Wenying Liu

Electrospun Nanofibers for Regenerative Medicine

This Progress Report reviews recent progress in applying electrospun nanofibers to the emerging field of regenerative medicine. It begins with a brief introduction to electrospinning and nanofibers, with a focus on issues related to the selection of materials, incorporation of bioactive molecules, degradation characteristics, control of mechanical properties, and facilitation of cell infiltration. Next, a number of approaches to fabricate scaffolds from electrospun nanofibers are discussed, including techniques for controlling the alignment of nanofibers and for producing scaffolds with complex architectures. The article also highlights applications of the nanofiber-based scaffolds in four areas of regenerative medicine that involve nerves, dural tissues, tendons, and the tendon-to-bone insertion site. The Progress Report concludes with perspectives on challenges and future directions for design, fabrication, and utilization of scaffolds based on electrospun nanofibers.

Radially Aligned, Electrospun Nanofibers as Dural Substitutes for Wound Closure and Tissue Regeneration Applications

This paper reports the fabrication of scaffolds consisting of radially aligned poly(ε-caprolactone) nanofibers by utilizing a collector composed of a central point electrode and a peripheral ring electrode. This novel class of scaffolds was able to present nanoscale topographic cues to cultured cells, directing and enhancing their migration from the periphery to the center. We also established that such scaffolds could induce faster cellular migration and population than nonwoven mats consisting of random nanofibers. Dural fibroblast cells cultured on these two types of scaffolds were found to express type I collagen, the main extracellular matrix component in dural mater. The type I collagen exhibited a high degree of organization on the scaffolds of radially aligned fibers and a haphazard distribution on the scaffolds of random fibers. Taken together, the scaffolds based on radially aligned, electrospun nanofibers show great potential as artificial dural substitutes and may be particularly useful as biomedical patches or grafts to induce wound closure and/or tissue regeneration.

Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair.

Outcomes after tendon repair are often unsatisfactory, despite improvements in surgical techniques and rehabilitation methods. Recent studies aimed at enhancing repair have targeted the paucicellular nature of tendon for enhancing repair; however, most approaches for delivering growth factors and cells have not been designed for dense connective tissues such as tendon. Therefore, we developed a scaffold capable of delivering growth factors and cells in a surgically manageable form for tendon repair. Platelet-derived growth factor BB (PDGF-BB), along with adipose-derived mesenchymal stem cells (ASCs), were incorporated into a heparin/fibrin-based delivery system (HBDS). This hydrogel was then layered with an electrospun nanofiber poly(lactic-co-glycolic acid) (PLGA) backbone. The HBDS allowed for the concurrent delivery of PDGF-BB and ASCs in a controlled manner, while the PLGA backbone provided structural integrity for surgical handling and tendon implantation. In vitro studies verified that the cells remained viable, and that sustained growth factor release was achieved. In vivo studies in a large animal tendon model verified that the approach was clinically relevant, and that the cells remained viable in the tendon repair environment. Only a mild immunoresponse was seen at dissection, histologically, and at the mRNA level; fluorescently labeled ASCs and the scaffold were found at the repair site 9days post-operatively; and increased total DNA was observed in ASC-treated tendons. The novel layered scaffold has the potential for improving tendon healing due to its ability to deliver both cells and growth factors simultaneously in a surgically convenient manner.

Neurite outgrowth on electrospun nanofibers with uniaxial alignment: The effects of fiber density, surface coating, and supporting substrate

Electrospun nanofibers with uniaxial alignment have recently gained its popularity as scaffolds for neural tissue engineering. Many studies have demonstrated that the nanofibers could guide the neurites to extend along the direction of alignment, resembling the native hierarchy of the nerve tissue. However, the contact cues provided by the nanofibers can be far more complicated than just guiding the neurites to extend along them. In the current study, we used dorsal root ganglia as a model system to systematically investigate the interactions between neurites and uniaxially aligned nanofibers. We demonstrated, for the first time, that the neurites could not only project along the nanofibers, but also be directed to grow along a direction perpendicular to the aligned nanofibers, depending on the following parameters: (i) the density of nanofibers, (ii) the protein deposited on the surfaces of the nanofibers, and (iii) surface properties of the substrate on which the nanofibers were supported. We also investigated the pharmacological effect of myosin II inhibition on the nanofiber-guided growth of neurites by adding blebbistatin to the culture medium. Our findings offer new insights into the design of nanofiber-based scaffolds for nerve injury repair and will provide new guidelines for the construction of well-defined neuronal network architecture (the so-called neural circuits).

Enhancing the stiffness of electrospun nanofiber scaffolds with a controlled surface coating and mineralization.

A new method was developed to coat hydroxyapatite (HAp) onto electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers for tendon-to-bone insertion site repair applications. Prior to mineralization, chitosan and heparin were covalently immobilized onto the surface of the fibers to accelerate the nucleation of bone-like HAp crystals. Uniform coatings of HAp were obtained by immersing the nanofiber scaffolds into a modified, 10-fold-concentrated simulated body fluid (m10SBF) for different periods of time. The new method resulted in thicker and denser coatings of mineral on the fibers compared to those produced by previously reported methods. Scanning electron microscopy measurements confirmed the formation of nanoscale HAp particles on the fibers. A mechanical property assessment demonstrated a higher stiffness with respect to previous coating methods. A combination of the nanoscale fibrous structure and bonelike mineral coating could mimic the structure, composition, and function of mineralized tissues.

Nanofiber Scaffolds with Gradients in Mineral Content for Spatial Control of Osteogenesis

Reattachment of tendon to bone has been a challenge in orthopedic surgery. The disparate mechanical properties of the two tissues make it difficult to achieve direct surgical repair of the tendon-to-bone insertion. Healing after surgical repair typically does not regenerate the natural attachment, a complex tissue that connects tendon and bone across a gradient in both mineral content and cell phenotypes. To facilitate the regeneration of the attachment, our groups have developed a nanofiber-based scaffold with a graded mineral coating to mimic the mineral composition of the native tendon-to-bone insertion. In the present work, we evaluated the ability of this scaffold to induce graded osteogenesis of adipose-derived mesenchymal stem cells (ASCs). Results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferating cell nuclear antigen staining indicated that cell proliferation was negatively correlated with the mineral content. In contrast, alkaline phosphatase staining, an indicator of osteogenesis, was positively correlated with the mineral content. Likewise, runt-related transcription factor 2 (an early marker of osteoblast differentiation) and osteocalcin (a late marker of osteoblast differentiation) immunostaining were both positively correlated with the mineral content. These results indicate that a gradient in mineral content on the surface of a nanofiber scaffold is capable of inducing graded differentiation of ASCs into osteoblasts for enthesis repair.

Nerve Guidance Conduits Based on Double-Layered Scaffolds of Electrospun Nanofibers for Repairing the Peripheral Nervous System

Compared to the nerve guidance conduits (NGCs) constructed from a single layer of aligned nanofibers, bilayer NGCs with random and aligned nanofibers in the outer and inner layers are more robust and tear-resistant during surgical procedures thanks to an isotropic mechanical property provided by the random nanofibers. However, it remains unclear whether the random nanofibers will interfere with the aligned nanofibers to alter the extension pattern of the neurites and impede regeneration. To answer this question, we seeded dorsal root ganglia (DRG) on a double-layered scaffold, with aligned and random nanofibers on the top and bottom layers, respectively, and evaluated the outgrowth of neurites. The random nanofibers in the bottom layer exerted a negative impact on the extension of neurites projecting from the DRG, giving neurites a less ordered structure compared to those cultured on a single layer of aligned nanofibers. The negative impact of the random nanofibers could be effectively mitigated by preseeding the double-layered scaffold with Schwann cells. DRG cultured on top of such a scaffold exhibited a neurite outgrowth pattern similar to that for DRG cultured on a single layer of aligned nanofibers. We further fabricated bilayer NGCs from the double-layered scaffolds and tested their ability to facilitate nerve regeneration in a rat sciatic nerve injury model. Both histomorphometric analysis and functional characterization demonstrated that bilayer NGCs with an inner surface that was preseeded with Schwann cells could reach 54%, 64.2%, and 74.9% of the performance of isografts in terms of nerve fiber number, maximum isometric tetanic force, and mass of the extensor digitorum longus muscle, respectively. It can be concluded that the bilayer NGCs hold great potential in facilitating motor axon regeneration and functional motor recovery.

Electrospun Nanofibers: New Concepts, Materials, and Applications

Electrospinning is a simple and versatile technique that relies on the electrostatic repulsion between surface charges to continuously draw nanofibers from a viscoelastic fluid. It has been applied to successfully produce nanofibers, with diameters down to tens of nanometers, from a rich variety of materials, including polymers, ceramics, small molecules, and their combinations. In addition to solid nanofibers with a smooth surface, electrospinning has also been adapted to generate nanofibers with a number of secondary structures, including those characterized by a porous, hollow, or core-sheath structure. The surface and/or interior of such nanofibers can be further functionalized with molecular species or nanoparticles during or after an electrospinning process. In addition, electrospun nanofibers can be assembled into ordered arrays or hierarchical structures by manipulation of their alignment, stacking, and/or folding. All of these attributes make electrospun nanofibers well-suited for a broad spectrum of applications, including those related to air filtration, water purification, heterogeneous catalysis, environmental protection, smart textiles, surface coating, energy harvesting/conversion/storage, encapsulation of bioactive species, drug delivery, tissue engineering, and regenerative medicine. Over the past 15 years, our group has extensively explored the use of electrospun nanofibers for a range of applications. Here we mainly focus on two examples: (i) use of ceramic nanofibers as catalytic supports for noble-metal nanoparticles and (ii) exploration of polymeric nanofibers as scaffolding materials for tissue regeneration. Because of their high porosity, high surface area to volume ratio, well-controlled composition, and good thermal stability, nonwoven membranes made of ceramic nanofibers are terrific supports for catalysts based on noble-metal nanoparticles. We have investigated the use of ceramic nanofibers made of various oxides, including SiO2, TiO2, SnO2, CeO2, and ZrO2, as supports for heterogeneous catalysts based on noble metals such as Au, Pt, Pd, and Rh. On the other hand, the diameter, composition, alignment, porosity, and surface properties of polymeric nanofibers can be engineered in a controllable fashion to mimic the hierarchical architecture of an extracellular matrix and help manipulate cell behaviors for tissue engineering and regenerative medicine. To this end, we can mimic the native structure and morphology of the extracellular matrix in tendon using uniaxially aligned nanofibers; we can use radially aligned nanofibers to direct the migration of cells from the periphery to the center in an effort to speed up wound healing; and we can also use uniaxially aligned nanofibers to guide and expedite the extension of neurites for peripheral nerve repair. Furthermore, we can replicate the anatomic structures at the tendon-to-bone insertion using nanofiber scaffolds with graded mineral coatings. In this Account, we aim to demonstrate the unique capabilities of electrospun nanofibers as porous supports for heterogeneous catalysis and as functional scaffolds for tissue regeneration by concentrating on some of the recent results.

Generation of Electrospun Nanofibers with Controllable Degrees of Crimping Through a Simple, Plasticizer‐Based Treatment

Electrospun nanofibers with controllable degrees of crimping are fabricated by simply exposing the samples to a plasticizer at preset shrinkage ratios. Compared with their straight counterparts, the crimped nanofibers are able to mechanically mimic native tendon tissue and better protect tendon fibroblasts under uniaxial strains.

Nanofiber membranes with controllable microwells and structural cues and their use in forming cell microarrays and neuronal networks.

There is a strong demand for cell culture substrates with arrayed microwells and controllable structural cues in many biological applications.[1,2] For example, such substrates are needed for fabricating cell microarrays to be used in high-throughput screening of a large number of test samples, and this approach is beneficial to drug screening, toxicology, stem cell research, and cell therapy.[3] Similarly, formation of neuronal networks could be studied in vitro through the use of arrayed microwells connected by structural cues, and this approach can be employed to examine synapse formation as well as the development and function of neuronal assemblies.[4,5] Most studies involving the formation of cell microarrays and neural networks have concentrated on the patterning of cell attractive or repulsive materials at pre-defined locations of a flat or contoured surface.[6] In general, the procedures are typically complex, costly, time-consuming, and require sophisticated techniques such as photolithography, e-beam writing, and focused ion beam (FIB) machining in order to generate the nanoscale structural cues.[7–10]