Electrospun Fibers for Drug Delivery after Spinal Cord Injury and the Effects of Drug Incorporation on Fiber Properties.

There is currently no cure for individuals with spinal cord injury (SCI). While many promising approaches are being tested in clinical trials, the complexity of SCI limits several of these approaches from aiding complete functional recovery. Several different categories of biomaterials are investigated for their ability to guide axonal regeneration, to deliver proteins or small molecules locally, or to improve the viability of transplanted stem cells. The purpose of this study is to provide a brief overview of SCI, present the different categories of biomaterial scaffolds that direct and guide axonal regeneration, and then focus specifically on electrospun fiber guidance scaffolds. Much like other polymer guidance approaches, electrospun fibers can retain and deliver therapeutic drugs. The experimental section presents new data showing the incorporation of two therapeutic drugs into electrospun poly-L-lactic acid fibers. Two different concentrations of either riluzole or neurotrophin-3 were loaded into the electrospun fibers to examine the effect of drug concentration on the physical characteristics of the fibers (fiber alignment and fiber diameter). Overall, the drugs were successfully incorporated into the fibers and the release was related to the loading concentration. The fiber diameter decreased with the inclusion of the drug, and the decreased diameter was correlated with a decrease in fiber alignment. Subsequently, the study includes considerations for successful incorporation of a therapeutic drug without changing the physical properties of the fibers.

Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate.

The versatility of natural killer cells has ignited growing interest in their therapeutic use for cancer and other immunotherapy treatments. However, NK cells compose a small portion of peripheral blood mononuclear cells (5%-20% of PBMCs) and clinical doses require billions of cells. Manufacturing suitable doses of NK cells remains a major challenge for NK immunotherapy. The current standard for expanding NK cells relies on feeder cells and fetal bovine serum to achieve large expansion, but both encounter regulatory concerns. We developed NK Cloudz, a dissolvable polymer-based microsphere platform, as an alternative to a feeder cell approach to expand NK cells. We demonstrated that a combination of NK Cloudz, a G-Rex6M culture vessel, and GMP Human Platelet Lysate expanded NK cells 387 ± 100-fold in 10 days from a PBMC starting population. The NK purity, viability, and cytotoxicity were similar to both a feeder cell protocol and an FBS-based protocol. Additionally, we found no significant differences between FBS and GMP Human Platelet Lysate and concluded that platelet lysate is a good xeno-free alternative to FBS for NK expansion. Overall, we demonstrated a feeder-cell-free and FBS-free protocol that leverages NK Cloudz as a promising step toward a commercial GMP manufacturing method to expand NK cells for therapeutic use.

Exploring the effects of electrospun fiber surface nanotopography on neurite outgrowth and branching in neuron cultures.

Three aligned, electrospun fiber scaffolds with unique surface features were created from poly-L-lactic acid (PLLA). Fibers without surface nanotopography (smooth fibers), fibers with surface divots (shallow pits), and fibers with surface pits (deeper pits) were fabricated, and fiber alignment, diameter, and density were characterized using scanning electron microscopy (SEM). Whole dorsal root ganglia (DRG) were isolated from rats and placed onto uncoated fibers or fibers coated with laminin. On uncoated fibers, neurite outgrowth was restricted by fibers displaying divoted or pitted nanotopography when compared to neurite outgrowth on smooth fibers. However, neurites extending from whole DRG cultured on laminin-coated fibers were not restricted by divoted or pitted surface nanotopography. Thus, neurites extending on laminin-coated fibers were able to extend long neurites even in the presence of surface divots or pits. To further explore this result, individual neurons isolated from dissociated DRG were seeded onto laminin-coated smooth, pitted, or divoted fibers. Interestingly, neurons on pitted or divoted fibers exhibited a 1.5-fold increase in total neurite length, and a 2.3 or 2.7-fold increase in neurite branching compared to neurons on smooth fibers, respectively. Based on these findings, we conclude that fiber roughness in the form of pits or divots can promote extension and branching of long neurites along aligned electrospun fibers in the presence of an extracellular matrix protein coating. Thus, aligned, electrospun fibers can be crafted to not only direct the extension of axons but to induce unique branching morphologies.

Multi-modal characterization of polymeric gels to determine the influence of testing method on observed elastic modulus.

Demand for materials that mechanically replicate native tissue has driven development and characterization of various new biomaterials. However, a consequence of materials and characterization technique diversity is a lack of consensus within the field, with no clear way to compare values measured via different modalities. This likely contributes to the difficulty in replicating findings across the research community; recent evidence suggests that different modalities do not yield the same mechanical measurements within a material, and direct comparisons cannot be made across different testing platforms. Herein, we examine whether "material properties" are characterization modality-specific by analyzing the elastic moduli determined by five typical biomaterial mechanical characterization techniques: unconfined-compression, tensiometry, rheometry, and micro-indentation at the macroscopic level, and microscopically using nanoindentation. These analyses were performed in two different polymeric gels frequently used for biological applications, polydimethylsiloxane (PDMS) and agarose. Each was fabricated to span a range of moduli, from physiologic to supraphysiologic values. All five techniques identified the same overall trend within each material group, supporting their ability to appreciate relative moduli differences. However, significant differences were found across modalities, illustrating a difference in absolute moduli values, and thereby precluding direct comparison of measurements from different characterization modalities. These observed differences may depend on material compliance, viscoelasticity, and microstructure. While determining the underlying mechanism(s) of these differences was beyond the scope of this work, these results demonstrate how each modality affects the measured moduli of the same material, and the sensitivity of each modality to changes in sample material composition.

The Effect of Electrospun Fiber Diameter on Astrocyte-Mediated Neurite Guidance and Protection.

The topography of electrospun fiber scaffolds modifies astrocytes toward in vivo-like morphologies and behaviors. However, little is known about how electrospun fiber diameter influences astrocyte behavior. In this work, aligned fibers with two distinct nanoscale fiber diameters (808 and 386 nm) were prepared, and the astrocyte response was measured over time. Astrocytes on the large diameter fibers showed significantly increased elongation as early as 2 h after seeding and remained significantly more elongated for up to 4 days compared to those on small diameter fibers. Astrocytes extending along larger diameter fibers were better equipped to support long neurite outgrowth from dorsal root ganglia neurons, and neurite outgrowth along these astrocytes was less branched than outgrowth along astrocytes cultured on small diameter fibers. The differences in astrocyte shape observed on the small or large diameter fibers did not translate into differences in GLT-1, GFAP, or GLAST protein expression. Thus, different fiber diameters were unable to influence astrocyte protein expression uniquely. Nevertheless, astrocytes cultured in either small or large fibers significantly increased their expression of GLT-1 compared to astrocytes cultured on nonfiber (film) controls. Fibrous-induced increases in astrocyte GLT-1 expression protected astrocyte/neuron cocultures from toxicity generated by high extracellular glutamate. Alternatively, astrocytes/neurons cultured on films were less able to protect these cells from culture conditions consisting of high glutamate levels. Biomaterials, such as the fibrous materials presented here, may help stimulate astrocytes to increase GLT-1 expression and uptake more glutamate, since astrocytes are less likely to uptake glutamate in neurodegenerative pathologies or following central nervous system injury.

Injectable, Magnetically Orienting Electrospun Fiber Conduits for Neuron Guidance.

Magnetic electrospun fibers are of interest for minimally invasive biomaterial applications that also strive to provide cell guidance. Magnetic electrospun fibers can be injected and then magnetically positioned in situ, and the aligned fiber scaffolds provide consistent topographical guidance to cells. In this study, magnetically responsive aligned poly-l-lactic acid electrospun fiber scaffolds were developed and tested for neural applications. Incorporating oleic acid-coated iron oxide nanoparticles significantly increased neurite outgrowth, reduced the fiber alignment, and increased the surface nanotopography of the electrospun fibers. After verifying neuron viability on two-dimensional scaffolds, the system was tested as an injectable three-dimensional scaffold. Small conduits of aligned magnetic fibers were easily injected in a collagen or fibrinogen hydrogel solution and repositioned using an external magnetic field. The aligned magnetic fibers provided internal directional guidance to neurites within a three-dimensional collagen or fibrin model hydrogel, supplemented with Matrigel. Neurites growing from dorsal root ganglion explants extended 1.4-3× farther on the aligned fibers compared with neurites extending in the hydrogel alone. Overall, these results show that magnetic electrospun fiber scaffolds can be injected and manipulated with a magnetic field in situ to provide directional guidance to neurons inside an injectable hydrogel. Most importantly, this injectable guidance system increased both neurite alignment and neurite length within the hydrogel scaffold.