The Incorporation of Etanercept into a Porous Tri-Layer Scaffold for Restoring and Repairing Cartilage Tissue.

Cartilage diseases currently affect a high percentage of the world's population. Almost all of these diseases, such as osteoarthritis (OA), cause inflammation of this soft tissue. However, this could be controlled with biomaterials that act as an anti-inflammatory delivery system, capable of dosing these drugs over time in a specific area. The objective of this study was to incorporate etanercept (ETA) into porous three-layer scaffolds to decrease the inflammatory process in this soft tissue. ETA is a blocker of pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). For this reason, the scaffold was built based on natural polymers, including chitosan and type I collagen. The scaffold was grafted next to subchondral bone using hydroxyapatite as filler. One of the biomaterials obtained was also crosslinked to compare its mechanical properties with the non-treated one. Both samples' physicochemical properties were studied with SEM, micro-CT and photoacoustic imaging, and their rheological properties were also compared. The cell viability and proliferation of the human chondrocyte C28/I2 cell line were studied in vitro. An in vitro and in vivo controlled release study was evaluated in both specimens. The ETA anti-inflammatory effect was also studied by in vitro TNF-α and IL-6 production. The crosslinked and non-treated scaffolds had rheological properties suitable for this application. They were non-cytotoxic and favoured the in vitro growth of chondrocytes. The in vitro and in vivo ETA release showed desirable results for a drug delivery system. The TNF-α and IL-6 production assay showed that this drug was effective as an anti-inflammatory agent. In an in vivo OA mice model, safranin-O and fast green staining was carried out. The OA cartilage tissue improved when the scaffold with ETA was grafted in the damaged area. These results demonstrate that this type of biomaterial has high potential for clinical applications in tissue engineering and as a controlled drug delivery system in OA articular cartilage.

The Effects of Crosslinking on the Rheology and Cellular Behavior of Polymer-Based 3D-Multilayered Scaffolds for Restoring Articular Cartilage.

Polymer-based tri-layered (bone, intermediate and top layers) scaffolds used for the restoration of articular cartilage were prepared and characterized in this study to emulate the concentration gradient of cartilage. The scaffolds were physically or chemically crosslinked. In order to obtain adequate scaffolds for the intended application, the impact of the type of calcium phosphate used in the bone layer, the polymer used in the intermediate layer and the interlayer crosslinking process were analyzed. The correlation among SEM micrographs, physical-chemical characterization, swelling behavior, rheological measurements and cell studies were examined. Storage moduli at 1 Hz were 0.3-1.7 kPa for physically crosslinked scaffolds, and 4-5 kPa (EDC/NHS system) and 15-20 kPa (glutaraldehyde) for chemically crosslinked scaffolds. Intrinsic viscoelasticity and poroelasticity were considered in discussing the physical mechanism dominating in different time/frequency scales. Cell evaluation showed that all samples are available as alternatives to repair and/or substitute cartilage in articular osteoarthritis.

Elastin-like recombinamers-based hydrogel modulates post-ischemic remodeling in a non-transmural myocardial infarction in sheep.

Ischemic heart disease is a leading cause of mortality due to irreversible damage to cardiac muscle. Inspired by the post-ischemic microenvironment, we devised an extracellular matrix (ECM)-mimicking hydrogel using catalyst-free click chemistry covalent bonding between two elastin-like recombinamers (ELRs). The resulting customized hydrogel included functional domains for cell adhesion and protease cleavage sites, sensitive to cleavage by matrix metalloproteases overexpressed after myocardial infarction (MI). The scaffold permitted stromal cell invasion and endothelial cell sprouting in vitro. The incidence of non-transmural infarcts has increased clinically over the past decade, and there is currently no treatment preventing further functional deterioration in the infarcted areas. Here, we have developed a clinically relevant ovine model of non-transmural infarcts induced by multiple suture ligations. Intramyocardial injections of the degradable ELRs-hydrogel led to complete functional recovery of ejection fraction 21 days after the intervention. We observed less fibrosis and more angiogenesis in the ELRs-hydrogel-treated ischemic core region compared to the untreated animals, as validated by the expression, proteomic, glycomic, and histological analyses. These findings were accompanied by enhanced preservation of GATA4+ cardiomyocytes in the border zone of the infarct. We propose that our customized ECM favors cardiomyocyte preservation in the border zone by modulating the ischemic core and a marked functional recovery. The functional benefits obtained by the timely injection of the ELRs-hydrogel in a clinically relevant MI model support the potential utility of this treatment for further clinical translation.

Self-assembly of Janus Au:Fe3O4 branched nanoparticles. From organized clusters to stimuli-responsive nanogel suprastructures.

Janus nanoparticles offer enormous possibilities through a binary selective functionalization and dual properties. Their self-assembly has attracted strong interest due to their potential as building blocks to obtain molecular colloids, supracrystals and well-organized nanostructures that can lead to new functionalities. However, this self-assembly has been focused on relatively simple symmetrical morphologies, while for complex nanostructures this process has been unexplored. Here, we study the assembly of plasmonic-magnetic Janus nanoparticles with a branched (nanostar) - sphere morphology. The branched morphology enhances their plasmonic properties in the near-infrared region and therefore their applicability, but at the same time constrains their self-assembly capabilities to obtain more organized or functional suprastructures. We describe the self-assembly of these nanoparticles after amphiphilic functionalization. The role of the nanoparticle branching, as well as the size of the polymer-coating, is explored. We show how the use of large molecular weight stabilizing polymers can overcome the anisotropy of the nanoparticles producing a change in the morphology from small clusters to larger quasi-cylindrical nanostructures. Finally, the Janus nanoparticles are functionalized with a thermo-responsive elastin-like recombinamer. These nanoparticles undergo reversible self-assembly in the presence of free polymer giving rise to nanoparticle-stabilized nanogel-like structures with controlled size, providing the possibility to expand their applicability to multi-stimuli controlled self-assembly.

Design, construction, and biological testing of an implantable porous trilayer scaffold for repairing osteoarthritic cartilage.

Various tissue engineering systems for cartilage repair have been designed and tested over the past two decades, leading to the development of many promising cartilage grafts. However, no one has yet succeeded in devising an optimal system to restore damaged articular cartilage. Here, the design, assembly, and biological testing of a porous, chitosan/collagen-based scaffold as an implant to repair damaged articular cartilage is reported. Its gradient composition and trilayer structure mimic variations in natural cartilage tissue. One of its layers includes hydroxyapatite, a bioactive component that facilitates the integration of growing tissue on local bone in the target area after scaffold implantation. The scaffold was evaluated for surface morphology; rheological performance (storage, loss, complex, and time-relaxation moduli at 1 kHz); physiological stability; in vitro activity and cytotoxicity (on a human chondrocyte C28 cell line); and in vivo performance (tissue growth and biodegradability), in a murine model of osteoarthritis. The scaffold was shown to be mechanically resistant and noncytotoxic, favored tissue growth in vivo, and remained stable for 35 days postimplantation in mice. These encouraging results highlight the potential of this porous chitosan/collagen scaffold for clinical applications in cartilage tissue engineering.

Protein disorder-order interplay to guide the growth of hierarchical mineralized structures.

A major goal in materials science is to develop bioinspired functional materials based on the precise control of molecular building blocks across length scales. Here we report a protein-mediated mineralization process that takes advantage of disorder-order interplay using elastin-like recombinamers to program organic-inorganic interactions into hierarchically ordered mineralized structures. The materials comprise elongated apatite nanocrystals that are aligned and organized into microscopic prisms, which grow together into spherulite-like structures hundreds of micrometers in diameter that come together to fill macroscopic areas. The structures can be grown over large uneven surfaces and native tissues as acid-resistant membranes or coatings with tuneable hierarchy, stiffness, and hardness. Our study represents a potential strategy for complex materials design that may open opportunities for hard tissue repair and provide insights into the role of molecular disorder in human physiology and pathology.

Development of tailored and self-mineralizing citric acid-crosslinked hydrogels for in situ bone regeneration.

Bone tissue engineering demands alternatives overcoming the limitations of traditional approaches in the context of a constantly aging global population. In the present study, elastin-like recombinamers hydrogels were produced by means of carbodiimide-catalyzed crosslinking with citric acid, a molecule suggested to be essential for bone nanostructure. By systematically studying the effect of the relative abundance of reactive species on gelation and hydrogel properties such as functional groups content, degradation and structure, we were able to understand and to control the crosslinking reaction to achieve hydrogels mimicking the fibrillary nature of the extracellular matrix. By studying the effect of polymer concentration on scaffold mechanical properties, we were able to produce hydrogels with a stiffness value of 36.13 ± 10.72 kPa, previously suggested to be osteoinductive. Microstructured and mechanically-tailored hydrogels supported the growth of human mesenchymal stem cells and led to higher osteopontin expression in comparison to their non-tailored counterparts. Additionally, tailored hydrogels were able to rapidly self-mineralize in biomimetic conditions, evidencing that citric acid was successfully used both as a crosslinker and a bioactive molecule providing polymers with calcium phosphate nucleation capacity.

Elastin-like recombinamers with acquired functionalities for gene-delivery applications.

In this work, well-defined elastin-like recombinamers (ELRs) were studied as a choice to the existing nonviral vectors due to their biocompatibility and ease of scale-up. Functional motifs, namely penetratin and LAEL fusogenic peptides were incorporated into a basic ELR sequence, and imidazole groups were subsequently covalently bound obtaining ELRs with new functionalities. Stable polyplexes composed of plasmid DNA and ELRs were formed. A particle size around 200 nm and a zeta potential up to nearly +24 mV made them suitable for gene delivery purposes. Additionally, viability and transfection assays with C6 rat glioma cell line showed an increase in the cellular uptake and transfection levels for the construction containing the LAEL motif. This study highlights the importance of controlling the polymer functionality using recombinant techniques and establishes the utility of ELRs as biocompatible nonviral systems for gene-therapy applications.

Elastin-like recombinamer-covered stents: Towards a fully biocompatible and non-thrombogenic device for cardiovascular diseases.

We explored the use of recently developed gels obtained by the catalyst free click reaction of elastin-like recombinamers (ELRs) to fabricate a new class of covered stents. The approach consists in embedding bare metal stents in the ELR gels by injection molding, followed by endothelialization under dynamic pressure and flow conditions in a bioreactor. The mechanical properties of the gels could be easily tuned by choosing the adequate concentration of the ELR components and their biofunctionality could be tailored by inserting specific sequences (RGD and REDV). The ELR-covered stents exhibited mechanical stability under high flow conditions and could undergo crimping and deployment without damage. The presence of RGD in the ELR used to cover the stent supported full endothelialization in less than 2weeks in vitro. Minimal platelet adhesion and fibrin adsorption were detected after exposure to blood, as shown by immunostaining and scanning electron microscopy. These results prove the potential of this approach towards a new and more effective generation of covered stents which exclude the atherosclerotic plaque from the blood stream and have high biocompatibility, physiological hemocompatibility and reduced response of the immune system.

Biomolecular functionalization for enhanced cell-material interactions of poly(methyl methacrylate) surfaces.

The integration of implants or medical devices into the body tissues requires of good cell-material interactions. However, most polymeric materials used for these applications lack on biological cues, which enhanced mid- and long-term implant failure due to weak integration with the surrounding tissue. Commonly used strategies for tissue-material integration focus on functionalization of the material surface by means of natural proteins or short peptides. However, the use of these biomolecules involves major drawbacks such as immunogenic problems and oversimplification of the constructs. Here, designed elastin-like recombinamers (ELRs) are used to enhance poly(methyl methacrylate) surface properties and compared against the use of short peptides. In this study, cell response has been analysed for different functionalization conditions in the presence and absence of a competing protein, which interferes on surface-cell interaction by unspecific adsorption on the interface. The study has shown that ELRs can induce higher rates of cell attachment and stronger cell anchorages than short peptides, being a better choice for surface functionalization.

Enhanced cell-material interactions through the biofunctionalization of polymeric surfaces with engineered peptides.

Research on surface modification of polymeric materials to guide the cellular activity in biomaterials designed for tissue engineering applications has mostly focused on the use of natural extracellular matrix (ECM) proteins and short peptides, such as RGD. However, the use of engineered proteins can gather the advantages of these strategies and avoid the main drawbacks. In this study, recombinant engineered proteins called elastin-like recombinamers (ELRs) have been used to functionalize poly(lactic) acid (PLA) model surfaces. The structure of the ELRs has been designed to include the integrin ligand RGDS and the cross-linking module VPGKG. Surface functionalization has been characterized and optimized by means of ELISA and atomic force microscopy (AFM). The results suggest that ELR functionalization creates a nonfouling canvas able to restrict unspecific adsorption of proteins. Moreover, AFM analysis reveals the conformation and disposition of ELRs on the surface. Biological performance of PLA surfaces functionalized with ELRs has been studied and compared with the use of short peptides. Cell response has been assessed for different functionalization conditions in the presence and absence of the bovine serum albumin (BSA) protein, which could interfere with the surface-cell interaction by adsorbing on the interface. Studies have shown that ELRs are able to elicit higher rates of cell attachment, stronger cell anchorages and faster levels of proliferation than peptides. This work has demonstrated that the use of engineered proteins is a more efficient strategy to guide the cellular activity than the use of short peptides, because they not only allow for better cell attachment and proliferation, but also can provide more complex properties such as the creation of nonfouling surfaces.

Efficient cell and cell-sheet harvesting based on smart surfaces coated with a multifunctional and self-organizing elastin-like recombinamer.

A wide range of smart surfaces with novel properties relevant for biomedical applications have been developed recently. Herein we focus on thermoresponsive surfaces that switch between cell-adherent and nonadherent states and their applications for cell harvesting. These smart surfaces are obtained by covalently coupling a tailored elastin-like recombinamer onto glass surfaces by means of the well-known and widely applied Click Chemistry methodology. The resulting recombinamer-functionalized surfaces have been characterized by means of water contact angle measurements, XPS and TOF-SIMS. A cell-based analysis of these surfaces with human fibroblasts showed a high degree of adhesion to the surface in its adherent state (37 °C), thus, promoting cell viability and proliferation. A temperature decrease triggers reorganization of the recombinamer, thus, markedly increasing the number of nonadherent domains and masking the adherent ones. This process allows a specific and efficient temporal control of cell adhesion and cell detachment. After determination of the properties required for a suitable cell-harvesting system, optimization of the process allows single cells or cell sheets from at least two types of cells (HFF-1 and ADSCs) to be rapidly harvested.

Nanostructured and thermoresponsive recombinant biopolymer-based microcapsules for the delivery of active molecules.

Multilayer capsules conceived at the nano- and microscales are receiving increasing interest due to their potential role as carriers of biomolecules for drug delivery and tissue engineering. Herein we report the construction of microcapsules by the sequential adsorption of chitosan and a biomimetic elastin-like recombinamer into nanostructured layers on inorganic microparticle templates. The release profile of bovine serum albumin, which was studied at 25 and 37 °C, shows higher retention and Fickian diffusion at physiological temperature. The self-assembled multilayers act as a barrier and allowed for sustained release over 14 days. The capsules studied are non-cytotoxic towards L929 cells, thereby suggesting multiple applications in the fields of biotechnology and bioengineering, where high control of the delivery of therapeutics and growth/differentiation factors is required. FROM THE CLINICAL EDITOR: In this paper, the construction of microcapsules by sequential adsorption of chitosan and a biomimetic, elastin-like recombinamer into nanostructured layers on inorganic microparticle templates is reported. The layers demonstrated sustained drug release over 14 days. These microcapsules are non-cytotoxic toward L929 cells, suggesting multiple applications where high control of drug or growth factor delivery is required.

A low elastic modulus Ti-Nb-Hf alloy bioactivated with an elastin-like protein-based polymer enhances osteoblast cell adhesion and spreading.

ß-type titanium alloys with low Young's modulus are desirable to reduce stress shielding effect and enhance bone remodeling for implants used to substitute failed hard tissue. For biomaterials application, the surface bioactivity is necessary to achieve optimal osseointegration. In the previous work, the low elastic modulus (43 GPa) Ti-25Nb-16Hf (wt %) alloy was mechanically and microstructurally characterized. In the present work, the biological behavior of Ti-25Nb-16Hf was studied. The biological response was improved by surface modification. The metal surface was modified by oxygen plasma and subsequently silanized with 3-chloropropyl(triethoxy)silane for covalent immobilization of the elastin-like polymer. The elastin-like polymer employed exhibits RGD bioactive motives inspired to the extracellular matrix in order to improve cell adhesion and spreading. Upon modification, the achieved surface presented different physical and chemical properties, such as surface energy and chemical composition. Subsequently, osteoblast adhesion, cell numbers, and differentiation studies were performed to correlate surface properties and cell response. The general tendency was that the higher surface energy the higher cell adhesion. Furthermore, cell culture and immunofluorescence microscopy images demonstrated that RGD-modified surfaces improved adhesion and spreading of the osteoblast cell type.

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.

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.

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 Bruch's membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.

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.

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 (10(4)) 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.