Rui A. Sousa

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.

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.

Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization

The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and organized into microcapillary-like structures. These vascular-like structures evolved from a cord-like configuration to a more complex branched morphology, had a lumen and stained in the perivascular region for type IV collagen. Genetic profiling of 84 osteogenesis-related genes was performed on co-culture vs. monoculture. Osteoblasts in co-culture showed a significant up-regulation of type I collagen and immunohistochemistry revealed that the scaffold was filled with a dense matrix stained for type I collagen. In direct contact with HDMEC hOBs secreted higher amounts of VEGF in relation to monoculture and the highest peak in the release profile correlated with the formation of microcapillary-like structures. The heterotypic communication between the two cell types was also assured by direct cell-cell contact as shown by the expression of the gap junction connexin 43. In summary, by making use of heterotypic cellular crosstalk this co-culture system is a strategy to form vascular-like structures in vitro on a 3D scaffold.

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

Gellan gum-based hydrogels for intervertebral disc tissue-engineering applications

Intervertebral disc (IVD) degeneration is a challenging clinical problem that urgently demands viable nucleus pulposus (NP) implant materials. The best suited biomaterial for NP regeneration has yet to be identified, but it is believed that biodegradable hydrogel‐based materials are promising candidates. In this work, we have developed ionic‐ and photo‐crosslinked methacrylated gellan gum (GG–MA) hydrogels to be used in acellular and cellular tissue‐engineering strategies for the regeneration of IVDs. The physicochemical properties of the developed hydrogels were investigated by Fourier‐transform infrared spectroscopy, 1H nuclear magnetic resonance and differential scanning calorimetry. The swelling ability and degradation rate of hydrogels were also analysed in phosphate‐buffered saline solution at physiological pH for a period of 30 days. Additionally, the morphology and mechanical properties of the hydrogels were assessed under a scanning electron microscope and dynamic compression, respectively. An in vitro study was carried out to screen possible cytotoxicity of the gellan gum‐based hydrogels by culturing rat lung fibroblasts (L929 cells) with hydrogel leachables up to 7 days. The results demonstrated that gellan gum was successfully methacrylated. We observed that the produced GG–MA hydrogels possess improved mechanical properties and lower water uptake ability and degradation rate as compared to gellan gum. This work also revealed that GG–MA hydrogels are non‐cytotoxic in vitro, thus being promising biomaterials to be used in IVD tissue‐engineering strategies. Copyright

The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.

In vitro model of vascularized bone: synergizing vascular development and osteogenesis.

Tissue engineering provides unique opportunities for regenerating diseased or damaged tissues using cells obtained from tissue biopsies. Tissue engineered grafts can also be used as high fidelity models to probe cellular and molecular interactions underlying developmental processes. In this study, we co-cultured human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs) under various environmental conditions to elicit synergistic interactions leading to the colocalized development of capillary-like and bone-like tissues. Cells were encapsulated at the 1∶1 ratio in fibrin gel to screen compositions of endothelial growth medium (EGM) and osteogenic medium (OM). It was determined that, to form both tissues, co-cultures should first be supplied with EGM followed by a 1∶1 cocktail of the two media types containing bone morphogenetic protein-2. Subsequent studies of HUVECs and MSCs cultured in decellularized, trabecular bone scaffolds for 6 weeks assessed the effects on tissue construct of both temporal variations in growth-factor availability and addition of fresh cells. The resulting grafts were implanted subcutaneously into nude mice to determine the phenotype stability and functionality of engineered vessels. Two important findings resulted from these studies: (i) vascular development needs to be induced prior to osteogenesis, and (ii) the addition of additional hMSCs at the osteogenic induction stage improves both tissue outcomes, as shown by increased bone volume fraction, osteoid deposition, close proximity of bone proteins to vascular networks, and anastomosis of vascular networks with the host vasculature. Interestingly, these observations compare well with what has been described for native development. We propose that our cultivation system can mimic various aspects of endothelial cell – osteogenic precursor interactions in vivo, and could find utility as a model for studies of heterotypic cellular interactions that couple blood vessel formation with osteogenesis.

Materials of marine origin: a review on polymers and ceramics of biomedical interest

Abstract Marine organisms are constituted by materials with a vast range of properties and characteristics that may justify their potential application within the biomedical field. Moreover, assuring the sustainable exploitation of natural marine resources, the valorisation of residues from marine origin, like those obtained from food processing, constitutes a highly interesting platform for development of novel biomaterials, with both economic and environmental benefits. In this perspective, an increasing number of different types of compounds are being isolated from aquatic organisms and transformed into profitable products for health applications, including controlled drug delivery and tissue engineering devices. This report reviews the work that is being developed on the isolation and characterisation of some polysaccharides, proteins, glycosaminoglycans and ceramics from marine raw materials. Emphasis is given to agar, alginates, carrageenans, chitin and chitosan, among other polysaccharides, collagen, glycosaminoglycans such as chondroitin sulphate, heparin and hyaluronic acid, calcium phosphorous compounds and biosilica. Finally, this report ends by reviewing the application of the previously mentioned materials on specific biomedical applications, in particular their participation on the development of controlled drug delivery systems and tissue engineering scaffolds.

The enhancement of the mechanical properties of a high‐density polyethylene

This paper describes the process optimization in injection molding of high- density polyethylene (HDPE). Both conventional injection molding and shear controlled orientation (SCORIM) were employed in processing. The process optimization was based on design of experiments and complemented with analysis of variance. Mechan- ical characterization was carried out by tensile testing. Wide-angle X-ray diffraction and differential scanning calorimetry were used for the structural characterization of the moldings. High-density polyethylene exhibits 7.2 GPa Youngs modulus and 155 MPa of ultimate tensile strength following the application of SCORIM processing. These results account for a fourfold increase in Youngs modulus and a fivefold increase in ultimate tensile strength compared to conventional injection molding. The mainte- nance of toughness while enhancing stiffness and strength of the SCORIM moldings is attributable to an oriented morphology developed during shear flow, i.e., shish-kebab structure. The frequency of shearing action has the strongest influence on the morphol- ogy development. It is also demonstrated that the studied parameters are very much interdependent. It is possible to achieve substantial gains in mechanical properties of HDPE in SCORIM processing without causing a substantial increase in cycle time.