Matthew R. MacEwan

THE DIFFERENTIATION OF EMBRYONIC STEM CELLS SEEDED ON ELECTROSPUN NANOFIBERS INTO NEURAL LINEAGES

Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.

Neurite outgrowth on nanofiber scaffolds with different orders, structures, and surface properties.

Electrospun nanofibers can be readily assembled into various types of scaffolds for applications in neural tissue engineering. The objective of this study is to examine and understand the unique patterns of neurite outgrowth from primary dorsal root ganglia (DRG) cultured on scaffolds of electrospun nanofibers having different orders, structures, and surface properties. We found that the neurites extended radially outward from the DRG main body without specific directionality when cultured on a nonwoven mat of randomly oriented nanofibers. In contrast, the neurites preferentially extended along the long axis of fiber when cultured on a parallel array of aligned nanofibers. When seeded at the border between regions of aligned and random nanofibers, the same DRG simultaneously expressed aligned and random neurite fields in response to the underlying nanofibers. When cultured on a double-layered scaffold where the nanofibers in each layer were aligned along a different direction, the neurites were found to be dependent on the fiber density in both layers. This biaxial pattern clearly demonstrates that neurite outgrowth can be influenced by nanofibers in different layers of a scaffold, rather than the topmost layer only. Taken together, these results will provide valuable information pertaining to the design of nanofiber scaffolds for neuroregenerative applications, as well as the effects of topology on neurite outgrowth, growth cone guidance, and axonal regeneration.

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.

Acellular nerve allografts in peripheral nerve regeneration: A comparative study

Introduction: Processed nerve allografts offer a promising alternative to nerve autografts in the surgical management of peripheral nerve injuries where short deficits exist. Methods: Three established models of acellular nerve allograft (cold‐preserved, detergent‐processed, and AxoGen‐processed nerve allografts) were compared with nerve isografts and silicone nerve guidance conduits in a 14‐mm rat sciatic nerve defect. Results: All acellular nerve grafts were superior to silicone nerve conduits in support of nerve regeneration. Detergent‐processed allografts were similar to isografts at 6 weeks postoperatively, whereas AxoGen‐processed and cold‐preserved allografts supported significantly fewer regenerating nerve fibers. Measurement of muscle force confirmed that detergent‐processed allografts promoted isograft‐equivalent levels of motor recovery 16 weeks postoperatively. All acellular allografts promoted greater amounts of motor recovery compared with silicone conduits. Conclusion: These findings provide evidence that differential processing for removal of cellular constituents in preparing acellular nerve allografts affects recovery in vivo. Muscle Nerve, 2011

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).

Fibrin Matrices With Affinity-Based Delivery Systems and Neurotrophic Factors Promote Functional Nerve Regeneration

Glial‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity‐based delivery of GDNF or NGF from fibrin‐filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity‐based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury. Biotechnol. Bioeng. 2010;106: 970–979.

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.

Neovascularization in Biodegradable Inverse Opal Scaffolds with Uniform and Precisely Controlled Pore Sizes

The formation of a stable vascular network in a scaffold is one of the most challenging tasks in tissue engineering and regenerative medicine. Despite the common use of porous scaffolds in these applications, little is known about the effect of pore size on the neovascularization in these scaffolds. Herein is fabricated poly(D, L-lactide-co-glycolide) inverse opal scaffolds with uniform pore sizes of 79, 147, 224, and 312 μm in diameter and which are then used to systematically study neovascularization in vivo. Histology analyses reveal that scaffolds with small pores (<200 μm) favor the formation of vascular networks with small vessels at high densities and poor penetration depth. By contrast, scaffolds with large pores (>200 μm) favor the formation of vascular networks with large blood vessels at low densities and deep penetration depth. Based on the different patterns of vessel ingrowth as regulated by the pore size, a model is proposed to describe vascularization in a 3D porous scaffold, which can potentially serve as a guideline for future design of porous scaffolds.

Mussel inspired protein-mediated surface modification to electrospun fibers and their potential biomedical applications.

Mussel inspired proteins have been demonstrated to serve as a versatile biologic adhesive with numerous applications. The present study illustrates the use of such Mussel inspired proteins (polydopamine) in the fabrication of functionalized bio-inspired nanomaterials capable of both improving cell response and sustained delivery of model probes. X-ray photoelectron spectroscopy analysis confirmed the ability of dopamine to polymerize on the surface of plasma-treated, electrospun poly(ε-caprolactone) (PCL) fiber mats to form polydopamine coating. Transmission electron microscopy images demonstrated that self-polymerization of dopamine was induced by pH shift and that the thickness of polydopamine coating was readily modulated by adjusting the concentration of dopamine and reaction time. Polydopamine coatings were noted to affect the mechanical properties of underlying fiber mats, as mechanical testing demonstrated a decrease in elasticity and increase in stiffness of polydopamine-coated fiber mats. Polydopamine coatings were also utilized to effectively immobilize extracellular matrix proteins (i.e., fibronectin) on the surface of polydopamine-coated, electrospun fibers, resulting in enhancement of NIH3T3 cell attachment, spreading, and cytoskeletal development. Comparison of release rates of rhodamine 6G encapsulated in coated and uncoated PCL fibers also confirmed that polydopamine coatings modulate the release rate of loaded payloads. The authors further demonstrate the significant difference of rhodamine 6G adsorption kinetics in water between PCL fibers and polydopamine-coated PCL fibers. Taken together, polydopamine-mediated surface modification to electrospun fibers may be an effective means of fabricating a wide range of bio-inspired nanomaterials with unique properties for use in tissue engineering, drug delivery, and advanced biomedical applications.

NERVE ALLOGRAFTS SUPPLEMENTED WITH SCHWANN CELLS OVEREXPRESSING GLIAL-CELL-LINE-DERIVED NEUROTROPHIC FACTOR

We sought to determine whether supplementation of acellular nerve allografts (ANAs) with Schwann cells overexpressing GDNF (G‐SCs) would enhance functional recovery after peripheral nerve injury.