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

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

Marine algae sulfated polysaccharides for tissue engineering and drug delivery approaches

Biomedical field is constantly requesting for new biomaterials, with innovative properties. Natural polymers appear as materials of election for this goal due to their biocompatibility and biodegradability. In particular, materials found in marine environment are of great interest since the chemical and biological diversity found in this environment is almost uncountable and continuously growing with the research in deeper waters. Moreover, there is also a slower risk of these materials to pose illnesses to humans. In particular, sulfated polysaccharides can be found in marine environment, in different algae species. These polysaccharides don’t have equivalent in the terrestrial plants and resembles the chemical and biological properties of mammalian glycosaminoglycans. In this perspective, are receiving growing interest for application on health-related fields. On this review, we will focus on the biomedical applications of marine algae sulfated polymers, in particular on the development of innovative systems for tissue engineering and drug delivery approaches.European Regional Development Fund (ERDF)Fundação para a Ciência e a Tecnologia (FCT

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

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

Materials of marine origin: a review on polymers and ceramics of biomedical interest

Marine organisms are constituted by materials with a vast range of properties and characteristics that may justify their potential application within the biomedical field. Moreover, assuring the sustainable exploitation of natural marine resources, the valorisation of residues from marine origin, like those obtained from food processing, constitutes a highly interesting platform for development of novel biomaterials, with both economic and environmental benefits. In this perspective, an increasing number of different types of compounds are being isolated from aquatic organisms and transformed into profitable products for health applications, including controlled drug delivery and tissue engineering devices. This report reviews the work that is being developed on the isolation and characterisation of some polysaccharides, proteins, glycosaminoglycans and ceramics from marine raw materials. Emphasis is given to agar, alginates, carrageenans, chitin and chitosan, among other polysaccharides, collagen, glycosaminoglycans such as chondroitin sulphate, heparin and hyaluronic acid, calcium phosphorous compounds and biosilica. Finally, this report ends by reviewing the application of the previously mentioned materials on specific biomedical applications, in particular their participation on the development of controlled drug delivery systems and tissue engineering scaffolds.European Fund for Regional Development (EFRD)Fundação para a Ciência e a Tecnologia (FCT

Revealing the potential of squid chitosan-based structures for biomedical applications

In recent years, much attention has been given to different marine organisms, namely as potential sources of valuable materials with a vast range of properties and characteristics. In this work, β-chitin was isolated from the endoskeleton of the giant squid Dosidicus gigas and further deacetylated to produce chitosan. Then, the squid chitosan was processed into membranes and scaffolds using solvent casting and freeze-drying, respectively, to assess their potential biomedical application. The developed membranes have shown to be stiffer and less hydrophobic than those obtained with commercial chitosan. On the other hand, the morphological characterization of the developed scaffolds, by SEM and micro-computed tomography, revealed that the matrices were formed with a lamellar structure. The findings also indicated that the treatment with ethanol prior to neutralization with sodium hydroxide caused the formation of larger pores and loss of some lamellar features. The in vitro cell culture study has shown that all chitosan scaffolds exhibited a non-cytotoxic effect over the mouse fibroblast-like cell line, L929 cells. Thus, chitosan produced from the endoskeletons of the giant squid Dosidicus gigas has proven to be a valuable alternative to existing commercial materials when considering its use as biomaterial.This work was partially funded by FEDER through INTERREG III A Project Proteus and POCTEP Project IBEROMARE. The Portuguese Foundation for Science and Technology is gratefully acknowledged for post-doctoral grants of THS, JMO and SSS. The authors would also like to acknowledge to Dr Julio Maroto from the Fundacion CETMAR and Roi Vilela from PESCANOVA S.A, Spain, for the kind offer of squid pens and to Dr Ramon Novoa, Professor Ricardo Riguera and Professor Mariana Landin from the University of Santiago of Compostela for the SEC-MALLS measurements

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

The use of ionic liquids in the processing of chitosan/silk hydrogels for biomedical applications

Natural polymers are adequate renewable resources for the processability of well-defined architectures for several applications. Combinations of polysaccharides and proteins may mimic the naturally occurring environment of certain tissues. The main goal of this work renders the application of green chemistry principles, namely the use of ionic liquids (ILs) and biorenewable sources, such as chitosan (CHT) and silkfibroin (SF), to process new hydrogel-based constructs. Although the solubilization of both materials in ILs has been studied individually, this work reports, for the first time, the role of ILs as solvent, for the production of hydrogels from blends of chitosan and silkfibroin (CSF). These systems offer the advantage of being homogeneous and presenting easy and short dissolution time of both biomacromolecules. Moreover, the use of chitosan obtained fromα- andβ-chitin allowed the production of blended hydrogels with distinct physical–chemical properties.In vitroassays demonstrated that these hydrogels supported the adhesion and growth of primary human dermalfibroblasts. Taken these properties together, the CSF hydrogels might be promising biomaterials to be explored for skin tissue engineering approaches.Fundação para a Ciência e a Tecnologia FCT - SFRH/BPD/45307/2008, SFRH/BPD/ 34704/2007, SFRH/BD/64601/2009, PTDC/QUI/68804/2006FEDER - POCTEP 0330_IBEROMARE_1_P

Investigation on the role of red fox in tuberculosis maintenance community ¿ second opus: experimental infection with a virulent field Mycobacterium bovis strain

Trabajo presentado al: 69th Wildlife Disease Association and 14th European Wildlife Disease Association Conference. Cuenca, Spain. p. 135. 31 agosto-2 septiembre