Current concepts and challenges in osteochondral tissue engineering and regenerative medicine

"Publication Date (Web): February 20, 2015"In the last few years, great progress has been made to validate tissue engineering strategies in preclinical studies and clinical trials on the regeneration of osteochondral defects. In the preclinical studies, one of the dominant strategies comprises the development of biomimetic/bioactive scaffolds, which are used alone or incorporated with growth factors and/or stem cells. Many new trends are emerging for modulation of stem cell fate towards osteogenic and chondrogenic differentiations, but bone/cartilage interface regeneration and physical stimulus have been showing great promise. Besides the matrix-associated autologous chondrocyte implantation (MACI) procedure, the matrix-associated stem cells implantation (MASI) and layered scaffolds in acellular or cellular strategy are also applied in clinic. This review outlines the progresses at preclinical and clinical levels, and identifies the new challenges in osteochondral tissue engineering. Future perspectives are provided, e.g., the applications of extracellular matrix-like biomaterials, computer-aided design/manufacture of osteochondral implant and reprogrammed cells for osteochondral regeneration.The authors thank the Portuguese Foundation for Science and Technology (FCT) through the projects TISSUE2TISSUE (PTDC/CTM/105703/2008) and OsteoCart (PTDC/CTM-BPC/115977/2009). We also acknowledge European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement REGPOT-CT2012-316331-POLARIS. L-P.Y. acknowledges the PhD scholarship from FCT (SFRH/BD/64717/2009). The FCT distinction attributed to J.M.O. and A.L.O. under the Investigator FCT program (IF/00423/2012) and (IF/00411/2013) are also greatly acknowledged

Production de vins mousseux a partir de "Vinhos Verdes" blancs monovarietaux

On a fait l'étude comparative de vins mousseux élaborés à partir de vins de base issus de cinq cépages recommandés pour la Région: Loureiro, Trajadura, Avesso, Pedernã et Azal blanc, en ce qui concerne les propriétés organoleptiques. Les vins de base ont été produits selon le processus utilisé dans la Région -égrappage, pressurage, débourbage et fermentation alcoolique-, ayant été soumis aussi à une fermentation malolactique. La prise de mousse a été effectuée en bouteille, avec des levures immobilisées en billes d'alginate, pendant cinq mois à 14 °C. Les propriétés sensorielles des vins ont été évaluées en utilisant des fiches classificatrices et descriptives, par une chambre de neuf dégustateurs expérimentés. Le traitement statistique des résultats, effectué par le logiciel SPSS, a été fait en recourrant à l'analyse de variance. On a trouvé quelques différences parmi les cinq vins mousseux surtout en ce qui concerne l'aspect du cordon et l'arôme. Cependant, tous ces vins ont obtenu des classifications globales au-dessus de l'acceptable, atteignant parfois l'excellent. Ces résultats préliminaires ainsi obtenus, basés sur les caractéristiques organoleptiques, suggèrent la possibilité d'obtenir des vins mousseux de qualité à partir des cépages de "Vinho Verde"

Development of a bilayered scaffold based on silk fibroin and silk fibroin/nano-calcium phosphate for osteochondral regeneration

Objectives: Osteochondral defect is a common condtion in clinic. Satisfactory outcomes are rarely achieved by traditional methods. Tissue engineering might be a promising strategy for this hinder. The aim of this study is to mimick the stratified structure of osteochondral tissue, by developing a bilayered scaffold for osteochondral regeneration. The developed bilayered scaffold is composed of a porous silk fibroin scaffold as the cartilage-like layer and a porous silk fibroin/nano-calcium phosphate (CaP) scaffold as the bone-like layer

Silk Fibroin/Nano-CaP Bilayered scaffolds for osteochondral tissue engineering

In this study, bilayered silk and silk/nano-CaP scaffolds were developed for osteochondral (OC) tissue engineering. Aqueous silk solution (16 wt.%) was used for preparation of the cartilage-like layer and, for generation of the silk/nano-CaP suspension and the bottom layer (CaP/Silk: 16 wt.%). The scaffolds were formed by using salt-leaching/lyophilization approach. The scanning electron microscopy revealed that the both layers presented porous structure and integrated well. Micro-computed tomography images confirmed that the CaP phase was only retained in the silk/nano-CaP layer. The hydration degree and mechanical properties of the bilayered scaffold were comparable to the ones of each single layer. The apatite crystal formation was limited to the silk/nano-CaP layer, when soaking the scaffold in a simulated body fluid solution, which is a must for the application of the developed scaffolds in OC tissue engineerin

Dendrimer nanoparticles for colorectal cancer applications

Accepted ManuscriptCancer nanotechnology is a prolific field of research, where nanotools are employed to diagnose and treat cancer with unprecedented precision. Targeted drug delivery is fundamental for more efficient cancer treatments. For this, nanoparticles have been extensively used during the last years in order to improve the specificity, selectivity and controlled release of drug delivery. It holds potential in minimizing systemic toxicity through the development of functionalized particles for targeted treatment. Among all the type of nanoparticles, dendrimers display several advantages, which make them ideal candidates for improved and targeted drug delivery in cancer research. Dendrimers can transport large amount of drug into specific areas. In addition, they can be employed for monitoring the progress of the treatment process, with an  unprecedented theranostic capability. Special emphasis is given in colorectal cancer, as well as the preferred employed strategies for producing drug-loaded/functionalized NPâ s for cancer therapy in the last years

New nanotechnology approaches using dendrimers modified with natural polymers for controlling stem cells behaviour in tissue engineering strategies

Tese de doutoramento em Ciência e Tecnologia de Materiais (ramo de conhecimento em Engenharia de Tecidos - Materiais Híbridos)In the recent years, great progress has been done in the emerging field of tissue engineering. Despite the important advances the performance of cells-scaffold constructs, one of the several tissue engineering approaches, remains limited in part due to the need for optimize cell culture techniques and culture media. Nanocarrier systems have generated a significant amount of interest in the ex vivo cell maintenance, and control of the cellular fate in vivo mainly due to their internalization efficiency, drug loading capacity, and to favorably modulate the solubility and pharmacokinetics of drugs. Dendrimers are synthetic, monodispersive, spherical and highly branched macromolecules that present unique advantages and fulfills most requirements as carriers for drug delivery; however, it has been found that high generation dendrimers are often cytotoxic. Thus, in this thesis we focused our attention in this fundamental problem and explore the development of novel nanobiomaterials based on the grafting of carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers, the socalled CMCht/PAMAM dendrimer nanoparticles. These macromolecular vehicles were developed to explore a new concept consisting on the intracellular and controlled delivery of bioactive molecules aimed at control stem cells functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. Thus, by combining nanotechnology-based systems and traditional tissue engineering strategies, we expect to develop a novel therapeutic solution for the efficient treatment of damage/diseased cells and tissues. To validate this new concept, there is the need to evaluate the performance of the developed nanocarriers, in vitro and in vivo. Firstly, the uptake efficiency and internalization mechanism of fluorescent-labeled CMCht/PAMAM dendrimer nanoparticles was investigated using different cell types. Fluorescence microscopy studies revealed that the developed nanocarriers could be efficiently internalized, either by cell lines and primary cultures, after a few hours. Flow cytometry studies revealed that rat bone marrow stromal cells (RBMSCs) cultured in the presence of colchicine, an alkaloid that inhibits endocytosis, decreased the internalization of the nanoparticles. These data showed that uptake by cells was primarily via an active endocytotic mechanism, but not exclusively. Preliminary studies were also carried out to evaluate the possible applicability of the CMCht/PAMAM dendrimer nanoparticles in the central nervous system. Internalization rate, cell viability and metabolic activity studies were performed using rat post-natal hippocampal neurons and cortical glial cells that revealed their ability for being taken up by these cells and its non cytotoxicity. Complementarily, dexamethasone (Dex), a glucocorticoid known to have important role on the proliferation and expression of osteoblastic differentiation markers, was used as a model drug and incorporated into the bulk of the nanoparticles. Physicochemical characterization and in vitro biological studies have demonstrated that the Dex-loaded CMCht/PAMAM dendrimer nanoparticles were successfully synthesized, were not cytotoxic in the range of concentration below 1 mg.mL-1 and promote osteogenesis (2-D system). To assess the true value of the Dex-loaded CMCht/PAMAM dendrimer nanoparticles systems for application in tissue engineering strategies, we use different biomaterials to develop a set of novel scaffolds both ceramic and polymeric or formulations. These scaffolds were found to be suitable for applications in bone, cartilage and osteochondral tissue engineering. In vitro studies have shown that combination of scaffolds, bone marrow stromal cells and Dex-loaded CMCht/PAMAM dendrimer nanoparticles (3-D system) enhanced osteogenesis. Finally, in vivo studies have shown that the novel Dex-loaded CMCht/PAMAM dendrimer nanoparticles may be beneficial as intracellular nanocarrier, supplying Dex in a regimented manner, while avoiding the need of culturing stem cells for long periods of time in vitro, towards promoting the osteogenic differentiation. Remarkably, the proposed strategy allow modulate and direct stem cells differentiation towards osteogenic phenotype, enhance in vivo proteoglycan extracellular matrix synthesis and promote superior de novo bone formation. This thesis mark the transition from the ‘proof-of-concept’ to useful intracellular nanocarrier tool, as the Dex-loaded CMCht/PAMAM dendrimer nanoparticles show promise for application in direct stem cell to a particular cell fate, in vitro and in vivo.Grandes progressos têm sido feitos nos últimos tempos no emergente campo científico da engenharia de tecidos. No entanto, a eficiência dos sistemas células-matriz tridimensional porosa, uma das estratégias usadas nas abordagens em engenharia de tecidos, tem sido limitada, em parte, pela necessidade de se optimizarem as técnicas e os meios de cultura usados. Sistemas de nanopartículas para o transporte de moléculas bioactivas têm suscitado grande interesse na área da biomedicina, nomeadamente no que respeita à possível aplicação como suplementos em meios de cultura ex vivo, e no controlo das funções celulares in vivo. Isto deve-se, principalmente, à sua eficiência de internalização, elevada capacidade de incorporação de fármacos, e ao facto de favorecerem a solubilidade de moléculas hidrofóbicas e de possibilitarem a modulação da sua farmacocinética. Os dendrímeros são sistemas macromoleculares sintéticos altamente ramificados que apresentam características únicas, tais como monodispersividade e uma estrutura esférica, e que preenchem a maioria dos requisitos para serem usados como veículos para a libertação controlada de fármacos. Não obstante, tem-se verificado que dendrímeros de elevada geração apresentam tipicamente uma citotoxicidade indesejada. Assim, nesta tese a atenção focou-se na resolução deste problema fundamental. Para tal foi explorado o desenvolvimento de novos nanobiomateriais tendo como estratégia a ligação química do polímero carboximetilquitosano (CMCht) a dendrímeros de poliamidoamina (PAMAM) de baixa geração, que se denominaram por nanopartículas de carboximetilquitosano/poliamidoamina (CMCht/PAMAM). Estas macromoléculas foram desenvolvidas e um novo conceito aplicativo foi testado relacionado com a sua aplicação em medicina regenerativa como veículos de libertação controlada e intracelular de moléculas bioactivas, de forma a ser possível controlar efectivamente as actividades celulares, tais como a proliferação e a diferenciação de células estaminais ex vivo, e a manutenção do fenótipo dessas mesmas células após o seu implante. Assim, recorrendo à nanotecnologia e a estratégias usadas na engenharia de tecidos, espera-se que seja possível desenvolver uma nova solução terapêutica que possa possibilitar, de uma forma eficiente, o tratamento de células e tecidos danificados ou que apresentem algum tipo de patologia. De forma a validar este novo conceito, é necessário avaliar o potencial dos nanosistemas desenvolvidos, in vitro e in vivo. Primeiramente, a eficiência e o mecanismo de internalização de nanopartículas de CMCht/PAMAM ligadas a um marcador fluorescente foram investigados, recorrendo a estudos celulares, in vitro. Estudos de microscopia de fluorescência revelaram que as nanopartículas desenvolvidas são internalizadas por diferentes tipos de células após algumas horas em cultura, incluindo linhas celulares e culturas primárias. Estudos envolvendo a citometria de fluxo mostraram que quando células multipotentes do estroma da medula óssea de rato (RBMSCs), foram cultivadas na presença de colchicina, um alcalóide inibidor da endocitose, exibiam menor capacidade de internalização das nanopartículas. Assim, a internalização das nanopartículas pelas células ocorre principalmente por um mecanismo de endocitose, mas que este não é o único. Foram também realizados estudos para determinar a taxa de internalização, viabilidade celular e actividade metabólica, recorrendo a neurónios isolados do hipocampo de rato pós-natais e células da glia. Estes estudos mostraram que as nanopartículas são internalizadas por estas células, e não afectam negativamente a viabilidade da cultura das células. A dexametasona (Dex), uma molécula pertencente à família dos glucocorticóides e conhecida pelo seu papel na modulação da proliferação e expressão dos marcadores de diferenciação osteoblástica, foi usada como fármaco modelo e incorporada nas nanopartículas de CMCht/PAMAM. A caracterização físico-química e estudos biológicos in vitro demonstraram que as nanopartículas de CMCht/PAMAM carregadas com Dex foram sintetizadas com sucesso, não apresentaram citotoxicidade em concentrações até 1 mg.mL-1 e promoveram a osteogénese (sistema de cultura 2-D). Por forma a avaliar o verdadeiro potencial aplicativo das nanopartículas de CMCht/PAMAM carregadas com Dex em estratégias de engenharia de tecidos, diferentes biomateriais foram usados no desenvolvimento de estruturas tridimensionais porosas, incluindo cerâmicos, polímeros e formulações contendo ambos. Estas estruturas tridimensionais porosas mostraram-se adequadas para serem usadas em engenharia de tecidos de osso, cartilagem e defeitos osteocondrais. Estudos in vitro revelaram que a abordagem constituída por estruturas tridimensionais porosas, RBMSCs e nanopartículas de CMCht/PAMAM carregadas com Dex (sistema de cultura 3-D) promoveu um aumento significativo da osteogénese. Por último, estudos in vivo mostraram que as nanopartículas de CMCht/PAMAM carregadas com Dex são um sistema de libertação intracelular altamente eficiente, uma vez que possibilitaram a libertação de Dex com um perfil cinético adequado, permitindo assim evitar os longos períodos de cultura in vitro, necessários à diferenciação osteogénica de células estaminais. A estratégia proposta permite modular e direccionar a diferenciação das células estaminais para o fenótipo osteogénico, aumentar a síntese de proteoglicanos da matriz extracelular e promover a formação de osso num estado de maturação mais avançado. Esta tese marca a transição de “prova de conceito” para uma ferramenta aplicativa das nanopartículas desenvolvidas na libertação intracelular de fármacos, uma vez que as nanopartículas de CMCht/PAMAM carregadas com Dex se mostraram promissoras no direccionamento das células estaminais para um determinado fenótipo, in vitro e in vivo.Fundação para a Ciência e a Tecnologia (FCT) - ref. SFRH/BD/21786/2005 através dos programas POCTI e FEDERRotary Club de Caldas das TaipasFundação para a Ciência e Tecnologia (FCT) - PhD grant Ref. SFRH/BD/21786/2005, POCTI, FEDER programsCanon Foundation in EuropeEuropean NoE EXPERTISSUES (NMP3-CT-2004-500283)European Union HIPPOCRATES STREP Project (NMP3-CT-2003-505758

Fundamentals and current strategies for Peripheral Nerve Repair and Regeneration

A body of evidence indicates that peripheral nerves have an extraordinary yet limited capacity to regenerate after an injury. Peripheral nerve injuries have confounded professionals in this field, from neuroscientists to neurologists, plastic surgeons, and the scientific community. Despite all the efforts, full functional recovery is still seldom. The inadequate results attained with the â gold standardâ autograft procedure still encourage a dynamic and energetic research around the world for establishing good performing tissue engineered alternative grafts. Resourcing to nerve guidance conduits, a variety of methods have been experimentally used to bridge peripheral nerve gaps of limited size, up to 30-40 mm in length, in humans. Herein, we aim to summarize the fundamentals related to peripheral nerve anatomy and overview the challenges and scientific evidences related to peripheral nerve injury and repair mechanisms. The most relevant reports dealing with the use of both synthetic and natural-based biomaterials used in tissue engineering strategies when treatment of nerve injuries is envisioned are also discussed in depth, along with the state-of-the-art approaches in this field.This work was supported by Cristiana Carvalho PhD scholarship (Norte-08-5369-FSE-000037). J. M. Oliveira also thanks the FCT for the funds provided under the program Investigador FCT 2015 (IF/01285/2015).The authors are also thankful to the FCT funded project NanoOptoNerv(ref. PTDC/NAN-MAT/29936/2017).The authors would also like to acknowledge the project: “Nano-accelerated nerve regeneration and optogenetic empowering of neuromuscular functionality” (ref.PTDC/NAN-MAT/29936/2017)

Finding the perfect match between nanoparticles and microfluidics to respond to cancer challenges

The clinical translation of new cancer theranostic has been delayed by inherent cancerâ s heterogeneity. Additionally, this delay has been enhanced by the lack of an appropriate in vitro model, capable to produce accurate data. Nanoparticles and microfluidic devices have been used to obtain new and more efficient strategies to tackle cancer challenges. On one hand, nanoparticles-based therapeutics can be modified to target specific cells, and/or molecules, and/or modified with drugs, releasing them over time. On the other hand, microfluidic devices allow the exhibition of physiologically complex systems, incorporation of controlled flow, and control of the chemical environment. Herein, we review the use of nanoparticles and microfluidic devices to address different cancer challenges, such as detection of CTCs and biomarkers, point-of-care devices for early diagnosis and improvement of therapies. The future perspectives of cancer challenges are also addressed herein.F.R. Maia acknowledges Portuguese Foundation for Scienceand Technology (FCT) for her work contract under theTransitional Rule DL 57/2016 (CTTI-57/18-I3BS5). J. M.Oliveira thanks FCT for his distinction attributed under theFCT Investigator program (IF/01285/2015)

Preparation of bioactive coatings on the surface of bioinert polymers through an innovative auto-catalytic electroless route

The aim of this research was to develop a new methodology to obtain bioactive coatings on bioinert and biodegradable polymers that are not intrinsically bioactive. In this study, three types of materials were used as substrates: (i) high molecular weight polyethylene (HMWPE) and two different types of starch based blends (ii) starch/ethylene vinyl alcohol blends, SEVA-C, and (iii) starch/cellulose acetate blends, SCA. These materials were obtained by injection moulding and by extrusion with blowing agents in order to obtain compact/porous 3D architectures. Three types of baths were developed in order to produce the newly proposed auto-catalytic Ca-P coatings: (i) alkaline, (ii) acid, and (iii) oxidant bath. The obtained results indicated that it was possible to coat the materials surfaces with calcium phosphate (Ca-P) layer with only 60 min of immersion in the different types of auto-catalytic solutions. These innovative auto-catalytic electroless route allows for the production of an adherent bioactive film on the polymeric surfaces. Furthermore, it was possible observe by SEM/EDS the clear bioactive nature of the Ca-P coatings after different immersion periods, in a simulated body fluid (SBF)

De novo bone formation on macro/microporous silk and silk/nano-sized calcium phosphate scaffolds

Macro/micro porous silk/nano-sized calcium phosphate scaffolds (SC16) with bioactive and superior physicochemical properties have been recently developed. In this study, we aim at evaluating the new bone formation ability of the SC 16 scaffolds in vivo, using silk fibroin scaffolds (S16) as control. The CaP distribution profile in the scaffolds was characterized by Micro-Computed Tomography. The in vitro mineralization behavior was examined by immersion in Simulated Body Fluid solution from 1 to 14 days. The long-term hydration degree and weight loss ratio of the scaffolds were evaluated by immersion in an Isotonic Saline Solution from 1 month to 1 year. In vivo osteogenesis properties of the scaffolds were screened by implantation into the rat femur defects for 3 weeks. The results showed that the CaP phase distributed homogeneously in the SC16 scaffolds. Mineralization was only observed in SC16 scaffolds, and both scaffolds gradually degraded with time. The staining of the explants showed that new bone formation with higher density was observed in the SC16 scaffolds as compared to S16 scaffolds, guiding the growth of new bone directly onto its surface. These results demonstrated that the SC16 hybrid scaffolds are osteoconductive and can be good candidates for bone tissue engineering as promoted superior de novo bone formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) projects OsteoCart (PTDC/CTM-BPC/115977/2009) and Tissue2Tissue (PTDC/CTM/105703/2008). Research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS. Le-Ping Yan is an FCT PhD scholarship holder (SFRH/BD/64717/2009)