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Abstract
The development of versatile tubular structures is a subject of broad interest in tissue engineering applications. Herein, we demonstrate the production of tubular structures based on chitosan through a combination of dipping, freeze-drying and supercritical technology approaches. The combination of these techniques yields versatile tubes with a perfectly defined hollow imprint, which upon chemical cross-linking with genipin acquire enhanced mechanical properties (Young Modulus (E) and ultimate tensile stress (σmax)), as well as improved stability in wet conditions. The biological performance reveals that cells remain attached, well-spread and viable on the surface of cross-linked tubes. As so, is envisioned that our methodology opens up new avenues on tissue engineering approaches, where the design of tubular structures with tuned length, diameter and elasticity is required.
Introduction
Large animals present tissues with a tubular geometry, such as the trachea, intestines, ureters, blood and lymph vessels. These tissues present a well-defined tri-dimensional shape with different types of cells along with them.1 The current available options for repair of vascular, urinary, gastrointestinal, nervous and respiratory systems are autologous grafts, xenografts, artificial prostheses or synthetic grafts. However, the lack of tissue donors, anatomical variability, shorter life span and rejection by the immune system is well known.2,3 Thus, the challenge in tissue engineering (TE) upon damage of these tissues relies on the ability to bioengineer suitable functional tissue analogs, which will be able to take over the function of the diseased tissue.4–7 Bearing such limitations in mind, many efforts have been done to successfully obtain functional tubular structures with the capability of promoting the regeneration, repair and replacement of damaged, injured or lost tissues.8
Up to now a large panel of methodologies were used to produce tubes such as rolling-up (membranes or cell sheets),4,6,9 decellularization,10 thermal induce phase separation,11 phase inversion /salt leaching/winding,4,12,13 electrospinning,14,15 inkjet printing,7,16 molding17–20 and dipping21–25 based approaches. Most of these approaches present some obstacles toward the creation of tubular structures such as the control over the rolling-up of membranes around a cylinder, the need of external materials (e.g. o-rings or biological glues) to fix the tubular shape, unprecise control over cell distribution, extensive material processing (e.g. the manual rolling of cell sheets requires almost three months), inability to obtain small-sized tubes, inadequate cell adhesion and penetration, limited donor ability (decellularization), lack of uniformity, low compliance, high swelling rate, low diffusion ability, use of harsh solvents, inadequate biochemical and mechanical cues.1,2,4,14,26,27 Additional concerns are associated with scaffold degradation, which may impact inflammatory response and induce hyperplasia,14 even though some drawbacks have been already addressed by using cross-linking approaches or by creating cell-friendly surfaces, e.g. coating with polyelectrolyte multilayers or by adsorption/immobilization of proteins from the extracellular matrix.12,28–30 However, other features remain unresolved, being a key challenge to the construction of stable and functional tubular structures with the potentiality to be applied in various TE applications (e.g. airway, intestine, blood vessel, bladder and nerve guided substitutes). Hence the need for the development of new methodologies that are able to solve the problems presented by conventional techniques.
In this work, we propose the development of versatile tubes based on chitosan (CHI) through a combination of dipping, freeze drying and supercritical technology approaches. To elucidate the methodology, schematic representation is given in Figure 1. CHI was selected due to its remarkable characteristics, namely biocompatibility, biodegradability, non-toxicity and remarkable affinity to proteins, along with anti-tumoral, anti-bacterial, anti-cholesteric, fungistatic and haemostatic properties.31,32 Tubular structures were subsequently cross-linked using genipin, which has the advantage of overcoming the inherent toxicity of most of the synthetic cross-linkers (e.g. glutaraldehyde, formaldehyde, epoxy compounds) while improving the mechanical and biological performance of the amine-based constructs.33–36 Genipin is derived from gardenia fruit, being able to cross-link macromolecules by binding amine groups between adjacent molecules. It has been proved to be 5000- to 10000-fold less cytotoxic than glutaraldehyde, while presenting similar mechanical and degradative properties. In addition, it has been reported for hydrogels, scaffolds and multilayered films that cell adhesion and viability increases as the concentration of genipin increased.33,37–40 After the production of these tubular structures, a series of physicochemical and biological tests were performed to fully validate the methodology used.
Figure 1. Production steps of tubular structures: (i) Dip the tubular templates in a concentrated CHI solution; (ii) freeze the templates coated with CHI; (iii) freeze dry the tubular templates coated with CHI; (iv) cross-link the tubes in a genipin solution, leading to a greenish tube; (v) Detach the tubes by using sodium hydroxide and dry them using critical point dryer.
Materials and methods
Buildup tubular structures
The tubular structures were built using CHI medium (molecular weight (Mw) 190.000–310.000 Da, 82.6 % degree of deacetylation, ref. 448877, Sigma Aldrich, USA). The CHI solution was freshly prepared at a concentration of 2.8% (w/v) in 2% (v/v) acetic acid. Tubular structures were produced using a dipping approach, where a glass template (Ø 5 mm) was immersed in a CHI solution (1 dipping step) to form a homogeneous layer. Upon freeze drying, the tubular templates were cross-linked with genipin (Wako chemical, USA) overnight. Briefly, a genipin solution (1 or 3.5 mg/mL−1) was prepared by dissolving the adequate amount of genipin in an ethanol/water mixture (1:2(v/v)). Afterward, the tubular templates were extensively washed with water and ethanol, before their detachment. The detachment of the tubes from their templates was achieved during the neutralization step with a sodium hydroxide at 2 M solution. This step was followed by a washing step with osmotized water. Similar procedure was used to detach the tubular templates without cross-linking. All the tubular formulations after dehydration were dried using critical point dryer (Autosamdri-815 Series A, Tousimis, USA). Using such approach, the samples were heated up to 37℃ and pressurized until 90 bar for 45 min.
Scanning electron microscopy
The morphology of the tubular structures was observed by scanning electron microscopy (SEM), using Jeol JSM-6010LV microscope operated in the secondary electron mode at an accelerating voltage of 15 kV. All the samples were fixed to the aluminum stubs by double-sided carbon conductive adhesive tape and gold-sputtered using a sputter coater 108 A (Cressington, UK). The images obtained were analyzed by Image J software to determine the mean pore size, thickness and diameter of the tubular structures.
Determination of the degree of cross-linking
The cross-linking degree of tubular structures was evaluated using the trypan blue, by a method previously described.41,42 Briefly, the test was performed by immersing the non-cross-linked and the cross-linked tubes in trypan blue 0.4% (Invitrogen, USA) diluted three-fold in MES buffer (0.5 M, pH 5) overnight at 37℃. The absorbance of the supernatant was measured at 580 nm in a Synergy HT microplate reader (Bio-TEK, USA). The cross-linking degree was calculated as follows
Weight loss
The effect of cross-linking degree over the degradation profile of CHI tubes was also studied. Weighed tubes were immersed in PBS solution at 37℃ on a shaker. At predetermined time points samples were removed and washed in ultrapure water, dehydrated, dried and weighed again to determine mass loss according to the following equation
The drying step was performed in critical point dryer, using an approach similar to the one used to dry the tubes upon chemical cross-linking, i.e. the samples were heated up to 37℃ and pressurized until 90 bar for 45 min.
Water uptake
The water-uptake ability of the tubes was measured soaking dry tubular structures of known weight in PBS (Sigma, USA) at 37℃. The swollen tubes were removed at predetermined periods of time. After removing the excess of PBS using a filter paper (Filter Lab, Spain), the tubes were weighed with an analytical balance (Denver Instrument, Germany). The water uptake was calculated as follows
The morphology of the tubes before and after the swelling equilibrium reaches the equilibrium (8 hours) was evaluated using a stereomicroscope (Zeiss, Germany) with a color camera (Nikon G12). The Image J software was used to determine the changes on the hollow imprint of the tube (inner space and wall thickness).
Mechanical tests
The mechanical properties of hollow tubes were evaluated in wet state using an INSTRON 5540 (Instron Int. Ltd, High Wycombe, UK) universal testing machine with a load cell of 1 kN. The dimensions of the specimens used were 60 mm in length, 4–5 mm width. The load was placed midway between the supports with a span (L) of 20 mm. The crosshead speed was 1 mm min−1. For each condition, the specimens were loaded until core break.
Cellular tests
To evaluate the biological performance of tubes with and without cross-linking, cell culture studies were performed with L929, a mouse fibroblast of connective tissue cell line (European Collection of Cell Cultures (ECCC), UK). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, USA), supplemented by 10% heat-inactivated fetal bovine serum (FBS; Biochrom AG, Germany) and 1% antibiotic-antimicotic (Gibco, USA). The cytotoxicity of the tubes was initially studied by analyzing the effect of their leachable on the cell’s metabolism, which is in accordance with ISO/EN 10993 guidelines. A latex rubber extract was used as positive control for cell death, while the culture medium was used as negative control representing the ideal situation for cell proliferation. After 72 h, cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulphofenyl)-2H-tetrazolium (MTS) assay. The amount of formazan was measured by absorbance at 490 nm in a microplate reader. After this preliminary assay, direct contact tests were also performed using the same cell line. Prior to cell seeding, tubes (diameter 1–2 mm and length 10 mm) were sterilized with 70% (v/v) ethanol and then rinsed three times in PBS. Cell seeding (30,000 cells per cm2) was performed in a Teflon mold, as previously described elsewhere.22 Cell density was optimized, and 70,000 cells were added per tube in 100 µL of the medium. After 6 h, the tubes were transferred to a 24-well plate. Hollow tubes with and without cross-linking were incubated for 3 days at 37℃ in a humidified 5% CO2 atmosphere. Cell viability was assessed after 3 days using MTS and live/dead assay (calcein AM/ propidium iodide (PI) staining). Briefly, the tubes were washed in PBS, followed by incubation with 2 µL calcein AM and 1 µL PI in 1 mL of PBS. After 15 min, tubes were washed with PBS to remove fluorescent residues and visualized by confocal laser microscopy (Leica TCS SP8, UK).
4,6-Diaminidino-2-phenylindole-dilactate (DAPI, 20 mg/mL–1, Sigma-Aldrich, USA) and fluorescein isothiocyanate labelled phalloidin from Amanita phalloides dyes (phalloidin, 10 mg/mL–1, Sigma-Aldrich, USA) were used to perform a DAPI–phalloidin assay. Briefly, at the predetermined time point, the culture medium was removed, and the samples were fixed in 10% (v/v) formalin. After 1 h, formalin was removed and replaced by PBS. Upon permeabilization, 1 mL of PBS containing 5 µL of phalloidin and 1 µL of DAPI was added for 40 min at room temperature and protected from light. After extensively washing with PBS, the tubes were visualized in the dark by confocal microscopy. The images obtained were analyzed by Image J software to determine cell area and circularity.
Statistical analysis
The experiments were carried out in triplicate if not otherwise specified. The results were presented as the mean ± standard error of mean (SEM). Statistical analysis were performed by Shapiro Wilk normality test using Graph Pad Prism 6.2 for Windows. After this analysis, non-parametric (Kruskal Wallis test) or parametric tests (one way Anova followed by Turkey test) were used depending on whether the samples were from normally distributed populations or not, respectively.
Results and discussion
Fabrication of CHI tubes – morphological characterization
CHI tubes were manufactured by dipping (1 dipping step) different templates in a concentrated solution of CHI as shown in Figure 1. The concentration of CHI was optimized to prevent the quick flow of polymeric solution along the templates. Different templates were employed such as polypropylene (PP), expanded polytetrafluoroethylene (e-PTFE) and glass with or without a sacrificial layer of paraffin – see Figure 2(a). Our main goal using this panel of different templates is to determine which one will lead to a uniform and cohesive deposition of a CHI layer. Using a CHI concentration of 2.8% (w/v) and a glass template with defined diameter, stable tubular structures can be obtained, since a homogeneous layer of CHI is anchored to the template by electrostatic interactions. At this stage, the tubes appear macroscopically smooth and without any defect, being the layer of CHI firmly adhered to the glass template. All other templates unmet the aforementioned pre-requisites due to the weak nature of the van der Walls forces between CHI and the template. Thus, to successfully achieve the fabrication of hollow tubes, a right balance between polymeric concentration and template has to be strictly evaluated, similar behavior has been previously reported in the literature.16,17,22,25,43
After dipping, the templates were immediately frozen using two different approaches, liquid nitrogen and –80℃ (Figure 2(b)). The results indicate that liquid nitrogen led to fractures while the –80℃ did not induce any defect on the tubular structures. Thus, the tubes were fabricated using the freeze-drying method, which allows us to obtain a spongy-like tubular structure firmly adhered to the glass tube. To promote the detachment of the tubular structures from the template, different solutions were tested, such as osmotized water and sodium hydroxide (Figure 2(c)). In this case, the detachment was successfully achieved by neutralization of charged layers using sodium hydroxide. During the neutralization step, the tubular structures were easily detached from the template due to the higher swelling rate of CHI at this pH (pH 13–14).44,45 The detached tubes were, then, washed, dehydrated and subjected to a solvent exchange step. In this process, ethanol replaces the water, and the material was dried using critical point dryer, which is based on supercritical technology, allowing us to avoid the collapse of the tubular structure. This is a simpler, cost-effective approach that is not likely to induce defects.
In the present work, to improve the stability of the tubular structures, a chemical cross-link with genipin was performed prior to the neutralization step. This methodology was used since CHI tubes present a high swelling when exposed to sodium hydroxide, which lead to the dissolution of CHI chains. The morphology of the developed tubular structures was evaluated by SEM (Figure 3). The results indicate a uniform tubular structure free of defects (i.e., cracks and holes), which indicates the efficiency of the approach used. Comparing all the formulations a decrease of roughness was obtained upon cross-linking. The micrographs also reveal porosity throughout the wall of the tubes, which is important for nutrient and oxygen diffusion. The average pore size of each formulation slightly increases with chemical cross-linker (i.e., 120.81 ± 8.82 µm (non-cross-linked tubes), 173.31 ±12.35 µm (cross-linked tubes at 1 mg/mL), 153.09 ±12.10 µm (cross-linked tubes at 3.5 mg/mL)). This behavior can be explained by the covalent bonds that were stablished in CHI chains upon cross-linking, which reduces the higher swelling ability during neutralization and, thus, the collapse of the pores in the drying step. These results are corroborated by the lower thickness of non-cross-linked tubes when compared with the cross-linked tubes (i.e., 209.04 ± 13.89 µm (non-cross-linked tubes), 360.68 ± 45.01 µm (cross-linked tubes at 1 mg/mL), 334.928 ± 25.75 µm (cross-linked tubes at 3.5 mg/mL)). It is also important to point out that in all the formulations the pore sizes are higher than 100 µm in diameter, which is considered a minimum requirement for cell attachment and proliferation as well as tissue growth and diffusion of nutrients and oxygen.46,47 Comparing the inner side of the tube with the external one, the results indicate that the inner side is smoother, which corroborated previous results.21,22,44 When compared with the alternative techniques, the developed methodology allows the creation of porous structure with ability to promote the diffusion of nutrients and oxygen, the tuning of the mechanical properties, swelling ability and degradation, as well as the fine control over the diameter, thickness and length of the tube.17,20,22–24 The ability to tailor the properties of tubes combined with a time-efficient and relative ease fabrication suggest the potential of this approach. Meanwhile, besides the higher yield of production of tubes, the automation of the process will allow the production of tubes even more uniform and reproducible in comparison with other techniques such as rolling up, which would be important for in vivo applications.2,4,48
Physicochemical characterization of tubes
After optimization of the fabrication process, tubes with and without cross-linking were physicochemically assessed. The chemical cross-linking was performed anticipating the weak features of CHI tubes. Using genipin as cross-linking, we aim to circumvent the potential limiting features, such as the stability of tubes at all the pH range, as well as their swelling ratio, degradation and mechanical strength.45
The cross-linking degree of tubes was determined by trypan blue which is a methodology reported in previous studies (Figure 4).41,42 Briefly, this dye has the ability to bind to free amine groups leading to changes in the blue color intensity of the samples and, consequently, of the supernatant solution.41,42,49 Upon reading the absorbance of the solution the cross-linking degree was determined. As expected, on increasing the concentration of cross-linker, the absorbance of the supernatant solution decreased, because the number of free amines also decreased. The results suggest that cross-linking degree could be varied from 0% to 65% by adjusting of the cross-linker concentration. Contrary to other cross-linkers, genipin does not lead to a ‘zero length” reaction due to the introduction of additional monomers and further dimerization, which greatly contributes to changes in surface chemistry and an increase in the tubes’ robustness and compliance.36,39,50
The stability of tubes at physiological-like conditions (PBS, 37℃) was evaluated up to 14 days to track their stability over the time (Figure 5(a)). In all the samples, any gross degradation was observed, and the tubes retain their hand ability. However, the CHI tubes present higher weight loss than the cross-linked tubes. The cross-linked tubes did not exhibit any significant mass degradation, and the tubes maintain their structural integrity. These results corroborated the ones reported in the literature, where it is well known that upon chemical cross-linking with genipin the stability in physiological-like conditions increases, which is usually associated with stronger covalent bonds that decreases the degradation and/or simple dissolution of the tubular structures.33,37,39,40 These results suggest that upon tuning the cross-linking degree or by additional dipped layers of CHI the degradation can be further prevented, resulting in a longer time period to obtain good cell infiltration within the tube.
The ability of a scaffold to preserve water and structural stability is critical to evaluate its practical use for TE applications.47 It is well reported that an initial swelling is desirable, however, continuous swelling would lead to loss of mechanical integrity and production of compressive stress to the surrounding tissue.47,51 Thus, the water-uptake of tubular structures was evaluated and the results show an increase during the first minutes (≈15 min), reaching a plateau afterward (Figure 5(b)). The water-binding ability of a material in this case is mainly attributed to its hydrophilicity, instead of to the porosity of the construct.46,51 Traditionally, CHI-based structures are expected to have a considerable water-uptake due to the hydrophilic groups such as the hydroxyl and amine ones.52,53 Besides an increase of hydrophilic groups upon cross-linking, the water uptake decreases due to the denser connection between the CHI chains.35,39,40 This finding is not surprising and is consistent with earlier reported studies where a decrease of water contact angle and water uptake was found with increasing cross-linker concentrations.39,54 Optical microscopy was also used to get insights about the behavior of all the tubular formulations during the water-uptake assay (Figure 5(c)). For that, the tubes were analyzed in Image J software in the dry and wet state to obtain further structural information about their inner space and thickness, which are related since an increase in thickness leads to a proportional decrease in the inner space. The results indicate that all the formulations in dry state present a tubular shape and did not present any defect. However, in wet state the lumen of the non-cross-linked tubes collapses due to the high water uptake and reduced robustness. Thus, a decrease in the inner space ratio and an increased thickness (3.82 ± 1.17 fold) was seen. On the other side, cross-linked tubes at different concentrations maintain a defined and stable tubular structure besides a slight increase in thickness and a decrease in inner side space. The cross-linked tubes at 1 mg/mL show a 1.97± 0.66-fold increase in thickness, whereas the cross-linked tubes at 3.5 mg/mL a 1.15 ± 1.17-fold increase. It should also be pointed out that on drying, the cross-linked tubes did not suffer any shrinkage, maintaining their geometric structure (i.e. open lumen), contrary to what happens in non-cross-linked tubes.
Mechanical properties
The mechanical properties of the constructs were evaluated in wet conditions by conventional tensile tests. CHI tubes present unsatisfactory mechanical properties due to the high hydration degree and gel-like behavior unlike the ones cross-linked with genipin. Thus, the tubes were cross-linked with different genipin concentrations, 1 mg/mL and 3.5 mg/mL. The representative curves of all the tubular formulations present different regions, linear being the one used to determine the Young modulus (E), while the region of failure was used to determine the ultimate tensile stress (σmax) and maximum extension (ɛmax) (Figure 6).
Tensile tests results show that all these parameters were influenced upon by chemical cross-linking. The covalent cross-linking with genipin guarantees an increase in the E and σmax, while the ɛmax decreases (Table 1). The increase in E suggests that there is a stiffening effect provided by genipin cross-linking, whereas the decrease in ɛmax indicates that the structures presented less ability to deform.
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Table 1. Ultimate tensile strength (σmax), maximum extension (ɛmax) and Young modulus (E) for all the tubular formulations.
Cell viability, adhesion and distribution along tubular structures
To assess the suitability of the construct TE applications, the cytotoxicity of the developed tubes was evaluated in accordance with ISO/EN 10-993 (Figure 7).55 The viability of the cells after contact with the extracts was determined, and no significant differences were verified when compared with TCPs (ideal culture conditions). Thus, the study moves forward for direct contact tests using the same cell line. L929 cells were grown on all the tubular formulations for 3 days, and cell viability and morphology was assessed. The cell viability was evaluated by MTS (Figure 7(b)), and the results indicate a statical enhancement of total cell viability as the concentration of genipin increases. Beside the interesting properties of CHI, scaffolds only based on this polysaccharide present low cell viability due to the limited mechanical properties, unamiable surface properties and higher swelling ability.38,39 Upon chemical cross-linking with genipin, the cell viability strongly increases, the results being in accordance with the ones previously reported in the literature, where it has been reported that the use of higher concentrations of genipin improves the cell viability. The results are also in accordance with the live-dead assay where upon increasing the concentration no detectable cell death can be seen, indicating that genipin did not induce any deleterious effect on cell viability (Figure 8). This behavior is a consequence of better cell adhesion and spreading upon increasing the concentration of cross-linker.33,37–40 It should also be noted that upon cross-linking with genipin non-fluorescing materials (non-cross-linked tubes) emit a red fluorescence.36,44,54 Thus, the cross-linked tubes present auto-fluorescence in red, which allow to get insights on the porosity of the tubular structures. The cell morphology and adhesion were evaluated with DAPI & phalloidin, the results indicate as expected that cells seeded on CHI tubes present a weak cell adhesion with a clusters-like organization, while the ones on cross-linked tubes are uniformly distributed, well spread and anchored to the surface of tubes, presenting a spindle-like morphology (Figure 8(a)). Thus, the cell spread area increases with an increase on cross-linker concentration, while the circularity decreases (Figure 8(b) and (c)). The cells seeded on non-cross-linked tubes present a circularity near 1 due to the formation of clusters. The clusters are well reported in the literature as a prominent evidence of a deficient cell-matrix interaction, which protects the cells against the so-called anoikis (apoptosis induced by poor adhesion).56 This behavior is in accordance with the literature for other types of cells (fibroblasts, smooth muscle cells, neurons, endothelial cells) as well as other types of materials (hydrogels, polyelectrolyte multilayers).30,39,50,57,58 The covalent cross-linking with genipin is well known to decrease the water uptake, increase the mechanical properties and change the surface chemistry.39,50 All these parameters strongly influence cellular processes such as spreading, proliferation, motility and differentiation.30,57,58 Gathering all the results the chemical cross-linking tune the cell adhesive properties of these tubes.
Figure 7. Cell cytotoxicity by MTS assay using (a) monolayers in contact with Dubecco's modified Eagle's medium (DMEM) extracts and (b) tubes seeded with cells. The inset images represent the experimental approach followed in each one of the experiments. Statistical differences were p < 0.05 (*) and (***) p < 0.001. (***) Denotes significant differences when compared with all the formulations.
Figure 8. (a) Cell behavior upon seeding on all the tubular formulations: Live & dead fluorescence (a, b, b) and DAPI & phalloidin (d, e, f) assay at 3 days of culture. (b) Quantification of cell-spread area (b) and cell circularity (c). Statistical analysis was performed and data were considered statistically different for p < 0.05 (*) and p < 0.001 (***).
Conclusion
In this study, we successfully create tubular structures based on CHI by using a dipping approach combined with freeze drying and supercritical technologies approaches. The covalent cross-linking of these tubular structures favor cell adhesion by changing the surface chemistry and also by improving their mechanical properties. Our approach opens new avenues to develop small hollow tubes that are difficult to manufacture by other conventional techniques. One of the most promising features of these tubular structures is the possibility to tailor different properties such as diameter, length and wall thickness in the same structure. We believe that this starting point approach would be amenable for other raw materials (polymers) and may also enable us to obtain multifunctional tubular substitutes for TE applications.
Acknowledgement
The authors acknowledge Diana Soares da Costa for her help in the confocal microscope analysis.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research leading to these results has received funding from the Portuguese Foundation for Science and Technology (FCT) by PTDC/CTM-BIO/4706/2014 and SFRH/BPD/ 93697/2013 and from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number REGPOT-CT2012- 316331-POLARIS.
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