Removal of Retained Electrospinning Solvent Prolongs Drug Release from Electrospun PLLA Fibers.

A major challenge in developing drug-releasing electrospun nanofibers is obtaining long-term drug release over many weeks with no burst release of drug. Here, we present new methods capable of prolonging the diffusive release of small molecule drugs from electrospun poly-L-lactic acid (PLLA) nanofibers. The methods focus on removal of retained electrospinning solvent through fiber heating, maintaining fibers in a laboratory setting, or a combination of these methods. These post-fabrication methods altered the release characteristics of a model small molecule drug, 6-aminonicotinamide (6AN), from PLLA fibers. Specifically, untreated fibers released 6AN over 9 days, and fibers that underwent a combined treatment of maintenance in a laboratory setting and heating released 6AN over 44 days. The unique and simple method presented here prolongs diffusive release of a small molecule drug from electrospun fibers and has potential to assist in lengthening small molecule drug release from a variety of polymeric nanomaterials.

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.

Using Solution Electrowriting to Control the Properties of Tubular Fibrous Conduits.

Multiple additive manufacturing techniques have been developed in recent years to produce structures with tunable physical, chemical, and mechanical properties and defined architecture. Solution electrospinning, although an older and more established technique, normally cannot achieve the pattern resolution and tunability of these newer manufacturing techniques. In this study, we present solution electrowriting as a method to produce fibrous conduits from various polymers with tunable patterns, dimensions, and scaffold porosity. We demonstrate the importance of solvent selection during solution electrowriting and discuss how solvent polarity and volatility can be exploited to controllably alter the structure of the resulting scaffolds. The technique can be readily implemented with equipment for conventional electrospinning and offers versatility, control, and customization that is uncommon in the solution electrospinning field.

TGFß3 is neuroprotective and alleviates the neurotoxic response induced by aligned poly-l-lactic acid fibers on naïve and activated primary astrocytes.

Following spinal cord injury, astrocytes at the site of injury become reactive and exhibit a neurotoxic (A1) phenotype, which leads to neuronal death. In addition, the glial scar, which is composed of reactive astrocytes, acts as a chemical and physical barrier to subsequent axonal regeneration. Biomaterials, specifically electrospun fibers, induce a migratory phenotype of astrocytes and promote regeneration of axons following acute spinal cord injury in preclinical models. However, no study has examined the potential of electrospun fibers or biomaterials in general to modulate neurotoxic (A1) or neuroprotective (A2) astrocytic phenotypes. To assess astrocyte reactivity in response to aligned poly-l-lactic acid microfibers, naïve spinal cord astrocytes or spinal cord astrocytes primed towards the neurotoxic phenotype (A1) were cultured on fibrous scaffolds. Gene expression analysis of the pan-reactive astrocyte makers (GFAP, Lcn2, SerpinA3), A1 specific markers (H2-D1, SerpinG1), and A2 specific makers (Emp1, S100a10) was done using quantitative polymerase chain reaction (qPCR). Electrospun fibers mildly increased the expression of the pan-reactive and A1-specific markers, showing the ability of fibrous materials to induce a more reactive, A1 phenotype. However, when naïve or activated astrocytes were cultured on fibers in the presence of transforming growth factor ß3 (TGFß3), the expression of A1-specific markers was greatly reduced, which in turn improved neuronal survival in culture.

Three-Dimensional Printing of Poly(glycerol sebacate) Acrylate Scaffolds via Digital Light Processing.

Digital light processing (DLP)-based three-dimensional (3D) printing offers large improvements in fabrication throughput and spatial resolution when compared to various other additive manufacturing techniques. Both properties are highly desirable when fabricating biomaterial scaffolds that require design precision. Poly(glycerol sebacate) acrylate (PGSA) is a degradable, biocompatible, and photocurable elastomer. In this work, PGSA ink was developed for DLP 3D printing of porous tubular structures. Ink formulations with varying prepolymer concentrations (10-60 wt %), diluent (dimethyl sulfoxide (DMSO), 2-butoxyethyl acetate (EGBEA), and 1:1 DMSO/EGBEA), and degree of PGSA acrylation (17-75%) were studied to optimize printing efficiency and bulk properties of the printed scaffolds. Prepolymer inks with viscosity (<5 Pa·s) and photopolymerization kinetics (exposure time <10 s) appropriate for DLP were developed. Photocrosslinked PGSA scaffolds were further exposed to postfabrication treatments including additional UV exposure or thermal curing (150 °C) to demonstrate tunability in scaffold degradation kinetics and mechanical properties. Complementary to this effort, a 3D model-generation tool was developed to enable user-friendly customization of tubular scaffold design by controlling the pore and strut size of the volumetric mesh. The resulting DLP-printed PGSA scaffolds present high mimicry to complex 3D models with a minimum feature thickness of 80 µm. The tunable properties of PGSA coupled with enhanced precision in microstructure geometry provide a fabrication platform for a variety of tissue regeneration applications.

Aligned Fingolimod-Releasing Electrospun Fibers Increase Dorsal Root Ganglia Neurite Extension and Decrease Schwann Cell Expression of Promyelinating Factors.

Researchers are investigating the use of biomaterials with aligned guidance cues, like those provided by aligned electrospun fibers, to facilitate axonal growth across critical-length peripheral nerve defects. To enhance the regenerative outcomes further, these aligned fibers can be designed to provide local, sustained release of therapeutics. The drug fingolimod improved peripheral nerve regeneration in preclinical rodent models by stimulating a pro-regenerative Schwann cell phenotype and axonal growth. However, the systemic delivery of fingolimod for nerve repair can lead to adverse effects, so it is necessary to develop a means of providing sustained delivery of fingolimod local to the injury. Here we created aligned fingolimod-releasing electrospun fibers that provide directional guidance cues in combination with the local, sustained release of fingolimod to enhance neurite outgrowth and stimulate a pro-regenerative Schwann cell phenotype. Electrospun fiber scaffolds were created by blending fingolimod into poly(lactic-co-glycolic acid) (PLGA) at a w/w% (drug/polymer) of 0.0004, 0.02, or 0.04%. We examined the effectiveness of these scaffolds to stimulate neurite extension in vitro by measuring neurite outgrowth from whole and dissociated dorsal root ganglia (DRG). Subsequently, we characterized Schwann cell migration and gene expression in vitro. The results show that drug-loaded PLGA fibers released fingolimod for 28 days, which is the longest reported release of fingolimod from electrospun fibers. Furthermore, the 0.02% fingolimod-loaded fibers enhanced neurite outgrowth from whole and dissociated DRG neurons, increased Schwann cell migration, and reduced the Schwann cell expression of promyelinating factors. The in vitro findings show the potential of the aligned fingolimod-releasing electrospun fibers to enhance peripheral nerve regeneration and serve as a basis for future in vivo studies.

Challenges of gene delivery to the central nervous system and the growing use of biomaterial vectors.

Gene therapy is a promising form of treatment for those suffering from neurological disorders or central nervous system (CNS) injury, however, obstacles remain that limit its translational potential. The CNS is protected by the blood brain barrier, and this barrier blocks genes from traversing into the CNS if administered outside of the CNS. Viral and non-viral gene delivery vehicles, commonly referred to as vectors, are modified to enhance delivery efficiency to target locations in the CNS. Still, there are few gene therapy approaches approved by the FDA for CNS disease or injury treatment. The lack of viable clinical approaches is due, in part, to the unpredictable nature of many vector systems. In particular, safety concerns exist with the use of viral vectors for CNS gene delivery. To seek some alternatives to viral vectors, development of new non-viral, biomaterial vectors is occurring at a rapid rate. This review discusses the challenges of delivering various forms of genetic material to the CNS, the use and limitations of current viral vector delivery systems, and the use of non-viral, biomaterial vectors for CNS applications.

Lactonic Sophorolipid Increases Surface Wettability of Poly-l-lactic Acid Electrospun Fibers.

The hydrophobicity of electrospun poly-l-lactic acid (PLLA) fibers hinders their integration with surrounding tissue for a variety of applications. In this study, we increased PLLA fiber hydrophilicity by incorporating the natural surfactant, lactonic sophorolipid (LSL). PLLA+LSL fibers had similar fiber morphology but significantly greater surface wettability, which suggested LSL accumulation on the fiber surface. Differential scanning calorimetry results also suggested that LSL was phase separated from PLLA. Despite the altered surface wettability of these fibers, there was no change in fibroblast adhesion. Future studies may explore the use of this natural surfactant to deliver bioactive factors to enhance fibroblast adhesion.

Vastly extended drug release from poly(pro-17ß-estradiol) materials facilitates in vitro neurotrophism and neuroprotection.

Central nervous system (CNS) injuries persist for years, and currently there are no therapeutics that can address the complex injury cascade that develops over this time-scale. 17ß-estradiol (E2) has broad tropism within the CNS, targeting and inducing beneficial phenotypic changes in myriad cells following injury. To address the unmet need for vastly prolonged E2 release, we report first-generation poly(pro-E2) biomaterial scaffolds that release E2 at nanomolar concentrations over the course of 1-10 years via slow hydrolysis in vitro. As a result of their finely tuned properties, these scaffolds demonstrate the ability to promote and guide neurite extension ex vivo and protect neurons from oxidative stress in vitro. The design and testing of these materials reported herein demonstrate the first step towards next-generation implantable biomaterials with prolonged release and excellent regenerative potential.

Stabilized Interleukin-4-Loaded Poly(lactic-co-glycolic) Acid Films Shift Proinflammatory Macrophages toward a Regenerative Phenotype in Vitro.

Macrophages are immune cells involved in wound healing and tissue regeneration; however, the sustained presence of proinflammatory macrophages in wound sites impairs healing. In this study, we shifted peritoneal macrophage polarization away from a proinflammatory (M1) phenotype through exposure to stabilized interleukin-4 (IL-4) in poly(lactic-co-glycolic acid) films in combination with topographical guidance from electrospun poly-L-lactic acid fibers. To our knowledge, this was the first study to stabilize IL-4 with bovine serum albumin (BSA) within a biomaterial. When IL-4 was coloaded with BSA for stabilization, we saw increased IL-4 bioactivity compared to no added stabilization, trehalose stabilization, or murine serum albumin stabilization. We observed increased elongation of peritoneal macrophages, increased RNA expression of anti-inflammatory marker arginase-1, increased ratio of interleukin-10/interleukin- 12 p40 RNA, and decreased protein expression of proinflammatory markers (interleukin-12 p40 and RANTES) compared to controls. Taken together, these results suggest the macrophages were less proinflammatory and were a more pro-resolving phenotype. When stabilized with BSA, IL-4-loaded films effectively shift macrophage polarization state and are thus promising scaffolds to reduce inflammation within in vivo injury models.

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.

Electrospun fiber surface nanotopography influences astrocyte-mediated neurite outgrowth.

Aligned, electrospun fiber scaffolds provide topographical guidance for regenerating neurons and glia after central nervous system injury. To date, no study has explored how fiber surface nanotopography affects astrocyte response to fibrous scaffolds. Astrocytes play important roles in the glial scar, the blood brain barrier, and in maintaining homeostasis in the central nervous system. In this study, electrospun poly L-lactic acid fibers were engineered with smooth, pitted, or divoted surface nanotopography. Cortical or spinal cord primary rat astrocytes were cultured on the surfaces for either 1 or 3 d to examine the astrocyte response over time. The results showed that cortical astrocytes were significantly shorter and broader on the pitted and divoted fibers compared to those on smooth fibers. However, spinal cord astrocyte morphology was not significantly altered by the surface features. These findings indicate that astrocytes from unique anatomical locations respond differently to the presence of nanotopography. Western blot results show that the differences in morphology were not associated with significant changes in glial fibrillary acidicprotein (GFAP) or vinculin in either astrocyte population, suggesting that surface pits and divots do not induce a reactive phenotype in either cortical or spinal cord astrocytes. Finally, astrocytes were co-cultured with dorsal root ganglia to determine how the surfaces affected astrocyte-mediated neurite outgrowth. Astrocytes cultured on the fibers for shorter periods of time (1 d) generally supported longer neurite outgrowth. Pitted and divoted fibers restricted spinal cord astrocyte-mediated neurite outgrowth, while smooth fibers increased 3 d spinal cord astrocyte-mediated neurite outgrowth. In total, fiber surface nanotopography can influence astrocyte elongation and influence the capability of astrocytes to direct neurites. Therefore, fiber surface characteristics should be carefully controlled to optimize astrocyte-mediated axonal regeneration.

Solvent retention in electrospun fibers affects scaffold mechanical properties.

Electrospinning is a robust material fabrication method allowing for fine control of mechanical, chemical, and functional properties in scaffold manufacturing. Electrospun fiber scaffolds have gained prominence for their potential in a variety of applications such as tissue engineering and textile manufacturing, yet none have assessed the impact of solvent retention in fibers on the scaffold's mechanical properties. In this study, we hypothesized that retained electrospinning solvent acts as a plasticizer, and gradual solvent evaporation, by storing fibers in ambient air, will cause significant increases in electrospun fiber scaffold brittleness and stiffness, and a significant decrease in scaffold toughness. Thermogravimetric analysis indicated solvent retention in PGA, PLCL, and PET fibers, and not in PU and PCL fibers. Differential scanning calorimetry revealed that polymers that were electrospun below their glass transition temperature (T g ) retained solvent and polymers electrospun above T g did not. Young's moduli increased and yield strain decreased for solventretaining PGA, PLCL, and PET fiber scaffolds as solvent evaporated from the scaffolds over a period of 14 days. Toughness and failure strain decreased for PGA and PET scaffolds as solvent evaporated. No significant differences were observed in the mechanical properties of PU and PCL scaffolds that did not retain solvent. These observations highlight the need to consider solvent retention following electrospinning and its potential effects on scaffold mechanical properties.

Poly-l-lactic acid-co-poly(pentadecalactone) Electrospun Fibers Result in Greater Neurite Outgrowth of Chick Dorsal Root Ganglia in Vitro Compared to Poly-l-lactic Acid Fibers.

Electrospun poly-l-lactic acid (PLLA) fiber scaffolds are used to direct axonal extension in neural engineering models. We aimed to improve the efficacy of these fibers in promoting neurite outgrowth by altering surface topography and reducing fiber elastic modulus through the incorporation of a compatibilized blend, poly-l-lactic acid-poly(pentadecalactone) (PLLA-PPDL) into the solution prior to electrospinning. PLLA+PLLA-PPDL fibers had a larger diameter, increased surface nanotopography, and lower glass transition temperature than PLLA fibers but had similar mechanical properties. Increases in neurite outgrowth on PLLA+PLLA-PPDL fibers were observed, potentially due to the significantly increased diameter and surface coverage with nanotopography. Ultimately, these results suggest that greater electrospun fiber diameter and surface topography may contribute to increases in neurite outgrowth.

The effect of engineered nanotopography of electrospun microfibers on fiber rigidity and macrophage cytokine production.

Currently, it is unknown how the mechanical properties of electrospun fibers, and the presentation of surface nanotopography influence macrophage gene expression and protein production. By further elucidating how specific fiber properties (mechanical properties or surface properties) alter macrophage behavior, it may be possible to create electrospun fiber scaffolds capable of initiating unique cellular and tissue responses. In this study, we determined the elastic modulus and rigidity of fibers with varying topographies created by finely controlling humidity and including a non-solvent during electrospinning. In total,five fiber scaffold types were produced. Analysis of fiber physical properties demonstrated no change in fiber diameter amongst the five different fiber groups. However, the four different fibrous scaffolds with nanopits or divots each possessed different numbers of pits with different nanoscale dimensions. Unpolarized bone marrow derived murine macrophages (M0), macrophages polarized towards a pro-inflammatory phenotype (M1), or macrophages polarized towards anti-inflammatory phenotype (M2b) were placed onto each of the scaffolds and cytokine RNA expression and protein production were analyzed. Specific nanotopographies did not appreciably alter cytokine production from undifferentiated macrophages (M0) or anti-inflammatory macrophages (M2b), but a specific fiber (with many small pits) did increase IL-12 transcript and IL-12 protein production compared to fibers with small divots. When analyzing the mechanical properties between fibers with divots or with many small pits,divoted fibers possessed similar elastic moduli but different stiffness values. In total,we present techniques capable of creating unique electrospun fibers. These unique fibers have varying fiber mechanical characteristics and modestly modulate macrophage cytokine expression.

Experimental and credentialing capital: an adaptable framework for facilitating science outreach for underrepresented youth.

Increasing the numbers of black, latino and native youth in STEM careers is both an important way to reduce poverty in low income communities, and a contribution to the diversity of thought and experience that drives STEM research. But underrepresented youth are often alienated from STEM. Two new forms of social capital have been identified that can be combined to create a learning environment in which students and researchers can meet and explore an area of shared interest. Experimental capital refers to the intrinsic motivation that students can develop when they learn inquiry techniques for exploring topics that they feel ownership over. Credentialing capital denotes a shared interest and ability between all parties engaged in the experimental endeavor. These two forms of social capital form an adaptable framework for researchers to use to create effective outreach programs. In this case study sports biomechanics was utilized as the area of shared interest and understanding the slam dunk was used as experimental capital.