João F. Mano

Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends

The fields of tissue engineering and regenerative medicine aim at promoting the regeneration of tissues or replacing failing or malfunctioning organs, by means of combining a scaffold/support material, adequate cells and bioactive molecules. Different materials have been proposed to be used as both three-dimensional porous scaffolds and hydrogel matrices for distinct tissue engineering strategies. Among them, polymers of natural origin are one of the most attractive options, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as typically good biological performance. In this review, the most studied and promising and recently proposed naturally derived polymers that have been suggested for tissue engineering applications are described. Different classes of such type of polymers and their blends with synthetic polymers are analysed, with special focus on polysaccharides and proteins, the systems that are more inspired by the ECM. The adaptation of conventional methods or non-conventional processing techniques for processing scaffolds from natural origin based polymers is reviewed. The use of particles, membranes and injectable systems from such kind of materials is also overviewed, especially what concerns the present status of the research that should lead towards their final application. Finally, the biological performance of tissue engineering constructs based on natural-based polymers is discussed, using several examples for different clinically relevant applications.

Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications

Chitosan is a natural based polymer, obtained by alkaline deacetylation of chitin, which presents excellent biological properties such as biodegradability and immunological, antibacterial and wound-healing activity. Recently, there has been a growing interest in the chemical modification of chitosan in order to improve its solubility and widen its applications. The main chemical modifications of chitosan that have been proposed in the literature are reviewed in this paper. Moreover, these chemical modifications lead to a wide range of derivatives with a broad range of applications. Recent and relevant examples of the distinct applications, with particular emphasis on tissue engineering, drug delivery and environmental applications, are presented.

Chitosan-Based Particles as Controlled Drug Delivery Systems

Chitosan, a natural-based polymer obtained by alkaline deacetylation of chitin, is nontoxic, biocompatible, and biodegradable. These properties make chitosan a good candidate for conventional and novel drug delivery systems. This article reviews the approaches aimed to associate bioactive molecules to chitosan in the form of colloidal structures and analyzes the evidence of their efficacy in improving the transport of the associated molecule through mucosae and epithelia. Chitosan forms colloidal particles and entraps bioactive molecules through a number of mechanisms, including chemical crosslinking, ionic crosslinking, and ionic complexation. A possible alternative of chitosan by the chemical modification also has been useful for the association of bioactive molecules to polymer and controlling the drug release profile. Because of the high affinity of chitosan for cell membranes, it has been used as a coating agent for liposome formulations. This review also examines the advances in the application of chitosan and its derivatives to nonviral gene delivery and gives an overview of transfection studies that use chitosan as a transfection agent. From the studies reviewed, we concluded that chitosan and its derivatives are promising materials for controlled drug and nonviral gene delivery.

Molecular Interactions Driving the Layer-by-Layer Assembly of Multilayers

This work received funding from the European Unions Seventh Framework Programme (FP7/2007-2013) under grant agreement no. REGPOT-CT2012-316331-POLARIS. The work was also funded by FEDER through the Competitive Factors Operational Program (COMPETE) and by National funds through the Portuguese Foundation for Science and Technology (FCT) in the scope of the projects PTDC/FIS/115048/2009 and PTDC/CTM-BIO/1814/2012. The authors gratefully acknowledge Dr. Luca Gasperini (3Bs Research Group, University of Minho, Portugal) for his help with the figures.

Three-dimensional plotted scaffolds with controlled pore size gradients: Effect of scaffold geometry on mechanical performance and cell seeding efficiency

Scaffolds produced by rapid prototyping (RP) techniques have proved their value for tissue engineering applications, due to their ability to produce predetermined forms and structures featuring fully interconnected pore architectures. Nevertheless, low cell seeding efficiency and non-uniform distribution of cells remain major limitations when using such types of scaffold. This can be mainly attributed to the inadequate pore architecture of scaffolds produced by RP and the limited efficiency of cell seeding techniques normally adopted. In this study we aimed at producing scaffolds with pore size gradients to enhance cell seeding efficiency and control the spatial organization of cells within the scaffold. Scaffolds based on blends of starch with poly(ε-caprolactone) featuring both homogeneously spaced pores (based on pore sizes of 0.75 and 0.1 mm) and pore size gradients (based on pore sizes of 0.1-0.75-0.1 and 0.75-0.1-0.75 mm) were designed and produced by three-dimensional plotting. The mechanical performance of the scaffolds was characterized using dynamic mechanical analysis (DMA) and conventional compression testing under wet conditions and subsequently characterized using scanning electron microscopy and micro-computed tomography. Osteoblast-like cells were seeded onto such scaffolds to investigate cell seeding efficiency and the ability to control the zonal distribution of cells upon seeding. Scaffolds featuring continuous pore size gradients were originally produced. These scaffolds were shown to have intermediate mechanical and morphological properties compared with homogenous pore size scaffolds. The pore size gradient scaffolds improved seeding efficiency from ∼35% in homogeneous scaffolds to ∼70% under static culture conditions. Fluorescence images of cross-sections of the scaffolds revealed that scaffolds with pore size gradients induce a more homogeneous distribution of cells within the scaffold.

Thermal properties of thermoplastic starch/synthetic polymer blends with potential biomedical applicability.

Previous studies shown that thermoplastic blends of corn starch with some biodegradable synthetic polymers (poly(ε-caprolactone), cellulose acetate, poly(lactic acid) and ethylene-vinyl alcohol copolymer) have good potential to be used in a series of biomedical applications. In this work the thermal behavior of these structurally complex materials is investigated by differential scanning calorimetry (DSC) and by thermogravimetric analysis (TGA). In addition, Fourier-transform infrared (FTIR) spectroscopy was used to investigate the chemical interactions between the different components. The endothermic gelatinization process (or water evaporation) observed by DSC in starch is also observed in the blends. Special attention was paid to the structural relaxation that can occur in the blends with poly(lactic acid) at body temperature that may change the physical properties of the material during its application as a biomaterial. At least three degradation mechanisms were identified in the blends by means of using TGA, being assigned to the mass loss due to the plasticizer leaching, and to the degradation of the starch and the synthetic polymer fractions. The non-isothermal kinetics of the decomposition processes was analyzed using two different integral methods. The analysis included the calculation of the activation energy of the correspondent reactions.

Starch-based biodegradable hydrogels with potential biomedical applications as drug delivery systems.

The design and preparation of novel biodegradable hydrogels developed by the free radical polymerization of acrylamide and acrylic acid, and some formulations with bis-acrylamide, in the presence of a corn starch/ethylene-co-vinyl alcohol copolymer blend (SEVA-C), is reported. The redox system benzoyl peroxide (BPO) and 4-dimethylaminobenzyl alcohol (DMOH) initiated the polymerization at room temperature. Xerogels were characterized by 1H NMR and FTIR spectroscopies. Swelling studies were performed as a function of pH in different buffer solutions determining the water-transport mechanism that governs the swelling behaviour. Degradation studies of the hydrogels were performed in simulated physiological solutions for time up to 90 days, determining the respective weight loss, and analyzing the solution residue by 1H NMR. The mechanical properties of the xerogels were characterized by tensile and compressive tests, as well as by dynamo-mechanical analysis (DMA). Dynamo-mechanical parameters are also reported for hydrated samples.

Controlling cell behavior through the design of polymer surfaces.

Polymers have gained a remarkable place in the biomedical field as materials for the fabrication of various devices and for tissue engineering applications. The initial acceptance or rejection of an implantable device is dictated by the crosstalk of the material surface with the bioentities present in the physiological environment. Advances in microfabrication and nanotechnology offer new tools to investigate the complex signaling cascade induced by the components of the extracellular matrix and consequently allow cellular responses to be tailored through the mimicking of some elements of the signaling paths. Patterning methods and selective chemical modification schemes at different length scales can provide biocompatible surfaces that control cellular interactions on the micrometer and sub-micrometer scales on which cells are organized. In this review, the potential of chemically and topographically structured micro- and nanopolymer surfaces are discussed in hopes of a better understanding of cell-biomaterial interactions, including the recent use of biomimetic approaches or stimuli-responsive macromolecules. Additionally, the focus will be on how the knowledge obtained using these surfaces can be incorporated to design biocompatible materials for various biomedical applications, such as tissue engineering, implants, cell-based biosensors, diagnostic systems, and basic cell biology. The review focusses on the research carried out during the last decade.

FTIR AND DSC STUDIES OF MECHANICALLY DEFORMED β-PVDF FILMS

Films of semicrystalline poly(vinylidene fluoride) (PVDF) in the β-phase were studied by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The main goal of this study was to improve the understanding of the structural changes that occur in β-PVDF during a mechanical deformation process. FTIR spectroscopy was used to examine the structural variations as a function of strain. DSC data allowed measurement of the melting temperatures and enthalpies of the material before and after deformation, providing information about the changes in the crystalline fraction. After the molecular vibrations were assigned to the corresponding vibrational modes, we investigated the energy and intensity variations of these vibrations at different deformations. A reorientation of the chains from perpendicular to parallel to the stress direction was observed to occur in the plastic region. During the deformation, a decrease in the degree of crystallinity of the material was observed, but the thickness of the lamellae did not change significantly.

Genipin-cross-linked collagen/chitosan biomimetic scaffolds for articular cartilage tissue engineering applications

In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.