Acellular Gellan-gum based bilayered structures for the regeneration of osteochondral defects: a preclinical study

 In orthopaedics, the management and treatment of osteochondral (OC) defects remains an ongoing clinical challenge. Autologous osteochondral mosaicplasty has been used as a valid option for OC treatments although donor site morbidity remains a source of concern [1]. Engineering a whole structure capable of mimicking different tissues (cartilage and subchondral bone) in an integrated manner could be a possible approach to regenerate OC defects. In our group we have been proposing the use of bilayered structures to regenerate osteochondral defects [2,3]. The present study aims to investigate the pre-clinical performance of bilayered hydrogels and spongy-like hydrogels in in vivo  models (mice and rabbit, respectively), in both subcutaneous and orthotopic models. The bilayered structures were produced from Low Acyl Gellan Gum (LAGG) from Sigma-Aldrich, USA. Cartilage-like layers were obtained from a 2wt% LAGG solution. The bone-like layers were made of 2wt% LAGG with incorporation of hydroxyapatite at 20% and 30% (w/v). Hydrogels and spongy-like were subcutaneouly implanted in mice to evaluate the inflammatory response. Then, OC defects were induced in rabbit knee to create a critical size defect (4 mm diameter and 5 mm depth), and then hydrogels and sponges implanted. Both structures followed different processing methods. The hydrogels were injected allowing in situ  crosslinking. Unlike, the spongy-like were pre-formed by freeze-drying. The studies concerning subcutaneous implantation and critical size OC defect were performed for 2 and 4 weeks time, respectively. Cellular behavior and inflammatory responses were assessed by means of histology staining and biochemical function and matrix deposition by immunohistochemistry. Additionally, both OC structures stability and new cartilage and bone formation were evaluated by using vivo- computed tomography (Scanco 80). The results showed no acute inflammatory response for both approaches. New tissue formation and integration in the adjacent tissues were also observed, which present different characteristic behaviors when comparing hydrogels and sponges response. As future insights, a novel strategy for regeneration of OC defects can be designed encompassing both, hydrogels and spongy-like structures and cellular approaches. References: 1. Espregueira-Mendes J. et al. Osteochondral transplantation using autografts from the upper tibio-fibular joint for the treatment of knee cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 20,1136, 2012. 2. Oliveira JM. et al, Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells. Biomaterials 27, 6123, 2006. 3. Pereira D R. et al. Gellan Gum-Based Hydrogel Bilayered Scaffolds for Osteochondral Tissue Engineering. Key Engineering Materials 587, 255, 2013.

Modulation of the secretome of hBMSCs by tailoring the macromolecular gradient in hydrogels to generate tissue-to-tissue interfaces

Tissue-to-tissue interfaces are commonly present in all tissues exhibiting structural, biological and chemical gradients serving a wide range of physiological functions. These interfaces are responsible for mediation of load transfer between two adjacent tissues. They are also important structures in sustaining the cellular communications to retain tissueâ s functional integration and homeostasis. [1] All cells have the capacity to sense and respond to physical and chemical stimulus and when cultured in three-dimensional (3D) environments they tend to perform their function better than in two-dimensional (2D) environments. Spatial and temporal 3D gradient hydrogels better resemble the natural environment of cells in mimicking their extracellular matrix. [2] In this study we hypothesize that differential functional properties can be engineered by modulation of macromolecule gradients in a cell seeded threedimensional hydrogel system. Specifically, differential paracrine secretory profiles can be engineered using human Bone Marrow Stem Cells (hBMSCâ s). Hence, the specific objectives of this study are to: assemble the macromolecular gradient hydrogels to evaluate the suitablity for hBMSCâ s encapsulation by cellular viability and biofunctionality by assessing the paracrine secretion of hBMSCâ s over time. The gradient hydrogels solutions were prepared by blend of macromolecules in one solution such as hyaluronic (HA) acid and collagen (Col) at different ratios. The gradient hydrogels were fabricated into cylindrical silicon moulds with higher ratio solutions assembled at the bottom of the mould and adding the two solutions consecutively on top of each other. The labelling of the macromolecules was performed to confirm the gradient through fluorescence microscopy. Additionally, AFM was conducted to assess the gradient hydrogels stiffness. Gradient hydrogels characterization was performed by HA and Col degradation assay, degree of crosslinking and stability. hBMSCâ s at P3 were encapsulated into each batch solution at 106 cells/ml solution and gradient hydrogels were produced as previously described. The hBMSCâ s were observed under confocal microscopy to assess viability by Live/Dead® staining. Cellular behaviour concerning proliferation and matrix deposition was also performed. Secretory cytokine measurement for pro-inflammatory and angiogenesis factors was carried out using ELISA. At genomic level, qPCR was carried out. The 3D gradient hydrogels platform made of different macromolecules showed to be a suitable environment for hBMSCâ s. The hBMSCâ s gradient hydrogels supported high cell survival and exhibited biofunctionality. Besides, the 3D gradient hydrogels demonstrated differentially secretion of pro-inflammatory and angiogenic factors by the encapsulated hBMSCâ s. References: 1. Mikos, AG. et al., Engineering complex tissues. Tissue Engineering 12,3307, 2006 2. Phillips, JE. et al., Proc Natl Acad Sci USA, 26:12170-5, 2008

Defining risk acceptance criteria in occupational settings: A case study in the furniture industrial sector

The use of appropriate acceptance criteria in the risk assessment process for occupational accidents is an important issue but often overlooked in the literature, particularly when new risk assessment methods are proposed and discussed. In most cases, there is no information on how or by whom they were defined, or even how companies can adapt them to their own circumstances. Bearing this in mind, this study analysed the problem of the definition of risk acceptance criteria for occupational settings, defining the quantitative acceptance criteria for the specific case study of the Portuguese furniture industrial sector. The key steps to be considered in formulating acceptance criteria were analysed in the literature review. By applying the identified steps, the acceptance criteria for the furniture industrial sector were then defined. The Cumulative Distribution Function (CDF) for the injury statistics of the industrial sector was identified as the maximum tolerable risk level. The acceptable threshold was defined by adjusting the CDF to the Occupational, Safety & Health (OSH) practitioners’ risk acceptance judgement. Adjustments of acceptance criteria to the companies’ safety cultures were exemplified by adjusting the Burr distribution parameters. An example of a risk matrix was also used to demonstrate the integration of the defined acceptance criteria into a risk metric. This work has provided substantial contributions to the issue of acceptance criteria for occupational accidents, which may be useful in overcoming the practical difficulties faced by authorities, companies and experts.

Evolution of the surface plasmon resonance of Au:TiO2 nanocomposite thin films with annealing temperature

This paper reports on the changes in the structural and morphological features occurring in a particular type of nanocomposite thin-film system, composed of Au nanoparticles (NPs) dispersed in a host TiO2 dielectric matrix. The structural and morphological changes, promoted by in-vacuum annealing experiments of the as-deposited thin films at different temperatures (ranging from 200 to 800 C), resulted in a well-known localized surface plasmon resonance (LSPR) phenomenon, which gave rise to a set of different optical responses that can be tailored for a wide number of applications, including those for optical-based sensors. The results show that the annealing experiments enabled a gradual increase of the mean grain size of the Au NPs (from 2 to 23 nm), and changes in their distributions and separations within the dielectric matrix. For higher annealing temperatures of the as-deposited films, a broad size distribution of Au NPs was found (sizes up to 100 nm). The structural conditions necessary to produce LSPR activity were found to occur for annealing experiments above 300 C, which corresponded to the crystallization of the gold NPs, with an average size strongly dependent on the annealing temperature itself. The main factor for the promotion of LSPR was the growth of gold NPs and their redistribution throughout the host matrix. On the other hand, the host matrix started to crystallize at an annealing temperature of about 500 C, which is an important parameter to explain the shift of the LSPR peak position to longer wavelengths, i.e. a red-shift.

Bacterial cellulose as a novel substrate for retinal pigment epithelium cells transplantation in age-related macular degeneration

Tese de Doutoramento em Engenharia Química e Biológica.

Poly-N-acetyl glucosamine expression in Staphylococcus epidermidis biofilm cells affects the immune response and promotes tissue pathology in infected hosts

Staphylococcus epidermidis is a biofilm - forming bacterium and a leading etiological agent of nosocomial infections. The ability to establish biofilms on indwelling medical devices is a key virulence factor for this bacterium. Still, the influence of poly - N - acetyl glucosamine (PNAG), the major component of the extracellular biofilm matrix, in the host immune response has been scarcely studied. Here, t h is influence was assessed in mice challenged i.p. with PNAG - p roducing (WT) and isogenic - mutant lacking PNAG (M10) bacteria grown in biofilm - inducing conditions. Faster bacterial clearance was observed in the mice infected with WT bacteria than in M10 - infected counterparts , which w as accompanied by earlier neutrophil recruitment and higher IL - 6 production. Interestingly, in the WT - infected mice, but not in those infected with M10 , elevated serum IL - 10 was detected . To further study the effe ct of PNAG in the immune response, mice were primed with WT or M10 biofilm bacteria and subsequently infected with WT biofilm - released cells. WT - primed mice presented a higher frequency of splenic IFN - γ + and IL - 17 + CD4 + T cells, and more severe liver patho logy than M10 - primed counterparts. Nevertheless, T reg cells obtained from the WT - primed mice presented a higher suppressive function than those obtained from M10 - primed mice. This effect was abrogated when IL - 10 - deficient mice were similarly primed and infected indicating that PNAG promotes the differentiati on of highly suppressive T reg cells by a mechanism dependent on IL - 10. Altogether, these results provide evidence help ing explain ing the coexistence of inflammation and bacterial persistence often observed in biofilm - originated S. epidermidis infections

The role of deviant-letter position in cognate word processing

Dissertação de mestrado integrado em Psicologia

Gd2O3: Eu Nanoparticle-Based Poly(vinylidene fluoride) Composites for Indirect X-ray Detection

Polymer based scintillator composites have been fabricated by combining poly(vinylidene fluoride) (PVDF) and Gd2O3:Eu nanoparticles (50nm). PVDF has been used since it is a flexible and stable binder matrix and highly resistance to thermal and light deterioration. Gd2O3:Eu has been selected as scintillator material due to its wide band gap, high density and suitable visible light yield. The structural, mechanical, thermal and electrical characteristics of the composites were studied as a function of filler content, together with their performance as scintillator material. The introduction of Gd2O3:Eu nanoparticles into the PVDF matrix does not influence the morphology of the polymer or the degree of crystallinity. On the other hand, an increase of the Young´s modulus with respect to PVDF matrix is observed for filler contents of 0.1-0.75 wt.%. The introduction of Gd2O3:Eu into the PVDF matrix increases dielectric constant and DC electrical conductivity as well as the visible light yield in the nanocomposite, being this increase dependent upon Gd2O3:Eu content and X-ray input power. In this way, Gd2O3:Eu/PVDF composites shows suitable characteristics to be used as X-ray radiation transducers, in particular for large area applications.

Energy harvesting performance of BaTiO3/poly(vinylidene fluoride-trifluoroethylene) spin coated nanocomposites

The energy harvesting efficiency of poly(vinylidene fluoride-trifluoroethylene) spin coated films and its nanocomposites with piezoelectric BaTiO3 have been investigated as a function of ceramic filler size and content. It is found that the best energy harvesting performance of ~0.28 W is obtained for the nanocomposite samples with 20% filler content of 10 nm size particles and for 5% filler content for the 100 and 500 nm size fillers. For the larger filler average sizes, the power decreases for filler contents above 5% due to increase of the mechanical stiffness of the samples. Due to the similar dielectric characteristics of the samples, the performance is mainly governed by the mechanical response. The obtained power values, easy processing and the low cost and robustness of the polymer, allow the implementation of the material for micro and nanogenerator applications.

Gd2O3: Eu3+/PPO/POPOP/PS composites for digital imaging radiation detectors

Polymer based scintillator composites have been produced by combining polystyrene (PS) and Gd2O3:Eu3+ scintillator nanoparticles. Polystyrene has been used since it is a flexible and stable binder matrix, resistant to thermal and light deterioration and with suitable optical properties. Gd2O3:Eu3+ has been selected as scintillator material due to its wide band gap, high density and visible light yield. The optical, thermal and electrical characteristics of the composites were studied as a function of filler content, together with their performance as scintillator material. Additionally 1wt.% of 2,5 dipheniloxazol (PPO) and 0.01wt.% of (1,4-bis(2-(5-phenioxazolil))-benzol (POPOP) were introduced in the polymer matrix in order to strongly improve light yield, i.e. the measured intensity of the output visible radiation, under X-ray irradiation. Whereas increasing scintillator filler concentration (from 0.25wt.% to 7.5wt.%) increases scintillator light yield, decreases the optical transparency of the composite. The addition of PPO and POPOP, strongly increased the overall 2 transduction performance of the composite due to specific absorption and re-emission processes. It is thus shown that Gd2O3:Eu3+/PPO/POPOP/PS composites in 0.25 wt.% of scintillator content with fluorescence molecules is suitable for the development of innovate large area X-ray radiation detectors with huge demand from the industries.

Effect of polymeric matrix on morphology and photoelectric properties of polymer-quantum dots nanocomposites

Recently, CdTe semiconductor quantum dots (QDs) have attracted great interest due to their unique properties [1]. Their dispersion into polymeric matrices would be very for several optoelectronics applications. Despite its importance, there has been relatively little work done on charge transport in the QD polymeric films [2], which is mainly affected by their structural and morphological properties. In the present work, polymer-quantum dot nanocomposites films based on optically transparent polymers in the visible spectral range and CdTe QDs with controlled particle size and emission wavelength, were prepared via solvent casting. Photoluminescent (PL) measurements indicate different emission intensity of the nanocomposites. A blue shift of the emission peak compared to that of QDs in solution occurred, which is attributed to the QDs environment changes. The morphological and structural properties of the CdTe nanocomposites were evaluated. Since better QDs dispersion was achieved, PMMA seemed to be the most promising matrix. Electrical properties measurements indicate an ohmic behavior.

Measurement of the inclusive jet cross-section in proton--proton collisions at s√=7 TeV using 4.5 fb−1 of data with the ATLAS detector

The inclusive jet cross-section is measured in proton--proton collisions at a centre-of-mass energy of 7 TeV using a data set corresponding to an integrated luminosity of 4.5 fb−1 collected with the ATLAS detector at the Large Hadron Collider in 2011. Jets are identified using the anti-kt algorithm with radius parameter values of 0.4 and 0.6. The double-differential cross-sections are presented as a function of the jet transverse momentum and the jet rapidity, covering jet transverse momenta from 100 GeV to 2 TeV. Next-to-leading-order QCD calculations corrected for non-perturbative effects and electroweak effects, as well as Monte Carlo simulations with next-to-leading-order matrix elements interfaced to parton showering, are compared to the measured cross-sections. A quantitative comparison of the measured cross-sections to the QCD calculations using several sets of parton distribution functions is performed.

Measurement of the tt¯ production cross-section as a function of jet multiplicity and jet transverse momentum in 7 TeV proton--proton collisions with the ATLAS detector

The tt¯ production cross-section dependence on jet multiplicity and jet transverse momentum is reported for proton--proton collisions at a centre-of-mass energy of 7 TeV in the single-lepton channel. The data were collected with the ATLAS detector at the CERN Large Hadron Collider and comprise the full 2011 data sample corresponding to an integrated luminosity of 4.6 fb−1. Differential cross-sections are presented as a function of the jet multiplicity for up to eight jets using jet transverse momentum thresholds of 25, 40, 60, and 80 GeV, and as a function of jet transverse momentum up to the fifth jet. The results are shown after background subtraction and corrections for all detector effects, within a kinematic range closely matched to the experimental acceptance. Several QCD-based Monte Carlo models are compared with the results. Sensitivity to the parton shower modelling is found at the higher jet multiplicities, at high transverse momentum of the leading jet and in the transverse momentum spectrum of the fifth leading jet. The MC@NLO+HERWIG MC is found to predict too few events at higher jet multiplicities.

Nonparametric estimation of the survival function for ordered multivariate failure time data: a comparative study

In longitudinal studies of disease, patients may experience several events through a follow-up period. In these studies, the sequentially ordered events are often of interest and lead to problems that have received much attention recently. Issues of interest include the estimation of bivariate survival, marginal distributions and the conditional distribution of gap times. In this work we consider the estimation of the survival function conditional to a previous event. Different nonparametric approaches will be considered for estimating these quantities, all based on the Kaplan-Meier estimator of the survival function. We explore the finite sample behavior of the estimators through simulations. The different methods proposed in this article are applied to a data set from a German Breast Cancer Study. The methods are used to obtain predictors for the conditional survival probabilities as well as to study the influence of recurrence in overall survival.

Thin films composed of gold nanoparticles dispersed in a dielectric matrix: the influence of the host matrix on the optical and mechanical responses

Gold nanoparticles were dispersed in two different dielectric matrices, TiO2 and Al2O3, using magnetron sputtering and a post-deposition annealing treatment. The main goal of the present work was to study how the two different host dielectric matrices, and the resulting microstructure evolution (including both the nanoparticles and the host matrix itself) promoted by thermal annealing, influenced the physical properties of the films. In particular, the structure and morphology of the nanocomposites were correlated with the optical response of the thin films, namely their localized surface plasmon resonance (LSPR) characteristics. Furthermore, and in order to scan the future application of the two thin film system in different types of sensors (namely biological ones), their functional behaviour (hardness and Young's modulus change) was also evaluated. Despite the similar Au concentrations in both matrices (~ 11 at.%), very different microstructural features were observed, which were found to depend strongly on the annealing temperature. The main structural differences included: (i) the early crystallization of the TiO2 host matrix, while the Al2O3 one remained amorphous up to 800 °C; (ii) different grain size evolution behaviours with the annealing temperature, namely an almost linear increase for the Au:TiO2 system (from 3 to 11 nm), and the approximately constant values observed in the Au:Al2O3 system (4–5 nm). The results from the nanoparticle size distributions were also found to be quite sensitive to the surrounding matrix, suggesting different mechanisms for the nanoparticle growth (particle migration and coalescence dominating in TiO2 and Ostwald ripening in Al2O3). These different clustering behaviours induced different transmittance-LSPR responses and a good mechanical stability, which opens the possibility for future use of these nanocomposite thin film systems in some envisaged applications (e.g. LSPR-biosensors).