Nuno M. Neves

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.

Chromosomal locus rearrangements are a rapid response to formation of the allotetraploid Arabidopsis suecica genome

Allopolyploidy is a significant evolutionary process, resulting in new species with diploid or greater chromosome complements derived from two or more progenitor species. We examined the chromosomal consequences of genomic merger in Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa. Fluorescence in situ hybridization with centromere, nucleolus organizer region (NOR), and 5S rRNA gene probes reveals the expected numbers of progenitor chromosomes in natural A. suecica, but one pair of A. thaliana NORs and one pair of A. arenosa-derived 5S gene loci are missing. Similarly, in newly formed synthetic A. suecica-like allotetraploids, pairs of A. thaliana NORs are gained de novo, lost, and/or transposed to A. arenosa chromosomes, with genotypic differences apparent between F3 siblings of the same F2 parent and between independent lines. Likewise, pairs of A. arenosa 5S genes are lost and novel linkages between 5S loci and NORs arise in synthetic allotetraploids. By contrast, the expected numbers of A. arenosa-derived NORs and A. thaliana-derived 5S loci are found in both natural and synthetic A. suecica. Collectively, these observations suggest that some, but not all, loci are unstable in newly formed A. suecica allotetraploids and can participate in a variety of alternative rearrangements, some of which resemble chromosomal changes found in nature.

Scaffolds Based Bone Tissue Engineering: The Role of Chitosan

As life expectancy increases, malfunction or loss of tissue caused by injury or disease leads to reduced quality of life in many patients at significant socioeconomic cost. Even though major progress has been made in the field of bone tissue engineering, present therapies, such as bone grafts, still have limitations. Current research on biodegradable polymers is emerging, combining these structures with osteogenic cells, as an alternative to autologous bone grafts. Different types of biodegradable materials have been proposed for the preparation of three-dimensional porous scaffolds for bone tissue engineering. Among them, natural polymers are one of the most attractive options, mainly due to their similarities with extracellular matrix, chemical versatility, good biological performance, and inherent cellular interactions. In this review, special attention is given to chitosan as a biomaterial for bone tissue engineering applications. An extensive literature survey was performed on the preparation of chitosan scaffolds and their in vitro biological performance as well as their potential to facilitate in vivo bone regeneration. The present review also aims to offer the reader a general overview of all components needed to engineer new bone tissue. It gives a brief background on bone biology, followed by an explanation of all components in bone tissue engineering, as well as describing different tissue engineering strategies. Moreover, also discussed are the typical models used to evaluate in vitro functionality of a tissue-engineered construct and in vivo models to assess the potential to regenerate bone tissue are discussed.

Electrospun nanostructured scaffolds for tissue engineering applications

Despite being known for decades (since 1934), electrospinning has emerged recently as a very widespread technology to produce synthetic nanofibrous structures. These structures have morphologies and fiber diameters in a range comparable with those found in the extracellular matrix of human tissues. Therefore, nanofibrous scaffolds are intended to provide improved environments for cell attachment, migration, proliferation and differentiation when compared with traditional scaffolds. In addition, the process versatility and the highly specific surface area of nanofiber meshes may facilitate their use as local drug-release systems. Common electrospun nanofiber meshes are characterized by a random orientation. However, in some special cases, aligned distributions of the fibers can be obtained, with an interconnected microporous structure. The characteristic pore sizes and the inherent planar structure of the meshes can be detrimental for the desired cell infiltration into the inner regions, and eventually compromise tissue regeneration. Several strategies can be followed to overcome these limitations, and are discussed in detail here.

Modified Gellan Gum hydrogels with tunable physical and mechanical properties.

Gellan Gum (GG) has been recently proposed for tissue engineering applications. GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations. However, physical crosslinking methods may yield hydrogels that become weaker in physiological conditions due to the exchange of divalent cations by monovalent ones. Hence, this work presents a new class of GG hydrogels crosslinkable by both physical and chemical mechanisms. Methacrylate groups were incorporated in the GG chain, leading to the production of a methacrylated Gellan Gum (MeGG) hydrogel with highly tunable physical and mechanical properties. The chemical modification was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR-ATR). The mechanical properties of the developed hydrogel networks, with Youngs modulus values between 0.15 and 148 kPa, showed to be tuned by the different crosslinking mechanisms used. The in vitro swelling kinetics and hydrolytic degradation rate were dependent on the crosslinking mechanisms used to form the hydrogels. Three-dimensional (3D) encapsulation of NIH-3T3 fibroblast cells in MeGG networks demonstrated in vitro biocompatibility confirmed by high cell survival. Given the highly tunable mechanical and degradation properties of MeGG, it may be applicable for a wide range of tissue engineering approaches.

Surface modification of electrospun polycaprolactone nanofiber meshes by plasma treatment to enhance biological performance

A critical aspect in the development of biomaterials is the optimization of their surface properties to achieve an adequate cell response. In the present work, electrospun polycaprolactone nanofiber meshes (NFMs) are treated by radio-frequency (RF) plasma using different gases (Ar or O(2)), power (20 or 30 W), and exposure time (5 or 10 min). Morphological and roughness analysis show topographical changes on the plasma-treated NFMs. X-ray photoelectron spectroscopy (XPS) results indicate an increment of the oxygen-containing groups, mainly --OH and --C==O, at the plasma-treated surfaces. Accordingly, the glycerol contact angle results demonstrate a decrease in the hydrophobicity of plasma-treated meshes, particularly in the O(2)-treated ones. Three model cell lines (fibroblasts, chondrocytes, and osteoblasts) are used to study the effect of plasma treatments over the morphology, cell adhesion, and proliferation. A plasma treatment with O(2) and one with Ar are found to be the most successful for all the studied cell types. The influence of hydrophilicity and roughness of those NFMs on their biological performance is discussed. Despite the often claimed morphological similarity of NFMs to natural extracellular matrixes, their surface properties contribute substantially to the cellular performance and therefore those should be optimized.

Gellan gum: A new biomaterial for cartilage tissue engineering applications

Gellan gum is a polysaccharide manufactured by microbial fermentation of the Sphingomonas paucimobilis microorganism, being commonly used in the food and pharmaceutical industry. It can be dissolved in water, and when heated and mixed with mono or divalent cations, forms a gel upon lowering the temperature under mild conditions. In this work, gellan gum hydrogels were analyzed as cells supports in the context of cartilage regeneration. Gellan gum hydrogel discs were characterized in terms of mechanical and structural properties. Transmissionelectron microscopy revealed a quite homogeneous chain arrangement within the hydrogels matrix, and dynamic mechanical analysis allowed to characterize the hydrogels discs viscoelastic properties upon compression solicitation, being the compressive storage and loss modulus of approximately 40 kPa and 3 kPa, respectively, at a frequency of 1 Hz. Rheological measurements determined the sol-gel transition started to occur at approximately 36 degrees C, exhibiting a gelation time of approximately 11 s. Evaluation of the gellan gum hydrogels biological performance was performed using a standard MTS cytotoxicity test, which showed that the leachables released are not deleterious to the cells and hence were noncytotoxic. Gellan gum hydrogels were afterwards used to encapsulate human nasal chondrocytes (1 x 10(6) cells/mL) and culture them for total periods of 2 weeks. Cells viability was confirmed using confocal calcein AM staining. Histological observations revealed normal chondrocytes morphology and the obtained data supports the claim that this new biomaterial has the potential to serve as a cell support in the field of cartilage regeneration.

Hierarchical starch-based fibrous scaffold for bone tissue engineering applications

Fibrous structures mimicking the morphology of the natural extracellular matrix are considered promising scaffolds for tissue engineering. This work aims to develop a novel hierarchical starch‐based scaffold. Such scaffolds were obtained by a combination of starch–polycaprolactone micro‐ and polycaprolactone nano‐motifs, respectively produced by rapid prototyping (RP) and electrospinning techniques. Scanning electron microscopy (SEM) and micro‐computed tomography analysis showed the successful fabrication of a multilayer scaffold composed of parallel aligned microfibres in a grid‐like arrangement, intercalated by a mesh‐like structure with randomly distributed nanofibres (NFM). Human osteoblast‐like cells were dynamically seeded on the scaffolds, using spinner flasks, and cultured for 7 days under static conditions. SEM analysis showed predominant cell attachment and spreading on the nanofibre meshes, which enhanced cell retention at the bulk of the composed/hierarchical scaffolds. A significant increment in cell proliferation and osteoblastic activity, assessed by alkaline phosphatase quantification, was observed on the hierarchical fibrous scaffolds. These results support our hypothesis that the integration of nanoscale fibres into 3D rapid prototype scaffolds substantially improves their biological performance in bone tissue‐engineering strategies. Copyright

Osteogenic induction of hBMSCs by electrospun scaffolds with dexamethasone release functionality.

Electrospun structures were proposed as scaffolds owing to their morphological and structural similarities with the extracellular matrix found in many native tissues. These fibrous structures were also proposed as drug release systems by exploiting the direct dependence of the release rate of a drug on the surface area. An osteogenic differentiation factor, dexamethasone (DEX), was incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (5, 10, 15 and 20 wt.% polymer), in a single-step process. The DEX incorporated into the polymeric carrier is in amorphous state, as determined by DSC, and does not influence the typical nanofibers morphology. In vitro drug release studies demonstrated that the dexamethasone release was sustained over a period of 15 days. The bioactivity of the released dexamethasone was assessed by cultivating human bone marrow mesenchymal stem cells (hBMSCs) on 15 wt.% DEX-loaded PCL NFMs, under dexamethasone-absent osteogenic differentiation medium formulation. An increased concentration of alkaline phosphatase and deposition of a mineralized matrix was observed. Phenotypic and genotypic expression of osteoblastic-specific markers corroborates the osteogenic activity of the loaded growth/differentiation factor. Overall data suggests that the electrospun biodegradable nanofibers can be used as carriers for the sustained release of growth/differentiation factors relevant for bone tissue engineering strategies.

Electrospinning: processing technique for tissue engineering scaffolding

Abstract Electrospinning has attracted tremendous interest in the research community as a simple and versatile technique to produce synthetic polymeric ultrafine fibres with diameters ranging from a few micrometres to tens of nanometres. Recently, some natural origin polymers have also been successfully electrospun. Owing to their very small diameter, polymeric nanofibres exhibit unusual properties such as high specific surface area, flexibility in surface functionalities and superior mechanical properties. In addition, electrospun non-woven meshes could physically mimic the extracellular matrix structure of native tissues. These remarkable properties render electrospun nanofibres useful for many applications, particularly those related to the field of biomedical engineering. The first part of this review is intended to provide a fundamental survey of the electrospinning process (apparatus, governing parameters) and of recent improvements of the technique, including associated structural modifications of polymeric nanofibre meshes. The prospective tissue engineering/biomedical applications of electrospun polymeric nanofibres are then reviewed, namely, wound dressings, medical prostheses, drug delivery systems, DNA release and tissue engineering scaffolds. The essential properties of scaffolds in terms of the structural features of electrospun nanofibre meshes are discussed. Finally, the future perspectives for applications of electrospun nanofibres, particularly in the field of tissue engineering, are considered.