Natália M. Alves

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.

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.

Chitosan/Poly(ɛ-caprolactone) blend scaffolds for cartilage repair

Chitosan (CHT)/poly(ɛ-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. μCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation.

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.

Glass transition and structural relaxation in semi-crystalline poly(ethylene terephthalate): a DSC study

The aim of this work is to determine the relaxation times of the cooperative conformational rearrangements of the amorphous phase in semi-crystalline poly(ethylene terephthalate) (PET) and compare them with those calculated in amorphous PET. Samples of nearly amorphous polymer were prepared by quenching and samples with different crystallinity fractions were prepared from the amorphous one using cold crystallisation to different temperatures. The differential scanning calorimetry (DSC) thermograms measured on samples rapidly cooled from temperatures immediately above the glass transition show a single glass transition which is much broader in the case of high-crystallinity samples than in the amorphous or low-crystallinity PET. To clarify this behaviour, the samples were subjected to annealing at different temperatures and for different periods prior to the DSC measuring heating scan. The thermograms measured in samples with low crystallinity clearly show the existence of two amorphous phases with different conformational mobility, these are called Phases I and II. Phase I contains polymer chains with a mobility similar to that in the purely amorphous polymer, while Phase II shows a much more restricted mobility, probably corresponding to conformational changes within the intraspherulitic regions. The model simulation allows to determine the temperature dependence of Phase II relaxation times, which are independent from the crystallinity fraction in the sample and around two decades longer than those of the amorphous polymer at the same temperature.

Self Assembling and Crosslinking of Polyelectrolyte Multilayer Films of Chitosan and Alginate Studied by QCM and IR Spectroscopy

The formation of novel biocompatible multilayer films based on the alternate deposition of CHI and ALG was investigated for the first time by QCM-D and FTIR-ATR. A linear increase of the thickness was found during the film build-up. GLUT was used to crosslink the films terminated with either CHI or ALG. A change in the QCM-D signal was observed just in the first case, indicating that crosslinking only takes place in the top CHI layer. The evolution of the dissipation factor during crosslinking was modelled with a first-order kinetics; this reaction was found to be faster for chitosan terminated films with a lower number of multilayers. It was also found that more robust films could be produced by crosslinking the intermediate CHI layers during the build-up.

New poly(e-caprolactone)/chitosan blend fibers for tissue engineering applications

This study reports for the first time on the production of poly(epsilon-caprolactone)/chitosan blend fibers for future application as tissue engineering scaffolds. Fibers of chitosan and poly(epsilon-caprolactone) were prepared by wet spinning from blend solutions, using a formic acid/acetone 70:30vol.% mixture as common solvent and methanol as coagulant. By this method, blend fibers with a wide compositional range and controllable diameters could be produced. Scanning electron microscopy shows the existence of roughness and porosity at the micron level scale in the blend fiber surface that could be potentially advantageous for cell attachment. Studies were also conducted using both conventional and innovative techniques to evaluate compatibility between the polymers, including FTIR imaging and investigation of the glass transition of chitosan using dynamic mechanical analysis on samples with controlled swelling. The data suggest that a certain degree of interaction exists, although it does not seem to be a result of chemical interaction. The designed fibers could be potentially used for the development of scaffolds for tissue engineering applications.

Bioinspired superhydrophobic poly(L-lactic acid) surfaces control bone marrow derived cells adhesion and proliferation

The aptitude of a cell to adhere, migrate, and differentiate on a compact substrate or scaffold is important in the field of tissue engineering and biomaterials. It is well known that cell behavior can be controlled and guided through the change in micro- and nano-scale topographic features. In this work, we intend to demonstrate that special topographic features that control wettability may also have an important role in the biological performance of biodegradable substrates. Poly(L-lactic acid) surfaces with superhydrophobic characteristics were produced, based on the so-called Lotus effect, exhibiting dual micro- and nano-scale roughness. The water contact angle could be higher than 150 degrees and a value of that order could be kept even upon immersion in a simulated body fluid solution for more than 20 days. Such water repellent surfaces were found to prevent adhesion and proliferation of bone marrow derived cells previously isolated from the femurs of 6-week-old male Wistar rats, when compared with smoother surfaces prepared by simple solvent casting. Such results demonstrate that these superhydrophobic surfaces may be used to control cell behavior onto biodegradable substrates.

Crosslink effect and albumin adsorption onto chitosan/alginate multilayered systems: an in situ QCM-D study.

The adsorption of HSA onto CHI/ALG multilayer assemblies was assessed in situ using QCM-D. It was found that the behavior of HSA on biomaterials surface can be tuned by adjusting parameters of the polyelectrolyte system such as pH, layer number, crosslinker and polymer terminal layer. Our results confirmed the key role of electrostatic interactions during HSA adsorption, since oppositely charged surfaces were more effective in promoting protein adhesion. QCM-D data revealed that crosslinking (CHI/ALG)(5) CHI films allows HSA to become adsorbed in physiological conditions. Our results suggested that the biological potential of biopolymers and the mild conditions of the LbL technique turn these natural nanoassemblies into a suitable choice to be used as pH-sensitive coatings.

Nanostructured Polymeric Coatings Based on Chitosan and Dopamine‐Modified Hyaluronic Acid for Biomedical Applications

In a marine environment, specific proteins are secreted by mussels and used as a bioglue to stick to a surface. These mussel proteins present an unusual amino acid 3,4-dihydroxyphenylalanine (known as DOPA). The outstanding adhesive properties of these materials in the sea harsh conditions have been attributed to the presence of the catechol groups present in DOPA. Inspired by the structure and composition of these adhesive proteins, dopamine-modified hyaluronic acid (HA-DN) prepared by carbodiimide chemistry is used to form thin and surface-adherent dopamine films. This conjugate was characterized by distinct techniques, such as nuclear magnetic resonance and ultraviolet spectrophotometry. Multilayer films are developed based on chitosan and HA-DN to form polymeric coatings using the layer-by-layer methodology. The nanostructured films formation is monitored by quartz crystal microbalance. The film surface is characterized by atomic force microscopy and scanning electron microscopy. Water contact angle measurements are also conducted. The adhesion properties are analyzed showing that the nanostructured films with dopamine promote an improved adhesion. In vitro tests show an enhanced cell adhesion, proliferation and viability for the biomimetic films with catechol groups, demonstrating their potential to be used in distinct biomedical applications.