The inhibitory effect of an RGD-human chitin-binding domain fusion protein on the adhesion of fibroblasts to reacetylated chitosan films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/27359/2006, POCTI/BIO/45356/200

Chitosan particles agglomerated scaffolds for cartilage and osteochondral tissue engineering approaches with adipose tissue derived stem cells

It is well accepted that natural tissue regeneration is unlikely to occur if the cells are not supplied with an extracellular matrix (ECM) substitute. With this goal, several different methodologies have been used to produce a variety of 3D scaffolds as artificial ECM substitutes suitable for bone and cartilage tissue engineering. Furthermore, osteochondral tissue engineering presents new challenges since the combination of scaffolding and co-culture requirements from both bone and cartilage applications is required in order to achieve a successful osteochondral construct. In this paper, an innovative processing route based on a chitosan particles aggregation methodology for the production of cartilage and osteochondral tissue engineering scaffolds is reported. An extensive characterization is presented including a morphological evaluation using Micro-Computed Tomography (μCT) and 3D virtual models built with an image processing software. Mechanical and water uptake characterizations were also carried out, evidencing the potential of the developed scaffolds for the proposed applications. Cytotoxicity tests show that the developed chitosan particles agglomerated scaffolds do not exert toxic effects on cells. Furthermore, osteochondral bilayered scaffolds could also be developed. Preliminary seeding of mesenchymal stem cells isolated from human adipose tissue was performed aiming at developing solutions for chondrogenic and osteogenic differentiation for osteochondral tissue engineering applications.Fundação para a Ciência e a Tecnologia (FCT)European NoE EXPERTISSUES (NMP3-CT-2004-500283)European STREP Project HIPPOCRATES (NMP3-CT-2003-505758

Next Generation of Electrosprayed Fibers for Tissue Regeneration

Electrospinning is a widely established polymer-processing technology that allows generation of fibers (in nanometer to micrometer size) that can be collected to form nonwoven structures. By choosing suitable process parameters and appropriate solvent systems, fiber size can be controlled. Since the technology allows the possibility of tailoring the mechanical properties and biological properties, there has been a significant effort to adapt the technology in tissue regeneration and drug delivery. This review focuses on recent developments in adapting this technology for tissue regeneration applications. In particular, different configurations of nozzles and collector plates are summarized from the view of cell seeding and distribution. Further developments in obtaining thick layers of tissues and thin layered membranes are discussed. Recent advances in porous structure spatial architecture parameters such as pore size, fiber size, fiber stiffness, and matrix turnover are summarized. In addition, possibility of developing simple three-dimensional models using electrosprayed fibers that can be utilized in routine cell culture studies is described