Novel Nanofiber Structures and Advanced Tissue Engineering Applications

Extracellular matrix (ECM) nanofibers such as collagen and elastin make up an important component of natural tissues. These structural components serve to impart mechanical strength and provide locations for cell attachment and biomolecule storage. Cells respond to their structural environment in a wide variety of ways beyond physical support, and it has been demonstrated that this environment directly modulates cell behaviors such as, morphology, differentiation, ECM production, attachment, and migration. ECM nanofibers also play an important role as a template for tissue formation during development, remodeling, and regeneration. Nanofiber based tissue engineering strategies aim to mimic the geometry of the natural fibrous component of the ECM to promote tissue regeneration. Nanofiber based approaches are of special interest in regeneration of aligned tissues such as, nerve, blood vessel, muscle, and connective tissue because they are able to promote aligned morphologies in resident cells. While there are many different nanofiber fabrication methods available, the electrospinning method may be the most promising due its simplicity, versatility and scalability. Many different types of materials can be easily electrospun into nanofibers with a wide variety of morphologies, sizes, and structural arrangements. However, the potential of the electrospinning method in tissue engineering applications is limited by the available assembly techniques. It was our goal to investigate new technologies that allow more precise assembly of electrospun nanofibers into useful complex structures. First, the parallel plate technique for collecting aligned nanofiber arrays was investigated systematically. Results of this study provided valuable insights into the relationships of fiber length to collection rate and collecting plate size, which were used in designing novel loose fiber collection technologies. One of our technologies utilizes parallel mobile tracks to collect and distribute aligned electrospun nanofibers into loose 3D arrays. Advantages of this technology include indefinitely continuous steady state nanofiber collection, and the capability to simultaneously collect nanofibers from an electrospinning jet in one location and assemble them into complex structures at another. In addition, nanofibers are allowed an indefinite amount of time to dry between collection and assembly, thus eliminating complications related to fiber-to-fiber adhesions. This technology demonstrates potential in complex nanofiber structure assembly, and in industrial scale up. Precision assembly, facilitated by the mobile track technology, led to the development of technologies to fabricate composite nanofiber/protein matrix thin films. These composites combined the strengths of each component as a scaffold for regenerating different types of tissues. Precision assembly technologies also facilitated the development of hybrid two components fibrous structures with finely tuned biomimetic microstructures. The mechanical properties of these structures were similar to those of natural tissues. It was demonstrated that the biomimetic mechanical properties of the hybrid materials were derived from precise nanofiber arrangement at the mechanical properties were highly responsive to subtle changes nanofiber arrangement. Nanofibrous structures were evaluated as tissue engineering scaffolds in vitro and in vitro. C2C12 myoblasts seeded on aligned nanofibers scaffolds attached, aligned, and grew to confluence to form thin cell/nanofiber sheets and cell/nanofiber/protein matrix films. Three dimensional skeletal muscle scaffolds were further assembled by stacking these constructs layer-by-layer or by assembling them into 3D bundled structures. Integration of multilayered grafts with natural muscle was evaluated in vivo. Tubular vascular grafts were also fabricated with biomimetic wavy stiff nanofibers and straight elastic fibers. These grafts demonstrated a remarkably similar mechanical profile to natural blood vessels when the microstructure was optimized. In vivo evaluation of vascular grafts was conducted in a rabbit carotid artery replacement model. Our studies indicate that advances in nanofiber assembly allow for the design of tissue engineering scaffolds with improved control over fiber density, placement, and microstructure. These advances offer the potential for the design of better tissue engineering scaffolds for regeneration of many tissues such as skeletal muscle, blood vessels, nerve, tendon, skin, and so on