Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate

Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet Lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P>5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL.Portuguese Foundation for Science and Technology is gratefully acknowledged for fellowships of Sara M. Oliveira. (SFRH/BD/70107/2010).r The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS and FP7-KBBE-2010-4-266033 - SPECIAL. This work was also supported by the European Research Council grant agreement ERC-2012-ADG 20120216-321266 for the project ComplexiTE. The authors acknowledge Rogerio P. Pirraco for the Flow cytometry analysis

Natural assembly of platelet lysate-loaded nanocarriers into enriched 3D hydrogels for cartilage regeneration

The role of Platelet Lysates (PLs) as a source of growth factors (GFs) and as main element of threedimensional (3D) hydrogels has been previously described. However, the resulting hydrogels usually suffer from high degree of contraction, limiting their usefulness. This work describes the development of a stable biomimetic 3D hydrogel structure based on PLs, through the spontaneous assembling of a high concentration of chitosan-chondroitin sulfate nanoparticles (CH/CS NPs) with PLs loaded by adsorption. The interactions between the NPs and the lysates resemble the ones observed in the extracellular matrix (ECM) native environment between glycosaminoglycans and ECM proteins. In vitro release studies were carried out focusing on the quantification of PDGF-BB and TGF-b1 GFs. Human adipose derived stem cells (hASCs) were entrapped in these 3D hydrogels and cultured in vitro under chondrogenic stimulus, in order to assess their potential use for cartilage regeneration. Histological, immunohistological and gene expression analysis demonstrated that the PL-assembled constructs entrapping hASCs exhibited results similar to the positive control (hASCS cultured in pellets), concerning the levels of collagen II expression and immunolocalization of collagen type I and II and aggrecan. Moreover, the deposition of new cartilage ECM was detected by alcian blue and safranin-O positive stainings. This work demonstrates the potential of PLs to act simultaneously as a source/carrier of GFs and as a 3D structure of support, through the application of a â â bottom-upâ â approach involving the assembly of NPs, resulting in an enriched construct for cartilage regeneration applications.The authors thank Fundacao para a Ciencia e Tecnologia for V.E. Santo and E.G. Popa's PhD grants (SFRH/BD/39486/2007 and SFRH/BD/64070/2009, respectively). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and it was partially supported by the European FP7 Project Find and Bind (NMP4-SL-2009-229292). We thank IPS for providing the human platelet donations and Hospital da Prelada for the human adipose tissue samples

Hybrid 3D structure of poly(d,l-lactic acid) loaded with chitosan/chondroitin sulfate nanoparticles to be used as carriers for biomacromolecules in tissue engineering

In the tissue engineering (TE) field, the concept of producing multifunctional scaffolds, capable not only of acting as templates for cell transplantation but also of delivering bioactive agents in a controlled manner, is an emerging strategy aimed to enhance tissue regeneration. In this work, a complex hybrid release system consisting in a three-dimensional (3D) structure based on poly(d,l-lactic acid) (PDLLA) impregnated with chitosan/chondroitin sulfate nanoparticles (NPs) was developed. The scaffolds were prepared by supercritical fluid foaming at 200 bar and 35 "C, and were then characterized by scanning electron microscopy (SEM) and micro-CT. SEM also allowed to assess the distribution of the NPs within the structure, showing that the particles could be found in different areas of the scaffold, indicating a homogeneous distribution within the 3D structure. Water uptake and weight loss measurements were also carried out and the results obtained demonstrated that weight loss was not significantly enhanced although the entrapment of the NPs in the 3D structure clearly enhances the swelling of the structure. Moreover, the hybrid porous biomaterial displayed adequate mechanical properties for cell adhesion and support. The possibility of using this scaffold as a multifunctional material was further evaluated by the incorporation of a model protein, bovine serum albumin (BSA), either directly into the PDLLA foam or in the NPs that were eventually included in the scaffold. The obtained results show that it is possible to achieve different release kinetics, suggesting that this system is a promising candidate for dual protein delivery system for TE applications

3D tumor models with defined cellular and physico- chemical components: Impact of recapitulative tumor microenvironment on disease progression

The high attrition rates observed in cancer drug discovery (up to 95% of failure of drugs tested in phase I trials) have raised the awareness of the scientific and industrial communities towards the need for more predictive pre-clinical models. These models should be more representative of the disease and consequently help to eliminate at pre-clinical stages drug candidates that lack efficacy or safety. Tumor microenvironment is composed by a network of fibroblasts, endothelial cells, immune-competent cells within the extracellular matrix (ECM). Interactions between these components are critical for tumor initiation, proliferation, migration and metastasis. The design of in vitro cancer cell models that recapitulate the tumor microenvironment and 3D architecture provides higher physiological relevance as they more closely resemble the in vivo cellular context. We have established methodologies for scalable generation of 3D cancer cell models in stirred-tank culture systems, and applied these to a large panel of tumor cell lines from different pathologies, including breast, colon, hepatic and lung tumor cell lines. Large numbers of spheroids were obtained per culture (typically 1000-1500 spheroids/mL) with representative characteristics of native tumors, such as morphology, proliferation and hypoxia gradients, in a cell-line dependent mode. With the aim of increasing the relevance of spheroids as tumor cell models, several aspects of tumor microenvironment were incorporated, such as the presence of stromal cells (fibroblasts and monocytes) and specific physico-chemical parameters, namely oxygen levels. Heterotypic 3D breast and Non-Small Cell Lung Carcinoma (NSCLC) cancer models, based on co-cultures of tumor cells with stromal cells were established by using an alginate matrix to provide physical support to cells. Tumor spheroids were microencapsulated alone or with fibroblasts and monocytes, thus allowing the establishment of an epithelial tumor compartment and a stromal compartment of increasing complexity. Cultures were performed in stirred-tank vessels for 15 days with continuous monitoring. In both breast and lung tumor models, the presence of fibroblasts was associated with secretion of pro-inflammatory cytokines and accumulation of collagen in the microcapsules. Long-term culture (up to 15 days) resulted in phenotypic alterations in co-cultured breast tumor spheroids, such as loss of cell polarity, reduced cell-cell adhesions, collective cell migration and increased angiogenic potential. In contrast, the effects of fibroblasts were not as significant in NSCLC co-cultures using H1650, H1437 and H157 cell lines suggesting that the effect of tumor-stroma cross-talk is cell line dependent. Moreover, these models have also been shown as feasible tools for drug screening by assessing the effect of chemotherapeutic and specific inhibitors compounds on mono- and co-cultures. In conclusion, we have developed scalable, robust and versatile methodologies for the generation and culture of 3D cancer models, enabling long-term in vitro recapitulation of tumor-stroma crosstalk, via reconstruction of key aspects of the tumor microenvironment, allowing continuous monitoring of disease progression events in vitro. In addition, it is easily transferable to industry for feeding high-throughput systems or miniaturized bioreactors used in drug development, target validation and target identification

Cell engineering by the internalization of bioinstructive micelles for enhanced bone regeneration

To direct precursor cells toward the osteoblastic lineage, by using an intracellular nanocarrier releasing dexamethasone. Materials & methods: Biodegradable gelatinbased micelles entrapped dexamethasone (dex-micelles). Internalization efficiency and biocompatibility of dex-micelles and their potency for in vitro osteogenic differentiation and in vivo bone regeneration were assessed. Results: Dex-micelles were internalized by rat bone marrow mesenchymal stem cells and demonstrated a pH-responsive release profile and an enhancement of 2D and 3D in vitro osteogenic differentiation. In vivo implantation of gelatin scaffolds seeded with rat bone marrow mesenchymal stem cells precultured for 24 h with dex-micelles promoted a significant enhancement of de novo bone formation in a rat ulna defect, in a dose-dependent manner. Conclusion: The proposed intracellular delivery system is a powerful tool to promote bone regeneration.The authors thank Fundacao para a Ciencia e Tecnologia and Japan Society for the Promotion of Science (JSPS) for VE Santo's PhD grant (SFRH/BD/39486/2007) and J Ratanavaraporn's post-doc fellowship, respectively. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

Development of new chitosan/carrageenan nanoparticles for drug delivery applications

The use of polymeric nanoparticles, especially those composed of natural polymers, has become a very interesting approach in drug delivery., mainly because of the advantages offered by their small dimensions. The aim of this work was to develop a novel formulation of nanoparticles comprised of two natural marine-derived polymers, namely chitosan and carrageenan, and to evaluate their potential for the association and controlled release of macromolecules. Nanoparticles were obtained in a hydrophilic environment, under very mild conditions, avoiding the use of organic solvents or other aggressive technologies for their preparation. The developed nanocarriers presented sizes within 350-650 run and positive zeta potentials of 50-60 mV. Polymeric interactions between nanoparticles' components were evaluated by Fourier transform infrared spectroscopy. Using ovalbumin as model protein, nanoparticles evidenced loading capacity varying from 4% to 17%, and demonstrated excellent capacity to provide a controlled release for up to 3 weeks. Furthermore, nanoparticles have demonstrated to exhibit a noncytotoxic behavior in biological in vitro tests performed using L929 fibroblasts, which is critical regarding the biocompatibility of those carriers. In summary, the developed chitosan-carrageenan nanoparticles have shown promising properties to be used as carriers of therapeutic macromolecules, with potential application not only strictly in drug delivery, but also in broader areas, such as tissue engineering and regenerative medicine. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 92A: 1265-1272, 2010Contract grant sponsor: Portuguese Foundation for Science and Technology (FCT) (POCTI/FEDER programmes)Contract grant sponsor: European Union STREP Project HIPPOCRATES; contract grant number: NMP3-CT-2003505758Contract grant sponsor: European NoE EXPERTISSUES; contract grant number: NMP3-CT-2004-50028

II Encontro Internacional de Língua Portuguesa e Relações Lusófonas: livro de resumos

O presente livro contém os resumos das comunicações (orais e em poster) enviados ao II Encontro Internacional de Língua Portuguesa e Relações Lusófonas (LUSOCONF2019), organizado pelo Instituto Politécnico de Bragança, através da sua Escola Superior de Educação de Bragança e em parceria com a Câmara Municipal de Bragança, nos dias 17, 18 e 19 de outubro de 2019. O LUSOCONF2019 continua a ser um espaço de discussão sobre a Língua Portuguesa no mundo e acerca de múltiplas problemáticas relevantes no âmbito dos espaços e relações lusófonos. Valorizando um olhar plural, o LUSOCONF apresenta-se e quer construir-se como um Encontro Internacional aberto a uma multiplicidade de intervenções (como se evidencia nos eixos temáticos propostos). A amplitude temática que carateriza o LUSOCONF está atestada nos seus eixos temáticos, sendo que, este ano, se acrescenta a abordagem a temas relativos à área da saúde. O leque de conferencistas convidados assegura a este encontro internacional uma diversidade de perspetivas, celebrando a multiculturalidade que nos enriquece e afirmando a vontade de vivermos em comum, ancorados no desejo de (nos) conhecer(mos) e no espírito de colaboração, como condições para um mundo mais humano. Esta visão holística do mundo lusófono e seus desafios leva-nos a reiterar a afirmação do LUSOCONF como um espaço desenhado para a partilha e a interação, pelo que voltamos a lançar um Call for arts, favorecendo a divulgação e a edição de trabalhos artísticos sobre o mundo lusófono. Entre outras reflexões, como a do papel da CPLP nas políticas relativas ao desenvolvimento sustentável e na nova configuração geopolítica do mundo, o LUSOCONF2019 propõe um debate acerca do valor internacional da língua portuguesa. Recorde-se que, de acordo com Simons e Fennig (2018), em Ethnologue: Languages of the World (21.ª edição, versão online: http://www.ethnologue.com), a Língua Portuguesa é atualmente falada por 236 512 000 indivíduos, sendo língua materna para 222 708 500 e língua segunda para 13 803 500 indivíduos. Como é claramente evidenciado na Mesa Redonda, o LUSOCONF assume como relevante o diálogo entre o discurso académico e o mundo dos negócios numa lógica de valorização do empreendedorismo, ético e responsável tanto em termos sociais como ambientais. Pretendemos, pois, um LUSOCONF2019 que, colocando o mundo lusófono numa indelével abertura ao multiculturalismo, reflita com profunda seriedade sobre a diversidade dos problemas humanos e naturais, fortalecendo a esperança de que o projeto lusófono, mais do que uma miragem (para usarmos um termo lourenciano), se consolide como efetiva rede – porisso sem centro nem periferias – em interação com a rede global. Valorizando a confraternidade que nos é conferida, antes de mais, pela partilha da língua portuguesa, tem de nos unir o reconhecimento e a clara aceitação de que somos legitimamente diferentes. Os investigadores da língua portuguesa e os que se debruçam sobre os problemas referentes ao seu ensino, bem como todos aqueles que se dedicam a áreas de relevância no âmbito das relações lusófonas, como: educação, cultura, literatura, artes, história, geografia, política, direito, economia, gestão, marketing, contabilidade, agricultura, turismo, ambiente, saúde e desenvolvimento sustentável, foram convidados a submeter trabalhos de reflexão e de investigação ao LUSOCONF2019. A diversidade e a qualidade de propostas apresentadas para os vários eixos temáticos constituem evidência da abrangência deste Encontro Internacional no qual se cruzam múltiplos olhares no mesmo desígnio de pensar, com rigor e profundidade, a lusofonia e os seus problemas, bem como as oportunidades de incremento das relações lusófonas. A finalizar esta breve nota de apresentação, a Comissão Organizadora agradece aos conferencistas convidados e a todos os investigadores que participaram no LUSOCONF2019.info:eu-repo/semantics/publishedVersio

Unleashing the potential of supercritical fluids for polymer processing in tissue engineering and regenerative medicine

One of the major scientific challenges that tissue engineering and regenerative medicine (TERM) faces to move from benchtop to bedside regards biomaterials development, despite the latest advances in polymer processing technologies. A variety of scaffolds processing techniques have been developed and include solvent casting and particles leaching, compression molding and particle leaching, thermally induced phase separation, rapid prototyping, among others. Supercritical fluids appear as an interesting alternative to the conventional methods for processing biopolymers as they do not require the use of large amounts of organic solvents and the processes can be conducted at mild temperatures. However, this processing technique has only recently started to receive more attention from researchers. Different processing methods based on the use of supercritical carbon dioxide have been proposed for the creation of novel architectures based on natural and synthetic polymers and these will be unleashed in this paper.The research leading to these results has recieved funding from the European Union Seventh Framework Programme (FP7) under grant agreement no. KBBE-2010-266033 (project SPECIAL), no. NMP4-SL-2009-229292 (project Find & Bind), from FEDER through POCTEP Project 0330_IBEROMARE_1_P, from the Sixth Framework Programme (FP6) under grant agreement NMP3-CT-2004-500283 (project EXPERTISSUES), Portuguese Foundation fo Science and Technology (FCT) is also acknowledged. for PhD and Post-Doc fellowships of Ana Rits C. Duarte, Vitor E. Santo, Anabela Alves, Simone S. Silva, Joana Moreira-Silva and Tiage H. Silva.Ana Rita C. Duarte would like to acknowledgs Fundacao Luso-Americana para o Desenvolvimento for the travel grant awarded to present this work at the international Symposium of Supercritical Fluids - ISSF 2012

II International Conference on Portuguese Language and Lusophone Relations: book of proceedings

O Lusoconf é um Encontro Internacional que tem a língua portuguesa como ponto charneira de múltiplas reflexões acerca do mundo lusófono. Este mundo, dentro de um mundo que a pós-modernidade vai desenhando como uma estrutura reticular, é inevitável e intrincadamente complexo com locais/momentos de turbulência e outros de silêncio. “O silêncio é que fabrica as janelas por onde o mundo se transparenta” – escreveu Mia Couto. Não noa atrevemos a contradizê-lo. Porém, temos de reconhecer a necessidade de uso da palavra e da sua partilha. A necessidade desse mergulho numa outra transparência que é a das palavras. Os textos que se reúnem neste volume constituem uma partilha do nosso saber acerca da língua portuguesa e acerca de múltiplos problemas que se levantam no âmbito das relações lusófonas. Os desafios colocados aos países de língua oficial portuguesa são grandes. A mobilidade de pessoas e particularmente de estudantes é um dos que têm relevância para as instituições de Ensino Superior. Na verdade, a relação entre estes povos tem de ser considerada na sua multiplicidade e, se queremos construir-nos como uma verdadeira comunidade de povos, temos de valorizar a língua portuguesa como um ativo comum e temos, igualmente, de reconhecer a necessidade de erguer outras pontes para além da identidade linguística (em muitos casos bem pouco firme e firmada). E, já agora, convém clarificar que a questão das relações lusófonas não se restringe aos países da CPLP, na medida em que há, em todos os continentes, comunidades de falantes de língua portuguesa, para as quais é necessário consertar um conjunto de ações, sempre inócuas se faltar o investimento no ensino do Português e uma sustentada estratégia de valorização da língua. Este é um desafio, tal como o é, por exemplo, a educação para o desenvolvimento sustentável. Estes são alguns dos tópicos que se desenvolvem ao longo destes textos. Os desafios são grandes e pode-nos parecer que a seara é sáfara. Importa, porém, ter a força, o engenho e a arte, para transformar dificuldades em oportunidade e para encarar a dissensão como oportunidade de diálogo e de conhecimento, de entendimento e de compromisso. O caminho para a multiculturalidade, responsável, livre, plena e eticamente assumida, é um dos nossos maiores desafios cuja concretização depende da proatividade de todos nós. Apesar das diversas e seguramente justas motivações, há um desígnio comum a unir-nos neste Encontro: o de lutarmos por um mundo de igualdade de oportunidades para que nenhum ser humano sinta culpa do nascer. Não podemos ser indiferentes aos sinais de alastramento de ideologias extremistas que fazem renascer movimentos nazis ou de extrema-esquerda. Estes são movimentos que vão muito para além da questão política – a qual não pode ser menosprezada – por serem fenómenos sociais e culturais de grande amplitude. Nós, que apaixonadamente procuramos o conhecimento e a compreensão do outro que é diferente, temos de ser forças de promoção da vivência democrática e de defesa de um verdadeiro humanismo universalista. Nesta linha, o Lusoconf tem, evidentemente, as suas especificidades. Em primeiro lugar, porque reúne várias áreas de investigação entre as quais o diálogo é tão raro (num mundo onde se supervalorizam as especializações). A leitura dos trabalhos aqui reunidos é prova deste encontro. A especificidade do Lusoconf passa também pelo conseguir trazer até nós representantes institucionais de outros países lusófonos, fortalecendo laços e reavivando a vontade de fazermos mais e melhor. O Lusoconf é ainda singular porque a sua organização, além de envolver as cinco escolas do IPB, é feita em colaboração com a Câmara Municipal de Bragança. Esta é uma parceria que queremos sustentar e ver crescer. Em Traço de União, Miguel Troga escreveu que “O universal é o local sem paredes”. É assim que apresentamos o Lusoconf. E, por falarmos em poetas, recordamos um excerto de um poema do escritor angolano João Melo que, em “Crónica verdadeira da língua portuguesa”, a apresenta assim: suave e discreta, debruçada sobre a varanda do tempo, o olhar estendendo-se com o mar e a memória, deliciando-se comovida com o sol despudorado ardendo nas vogais abertas da língua, violentando com doçura os surdos limites das consoantes e ampliando-os para lá da História. Não sabemos o que nos oferecerá a História. Afinal, os humanos nunca foram bons a prever o futuro. Parafraseando as palavras que o Sr. diretor da ESE escreveu para a página do Encontro, esperamos que o LUSOCONF possa prosseguir a aventura do conhecimento, compreendendo a diferença e a semelhança de ser-se humano, neste mundo que é de todos. A finalizar esta breve nota de apresentação, a Comissão Organizadora agradece a todos os que tornaram possível o LUSOCONF2019 e, em particular, aos conferencistas convidados e a todos os investigadores que nele participaram.info:eu-repo/semantics/publishedVersio