Jorge Padrão

Exploiting the Sequence of Naturally Occurring Elastin: Construction, Production and Characterization of a Recombinant Thermoplastic Protein-Based Polymer

Genetic engineering was used to produce an elastin-like polymer (ELP) with precise amino acid composition, sequence and length, resulting in the absolute control of MW and stereochemistry. A synthetic monomer DNA sequence encoding for (VPAVG)20, was used to build a library of concatemer genes with precise control on sequence and size. The higher molecular weight polymer with 220 repeats of VPAVG was biologically produced in Escherichia coli and purified by hot and cold centrifugation cycles, based on the reversible inverse temperature transition property of ELPs. The use of low cost carbon sources like lactose and glycerol for bacteria cells culture media was explored using Central Composite Design approach allowing optimization of fermentation conditions. Due to its self-assembling behaviour near 33 °C stable spherical microparticles with a size ~ 1µm were obtained, redissolving when a strong undercooling is achieved. The polymer produced showed hysteresis behaviour with thermal absorbing/releasing components depending on the salt concentration of the polymer solution.

High Level Biosynthesis of a Silk-Elastin-like Protein in E. coli

Silk-elastin-like proteins (SELPs) have enormous potential for use as customizable biomaterials in numerous biomedical and materials applications, yet success in harnessing this potential has been limited by the lack of a commercially viable industrially relevant production process. We have developed a scalable fed-batch production approach which enables a SELP volumetric productivity of 4.3 g L(-1) with E. coli BL21(DE3). This is the highest SELP productivity reported to date and is 50-fold higher than that reported by other groups. As compared to typical fed-batch processes, high preinduction growth rates and low inducer and oxygen concentrations are allowed whereas reduced postinduction feeding rates are preferred. Limiting factors were identified and productivity was found to be strongly influenced by a trade-off between the rate of production and plasmid stability. The process developed is robust, reproducible, and applicable to scale up to the industrial level and moves these biopolymers a step closer to the marketplace.

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.

Antibacterial performance of bovine lactoferrin-fish gelatine electrospun membranes

The increase of antibiotic resistant microorganisms urged the development and synthesis of novel antimicrobial biomaterials to be employed in a broad range of applications, ranging from food packaging to medical devices. This work describes the production and characterization of a protein-based electrospun fibrous membranes bearing antimicrobial properties. Its composition is exclusively comprised of proteins, with fish gelatine as structural matrix and bovine lactoferrin (bLF) as the active antimicrobial agent. The bLF bactericidal effect was determined against clinical isolates of Escherichia coli and Staphylococcus aureus through microdilution assays. Two distinctive methods were used to incorporate bLF into the fish gelatine nanofibres: (i) as a filler in the electrospinning formulation with concentrations of 2, 5 and 10 (wt%), and cross-linked with glutaraldehyde vapour, in order to achieve stability in aqueous solution; and (ii) through adsorption in a solution with 40mgmL(-1) bLF. Fourier transform infrared spectroscopy analysis showed that the structure of both proteins remained intact through the electrospinning blending and cross-linking procedure. Remarkable antibacterial properties were obtained with membranes containing 5% and 10% bLF with a bacterial reduction of approximately 90% and 100%, respectively.

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

Retinal Pigment Epithelium Sara Goncalves,† Jorge Padrao,† Ineŝ Patricio Rodrigues,‡,§ Joao Pedro Silva,† Vitor Sencadas, Senentxu Lanceros-Mendez, Henrique Girao,‡ Fernando Dourado,† and Ligia R. Rodrigues*,† †Centre of Biological Engineering and Center/Department of Physics, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal ‡Centre of Ophthalmology and Vision Sciences, IBILI-Faculty of Medicine, University of Coimbra, 3000-354, Coimbra, Portugal CNC Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal