Evaluation of the potential of collagen from codfish skin as a biomaterial for biomedical applications

Collagen is one of the most widely used biomaterials, not only due its biocompatibility, biodegradability and weak antigenic potential, but also due to its role in the structure and function of tissues. Searching for alternative collagen sources, the aim of this study was to extract collagen from the skin of codfish, previously obtained as a by-product of fish industrial plants, and characterize it regarding its use as a biomaterial for biomedical application, according to American Society for Testing and Materials (ASTM) Guidelines. Collagen type I with a high degree of purity was obtained through acid-extraction, as confirmed by colorimetric assays, SDS-PAGE and amino acid composition. Thermal analysis revealed a denaturing temperature around 16 C. Moreover, collagen showed a concentration-dependent effect in metabolism and on cell adhesion of lung fibroblast MRC-5 cells. In conclusion, this study shows that collagen can be obtained from marine-origin sources, while preserving its bioactivity, supporting its use in biomedical applications.European Research Council grant agreement ERC-2012-ADG 20120216-321266 for the project ComplexiTEinfo:eu-repo/semantics/publishedVersio

Evaluation of the potential of fucoidan-based microparticles for diabetes treatment

Abstract INTRODUCTION: Marine organisms have in their constitution materials with a wide range of properties and characteristics inspiring their application within the biomedical field. One important example is fucoidan (Fu), an underexploited sulfated polysaccharide extracted from the cell wall of the brown seaweeds, with high solubility in water1. Fucoidan is composed of L- fucose and glucuronic acid including sulfate groups and has important bioactive properties such as antioxidative, anticoagulant, anticancer and in the reduction of blood glucose1,2. In this work, the biomedical potential of fucoidan was assessed by processing modified fucoidan (MFu) into microparticles by photocrosslinking using superhydrophobic surfaces and visible light3,4. Biological performance on the developed constructs using human pancreatic beta cells is currently under investigation. METHODS: To design the materials structures, fucoidan was modified by methacrylation reaction3. Briefly, Fu aqueous solution 4% w/v was mixed with methacrylated anhydride (MA) in volume of 12% v/v at 50oC to react for 6h. Further, MFu particles with and without insulin (0.5% w/v) were produced by pipetting a solution of 5% MFu v/v with triethanolamine and eosin-y (photoinitiators) onto superhydrophobic surfaces4 (Fig. 1A) and then photocrosslinking using visible light4. MFu and developed particles were characterized using 1HNMR, turbidimetry and SEM to assess their chemistry and morphology, respectively. Moreover, the insulin release was evaluated in phosphate buffered saline (PBS) solution at pH 7and simulated intestinal fluid (SIF) at pH 5. The ability of the developed materials to support adhesion and proliferation of cells was assessed by suspension culture of human pancreatic cells 1.1B4 (3.5x105 cells/ml) in contact with MFu microparticles during up to 7 days. RESULTS: The chemical modification performed on Fu was confirmed by the presence of vinyl and additional methyl peaks in the 1HNMR of modified fucoidan, not present in Fu spectrum. Methacrylated fucoidan was obtained with a methacrylation degree of 17%. The produced fucoidan particles have round shape and average diameter of 1.53 mm (Fig. 1B). The insulin release in PBS and SIF demonstrate that the particles can release insulin in a sustained manner under the studied period. It seems that the insulin release is slower for SIF (pH5, Fig. 1C), than for PBS. The biological tests regarding the culture of pancreatic beta cells demonstrate that cells show a round-like shape and tend to form pseudo-islets during the culture period studied (Fig. 1D). DISCUSSION & CONCLUSIONS: This work demonstrates the successful production of fucoidan- based-microparticles through the methacrylation of fucoidan, using visible light and superhydrophobic surfaces. The covalent crosslinking methacrylated fucoidan through visible light represents a promising method to obtain biocompatible fucoidan particles with a uniform round shape. The obtained insulin release profiles are sensitive to different pH (pH7 and pH5), mimicking the normal physiological pathway for insulin release. Furthermore, the results suggest these systems could be used for treatment of type I diabetes mellitus as they sustain beta cells viability and proliferation. The response also suggested, that the MFu particles could be a good candidate as drug delivery vehicles for the diabetes mellitus treatment. REFERENCES: 1 Silva TH et al (2012), Biomatter 2(4): 278:289. 2Sezer Alidemir et al (2011), Fucoidan: A versatile biopolymer for biomedical applicatons (Springer Ber.Heid).pp377-406. 3Mihaila S.et al (2013), Adv. Health. Mat. 2(6): 895-907. 4Rial Hermida et al, Acta Biomater.(2014) 10(10) 4314-4322. ACKNOWLEDGEMENTS: This work was partially funded by projects 0687_NOVOMAR_1_P (POCTEP), CarbPol_u_Algae (EXPL/MAR- BIO/0165/2013), ComplexiTE (ERC-2012-ADG 20120216-321266). Portuguese Foundation for Science and Technology is also gratefully acknowledged for doctoral grants of L. Reys and N. Oliveira and post- doctoral grants of S.S. Silva and D. Soares da Costafunded by projects 0687_NOVOMAR_1_P (POCTEP), CarbPol_u_Algae (EXPL/MARBIO/0165/2013) , ComplexiTE(ERC-2012-ADG 20120216-321266). Portuguese Foundation for Science and Technologyinfo:eu-repo/semantics/publishedVersio

Acid and enzymatic extraction of collagen from Atlantic cod (Gadus Morhua) swim bladders envisaging health-related applications

Atlantic cod is processed industrially for food purposes, with several by-products being directed to animal feed and other ends. Looking particularly into swim bladders, the extraction of collagen can be a valuable strategy for by-product valorization, explored in the present work for the first time. Collagen was extracted using acetic acid (ASCsb) and pepsin (PSCsb) with yields of 5.72% (w/w) and 11.14% (w/w), respectively. SDS-PAGE profile showed that the extracts were compatible with type I collagen. FTIR, CD and XRD results suggest that the PSCsb structure underwent partial denaturation, with microDSC showing a band at 54 ºC probably corresponding to a melting process, while ASCsb structure remained intact, with preserved triple helix and a denaturation temperature of 29.6 ºC. Amino acid composition indicates that the total content of proline-like amino acids was 148/1000 residues for ASCsb and 141/1000 residues for PSCsb, with a hydroxylation degree of about 37%. The extracts exhibited a typical shear thinning behavior, interesting property regarding their further processing toward the development of biomaterials. In this regard, assessment of metabolic activity of human fibroblast cells cultured in the presence of collagen extracts with concentrations up to 3mg/mL revealed the absence of cytotoxic behavior. Collagen extracts obtained from Atlantic cod swim bladders shown attractive properties regarding their use in cosmetic or biomedical applications.The authors would like to acknowledge to European Union for the financial support under the scope of European Regional Development Fund (ERDF) through the Structured Project NORTE-01-0145-FEDER-000021 (Norte2020) and under the scope of the European Union Seventh Framework Programme (FP7/2007-2013) through grant agreement ERC-2012-ADG 20120216-321266 (ERC Advanced Grant ComplexiTE). The Portuguese Foundation for Science and Technology is also acknowledged for the PhD grant of A. L. A under Doctoral Programme Do ~ Mar (PD/BD/127995/2016), as well as Norte2020 Regional Programme for the PhD grant of C. O. (Norte-08-5369-000037)

Biomedical potential of fucoidan, a seaweed sulfated polysaccharide: from a anticancer agent to a building block of cell encapsulating systems for regenerative medicine

Marine macroalgae or seaweeds synthesize a wide variety of polymers and smaller compounds with several bioactivities, among which the sulfated polysaccharides acquire greater relevance not only due to the reported antioxidant, antiviral and anticancer[1] activities, but also to the resemblance of extracellular matrix glycosaminoglycans found in the human body[2]. In this study, the potential of fucoidan (Fu) isolated from brown seaweed Fucus vesiculosus for therapeutical use has been evaluated, focusing in its performance as antitumoral agent (bioactive role) or as building block of cell encapsulating systems (structural role). Materials and Methods: The anticancer activity of Fu extracts was assessed by evaluating the cytotoxic behavior over two human breast cancer cell lines (MCF-7 and MDA-MB-231) in in-vitro culture, using human fibroblasts and endothelial cells (HPMEC-ST1 and MRC-5, respectively) as reference. Regarding the structural role, Fu was modified by methacrylation reaction (MFu) using methacrylic acid and further crosslinked using visible radiation and triethanolamine and eosin-y as photoinitiators. The photocrosslinking was performed on MFu solution droplets placed in a silica-based superhydrophobic surface[3], allowing the formation of particles[4] (since natural Fu is highly soluble in water and ion gelation is not effective). Biological performance of the developed particles was assessed by in vitro culture of fibroblasts and pancreatic cells (L929 and 1.1B4, respectively) in contact with MFu particles, up to 7 days. The ability of the developed materials to support adhesion and proliferation of cells was evaluated for both types of cells. Results and Discussion: The tested anticancer activity is not ubiquitous on Fu extracts, being dependent on its chemical features, with molecular weight (Mw) representing a particular role. Specifically, Mw values around 60 kDa exhibited cytotoxic effects to human breast cancer cell lines, while not affecting normal fibroblasts or endothelial cells (which represent the cells of the healthy tissue that would be closer to the tumor in a real situation). A concentration range of 0.2 to 0.3 mg mL-1 from the selected Fu extract could be considered as the therapeutic window for further studies. Regarding fucoidanâ s role on innovative biomaterials, the developed MFu particles could support the proliferation of fibroblasts (L929), but also of human pancreatic beta cells (1.1B4), which tend to form pseudo-islets after 7 days in culture (Fig. 1). This pancreatic cells could be also successfully encapsulated, opening a new route for a diabetes mellitus type 1 therapeutic approach. Fig. 1: Confocal microscopy images of 1.1B4 cells cultured in the presence of fucoidan-based particles and organized in pseudo-islets (red â actin; blue â nuclei). Conclusion: The present work establishes fucoidan as a high performance building block for the development of advanced therapies for cancer (targeted therapy) or tissue and organ regeneration. It shed light on the relation between chemical structure and biological activity towards anti-cancer effect and proposes novel beta cell laden particles as injectable insulin producing systems to tackle diabetes.Funding from projects 0687_NOVOMAR_1_P (co-funded by INTERREG 2007-2013 / POCTEP), CarbPol_u_Algae (EXPL/MAR-BIO/0165/2013, funded by the Portuguese Foundation for Science and Technology, FCT), POLARIS (FP7-REGPOT-CT2012-316331) and ComplexiTE (ERC-2012-ADG 20120216-321266), funded by the European Union’s Seventh Framework Programme for Research and Development is acknowledged. ASF, SSS, NMO and DSC are also thankful to FCT for their individual fellowships

Peripheral nerve injury: current challenges, conventional treatment approaches, and new trends in biomaterials-based regenerative strategies

Damage to peripheral nerves is a widely extended health problem, causing important socio-economic costs worldwide. Indeed, peripheral nerve injuries (PNI) have been concerning the medical community for many decades. Nevertheless, despite the increase in knowledge in the injury physiopathology and the great research efforts being undertaken, the current standard grafting strategies used to repair PNI are not as efficient as desired. Although alternative engineered nerve grafts are already commercialized, their clinical performance is suboptimal. In this review, a general description of the circumstances and repercussions surrounding the PNI pathological state are presented, together with the treatment limitations and current challenges when addressing both short- and long-gap defects. In addition, potential therapeutic molecules are considered, while innovative regenerative strategies have been identified. Finally, the most relevant advances on the use of a wide range of biomaterials for the development of novel medical devices are also overviewed in depth, either considering strategies making use of empty or filled nerve conduits for guided tissue regeneration.This work has received funding from the European Community’s Seventh Framework Programme (FP7- HEALTH-2011) under grant agreement 278612 (BIOHYBRID), from ERDF/POCTEP 2007-2013 under project 0687_NOVOMAR_1_P, from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number REGPOT-CT2012-316331-POLARIS, and from the ComplexiTE − An integrated multidisciplinary tissue engineering approach combining novel high-throughput screening and advanced methodologies to create complex biomaterials-stem cells constructs (ERC-2012-ADG_20120216- 321266). Portuguese Foundation for Science and Technology (FCT) is also acknowledged for the funds provided under the program Investigador FCT to J.S.C. (IF/00115/2015) and J.M.O. (IF/00423/2012 and IF/01285/2015).info:eu-repo/semantics/publishedVersio

Polissacarídeos de origem marinha para aplicação em estratégias de engenharia de tecidos

Os polissacarídeos de origem marinha tem recebido atenção crescente, quer dos cientistas, quer dos engenheiros a nível industrial, pelas várias propriedades que lhes são conhecidas, bem como pela variedade química que evidenciam. São originários de um meio ainda pouco explorado, mas ao qual se tem procurado aplicar estratégias sustentáveis de utilização, pelo que o seu potencial de aplicação é, de facto, impressionante. Neste capítulo, será dada particular ênfase a dois polissacarídeos – quitosano e fucoidana – a título de exemplo, em ambos os casos atendendo à sua semelhança aos glicosaminoglicanos presentes na matriz extracelular e por isso prometendo interesse muito significativo para a regeneração de tecidos

Fucoidan hydrogels photo-cross-linked with visible radiation as matrices for cell culture

Algae are abundant sources of bioactive components with extensive therapeutic properties, receiving much interest in recent years. The research on marine brown algae, namely one of its polysaccharide-fucoidan, has increased exponentially. Fucoidan is a sulfated cell-wall polysaccharide with several reported biological properties including anticancer, antivirus, anticoagulant, antioxidant and anti-inflammatory effects. In this study, fucoidan was functionalized by grafting methacrylic groups in the chain backbone, photo-cross-linkable under visible light to obtain biodegradable structures for tissue engineering. The functionalization reaction was carried out by concentrations (8 and 12%) of methacrylic anhydride (MA). The modified fucoidan (MFu) was characterized by FTIR and 1HNMR spectroscopy to confirm the functionalization. Further, modified fucoidan was photo-cross-linked under visible light and using superhydrophobic surfaces, to obtain spherical particles with controlled geometries benefiting from the high repellence of the surfaces. When using higher concentrations of MA, the particles were observed to exhibit a smaller average diameter. Moreover, the behavior of L929 mouse fibroblast-like cells was evaluated when cultured in contact with photo-cross-linked particles was investigated, being observed up to 14 days in culture. The photo-cross-linking of MFu under visible light enables thus the formation of particles here suggested as potentially relevant in a wide range of biomedical applications.ERDF: Atlantic Area Project 2011-1/164 MARMED and POCTEP Project 0687_NOVOMAR_1_P FP7: Project POLARIS (FP7-REGPOT_CT2012-316331) Portuguese Foundation for Science and Technology (FCT): project CarbPol_mu_Algae (EXPL/MAR-BIO/0165/2013) and scholarships (SFRH/BD/73172/2010, SFRH/BD/112139/2015, and SFRH/BPD/85790/2012, SFRH/BPD/112140/2015)

Sponge-derived silica for tissue regeneration: Bioceramics of deep-sea sponge

European Union H2020 programme (SponGES – 679849, CHEM2NATURE – 692333 and FORECAST – 668983) and FP7 programme (ERC Advanced Grant COMPLEXITE, ERC-2012-ADG-20120216-321266)info:eu-repo/semantics/publishedVersio

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