Combined remediation technology for the reduction and bioleaching of hexavalent chromium from soils using Acidithiobacillus thiooxidans

Contamination of soils due to the release of effluents or deposition of wastes containing hexavalent chromium has been arising serious environmental problems. Therefore, the development of cost effectiveness but also ecological cleaning techniques is a matter of great concern among the scientific community. Bioremediation is attracting more and more attention due to its efficiency, low impact in the ecosystems and low cost. In particular, this study approaches a bioleaching technique using an Acidithiobacillus thiooxidans DSM504 pure culture to clean a soil contaminated with hexavalent chromium. Eight batch tests were performed in order to evaluate the effect of combined parameters: operational temperature (26ºC and Troom), hexavalent chromium concentration (50 mg kg-1 and 100 mg kg-1) and pH of the contaminant solution (2 and pHfree). The bioleaching technique herein exposed presented removal values between 33.3% and 83.3%, undergoing higher deviations due to changes on the contamination pH. Generally, it was more efficient when applied to soils contaminated with acid solutions. The lowest and highest values were both observed for operational temperatures of 26ºC and hexavalent chromium concentrations of 50 mg kg-1. Moreover, the highest value was observed for the soil contaminated with a hexavalent chromium solution of pH 2

Regeneration of the intervertebral disc

Degeneration of intervertebral disc (IVD) seems to be one of the main causes associated to lower back pain (LBP), one of the most common painful conditions that lead to work absenteeism, medical visits, and hospitalization in actual society [1,2]. This complex fibro-cartilaginous structure is composed by two structures, an outer multilayer fiber structure (annulus fibrosus, AF) and a gel-like inner core (nucleus pulposus, NP), which are sandwiched in part between two cartilage endplates (CEP) [1]. Existing conservative and surgical treatments for LBP are directed to pain relief and do not adequately restore disc structure and mechanical function [2]. In the last years, several studies have been focusing on the development of tissue engineering (TE) approaches aiming to substitute/regenerate the AF or NP, or both by developing an artificial disc that could be implanted in the body thus replacing the damaged disc [3]. TE strategies aiming to regenerate NP tissue often rely on the use of natural hydrogels, due to the number of advantages that these highly hydrated networks can offer. Nevertheless, several of the hydrogel systems developed still present numerous problems, such as variability of production, and inappropriate mechanical and degradation behaviour. Recently, our group has proposed the use of gellan gum (GG) and its derivatives, namely the ionic- and photo-crosslinked methacrylated gellan gum (GG-MA) hydrogels, as potential injectable scaffolds for IVD regeneration [4,5]. Work has been conducted regarding the improvement of GG mechanical properties either by chemically modifying the polymer (allowing to better control in situ gelation and hydrogel stability) [4] or by reinforcing it with biocompatible and biodegradable GG microparticles (enabling the control of degradation rate and cell distribution) [5]. Another strategy currently under investigation relies on the development of a biphasic scaffold that mimics the total disc by using a reverse engineering approach

Evaluation of different formulations of gellan gum-based hydrogels for tissue engineering of intervertebral disc

Gellan gum based-hydrogels present advantageous features for application as acellular and cellular nucleus pulposus (NP) substitutes due to the possibility of fine-tuning its physico-chemical and biological properties. In this study, ionic-crosslinked hydrogel discs were produced by means of mixing a raw and chemically modified material, i.e., high acyl gellan gum (HAGG) and methacrylated low acyl gellan gum (GG-MA), respectively. The hydrogel discs were characterized in terms of its mechanical properties and degradation/swelling ability. The biocompatibility of the different hydrogel formulations was assessed in vitro using NP rabbit cells isolated from the intervertebral disc. The biological performance of the developed gellan gum-based hydrogels formulations was evaluated by: (i) culturing of NP cells in the presence of the hydrogel leachables, and (ii) seeding or encapsulation of the NP cells within the hydrogels. The present work demonstrated that as HAGG content increases, the modulus of the hydrogels decreases. Moreover, the increase of the HAGG content induces a higher weight loss of the GG-MA/HA-GG formulation as compared to GG-MA hydrogel. The in vitro study revealed that hydrogels are non-cytotoxic and support the encapsulation of rabbit NP cells. The methacrylated gellan gum and formulations possessing high acyl gellan gum present tunable properties that may be interesting for application as NP substitutes

Foraging behaviour and diet of Brown boobies Sula leucogaster from Tinhosas Islands, Gulf of Guinea

Seabirds are a highly threatened group, yet the foraging ecology of several species remains poorly understood. Brown boobies breed in all oceans in the tropical region and are common across their range. In Tinhosa Grande (São Tomé and Príncipe), this species breeds in one of the largest colonies of seabirds in the east tropical Atlantic. We studied the foraging ecology of Brown boobies from this colony during the chick-rearing period. Thirty-three birds were tracked with GPS loggers and their diet was analysed from 11 regurgitations, using traditional and DNA barcoding techniques for prey identifcation. A total of 127 completed foraging trips were identifed, 89% of which lasted less than 24 h. Females performed signifcantly longer trips and both sexes foraged preferentially over deep oceanic waters. The diet of Brown boobies included juvenile fsh and squid (Sthenoteuthis pteropus), comprising mostly fsh species whose juvenile phases live in the pelagic environment, and only migrate to coastal waters when adults. The most frequent of those prey found was Flying gurnard (Dactylopterus volitans). The relevance of such prey shows that Brown booby conservation depends not only on the management of their foraging areas and breeding sites but also on the correct management of the coastal adjacent areas that support the adult individuals of some of their prey. Our results suggest that the areas closest to the colony do not have available resources for these birds to feed on and that Brown boobies may be associate with subsurface marine predators or with sargassum patches to forage.Fundação para a Ciência e Tecnologia - FCTinfo:eu-repo/semantics/publishedVersio

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

Anti-angiogenic potential of VEGF blocker dendron-laden gellan gum hydrogels for tissue engineering applications

Damage of non-vascularised tissues such as cartilage and cornea can result in healing processes accompanied by a non-physiological angiogenesis. Peptidic aptamers have recently been reported to block the vascular endothelial growth factor (VEGF). However, the therapeutic applications of these aptamers is limited due to their short half-life in vivo. In this work, an enhanced stability and bioavailability of a known VEGF blocker aptamer sequence (WHLPFKC) was pursued through its tethering of molecular scaffolds based on hyperbranched peptides, the poly(É -lysine) dendrons, bearing three branching generations. The proposed design allowed simultaneous and orderly-spaced exposure of sixteen aptamers per dendrimer to the surrounding biological microenvironent, as well as a relatively hydrophobic core based on di-phenylalanine aiming to promote an hydrophobic interaction with the hydrophobic moieties of ionically-crosslinked metacrylated gellan gum (iGG-MA) hydrogels. The VEGF blocker dendrons were entrapped in iGG-MA hydrogels and their capacity to prevent endothelial cell sprouting was assessed qualitatively and quantitatively using 3D in vitro models and the in vivo chick chorioallantoic membrane (CAM) assay. The data demonstrate that at nanoscale concentrations, the dendronised structures were able to enhance control of the biological actvity of WHLPFKC at the material/tissue interface and hence the anti-angiogenic capacity of iGG-MA hydrogels not only preventing blood vessel invasion, but also inducing their regression at the tissue/iGG-MA interface. The in ovo study confirmed that iGG-MA functionalised with the dendron VEGF blockers do inhibit angiogenesis by controlling both size and ramifications of blood vessels in proximity of the implanted gel surface.This work was mainly supported by the EC FP7 project Disc Regeneration (Contract No NMP3-LA-2008-213904). This study was also funded by the EC FP7 Programme contract agreement no REGPOT-CT2012-316331-POLARIS

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

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

Microparticles loaded gellan gum hydrogel matrices: engineering tissues for nucleus pulposus regeneration

The intervertebral disc central core is made by a gel-like tissue structure composed of more than 80% of water, Nucleus Pulposus (NP). Proteoglycans such as versican and especially aggrecan are the main constituents of the NP matrix as well as collagen type II. The purpose of this work is creating novel Gellan gum-based (GG) hydrogel formulations. GG microparticles (MPs) dispersed in a GG matrix are the novelty for finding application as NP substitute. The ongoing experiment comprises de GG functionalization through methacrylated groups addition. In order to optimize some properties of GG, the functionalization will allow us to improve the water solubility and photopolymerization in situ of the biomaterial. High acyl (HA) and Low acyl (LA) Gellan Gum (GG) at different ratio [75%:25% (v/v); 50%:50% (v/v), 25%:75% (v/v)], HAGG 0.75% and LAGG 2%, were mixed in order to prepare solutions to be used as formulations of GG MPs/hydrogels matrix. The GG MP/hydrogel matrix formulations were characterized by dynamic mechanical analysis (DMA), swelling behaviour and degradation rate. The toxic effect of GG MPs/hydrogel discs leachables onto the cells was investigated in vitro using a mouse lung fibroblast-like cell (L929 cells) line. Live/Dead cell viability assay was performed to assess the encapsulation efficacy; meanwhile DAPI/Phalloidin staining was performed to evaluate cell morphology. The Methacrylated Gellan Gum (GG-MA) was prepared following the protocol [1]. Mechanism reactions occurred in presence of glycidyl methacrylate by addition to a solution of LAGG at 2% (w/v). The reaction was running over 24 h at room temperature controlling the pH at 8.5 with sodium hydroxide 1 M. GG MPs size was measured using a stereo microscope by staining the MPs with Toluidine Blue-O. This method also allowed evaluating the MPs dispersion and matrix cohesion. From DMA analysis it was observed that the range of 50–500 mg/mL of incorporated MPs is the optimal concentration to reinforce GG matrices. It was demonstrated the non–cytotoxic effect of MPs/hydrogels over L929 cells. In fact, L929 cells were successfully encapsulated in all GG formulations GG MPs and remaining viable over 72 h of culturing. The resulting product from methacrylation reaction was evaluated by nuclear magnetic resonance to assess the reaction efficiency and the degree of substitution. Methacrylated Gellan gum and GG MP/hydrogel matrix are promising hydrogels to be used in tissue engineering strategies for treatment of the degenerated NP

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)