The development of biomaterials for medical applications envisages the design of three-dimensional structures – the
scaffolds. These structures, mimicking the biological structures and interacting with the surrounding tissues through
biomolecular recognition, elicit cellular responses mediated by specific interactions. Among the different scaffolds used in
biomedicine, the materials based on polysaccharides present promising characteristics, due to their biocompatibility,
hydrophilicity, degradability and appropriate mechanical properties, allowing for a favorable controlled interaction with
Recombinant proteins are widely used in the biomedical field, namely in the fuctionalization of biomaterials. It is well
established that Carbohydrate-Binding Modules (CBMs) present in several glycanases are structural and functionally
independent of the catalytic module; therefore, their application as fusion partners may be exploited, contributing to
protein expression, solubilization, purification, and finally for the functionalization of polysaccharide-based materials. This
is the main subject of this thesis: the evaluation of the potential of CBMs as tools for the improvement of the
biocompatibility of polysaccharides.
One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence,
present in several proteins of the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this
sequence was recognized as a major functional group responsible for cellular adhesion. Several polysaccharide-based
materials have been produced recently at the DEB-UM laboratories, namely dextrin based hydrogels and bacterial
cellulose scaffolds. In this study, recombinant proteins containing a CBM with starch affinity were fused to the bioactive
molecule RGD, using recombinant DNA technology, in order to functionalize dextrin-based hydrogels.
The general introduction of this thesis is presented in chapter 1 and includes a bibliographic revision of: 1) the
applications of polysaccharides as biomedical biomaterials (this revision is restricted to the dextrin and bacterial cellulose
(BC) based materials, the ones that were used in this work); 2) the strategies available for the production of recombinant
proteins, using bacterial systems; and 3) a state of the art on the CBMs and their applications.
The chapter 2 describes the development of a methodology for the expression and purification of the recombinant protein
CBM-RGD, which has a CBM from the human protein laforin fused to a RGD sequence. Different commercial
heterologous Escherichia coli expression systems (pET 29a, pET 25b and pGEXT41) were used in order to obtain high
levels of soluble protein. Despite the use of the periplasmatic secretion approach (pET25) or the fusion of CBM with
enhancing solubility tag (GST), the recombinant proteins were always obtained in the insoluble fraction. The utilization of
CHAPS and arginine allowed the protein solubilization and purification, but not the production of functional protein with
starch binding ability. Using the pET29a vector, the recombinant proteins were obtained in inclusion bodies (IB). After solubilization and refolding, the CBM was recovered and showed starch affinity. This is the first report on the expression
of the functional CBM from the human protein laforin.
The chapter 3 describes the production of recombinant proteins containing a bacterial CBM, which belongs to an α-
amylase from Bacillus sp. TS-23. This protein, like the laforin CBM, also has starch affinity, being designated a Starch-
Binding Module (SBM). The recombinant SBM and RGD-SBM proteins were cloned, expressed, purified and tested in
vitro. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with
recombinant proteins were performed using mouse embryo fibroblasts 3T3. The results showed that the RGD-SBM
recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell
spreading on the hydrogel surface was observed only in the presence of the RGD-SBM. The fusion protein RGD-SBM
provides an efficient way to functionalize the dextrin-based hydrogel, improving the interaction with cells.
The characterization of dextrin-vinyl acrylate (dextrin-VA) and dextrin-hydroxyethylmethacrylate (dextrin-HEMA)
hydrogels was presented in a previous study carried out at the DEB-UM laboratories. In this work (chapter 4) the in vivo
biocompatibility and degradability of these hydrogels are reported. The histological analysis of subcutaneous implants of
these hydrogels, featuring inflammatory and resorption events in mice, was carried out over a period of 16 weeks. While
dextrin-HEMA hydrogel was quickly and completely degraded and reabsorbed, dextrin-VA degradation occurred slowly,
apparently through an erosion controlled process. A thin fibrous capsule was observed 16 weeks post-implantation,
surrounding the non-degradable hydrogel. In the case of the degradable material, only a mild inflammatory reaction was
observed, with few foamy macrophages being detected around the implant. This reaction was followed by complete
resorption, with no signs of capsule formation or fibrosis associated with the implants. Altogether, these results strongly
suggest that the dextrin hydrogels are fully biocompatible, since no toxicity on the tissues surrounding the implants was
found. Moreover, it may be speculated that a controlled degradation rate of the hydrogels may be obtained, using dextrin
with grafted HEMA and VA in different proportions.
Chapter 5 presents the evaluation of Bacterial Cellulose – NanoFibers (BC-NFs) nanotoxicology. BC is a promising
material for biomedical applications, namely due its biocompatibility. Although BC has been shown to be neither cytotoxic
nor genotoxic, the properties of isolated BC-NFs on cells and tissues has never been analysed. Considering the toxicity
associated to other fibre-shaped nanoparticles, it seems crucial to evaluate the toxicity associated to the BC-NFs. The
results from single cell gel electrophoresis (also known as comet assay) and the Salmonella reversion assay showed that
NFs, produced from BC by a combination of acid and ultrasonic treatment, are not genotoxic under the conditions tested.
A proliferation assay using fibroblasts and CHO cells reveals a slight reduction in the proliferation rate, although no
modification in the cell morphology is observed.
Overall, this work reports the successful expression and isolation of the atypical human CBM, from the protein laforin. It
provides a contribution to the development of a strategy based on the use of CBMs as tools for the modification of the
surface properties of biomaterials, improving the interaction with cells. Finally, this work characterizes biocompatibility
aspects of biomaterials currently under development at DEB-UM laboratories.
Publication Type: PhD Theses