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Electrospinning Nanofibers for Neural Tissue Engineering Term paper for BMEN 5325 by name Department of Biomedical Engineering University of North Texas April 23rd, 2021 Introduction: 8.5/10 Research Summary: 10/10 Critical Review: 45/50 Recommendation: 9.5/10 Conclusion: 5/5 Structure: 9/10 Format: 3/5 Total: 90/100 1. Introduction Neural tissue is primarily composed of neurons and supporting glial cells. The complexity and specialization of the nervous system makes developing neural scaffolds for neural tissue trauma and diseases a challenging task for scientists and clinicians. Peripheral nerves have a limited capacity for regeneration following physical damage and current treatment options such as nerve grafting has limitations [1]. Autologous grafts involve the harvesting and implantation of a patient-derived donor nerve. This is mostly reserved for large nerve defects and lead to functional recovery [1]. However, this procedure requires multiple surgeries, leaves the donor nerve site nonfunctional, and there is a limited availability of donor nerves. Allografts are nerves harvested from other humans, or animals, but are also limited due to possible immune system rejection and disease related complications. Thus, the need for a nano engineered biomimetic neural structure is imperative to overcome the limitations of nerve grafting [1]. The application of nanotechnology in neural tissue engineering has great potential to overcoming the limitations seen with nerve grafting. By fabricating an implantable and biodegradable neural scaffold seeded with a variety of cellular or protein therapies, the possibility of complete nerve regeneration may be feasible in the future. A promising avenue of research in neural tissue engineering is the process of electrospinning polymer nanofibers. Electrospinning nanofibers is a relatively simple and versatile procedure and has been applied successfully to a variety of tissue engineering studies. Figure 1 shows a typical apparatus for electrospinning which consist of three main components: a high voltage power supply, a syringe pump and an electroconductive collecting surface. A wide range of natural or synthetic polymers can be used to fabricate the nanofibers. The electrospinning of nanofibers has tunable properties, and the morphology of an individual nanofiber is determined by the setting of certain variables. The syringe pump is the main control unit of the electrospinning process, and sets the parameters for the flow rate of the polymer solution/melts. The high voltage power supply is then applied to syringe needle, which produces the whipping jet motion of the pumped polymer solution and accumulates on the grounded collector. The small diameter of the electrospun nanofibers is a result of the whipping motion that exerts a strong axial force [1]. The polymer solution of choice must have the optimum viscoelastic properties to maintain its morphology during this whipping process. This continuous acceleration and stretching of the polymer solution results in the electrospun nanofibers, which are generally as thin as tens of nanometers in diameter [1]. The geometry and kinetics of the grounded collector also plays a crucial role in determining the overall orientation of the produced nanofibers. A rapidly rotating collector results in more aligned nanofibers, while a stationary collector produces randomly oriented nanofibers. Figure 1. Schematic view of electrospinning technique Nanofibers aligned into uniaxial arrays provide effective cues to direct and enhance neurite outgrowth, which is more advantageous than other materials such as hydrogels or nonaligned nanofibers. By manipulating the alignment, morphology, and stacking, the polymer nanofibers can be fabricated into a nerve guidance conduit (NGC) which can be fabricated to mimic the native extracellular matrix (ECM) of neural tissues. NGCs can also be seeded with a variety of bioactive molecules or growth factors to facilitate nerve regeneration, which make electrospun nanofibers a prime target of research in neural tissue engineering. A derivative of electrospinning is known as coaxial electrospraying. Coaxial electrospraying produces multilayer nanoparticles by introducing coaxial electrified jets [2]. Polymeric nanoparticles can be used to encapsulate, deliver, and release various therapeutic agents such as proteins, drugs, and gene therapies. Advantages of this process include high encapsulation efficiency, protection from bioactivity, and uniform size distribution [2]. This paper will discuss recent research in neural tissue engineering and summarize the ideal properties of a neural scaffold. It will also elaborate different strategies researchers have taken to fabricate nanofibers for neural tissue engineering applications. In addition, recommendations for future research will be discussed for the regenerating injured neural tissue using electrospun nanofibers. 2. The Ideal Neural Scaffold The ideal neural scaffold should be biocompatible and optimally improve cell adhesion, proliferation, migration, and axonal extension [3]. The scaffold should also provide the mechanical and chemical cues to promote new neural tissue formation. It should also be biodegradable in vivo, so there is no need for an additional removal surgery. Neural scaffolds can also be seeded with bioactive proteins and growth factors, but have ongoing limitations such as short-term retention, rapid half-life in circulation and rapid loss of biological activity in vivo [3]. Figure 2 shows the important properties for an ideal neural scaffold. Figure 2. The ideal properties of a tissue engineered neural construct [3]. 3. Aligned nanocomposite scaffold for neural regeneration To overcome the limitations seen in previous reports utilizing bioactive factor seeded neural scaffolds, Zhu et al, developed a sustained biodegradable core-shell nanospheres made of poly lactic-co-glycolic acid (PLGA), encapsulated with bovine serum albumin (BSA). This was done via a coaxial electrospraying technique. Coaxial electrospraying allows the fabrication of a controllable core-shell nanosphere with bioactive factors within the core and outer shell [3]. BSA is a large globular protein and was used as a nutrient to improve neural cell behavior [3]. Following the fabrication of the core-shell nanosphere, Zhu et al, electrospun polycaprolactone (PCL) microfibers to create the nanocomposite 3D scaffold that directed neural cell growth. In vitro analysis of the cell-scaffold interaction was performed using PC-12 cells. Figure 3 shows that PC-12 grew well on all the scaffolds used in this study, but cell proliferation was significantly higher in the PCL with BSA embedded nanospheres than PCL controls, and PCL scaffolds sprayed directly with BSA after 4 and 6 days. Figure 3. PC-12 cell proliferation in nanocomposite scaffold at 2, 4, and 6 days [3]. Confocal micrographs of PC-12 cells cultured with nerve growth factor (NGF) on aligned and random scaffolds with and without nanospheres are shown below in Figure 4. Outgrowth and extension of neurites were seen on both aligned and random scaffolds. Neuronal markers TuJ1 and MAP2 were stained to indicate neural differentiation of PC-12 cells. After seven days, all the scaffolds demonstrated differentiation. The orientation of the differentiated neurites extended along the axis of the aligned fibers, parallel with neighboring cells [3]. Axons on the randomly aligned fibers extended radially with no specific direction. This study showed that aligned fibrous scaffolds with topographical cues show a superior ability to direct neurite outgrowth and extension. Aligned fibrous neural conduits influenced endogenous neural repair mechanisms and are much more conducive to neurite growth with no need for additional exogenous growth factors. In addition, the aligned fibers benefit the formation of longitudinally oriented Bunger bands. The Bunger bands include aligned strands of Schwann cells and laminin, which are a key element in nerve repair [3]. This study demonstrated the advantages of aligned nanocomposite nanofibers over randomly aligned nanofibers. Figure 4. Confocal microscopy images of PC-12 cell line. Staining of MAP2 and TuJ1 detected PC-12 differentiation on various scaffolds following 7 days of culture. [3]. In another study, random and aligned nanofiber scaffolds were also fabricated from PCL. However, an emulsion electrospinning technique was used where BSA and NGF formed the core, while PCL formed the shell. Emulsion electrospinning has been developed to prepare core-shell structured nanofibers as drug delivery vehicles [4]. Random and aligned pure PCL, PCL-BSA-NGF, PCL-BSA, and PCL-NGF nanofibers were produced for comparison. Figure 4 shows a schematic illustration of the emulsion preparation and electrospinning set up. Figure 4. Schematic illustration of (A) emulsion preparation process, and the electrospinning set-up to produce (B) random, and (C) aligned nanofibers. Red = water phase. Green = oil phase [4]. PC-12 cells were cultured for eight days on the surface of each nanofiber created for this study. After 8 days of culture, some PC12 cells grown on NGF-added (R/A)-PCL, (R/A)-PCL-BSA scaffolds showed elongation. PC12 cells on (R/A)-PCL-NGF and (R/A)-PCL-NGF&BSA scaffolds projected the neurites [4]. These results suggest that NGF stimulated PC12 differentiation [4]. However, NGF released from the NGF encapsulated nanofibers were more effective on PC12 differentiation compared to NGF that was only added directly in the culture medium [4]. 4. Blended electrospun nanofibers In another study by Lins et al, they developed electrospun nanofiber scaffolds that showed similar structure to the ECM present in neural tissues. This group used poly(lactic acid) (PLA)/poly(lactide-b-ethylene glycol-b-lactide) block copolymer (PELA) and PLA/polyethylene glycol (PEG) as the blended polymer materials. An advantage for PLA in neural tissue engineering applications is its hydrolytic degradation kinetics [5]. This allows PLA to be eliminated as carbon dioxide and water in the Krebs’ cycle. The limitations of PLA include, slow biodegradation process, high stiffness, and is hydrophobic. To increase the hydrophilicity and reduce the brittleness of the PLA-based scaffold, soft PEG was blended with PLA to increase the biocompatibility of the scaffold. PEG has good biocompatibility and low toxicity and has been investigated for its treatment of injury to neuronal membranes [5]. PELA is a block copolymer that is good for tissue engineering applications because it possesses intermediate physicochemical characteristics and has a good balance between degradation rate and hydrophilicity. The PLA, PLA/PELA, and PLA/PEG-based membranes were prepared by electrospinning. This paper focused on the comparison between the blends of PLA with varying molecular weights of PEG and PELA. Following the electrospinning of the polymer blends, 3D interconnected fibers with smooth and round shapes were formed. The PLA/PELA2k and PLA/PEG2k blended nanofibers showed more homogenous morphologies than PLA, PLA/PELA20k and PLA/PEG20k blended nanofibers. To test the cell affinity of the electrospun fibers, monkey embryonic stem cells (ESCs) and neural stem cells (NSCs) were used. Monkey ESCs and NSCs share the same properties as human ESCs and NSCs, which made them an appropriate cell type for this study. NSCs were first cultured on the PLA, PLA/PELA, and PLA/PEG blended scaffolds, and immunostained for NSC marker SOX2 (Figure 6B). TAU-green fluorescent protein (TAU-GFP) allowed the visualization of cell morphology, which bind GFP to microtubules [5]. Neat PLA expressed SOX2 at 71%, and PLA/PELA20k expressed SOX2 at 67% (Fig 7C). The cell density on the PLA and PLA/PELA20k was similar as well at 541 and 494 cells/mm2 respectively. Cell density was quantified by counting the number of living cells per mm2. The PLA/PELA2k cell density was reduced at 339 cells/mm2 and SOX2 expression was lower at 57%. In contrast to the previously described fibers, the PLA/PEG2k and 20k resulted in very low cell density. These results indicated that PLA/PEG2K and 20K were not suitable scaffolds for neural cell maintenance and differentiation. Figure 6. ESC-NSC characterization upon culture on PLA, PLA/PEG2K, PLA/PEG20K, PLA/PELA2K, and PLA/PELA 20K scaffolds. (B) Immunostaining for TAU-GFP, SOX2, and Ki67 in EXS-NSCs cultured for 2 and 5 DIV on the different scaffolds. Scale bars, 50um (2DIV); 20 um (5 DIV). (C) Cell density and SOX-2 positive cell percentage after 2 DIV. DIV = days in vitro. [5] In addition, PGS, PMMA, and gelatin were electrospun to form blended nanofibers for neural tissue engineering. PGS is a biodegradable and elastic polymer, but uncured PGS is difficult to electrospin into nanofibers. PGS was modified by using atom transfer radical polymerization (ATRP) to synthesize PGS-based copolymers with MMA. PGS-PMMA was easily electrospun into nanofibers with a diameter of 167 +/- 33 nm [6]. Rat PC12 cells were seeded onto the PGS- PMMA/gelatin nanofibers and analyzed for its efficacy in nerve regeneration. As shown in Figure 8 below, the gelatin-containing PGS based nanofibers showed the greatest amount of PC-12 cell proliferation after 8 days. This was most likely due to the preferential proliferation of neuronal cell on nanofibers containing natural polymers [6]. Gelatin is a derivative of collagen, and present amide functional groups on the surface of the nanofibrous scaffolds, which provide chemical and biological cues for cell adhesion and growth [6]. This study indicated that by modifying PGS and synthesizing a new PGS-PMMA copolymer material, may be a promising approach for novel biomaterial applications. Figure 8. Proliferation of rat PC12 cells on PGS-PMMA/gelatin nanofibers as determined by alamarBlue. #p



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