Layer-by-layer assembly of 3D tissue constructs with functionalized graphene.

Carbon-based nanomaterials have been considered as promising candidates to mimic certain structure and function of native extracellular matrix materials for tissue engineering. Significant progress has been made in fabricating carbon nanoparticle-incorporated cell culture substrates, but limited studies have been reported on the development of three-dimensional (3D) tissue constructs using these nanomaterials. Here, we present a novel approach to engineer 3D multi-layered constructs using layer-by-layer (LbL) assembly of cells separated with self-assembled graphene oxide (GO)-based thin films. The GO-based structures are shown to serve as cell adhesive sheets that effectively facilitate the formation of multi-layer cell constructs with interlayer connectivity. By controlling the amount of GO deposited in forming the thin films, the thickness of the multi-layer tissue constructs could be tuned with high cell viability. Specifically, this approach could be useful for creating dense and tightly connected cardiac tissues through the co-culture of cardiomyocytes and other cell types. In this work, we demonstrated the fabrication of stand-alone multi-layer cardiac tissues with strong spontaneous beating behavior and programmable pumping properties. Therefore, this LbL-based cell construct fabrication approach, utilizing GO thin films formed directly on cell surfaces, has great potential in engineering 3D tissue structures with improved organization, electrophysiological function, and mechanical integrity.

Fabricated tropoelastin-silk yarns and woven textiles for diverse tissue engineering applications.

Electrospun yarns offer substantial opportunities for the fabrication of elastic scaffolds for flexible tissue engineering applications. Currently available yarns are predominantly made of synthetic elastic materials. Thus scaffolds made from these yarns typically lack cell signaling cues. This can result in poor integration or even rejection on implantation, which drive demands for a new generation of yarns made from natural biologically compatible materials. Here, we present a new type of cell-attractive, highly twisted protein-based yarns made from blended tropoelastin and silk fibroin. These yarns combine physical and biological benefits by being rendered elastic and bioactive through the incorporation of tropoelastin and strengthened through the presence of silk fibroin. Remarkably, the process delivered multi-meter long yarns of tropoelastin-silk mixture that were conducive to fabrication of meshes on hand-made frames. The resulting hydrated meshes are elastic and cell interactive. Furthermore, subcutaneous implantation of the meshes in mice demonstrates their tolerance and persistence over 8 weeks. This combination of mechanical properties, biocompatibility and processability into diverse shapes and patterns underscores the value of these materials and platform technology for tissue engineering applications. STATEMENT OF SIGNIFICANCE: Synthetic yarns are used to fabricate textile materials for various applications such as surgical meshes for hernia repair and pelvic organ prolapse. However, synthetic materials lack the attractive biological and physical cues characteristic of extracellular matrix and there is a demand for materials that can minimize postoperative complications. To address this need, we made yarns from a combination of recombinant human tropoelastin and silk fibroin using a modified electrospinning approach that blended these proteins into functional yarns. Prior to this study, no protein-based yarns using tropoelastin were available for the fabrication of functional textile materials. Multimeter-long, uniform and highly twisted yarns based on these proteins were elastic and cell interactive and demonstrated processing to yield textile fabrics. By using these yarns to weave fabrics, we demonstrate that an elastic human matrix protein blend can deliver a versatile platform technology to make textiles that can be explored for efficacy in tissue repair.

Freestanding hierarchical vascular structures engineered from ice.

The ability to engineer a synthetic hierarchical vascular network is one of the most demanding and unaddressed challenges in tissue engineering and regenerative medicine. A material that is both structurally rigid and biocompatible is needed to fabricate freestanding hierarchical vascular structures with defined dimensions and geometry. This is particularly important for creating commercially viable and easily suturable synthetic vasculature. Here, we present the surprising discovery that ice is a versatile material which satisfies these requirements. We demonstrate utilizing ice as a sacrificial scaffold, onto which a diverse range of materials were coated, including tropoelastin, polycaprolactone (PCL), silk, and polydimethylsiloxane (PDMS). We present ice facilitating the fabrication of freestanding hierarchical vascular structures with variable lumen dimensions, and validate the vascular application of these vessels by demonstrating their mechanical tunability, biocompatibility, and permeability to nutrient diffusion. This adaptable technology delivers engineered synthetic vasculature and has potential wider applications encompassing tissue engineering bespoke structures.

Optically robust, highly permeable and elastic protein films that support dual cornea cell types.

Damaged corneas can lead to blindness. Due to the worldwide shortage of donor corneas there is a tremendous unmet demand for a robust corneal replacement that supports growth of the major corneal cell types. Commercial artificial corneas comprise plastic polymers that do not adequately support diverse cell growth. We present a new class of protein elastomer-dominated synthetic corneas with attractive performance that intimately couple biologically active tropoelastin to mechanically robust and durable protein silk. Fabricated films substantially replicate the natural cornea physically and by interacting with both key cells types used in cornea repair. Performance encompasses optical clarity at high transmittance, compatible refractive index, substantial glucose permeability, compliant mechanical properties, and support of both growth and function of corneal epithelial and endothelial cells.

Tropoelastin Implants That Accelerate Wound Repair.

A novel, pure, synthetic material is presented that promotes the repair of full-thickness skin wounds. The active component is tropoelastin and leverages its ability to promote new blood vessel formation and its cell recruiting properties to accelerate wound repair. Key to the technology is the use of a novel heat-based, stabilized form of human tropoelastin which allows for tunable resorption. This implantable material contributes a tailored insert that can be shaped to the wound bed, where it hydrates to form a conformable protein hydrogel. Significant benefits in the extent of wound healing, dermal repair, and regeneration of mature epithelium in healthy pigs are demonstrated. The implant is compatible with initial co-treatment with full- and split-thickness skin grafts. The implant's superiority to sterile bandaging, commercial hydrogel and dermal regeneration template products is shown. On this basis, a new concept for a prefabricated tissue repair material for point-of-care treatment of open wounds is provided.

Elastic proteins and elastomeric protein alloys.

The elastomeric proteins elastin and resilin have been used extensively in the fabrication of biomaterials for tissue engineering applications due to their unique mechanical and biological properties. Tropoelastin is the soluble monomer component of elastin. Tropoelastin and resilin are both highly elastic with high resilience, substantial extensibility, high durability and low energy loss, which makes them excellent candidates for the fabrication of elastic tissues that demand regular and repetitive movement like the skin, lung, blood vessels, muscles and vocal folds. Combinations of these proteins with silk fibroin further enhance their biomechanical and biological properties leading to a new class of protein alloy materials with versatile properties. In this review, the properties of tropoelastin-based and resilin-based biomaterials with and without silk are described in concert with examples of their applications in tissue engineering.

Fabricated Elastin.

The mechanical stability, elasticity, inherent bioactivity, and self-assembly properties of elastin make it a highly attractive candidate for the fabrication of versatile biomaterials. The ability to engineer specific peptide sequences derived from elastin allows the precise control of these physicochemical and organizational characteristics, and further broadens the diversity of elastin-based applications. Elastin and elastin-like peptides can also be modified or blended with other natural or synthetic moieties, including peptides, proteins, polysaccharides, and polymers, to augment existing capabilities or confer additional architectural and biofunctional features to compositionally pure materials. Elastin and elastin-based composites have been subjected to diverse fabrication processes, including heating, electrospinning, wet spinning, solvent casting, freeze-drying, and cross-linking, for the manufacture of particles, fibers, gels, tubes, sheets and films. The resulting materials can be tailored to possess specific strength, elasticity, morphology, topography, porosity, wettability, surface charge, and bioactivity. This extraordinary tunability of elastin-based constructs enables their use in a range of biomedical and tissue engineering applications such as targeted drug delivery, cell encapsulation, vascular repair, nerve regeneration, wound healing, and dermal, cartilage, bone, and dental replacement.

Cell-laden microengineered and mechanically tunable hybrid hydrogels of gelatin and graphene oxide.

Incorporating graphene oxide inside GelMA hydrogels enhances their mechanical properties and reduces UV-induced cell damage while preserving their favorable characteristics for 3D cell encapsulation. NIH-3T3 fibroblasts encapsulated in GO-GelMA microgels demonstrate excellent cellular viability, proliferation, spreading, and alignment. GO reinforcement combined with a multi-stacking approach offers a facile engineering strategy for the construction of complex artificial tissues.