PT Government Associated Laboratory

Braga, Portugal

PT Government Associated Laboratory

Braga, Portugal
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Costa R.R.,University of Minho | Costa R.R.,PT Government Associated Laboratory | Alatorre-Meda M.,University of Minho | Alatorre-Meda M.,PT Government Associated Laboratory | And 3 more authors.
Biotechnology Advances | Year: 2015

The pharmaceutical industry has been able to tackle the emergence of new microorganisms and diseases by synthesizing new specialized drugs to counter them. Their administration must ensure that a drug is effectively encapsulated and protected until it reaches its target, and that it is released in a controlled way. Herein, the potential of layer-by-layer (LbL) structures to act as drug reservoirs is presented with an emphasis to "nano"-devices of various geometries, from planar coatings to fibers and capsules. The inherent versatile nature of this technique allows producing carriers resorting to distinct classes of materials, variable geometry and customized release profiles that fit within adequate criteria required for disease treatment or for novel applications in the tissue engineering field. The production methods of LbL reservoirs are varied and allow for different kinds of molecules to be incorporated, such as antibiotics, growth factors and biosensing substances, not limited to water-soluble molecules but including hydrophobic drugs. We will also debate the future of LbL in the pharmaceutical industry. Currently, multilayered structures are yet to be covered by the regulatory guidelines that govern the fabrication of nanotechnology products. However, as they stand now, LbL nanodevices have already shown usefulness for antifouling applications, gene therapy, nanovaccines and the formation of de novo tissues. © 2015 Elsevier Inc.


Custodio C.A.,University of Minho | Custodio C.A.,PT Government Associated Laboratory | Santo V.E.,University of Minho | Santo V.E.,PT Government Associated Laboratory | And 8 more authors.
Advanced Functional Materials | Year: 2014

The development of biologically instructive biomaterials with application for tissue regeneration has become the focus of intense research over the last years. This work reports a novel approach for the production of three-dimensional constructs for tissue engineering applications based on the assembly of chitosan microparticles exhibiting specific biological response with cells. Chitosan microparticles with a size range between 20 and 70 μm are functionalized with platelet derived growth factor (PDFG-BB). The functionalization is achieved by previous immobilization of an anti-PDGF-BB antibody, using a water-soluble carbodiimide. When incubated with a cocktail of growth factors-platelet lysates, the previously functionalized particles are able to target PDGF-BB from the protein mixture. In vitro studies are carried out focusing on the ability of these systems to promote the assembly into a stable 3D construct triggered by the presence of human adipose stem cells, which act as crosslinker agents and induce the formation of a hydrogel network. The presence of immobilized growth factors gives to this system a biological functionality towards control on cell function. It is also bioresponsive, as cells drive the assembly process of the microgel. These versatile biomimetic microgels may provide a powerful tool to be used as an injectable system for non-invasive tissue engineering applications with additional control over cellular function by creating specific microenvironments for cell growth. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Custodio C.A.,University of Minho | Custodio C.A.,PT Government Associated Laboratory | Reis R.L.,University of Minho | Reis R.L.,PT Government Associated Laboratory | And 2 more authors.
Advanced Healthcare Materials | Year: 2014

Engineered cell instructive microenvironments with the ability to stimulate specific cellular responses are a topic of high interest in the fabrication and development of biomaterials for application in tissue engineering. Cells are inherently sensitive to the in vivo microenvironment that is often designed as the cell "niche." The cell "niche" comprising the extracellular matrix and adjacent cells, influences not only cell architecture and mechanics, but also cell polarity and function. Extensive research has been performed to establish new tools to fabricate biomimetic advanced materials for tissue engineering that incorporate structural, mechanical, and biochemical signals that interact with cells in a controlled manner and to recapitulate the in vivo dynamic microenvironment. Bioactive tunable microenvironments using micro and nanofabrication have been successfully developed and proven to be extremely powerful to control intracellular signaling and cell function. This Review is focused in the assortment of biochemical signals that have been explored to fabricate bioactive cell microenvironments and the main technologies and chemical strategies to encode them in engineered biomaterials with biological information. This Review is focused on the assortment of biochemical signals that have been explored to fabricate bioactive cell microenvironments. The main receptor molecules that the cell has available on its surface that interacts with external ligands are also considered. Existing tools to pattern and functionalize cell microenvironments with active biologic motifs are described in the last part. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Costa R.R.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Costa R.R.,PT Government Associated Laboratory | Testera A.M.,University of Valladolid | Testera A.M.,CIBER ISCIII | And 6 more authors.
Journal of Physical Chemistry B | Year: 2013

Nanostructured films consisting of polysaccharides and elastin-like recombinamers (ELRs) are fabricated in a layer-by-layer manner. A quartz-crystal microbalance with dissipation monitoring (QCM-D) is used to follow the buildup of hybrid films containing one polysaccharide (chitosan or alginate) and one of several ELRs that differ in terms of amino acid content, length, and biofunctionality in situ at pH 4.0 and pH 5.5. The charge density of the ingredients at each pH is determined by measuring their ζ-potential, and the thickness of a total of 36 different films containing five bilayers is estimated using the Voigt-based viscoelastic model. A comparison of the values obtained reveals that thicker films can be obtained when working at a pH close to the acidity constant of the polysaccharide used (near-pKa conditions), suggesting that the construction of such films is more favorable when based on the presence of hydrophobic interactions between ELRs and partially neutralized polysaccharides. Further analysis shows that the molecular weight of the ELRs plays only a minor role in defining the growth tendency. When taken together, these results point to the most favorable conditions for constructing nanostructured films from natural and distinct recombinant polypeptides that can be tuned to exhibit specialized biofunctionality for tissue-engineering, drug-delivery, and biotechnological applications. © 2013 American Chemical Society.


Santo V.E.,University of Minho | Santo V.E.,PT Government Associated Laboratory | Rodrigues M.T.,University of Minho | Rodrigues M.T.,PT Government Associated Laboratory | And 2 more authors.
Expert Review of Molecular Diagnostics | Year: 2013

The current limitations of regenerative medicine strategies may be overcome through the use of magnetic nanoparticles (MNPs), a class of nanomaterial typically composed of magnetic elements that can be manipulated under the influence of an external magnetic field. Cell engineering approaches following the internalization of these MNPs by cells and/or the incorporation of these nanosystems within 3D constructs (scaffolds or hydrogels) may constitute a new attractive approach to achieve a magnetically responsive system enabling remote control over tissue-engineered constructs in an in vivo scenario. Moreover, the incorporation of bioactive factors within these MNPs also enables a targeted and smart delivery of biomolecules to specific regions and/or triggering specific cell responses upon external magnetic stimulation. Certainly, one of the most attractive properties of MNPs is their ability to be used as theranostic tools for regenerative medicine applications, enabling live monitoring and tracking of the system while simultaneously acting as a therapeutic stimulation. © 2013 Informa UK Ltd.


Pereira P.,University of Aveiro | Pereira P.,IPMA Portuguese Institute for the Sea and Atmosphere | Pereira P.,University of Minho | Pereira P.,PT Government Associated Laboratory | And 5 more authors.
Science of the Total Environment | Year: 2014

Fish eyes and brain are highly susceptible to environmental Hg exposure but this issue is still scarcely investigated, mainly regarding methylmercury (MeHg) accumulation. Yet, Hg levels in fish lens have not been previously examined under field conditions. Total Hg (tHg), MeHg and inorganic Hg (iHg) levels were assessed in the brain, eye wall and lens of the golden grey mullet (Liza aurata) from an Hg contaminated area, both in winter and summer, together with water and sediment levels. Sampling was performed at Aveiro lagoon (Portugal) where a confined area (LAR) is severely contaminated by Hg. Fish brain, eye wall and lens accumulated higher levels of tHg, MeHg and iHg at LAR than the reference site, reflecting faithfully environmental spatial differences. The brain and eye wall responded also to the winter-summer changes found in water and sediment, accumulating higher levels of MeHg (and tHg) in winter. Contrarily, lens was unable to reflect seasonal changes, probably due to its composition and structural stability over time. The three neurosensory structures accumulated preferentially MeHg than iHg (MeHg was higher than 77% of tHg). Lens exhibited a higher retention capacity of MeHg (mean around 1μgg-1 at LAR), accumulating higher levels than the other two tissues. Interestingly, MeHg and iHg levels were significantly correlated for the brain and eye wall but poorly associated within the two analysed eye components. The high levels of MeHg found in the brain, eye wall and lens could compromise their functions and this needs further research. © 2014.


Costa R.R.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Costa R.R.,PT Government Associated Laboratory | Custodio C.A.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Custodio C.A.,PT Government Associated Laboratory | And 6 more authors.
Nanomedicine: Nanotechnology, Biology, and Medicine | Year: 2013

Multilayer capsules conceived at the nano- and microscales are receiving increasing interest due to their potential role as carriers of biomolecules for drug delivery and tissue engineering. Herein we report the construction of microcapsules by the sequential adsorption of chitosan and a biomimetic elastin-like recombinamer into nanostructured layers on inorganic microparticle templates. The release profile of bovine serum albumin, which was studied at 25 and 37. °C, shows higher retention and Fickian diffusion at physiological temperature. The self-assembled multilayers act as a barrier and allowed for sustained release over 14. days. The capsules studied are non-cytotoxic towards L929 cells, thereby suggesting multiple applications in the fields of biotechnology and bioengineering, where high control of the delivery of therapeutics and growth/differentiation factors is required. From the Clinical Editor: In this paper, the construction of microcapsules by sequential adsorption of chitosan and a biomimetic, elastin-like recombinamer into nanostructured layers on inorganic microparticle templates is reported. The layers demonstrated sustained drug release over 14 days. These microcapsules are non-cytotoxic toward L929 cells, suggesting multiple applications where high control of drug or growth factor delivery is required. © 2013 Elsevier Inc.


Costa R.R.,University of Minho | Costa R.R.,PT Government Associated Laboratory | Castro E.,University of Minho | Castro E.,PT Government Associated Laboratory | And 6 more authors.
Biomacromolecules | Year: 2013

Inspired by the cells' structure, we present compartmentalized capsules with temperature and magnetic-based responsiveness and hierarchical organization ranging from the nano-to the visible scales. Liquefied alginate macroscopic beads coated with a layer-by-layer (LbL) chitosan/alginate shell served as containers both for model fluorophores and microcapsules, which in their turn encapsulated either another fluorophore or magnetic nanoparticles (MNPs). The microcapsules were coated with a temperature-responsive chitosan/elastin-like recombinamer (ELR) nanostructured shell. By varying the temperature from 25 to 37 C, the two-hour release of rhodamine encapsulated within the microcapsules and its diffusion through the external compartment decreased from 84% and 71%. The devices could withstand handling and centrifugal stress, with 50% remaining intact at a rotation speed of 2000g. MNPs attributed magnetic responsiveness toward external magnetic fields. Such a customizable system can be envisaged to transport bioactive agents and cells in tissue engineering applications. © 2013 American Chemical Society.


Silva N.A.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Silva N.A.,PT Government Associated Laboratory | Silva N.A.,University of Minho | Silva N.A.,University of Toronto | And 13 more authors.
Biomaterials | Year: 2012

The regenerative capacity of injured adult central nervous system (CNS) tissue is very limited. Specifically, traumatic spinal cord injury (SCI) leads to permanent loss of motor and sensory functions below the site of injury, as well as other detrimental complications. A potential regenerative strategy is stem cell transplantation; however, cell survival is typically less than 1%. To improve cell survival, stem cells can be delivered in a biomaterial matrix that provides an environment conducive to survival after transplantation. One major challenge in this approach is to define the biomaterial and cell strategies in vitro. To this end, we investigated both peptide-modification of gellan gum and olfactory ensheathing glia (OEG) on neural stem/progenitor cell (NSPC) fate. To enhance cell adhesion, the gellan gum (GG) was modified using Diels-Alder click chemistry with a fibronectin-derived synthetic peptide (GRGDS). Amino acid analysis demonstrated that approximately 300 nmol of GRGDS was immobilized to each mg of GG. The GG-GRGDS had a profound effect on NSPC morphology and proliferation, distinct from that of NSPCs in GG alone, demonstrating the importance of GRGDS for cell-GG interaction. To further enhance NSPC survival and outgrowth, they were cultured with OEG. Here NSPCs interacted extensively with OEG, demonstrating significantly greater survival and proliferation relative to monocultures of NSPCs. These results suggest that this co-culture strategy of NSPCs with OEG may have therapeutic benefit for SCI repair. © 2012 Elsevier Ltd.


PubMed | European Institute of Excellence on Tissue Engineering and Regenerative Medicine, PT Government Associated Laboratory and University of Algarve
Type: | Journal: Scientific reports | Year: 2016

The ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the mechanisms underlying de novo bone formation and to develop screening methods towards the discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely depends on successfully measuring new tissue formation through methods that require optimization and standardization. Here, we present an improved methodology to characterize and analyse overall caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/volume. The applicability of this methodology on live specimens, to reduce animal experimentation and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated. Finally, the methodology was validated on retinoic acid- and warfarin-treated specimens, and further confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not require sophisticated equipment, the present methodology will certainly provide valuable technical standardization for research in tissue engineering, regenerative medicine and skeletal biology.

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