwise isolation and identification of chitin from Cirrhipathes sp. (Antipatharia, Antipathidae) for the first time. The proposed method, aiming at the extraction of the chitinous scaffold from the skeleton of black coral species, combined a well-known chemical treatment with in situ electrolysis, using a concentrated Na2SO4 aqueous solution as the electrolyte. This novel method allows the isolation of α-chitin in the form of a microporous membrane-like material. Moreover, the extracted chitinous scaffold, with a well-preserved, unique pore distribution, has been extracted in an astoundingly short time (12 h) compared to the earlier reported attempts at chitin isolation from Antipatharia corals.

3D Chitin Scaffolds of Marine Demosponge Origin for Biomimetic Mollusk Hemolymph-Associated Biomineralization Ex-Vivo.

Structure-based tissue engineering requires large-scale 3D cell/tissue manufacture technologies, to produce biologically active scaffolds. Special attention is currently paid to naturally pre-designed scaffolds found in skeletons of marine sponges, which represent a renewable resource of biomaterials. Here, an innovative approach to the production of mineralized scaffolds of natural origin is proposed. For the first time, a method to obtain calcium carbonate deposition ex vivo, using living mollusks hemolymph and a marine-sponge-derived template, is specifically described. For this purpose, the marine sponge Aplysin aarcheri and the terrestrial snail Cornu aspersum were selected as appropriate 3D chitinous scaffold and as hemolymph donor, respectively. The formation of calcium-based phase on the surface of chitinous matrix after its immersion into hemolymph was confirmed by Alizarin Red staining. A direct role of mollusks hemocytes is proposed in the creation of fine-tuned microenvironment necessary for calcification ex vivo. The X-ray diffraction pattern of the sample showed a high CaCO3 amorphous content. Raman spectroscopy evidenced also a crystalline component, with spectra corresponding to biogenic calcite. This study resulted in the development of a new biomimetic product based on ex vivo synthetized ACC and calcite tightly bound to the surface of 3D sponge chitin structure.

Functionalization of 3D Chitinous Skeletal Scaffolds of Sponge Origin Using Silver Nanoparticles and Their Antibacterial Properties.

Chitin, as one of nature's most abundant structural polysaccharides, possesses worldwide, high industrial potential and a functionality that is topically pertinent. Nowadays, the metallization of naturally predesigned, 3D chitinous scaffolds originating from marine sponges is drawing focused attention. These invertebrates represent a unique, renewable source of specialized chitin due to their ability to grow under marine farming conditions. In this study, the development of composite material in the form of 3D chitin-based skeletal scaffolds covered with silver nanoparticles (AgNPs) and Ag-bromide is described for the first time. Additionally, the antibacterial properties of the obtained materials and their possible applications as a water filtration system are also investigated.

Naturally pre-designed biomaterials: Spider molting cuticle as a functional crude oil sorbent.

Diverse fields of modern environmental technology are nowadays focused on the discovery and development of new sources for oil spill removal. An especially interesting type of sorbents is those of natural origin-biosorbents-as ready-to-use constructs with biodegradable, nontoxic, renewable and cost-efficient properties. Moreover, the growing problem of microplastic-related contamination in the oceans further encourages the use of biosorbents. Here, for the first time, naturally pre-designed molting cuticles of the Theraphosidae spider Avicularia sp. "Peru purple", as part of constituting a large-scale spider origin waste material, were used for efficient sorption of crude oil. Compared with currently used materials, the proposed biosorbent of spider cuticular origin demonstrates excellent ability to remain on the water surface for a long time. In this study the morphology and hydrophobic features of Theraphosidae cuticle are investigated for the first time. The unique surface morphology and very low surface free energy (4.47 ± 0.08 mN/m) give the cuticle-based, tube-like, porous biosorbent excellent oleophilic-hydrophobic properties. The crude oil sorption capacities of A. sp. "Peru purple" molt structures in sea water, distilled water and fresh water were measured at 12.6 g/g, 15.8 g/g and 16.6 g/g respectively. These results indicate that this biomaterial is more efficient than such currently used fibrous sorbents as human hairs or chicken feathers. Four cycles of desorption were performed and confirmed the reusability of the proposed biosorbent. We suggest that the oil adsorption mechanism is related to the brush-like and microporous structure of the tubular spider molting cuticles and may also involve interaction between the cuticular wax layers and crude oil.

Express Method for Isolation of Ready-to-Use 3D Chitin Scaffolds from Aplysina archeri (Aplysineidae: Verongiida) Demosponge.

Sponges are a valuable source of natural compounds and biomaterials for many biotechnological applications. Marine sponges belonging to the order Verongiida are known to contain both chitin and biologically active bromotyrosines. Aplysina archeri (Aplysineidae: Verongiida) is well known to contain bromotyrosines with relevant bioactivity against human and animal diseases. The aim of this study was to develop an express method for the production of naturally prefabricated 3D chitin and bromotyrosine-containing extracts simultaneously. This new method is based on microwave irradiation (MWI) together with stepwise treatment using 1% sodium hydroxide, 20% acetic acid, and 30% hydrogen peroxide. This approach, which takes up to 1 h, made it possible to isolate chitin from the tube-like skeleton of A. archeri and to demonstrate the presence of this biopolymer in this sponge for the first time. Additionally, this procedure does not deacetylate chitin to chitosan and enables the recovery of ready-to-use 3D chitin scaffolds without destruction of the unique tube-like fibrous interconnected structure of the isolated biomaterial. Furthermore, these mechanically stressed fibers still have the capacity for saturation with water, methylene blue dye, crude oil, and blood, which is necessary for the application of such renewable 3D chitinous centimeter-sized scaffolds in diverse technological and biomedical fields.

New Source of 3D Chitin Scaffolds: The Red Sea Demosponge Pseudoceratina arabica (Pseudoceratinidae, Verongiida).

The bioactive bromotyrosine-derived alkaloids and unique morphologically-defined fibrous skeleton of chitin origin have been found recently in marine demosponges of the order Verongiida. The sophisticated three-dimensional (3D) structure of skeletal chitinous scaffolds supported their use in biomedicine, tissue engineering as well as in diverse modern technologies. The goal of this study was the screening of new species of the order Verongiida to find another renewable source of naturally prefabricated 3D chitinous scaffolds. Special attention was paid to demosponge species, which could be farmed on large scale using marine aquaculture methods. In this study, the demosponge Pseudoceratina arabica collected in the coastal waters of the Egyptian Red Sea was examined as a potential source of chitin for the first time. Various bioanalytical tools including scanning electron microscopy (SEM), fluorescence microscopy, FTIR analysis, Calcofluor white staining, electrospray ionization mass spectrometry (ESI-MS), as well as a chitinase digestion assay were successfully used to confirm the discovery of α-chitin within the skeleton of P. arabica. The current finding should make an important contribution to the field of application of this verongiid sponge as a novel renewable source of biologically-active metabolites and chitin, which are important for development of the blue biotechnology especially in marine oriented biomedicine.

Naturally Drug-Loaded Chitin: Isolation and Applications.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Naturally Prefabricated Marine Biomaterials: Isolation and Applications of Flat Chitinous 3D Scaffolds from Ianthella labyrinthus (Demospongiae: Verongiida).

Marine sponges remain representative of a unique source of renewable biological materials. The demosponges of the family Ianthellidae possess chitin-based skeletons with high biomimetic potential. These three-dimensional (3D) constructs can potentially be used in tissue engineering and regenerative medicine. In this study, we focus our attention, for the first time, on the marine sponge Ianthella labyrinthus Bergquist & Kelly-Borges, 1995 (Demospongiae: Verongida: Ianthellidae) as a novel potential source of naturally prestructured bandage-like 3D scaffolds which can be isolated simultaneously with biologically active bromotyrosines. Specifically, translucent and elastic flat chitinous scaffolds have been obtained after bromotyrosine extraction and chemical treatments of the sponge skeleton with alternate alkaline and acidic solutions. For the first time, cardiomyocytes differentiated from human induced pluripotent stem cells (iPSC-CMs) have been used to test the suitability of I. labyrinthus chitinous skeleton as ready-to-use scaffold for their cell culture. Results reveal a comparable attachment and growth on isolated chitin-skeleton, compared to scaffolds coated with extracellular matrix mimetic Geltrex®. Thus, the natural, unmodified I. labyrinthus cleaned sponge skeleton can be used to culture iPSC-CMs and 3D tissue engineering. In addition, I. labyrinthus chitin-based scaffolds demonstrate strong and efficient capability to absorb blood deep into the microtubes due to their excellent capillary effect. These findings are suggestive of the future development of new sponge chitin-based absorbable hemostats as alternatives to already well recognized cellulose-based fabrics.

Spider Chitin: An Ultrafast Microwave-Assisted Method for Chitin Isolation from Caribena versicolor Spider Molt Cuticle.

Chitin, as a fundamental polysaccharide in invertebrate skeletons, continues to be actively investigated, especially with respect to new sources and the development of effective methods for its extraction. Recent attention has been focused on marine crustaceans and sponges; however, the potential of spiders (order Araneae) as an alternative source of tubular chitin has been overlooked. In this work, we focused our attention on chitin from up to 12 cm-large Theraphosidae spiders, popularly known as tarantulas or bird-eating spiders. These organisms "lose" large quantities of cuticles during their molting cycle. Here, we present for the first time a highly effective method for the isolation of chitin from Caribena versicolor spider molt cuticle, as well as its identification and characterization using modern analytical methods. We suggest that the tube-like molt cuticle of this spider can serve as a naturally prefabricated and renewable source of tubular chitin with high potential for application in technology and biomedicine.

Collagens of Poriferan Origin.

The biosynthesis, structural diversity, and functionality of collagens of sponge origin are still paradigms and causes of scientific controversy. This review has the ambitious goal of providing thorough and comprehensive coverage of poriferan collagens as a multifaceted topic with intriguing hypotheses and numerous challenging open questions. The structural diversity, chemistry, and biochemistry of collagens in sponges are analyzed and discussed here. Special attention is paid to spongins, collagen IV-related proteins, fibrillar collagens from demosponges, and collagens from glass sponge skeletal structures. The review also focuses on prospects and trends in applications of sponge collagens for technology, materials science and biomedicine.

Marine Spongin: Naturally Prefabricated 3D Scaffold-Based Biomaterial.

The biosynthesis, chemistry, structural features and functionality of spongin as a halogenated scleroprotein of keratosan demosponges are still paradigms. This review has the principal goal of providing thorough and comprehensive coverage of spongin as a naturally prefabricated 3D biomaterial with multifaceted applications. The history of spongin's discovery and use in the form of commercial sponges, including their marine farming strategies, have been analyzed and are discussed here. Physicochemical and material properties of spongin-based scaffolds are also presented. The review also focuses on prospects and trends in applications of spongin for technology, materials science and biomedicine. Special attention is paid to applications in tissue engineering, adsorption of dyes and extreme biomimetics.

First Report on Chitin in a Non-Verongiid Marine Demosponge: The Mycale euplectellioides Case.

Sponges (Porifera) are recognized as aquatic multicellular organisms which developed an effective biochemical pathway over millions of years of evolution to produce both biologically active secondary metabolites and biopolymer-based skeletal structures. Among marine demosponges, only representatives of the Verongiida order are known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. The unique three-dimensional (3D) architecture of such chitinous skeletons opens the widow for their recent applications as adsorbents, as well as scaffolds for tissue engineering and biomimetics. This study has the ambitious goal of monitoring other orders beyond Verongiida demosponges and finding alternative sources of naturally prestructured chitinous scaffolds; especially in those demosponge species which can be cultivated at large scales using marine farming conditions. Special attention has been paid to the demosponge Mycale euplectellioides(Heteroscleromorpha: Poecilosclerida: Mycalidae) collected in the Red Sea. For the first time, we present here a detailed study of the isolation of chitin from the skeleton of this sponge, as well as its identification using diverse bioanalytical tools. Calcofluor white staining, Fourier-transform Infrared Spcetcroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS), scanning electron microscopy (SEM), and fluorescence microscopy, as well as a chitinase digestion assay were applied in order to confirm with strong evidence the finding of a-chitin in the skeleton of M. euplectellioides. We suggest that the discovery of chitin within representatives of the Mycale genus is a promising step in their evaluation of these globally distributed sponges as new renewable sources for both biologically active metabolites and chitin, which are of prospective use for pharmacology and biomaterials oriented biomedicine, respectively.

Applicability of a Green Nanocomposite Consisted of Spongin Decorated Cu2WO4(OH)2 and AgNPs as a High-Performance Aptasensing Platform in Staphylococcus aureus Detection.

This study reports the synthesis of a nanocomposite consisting of spongin and its applicability in the development of an aptasensing platform with high performance. The spongin was carefully extracted from a marine sponge and decorated with copper tungsten oxide hydroxide. The resulting spongin-copper tungsten oxide hydroxide was functionalized by silver nanoparticles and utilized in electrochemical aptasensor fabrication. The nanocomposite covered on a glassy carbon electrode surface amplified the electron transfer and increased active electrochemical sites. The aptasensor was fabricated by loading of thiolated aptamer on the embedded surface via thiol-AgNPs linkage. The applicability of the aptasensor was tested in detecting the Staphylococcus aureus bacterium as one of the five most common causes of nosocomial infectious diseases. The aptasensor measured S. aureus under a linear concentration range of 10-108 colony-forming units per milliliter and a limit of quantification and detection of 12 and 1 colony-forming unit per milliliter, respectively. The highly selective diagnosis of S. aureus in the presence of some common bacterial strains was satisfactorily evaluated. The acceptable results of the human serum analysis as the real sample may be promising in the bacteria tracking in clinical samples underlying the green chemistry principle.

High-Performance Three-Dimensional Spongin-Atacamite Biocomposite for Electrochemical Nonenzymatic Glucose Sensing.

The design of sensitive and cost-effective biocomposite materials with high catalytic activity for the effective electrooxidation of glucose plays a key role in developing enzyme-free glucose sensors. The porous three-dimensional (3D) spongin scaffold of marine sponge origin provides an excellent template for the growth of atacamite crystals and improves the activity of atacamite as a catalyst. By using the design of experiment method, the influence of different parameters on the electrode efficiency was optimized. The optimized sensor based on spongin-atacamite showed distinguished performance toward glucose with two linear ranges of 0.4-200 µM and 0.2-10 mM and high sensitivities of 3908.4 and 600.5 µA mM-1 cm-2, respectively. Importantly, the designed sensor exhibited strong selectivity and favorable stability, reproducibility, and repeatability. The performance in the real application was estimated by glucose detection in spiked human blood serum samples, which verified its great potential as a reliable platform for enzyme-free glucose sensing.

Arrested in Glass: Actin within Sophisticated Architectures of Biosilica in Sponges.

Actin is a fundamental member of an ancient superfamily of structural intracellular proteins and plays a crucial role in cytoskeleton dynamics, ciliogenesis, phagocytosis, and force generation in both prokaryotes and eukaryotes. It is shown that actin has another function in metazoans: patterning biosilica deposition, a role that has spanned over 500 million years. Species of glass sponges (Hexactinellida) and demosponges (Demospongiae), representatives of the first metazoans, with a broad diversity of skeletal structures with hierarchical architecture unchanged since the late Precambrian, are studied. By etching their skeletons, organic templates dominated by individual F-actin filaments, including branched fibers and the longest, thickest actin fiber bundles ever reported, are isolated. It is proposed that these actin-rich filaments are not the primary site of biosilicification, but this highly sophisticated and multi-scale form of biomineralization in metazoans is ptterned.