Susana Faria

Surface modification of electrospun polycaprolactone nanofiber meshes by plasma treatment to enhance biological performance

A critical aspect in the development of biomaterials is the optimization of their surface properties to achieve an adequate cell response. In the present work, electrospun polycaprolactone nanofiber meshes (NFMs) are treated by radio-frequency (RF) plasma using different gases (Ar or O(2)), power (20 or 30 W), and exposure time (5 or 10 min). Morphological and roughness analysis show topographical changes on the plasma-treated NFMs. X-ray photoelectron spectroscopy (XPS) results indicate an increment of the oxygen-containing groups, mainly --OH and --C==O, at the plasma-treated surfaces. Accordingly, the glycerol contact angle results demonstrate a decrease in the hydrophobicity of plasma-treated meshes, particularly in the O(2)-treated ones. Three model cell lines (fibroblasts, chondrocytes, and osteoblasts) are used to study the effect of plasma treatments over the morphology, cell adhesion, and proliferation. A plasma treatment with O(2) and one with Ar are found to be the most successful for all the studied cell types. The influence of hydrophilicity and roughness of those NFMs on their biological performance is discussed. Despite the often claimed morphological similarity of NFMs to natural extracellular matrixes, their surface properties contribute substantially to the cellular performance and therefore those should be optimized.

Osteogenic induction of hBMSCs by electrospun scaffolds with dexamethasone release functionality.

Electrospun structures were proposed as scaffolds owing to their morphological and structural similarities with the extracellular matrix found in many native tissues. These fibrous structures were also proposed as drug release systems by exploiting the direct dependence of the release rate of a drug on the surface area. An osteogenic differentiation factor, dexamethasone (DEX), was incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (5, 10, 15 and 20 wt.% polymer), in a single-step process. The DEX incorporated into the polymeric carrier is in amorphous state, as determined by DSC, and does not influence the typical nanofibers morphology. In vitro drug release studies demonstrated that the dexamethasone release was sustained over a period of 15 days. The bioactivity of the released dexamethasone was assessed by cultivating human bone marrow mesenchymal stem cells (hBMSCs) on 15 wt.% DEX-loaded PCL NFMs, under dexamethasone-absent osteogenic differentiation medium formulation. An increased concentration of alkaline phosphatase and deposition of a mineralized matrix was observed. Phenotypic and genotypic expression of osteoblastic-specific markers corroborates the osteogenic activity of the loaded growth/differentiation factor. Overall data suggests that the electrospun biodegradable nanofibers can be used as carriers for the sustained release of growth/differentiation factors relevant for bone tissue engineering strategies.

Cartilage Tissue Engineering Using Electrospun PCL Nanofiber Meshes and MSCs

Mesenchymal stem cells (MSCs) have been recognized for their ability to differentiate into cells of different tissues such as bone, cartilage, or adipose tissue, and therefore are of great interest for potential therapeutic strategies. Adherent, colony-forming, fibroblastic cells were isolated from human bone marrow aspirates, from patients undergoing knee arthroplasties, and the MSCs phenotype characterized by flow cytometry. Afterward, cells were seeded onto electrospun polycaprolactone nanofiber meshes and cultured in a multichamber flow perfusion bioreactor to determine their ability to produce cartilagineous extracellular matrix. Results indicate that the flow perfusion bioreactor increased the chondrogenic differentiation of hBM-MSCs, as confirmed either by morphological and RT-PCR analysis. Cartilage-related genes such as aggrecan, collagen type II, and Sox9 were expressed. ECM deposition was also detected by histological procedures. Collagen type II was present in the samples, as well as collagen type I. Despite no statistically significant values being obtained for gene expression, the other results support the choice of the bioreactor for this type of culture.

Chondrogenic differentiation of human bone marrow mesenchymal stem cells in chitosan-based scaffolds using a flow-perfusion bioreactor.

Native articular cartilage is subjected to synovial fluid flow during normal joint function. Thus, it is believed that the morphogenesis of articular cartilage may be positively regulated by the application of similar stimulation in vitro. In the present study, the effect of fluid flow over the chondrogenic differentiation of human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) was investigated. We intended to find out whether the shear stress caused by perfusion of the medium through the constructs was capable of augmenting the differentiation process. Human BMSCs were isolated from bone marrow aspirates and were characterized by flow cytometry. After expansion, hBM‐MSCs were seeded statically onto fibre mesh scaffolds, consisting of a blend of 50:50 chitosan:poly(butylene terephthalate adipate) (CPBTA). Constructs were cultured in a flow‐perfusion bioreactor for 28 days, using complete medium for chondrogenesis supplemented by TGFβ3. An enhanced ECM deposition and collagen type II production was observed in the bioreactor samples when compared to the static controls. Moreover, it was observed that hBM‐MSCs, in static cultures, take longer to differentiate. ECM accumulation in these samples is lower than in the bioreactor sections, and there is a significant difference in the expression of collagen type I. We found that the flow‐induced shear stress has a beneficial effect on the chondrogenic differentiation of hMSCs. Copyright

Solving cell infiltration limitations of electrospun nanofiber meshes for tissue engineering applications

AIM Utilize the dual composition strategy to increase the pore size and solve the low cell infiltration capacity on random nanofiber meshes, an intrinsic limitation of electrospun scaffolds for tissue engineering applications. MATERIALS & METHODS Polycaprolactone and poly(ethylene oxide) solutions were electrospun simultaneously to obtain a dual composition nanofiber mesh. Selective dissolution of the poly(ethylene oxide) nanofiber fraction was performed. The biologic performance of these enhanced pore size nanofibrous structures was assessed with human osteoblastic cells. RESULTS The electrospun nanofiber meshes, after the poly(ethylene oxide) dissolution, showed statistically significant larger pore sizes when compared with polycaprolactone nanofiber meshes with a similar polycaprolactone volume fraction. This was also confirmed by interferometric optical profilometry. Using scanning electron microscopy and laser scanning confocal microscopy, it was observed that osteoblastic cells could penetrate into the nanofibrous structure and migrate into the opposite and unseeded side of the mesh. CONCLUSION An electrospun mesh was created with sufficient pore size to allow cell infiltration into its structure, thus resulting in a fully populated construct appropriate for 3D tissue engineering applications.

The Influence of Patterned Nanofiber Meshes on Human Mesenchymal Stem Cell Osteogenesis

A specially designed electroconductive collector enables the electrospinning of P-NFM composed of areas of parallel/uniaxially aligned fibers and areas of random/orthogonal nanofiber distribution. The biological relevance of P-NFM is demonstrated using hBMSCs as an autologous cell source. The structures induce cell orientation along the uniaxially aligned fibers, mainly during earlier culturing periods under basal and osteogenic differentiation conditions. The microtopography of the P-NFM also controls the deposition of mineralized extracellular matrix along the pre-defined fiber direction. Genotypic characterization confirms the successful differentiation into the osteogenic lineage.

Fitting mixtures of linear regressions

In most applications, the parameters of a mixture of linear regression models are estimated by maximum likelihood using the expectation maximization (EM) algorithm. In this article, we propose the comparison of three algorithms to compute maximum likelihood estimates of the parameters of these models: the EM algorithm, the classification EM algorithm and the stochastic EM algorithm. The comparison of the three procedures was done through a simulation study of the performance (computational effort, statistical properties of estimators and goodness of fit) of these approaches on simulated data sets. Simulation results show that the choice of the approach depends essentially on the configuration of the true regression lines and the initialization of the algorithms.

Immobilization of bioactive factor-loaded liposomes on the surface of electrospun nanofibers targeting tissue engineering

Electrospun nanofiber meshes (NFM), due to their morphology and fibrous structure, are extensively proposed as biomedical devices, for tissue engineering on scaffolds and also as drug delivery systems. Liposomes are nanoparticles prepared from a biologically derived material (phospholipid), which are already in clinical use as a drug release device. Liposomes may be combined with biomaterial scaffolds to promote a local and sustained delivery of loaded bioactive agents. The main objective of the present study is to evaluate the efficacy of dexamethasone (Dex)-loaded liposomes immobilized on the surface of electrospun polycaprolactone (PCL) NFM for promoting the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). The in vitro release profile demonstrates a sustained release of Dex over 21 days, after an initial burst release over 12 h. Biological assays show that Dex-loaded liposomes immobilized on the surface of electrospun PCL NFMs do not exhibit any cytotoxic effect, being able to successfully promote the osteogenic differentiation of hBMSCs. We herein validate the concept of using liposomes immobilized on the surface of a nanostructured fibrous system to be used as an advanced cell carrier device with autonomous release of growth/differentiation factors relevant for tissue engineering and regenerative medicine strategies.

Cognitive Appraisal as a Mediator in the Relationship Between Stress and Burnout

The relation between job stressors and burnout is well established in the literature. However, the mechanisms behind this relationship are still not clear. This study has the main goal of analysing the mediating role of cognitive appraisal in the relation between occupational stress and burnout. Cognitive appraisals comprise primary appraisals, which are of the significance of a situation to the individual, and secondary appraisals, which involve evaluating the individuals ability to cope with stressors. To test the relationship between appraisals, stress and burnout, structural equation modelling was used in a sample of academic teaching staff (N = 333) working at a public university in Portugal. The participants completed a survey with measures that included their level of stress, cognitive appraisals of aspects of their work, and the Maslach Burnout Inventory–Educators Survey. The results indicated perceptions of distinct sources of stress on their work activity and a relation between stress, cognitive appraisal and burnout. Most importantly, the results showed that primary and secondary cognitive appraisals partially mediated the relationship between occupational stress and burnout at work, making the relationship between these variables a promising underlying mechanism for explaining responses to work-related stress.

Hot pressing effect on the shear bond strength of dental porcelain to CoCrMoSi alloy substrates with different surface treatments

The purpose of this study was to evaluate the effect of hot pressing on the shear bond strength of a CoCrMoSi alloy to a low-fusing feldspathic porcelain, for two types of surface treatments: polished and grit-blasted. Moreover, the shear strength of hot pressed porcelain was also compared with that of conventional vacuum sintered porcelain. Bond strength of metal-porcelain composites were assessed by the means of a shear test performed in a universal test machine until fracture. Fracture surfaces and interfaces were investigated by optical microscope, stereomicroscope and SEM/EDS. Data was analyzed with Shapiro-Wilk test to test the assumption of normality. The 2-way ANOVA followed by Tukey HSD multiple comparison test was used to compare shear bond strength results and the t-test was used to compare the porcelain shear strength (p<0.05). Hot pressed specimens exhibited significantly (p<0.001) higher bond strength values than those obtained by conventional PFM technique. Significant differences (p<0.001) were found in the shear bond strength between grit-blasted and polished specimens. Significant differences (p<0.05) were also found between the shear strength of vacuum sintered and hot pressed porcelain. This study revealed that metal-ceramic bond strength is maximized for hot pressed porcelain onto rough metal substrates, with lower variability in results. Hot pressing technique was also shown to enhance the cohesion of porcelain.