Fernando Dourado

Characterization of cellulose surface free energy

Junta Nacional de Investigacao Cientifica e Tecnologica (JNICT) - INTERREG II/BM/03/REG II/3/97.

Studies on the properties of Celluclast/Eudragit L-100 conjugate

A cellulase from Trichoderma reesei was immobilized on Eudragit L-100, a reversibly soluble polymer depending on the pH of the medium. The solubility of the modified cellulase was studied at different pH values. By changing the pH, the adsorption equilibrium of the derivatized proteins is switched towards the liquid phase, thus making recycling possible. This method allows for improved stability, without major loss of specific activity. The adsorption of cellulase on Eudragit lowers the enthalpy of denaturation, but affects only slightly the denaturation temperature. The use of carbodiimide was ineffective on linking the enzymes covalently to the polymer, since the immobilization process was found to be only mediated by non-covalent forces.

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.

Neuronal cells’ behavior on polypyrrole coated bacterial nanocellulose three-dimensional (3D) scaffolds

In this work, polypyrrole (PPy) was in situ polymerized onto the surface of bacterial nanocellulose (BNC) produced by Gluconacetobacter xylinus, by chemical oxidation in aqueous medium using ammonium persulfate. Composites (BNC/PPy) were produced with varying concentrations of pyrrole (Py). The produced BNC/PPy membranes were used as a template for the seeding of PC12 rat neuronal cells. Cell suspensions were directly seeded onto the surfaces of the BNC/PPy membranes. The Py concentration affected the behavior of neuronal cells that adhered and grew significantly more on BNC/PPy comparatively to BNC. Scanning electron microscopy (SEM) micrographs revealed that PC12 cells adhered on the surface of the BNC and BNC/PPy membranes. Conductive PPy coatings on nanofibers acting as an active interface for tissue engineering may be used to regulate cell activity through electrical stimulations.

Production and Characterization of a New Bacterial Cellulose/Poly(Vinyl Alcohol) Nanocomposite

Bacterial cellulose (BC) is characterized for its high water holding capacity, high crystallinity, an ultrafine fiber network and high tensile strength. This work demonstrates the production of a new interpenetrated polymer network nanocomposite obtained through the incorporation of poly(vinyl alcohol) (PVA) on the BC matrix and evaluates the effect of oven drying on the morphological, mechanical and mass transfer properties of the composite membranes. Both the addition of PVA and oven drying induce the appearance of larger pores (circa 1–3 µm in average diameter) in dried BC/PVA membranes. Both types of treatments also affect the permeability of the composite, as assessed by the diffusion coefficients of polyethylene glycol (PEG) molecules (900, 8,000, 35,000 and 100,000 Da) across the membranes. Finally, the Young’s modulus of dry pristine BC decreases following PVA incorporation, resulting in a change from 3.5 GPa to 1 GPa and a five-fold loss in tensile strength.

Physicochemical and biological evaluation of poly(ethylene glycol) methacrylate grafted onto poly(dimethyl siloxane) surfaces for prosthetic devices

Poly(dimethyl siloxane) (PDMS) was surface-polymerized with poly(ethylene glycol)methacrylate (PEGMA) by surface-initiated atom transfer radical polymerization (SI-ATRP) in aqueous media at room temperature. Modification of the PDMS surface followed a three-step procedure: (i) PDMS surface hydroxylation by UV/ozone exposure, immediately followed by (ii) covalent attachment of the initiator, 1-trichlorosilyl-2-(chloromethylphenyl)ethane, onto the hydroxylated PDMS, via chemical vapor deposition; finally (iii) PDMS surface-polymerization of PEGMA by ATRP. Modified PDMS was characterized by water contact angle measurement, SEM, FTIR-ATR, and XPS. Results showed that modified surfaces had a hydrophilic character, given the water contact angles around 60°; FTIR-ATR and XPS analysis confirmed the presence of polymerized PEGMA on the surface of PDMS and the adhesion of Staphylococcus aureus GB 2/1 and Streptococcus salivarius GB 24/9 onto the modified surfaces was inhibited 94% and 81%, respectively. Finally, the modified PDMS showed no evidence of cytotoxic effects in in vitro assays using human skin fibroblasts.

Effect of cellulase adsorption on the surface and interfacial properties of cellulose

The surface properties of several purified cellulose (Sigmacell 101, Sigmacell 20, Avicel pH 101, and Whatman CF 11) were characterised, before and after cellulase adsorption. The following techniques were used: thin-layer wicking (except for the cellulose Whatman), thermogravimetry, and differential scanning calorimetry (for all of the above celluloses). The results obtained from the calorimetric assays were consistent with those obtained from thin-layer wicking – Sigmacell 101, a more amorphous cellulose, was the least hydrophobic of the analysed celluloses, and had the highest specific heat of dehydration. The other celluloses showed less affinity for water molecules, as assessed by the two independent techniques. The adsorption of protein did not affect the amount of water adsorbed by Sigmacell 101. However, this water was more strongly adsorbed, since it had a higher specific heat of dehydration. The more crystalline celluloses adsorbed a greater amount of water, which was also more strongly bound after the treatment with cellulases. This effect was more significant for Whatman CF-11. Also, the more crystalline celluloses became slightly hydrophilic, following protein adsorption, as assessed by thin-layer wicking. However, this technique is not reliable when used with cellulase treated celluloses.

Bacterial Cellulose As a Support for the Growth of Retinal Pigment Epithelium

The feasibility of bacterial cellulose (BC) as a novel substrate for retinal pigment epithelium (RPE) culture was evaluated. Thin (41.6 ± 2.2 μm of average thickness) and heat-dried BC substrates were surface-modified via acetylation and polysaccharide adsorption, using chitosan and carboxymethyl cellulose. All substrates were characterized according to their surface chemistry, wettability, energy, topography, and also regarding their permeability, dimensional stability, mechanical properties, and endotoxin content. Then, their ability to promote RPE cell adhesion and proliferation in vitro was assessed. All surface-modified BC substrates presented similar permeation coefficients with solutes of up to 300 kDa. Acetylation of BC decreased its swelling and the amount of endotoxins. Surface modification of BC greatly enhanced the adhesion and proliferation of RPE cells. All samples showed similar stress-strain behavior; BC and acetylated BC showed the highest elastic modulus, but the latter exhibited a slightly smaller tensile strength and elongation at break as compared to pristine BC. Although similar proliferation rates were observed among the modified substrates, the acetylated ones showed higher initial cell adhesion. This difference may be mainly due to the moderately hydrophilic surface obtained after acetylation.

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium.

This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of retinal pigment epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.5-2.0 swelling/mm(3)), high mechanical strength (2048MPa) and non-pyrogenicity (2.12EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored.

Modifying fish gelatin electrospun membranes for biomedical applications: cross-linking and swelling behavior

Development of suitable membranes is a fundamental requisite for tissue and biomedical engineering applications. This work presents fish gelatin random and aligned electrospun membranes cross-linked with glutaraldehyde (GA). It was observed that the fiber average diameter and the morphology is not influenced by the GA exposure time and presents fibers with an average diameter around 250 nm. Moreover, when the gelatin mats are immersed in a phosphate buffered saline solution (PBS), they can retain as much as 12 times its initial weight of solution almost instantaneously, but the material microstructure of the fiber mats changes from the characteristic fibrous to an almost spherical porous structure. Cross-linked gelatin electrospun fiber mats and films showed a water vapor permeability of 1.37 ± 0.02 and 0.13 ± 0.10 (g.mm)/(m2.h.kPa), respectively. Finally, the processing technique and cross-linking process does not inhibit MC-3T3-E1 cell adhesion. Preliminary cell culture results showed good cell adhesion and proliferation in the cross-linked random and aligned gelatin fiber mats.