Extraction and characterization of collagen from elasmobranch byproducts for potential biomaterial use

With the worldwide increase of fisheries, fish wastes have had a similar increase, alternatively they can be seen as a source of novel substances for the improvement of societyâ s wellbeing. Elasmobranchs are a subclass fished in high amounts, with some species being mainly bycatch. They possess an endoskeleton composed mainly by cartilage, from which chondroitin sulfate is currently obtained. Their use as a viable source for extraction of type II collagen has been hypothesized with the envisaging of a biomedical application, namely in biomaterials production. In the present work, raw cartilage from shark (Prionace glauca) and ray (Zeachara chilensis and Bathyraja brachyurops) was obtained from a fish processing company and submitted to acidic and enzymatic extractions, to produce acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC). From all the extractions, P. glauca PSC had the highest yield (3.5%), followed by ray ASC (0.92%), ray PSC (0.50%), and P. glauca ASC (0.15%). All the extracts showed similar properties, with the SDS-PAGE profiles being compatible with the presence of both type I and type II collagens. Moreover, the collagen extracts exhibited the competence to maintain their conformation at human basal temperature, presenting a denaturation temperature higher than 3 C. Hydrogels were produced using P. glauca PSC combined with shark chondroitin sulfate, with the objective of mimicking the human cartilage extracellular matrix. These hydrogels were cohesive and structurally-stable at 37 C, with rheological measurements exhibiting a conformation of an elastic solid when submitted to shear strain with a frequency up to 4 Hz. This work revealed a sustainable strategy for the valorization of fisheriesâ by-products, within the concept of a circular economy, consisting of the use of P. glauca, Z. chilensis, and B. brachyurops cartilage for the extraction of collagen, which would be further employed in the development of hydrogels as a proof of concept of its biotechnological potential, ultimately envisaging its use in marine biomaterials to regenerate damaged cartilaginous tissues.The authors acknowledge the fish-processing industry Nigel (Peniche, Portugal) for the kind offer of shark and ray by-products, Marco Lemos (MARE-Leiria, IPLeiria, Portugal) for valuable discussions, and Filipe Costa and Sofia Duarte (CBMA, University of Minho, Portugal) for the DNA barcoding analysis for identification/confirmation of elasmobranchii species

Técnicas avançadas no estudo da corrosão

RESUMO: A M.T. Brandão, constituída em 1984, atua no setor tecnológico tendo como principais clientes as indústrias petrolíferas, alimentares e químicas, entre outras empresas industriais e tecnológicas, bem como instituições de ensino e investigação científica, institutos e organismos públicos. Com uma linha completa de produtos e soluções dedicadas à caraterização de materiais, a MT Brandão tem como missão responder às necessidades dos nossos clientes de forma a impulsionar os seus negócios, através da inovação e da melhoria contínua. Esta apresentação pretende assim dar a conhecer os equipamentos que a MT Brandão comercializa que permitem não só o estudo do processo de corrosão como o estudo de revestimentos protetivos e sua caracterização, em particular através de técnicas de Tribocorrosão e Espectroscopia de Raman.info:eu-repo/semantics/publishedVersio

Nanostructured natural-based polyelectrolyte multilayers to agglomerate chitosan particles into scaffolds for tissue engineering

The Layer-by-Layer (LbL) deposition technique is a self-assembly process that allows the coating of material's surface with nanostructured layers of polyelectrolytes, allowing to control several surface properties. This technique presents some advantages when compared with other thin film assembly techniques like having the possibility to coat surfaces with complex geometries in mild conditions or to incorporate active compounds. Tissue engineering involves typically the use of porous biodegradable scaffolds for the temporary support of cells. Such structures can be produced by agglomeration of micro-spheres that needs to be fixed into a three dimensional structure. In this work we suggest the use of LbL to promote such mechanical fixation in free-formed micro-spheres assemblies and simultaneously to control the properties of its surface. For the proof of concept the biological performance of chitosan/alginate multilayers is first investigated in two-dimensional models in which the attachment and proliferation of L929 and ATDC5 cells are studied in function of the number of layers and the nature of the final layer. Scaffolds prepared by agglomeration of chitosan particles using the same multilayered system were processed and characterized; it was found that they could support the attachment and proliferation of ATDC5 cells. This study suggests that LbL can be used as a versatile methodology to prepare scaffolds by particle agglomeration that could be suitable for tissue engineering applications.Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional de Cooperação Transfronteiriça Espanha Portugal 2007-2013 (POCTEP)Fundação para a Ciência e a Tecnologia (FCT

Development of marine-based nanocomposite scaffolds for biomedical applications

Despite the increasing attention that marine organisms are receiving, many of those are not efficiently exploited and subproducts with valuable compounds are being discarded. Two examples of those subproducts are the endoskeleton of squid, from which β-­‐chitin and consecutively chitosan can be obtained; and fish-­‐bones, as a source for the production of nano-­‐ hydroxyapatite. In this work, inspired in the nanocomposite structure of human bone, marine-­‐ based nanocomposite scaffolds composed by chitosan and nano-­‐hydroxyapatite (nHA) were developed using particle aggregation methodology. Chitosan was obtained from endoskeleton of giant squid Dosidicus Gigas while fish hydroxyapatite nanoparticles were synthesized from fish-­‐bones by pulsed laser in deionized water. An innovative methodology was used based on the agglomeration of prefabricated microspheres of chitosan/nHA, generally based on the random packing of microspheres with further aggregation by physical or thermal means to create a marine nanocomposite (CHA) .The morphological analysis of the developed nanocomposites revealed a low porosity structure, but with high interconnectivity, for all produced scaffolds. Furthermore, the nanocomposite scaffolds were characterized in terms of their mechanical properties, bioactivity, crystallinity and biological behavior. The obtained results highlight that the chitosan/nHA-­‐based marine nanocomposite can be a good candidate for biomedical applications, namely on bone regeneration

Cartilage regeneration approach based on squid chitosan scaffolds : in-vitro assessment

During the past decades, marine organisms have been the focus of considerable attention as potential source of valuable materials. For instance, chitosan is a biopolymer with high potential in the biomedical field and can be produced from crustacean shells and squid pens [1]. In this sense, we propose the use of chitosan to produce scaffolds for regenerative medicine purposes. An alkaline solution was used to deproteinize squid pens and isolate β- chitin (Chaussard 2004), which was further converted into chitosan through a deacetylation reaction. Chitosan was then processed into porous structures by freeze-drying [3], where chitosan solutions (4%) were submitted to different freezing temperature of -80ºC and - 196ºC. The produced structures were further submitted to neutralization methods with 4% NaHO, including in some cases a pre-washing step using ethanol/water solutions (100:0; 90:10, 80:20; 70:30 and 50:50) [4]. The morphology of scaffolds produced using either squid or commercial chitosan revealed a lamellar structure, independent of the source and/or freezing temperature. All chitosan scaffolds produced exhibited no-cytotoxic behaviour over L929 cells. To test the in vitro functionality of the scaffolds, cells from the mouse chondrogenic cell line ATDC-5 were seeded in the scaffolds and cultured for different time periods. Scaffolds made from squid chitosan were shown to promote better cell adhesion than commercial chitosan scaffolds and comparable or better cell proliferation. This demonstrates that squid chitosan is a valuable alternative to produce scaffolds for different applications in regenerative medicine, namely the regeneration of cartilage

Valorization of chitosan from squid pens and further use on the development of scaffolds for biomedical applications

Objectives: The aim of the present work is the valorization of squid pens through the production of chitosan that can be used for the development of biomedical applications. The present work is focused on !-chitin extraction from squid pens of the species Dosidicus gigas and its further conversion into chitosan. The biomedical potential of the isolated squid chitosan was assessed by processing this polymer as scaffolds for tissue engineering strategies. Methods: Alkali solution was used to deproteinized squid pens and thus isolate !-chitin, which was further converted into chitosan through a deacetylation reaction. The chitosan scaffolds were developed using a freeze-drying process, from 3% and 4% chitosan solutions in acetic acid and freezing at temperatures of -80ºC and -196ºC. Chitosan scaffolds were neutralized using two different methods: M1 – NaHO solution; and M2 – ethanol/water and NaHO solution. Morphology, Mechanical properties, degradation, cytotoxicity (L929 cells) and cellular adhesion (ATDC5 Chondrocytes like cells) of squid chitosan scaffolds were assessed and compared with the properties of scaffolds produced with commercial chitosan. Results: The morphology of scaffolds revealed a lamellar structure for all produced scaffolds, independent of the origin and concentration of chitosan. The treatment with sodium hydroxide and ethanol caused the formation of larger pores and loose of some lamellar features. Different freezing temperatures gave different pore morphology and the lower temperature a smaller pore size. The in vitro cell culture and cell adhesion studies showed that all chitosan scaffolds exhibited a non-cytotoxic effect over the mouse fibroblast-like cell line, L929 cells. Conclusions: The chitosan produced from the endoskeletons of giant squid Dosidicus Gigas has proven to be a valuable alternative to the commercial one when considering its use as biomaterial for different biomedical applications

Supporting the design of an ambient assisted living system using virtual reality prototypes

APEX, a framework for prototyping ubiquitous environments, is used to design an Ambient Assisted Living (AAL) system to enhance a care home for older people. The environment allows participants in the design process to experience the proposed design and enables developers to explore the design by rapidly developing alternatives. APEX provided the means to explore alternative designs through a virtual environment. It provides a mediating representation (a boundary object) allowing users to be involved in the design process. A group of residents in a city-based care home were involved in the design. The paper describes the design process and lessons learnt for the design of AAL systems.EPSRC - Engineering and Physical Sciences Research Council(EP/G059063/1)Jose C. Campos acknowledges support by the FCT – Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) within project UID/EEA/50014/2013. José Luís Silva acknowledges support from project PEST-OE/EEI/LA0009/2015. Michael Harrison was also funded by EPSRC research grant EP/G059063/1: CHI+MED (Computer–Human Interaction for Medical Devices)

Influence of freezing temperature and deacetylation degree on the performance of freeze-dried chitosan scaffolds towards cartilage tissue engineering

Chitosan-based porous structures have been significantly studied across the world as potential tissue engineering scaffolds. Despite the differences in chitosan produced from squid pens or crustacean shells, with the former being more reactive and easily available with a higher degree of deacetylation (DD), most of the studies report the use of crab or shrimp chitosan as they are readily available commercial sources. The aim of this work was to highlight the great potential of chitosan produced from squid pens for biomedical application. From freeze-dried scaffolds for soft tissue engineering, we investigated the influence of the DD of chitosan and the freezing temperature during processing on their performance. Chitosan was obtained by deacetylation of β-chitin previously isolated from endoskeleton of giant squid Dosidicus gigas (DD 91.2%) and compared with a commercially available batch obtained from crab shells (DD 76.6%). Chitosan solutions were frozen at â 80° C or â 196° C and further freeze-dried to obtain 3D porous structures (scaffolds). Scaffolds prepared at â 196° C have a compact structure with smaller pores, while those prepared at â 80° C showed a lamellar structure with larger pores. The compressive modulus varied from 0.7 up to 8.8 MPa. Both types of scaffolds were stable on PBS, including in the presence of lysozyme, up to 4 weeks. Furthermore, the squid chitosan scaffolds processed at â 80° C promoted ATDC5 chondrocyte-like cells adhesion and proliferation. The results suggest that the developed squid chitosan scaffolds might be further exploited for ap- plications in cartilage tissue engineering.This work was partially funded by ERDF through POCTEP Projects 0330_IBEROMARE_1_P and 0687_NOVOMAR_1_P, Atlantic Area Project 2011-1/164 MARMED and by European Union through European Research Council – Project ComplexiTE (ERC-2012-ADG 20120216-321266). Portuguese Foundation for Science and Technology is gratefully acknowledged for post-doc grants of R.P. Pirraco (SFRH/BPD/101886/2014) and S.S. Silva (SFRH/BPD/112140/2015) and PhD grant of Lara L. Reys (SFRH/BD/112139/2015). The authors would also like to acknowledge to Dr. Julio Maroto, from Fundación CETMAR (Spain) and Roi Vilela, from PESCANOVA S.A. (Spain), for the kind offer of squid pens.info:eu-repo/semantics/publishedVersio

Semiconductor gellan gum based composite hydrogels for tissue engineering applications

Publicado em "Journal of Tissue Engineering and Regenerative Medicine", vol. 7, supp. 1 (2013)Semiconductor hydrogels can be developed by combining the intrinsic electrical properties of semiconductors with the specific characteristics of hydrogels. These hydrogels have recently attracted much attention for applications in tissue engineering, especially formulations incorporating pyrrole and excellent biocompatibility. Several studies have reported that electrical stimulation influences the migration, proliferation and differentiation of stem cells and other cell lines [1]. The goal of this work is to use in situ chemical polymerization of polypyrrole (PPy) with gellan gum (GG) in order to obtain a new generation of semiconductor composite hydrogels. For the synthesis of GG/PPy composites, GG at 1.25% (w/v) final concentration was prepared in distilled water at room temperature. The solution was then heated under stirring at 90°C for 20 min. Temperature was decreased to 65°C and Py was added under vigorous agitation. The crosslinker solution (CaCl2, 0.18%) was added at 50°C. After 2 h, GG/Py composite hydrogels were obtained. In a further step, GG/Py samples were immersed in a solution of oxidizing agent in PBS and the reaction was carried out for 18 h under agitation at room temperature. Finally, the samples were frozen at -80°C for 48 h and lyophilized. The characterization of GG, GG/PPy and PPy samples was performed by scanning electron microscopy (SEM). The incorporation of PPy in the gellan gum was confirmed by SEM analysis. The coating with PPy increases the thickness of each sheet in 3 fold when compared with GG samples. Conductivity tests were also performed. For cytotoxicity assay, the samples were rehydrated with complete culture medium. MTS and DNA quantification assays were performed to evaluate the metabolic activity and proliferation of L929 fibroblast cells after 1, 3 and 7 days in culture with GG, GG/PPy and PPy samples. MTS assays clearly indicate a proportional relation between the cell viability and the PPy concentration: higher concentrations of PPy resulted in lower cell viability. These results show that lower concentration of PPy incorporated in the GG hydrogels can provide an adequate electrical stimulus to improve cell behavior. In conclusion, semiconductor hydrogels can be an excellent platform for tissue engineering and electrochemical therapy application