Helena S. Azevedo

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.

Self-Assembly of Large and Small Molecules into Hierarchically Ordered Sacs and Membranes

We report here the self-assembly of macroscopic sacs and membranes at the interface between two aqueous solutions, one containing a megadalton polymer and the other, small self-assembling molecules bearing opposite charge. The resulting structures have a highly ordered architecture in which nanofiber bundles align and reorient by nearly 90° as the membrane grows. The formation of a diffusion barrier upon contact between the two liquids prevents their chaotic mixing. We hypothesize that growth of the membrane is then driven by a dynamic synergy between osmotic pressure of ions and static self-assembly. These robust, self-sealing macroscopic structures offer opportunities in many areas, including the formation of privileged environments for cells, immune barriers, new biological assays, and self-assembly of ordered thick membranes for diverse applications.

Designing biomaterials based on biomineralization of bone

In nature, organisms control crystal nucleation and growth using organic interfaces as templates. Scientists, in the last decades, have tried to learn from nature how to design biomimetic biomaterials inspired by the hierarchical complex structure of bone and other natural mineralised tissues or to control the biomineralization process onto biomaterials substrates to promote the osteoconductive properties of implantable devices. The design of synthetic bone analogues, i.e., with a structure and properties similar to bone, would certainly constitute a major breakthrough in bone tissue engineering. Moreover, many strategies have been proposed in the literature to develop bioactive bone-like materials, for instance using bioactive glasses. Fundamental aspects of biomineralization may be also important in order to propose new methodologies to improve calcification onto the surface of biomaterials or to develop bioactive tridimensional templates that could be used in regenerative medicine. In particular, it has been shown that some chemical groups and proteins, as well as the tridimensional matrix in which calcification would occur, play a fundamental role on the nucleation and growth of hydroxyapatite. All these distinct aspects will be reviewed and discussed in this paper.

Silk fibroin microparticles as carriers for delivery of human recombinant BMPs. Physical characterization and drug release

Bone morphogenetic proteins (BMPs) are cytokines with strong ability to promote new bone formation. Herein, we report the use of silk fibroin microparticles as carriers for the delivery of BMP‐2, BMP‐9 or BMP‐14. BMP‐containing fibroin microparticles were prepared by a mild methodology using dropwise addition of ethanol, exhibiting mean diameters of 2.7 ± 0.3 µm. Encapsulation efficiencies varied between 67.9 ± 6.1 % and 97.7 ± 2.0 % depending on the type and the amount of BMP loaded. Release kinetics showed that BMP‐2, BMP‐9 and BMP‐14 were released in two phases profile, with a burst release in the first two days followed by a slower release, for a period of 14 days. The release data were best explained by Korsmeyers model and the Fickian model of drug diffusion. Silk fibroin microparticles can offer a promising approach for the sustained delivery of different BMPs in tissue engineering applications. Copyright

Self-assembly in nature: using the principles of nature to create complex nanobiomaterials

Self-assembly is a ubiquitous process in biology where it plays numerous important roles and underlies the formation of a wide variety of complex biological structures. Over the past two decades, materials scientists have aspired to exploit natures assembly principles to create artificial materials, with hierarchical structures and tailored properties, for the fabrication of functional devices. Toward this goal, both biological and synthetic building blocks have been subject of extensive research in self-assembly. In fact, molecular self-assembly is becoming increasingly important for the fabrication of biomaterials because it offers a great platform for constructing materials with high level of precision and complexity, integrating order and dynamics, to achieve functions such as stimuli-responsiveness, adaptation, recognition, transport, and catalysis. The importance of peptide self-assembling building blocks has been recognized in the last years, as demonstrated by the literature available on the topic. The simple structure of peptides, as well as their facile synthesis, makes peptides an excellent family of structural units for the bottom-up fabrication of complex nanobiomaterials. Additionally, peptides offer a great diversity of biochemical (specificity, intrinsic bioactivity, biodegradability) and physical (small size, conformation) properties to form self-assembled structures with different molecular configurations. The motivation of this review is to provide an overview on the design principles for peptide self-assembly and to illustrate how these principles have been applied to manipulate their self-assembly across the scales. Applications of self-assembling peptides as nanobiomaterials, including carriers for drug delivery, hydrogels for cell culture and tissue repair are also described.

Preparation and characterization of starch-poly-ε-caprolactone microparticles incorporating bioactive agents for drug delivery and tissue engineering applications

One limitation associated with the delivery of bioactive agents concerns the short half-life of these molecules when administered intravenously, which results in their loss from the desired site. Incorporation of bioactive agents into depot vehicles provides a means to increase their persistence at the disease site. Major issues are involved in the development of a proper carrier system able to deliver the correct drug, at the desired dose, place and time. In this work, starch-poly-epsilon-caprolactone (SPCL) microparticles were developed for use in drug delivery and tissue engineering (TE) applications. SPCL microparticles were prepared by using an emulsion solvent extraction/evaporation technique, which was demonstrated to be a successful procedure to obtain particles with a spherical shape (particle size between 5 and 900 microm) and exhibiting different surface morphologies. Their chemical structure was confirmed by Fourier transform infrared spectroscopy. To evaluate the potential of the developed microparticles as a drug delivery system, dexamethasone (DEX) was used as model drug. DEX, a well-known component of osteogenic differentiation media, was entrapped into SPCL microparticles at different percentages up to 93%. The encapsulation efficiency was found to be dependent on the polymer concentration and drug-to-polymer ratio. The initial DEX release seems to be governed mainly by diffusion, and it is expected that the remaining DEX will be released when the polymeric matrix starts to degrade. In this work it was demonstrated that SPCL microparticles containing DEX can be successfully prepared and that these microparticular systems seem to be quite promising for controlled release applications, namely as carriers of important differentiation agents in TE.

Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulomonas fimi).

The activities (at pH 7 and 50 degrees C) of purified EGV (Humicola insolens) and CenA (Cellulomonas fimi) were determined on cotton fabrics at high and low levels of mechanical agitation. Similar activity measurements were also made by using the core domains of these cellulases. Activity experiments suggested that the presence of cellulose binding domains (CBDs) is not essential for cellulase performance in the textile processes, where high levels of mechanical agitation are applied. The binding reversibilities of these cellulases and their cores were studied by dilution of the treatment liquor after equilibrium adsorption. EGV showed low percentage of adsorption under both levels of agitation. It was observed that the adsorption/desorption processes of cellulases are enhanced by higher mechanical agitation levels and that the binding of cellulase with CBD of family I (EGV) is more reversible than that of CBD of the cellulase of family II (CenA).

Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration

We developed a novel injectable carrageenan/fibrin/hyaluronic acid‐based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage‐specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering. Copyright

Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications

This work describes the development of a biodegradable matrix, based on chitosan and starch, with the ability to form a porous structure in situ due to the attack by specific enzymes present in the human body (alpha-amylase and lysozyme). Scaffolds with three different compositions were developed: chitosan (C100) and chitosan/starch (CS80-20, CS60-40). Compressive test results showed that these materials exhibit very promising mechanical properties, namely a high modulus in both the dry and wet states. The compressive modulus in the dry state for C100 was 580+/-33MPa, CS80-20 (402+/-62MPa) and CS60-40 (337+/-78MPa). Degradation studies were performed using alpha-amylase and/or lysozyme at concentrations similar to those found in human serum, at 37 degrees C for up to 90 days. Scanning electron micrographs showed that enzymatic degradation caused a porous structure to be formed, indicating the potential of this methodology to obtain in situ forming scaffolds. In order to evaluate the biocompatibility of the scaffolds, extracts and direct contact tests were performed. Results with the MTT test showed that the extracts of the materials were clearly non-toxic to L929 fibroblast cells. Analysis of cell adhesion and morphology of seeded osteoblastic-like cells in direct contact tests showed that at day 7 the number of cells on CS80-20 and CS60-40 was noticeably higher than that on C100, which suggests that starch containing materials may promote cell adhesion and proliferation. This combination of properties seems to be a very promising approach to obtain scaffolds with gradual in vivo pore forming capability for bone tissue engineering applications.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein–peptide system

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.