Extracellular matrix type 0: From ancient collagen lineage to a versatile product pipeline - JellaGel™.

Extracellular matrix type 0 is reported. The matrix is developed from a jellyfish collagen predating mammalian forms by over 0.5 billion years. With its ancient lineage, compositional simplicity, and resemblance to multiple collagen types, the matrix is referred to as the extracellular matrix type 0. Here we validate the matrix describing its physicochemical and biological properties and present it as a versatile, minimalist biomaterial underpinning a pipeline of commercialised products under the collective name of JellaGelTM. We describe an extensive body of evidence for folding and assembly of the matrix in comparison to mammalian matrices, such as bovine collagen, and its use to support cell growth and development in comparison to known tissue-derived products, such as Matrigel™. We apply the matrix to co-culture human astrocytes and cortical neurons derived from induced pluripotent stem cells and visualise neuron firing synchronicity with correlations indicative of a homogenous extracellular material in contrast to the performance of heterogenous commercial matrices. We prove the ability of the matrix to induce spheroid formation and support the 3D culture of human immortalised, primary, and mesenchymal stem cells. We conclude that the matrix offers an optimal solution for systemic evaluations of cell-matrix biology. It effectively combines the exploitable properties of mammalian tissue extracts or top-down matrices, such as biocompatibility, with the advantages of synthetic or bottom-up matrices, such as compositional control, while avoiding the drawbacks of the two types, such as biological and design heterogeneity, thereby providing a unique bridging capability of a stem extracellular matrix.

Marine collagen-chitosan-fucoidan/chondroitin sulfate cryo-biomaterials loaded with primary human cells envisaging cartilage tissue engineering.

Cartilage repair after a trauma or a degenerative disease like osteoarthritis (OA) continues to be a big challenge in current medicine due to the limited self-regenerative capacity of the articular cartilage tissues. To overcome the current limitations, tissue engineering and regenerative medicine (TERM) and adjacent areas have focused their efforts on new therapeutical procedures and materials capable of restoring normal tissue functionalities through polymeric scaffolding and stem cell engineering approaches. For this, the sustainable exploration of marine origin materials has emerged in the last years as a natural alternative to mammal sources, benefiting from their biological properties (e.g., biocompatibility, biodegradability, no toxicity, among others) for the development of several types of scaffolds. In this study, marine collagen(jCOL)-chitosan(sCHT)-fucoidan(aFUC)/chondroitin sulfate(aCS) were cryo-processed (-20 °C, -80 °C, and -196 °C) and a chemical-free crosslinking approach was explored to establish cohesive and stable cryogel materials. The cryogels were intensively characterized to assess their oscillatory behavior, thermal structural stability, thixotropic properties (around 45 % for the best formulations), injectability, and surface structural organization. Additionally, the cryogels demonstrate an interesting microenvironment in in vitro studies using human adipose-derived stem cells (hASCs), supporting their viability and proliferation. In both physic-chemical and in vitro studies, the systems that contain fucoidan in their formulations, i.e., C1 (jCOL, sCHT, aFUC) and C3 (jCOL, sCHT, aFUC, aCS), submitted at -80 °C, are those that demonstrated most promising results for future application in articular cartilage tissues.

Advanced Polymeric Membranes as Biomaterials Based on Marine Sources Envisaging the Regeneration of Human Tissues.

The self-repair capacity of human tissue is limited, motivating the arising of tissue engineering (TE) in building temporary scaffolds that envisage the regeneration of human tissues, including articular cartilage. However, despite the large number of preclinical data available, current therapies are not yet capable of fully restoring the entire healthy structure and function on this tissue when significantly damaged. For this reason, new biomaterial approaches are needed, and the present work proposes the development and characterization of innovative polymeric membranes formed by blending marine origin polymers, in a chemical free cross-linking approach, as biomaterials for tissue regeneration. The results confirmed the production of polyelectrolyte complexes molded as membranes, with structural stability resulting from natural intermolecular interactions between the marine biopolymers collagen, chitosan and fucoidan. Furthermore, the polymeric membranes presented adequate swelling ability without compromising cohesiveness (between 300 and 600%), appropriate surface properties, revealing mechanical properties similar to native articular cartilage. From the different formulations studied, the ones performing better were the ones produced with 3 % shark collagen, 3% chitosan and 10% fucoidan, as well as with 5% jellyfish collagen, 3% shark collagen, 3% chitosan and 10% fucoidan. Overall, the novel marine polymeric membranes demonstrated to have promising chemical, and physical properties for tissue engineering approaches, namely as thin biomaterial that can be applied over the damaged articular cartilage aiming its regeneration.

Larynx proteomics after jellyfish collagen IL: Increased ECM/collagen and suppressed inflammation.

Objectives/Hypothesis: Compare proteomic profiles of rabbit vocal folds (VFs) injected with micronized cross-linked jellyfish collagen "collagen Type 0" (MX-JC) against two clinical products for injection medialization laryngoplasty (IL). Study Design: Animal model. Methods: Left recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received (MX-JC) and adipose-derived stem cells (ADSCs), Group 2, MX-JC alone; Group 3, cross-linked hyaluronic acid; and Group 4, micronized acellular dermis. Animals were sacrificed at 4 and 12 weeks. Proteomic profiling of injected versus noninjected VFs by nano-liquid chromatography, tandem mass spectrometry, and reactome gene ontology analysis was performed. Results: Overall, 37-61 proteins were found to be upregulated and 60-284 downregulated in injected versus non-injected VFs (>1.5 fold, false discovery rate-adjusted p < .05). Over-representation analysis (% of total) revealed top up-regulated pathways at 4 and 12 weeks, respectively: Group 1, keratan sulfate metabolism (46%) and cellular processes (29%); Group 2, extracellular matrix (ECM)/collagen processes (33%) and beta oxidation (39%); Group 3, cellular processes (50%) and energy metabolism (100%); and Group 4, keratan sulfate metabolism (31%) and inflammation (50%). Top downregulated pathways were: Group 1, Inflammation (36%) and glucose/citric acid metabolism (42%); Group 2, cell signaling (38%) and glucose/citric acid metabolism (35%); Group 3, keratan sulfate metabolism (31%) and ECM/collagen processes (48%); and Group 4, glucose/citric acid metabolism (33%) and ECM/collagen processes (43%). Conclusions: MX-JC "collagen Type 0" upregulates pathways related to ECM/collagen formation and downregulates pathways related to inflammation suggesting that it is promising biomaterial for IL. Level of Evidence: NA.

Marine origin biomaterials using a compressive and absorption methodology as cell-laden hydrogel envisaging cartilage tissue engineering.

In the recent decade, marine origin products have been growingly studied as building blocks complying with the constant demand of the biomedical sector regarding the development of new devices for Tissue Engineering and Regenerative Medicine (TERM). In this work, several combinations of marine collagen-chitosan-fucoidan hydrogel were formed using a newly developed eco-friendly compressive and absorption methodology to produce hydrogels (CAMPH), which consists of compacting the biopolymers solution while removing the excess of water. The hydrogel formulations were prepared by blending solutions of 5% collagen from jellyfish and/or 3% collagen from blue shark skin, with solutions of 3% chitosan from squid pens and solutions of 10% fucoidan from brown algae, at different ratios. The biopolymer physico-chemical characterization comprised Amino Acid analysis, ATR-FTIR, CD, SDS-PAGE, ICP, XRD, and the results suggested the shark/jellyfish collagen(s) conserved the triple helical structure and had similarities with type I and type II collagen, respectively. The studied collagens also contain a denaturation temperature of around 30-32 °C and a molecular weight between 120 and 125 kDa. Additionally, the hydrogel properties were determined by rheology, water uptake ability, degradation rate, and SEM, and the results showed that all formulations had interesting mechanical (strong viscoelastic character) and structural stability properties, with a significant positive highlight in the formulation of H3 (blending all biopolymers, i.e., 5% collagen from jellyfish, 3% collagen from skin shark, 3% chitosan and 10% of fucoidan) in the degradation test, that shows a mass loss around 18% over the 30 days, while the H1 and H2, present a mass loss of around 35% and 44%, respectively. Additionally, the in vitro cellular assessments using chondrocyte cells (ATDC5) in encapsulated state revealed, for all hydrogel formulations, a non-cytotoxic behavior. Furthermore, Live/Dead assay and Phalloidin/DAPI staining, to assess the cytoskeletal organization, proved that the hydrogels can provide a suitable microenvironment for cell adhesion, viability, and proliferation, after being encapsulated. Overall, the results show that all marine collagen (jellyfish/shark)-chitosan-fucoidan hydrogel formulations provide a good structural architecture and microenvironment, highlighting the H3 biomaterial due to containing more polymers in their composition, making it suitable for biomedical articular cartilage therapies.

A Design of Experiments (DoE) Approach to Optimize Cryogel Manufacturing for Tissue Engineering Applications.

Marine origin polymers represent a sustainable and natural alternative to mammal counterparts regarding the biomedical application due to their similarities with proteins and polysaccharides present in extracellular matrix (ECM) in humans and can reduce the risks associated with zoonosis and overcoming social- and religious-related constraints. In particular, collagen-based biomaterials have been widely explored in tissue engineering scaffolding applications, where cryogels are of particular interest as low temperature avoids protein denaturation. However, little is known about the influence of the parameters regarding their behavior, i.e., how they can influence each other toward improving their physical and chemical properties. Factorial design of experiments (DoE) and response surface methodology (RSM) emerge as tools to overcome these difficulties, which are statistical tools to find the most influential parameter and optimize processes. In this work, we hypothesized that a design of experiments (DoE) model would be able to support the optimization of the collagen-chitosan-fucoidan cryogel manufacturing. Therefore, the parameters temperature (A), collagen concentration (B), and fucoidan concentration (C) were carefully considered to be applied to the Box-Behnken design (three factors and three levels). Data obtained on rheological oscillatory measurements, as well as on the evaluation of antioxidant concentration and adenosine triphosphate (ATP) concentration, showed that fucoidan concentration could significantly influence collagen-chitosan-fucoidan cryogel formation, creating a stable internal polymeric network promoted by ionic crosslinking bonds. Additionally, the effect of temperature significantly contributed to rheological oscillatory properties. Overall, the condition that allowed us to have better results, from an optimization point of view according to the DoE, were the gels produced at -80 °C and composed of 5% of collagen, 3% of chitosan, and 10% fucoidan. Therefore, the proposed DoE model was considered suitable for predicting the best parameter combinations needed to develop these cryogels.

Jellyfish Collagen: A Biocompatible Collagen Source for 3D Scaffold Fabrication and Enhanced Chondrogenicity.

Osteoarthritis (OA) is a multifactorial disease leading to degeneration of articular cartilage, causing morbidity in approximately 8.5 million of the UK population. As the dense extracellular matrix of articular cartilage is primarily composed of collagen, cartilage repair strategies have exploited the biocompatibility and mechanical strength of bovine and porcine collagen to produce robust scaffolds for procedures such as matrix-induced chondrocyte implantation (MACI). However, mammalian sourced collagens pose safety risks such as bovine spongiform encephalopathy, transmissible spongiform encephalopathy and possible transmission of viral vectors. This study characterised a non-mammalian jellyfish (Rhizostoma pulmo) collagen as an alternative, safer source in scaffold production for clinical use. Jellyfish collagen demonstrated comparable scaffold structural properties and stability when compared to mammalian collagen. Jellyfish collagen also displayed comparable immunogenic responses (platelet and leukocyte activation/cell death) and cytokine release profile in comparison to mammalian collagen in vitro. Further histological analysis of jellyfish collagen revealed bovine chondroprogenitor cell invasion and proliferation in the scaffold structures, where the scaffold supported enhanced chondrogenesis in the presence of TGFß1. This study highlights the potential of jellyfish collagen as a safe and biocompatible biomaterial for both OA repair and further regenerative medicine applications.

In vivo comparison of jellyfish and bovine collagen sponges as prototype medical devices.

Jellyfish have emerged as a source of next generation collagen that is an attractive alternative to existing sources, such as bovine and porcine, due to a plentiful supply and providing a safer source through lack of bovine spongiform encephalopathy (BSE) transmission risk and potential viral vectors, both of which could be transmitted to humans. Here we compare collagen implantable sponges derived for the first time from the Rhizostoma pulmo jellyfish. A further novelty for the research was that there was a comparison for sponges that were either uncrosslinked or crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and an assessment on how this affected resorption, as well as their biocompatibility compared to bovine type I collagen sponges. The scaffolds were prepared and examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and scanning electron microscopy (SEM). The samples were implanted in adult male Wistar rats for in vivo experimentation. Both crosslinked and uncrosslinked jellyfish collagen sponges showed a significant reduction in histopathology scores over the course of the study, whereas the bovine collagen sponge scores were not significantly reduced. Both jellyfish collagen sponges and the bovine sponge were tolerated well by the hosts, and a recovery was visible in all samples, suggesting that R. pulmo jellyfish-derived collagen could offer compelling biocompatibility with wound healing applications. We also demonstrate that noncrosslinked samples could be safer with better resorption times than crosslinked samples. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1524-1533, 2018.

Prospecting for new bacterial metabolites: a glossary of approaches for inducing, activating and upregulating the biosynthesis of bacterial cryptic or silent natural products.

Covering: up to 2015. Over the centuries, microbial secondary metabolites have played a central role in the treatment of human diseases and have revolutionised the pharmaceutical industry. With the increasing number of sequenced microbial genomes revealing a plethora of novel biosynthetic genes, natural product drug discovery is entering an exciting second golden age. Here, we provide a concise overview as an introductory guide to the main methods employed to unlock or up-regulate these so called 'cryptic', 'silent' and 'orphan' gene clusters, and increase the production of the encoded natural product. With a predominant focus on bacterial natural products we will discuss the importance of the bioinformatics approach for genome mining, the use of first different and simple culturing techniques and then the application of genetic engineering to unlock the microbial treasure trove.

A fatty acid from the diatom Phaeodactylum tricornutum is antibacterial against diverse bacteria including multi-resistant Staphylococcus aureus (MRSA).

Pathogenic bacteria, such as multidrug-resistant Staphylococcus aureus (MRSA), which are not susceptible to most conventional antibiotics, are causing increased concern in healthcare institutions worldwide. The discovery of novel antibacterial compounds for biomedical exploitation is one avenue that is being pursued to combat these problematic bacteria. Marine eukaryotic microalgae are known to produce numerous useful products but have attracted little attention in the search for novel antibiotic compounds. Cell lysates of the marine diatom, Phaeodactylum tricornutum Bohlin, have been reported to display antibacterial activity in vitro, but the compounds responsible have not been fully identified. In this paper, using column chromatography and reversed-phase high-performance liquid chromatography, we report the isolation of an antibacterial fatty acid. Mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy revealed it to be the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). We show that EPA is active against a range of both Gram-positive and Gram-negative bacteria, including MRSA, at micromolar concentrations. These data indicate that it could find application in the topical and systemic treatment of drug-resistant bacterial infections.