Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate

Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet Lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P>5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL.Portuguese Foundation for Science and Technology is gratefully acknowledged for fellowships of Sara M. Oliveira. (SFRH/BD/70107/2010).r The 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 and FP7-KBBE-2010-4-266033 - SPECIAL. This work was also supported by the European Research Council grant agreement ERC-2012-ADG 20120216-321266 for the project ComplexiTE. The authors acknowledge Rogerio P. Pirraco for the Flow cytometry analysis

Natural assembly of platelet lysate-loaded nanocarriers into enriched 3D hydrogels for cartilage regeneration

The role of Platelet Lysates (PLs) as a source of growth factors (GFs) and as main element of threedimensional (3D) hydrogels has been previously described. However, the resulting hydrogels usually suffer from high degree of contraction, limiting their usefulness. This work describes the development of a stable biomimetic 3D hydrogel structure based on PLs, through the spontaneous assembling of a high concentration of chitosan-chondroitin sulfate nanoparticles (CH/CS NPs) with PLs loaded by adsorption. The interactions between the NPs and the lysates resemble the ones observed in the extracellular matrix (ECM) native environment between glycosaminoglycans and ECM proteins. In vitro release studies were carried out focusing on the quantification of PDGF-BB and TGF-b1 GFs. Human adipose derived stem cells (hASCs) were entrapped in these 3D hydrogels and cultured in vitro under chondrogenic stimulus, in order to assess their potential use for cartilage regeneration. Histological, immunohistological and gene expression analysis demonstrated that the PL-assembled constructs entrapping hASCs exhibited results similar to the positive control (hASCS cultured in pellets), concerning the levels of collagen II expression and immunolocalization of collagen type I and II and aggrecan. Moreover, the deposition of new cartilage ECM was detected by alcian blue and safranin-O positive stainings. This work demonstrates the potential of PLs to act simultaneously as a source/carrier of GFs and as a 3D structure of support, through the application of a â â bottom-upâ â approach involving the assembly of NPs, resulting in an enriched construct for cartilage regeneration applications.The authors thank Fundacao para a Ciencia e Tecnologia for V.E. Santo and E.G. Popa's PhD grants (SFRH/BD/39486/2007 and SFRH/BD/64070/2009, respectively). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and it was partially supported by the European FP7 Project Find and Bind (NMP4-SL-2009-229292). We thank IPS for providing the human platelet donations and Hospital da Prelada for the human adipose tissue samples

Hybrid 3D structure of poly(d,l-lactic acid) loaded with chitosan/chondroitin sulfate nanoparticles to be used as carriers for biomacromolecules in tissue engineering

In the tissue engineering (TE) field, the concept of producing multifunctional scaffolds, capable not only of acting as templates for cell transplantation but also of delivering bioactive agents in a controlled manner, is an emerging strategy aimed to enhance tissue regeneration. In this work, a complex hybrid release system consisting in a three-dimensional (3D) structure based on poly(d,l-lactic acid) (PDLLA) impregnated with chitosan/chondroitin sulfate nanoparticles (NPs) was developed. The scaffolds were prepared by supercritical fluid foaming at 200 bar and 35 "C, and were then characterized by scanning electron microscopy (SEM) and micro-CT. SEM also allowed to assess the distribution of the NPs within the structure, showing that the particles could be found in different areas of the scaffold, indicating a homogeneous distribution within the 3D structure. Water uptake and weight loss measurements were also carried out and the results obtained demonstrated that weight loss was not significantly enhanced although the entrapment of the NPs in the 3D structure clearly enhances the swelling of the structure. Moreover, the hybrid porous biomaterial displayed adequate mechanical properties for cell adhesion and support. The possibility of using this scaffold as a multifunctional material was further evaluated by the incorporation of a model protein, bovine serum albumin (BSA), either directly into the PDLLA foam or in the NPs that were eventually included in the scaffold. The obtained results show that it is possible to achieve different release kinetics, suggesting that this system is a promising candidate for dual protein delivery system for TE applications

Cell engineering by the internalization of bioinstructive micelles for enhanced bone regeneration

To direct precursor cells toward the osteoblastic lineage, by using an intracellular nanocarrier releasing dexamethasone. Materials & methods: Biodegradable gelatinbased micelles entrapped dexamethasone (dex-micelles). Internalization efficiency and biocompatibility of dex-micelles and their potency for in vitro osteogenic differentiation and in vivo bone regeneration were assessed. Results: Dex-micelles were internalized by rat bone marrow mesenchymal stem cells and demonstrated a pH-responsive release profile and an enhancement of 2D and 3D in vitro osteogenic differentiation. In vivo implantation of gelatin scaffolds seeded with rat bone marrow mesenchymal stem cells precultured for 24 h with dex-micelles promoted a significant enhancement of de novo bone formation in a rat ulna defect, in a dose-dependent manner. Conclusion: The proposed intracellular delivery system is a powerful tool to promote bone regeneration.The authors thank Fundacao para a Ciencia e Tecnologia and Japan Society for the Promotion of Science (JSPS) for VE Santo's PhD grant (SFRH/BD/39486/2007) and J Ratanavaraporn's post-doc fellowship, respectively. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

Development of new chitosan/carrageenan nanoparticles for drug delivery applications

The use of polymeric nanoparticles, especially those composed of natural polymers, has become a very interesting approach in drug delivery., mainly because of the advantages offered by their small dimensions. The aim of this work was to develop a novel formulation of nanoparticles comprised of two natural marine-derived polymers, namely chitosan and carrageenan, and to evaluate their potential for the association and controlled release of macromolecules. Nanoparticles were obtained in a hydrophilic environment, under very mild conditions, avoiding the use of organic solvents or other aggressive technologies for their preparation. The developed nanocarriers presented sizes within 350-650 run and positive zeta potentials of 50-60 mV. Polymeric interactions between nanoparticles' components were evaluated by Fourier transform infrared spectroscopy. Using ovalbumin as model protein, nanoparticles evidenced loading capacity varying from 4% to 17%, and demonstrated excellent capacity to provide a controlled release for up to 3 weeks. Furthermore, nanoparticles have demonstrated to exhibit a noncytotoxic behavior in biological in vitro tests performed using L929 fibroblasts, which is critical regarding the biocompatibility of those carriers. In summary, the developed chitosan-carrageenan nanoparticles have shown promising properties to be used as carriers of therapeutic macromolecules, with potential application not only strictly in drug delivery, but also in broader areas, such as tissue engineering and regenerative medicine. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 92A: 1265-1272, 2010Contract grant sponsor: Portuguese Foundation for Science and Technology (FCT) (POCTI/FEDER programmes)Contract grant sponsor: European Union STREP Project HIPPOCRATES; contract grant number: NMP3-CT-2003505758Contract grant sponsor: European NoE EXPERTISSUES; contract grant number: NMP3-CT-2004-50028

Unleashing the potential of supercritical fluids for polymer processing in tissue engineering and regenerative medicine

One of the major scientific challenges that tissue engineering and regenerative medicine (TERM) faces to move from benchtop to bedside regards biomaterials development, despite the latest advances in polymer processing technologies. A variety of scaffolds processing techniques have been developed and include solvent casting and particles leaching, compression molding and particle leaching, thermally induced phase separation, rapid prototyping, among others. Supercritical fluids appear as an interesting alternative to the conventional methods for processing biopolymers as they do not require the use of large amounts of organic solvents and the processes can be conducted at mild temperatures. However, this processing technique has only recently started to receive more attention from researchers. Different processing methods based on the use of supercritical carbon dioxide have been proposed for the creation of novel architectures based on natural and synthetic polymers and these will be unleashed in this paper.The research leading to these results has recieved funding from the European Union Seventh Framework Programme (FP7) under grant agreement no. KBBE-2010-266033 (project SPECIAL), no. NMP4-SL-2009-229292 (project Find & Bind), from FEDER through POCTEP Project 0330_IBEROMARE_1_P, from the Sixth Framework Programme (FP6) under grant agreement NMP3-CT-2004-500283 (project EXPERTISSUES), Portuguese Foundation fo Science and Technology (FCT) is also acknowledged. for PhD and Post-Doc fellowships of Ana Rits C. Duarte, Vitor E. Santo, Anabela Alves, Simone S. Silva, Joana Moreira-Silva and Tiage H. Silva.Ana Rita C. Duarte would like to acknowledgs Fundacao Luso-Americana para o Desenvolvimento for the travel grant awarded to present this work at the international Symposium of Supercritical Fluids - ISSF 2012

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono-and stromal cocultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.Peer reviewe

Emergências hiperglicêmicas - aspectos epidemiológicos, fisiopatológicos e manejo terapêutico: Hyperglycemic emergencies - epidemiological, physiopathological aspects and therapeutic management

Diabetes mellitus (DM) corresponde a uma doença metabólica na qual há produção inadequada de insulina ou resistência periférica a este hormônio, correspondendo tais definições à DM do tipo 1 e 2, respectivamente. A doença apresenta alta prevalência e incidência em todo o mundo e pode levar à inúmeras complicações, tanto agudas quanto crônicas; nesse sentido, a cetoacidose diabética (CAD) e o estado hiperosmolar hiperglicêmico (EHH) correspondem às principais complicações agudas da doença; requerem diagnóstico e tratamento imediato devido à gravidade do quadro gerado pelas mesmas. Ademais, ambas as complicações são denominadas de emergências hiperglicêmicas, justamente por seus sinais e sintomas acontecerem na vigência de uma glicemia elevada em um paciente diabético. Outrossim, são diversas as causas da CAD e do EHH; no entanto, as principais causas estabelecidas pela literatura são a presença de infecções e o uso inadequado de insulina em pacientes portadores de DM. Além disso, o manejo adequado do paciente nestas circunstâncias envolve a administração precoce de fluidos intravenosos, terapia com insulina, reposição de eletrólitos e reconhecimento e tratamento das causas precipitantes. Contudo, a despeito da existências de inúmeros protocolos envolvidos no diagnóstico e tratamento da CAD e do EHH, pouco se tem feito na prevenção destas complicações; dessa forma, urge a necessidade de melhor esclarecer pacientes diabéticos a respeito de sua doença e suas prováveis complicações, bem como alertá-los sobre possíveis sinais de alerta, para que possam buscar assistência o mais precoce possível, diminuindo a probabilidade de desfechos negativos

Capturing tumor complexity in vitro : Comparative analysis of 2D and 3D tumor models for drug discovery

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono-and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.Peer reviewe