José Carlos Rodríguez-Cabello

Layer-by-Layer Assembly of Chitosan and Recombinant Biopolymers into Biomimetic Coatings with Multiple Stimuli-Responsive Properties

In this work, biomimetic smart thin coatings using chitosan and a recombinant elastin-like recombinamer (ELR) containing the cell attachment sequence arginine-glycine-(aspartic acid) (RGD) are fabricated through a layer-by-layer approach. The synthetic polymer is characterized for its molecular mass and composition using mass spectroscopy and peptide sequencing. The adsorption of each polymeric layer is followed in situ at room temperature and pH 5.5 using a quartz-crystal microbalance with dissipation monitoring, showing that both polymers can be successfully combined to conceive nanostructured, multilayered coatings. The smart properties of the coatings are tested for their wettability by contact angle (CA) measurements as a function of external stimuli, namely temperature, pH, and ionic strength. Wettability transitions are observed from a moderate hydrophobic surface (CAs approximately from 62° to 71°) to an extremely wettable one (CA considered as 0°) as the temperature, pH, and ionic strength are raised above 50 °C, 11, and 1.25 M, respectively. Atomic force microscopy is performed at pH 7.4 and pH 11 to assess the coating topography. In the latter, the results reveal the formation of large and compact structures upon the aggregation of ELRs at the surface, which increase water affinity. Cell adhesion tests are conducted using a SaOs-2 cell line. Enhanced cell adhesion is observed in the coatings, as compared to a coating with a chitosan-ending film and a scrambled arginine-(aspartic acid)-glycine (RDG) biopolymer. The results suggest that such films could be used in the future as smart biomimetic coatings of biomaterials for different biomedical applications, including those in tissue engineering or in controlled delivery systems.

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

Genetically Engineered Elastin-Like Polymer as a Substratum to Culture Cells from the Ocular Surface

Purpose: To investigate epithelial cell adhesion and proliferation on a newly developed elastin-like polymer (ELP) that mimics the functional characteristics of extracellular matrices. Materials and Methods: A genetically engineered ELP with cell attachment sequences was adsorbed onto glass coverslips as 1, 2, or 3 molecular films. Conjunctival epithelial cells from a human cell line and human skin fibroblast cells (as controls) were plated onto coverslips with three different substrata: plain glass, Thermanox®, and ELP-coated. Cells (104) were plated after EDTA- or trypsin-based detachment. To test adhesion, epithelial and fibroblast cells were incubated for 4 hr, stained with hematoxylin, and counted. To study proliferation, Ki-67-positive epithelial cells were counted after 1, 3, and 5 days in culture. Immunostaining for conjunctival and adhesion markers was performed. Results: Epithelial cell, but not fibroblast, adhesion on ELP was significantly enhanced compared to that of control substrata. Epithelial cells detached with EDTA alone adhered significantly better than those detached with trypsin. By day 5, epithelial cell proliferation on ELP was significantly greater than that on plain glass. Epithelial cells grown on ELP expressed conjunctival and adhesion markers. Conclusions: The recombinant ELP resembling the ocular surface extracellular matrix was a suitable substratum to sustain epithelial cell attachment and growth. This type of polymer may be suitable for tissue engineering to restore vision by reconstructing the ocular surface.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein–peptide system

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Dynamic cell culturing and its application to micropatterned, elastin-like protein-modified poly(N-isopropylacrylamide) scaffolds

In this study a tissue engineering scaffold was constructed from poly(N-isopropylacrylamide) (pNIPAM) to study the influence of strain on cell proliferation and differentiation. The effect of surface chemistry and topography on bone marrow mesenchymal stem cells was also investigated. Micropatterned pNIPAM films (channels with 10 microm groove width, 2 microm ridge width, 20 microm depth) were prepared by photopolymerization. The films were chemically modified by adsorption of a genetically engineered and temperature sensitive elastin-like protein (ELP). Dynamic conditions were generated by repeated temperature changes between 29 degrees C and 37 degrees C. ELP presence on the films enhanced initial cell attachment two fold (Day 1 cell number on films with ELP and without ELP were 27.6 x 10(4) and 13.2 x 10(4), respectively) but had no effect on proliferation in the long run. ELP was crucial for maintaining the cells attached on the surface in dynamic culturing (Day 7 cell numbers on the films with and without ELP were 81.4 x 10(4) and 12.1 x 10(4), respectively) and this enhanced the ability of pNIPAM films to transfer mechanical stress on the cells. Dynamic conditions improved cell proliferation (Day 21 cell numbers with dynamic and with static groups were 180.4 x 10(4) and 157.7 x 10(4), respectively) but decreased differentiation (Day 14 specific ALP values on the films of static and dynamic groups were 6.6 and 3.5 nmol/min/cell, respectively). Thus, a physically and chemically modified pNIPAM scaffold had a positive influence on the population of the scaffolds under dynamic culture conditions.

Structural analysis of injection-moulded semicrystalline polymers by Fourier transform infra-red spectroscopy with photoacoustic detection and differential scanning calorimetry: 1. Poly(ethylene terephthalate)

Abstract Fourier transform infra-red spectroscopy coupled with photoacoustic detection (PA FT i.r.) has proved to be a useful tool for finding out about quantitative structural changes in solid materials. Poly(ethylene terephthalate) (PET) is a well known semicrystalline polymer that shows important changes on annealing. In order to obtain a complete picture of isomer distributions in industrial PET samples, spectroscopic measurements were correlated with differential scanning calorimetry (d.s.c.). The results obtained indicate that the structural characteristics of the thermally treated samples are related to the fabrication process. Two different strata in the plates can be distinguished: a skin layer and the core. The correlation between the apparent degree of crystallinity of the surface obtained by d.s.c. and the percentage of trans isomer obtained by PA FT i.r. allows the latter parameter to be separated into crystalline and amorphous trans isomer and to follow its evolution with the annealing process. Amorphous trans isomer vanishes at the primary isomerization (∼ 100°C) while crystalline trans and gauche conformations show sigmoidal evolution. At higher annealing temperatures (> 140°C) the ordered trans conformation shows an approximately linear increase at the expense of the gauche conformation. Finally, a direct correlation between the ordered trans isomer and the apparent degree of surface crystallinity can be made.

Elastin-like recombinamers: Biosynthetic strategies and biotechnological applications

The past few decades have witnessed the development of novel naturally inspired biomimetic materials, such as polysaccharides and proteins. Likewise, the seemingly exponential evolution of genetic‐engineering techniques and modern biotechnology has led to the emergence of advanced protein‐based materials with multifunctional properties. This approach allows extraordinary control over the architecture of the polymer, and therefore, monodispersity, controlled physicochemical properties, and high sequence complexity that would otherwise be impossible to attain. Elastin‐like recombinamers (ELRs) are emerging as some of the most prolific of these protein‐based biopolymers. Indeed, their inherent properties, such as biocompatibility, smart nature, and mechanical qualities, make these recombinant polymers suitable for use in numerous biomedical and nanotechnology applications, such as tissue engineering, “smart” nanodevices, drug delivery, and protein purification. Herein, we present recent progress in the biotechnological applications of ELRs and the most important genetic engineering‐based strategies used in their biosynthesis.

Structural analysis of injection-moulded semicrystalline polymers by Fourier-transform infra-red spectroscopy with photoacoustic detection and differential scanning calorimetry: 2. Polyamide-6,6

Abstract In this paper, a study of the structural changes due to annealing of injection-moulded polyamide-6,6 has been carried out. The spectroscopic behaviour of polyamide-6,6 is quite similar to that of poly(ethylene terephthalate) (PET). Using the band at 1650 cm −1 as an internal reference band, the intensity changes of the bands situated at 1146 and 936 cm −1 were followed. The former decreases when the annealing temperature exceeds 180°C whereas the latter increases. Furthermore, the bandwidth of the band at 936 cm −1 decreases from 23 to 20 cm −1 for annealing above 180°C. These spectroscopic changes were related to gauche/trans isomerism induced by the thermal treatment. Moreover, polyamide-6,6 verifies a two-phase conformational model, similarly to PET. As far as the thermal behaviour is concerned, two endothermic peaks at low (LM peak) and high (HM peak) temperature were found in thermograms of samples annealed above 150°C, and these displayed similar behaviour to those found in PET. The LM peak can be attributed to melting of crystals with increasing perfection and fold-surface smoothing of the crystalline layers due to the annealing treatment, and the HM peak to melting of the recrystallized crystalline structure during heating in differential scanning calorimetry (d.s.c.). On the other hand, the influence of the fabrication process in polyamide-6,6 seems not to be as important as in PET. The correlation between Fourier-transform infra-red spectroscopy with photoacoustic detection (p.a.- FT i.r.) and d.s.c. measurements show that the amide group works as an important constraint that limits the mobility of the crystalline molecules, and most of the conformational changes occur in the amorphous phase for annealing temperatures above 180°C. In the ordered phase, a slight crystallinity increase beyond this temperature can be related to crystalline perfection and fold-surface smoothing.

3D microstructuring of smart bioactive hydrogels based on recombinant elastin-like polymers

We describe a simple method of replica moulding to obtain novel 3D microstructured smart hydrogels based on protein polymers mimicking the structure and behaviour of the extracellular matrix (ECM).

Elastin-like recombinamers as substrates for retinal pigment epithelial cell growth

The aim of this study is to investigate the use of elastin-like recombinamers (ELRs) as a substrate that can maintain the growth, phenotype, and functional characteristics of retinal pigment epithelial (RPE) cells efficiently and as a suitable carrier for the transplantation of autologous RPE cells for treatment of age-related macular degeneration (AMD). ELR films containing a bioactive sequence, RGD (ELR-RGD), and one with no specific sequence (ELR-IK) as control, were obtained by solvent-casting onto glass and subsequent cross-linking. ARPE19 cells were seeded on sterilized ELR films as well as on the control surfaces. Cells were analysed after 4, 24, 72, and 120 h to study cell adhesion, proliferation, cell viability, morphology, and specificity by staining with Trypan blue, DAPI, Rhodamin-Phalloidin and RPE65, ZO-1 antibodies and observing under fluorescence as well as electron microscope. ARPE19 cells seeded on both ELR films and controls were 100% viable and maintained their morphology and set of characteristics at the different time points studied. Cell proliferation on ELR-RGD was significantly higher than that found on ELR-IK at all time points, although it was less than the growth rate on polystyrene. ARPE19 cells grow well on ELR-RGD maintaining their phenotype. These results should be extended to further studies with fresh human RPE cells and in vivo studies to determine whether this ELR-RGD matrix could be used as a Bruchs membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.