Single step fabrication of antimicrobial fibre mats from a bioengineered protein-based polymer.

Genetically engineered protein polymers functionalized with bioactive domains have potential as multifunctional versatile materials for biomedical use. The present work describes the fabrication and characterisation of antimicrobial fibre mats comprising the antimicrobial elastin-like recombinamer (ELR) CM4-A200. The CM4-A200 protein polymer derives from the genetic fusion of the ABP-CM4 antimicrobial peptide from Bombyx mori with 200 repetitions of the pentamer VPAVG. This is the first report on non-crosslinked fibre mats fabricated with an antimicrobial ELR stable in solution. Thermal gravimetric analysis of CM4-A200 fibre mats shows one single degradation step at temperatures above 300 °C, with fibres displaying a higher thermal degradation activation. The electrospun CM4-A200 fibres display high antimicrobial activity against Gram-positive and Gram-negative bacteria with no detectable cytotoxic effects against normal human skin fibroblasts and keratinocytes, revealing the great potential of these polymers for the fabrication of biomedical materials.

Kinetic study of thermal degradation of chitosan as a function of deacetylation degree.

Thermal degradation of chitosan with varying deacetylation degree (DD) ranging between 50 and 85% was analyzed by dynamic thermogravimetric analysis at different heating rates. The present study focused on the temperature range between 500 and 800K, above water evaporation. Thermal degradation showed a main degradation stage in this temperature interval with a second stage that appeared in the weight derivative curves as a shoulder in the high temperature side of the main peak with increasing intensity as the DD decreased. The Kissinger and isoconversional Ozawa-Flynn-Wall models were employed to evaluate the Ea of both thermal degradation processes. Different kinetic models were tested to computer simulate the thermogravimetric traces calculating the model parameters with a non-linear least squares fitting routine. The Sestack-Berggren model allowed reproducing accurately the overlapping of the two degradation mechanisms and calculating the mass fraction lost in each of them revealing the coupling between the two degradation mechanisms.

Processing and size range separation of pristine and magnetic poly(l-lactic acid) based microspheres for biomedical applications.

Biodegradable poly(l-lactic acid) (PLLA) and PLLA/CoFe2O4 magnetic microspheres with average sizes ranging between 0.16-3.9µm and 0.8-2.2µm, respectively, were obtained by an oil-in-water emulsion method using poly(vinyl alcohol) (PVA) solution as the emulsifier agent. The separation of the microspheres in different size ranges was then performed by centrifugation and the colloidal stability assessed at different pH values. Neat PLLA spheres are more stable in alkaline environments when compared to magnetic microspheres, both types being stable for pHs higher than 4, resulting in a colloidal suspension. On the other hand, in acidic environments the microspheres tend to form aggregates. The neat PLLA microspheres show a degree of crystallinity of 40% whereas the composite ones are nearly amorphous (17%). Finally, the biocompatibility was assessed by cell viability studies with MC3T3-E1 pre-osteoblast cells.

Effect of Sterilization Methods on Electrospun Poly(lactic acid) (PLA) Fiber Alignment for Biomedical Applications.

Medically approved sterility methods should be a major concern when developing a polymeric scaffold, mainly when commercialization is envisaged. In the present work, poly(lactic acid) (PLA) fiber membranes were processed by electrospinning with random and aligned fiber alignment and sterilized under UV, ethylene oxide (EO), and γ-radiation, the most common ones for clinical applications. It was observed that UV light and γ-radiation do not influence fiber morphology or alignment, while electrospun samples treated with EO lead to fiber orientation loss and morphology changing from cylindrical fibers to ribbon-like structures, accompanied to an increase of polymer crystallinity up to 28%. UV light and γ-radiation sterilization methods showed to be less harmful to polymer morphology, without significant changes in polymer thermal and mechanical properties, but a slight increase of polymer wettability was detected, especially for the samples treated with UV radiation. In vitro results indicate that both UV and γ-radiation treatments of PLA membranes allow the adhesion and proliferation of MG 63 osteoblastic cells in a close interaction with the fiber meshes and with a growth pattern highly sensitive to the underlying random or aligned fiber orientation. These results are suggestive of the potential of both γ-radiation sterilized PLA membranes for clinical applications in regenerative medicine, especially those where customized membrane morphology and fiber alignment is an important issue.

Design and validation of a biomechanical bioreactor for cartilage tissue culture.

Specific tissues, such as cartilage, undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation--amplitude and frequency--to three-dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

Magnetoelectric CoFe2O4/polyvinylidene fluoride electrospun nanofibres.

Magnetoelectric 0-1 composites comprising CoFe2O4 (CFO) nanoparticles in a polyvinylidene fluoride (PVDF) polymer-fibre matrix have been prepared by electrospinning. The average diameter of the electrospun composite fibres is ∼325 nm, independent of the nanoparticle content, and the amount of the crystalline polar ß phase is strongly enhanced when compared to pure PVDF polymer fibres. The piezoelectric response of these electroactive nanofibres is modified by an applied magnetic field, thus evidencing the magnetoelectric character of the CFO/PVDF 0-1 composites.

In vitro mechanical fatigue behavior of poly-ɛ-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel.

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and submitted to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long-term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behavior of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow's criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.

Fatigue prediction in fibrin poly-ε-caprolactone macroporous scaffolds.

Tissue engineering applications rely on scaffolds that during its service life, either for in-vivo or in vitro applications, are under loading. The variation of the mechanical condition of the scaffold is strongly relevant for cell culture and has scarcely been addressed. The fatigue life cycle of poly-ε-caprolactone, PCL, scaffolds with and without fibrin as filler of the pore structure were characterized both dry and immersed in liquid water. It is observed that the there is a strong increase from 100 to 500 in the number of loading cycles before collapse in the samples tested in immersed conditions due to the more uniform stress distributions within the samples, the fibrin loading playing a minor role in the mechanical performance of the scaffolds.

Osteoblast, fibroblast and in vivo biological response to poly(vinylidene fluoride) based composite materials.

Electroactive materials can be taken to advantage for the development of sensors and actuators as well as for novel tissue engineering strategies. Composites based on poly(vinylidene fluoride), PVDF, have been evaluated with respect to their biological response. Cell viability and proliferation were performed in vitro both with Mesenchymal Stem Cells differentiated to osteoblasts and Human Fibroblast Foreskin 1. In vivo tests were also performed using 6-week-old C57Bl/6 mice. It was concluded that zeolite and clay composites are biocompatible materials promoting cell response and not showing in vivo pro-inflammatory effects which renders both of them attractive for biological applications and tissue engineering, opening interesting perspectives to development of scaffolds from these composites. Ferrite and silver nanoparticle composites decrease osteoblast cell viability and carbon nanotubes decrease fibroblast viability. Further, carbon nanotube composites result in a significant increase in local vascularization accompanied an increase of inflammatory markers after implantation.

Fabrication of poly(lactic acid)-poly(ethylene oxide) electrospun membranes with controlled micro to nanofiber sizes.

Biodegradable poly(L-lactide acid) (PLLA) nanofiber membranes were prepared by electrospinning of PLLA and poly(ethylene oxide) (PEO). The selective removal of PEO by water allows to obtain smaller fiber diameters and to increase the porosity of the membranes in comparison to PLLA membranes obtained under the same electrospinning conditions. After removal of PEO membranes with fiber sizes of 260 nm and average porosity close to 80% are obtained. Thermal and infrared results confirm the poor miscibility of PLLA and PEO, with the PEO randomly distributed along the PLLA fibers. On the other, PLLA and PEO mixing strongly affect their respective degradation temperatures. The influence of the PEO in the electrospinning process is discussed and the results are correlated to the evolution of the PLLA fiber diameter.

Relaxation dynamics of poly(vinylidene fluoride) studied by dynamical mechanical measurements and dielectric spectroscopy.

The aim of this study is to analyze the mobility of polymer chains in semicrystalline poly(vinylidene fluoride) (PVDF). PVDF crystallizes from the melt in the α crystalline phase. The transformation from the α phase to the electroactive ß phase can be induced by stretching at temperatures in the range between 80 and 140 °C. The spherulitic structure of the crystalline phase is deformed during stretching to form fibrils oriented in the direction of the strain. The amorphous phase confined among the crystalline lamellae is distorted as well and some degree of orientation of the polymer chains is expected. Dynamic-mechanical and dielectric spectroscopy measurements were performed in PVDF films stretched to strain ratios up to 5 at temperatures between 80 and 140 °C. Dynamic-mechanical measurements were conducted between -60 °C and melting and in this temperature range the relaxation spectra show the main relaxation of the amorphous phase (called ß-relaxation) and at higher temperatures a relaxation related to crystallites motions (α (c)-relaxation). Although the mean relaxation times of the ß-relaxation are nearly equal in PVDF before and after crystal phase transformation, a significant change of shape of the relaxation spectrum proves the effect of chain distortion due to crystal reorganization. In stretched PVDF the elastic modulus of the polymer in the direction of deformation is significantly higher than in the transversal one, as expected by chain and crystals fibril orientation. The recovery of the deformation when the sample is heated is related with the appearance of the α (c)-relaxation. Dielectric spectroscopy spectrum shows the main relaxation of the amorphous phase and a secondary process (γ-relaxation) at lower temperatures. Stretching produces significant changes in the relaxation processes, mainly in the strength and shape of the main relaxation ß. The Havriliak-Negami function has been applied to analyze the dielectric response.

Fibronectin adsorption and cell response on electroactive poly(vinylidene fluoride) films.

Due to the large potential of electroactive materials in novel tissue engineering strategies, the aim of this work is to determine if the crystalline phase and/or the surface electrical charge of electroactive poly(vinylidene fluoride), PVDF, have influence on the biological response in monolayer cell culture. Non-polar α-PVDF and electroactive ß-PVDF were prepared. The ß-PVDF films were poled by corona discharge to show negative or positive electrical surface charge density. It has been concluded that hydrophilicity of the PVDF substrates depends significantly on crystalline phase and polarity. Furthermore, by means of atomic force microscopy and an enzyme-linked immunosorbent assay test, it has been shown that positive or negative poling strongly influences the behavior of ß-PVDF supports with respect to fibronectin (FN) adsorption, varying the exhibition of adhesion ligands of adsorbed FN. Culture of MC3T3-E1 pre-osteoeblasts proved that cell proliferation depends on surface polarity as well. These results open the viability of cell culture stimulation by mechanical deformation of a piezoelectric substrate that results in varying electrical charge densities on the substrate surface.

Influence of silver nanoparticles concentration on the alpha- to beta-phase transformation and the physical properties of silver nanoparticles doped poly(vinylidene fluoride) nanocomposites.

This paper describes the processing of silver nanoparticle doped poly(vinylidene fluoride). The effect of the dopant concentration on the alpha- to beta-phase transformation of the polymer as well as in the morphological, thermal, optical and dielectric properties of the nanocomposites was investigated. Spherical silver nanoparticles were incorporated into the poly(vinylidene fluoride) polymeric matrix by the solvent casting method, with different Ag concentrations. Well-dispersed Ag nanoparticles act as nucleation centers, increasing the degree of crystallinity of the nanocomposites. Homogeneous dispersion of silver nanoparticles is demonstrated through the presence of surface plasmon resonance absorption in the nanocomposites. The alpha- to beta-phase transformation was achieved in the polymer matrix and a maximum of approximately equal to 70% of beta-PVDF was reached at 80 degrees C and a stretching ratio of 400%. The dielectric constant of the nanocomposites increases with increasing metal nanoparticle concentration, up to approximately equal to 26 at 0.020 wt% Ag content. The alpha- to beta-phase transformation affects both the dielectric response and the surface plasmon resonance.