RESUMEN
Resilin, a protein found in insect cuticles, is renowned for its outstanding elastomeric properties. The authors' laboratory previously developed a recombinant protein, which consisted of consensus resilin-like repeats from Anopheles gambiae, and demonstrated its potential in cartilage and vascular engineering. To broaden the versatility of the resilin-like protein, this study utilizes a cleavable crosslinker, which contains a disulfide bond, to develop smart resilin-like hydrogels that are redox-responsive. The hydrogels exhibit a porous structure and a stable storage modulus (G') of ≈3 kPa. NIH/3T3 fibroblasts cultured on hydrogels for 24 h have a high viability (>95%). In addition, the redox-responsive hydrogels show significant degradation in a reducing environment (10 mm glutathione (GSH)). The release profiles of fluorescently labeled dextrans encapsulated within the hydrogels are assessed in vitro. For dextran that is estimated to be larger than the mesh size of the gel, faster release is observed in the presence of reducing agents due to degradation of the hydrogel networks. These studies thus demonstrate the potential of using these smart hydrogels in a variety of applications ranging from scaffolds for tissue engineering to drug delivery systems that target the intracellular reductive environments of tumors.
Asunto(s)
Materiales Biocompatibles/síntesis química , Sistemas de Liberación de Medicamentos/métodos , Hidrogeles/síntesis química , Proteínas de Insectos/química , Proteínas Recombinantes/química , Ingeniería de Tejidos/métodos , Secuencia de Aminoácidos , Animales , Anopheles/química , Anopheles/fisiología , Materiales Biocompatibles/farmacología , Vasos Sanguíneos/citología , Vasos Sanguíneos/fisiología , Cartílago/citología , Cartílago/fisiología , Supervivencia Celular/efectos de los fármacos , Dextranos/metabolismo , Composición de Medicamentos/métodos , Liberación de Fármacos , Elasticidad , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Expresión Génica , Hidrogeles/farmacología , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Cinética , Ratones , Células 3T3 NIH , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , ReologíaRESUMEN
Resilin is an elastomeric protein abundant in insect cuticle. Its exceptional properties, which include high resilience and efficient energy storage, motivate its potential use in tissue engineering and drug delivery applications. Our lab has previously developed recombinant proteins based on the resilin-like sequence derived from Anopheles gambiae and demonstrated their promise as a scaffold for cartilage and vascular engineering. In this work, we describe a more thorough investigation of the physical properties of crosslinked resilin-like hydrogels. The resilin-like proteins rapidly form crosslinked hydrogels in physiological conditions. We also show that the mechanical properties of these resilin-like hydrogels can be modulated simply by varying the protein concentration or the stoichiometric ratio of crosslinker to crosslinking sites. Crosslinked resilin-like hydrogels were hydrophilic and had a high water content when swollen. In addition, these hydrogels exhibited moderate resilience values, which were comparable to those of common synthetic rubbers. Cryo-scanning electron microscopy showed that the crosslinked resilin-like hydrogels at 16â¯wt% featured a honeycomb-like structure. These studies thus demonstrate the potential to use recombinant resilin-like proteins in a wide variety of applications such as tissue engineering and drug delivery due to their tunable physical properties.
Asunto(s)
Materiales Biocompatibles , Proteínas de Insectos , Fenómenos Mecánicos , Materiales Biocompatibles/química , Fuerza Compresiva , Hidrogeles/química , Proteínas de Insectos/química , Reología , Ingeniería de TejidosRESUMEN
Resilin is an elastomeric protein found in insect cuticles and is remarkable for its high strain, low stiffness, and high resilience. Since the first resilin sequence was identified in Drosophilia melanogaster (fruit fly), researchers have utilized molecular cloning techniques to construct resilin-based proteins for a number of different applications. In addition to exhibiting the superior mechanical properties of resilin, resilin-based proteins are autofluorescent, display self-assembly properties, and undergo phase transitions in response to temperature. These properties have potential application in designing biosensors or environmentally responsive materials for use in tissue engineering or drug delivery. Furthermore, the capability of resilin-based biomaterials has been expanded by designing proteins that include both resilin-based sequences and bioactive domains such as cell-adhesion or matrix metalloproteinase sequences. These new materials maintain the superior mechanical and physical properties of resilin and also have the added benefit of controlling cell response. Because the mechanical and biological properties can be tuned through protein engineering, a wide range of properties can be achieved for tissue engineering applications including muscles, vocal folds, cardiovascular tissues, and cartilage.
Asunto(s)
Materiales Biocompatibles/farmacología , Elasticidad , Proteínas de Insectos/química , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Ingeniería de Tejidos , Temperatura de TransiciónRESUMEN
Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II ß-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.
Asunto(s)
Fragmentos de Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Animales , Adhesión Celular , Movimiento Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Escherichia coli , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Ensayo de Materiales , Pectinidae , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/toxicidad , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/toxicidad , Propiedades de SuperficieRESUMEN
Modular proteins have emerged as powerful tools in tissue engineering because both the mechanical and biochemical properties can be precisely controlled through amino acid sequence. Resilin is an attractive candidate for use in modular proteins because it is well-known for having low stiffness, high fatigue lifetime, and high resilience. However, no studies have been conducted to assess resilin's compressive properties, cytocompatibility with clinically relevant cells, or effect on cell spreading. We designed a modular protein containing repeating sequences of a motif derived from Anopheles gambiae and cell-binding domains derived from fibronectin. Rapid cross-linking with tris(hydroxymethyl)phosphine was observed. The hydrogels had a complex modulus of 22 ± 1 kPa and yield strain of 63%. The elastic modulus in compression, or unconfined compressive modulus, was 2.4 ± 0.2 MPa, which is on the same order as human cartilage. A LIVE/DEAD assay demonstrated that human mesenchymal stem cells cultured on the resilin-based protein had a viability of 95% after three days. A cell-spreading assay revealed that the cells interacted with the fibronectin-derived domain in a sequence-specific manner and resulted in a mean cell area ~1.4-fold larger than when cells were seeded on a sequence-scrambled negative control protein. These results demonstrate that our resilin-based biomaterial is a promising biomaterial for cartilage tissue engineering.