The first 8-13 micron spectra of globular cluster red giants: circumstellar silicate dust grains in 47 Tucanae (NGC 104)

We present 8-13 micron spectra of eight red giants in the globular cluster 47 Tucanae (NGC 104), obtained at the European Southern Observatory 3.6m telescope. These are the first mid-infrared spectra of metal-poor, low-mass stars. The spectrum of at least one of these, namely the extremely red, large-amplitude variable V1, shows direct evidence of circumstellar grains made of amorphous silicate.Comment: Accepted for publication in Astronomy and Astrophysics, 5 page

Rheological and mechanical properties of acellular and cellladen methacrylated gellan gum hydrogels

Tissue engineered hydrogels hold great potential as nucleus pulposus substitutes (NP), as they promote intervertebral disc (IVD) regeneration and re-establish its original function. But, the key to their success in future clinical applications greatly depends on its ability to replicate the native 3D micro-environment and circumvent their limitation in terms of mechanical performance. In the present study, we investigated the rheological/mechanical properties of both ionic- (iGG-MA) and photo-crosslinked methacrylated gellan gum (phGG-MA) hydrogels. Steady shear analysis, injectability and confined compression stress-relaxation tests were carried out. The injectability of the reactive solutions employed for the preparation of iGG-MA and phGG-MA hydrogels was firstly studied, then the zero-strain compressive modulus and permeability of the acellular hydrogels were evaluated.In addition, human intervertebral disc (hIVD) cells encapsulated in both iGG-MA and phGG-MA hydrogels were cultured in vitro, and its mechanical properties also investigated under dynamic mechanical analysis at 37ºC and pH 7.4. After 21 d of culturing, hIVD cells were alive (Calcein AM) and the E’ of ionic-crosslinked hydrogels and photo-crosslinked was higher than that observed for acellular hydrogels. Our study suggests that methacrylated gellan gum hydrogels present promising mechanical and biological performance as hIVD cells were producing extracellular matrix

Injectable gellan gum-based hydrogels for intervertebral disc regeneration

Intervertebral disc (IVD) degeneration is a challenging pathology that, due to the inefficiency of the current treatments, urgently demands for the development of new regenerative approaches[1]. The best viable implant material for nucleus pulposus (NP) regeneration has yet to be identified, but it is believed that biodegradable hydrogelbased materials are promising candidates[2]. In this work, we are proposing the use of ionic- and photocrosslinked methacrylated gellan gum (GG-MA) hydrogels as potential acellular and cellular injectable scaffolds for IVD regeneration

Gellan gum-based hydrogels for intervertebral disc tissue engineering applications

Intervertebral disc (IVD) degeneration is a challenging clinical problem that urgently demands viable nucleus pulposus (NP) implant materials. The best suited biomaterial for NP regeneration has yet to be identified, but it is believed that biodegradable hydrogel-based materials are promising candidates. In this work, we have developed ionic- and photo-crosslinked methacrylated gellan gum (GG–MA) hydrogels to be used in acellular and cellular tissue-engineering strategies for the regeneration of IVDs. The physicochemical properties of the developed hydrogels were investigated by Fourier-transform infrared spectroscopy, 1H nuclear magnetic resonance and differential scanning calorimetry. The swelling ability and degradation rate of hydrogels were also analysed in phosphate-buffered saline solution at physiological pH for a period of 30 days. Additionally, the morphology and mechanical properties of the hydrogels were assessed under a scanning electron microscope and dynamic compression, respectively. An in vitro study was carried out to screen possible cytotoxicity of the gellan gum-based hydrogels by culturing rat lung fibroblasts (L929 cells) with hydrogel leachables up to 7 days. The results demonstrated that gellan gum was successfully methacrylated. We observed that the produced GG–MA hydrogels possess improved mechanical properties and lower water uptake ability and degradation rate as compared to gellan gum. This work also revealed that GG–MA hydrogels are non-cytotoxic in vitro, thus being promising biomaterials to be used in IVD tissue-engineering strategies.The authors are grateful for funds provided by the Portuguese Foundation for Science and Technology (FCT) through the POCTI and FEDER programmes, including Project ProteoLight (Grant No. PTDC/FIS/68517/2006). This work was also carried outwith the support of the European Union-funded Collaborative Project Disc Regeneration (Grant No. NMP3-LA-2008-213904)

Functionalisation of methacrylated gellan gum hydrogels by anti-angiogenic dendrons

The regeneration of cartilage in the intervertebral disc nucleus pulposus and joints is impaired by the formation of fibrocartilage that is caused by the invasion of the tissue by blood vessels. Peptides have been identified by phage display technique which are able to bind VEGF thus inhibiting angiogenesis. The present works focusses on the synthesis of poly(epsilon-lysine) dendrons of three branching generations in which the molecular root of the dendron bears a di-phenylalanine sequence to promote hydrophobic interactions with material surfaces and the uppermost molecular branches are functionalised with the amino acid sequenceWHLPFKC that is known to block VEGF. These biofunctionalised dendrons were entrapped in methacrylated Gellan Gum (GG-MA) hydrogels and tested for their ability to inhibit endothelial cell sprouting by both a 3D in vitro cell models and an in ovo model. The results show that when GG-MA is functionalised with the dendronised VEGF blockers, a regression of angiogenesis takes place around the hydrogel boundary. The in ovo study supports these findings as the GG-MA functionalised with the dendronised VEGF blockers did not elicit any acute inflammatory response, and decrease the number of converging macroscopic blood vessels as compared to positive controls. Moreover, the hydrogels prevented the infiltration of blood vessels, after 4 days of implantation.Fundação para a Ciência e TecnologiaEU 7th Framework ProgrammeMICINNJunta de Castilla y LeónCIBER-BBNFundo Social EuropeuPrograma Operacional Potencial Human

Advanced mimetic materials for meniscus tissue engineering : targeting segmental vascularization

Meniscus lesions are among the most common orthopaedic injuries which can ultimately lead to degeneration of the knee articular cartilage. The human meniscus has a limited healing potential, partly due to a poor vasculature, and thus meniscus regeneration using tissue engineering strategies has recently been investigated as a promising alternative to total/partial meniscectomy [1]. Advanced scaffolds for tissue engineering of meniscus should be able to mimic and preserve the asymmetric vascular network of this complex tissue, i.e. enable controlling the segmental vascularization during the regeneration process. Novel scaffolds were produced combining a silk polymeric matrix (12 wt%) [2] and the methacrylated gellan gum hydrogel (iGG-MA), which has been shown to be able to prevent the ingrowth of endothelial cells and blood vessels into the hydrogels [3,4]. The angiogenic/ anti-angiogenic potential of acellular and cell-laden silk-12 scaffolds combined with iGG-MA hydrogel was investigated in vivo, using the chick embryo chorioallantoic membrane (CAM) assay. For producing the cell-laden scaffolds, human meniscus cells (HMC¢s) were isolated from morphologically intact human menisci using an enzymatic-based digestion and expanded using standard culture conditions. The HMC’sladen hydrogel/silk scaffolds were produced by encapsulating the HMC’s into a 2 wt% GG-MA hydrogel, followed by impregnation onto the 12 wt% silk scaffold and ionic-crosslinking in a saline solution. A CAM assay was used to investigate the control of segmental vascularization of the acellular and HMC¢s-laden hydrogel/silk scaffolds by the effect of GG-MA hydrogel, until day 14 of embryonic development. The in vivo study allowed investigating the number of macroscopic blood vessels converging to the implants. The evaluation of possible inflammation and endothelial cells ingrowths was performed by histological (haematoxylin and eosin - H&E - staining) and immunohistochemical methods (SNA-lectin staining). When the silk-12 scaffold was combined with the hydrogel, an inhibitory effect was observed as demonstrated by the low number of convergent blood vessels. Results have shown that iGG-MA hydrogel prevented the endothelial cells adhesion and blood vessels infiltration into the HMC’s hydrogel/silk scaffolds, after 4 days of implantation. This study showed that the hydrogel/silk scaffolds enabled controlling the segmental vascularization, thus it can possibly mimic the native vasculature architecture during meniscus regeneration

Sympatric threatened Iberian leuciscids exhibit differences in Aeromonas diversity and skin lesions’ prevalence

Funding: This research was supported by CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Project UIDB/00276/2020 (funded by FCT - Fundação para a Ciência e Tecnologia IP) and by MARE (MARE-ISPA), MARE/UIDB/MAR/04292/2020 and strategic project MARE/UIDP/MAR/04292/2020 (also funded by FCT). MLG thanks funding by the University of Lisbon (PhD fellowship C10571K). TAM thanks partial support by CEAUL (funded by FCT, Portugal, through the project UIDB/00006/2020).Assessments regarding health aspects of Iberian leuciscids are limited. There is currently an information gap regarding effects of infectious diseases on these populations and their role as a possible conservation threat. Moreover, differences in susceptibility to particular agents, such as Aeromonas spp., by different species/populations is not clear. To understand potential differences in Aeromonas diversity and load, as well as in the prevalence and proportion of skin lesions, in fishes exposed to similar environmental conditions, an observational study was implemented. Using a set of 12 individuals belonging to two sympatric Iberian leuciscid species (Squalius pyrenaicus and Iberochondrostoma lusitanicum), the skin lesion score in each individual was analyzed. Furthermore, a bacterial collection of Aeromonas spp. isolated from each individual was created and isolates’ load was quantified by plate counting, identified at species level using a multiplex-PCR assay and virulence profiles established using classical phenotypic methods. The similarity relationships of the isolates were evaluated using a RAPD analysis. The skin lesion score was significantly higher in S. pyrenaicus, while the Aeromonas spp. load did not differ between species. When analyzing Aeromonas species diversity between fishes, different patterns were observed. A predominance of A. hydrophila was detected in S. pyrenaicus individuals, while I. lusitanicum individuals displayed a more diverse structure. Similarly, the virulence index of isolates from S. pyrenaicus was higher, mostly due to the isolated Aeromonas species. Genomic typing clustered the isolates mainly by fish species and skin lesion score. Specific Aeromonas clusters were associated with higher virulence indexes. Current results suggest potential differences in susceptibility to Aeromonas spp. at the fish species/individual level, and constitute important knowledge for proper wildlife management through the signalization of at-risk fish populations and hierarchization of conservation measures.Publisher PDFPeer reviewe

Silk-Fibroin/Methacrylated Gellan Gum Hydrogel as an novel scaffold for application in meniscus cell-based tissue engineering

Introduction: Knee meniscus injury is highly prevalent and there is a demand for new cost-effective treatment solutions. Tissue engineering (TE) and regenerative medicine strategies using acellular scaffolds are being used in clinical application for total or partial meniscus replacement [1]. Although this strategy has been considered as a safe and promising approach, progressive volume reduction of the implant and early failure have been described. Advances in the field of meniscus TE are required and greatly depend on increased knowledge of meniscus biology, improvement of biomaterials and cell-based therapies [2]. Advanced scaffolds for meniscus TE should possess adequate mechanics, biodegradability and biocompatibility, and also be able to mimic and preserve the asymmetric vascular network of this complex tissue, i.e. enable controlling the segmental vascularization during the regeneration process. Silk fibroin scaffolds derived from Bombyx mori cocoon have been recognized as a versatile biomaterial for application in meniscus TE [3]. The purpose of this study is to: 1) contribute to the knowledge of meniscus aiming future clinical applications (namely, the aspects dealing with the characterization of cellular phenotypes and density, biomechanics and extracellular matrix composition) and 2) to develop an alternative and viable silk fibroin scaffold possessing adequate properties for either use in acellular or cellular approaches for partial and/or total meniscus replacement, and combine it with the methacrylated gellan gum hydrogel (iGG-MA) hydrogel, which is able to prevent the ingrowth of endothelial cells and blood vessels into the hydrogels [4,5]. Patients & Methods: Morphologically intact menisci were collected from 44 human donors (12 male, 32 female). All menisci (30 lateral and 14 medial) were divided into anterior, middle and posterior segments prior to mechanical, biological or histological characterization. Human meniscus cells (HMC´s) were isolated using an enzymatic standard protocol. HMC´s phenotype was characterized by flow cytometry analysis. Haematoxylin and eosin (H&E), safranin-O and collagen I staining were performed for segmental characterization of the extracellular matrix. For the evaluation of the viscoelastic properties, dynamic mechanical analysis (DMA) was performed using fresh tissue samples. The three segments of menisci were cut in cylindrical shapes with 4 mm diameter and analyzed at 37ºC in PBS (pH 7.4). The microstructure of freeze-dried meniscus was investigated by micro-computed tomography (micro-CT) analysis. Silk-based scaffolds (10 and 12 wt%) were produced by means of combining salt leaching and freeze-drying methods [3], in order to match human tissue biological and biomechanical features. HMC’s were seeded onto the different silk scaffolds at a cell density of 5x104 cells/disc. Then, the cell-laden scaffolds were cultured in static conditions, for times of culturing up to 21 days. After specific times of culturing (from 1 day up to 21 days), HMC´s adhesion, viability and proliferation were investigated by scanning electron microscopy (SEM), calcein-AM assay and DNA quantification tests, respectively. In addition, the mechanical properties of the cell-loaded scaffolds were evaluated by DMA. The HMC’s-laden hydrogel/silk scaffolds were produced by encapsulating the HMC’s into a 2 wt% iGG-MA hydrogel, followed by impregnation onto the 12 wt% silk scaffold. A chorioallantoic membrane (CAM) assay was used to investigate in vivo the control of segmental vascularization of the acellular and cell-laden hydrogel/silk scaffolds by the effect of iGG-MA hydrogel, until day 14 of embryonic development. Results & Discussion: The biological characterization of this meniscus tissue, although not yet completely accomplished, has evolved significantly in the last few years. In this work, DMA analysis has shown that medial meniscus has significantly higher stiffness (E' and Tan d) than lateral meniscus. There is also significant regional variation form anterior to posterior menisci segments regarding biomechanical features. Age, gender and bone mass index (BMI) also influences meniscus stiffness. The FACS analysis revealed that cells isolated from the human meniscus are a mixed population of cells, i.e. fibrochondrocyte-like and MSCs (cells are positive for CD105, CD73 and CD90, and lack CD34 and CD45). HMC’s maintained their phenotype for 21 days when cultured in tissue culture polystyrene plate (2D). The micro-CT analysis revealed that the human freeze-dried meniscus possessed a mean porosity of 58.0±20.3% and interconectivity of 41.9%±53.7. The mean pore size and trabeculae thickness was 220.7±111.5 µm and 159.7±78.6 µm, respectively. The knowledge arising from the present study allowed us to develop a novel polymeric scaffold made of silk fibroin, which was subsequently characterized without cells and after cell-loading. SEM analysis revealed that the HMC´s adhered to the surface of the scaffolds. The viability assay and DNA quantification showed that HMC´s were viable and proliferated well when cultured onto both silk-10 and silk-12 scaffolds, until 21 days. DMA analysis has shown that the moduli of the acellular scaffolds immersed in culture medium for 14 days were 27.6 ± 7.9 kPa and 61.1 ± 0.4 at 10 Hz, for silk-10 e silk-12, respectively. By its turn, the moduli determined at 10 Hz of the cell-laden scaffolds cultured after 14 days of culturing were 48.2± 19.8 and 140.1 ± 15.6 kPa, for silk-10 and silk-12, respectively. The in vivo study allowed investigating the number of macroscopic blood vessels converging to the implants. The evaluation of possible inflammation and endothelial cells ingrowths was performed by histological (H&E staining) and immunohistochemical methods (SNA-lectin staining). Results have shown that iGG-MA hydrogel prevented the endothelial cells adhesion and blood vessels infiltration into the HMC’s hydrogel/silk scaffolds, after 4 days of implantation, even in the presence of VEGF

Bioactive macro/micro porous silk fibroin/Nano-sized calcium phosphate scaffolds with potential for bone tissue engineering applications

Aim: The development of novel silk/nano-sized calcium phosphate (silk/nano-CaP) scaffolds with highly dispersed CaP nanoparticles in the silk fibroin (SF) matrix for bone tissue engineering. Materials & methods: Nano-CaP was incorporated in a concentrated aqueous SF solution (16 wt.%) by using an in situ synthesis method. The silk/nano-CaP scaffolds were then prepared through a combination of salt-leaching/ lyophilization approaches. Results: The CaP particles presented good affinity to SF and their size was inferior to 200 nm when theoretical CaP/silk ratios were between 4 and 16 wt.%, as determined by scanning electron microscopy. The CaP particles displayed a uniform distribution in the scaffolds at both microscopic and macroscopic scales as observed by backscattered scanning electron microscopy and micro-computed tomography, respectively. The prepared scaffolds presented self-mineralization capability and no cytotoxicity confirmed by in vitro bioactivity tests and cell viability assays, respectively. Conclusion: These results indicated that the produced silk/nano-CaP scaffolds could be suitable candidates for bone-tissueengineering applications.This study was funded by the Portuguese Foundation for Science and Technology (FCT) through the projects Tissue2Tissue (PTDC/CTM/105703/2008) and Osteo Cart (PTDC/CTM-BPC/115977/2009). The funding from Foundation Luso-Americana is greatly acknowledged. L-P Yan gives thanks for his PhD scholarship from FCT (SFRHIBD/64717/2009). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or potions, expert testimony, grants or patents received or pending, or royalties

Optimal design of THEDES based on Perillyl Alcohol and Ibuprofen

Therapeutic deep eutectic systems (THEDES) have dramatically expanded their popularity in the pharmaceutical field due to their ability to increase active pharmaceutical ingredients (APIs) bioavailability. However, their biological performance has not yet been carefully scrutinized. Herein, THEDES based on the binary mixture of perillyl alcohol (POH) and ibuprofen (IBU) were prepared using different molar ratios. Our comprehensive strategy includes the characterization of their thermal and structural behavior to identify the molar ratios that successfully form deep eutectic systems. The in vitro solubility of the different systems prepared has demonstrated that, unlike other reported examples, the presence of the terpene did not affect the solubility of the anti-inflammatory agent in a physiological simulated media. The biological performance of the systems was studied in terms of their antimicrobial activity against a wide panel of microorganisms. The examined THEDES showed relevant antimicrobial activity against all tested microbial strains, with the exception of P. aeruginosa. A synergistic effect from the combination of POH and IBU as a eutectic system was verified. Furthermore, the cytotoxic profile of these eutectic systems towards colorectal cancer (CRC) in vitro cell models was also evaluated. The results provide the indication that the cell viability varies in a dose-dependent manner, with a selective THEDES action towards CRC cells. With tunable bioactivities in a ratio-dependent manner, THEDES enhanced the antimicrobial and anticancer properties, representing a possible alternative to conventional therapies. Therefore, this study provides foreseeable indications about the utility of THEDES based on POH and IBU as strong candidates for novel active pharmaceutical systems.Foundation for Science and Technology (FCT), through project PTDC/BBB-490 EBB/1676/2014–Des.Zyme, Light2Skin-PTDC/CTM-CTM/29813/2017 and ERC-2016-CoG 725034 (ERC Consolidator Grant Des.solve). E.S. would also like to acknowledge the financial support by the FCT through the doctoral grant with reference number SFHR/BD/143902/2019. J.M.S. would also like to acknowledge the financial support by the FCT through the post-doctoral grant with reference number SFRH/BPD/116779/201