Chondrogenic differentiation within magnetic-responsive multilayered liquified capsules containing collagen II/TFG-β3 coated microparticles

The use of stem cells is a promising therapeutic approach for the substantial challenge to regenerate cartilage. Considering the two prerequisites, namely the use of a 3D system to enable the chondrogenic differentiation and growth factors to avoid dedifferentiation, the diffusion efficiency of essential biomolecules is an intrinsic issue. We already proposed a liquified bioencapsulation system containing solid microparticles as cell adhesion sites1. Here, we intend to use the optimized system towards chondrogenic differentiation by encapsulating stem cells and collagenII-TGF-β3 PLLA microparticles. As a proof-of-concept, magnetite-nanoparticles were incorporated into the multilayered membrane. This can be a great advantage after implantation procedures to fixate the capsules in situ with the held of an external magnetic patch and for the follow-up through imaging. Results showed that the production of glycosaminoglycans and the expression of cartilage-relevant markers (collagen II, Sox9, aggrecan, and COMP) increased up to 28 days, while hypertrophic (collagen X) and fibrotic (collagen I) markers were downregulated. The presence of nanofibers in the newly deposited ECM was visualized by SEM, which resembles the collagen fibrils of native cartilage. The presence of the major constituent of cartilage, collagen II, was detected by immunocytochemistry and afranin-O and alcian blue stainings revealed a basophilic ECM deposition, which is characteristic of neocartilage. These findings suggest that the proposed system may provide a suitable environment for chondrogenic differentiation.

Osteogenic differentiation of adipose stem cells by endothelial cells co-culture within liquified capsules

Inspired by the native co-existence of multiple cell types and from the concept of deconstructing the stem cell niche, we propose a co-encapsulation strategy within liquified capsules. The present team has already proven the application of liquified capsules as bioencapsulation systems1. Here, we intend to use the optimized system towards osteogenic differentiation. Capsules encapsulating adipose stem cells alone (MONO-capsules) or in co-culture with endothelial cells (CO-capsules) were maintained in endothelial medium with or without osteogenic differentiation factors. The suitability of the capsules for living stem and endothelial cells encapsulation was demonstrated by MTS and DNA assays. The osteogenic differentiation was assessed by quantifying the deposition of calcium and the activity of ALP up to 21 days. CO capsules had an enhanced osteogenic differentiation, even when cultured in the absence of osteogenic factors. Furthermore, osteopontin and CD31 could be detected, which respectively indicate that osteogenic differentiation had occurred and endothelial cells maintained their phenotype. An enhanced osteogenic differentiation by co-encapsulation was also confirmed by the upregulation of osteogenic markers (BMP-2, RUNX2, BSP) while the expression of angiogenic markers (VEGF, vWF, CD31) revealed the presence of endothelial cells. The proposed capsules can also act as a growth factor release system upon implantation, as showed by VEGF and BMP-2 quantification. These findings demonstrate that the co-encapsulation of stem and endothelial cells within liquified injectable capsules provides a promising strategy for bone tissue engineering.  

Silica nanoparticles as dexamethasone delivery systems able to induce the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Bioactive glasses, especially silica-based materials, are reported to pres- ent osteoconductive and osteoinductive properties, fundamental char- acteristics in bone regeneration [1,2]. Additionally, dexamethasone (Dex) is one of the bioactive agents able to induce the osteogenic differ- entiation of mesenchymal stem cells by increasing the alkaline phos- phatase activity, and the expression levels of Osteocalcin and Bone Sialoprotein [3]. Herein, we synthesised silica (SiO2) nanoparticles (that present inherent bioactivity and ability to act as a sustained drug delivery system), and coated their surface using poly-L-lysine (PLL) and hyaluronic acid (HA) using the layer-by-layer processing technique. Further on, we studied the influence of these new SiO2-polyelectrolyte coated nanoparticles as Dex sustained delivery systems. The SiO2 nanoparticles were loaded with Dex (SiO2-Dex) and coated with PLL and HA (SiO2-Dex-PLL-HA). Their Dex release profile was evaluated and a more sustained release was obtained with the SiO2-Dex-PLL-HA. All the particles were cultured with human bone marrow-derived mes- enchymal stem cells (hBMSCs) under osteogenic differentiation culture conditions. hBMSCs adhered, proliferated and differentiated towards the osteogenic lineage in the presence of SiO2 (DLS 174nm), SiO2-Dex (DLS 175nm) and SiO2-Dex-PLL-HA (DLS 679nm). The presence of these materials induced the overexpression of osteogenic transcripts, namely of Osteocalcin, Bone Sialoprotein and Runx2. Scanning Elec- tron Microscopy/Electron Dispersive Spectroscopy analysis demon- strated that hBMSCs synthesised calcium phosphates when cultured with SiO2-Dex and SiO2-Dex-PLL-HA nanoparticles. These results indi- cate the potential use of these SiO2-polyelectrolytes coated nanoparti- cles as dexamethasone delivery systems capable of promoting osteogenic differentiation of hBMSCs.

Enhancement of adhesion and promotion of osteogenic differentiation of human adipose stem cells by poled electroactive poly(vinylidene fluoride)

Poly(vinylidene fluoride) (PVDF) is a biocompatible material with excellent electroactive properties. Non-electroactive α-PVDF and electroactive β-PVDF were used to investigate the substrate polarization and polarity influence on the focal adhesion size and number as well as on human adipose stem cells (hASCs) differentiation. hASCs were cultured on different PVDF surfaces adsorbed with fibronectin and focal adhesion size and number, total adhesion area, cell size, cell aspect ratio and focal adhesion density were estimated using cells expressing EGFP-vinculin. Osteogenic differentiation was also determined using a quantitative alkaline phosphatase assay. The surface charge of the poled PVDF films (positive or negative) influenced the hydrophobicity of the samples, leading to variations in the conformation of adsorbed extracellular matrix (ECM) proteins, which ultimately modulated the stem cell adhesion on the films and induced their osteogenic differentiation.

Dynamic piezoelectric stimulation enhances osteogenic differentiation of human adipose stem cells

This work reports on the influence of the substrate polarization of electroactive β-PVDF on human adipose stem cells (hASCs) differentiation under static and dynamic conditions. hASCs were cultured on different β-PVDF surfaces (non-poled and “poled -”) adsorbed with fibronectin and osteogenic differentiation was determined using a quantitative alkaline phosphatase assay. “Poled -” β-PVDF samples promote higher osteogenic differentiation, which is even higher under dynamic conditions. It is thus demonstrated that electroactive membranes can provide the necessary electromechanical stimuli for the differentiation of specific cells and therefore will support the design of suitable tissue engineering strategies, such as bone tissue engineering.

Biochemical and physical surface functionalization of polycaprolactone as a key mediator of osteogenic differentiation and vascularized tissue morphogenesis

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais.

Examining mindfulness and its relation to self-differentiation and alexithymia

Published online first in 10 July 2013

Osteogenic differentiation of human mesenchymal stem cells in the absence of osteogenic supplements: a surface-roughness gradient study

The use of biomaterials to direct osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of osteogenic supplements is thought to be part of the next generation of orthopedic implants. We previously engineered surface-roughness gradients of average roughness (Ra) varying from the sub-micron to the micrometer range ( 0.5–4.7 lm), and mean distance between peaks (RSm) gradually varying from 214 lm to 33 lm. Here we have screened the ability of such surface-gradients of polycaprolactone to influence the expression of alkaline phosphatase (ALP), collagen type 1 (COL1) and mineralization by hMSCs cultured in dexamethasone (Dex)-deprived osteogenic induction medium (OIM) and in basal growth medium (BGM). Ra 1.53 lm/RSm 79 lm in Dex-deprived OI medium, and Ra 0.93 lm/RSm 135 lm in BGM consistently showed higher effectiveness at supporting the expression of the osteogenic markers ALP, COL1 and mineralization, compared to the tissue culture polystyrene (TCP) control in complete OIM. The superior effectiveness of specific surface-roughness revealed that this strategy may be used as a compelling alternative to soluble osteogenic inducers in orthopedic applications featuring the clinically relevant biodegradable polymer polycaprolactone.

Semipermeable capsules wrapping a multifunctional and self-regulated co-culture microenvironment for osteogenic differentiation

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the â stem cell nicheâ , the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

Self-esteem, assertiveness and resilience in adolescents institutionalized

The institutionalization of children and adolescents has been an increasingly visible problem in modern society. Unfavourable socio-economic conditions have been joining the behavior problems and school absenteeism. When the family fails in its competence for education, social security or the Court withdraws the child or adolescent to a host institution. The aim of this research was to characterize self-esteem, assertiveness and resilience of institutionalized adolescents in the northern region of Portugal and to establish associations with these dependent variables and gender, scholar level and duration of the institutionalization. For the purpose of this study a wider questionnaire was carried out, and validated with a smaller group. It was a transversal study following a predominantly quantitative methodology, with a convenience sample. The sample included 101 adolescents (55 female and 46 males) from eight institutions, aged between 11 to 21 years old (average 15.45). For self-esteem the Rosenberg Self-Esteem Scale (Rosenberg, 1965), already validate for Portuguese adolescents, was used. For assertiveness and resilience it was applied the Global Evaluation Scale of Assertiveness and the Global Evaluation Scale of Resilience (Jardim & Pereira, 2006) we previously adapted and validated for adolescents. Collected data was introduced in a SPSS database. A descriptive analysis was done to characterize the sample concerning all the variables. To establish associations between individual factors and dependent variables t test, correlations and non-parametric test were applied. Results indicated a relatively low self-esteem (28.03), with girls having a lower value than boys, without significant differences. No correlations were found between self-esteem and the time in the institution. Assertiveness of the sample is average (23.97) and higher for girls than boys, with a positive significant correlation with the scholar grade. Also the resilience is average (25.97), having girls a little lower mean than boys and no significant differences or correlations were found.

Intervir através da educação física na coeducação: realidade ou intenção?

Intervir de forma consciente e organizada em prol da promoção da igualdade de oportunidades na escola tem sido, nas últimas décadas, um dos propósitos da Educação Física (EF) na Europa. Através das rotinas e das oportunidades que se proporcionam, rapazes e raparigas têm acesso a uma prática de atividade física e desportiva (AFD) que lhes possibilita aperfeiçoar capacidades, competências e atitudes. Consideramos que o contributo da EF na educação é de um valor significativo, viabilizando às crianças e jovens construir a sua imagem corporal e os estereótipos de género, desenvolver afectos e emoções, cultivar a sua relação com os outros, criar capacidades e valores, ajudando deste modo uma formação das atitudes individuais ou coletivas, que respeitem a igualdade de género e, por isso, os direitos humanos. Este estudo foi realizado a partir da informação recolhida, através da aplicação de um questionário, a uma amostra de 993 crianças, 57.5% raparigas e 42.5% rapazes, com idades compreendidas entre os 10 e 12 anos, pertencentes a várias escolas do ensino público e ambicionou comparar as perceções e preferências de rapazes e raparigas relativamente às práticas AFD que decorrem na disciplina de EF, para se poderem apresentar alternativas de práticas mais flexíveis e integradoras. A partir dos nossos resultados podemos concluir que a AFD na escola continua a ser muito marcada por práticas de técnicas de movimento, jogos e competições desportivas, vivências muito identificadas com os estereótipos masculinos e que, nem sempre ajudam à formação de um bom esquema corporal e de comportamentos que se tendem a manter ao longo da vida, para muita(o)s jovens. Sugerimos que as atividades oferecidas às crianças e jovens nas escolas sejam mais diversificadas e inclusivas, impondo um nível de desempenho e de satisfação mais adequado a cada caso em particular, devendo estar mais centradas quer em atividades de ar livre, quer em práticas que desenvolvem também as dimensões sensitiva, expressiva e corporal do(a)s aluno(a)s.

O uso do Facebook e as emoções experienciadas pelas crianças na óptica dos seus professores

Atualmente a rede social mais visitada em todo o mundo é o Facebook. Além de nos permitir ter um perfil pessoal podemos partilhar fotos, vídeos, mensagens e outros ficheiros. No Facebook também é permitido criar grupos fechados onde os seus membros podem interagir através de mensagens escritas, de áudio, de vídeo e/ou imagens. Tanto esta rede social quanto os grupos dentro da mesma podem ser usados de forma indevida, mas, por outro lado, também podem ser utilizados para aprender, aprender a fazer, conhecer, desenvolver a linguagem escrita e outras competências. O nosso estudo teve como objetivo conhecer a perceção de educadores de infância e professores de primeiro ciclo do ensino básico relativamente ao uso do Facebook por parte das crianças, bem como às emoções que as crianças manifestam neste uso. Foi um estudo transversal, para o qual construímos e validámos um questionário específico, com questões fechadas sobre o uso da respetiva rede social. O instrumento foi disponibilizado e preenchido online através do Google Forms. A amostra foi de conveniência, tendo sido constituída por 75 indivíduos (69 mulheres, 6 homens) com idades compreendidas entre os 25 e os 56 anos. Os resultados evidenciam que os professores e educadores discordam essencialmente que as crianças aceitem pedidos de amizade e comuniquem pelo chat do Facebook com pessoas que não conhecem, concordando mais que usem para comunicar com familiares e amigos, para aderir a grupos com o objetivo de aprender e que permitem o acesso dos adultos à sua conta. No respeitante as emoções e sentimentos registou-se maior discordância quanto ao facto de as crianças preferirem estar no Facebook do que na presença dos seus amigos e de se sentirem dependentes do uso do mesmo. Porém, observou-se o maior nível de concordância para o facto de ficarem aborrecidas se as obrigarem a sair do Facebook.

All different or all the same? Exploring the diversity of professional practices in Portuguese school psychology

"Published online: 29 March 2016"

Study of validation of the Portuguese version of the inventory «Experiences in Close Relationships»

The Experiences in Close Relationships Inventory permits to evaluate attachment in close relationships during adulthood based on two dimensions able to be present in this kind of relationships: the avoidance of proximity and the anxiety related with to abandonment. It is a self-report 7- points likert scale composed by 36 items. The Portuguese version was administered to a sample of 551 university students (60% female), the majority with ages between 19 and 24 years old (88%) in a dating relationship (86%). The principal components analysis with oblimin rotation was performed. The total scale has good internal consistency (α=.86), as also has the 2 sub-scales: anxiety (α=.86) and avoidance (α=.88). The two dimensions evaluated are significantly correlated with socio-demographics, relational characteristics (jealousy, relationship distress, and compromise), wishes (enmeshment versus differentiation) and fears (abandonment versus control) related to attitudes in significant relationships, which testify the construct validity of the instrument. The results obtained are coherent with the original version and other ECR‘s adaptations. Practitioners and researchers in the context of clinical psychology and related areas have now at their disposal the Portuguese version of the ECR inventory, which has shown its very high usefulness in the study of close relationships, and specifically attachment in adulthood.