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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. Source

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. Source

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. Source

Requicha J.F.,University of Tras 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 Tras 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. Source

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. Source

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