ICVS 3Bs PT Government Associated Laboratory

Braga, Portugal

ICVS 3Bs PT Government Associated Laboratory

Braga, Portugal
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Duarte A.R.C.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Duarte A.R.C.,ICVS 3Bs PT Government Associated Laboratory | Ferreira A.S.D.,New University of Lisbon | Barreiros S.,New University of Lisbon | And 4 more authors.
European Journal of Pharmaceutics and Biopharmaceutics | Year: 2017

THEDES, so called therapeutic deep eutectic solvents are here defined as a mixture of two components, which at a particular molar composition become liquid at room temperature and in which one of them is an active pharmaceutical ingredient (API). In this work, THEDES based on menthol complexed with three different APIs, ibuprofen (ibu), BA (BA) and phenylacetic acid (PA), were prepared. The interactions between the components that constitute the THEDES were studied by NMR, confirming that the eutectic system is formed by H-bonds between menthol and the API. The mobility of the THEDES components was studied by PFGSE NMR spectroscopy. It was determined that the self-diffusion of the species followed the same behavior as observed previously for ionic liquids, in which the components migrate via jumping between voids in the suprastructure created by punctual thermal fluctuations. The solubility and permeability of the systems in an isotonic solution was evaluated and a comparison with the pure APIs was established through diffusion and permeability studies carried out in a Franz cell. The solubility of the APIs when in the THEDES system can be improved up to 12 fold, namely for the system containing ibu. Furthermore, for this system the permeability was calculated to be 14 × 10−5 cm/s representing a 3 fold increase in comparison with the pure API. With the exception of the systems containing PA an increase in the solubility, coupled with an increase in permeability was observed. In this work, we hence demonstrate the efficiency of THEDES as a new formulation for the enhancement of the bioavailability of APIs by changing the physical state of the molecules from a solid dosage to a liquid system. © 2017 Elsevier B.V.


Faia-Torres A.B.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Faia-Torres A.B.,ICVS 3Bs PT Government Associated Laboratory | Faia-Torres A.B.,ETH Zurich | Guimond-Lischer S.,Empa - Swiss Federal Laboratories for Materials Science and Technology | And 11 more authors.
Biomaterials | Year: 2014

Tissue engineering using scaffold-cell constructs holds the potential to develop functional strategies to regenerate bone. The interface of orthopedic implants with the host tissues is of great importance for its later performance. Thus, the optimization of the implant surface in a way that could stimulate osteogenic differentiation of mesenchymal stem cells (MSCs) is of significant therapeutic interest. The effect of surface roughness of polycaprolactone (PCL) on the osteogenic differentiation of human bone-marrow MSCs was investigated. We prepared surface roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range (~0.5-4.7μm), and mean distance between peaks (RSm) gradually varying from ~214μm to 33μm. We analyzed the degree of cytoskeleton spreading, expression of alkaline phosphatase, collagen type 1 and mineralization. The response of cells to roughness divided the gradient into three groups of elicited stem cell behavior: 1) faster osteogenic commitment and strongest osteogenic expression; 2) slower osteogenic commitment but strong osteogenic expression, and 3) similar or inferior osteogenic potential in comparison to the control material. The stem-cell modulation by specific PCL roughness surfaces highlights the potential for creating effective solutions for orthopedic applications featuring a clinically relevant biodegradable material. © 2014 Elsevier Ltd.


Oliveira M.B.,University of Minho | Custodio C.A.,University of Minho | Gasperini L.,University of Minho | Reis R.L.,ICVS 3Bs PT Government Associated Laboratory | Mano J.F.,ICVS 3Bs PT Government Associated Laboratory
Acta Biomaterialia | Year: 2016

Methacrylated gellan-gum (GG-MA) alone and combined with collagen type I (Coll) is suggested here for the first time as a cell-laden injectable biomaterial for bone regeneration. On-chip high-throughput studies allowed rapidly assessing the suitability of 15 biomaterials/media combinations for the osteodifferentiation of human adipose stem cells (hASCs). Hydrogels composed solely of GG-MA (GG100:0Coll) led hASCs from three different donors into the osteogenic lineage after 21. days of cell culture, in the absence of any osteogenic or osteoconductive factors. Hydrogels containing more than 30% of Coll promoted increased cellular proliferation and led hASCs into osteogenic differentiation under basal conditions. Studies using isolated individual hydrogels - excluding eventual on-chip crosstalk - and standard biochemical assays corroborated such findings. The formation of focal adhesions of hASCs on GG100:0Coll hydrogels was verified. We hypothesize that the hydrogels osteogenic effect could be guided by mechanotransduction phenomena. Indeed, the hydrogels showed elastic modulus in ranges previously reported as osteoinductive and the inhibition of the actin-myosin contractility pathway impaired hASCs' osteodifferentiation. GG-MA hydrogels also did not promote hASCs' adipogenesis while used in basal conditions. Overall, GG-MA showed promising properties as an innovative and off-the shelf self-inducing osteogenic injectable biomaterial. Statement of Significance: Methacrylated gellan gum (GG-MA) is here suggested for the first time as a widely available polysaccharide to easily prepare hydrogels with cell adhesion properties and capability of inducing the autonomous osteogenic differentiation of human adipose-derived stem cells (hASCs). GG-MA was processed as stand-alone hydrogels or in different combinations with collage type I. All hydrogel formulations elicited the osteogenic differentiation of hASCs, independently of the addition of any osteoconductive or osteogenic stimuli, i.e. in basal/growth medium. Effective cellular adhesion to methacrylated gellan gum hydrogels in the absence of any cell-ligand peptide/protein was here proved for the first time. Moreover, we showed that the encapsulated hASCs underwent osteogenic differentiation due to a mechanotransduction phenomenon dependent on the actin-myosin contractility pathway. © 2016 Acta Materialia Inc.


Custodio C.A.,University of Minho | Reis R.L.,University of Minho | Mano J.F.,University of Minho | Mano J.F.,ICVS 3Bs PT Government Associated Laboratory
Biomacromolecules | Year: 2016

Laminarin is a low-molecular-weight (<10 kDa) glucan found in brown algae made up of β(1→3)-glucan with β(1→6)-branches. This is one of the most abundant carbon sources in the marine ecosystem. Laminarin has been found to possess various biological interesting properties, such as antioxidant and antimicrobial activities. An attractive feature of laminarin is its inherently low viscosity and high solubility in organic and aqueous solvents that facilitate processing. This makes laminarin an appealing material for the development of new hydrogels that can be easily injected through minimally invasive procedures or used for microfabrication of hydrogels. An approach for synthesizing photo-cross-linkable laminarin hydrogels is presented in this work for the first time. Photo-cross-linkable laminarin was prepared by chemical modification with acrylate groups. The synthesized photo-cross-linkable laminarin material provides the basis for the development of a new injectable system for biomedical purposes that could be used alone or with encapsulated cells or biological molecules. The cross-linking of the methacrylated laminarin is straightforward via photoinitiated polymerization. The possibility to control the methacrylation degree of laminarin and to prepare solutions up to at least 15% w/v permits us to obtain hydrogels with tuned and wide range of stiffness and swelling. Furthermore, the encapsulation of human-adipose-derived stem cells encapsulated in the photo-cross-linked hydrogels demonstrated in vitro biocompatibility. © 2016 American Chemical Society.


Martins M.,University of Minho | Martins M.,ICVS 3Bs PT Government Associated Laboratory | Craveiro R.,New University of Lisbon | Paiva A.,New University of Lisbon | And 4 more authors.
Chemical Engineering Journal | Year: 2014

Some approaches have been developed in our group to investigate the role of novel ionic liquids as process and property modifiers of natural-based polymers. In our previous work, we proposed the use of ionic liquids as plasticizing agents for the creation of porous structures from a semi-crystalline natural-based polymer. The current work intended to complement the previous studies, evaluating the ability of ionic liquid (IL) to plasticize polymers such as blends of starch-poly-lactic acid (SPLA) and its effect on supercritical fluid foaming process (SCF) and providing more insights on the mechanisms involved. For this purpose, blends of starch with poly (lactic) acid, with different ratios of starch and poly-lactic acid of 50:50 and 30:70 were modified and processed using 1-butyl-3-methylimidazolium chloride ([bmim]Cl). Supercritical fluid foaming was studied at different soaking times (1, 3 and 6h) using carbon dioxide at 20.0MPa and 40°C. The blends were characterized by different techniques, such as infra-red spectroscopy, differential scanning calorimetry and compression and tensile mechanical analysis. The morphology of the foamed structures was analyzed by scanning electron microscopy and micro-computed tomography. The results suggest that after 3h of soaking time an equilibrium state of carbon dioxide into the bulk samples is attained, yielding structures with 6% and 15% of porosity, for SPLA70 and SPLA50 respectively. The solubility of carbon dioxide within the matrices was studied for the same conditions and the results demonstrate a higher sorption degree in the samples doped with ionic liquid. Sorption and desorption diffusion coefficients of supercritical CO2 in the SPLA matrix were determined for the raw polymer and for the SPLA doped with [bmim]Cl. It was found that the lower desorption diffusion coefficients are related with the higher porosity obtained by the foaming process. © 2013.


Rodrigues A.J.,University of Minho | Rodrigues A.J.,ICVS 3Bs PT Government Associated Laboratory | Leao P.,University of Minho | Leao P.,ICVS 3Bs PT Government Associated Laboratory | And 21 more authors.
Molecular Psychiatry | Year: 2012

Stress and exposure to glucocorticoids (GC) during early life render individuals vulnerable to brain disorders by inducing structural and chemical alterations in specific neural substrates. Here we show that adult rats that had been exposed to in utero GCs (iuGC) display increased preference for opiates and ethanol, and are more responsive to the psychostimulatory actions of morphine. These animals presented prominent changes in the nucleus accumbens (NAcc), a key component of the mesolimbic reward circuitry; specifically, cell numbers and dopamine (DA) levels were significantly reduced, whereas DA receptor 2 (Drd2) mRNA expression levels were markedly upregulated in the NAcc. Interestingly, repeated morphine exposure significantly downregulated Drd2 expression in iuGC-exposed animals, in parallel with increased DNA methylation of the Drd2 gene. Administration of a therapeutic dose of L-dopa reverted the hypodopaminergic state in the NAcc of iuGC animals, normalized Drd2 expression and prevented morphine-induced hypermethylation of the Drd2 promoter. In addition, L-dopa treatment promoted dendritic and synaptic plasticity in the NAcc and, importantly, reversed drug-seeking behavior. These results reveal a new mechanism through which drug-seeking behaviors may emerge and suggest that a brief and simple pharmacological intervention can restrain these behaviors in vulnerable individuals. © 2012 Macmillan Publishers Limited.


Requicha J.F.,University of Trás os Montes e Alto Douro | Requicha J.F.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Requicha J.F.,ICVS 3Bs PT Government Associated Laboratory | Moura T.,University of Trás os Montes e Alto Douro | And 13 more authors.
Journal of Orthopaedic Research | Year: 2014

Damages in the maxillofacial bones are frequent in humans following trauma, metabolic diseases, neoplasia, or inflammatory processes. Many of the available treatments to regenerate bone are often ineffective. The goal of this work was to assess the in vivo behavior of an innovative double-layered scaffold based on a blend of starch and polycaprolactone (SPCL) that comprises a membrane obtained by solvent casting, which aims to act as a guided tissue regeneration membrane, and a wet-spun fiber mesh (in some cases functionalized with osteoconductive silanol groups) targeting bone regeneration. The behavior of the double layer scaffold, functionalized with silanol groups (SPCL-Si) or without (SPCL), was assessed in a mandibular rodent model and compared to a commercial collagen membrane (positive control) and to empty defects (negative control). After 8 weeks of implantation, the micro-computed tomography and the histomorphometric analysis revealed that the SPCL-Si scaffolds induced significantly higher new bone formation compared to the collagen membrane and to the empty defects, although they had a similar performance when compared to the SPCL scaffolds. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.


Requicha J.F.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Requicha J.F.,University of Trás os Montes e Alto Douro | Requicha J.F.,ICVS 3Bs PT Government Associated Laboratory | Viegas C.A.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | And 8 more authors.
Stem Cell Reviews and Reports | Year: 2012

Mesenchymal stem cells have a great potential for application in cell based therapies, such as tissue engineering. Adipose derived stem cells have shown the capacity to differentiate into several lineages, and have been isolated in many animal species. Dog is a very relevant animal model to study several human diseases and simultaneously an important subject in veterinary medicine. Thus, in this study we assessed the potential of canine adipose tissue derived stem cells (cASCs) to differentiate into the osteogenic and chondrogenic lineages by performing specific histological stainings, and studied the cell passaging effect on the cASCs stemness and osteogenic potential. We also evaluated the effect of the anatomical origin of the adipose tissue, namely from abdominal subcutaneous layer and from greater omentum. The stemness and osteogenic differentiation was followed by real time RT-PCR analysis of typical markers of mesenchymal stem cells (MSCs) and osteoblasts. The results obtained revealed that cASCs exhibit a progressively decreased expression of the MSCs markers along passages and also a decreased osteogenic differentiation potential. In the author's knowledge, this work presents the first data about the MSCs markers profile and osteogenic potential of cASCs along cellular expansion. Moreover, the obtained data showed that the anatomical origin of the adipose tissue has an evident effect in the differentiation potential of the ASCs. Due to the observed resemblances with the human ASCs, we conclude that canine ASCs can be used as a model cells in tissue engineering research envisioning human applications. © 2012 Springer Science+Business Media, LLC.


Pirraco R.P.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Melo-Ferreira B.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Melo-Ferreira B.,ICVS 3Bs PT Government Associated Laboratory | Santos T.C.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | And 7 more authors.
Acta Biomaterialia | Year: 2013

Vascularization is the most pressing issue in tissue engineering (TE) since ensuring that engineered constructs are adequately perfused after in vivo transplantation is essential for the construct's survival. The combination of endothelial cells with current TE strategies seems the most promising approach but doubts persist as to which type of endothelial cells to use. Umbilical cord blood (UCB) cells have been suggested as a possible source of endothelial progenitors. Osteoblasts obtained from human adipose-derived stem cells (hASCs) were co-cultured with the mononuclear fraction of human UCB for 7 and 21 days on carrageenan membranes. The expression of vWF and CD31, and the DiI-AcLDL uptake ability allowed detection of the presence of endothelial and monocytic lineages cells in the co-culture for all culture times. In addition, the molecular expression of CD31 and VE-cadherin increased after 21 days of co-culture. The functionality of the system was assessed after transplantation in nude mice. Although an inflammatory response developed, blood vessels with cells positive for human CD31 were detected around the membranes. Furthermore, the number of blood vessels in the vicinity of the implants increased when cells from the mononuclear fraction of UCB were present in the transplants compared to transplants with only hASC-derived osteoblasts. These results show how endothelial progenitors present in the mononuclear fraction of UCB can be sustained by hASC-derived osteoblast co-culture and contribute to angiogenesis even in an in vivo setting of inflammatory response. © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.


Carvalho P.P.,European Institute of Excellence on Tissue Engineering and Regenerative Medicine | Carvalho P.P.,ICVS 3Bs PT Government Associated Laboratory | Carvalho P.P.,Louisiana State University | Gimble J.M.,Louisiana State University | And 7 more authors.
Tissue Engineering - Part C: Methods | Year: 2013

Human adipose-derived stromal/stem cells (ASCs) are an abundant, readily available population of adult stem cells that reside in adipose tissue and that have a great potential utility for tissue engineering and regenerative medicine therapeutic applications. Several preclinical studies have shown that ASCs have therapeutic applicability, but a standardized isolation and expansion methodology for clinical cell therapy has yet to be established. ASC are typically isolated and expanded using reagents with xenogenic components and this may pose certain risks and safety issues, such as exposure to infectious agents and immune reactions, creating further obstacles to the translation of ASC-based therapies to clinical scenarios. The objective of this study was to determine the suitability and efficacy of various alternative enzymatic products, CLS1 (Worthington), CLSAFA (Worthington), NB4 (SERVA), and Liberase (Roche), for the digestion of adipose tissue and subsequent isolation of ASCs, assessing cell functionality concerning their proliferation and differentiation ability. Results show that there are no statistically significant differences on yield and proliferation of cells isolated after enzymatic digestion with any of the studied products. The differentiation potential of the cells was not affected, and cell surface marker expression was similar among all products. We concluded that clinical grade products can replace current research-grade products effectively in our cell isolation protocols without any negative effect in the yield or function of human ASCs. © 2013, Mary Ann Liebert, Inc.

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