Current concepts and challenges in osteochondral tissue engineering and regenerative medicine

"Publication Date (Web): February 20, 2015"In the last few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate towards osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cells implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant and reprogrammed cells for osteochondral regeneration.The authors thank the Portuguese Foundation for Science and Technology (FCT) through the projects TISSUE2TISSUE (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009). We also acknowledge European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS. L-P.Y. acknowledges the PhD scholarship from FCT (SFRH/BD/64717/2009). The FCT distinction attributed to J.M.O. and A.L.O. under the Investigator FCT program (IF/00423/2012) and (IF/00411/2013) are also greatly acknowledged

Production de vins mousseux a partir de "Vinhos Verdes" blancs monovarietaux

On a fait l'étude comparative de vins mousseux élaborés à partir de vins de base issus de cinq cépages recommandés pour la Région: Loureiro, Trajadura, Avesso, Pedernã et Azal blanc, en ce qui concerne les propriétés organoleptiques. Les vins de base ont été produits selon le processus utilisé dans la Région -égrappage, pressurage, débourbage et fermentation alcoolique-, ayant été soumis aussi à une fermentation malolactique. La prise de mousse a été effectuée en bouteille, avec des levures immobilisées en billes d'alginate, pendant cinq mois à 14 °C. Les propriétés sensorielles des vins ont été évaluées en utilisant des fiches classificatrices et descriptives, par une chambre de neuf dégustateurs expérimentés. Le traitement statistique des résultats, effectué par le logiciel SPSS, a été fait en recourrant à l'analyse de variance. On a trouvé quelques différences parmi les cinq vins mousseux surtout en ce qui concerne l'aspect du cordon et l'arôme. Cependant, tous ces vins ont obtenu des classifications globales au-dessus de l'acceptable, atteignant parfois l'excellent. Ces résultats préliminaires ainsi obtenus, basés sur les caractéristiques organoleptiques, suggèrent la possibilité d'obtenir des vins mousseux de qualité à partir des cépages de "Vinho Verde"

Insulin and IGF-1 improve mitochondrial function in a PI-3K/Akt-dependent manner and reduce mitochondrial generation of reactive oxygen species in Huntington’s disease knock-in striatal cells

Akt, protein kinase B; ARE, antioxidant response element; Erk, extracellular signal-regulated kinase; CBP, CREB-binding protein; CREB, cAMP response-element (CRE) binding protein; CDK, cyclin-dependent kinase; DHE, dihydroethidium; Drp1, dynamin-related protein 1 or dynamin 1-like (DNM1L); GCL, glutamate-cysteine ligase; GCLc, glutamate-cysteine catalytic subunit; GPx, glutathione peroxidase; GSH, glutathione, reduced form; GSSG, glutathione oxidized form; IGF-1, Insulin-like growth factor 1; IGF1R, insulin-like growth factor 1 receptor; IR, insulin receptor; IRS, insulin receptor substrate; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; HKII, hexokinase type II; HD, Huntington’s disease; HO-1, heme oxygenase; Hsp60, heat shock 60 kDa protein 1 (chaperonin); mHtt, mutant huntingtin; mtDNA, mitochondrial DNA; MT-COII, mitochondrial-encoded cytochrome c oxidase II; mTOR, mammalian target of rapamycin; NDUFS3, NADH dehydrogenase (ubiquinone) Fe–S protein 3, 30 kDa (NADH-coenzyme Q reductase); NQO1, NAD(P)H dehydrogenase [quinone] 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; PI-3K, phosphatidylinositol 3-kinase; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator 1α; ROS, reactive oxygen species; SDHA, succinate dehydrogenase complex, subunit A, flavoprotein (Fp); SOD, superoxide dismutase; Tfam, transcription factor A, mitochondrial; TMRM, tetramethylrhodamine methyl ester; Tom20, translocase of outer mitochondrial membrane 20 homolog (yeast); Tom40, translocase of outer mitochondrial membrane 40 homolog (yeast)

Alternative products to carbazoles in the oxidation of diphenylamines with palladium (II) acetate

Although simple diphenylamines are conveniently oxidised with Palladium (II) acetate to give carbazoles, for more complex examples, carbazoles are minor products amongst many.CRUP (Portugal). British Council - Treaty of Windsor Programme. Junta Nacional de Investigação Científica e Tecnológica

Cleavages of photochromic compounds derived from heterocycles under electrospray tandem mass spectrometry : study of the influence of the heteroatom in fragmentation mechanisms

In this paper we report the fragmentation pathways of chromenes derived from carbazole, dibenzofuran and dibenzothiophene, under ESI-MS/MS experimental conditions, and their relationship with structural features, specially focused on the heteroatom’s effect on the fragmentation mechanisms.Fundação para a Ciência e Tecnologia (FCT

FTIR spectroscopic and theoretical study of matrix-isolated (E)-1-(cyclopropyldiazenyl)naphthalen-2-ol

Photochromic systems are important due to their industrial applications in variable optical transmission materials and optobioelectronic devices. For such applications, the organic photochromic compounds involved are usually incorporated in polymers, liquid crystalline materials, or other convenient host matrices [1, 2]. Herein, a photochromic compound, (E)-1-(cyclopropyldiazenyl)naphthalen-2-ol (show in Figure 1), which was synthesized by a published method [3] and characterized, was isolated in a cryogenic argon matrix and its structure as well as UV-induced phototransformations were characterized by IR spectroscopy. The structures of the starting compound and of the generated photoproducts were identified by comparison of their experimental IR spectra with the spectra theoretically calculated at the DFT (B3LYP)/6-311++G(d,p) level for several possible tautomeric and rotameric forms.Fundação para a Ciência e a Tecnologia (FCT

Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications

This study describes the developmental physicochemical properties of silk fibroin scaffolds derived from high concentration aqueous silk fibroin solutions. The silk fibroin scaffolds were prepared with different initial concentrations (8%, 10%, 12% and 16% (wt%)) and obtained by combining the salt-leaching and freeze-drying methodologies. The results indicated that the antiparallel β-pleated sheet (silk-II) conformation was present in the silk fibroin scaffolds. All the scaffolds possessed macro/micro porous structure. Homogeneous porosity distribution was achieved in all the groups of samples. As the silk fibroin concentration increased from 8% to 16%, the mean porosity decreased from 90.8±0.9% to 79.8±0.3%, and the mean interconnectivity decreased from 97.4±0.5% to 92.3±1.3%. The mechanical properties of the scaffolds exhibited a concentration dependence. The dry state compressive modulus increased from 0.81±0.29 MPa to 15.14±1.70 MPa, and the wet state dynamic storage modulus increased around 20-30 folds at each testing frequencies when the silk fibroin concentration increased from 8% to 16%. The water-uptake ratio decreased by means of increasing silk fibroin concentration. The scaffolds present favorable stability as their structure integrity, morphology and mechanical properties were maintained after in vitro degradation for 30 days. Based on these results, the scaffolds developed in this study are herein proposed to be used in meniscus and cartilage tissue engineering scaffolding.Tissue2Tissue project (PTDC/CTM/105703/2008

Combinatory approach for developing silk fibroin-based scaffolds with hierarchical porosity and enhanced performance for cartilage tissue engineering applications

Introduction: The combination of several processing technologies can open the possibility for producing scaffolds with superior performance for tissue engineering (TE) applications. Hydrogels are structurally similar to the natural extracellular matrix microenvironment presenting high elasticity and resistance to compression forces. They have been extensively used in biomedical devices fabrication and for TE applications, including for cartilage defects repair[1]. Recently, it was found that proteins like silk fibroin (SF), presenting tyrosine groups can be used to prepare fast formed hydrogels with controlled gelation properties, via an enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2)[2],[3]. Moreover, the high versatility, processability and tailored mechanical properties of SF, make this natural polymer attractive for the development of innovative scaffolding strategies for cartilage TE applications[4],[5]. Materials and Methods: The present work proposes a novel route for developing SF-based scaffolds derived from high- concentrated SF (16wt%) enzymatically cross-linked by a HRP/H2O2 complex. The combination of salt-leaching and freeze-drying methodologies was used to prepare macro/microporous SF scaffolds with an interconnected structure and specific features regarding biodegradation and mechanical properties (Fig. 1a). The scaffolds morphology and porosity were analyzed by SEM and micro-CT. The mechanical properties (Instron) and protein conformation (FTIR, XRD) were also assessed. In order to evaluate the scaffolds structural integrity, swelling ratio and degradation profile studies were performed for a period of 30 day. This work also aims to evaluate the in vitro chondrogenic differentiation response by culturing human adipose derived stem cells (hASCs) over 21 days in basal and chondrogenic conditions. Cell behaviour in the presence of the macro/microporous structures will be evaluated through different quantitative (Live/Dead, DNA, GAGs, RT PCR) and qualitative (SEM, histology, immunocytochemistry) assays. Results and Discussion: The macro/microporous SF scaffolds showed high porosity and interconnectivity with the trabecular structures evenly distributed (Fig. 1b,c). A dramatic decrease of compressive modulus was observed for samples in hydrated state. Chemical analysis revealed that SF scaffolds displayed the characteristic peaks for β-sheet conformation. Swelling ratio data demonstrated a large swelling capacity, maintaining their structural integrity for 30 days. As expected, when immersed in protease XIV the degradation rate of SF scaffolds increased. Based on the promising morphology and physicochemical properties of the developed SF scaffolds, in vitro chondrogenic differentiation studies with hASCs are envisioned in order to validate their performance for cartilage regeneration applications. Conclusion: This study proposes an innovative approach to produce fast-formed porous SF scaffolds using enzymatically cross- linked SF hydrogels structured by the combination of salt-leaching and freeze-drying methodologies. The obtained results can provide a valuable reference of SF as a tunable and versatile biomaterial with great potential for applications in cartilage TE scaffolding. Portuguese Foundation for Science and Technology (FCT) project PEst-C/SAU/LA0026/201; ERDP funding through POCTEP Project 0687_NOVOMAR_1_P; Investigator FCT program IF/00423/2012 and IF/00411/2013 References: [1] Xia, L.-W., R. Xie, X.-J. Ju, W. Wang, Q. Chen, and L.-Y. Chu, Nano-structured smart hydrogels with rapid response and high elasticity. Nature communications, 2013. 4. [2] Sofia, S.J., A. Singh, and D.L. Kaplan, Peroxidase-catalyzed crosslinking of functionalized polyaspartic acid polymers. Journal of Macromolecular Science, Part A, 2002. 39(10): p. 1151-1181. [3] Reis, R.L., L.-P. Yan, A.L. Oliveira, J.M. Oliveira, D.R. Pereira, C. Correia, and R.A. Sousa, Hydrogels derived from silk fibroin: Methods and uses thereof. 2014. 107426. [4] Chen, C.-H., J.M.-J. Liu, C.-K. Chua, S.-M. Chou, V.B.-H. Shyu, and J.-P. Chen, Cartilage tissue engineering with silk fibroin scaffolds fabricated by indirect additive manufacturing technology. Materials, 2014. 7(3): p. 2104-2119. [5] Yan, L.-P., J.M. Oliveira, A.L. Oliveira, S.G. Caridade, J.F. Mano, and R.L. Reis, Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications. Acta biomaterialia, 2012. 8(1): p. 289-301.Â

Loss of proteostasis induced by amyloid beta peptide in brain endothelial cells

AbstractAbnormal accumulation of amyloid-β (Aβ) peptide in the brain is a pathological hallmark of Alzheimer's disease (AD). In addition to neurotoxic effects, Aβ also damages brain endothelial cells (ECs) and may thus contribute to the degeneration of cerebral vasculature, which has been proposed as an early pathogenic event in the course of AD and is able to trigger and/or potentiate the neurodegenerative process and cognitive decline. However, the mechanisms underlying Aβ-induced endothelial dysfunction are not completely understood. Here we hypothesized that Aβ impairs protein quality control mechanisms both in the secretory pathway and in the cytosol in brain ECs, leading cells to death. In rat brain RBE4 cells, we demonstrated that Aβ1–40 induces the failure of the ER stress-adaptive unfolded protein response (UPR), deregulates the ubiquitin–proteasome system (UPS) decreasing overall proteasome activity with accumulation of ubiquitinated proteins and impairs the autophagic protein degradation pathway due to failure in the autophagic flux, which culminates in cell demise. In conclusion, Aβ deregulates proteostasis in brain ECs and, as a consequence, these cells die by apoptosis

Recent progress on gellan gum hydrogels provided by functionalization strategies

Gellan gum, a microbial exopolysaccharide fermentation product of Pseudomonas elodea, is a natural biomaterial that has shown promise for tissue engineering and regenerative medicine applications. Although this exopolysaccharide possesses many advantages, such interesting physicochemical properties and non-cytotoxicity, the mechanical properties and processability of gellan gum are not totally satisfactory in different tissue engineering contexts, i.e. gellan gum hydrogels are mechanically weak and the high gelling temperature is also unfavourable. An additional critical limitation is the lack of specific attachment sites for anchorage-dependent cells. However, the multiple hydroxyl groups and the free carboxyl per repeating unit of gellan gum can be used for chemical modification and functionalization in order to optimize its physicochemical and biological properties. A number of physical modification approaches have also been employed. This review outlines the recent progresses for gellan gum hydrogels and derivatives, and identifies the new challenges in tissue engineering, provided by blending and/or chemical modifications.Portuguese Foundation for Science and Technology (FCT) through the project EPIDisc (UTAP-EXPL/BBBECT/0050/2014), funded in the Framework of the ‘‘International Collaboratory for Emerging Technologies, CoLab’’, UT Austin Portugal Program.FCT for the fellowship grant SFRH/BPD/100590/2014FCT program (IF/00423/2012