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Maia F.R.,INEB Institute Engineering Biomedica | Maia F.R.,University of Porto | Bidarra S.J.,INEB Institute Engineering Biomedica | Granja P.L.,INEB Institute Engineering Biomedica | And 3 more authors.
Acta Biomaterialia | Year: 2013

Human mesenchymal stem cells (MSCs) are currently recognized as a powerful cell source for regenerative medicine, notably for their capacity to differentiate into multiple cell types. The combination of MSCs with biomaterials functionalized with instructive cues can be used as a strategy to direct specific lineage commitment, and can thus improve the therapeutic efficacy of these cells. In terms of biomaterial design, one common approach is the functionalization of materials with ligands capable of directly binding to cell receptors and trigger specific differentiation signaling pathways. Other strategies focus on the use of moieties that have an indirect effect, acting, for example, as sequesters of bioactive ligands present in the extracellular milieu that, in turn, will interact with cells. Compared with complex biomolecules, the use of simple compounds, such as chemical moieties and peptides, and other small molecules can be advantageous by leading to less expensive and easily tunable biomaterial formulations. This review describes different strategies that have been used to promote substrate-mediated guidance of osteogenic differentiation of immature osteoblasts, osteoprogenitors and MSCs, through chemically conjugated small moieties, both in two- and three-dimensional set-ups. In each case, the selected moiety, the coupling strategy and the main findings of the study were highlighted. The latest advances and future perspectives in the field are also discussed. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.


Gomes M.J.,INEB Institute Engineering Biomedica | Martins S.,University of Southern Denmark | Sarmento B.,INEB Institute Engineering Biomedica | Sarmento B.,Institute Investigacao e Formacao Avancada em Ciencias e Tecnologias da Saude
Ageing Research Reviews | Year: 2015

As the population ages, brain pathologies such as neurodegenerative diseases and brain cancer increase their incidence, being the need to find successful treatments of upmost importance. Drug delivery to the central nervous system (CNS) is required in order to reach diseases causes and treat them. However, biological barriers, mainly blood-brain barrier (BBB), are the key obstacles that prevent the effectiveness of possible treatments due to their ability to strongly limit the perfusion of compounds into the brain. Over the past decades, new approaches towards overcoming BBB and its efflux transporters had been proposed. One of these approaches here reviewed is through small interfering RNA (siRNA), which is capable to specifically target one gene and silence it in a post-transcriptional way. There are different possible functional proteins at the BBB, as the ones responsible for transport or just for its tightness, which could be a siRNA target. As important as the effective silence is the way to delivery siRNA to its anatomical site of action. This is where nanotechnology-based systems may help, by protecting siRNA circulation and providing cell/tissue-targeting and intracellular siRNA delivery. After an initial overview on incidence of brain diseases and basic features of the CNS, BBB and its efflux pumps, this review focuses on recent strategies to reach brain based on siRNA, and how to specifically target these approaches in order to treat brain diseases. © 2015 Elsevier B.V.


Nascimento A.V.,Institute Investigacao e Formacao Avancada em Ciencias e Tecnologias da Saude | Nascimento A.V.,University of Porto | Singh A.,Northeastern University | Bousbaa H.,Institute Investigacao e Formacao Avancada em Ciencias e Tecnologias da Saude | And 5 more authors.
Molecular Pharmaceutics | Year: 2014

RNA interference has emerged as a powerful strategy in cancer therapy because it allows silencing of specific genes associated with tumor progression and resistance. Mad2 is an essential mitotic checkpoint component required for accurate chromosome segregation during mitosis, and its complete abolition leads to cell death. We have developed an epidermal growth factor receptor (EGFR)-targeted chitosan system for silencing the Mad2 gene as a strategy to efficiently induce cell death in EGFR overexpressing human A549 non-small cell lung cancer cells. Control and EGFR-targeted chitosan nanoparticles loaded with small interfering RNAs (siRNAs) against Mad2 were formulated and characterized for size, charge, morphology, and encapsulation efficiency. Qualitative and quantitative intracellular uptake studies by confocal imaging and flow cytometry, respectively, showed time-dependent enhanced and selective intracellular internalization of EGFR-targeted nanoparticles compared to nontargeted system. Targeted nanoparticles showed nearly complete depletion of Mad2 expression in A549 cells contrasting with the partial depletion in the nontargeted system. Accordingly, Mad2-silencing-induced apoptotic cell death was confirmed by cytotoxicity assay and flow cytometry. Our results demonstrate that EGFR-targeted chitosan loaded with Mad2 siRNAs is a potent delivery system for selective killing of cancer cells. © 2014 American Chemical Society.


Bidarra S.J.,INEB Institute Engineering Biomedica | Bidarra S.J.,University of Porto | Barrias C.C.,University of Porto | Barbosa M.A.,INEB Institute Engineering Biomedica | And 6 more authors.
Stem Cell Research | Year: 2011

The purpose of this work was to investigate if a coculture system of human mesenchymal stem cells (hMSC) with endothelial cells (human umbilical vein endothelial cells, HUVEC) could modulate the phenotype and proliferation of harvested MSCs. In addition to previous investigations on the crosstalk between these two cell types, in the present work different relative cell ratios were analyzed for long, therapeutically relevant, culture periods. Moreover, MSCs osteogenic commitment was assessed in a non-osteogenic medium and in the presence of HUVECs through magnetic cell separation, cell quantification by flow cytometry, morphology by fluorescent microscopy, metabolic activity and gene expression of osteogenic markers. Collectively, the present findings demonstrate that, by coculturing MSCs with HUVECs, there was not only the promotion of osteogenic differentiation (and its enhancement, depending on the relative cell ratios used), but also a significant increase on MSCs proliferation. This augmentation in cell proliferation occurred independently of relative cell ratios, but was favored by higher relative amounts of HUVECs. Taken together, this data suggests that HUVECs not only modulate MSC phenotype but also their proliferation rate. Therefore, a coculture system of MSCs and HUVECs can a have a broad impact on bone tissue engineering approaches. © 2011 Elsevier B.V.


Das Neves J.,University of Porto | Das Neves J.,Instituto Superior Of Ciencias Da Saude Norte | Rocha C.M.R.,University of Porto | Goncalves M.P.,University of Porto | And 6 more authors.
Molecular Pharmaceutics | Year: 2012

The interaction with cervicovaginal mucus presents the potential to impact the performance of drug nanocarriers. These systems must migrate through this biological fluid in order to deliver their drug payload to the underlying mucosal surface. We studied the ability of dapivirine-loaded polycaprolactone (PCL)-based nanoparticles (NPs) to interact with a simulated vaginal fluid (SVF) incorporating mucin. Different surface modifiers were used to produce NPs with either negative (poloxamer 338 NF and sodium lauryl sulfate) or positive (cetyltrimethylammonium bromide) surface charge. Studies were performed using the mucin particle method, rheological measurements, and real-time multiple particle tracking. Results showed that SVF presented rheological properties similar to those of human cervicovaginal mucus. Analysis of NP transport indicated mild interactions with mucin and low adhesive potential. In general, negatively charged NPs underwent subdiffusive transport in SVF, i.e., hindered as compared to their diffusion in water, but faster than for positively charged NPs. These differences were increased when the pH of SVF was changed from 4.2 to 7.0. Diffusivity was 50- and 172-fold lower in SVF at pH 4.2 than in water for negatively charged and positively charged NPs, respectively. At pH 7.0, this decrease was around 20- and 385-fold, respectively. The estimated times required to cross a layer of SVF were equal to or lower than 1.7 h for negatively charged NPs, while for positively charged NPs these values were equal to or higher than 7 h. Overall, our results suggest that negatively charged PCL NPs may be suitable to be used as carriers in order to deliver dapivirine and potentially other antiretroviral drugs to the cervicovaginal mucosal lining. Also, they further reinforce the importance in characterizing the interactions of nanosystems with mucus fluids or surrogates when considering mucosal drug delivery. © 2012 American Chemical Society.


Bidarra S.J.,INEB Institute Engineering Biomedica | Bidarra S.J.,University of Porto | Barrias C.C.,INEB Institute Engineering Biomedica | Fonseca K.B.,INEB Institute Engineering Biomedica | And 6 more authors.
Biomaterials | Year: 2011

Cell-based therapies offer an attractive approach for revascularization and regeneration of tissues. However, and despite the pressing clinical needs for effective revascularization strategies, the successful immobilization of viable vascular cells within 3D matrices has been difficult to achieve. In this paper the in vitro potential of a natural, injectable RGD-alginate hydrogel as an in situ forming matrix to deliver endothelial cells was evaluated. Several techniques were employed to investigate how these microenvironments could influence the behavior of vascular cells, namely their ability to promote the outward migration of viable, proliferative cells, retaining the ability to form a 3D arrangement. Cells within RGD-grafted alginate hydrogel were able to proliferate and maintained 80% of viability for at least 48 h post-immobilization. Additionally, entrapped cells created a 3D organization into cellular networks and, when put in contact with matrigel, cells migrated out of the RGD-matrix. Overall, the obtained results support the idea that the RGD peptides conjugated to alginate provide a 3D environment for endothelial cells adhesion, survival, migration and organization. © 2011 Elsevier Ltd.


Bidarra S.J.,INEB Institute Engineering Biomedica | Bidarra S.J.,University of Porto | Barrias C.C.,INEB Institute Engineering Biomedica | Barbosa M.A.,INEB Institute Engineering Biomedica | And 4 more authors.
Biomacromolecules | Year: 2010

In this work, human mesenchymal stem cells (hMSC) immobilized in RGD-coupled alginate microspheres, with a binary composition of high and low molecular weight alginate, were investigated. Cells immobilized within RGD-alginate microspheres (during 21 days) showed metabolic activity, with an overall viability higher than 90%, short cell extensions, and, when induced, they were able to differentiate into the osteogenic lineage. In osteogenic conditions (comparing to basal conditions), immobilized cells presented alkaline phosphatase (ALP) activity and an upregulation of ALP, collagen type I, and Runx 2 expression. Moreover, mineralization was also detected in immobilized cells under osteogenic stimulus. In addition, it was demonstrated for the first time that MSCs immobilized in this 3D matrix were able to enhance the ability of neighboring endothelial cells to form tubelike structures. Overall, these findings represent a step forward in the development of injectable stem cell carriers for bone tissue engineering. © 2010 American Chemical Society.


Debasu M.L.,University of Aveiro | Ananias D.,University of Aveiro | Pinho S.L.C.,INEB Institute Engineering Biomedica | Geraldes C.F.G.C.,University of Coimbra | And 2 more authors.
Nanoscale | Year: 2012

Up-conversion (Gd,Yb,Tb)PO 4 materials and their potential for bimodal imaging have received little attention in the literature. Herein, we report the first study on the up-conversion emission of (Gd,Yb,Tb)PO 4 nanocrystals synthesized via a hydrothermal method at 150°C. These materials exhibit ultraviolet, blue and green up-conversion emissions upon excitation with a 980 nm continuous wave laser diode. The intensity of the blue-emission band at 479 nm, ascribed to the cooperative up-conversion emission of a pair of excited Yb 3+ ions, depends on the Yb 3+/Tb 3+ concentration ratio, calcination temperature and particle size. Strong green up-conversion emission of Tb 3+ is observed at 543 nm for the 5D 4 → 7F 5 transition. Relaxometry measurements reveal that the nanocrystals are efficient T 2-weighted (negative) contrast agents which, combined with visible-light emission generated by infrared excitation, affords them considerable potential for being used in bimodal, photoluminescence-magnetic resonance, imaging. © 2012 The Royal Society of Chemistry.


Almeida C.R.,INEB Institute Engineering Biomedica | Vasconcelos D.P.,INEB Institute Engineering Biomedica | Vasconcelos D.P.,Abel Salazar Biomedical Sciences Institute | Goncalves R.M.,INEB Institute Engineering Biomedica | And 2 more authors.
Journal of the Royal Society Interface | Year: 2012

An exacerbated inflammatory response questions biomaterial biocompatibility, but on the other hand, inflammation has a central role in the regulation of tissue regeneration. Therefore, it may be argued that an 'ideal' inflammatory response is crucial to achieve efficient tissue repair/regeneration. Natural killer (NK) cells, being one of the first populations arriving at an injury site, can have an important role in regulating bone repair/regeneration, particularly through interactions with mesenchymal stem/stromal cells (MSCs). Here, we studied how biomaterials designed to incorporate inflammatory signals affected NK cell behaviour and NK cell-MSC interactions. Adsorption of the pro-inflammatory molecule fibrinogen (Fg) to chitosan films led to a 1.5-fold increase in adhesion of peripheral blood human NK cells, without an increase in cytokine secretion. Most importantly, it was found that NK cells are capable of stimulating a threefold increase in human bone marrow MSC invasion, a key event taking place in tissue repair, but did not affect the expression of the differentiation marker alkaline phosphatase (ALP). Of significant importance, this NK cell-mediated MSC recruitment was modulated by Fg adsorption. Designing novel biomaterials leading to rational modulation of the inflammatory response is proposed as an alternative to current bone regeneration strategies. © 2011 The Royal Society.


Rodrigues F.,University of Porto | Almeida I.,University of Porto | Sarmento B.,IINFACTS Institute Investigacao e Formacao Avancada em Ciencias e Tecnologias da Saude | Sarmento B.,INEB Institute Engineering Biomedica | And 2 more authors.
Industrial Crops and Products | Year: 2014

This study focuses on the isoflavones composition of different extracts of Medicago species (M. minima, M. tornata, M. truncatula, M. rigidula, M. scutelata, M. segitalis and M. sativa), a leguminous crop used as animal feeding. There are some research studies on the isoflavones composition of Medicago sativa, but no comprehensive profile of isoflavones has been reported for these other six species. In this work, two glycosides (daidzin and genistin) and six aglycones (daidzein, glycitein, genistein, formononetin, prunetin and biochanin A) were determined by high performance liquid chromatography (HPLC/DAD) in three different extracts of these species: aqueous, hydroalcoholic and alcoholic. In all samples genistin, daidzein and genistein were the most abundant isoflavone compounds. The highest concentration of genistin was observed in the extracts of M. scutelata (5.2 and 9.6. mg/kg. db, respectively, for hydroalcoholic and alcoholic extract), in amounts 2-4.5-fold higher than the other samples evaluated. Daidzein is also present in high amounts in M. scutelata (19.2. mg/kg. db, alcoholic extract). Genistein is detected in several species at concentrations ranging from 0.7 (aqueous extract of M. tornata) to 16.1. mg/kg. db (alcoholic extract of M. scutelata). In terms of total isoflavones content per species, results showed significant differences between M. rigidula and M. sativa, considering all type of solvents, being the highest and the lowest one, respectively. In a general way, the extractability of compounds significantly increased from aqueous to alcoholic extracts. The study demonstrates that some Medicago species possess an excellent potential as a source of these health promoting bioactive compounds. © 2014 Elsevier B.V.

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