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

Fabrication of three dimensional aligned nanofiber array

Disclosed are methods of forming three dimensional arrays of aligned nanofibers in an open, loose structure of any desired depth. The arrays are formed according to an electrospinning process utilizing two parallel conducting plates to align the fibers and rotating tracks to distribute the fibers throughout the array. Arrays can be used as formed, for instance in tissue engineering applications as three dimensional scaffolding constructs. As-formed arrays can be combined with other materials to form a composite 3-D structure. For instance, composite polymeric materials can be electrospun to form composite nanofibers within the array. Multiple polymeric materials can be electrospun at different areas of the array to form a composite array including materially different nanofibers throughout the array. The arrays can be loaded with other fibrous or non-fibrous materials to form a composite array. Arrays can also be rolled to form a uniaxial fiber bundle

Fabrication of Three Dimensional Aligned Nanofiber Array

Disclosed are methods of forming three dimensional arrays of aligned nanofibers in an open, loose structure of any desired depth. The arrays are formed according to an electrospinning process utilizing two parallel conducting plates to align the fibers and rotating tracks to distribute the fibers throughout the array. Arrays can be used as formed, for instance in tissue engineering applications as three dimensional scaffolding constructs. As-formed arrays can be combined with other materials to form a composite 3-D structure. For instance, composite polymeric materials can be electrospun to form composite nanofibers within the array. Multiple polymeric materials can be electrospun at different areas of the array to form a composite array including materially different nanofibers throughout the array. The arrays can be loaded with other fibrous or non-fibrous materials to form a composite array. Arrays can also be rolled to form a uniaxial fiber bundle

Precisely Assembled Nanofiber Arrays as a Platform to Engineer Aligned Cell Sheets for Biofabrication

A hybrid cell sheet engineering approach was developed using ultra-thin nanofiber arrays to host the formation of composite nanofiber/cell sheets. It was found that confluent aligned cell sheets could grow on uniaxially-aligned and crisscrossed nanofiber arrays with extremely low fiber densities. The porosity of the nanofiber sheets was sufficient to allow aligned linear myotube formation from differentiated myoblasts on both sides of the nanofiber sheets, in spite of single-side cell seeding. The nanofiber content of the composite cell sheets is minimized to reduce the hindrance to cell migration, cell-cell contacts, mass transport, as well as the foreign body response or inflammatory response associated with the biomaterial. Even at extremely low densities, the nanofiber component significantly enhanced the stability and mechanical properties of the composite cell sheets. In addition, the aligned nanofiber arrays imparted excellent handling properties to the composite cell sheets, which allowed easy processing into more complex, thick 3D structures of higher hierarchy. Aligned nanofiber array-based composite cell sheet engineering combines several advantages of material-free cell sheet engineering and polymer scaffold-based cell sheet engineering; and it represents a new direction in aligned cell sheet engineering for a multitude of tissue engineering applications

Synthetic vascular tissue and method of forming same

Disclosed are composite materials that can more closely mimic the mechanical characteristics of natural elastic tissue, such as vascular tissue. Disclosed materials include a combination of elastic nanofibers and non-elastic nanofibers. Also disclosed are a variety of methods for forming the composite materials. Formation methods generally include the utilization of electrospinning methods to form a fibrous composite construct including fibers of different mechanical characteristics

Biocompatible Silk/Polymer Energy Harvesters Using Stretched Poly (vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) Nanofibers

Energy harvested from human body movement can produce continuous, stable energy to portable electronics and implanted medical devices. The energy harvesters need to be light, small, inexpensive, and highly portable. Here we report a novel biocompatible device made of poly (vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) nanofibers on flexible substrates. The nanofibers are prepared with electrospinning followed by a stretching process. This results in aligned nanofibers with diameter control. The assembled device demonstrates high mechanical-to-electrical conversion performance, with stretched PVDF-HFP nanofibers outperforming regular electrospun samples by more than 10 times. Fourier transform infrared spectroscopy (FTIR) reveals that the stretched nanofibers have a higher β phase content, which is the critical polymorph that enables piezoelectricity in polyvinylidene fluoride (PVDF). Polydimethylsiloxane (PDMS) is initially selected as the substrate material for its low cost, high flexibility, and rapid prototyping capability. Bombyx Mori silkworm silk fibroin (SF) and its composites are investigated as promising alternatives due to their high strength, toughness, and biocompatibility. A composite of silk with 20% glycerol demonstrates higher strength and larger ultimate strain than PDMS. With the integration of stretched electrospun PVDF-HFP nanofibers and flexible substrates, this pilot study shows a new pathway for the fabrication of biocompatible, skin-mountable energy devices

The fusion of tissue spheroids attached to pre-stretched electrospun polyurethane scaffolds

Publisher Copyright: © 2014, © The Author(s) 2014. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.Effective cell invasion into thick electrospun biomimetic scaffolds is an unsolved problem. One possible strategy to biofabricate tissue constructs of desirable thickness and material properties without the need for cell invasion is to use thin (<2 µm) porous electrospun meshes and self-assembling (capable of tissue fusion) tissue spheroids as building blocks. Pre-stretched electrospun meshes remained taut in cell culture and were able to support tissue spheroids with minimal deformation. We hypothesize that elastic electrospun scaffolds could be used as temporal support templates for rapid self-assembly of cell spheroids into higher order tissue structures, such as engineered vascular tissue. The aim of this study was to investigate how the attachment of tissue spheroids to pre-stretched polyurethane scaffolds may interfere with the tissue fusion process. Tissue spheroids attached, spread, and fused after being placed on pre-stretched polyurethane electrospun matrices and formed tissue constructs. Efforts to eliminate hole defects with fibrogenic tissue growth factor-β resulted in the increased synthesis of collagen and periostin and a dramatic reduction in hole size and number. In control experiments, tissue spheroids fuse on a non-adhesive hydrogel and form continuous tissue constructs without holes. Our data demonstrate that tissue spheroids attached to thin stretched elastic electrospun scaffolds have an interrupted tissue fusion process. The resulting tissue-engineered construct phenotype is a direct outcome of the delicate balance of the competing physical forces operating during the tissue fusion process at the interface of the pre-stretched elastic scaffold and the attached tissue spheroids. We have shown that with appropriate treatments, this process can be modulated, and thus, a thin pre-stretched elastic polyurethane electrospun scaffold could serve as a supporting template for rapid biofabrication of thick tissue-engineered constructs without the need for cell invasion.publishersversionPeer reviewe

An In Vitro Model of the Glomerular Capillary Wall Using Electrospun Collagen Nanofibres in a Bioartificial Composite Basement Membrane

The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease

A vertically translating collection system to facilitate roll-to-roll centrifugal spinning of highly aligned polyacrylonitrile nanofibers

Abstract Centrifugal spinning is a fiber spinning method capable of producing fibers in the nanoscale diameter range from a multitude of polymers, including polyacrylonitrile (PAN). With a traditional centrifugal spinner, fiber can be rapidly spun and collected on static collection posts. However, the use of posts inevitably forms a dense fiber “ring” that is incompatible with roll-to-roll manufacturing processes. In this work, factors that influence throughput and scalability of highly aligned centrifugally spun PAN fibers are explored. A custom centrifugal setup is used to vertically translate collected fibers during the spinning process to distribute them over a large surface area. In addition, factors that affect PAN fiber diameter during the spinning process are investigated, including spinneret to collector distance, rotational speed, and humidity. Resulting data demonstrates that these factors can be independently optimized to reliably produce quality PAN fiber in the nanoscale diameter range. Furthermore, the fiber mass collection rate can be increased without affecting sample quality when the vertical translation speed is increased. This work demonstrates the potential scalability of centrifugal spinning to quickly produce large amounts of highly aligned nanofiber in a cheap, efficient, and reliable manner, and also lends the ability to be collected in a roll-to-roll fashion