Evaluating glioblastoma tumour sphere growth and migration in interaction with astrocytes using 3D collagen-hyaluronic acid hydrogels.

Glioblastoma (GB) is an astrocytic brain tumour with a low survival rate, partly because of its highly invasive nature. The GB tumour microenvironment (TME) includes its extracellular matrix (ECM), a variety of brain cell types, unique anatomical structures, and local mechanical cues. As such, researchers have attempted to create biomaterials and culture models that mimic features of TME complexity. Hydrogel materials have been particularly popular because they enable 3D cell culture and mimic TME mechanical properites and chemical composition. Here, we used a 3D collagen I-hyaluronic acid hydrogel material to explore interactions between GB cells and astrocytes, the normal cell type from which GB likely derives. We demonstrate three different spheroid culture configurations, including GB multi-spheres (i.e., GB and astrocyte cells in spheroid co-culture), GB-only mono-spheres cultured with astrocyte-conditioned media, and GB-only mono-spheres cultured with dispersed live or fixed astrocytes. Using U87 and LN229 GB cell lines and primary human astrocytes, we investigated material and experiment variability. We then used time-lapse fluorescence microscopy to measure invasive potential by characterizing the sphere size, migration capacity, and weight-averaged migration distance in these hydrogels. Finally, we developed methods to extract RNA for gene expression analysis from cells cultured in hydrogels. U87 and LN229 cells displayed different migration behaviors. U87 migration occurred primarily as single cells and was reduced with higher numbers of astrocytes in both multi-sphere and mono-sphere plus dispersed astrocyte cultures. In contrast, LN229 migration exhibited features of collective migration and was increased in monosphere plus dispersed astrocyte cultures. Gene expression studies indicated that the most differentially expressed genes in these co-cultures were CA9, HLA-DQA1, TMPRSS2, FPR1, OAS2, and KLRD1. Most differentially expressed genes were related to immune response, inflammation, and cytokine signalling, with greater influence on U87 than LN229. These data show that 3D in vitro hydrogel co-culture models can be used to reveal cell line specific differences in migration and to study differential GB-astrocyte crosstalk.

One step closer to cancer nanomedicine.

High-throughput tool uncovers links between cell signaling and nanomaterial uptake.

Comparative Encapsulation Efficiency of Lutein in Micelles Synthesized via Batch and High Throughput Methods.

PURPOSE: Black raspberries (BRBs) and their anthocyanin-rich hydrophilic fractions (BRB-H) have exhibited significant chemopreventative activity across aerodigestive cancers. Lutein, the primary component of the BRB lipophilic fraction (BRB-L), also demonstrates bioactivity potential, but is less well characterized, in part because of its poor, innate bioavailability. For these lipophilic compounds to be accurately evaluated for anticancer efficacy, it is necessary to increase their functional bioavailability using delivery vehicles. Lutein has been delivered in commercial settings in emulsion form. However, emulsions are unstable, particularly in the gastrointestinal tract, which limit their use as an oral nutraceutical. Here, we evaluated lutein encapsulation and cellular uptake for nanoparticle (NP) delivery vehicles composed of three different materials synthesized via two different approaches. METHODS: Specifically, NPs were synthesized via smaller scale batch interfacial instability (II) sonication and semi-continuous high throughput electrohydrodynamic-mediated mixing nanoprecipitation (EM-NP) methods using polystyrene-polyethylene oxide (PSPEO) or polycaprolactone-polyethylene glycol (PCLPEG) block copolymers and PHOSPHOLIPON 90G® (P90G, Lipoid GmbH) lipids. Size distribution, lutein encapsulation efficiency (EE), and cellular uptake and delivery were evaluated for each NP formulation. RESULTS: NPs produced via high throughput EM-NP had higher EEs than NPs produced via batch II sonication, and P90G had the greatest EE (55%) and elicited faster cellular uptake in premalignant oral epithelial cells (SCC83) compared to other delivery systems. CONCLUSION: These qualities suggest P90G could be a beneficial candidate for future lutein in vitro delivery research and clinical translation for oral cancer prevention.

Hyaluronic acid induces ROCK-dependent amoeboid migration in glioblastoma cells.

Glioblastoma (GBM) is the most aggressive and deadly adult brain tumor, primarily because of its high infiltrative capacity and development of resistance to therapy. Although GBM cells are typically believed to migrate via mesenchymal (e.g., fibroblast-like) migration modes, amoeboid (e.g., leucocyte-like) migration modes have been identified and may constitute a salvage pathway. However, the mesenchymal to amoeboid transition (MAT) process in GB is not well characterized, most likely because most culture models induce MAT via pharmacological or genetic inhibition conditions that are far from physiological. In this study, we examined the ability of hyaluronic acid (HA) content in three-dimensional collagen (Col) hydrogels to induce MAT in U87 GBM cells. HA and Col are naturally-occurring components of the brain extracellular matrix (ECM). In pure Col gels, U87 cells displayed primarily mesenchymal behaviors, including elongated cell morphology, clustered actin and integrin expression, and crawling migration behaviors. Whereas an increasing population of cells displaying amoeboid behaviors, including rounded morphology, cortical actin expression, low/no integrin expression, and squeezing or gliding motility, were observed with increasing HA content (0.1-0.2 wt% in Col). Consistent with amoeboid migration, these behaviors were abrogated by ROCK inhibition with the non-specific small molecule inhibitor Y27632. Toward identification of histological MAT classification criteria, we also examined the correlation between cell and nuclear aspect ratio (AR) in Col and Col-HA gels, finding that nuclear AR has a small variance and is not correlated to cell AR in HA-rich gels. These results suggest that HA may regulate GBM cell motility in a ROCK-dependent manner.

MicroRNA-mRNA Interactions at Low Levels of Compressive Solid Stress Implicate mir-548 in Increased Glioblastoma Cell Motility.

Glioblastoma (GBM) is an astrocytic brain tumor with median survival times of <15 months, primarily as a result of high infiltrative potential and development of resistance to therapy (i.e., surgical resection, chemoradiotherapy). A prominent feature of the GBM microenvironment is compressive solid stress (CSS) caused by uninhibited tumor growth within the confined skull. Here, we utilized a mechanical compression model to apply CSS (<115 Pa) to well-characterized LN229 and U251 GBM cell lines and measured their motility, morphology, and transcriptomic response. Whereas both cell lines displayed a peak in migration at 23 Pa, cells displayed differential response to CSS with either minimal (i.e., U251) or large changes in motility (i.e., LN229). Increased migration of LN229 cells was also correlated to increased cell elongation. These changes were tied to epigenetic signaling associated with increased migration and decreases in proliferation predicted via Ingenuity® Pathway Analysis (IPA), characteristics associated with tumor aggressiveness. miRNA-mRNA interaction analysis revealed strong influence of the miR548 family (i.e., mir-548aj, mir-548az, mir-548t) on differential signaling induced by CSS, suggesting potential targets for pharmaceutical intervention that may improve patient outcomes.

Biomolecular detection, tracking, and manipulation using a magnetic nanoparticle-quantum dot platform.

Fluorescent and magnetic materials play a significant role in biosensor technology, enabling sensitive quantification and separations with applications in diagnostics, purification, quality control, and therapeutics. Here, we present a magneto-fluorescent biosensor/separations platform consisting of quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs) that are separately encapsulated in amphiphilic block co-polymer micelles conjugated to DNA or protein (i.e., single-stranded (ss) DNA derived from the mRNA of the tumor suppressor protein p53 or avidin protein). Analytes were detected via an aggregation sandwich assay upon binding of at least 1 QD and 1 SPION-containing micelle to result in a fluorescent/magnetic composite. Multiplexed isolation of protein and DNA biomolecules was demonstrated by using QDs of varying emission wavelength; QD fluorescence intensity could be correlated with analyte concentration. Sequential or parallel biomolecule separation was achieved by adding appropriately functionalized SPION-containing micelles and applying user-controlled magnetic fields via patterned magnetic disks and wires. QD fluorescence was used to continuously visualize analyte separation during this process. This QD/SPION platform is simple to use, demonstrates ∼10-16 M sensitivity in analyte detection (comparable to competing QD biosensors based on energy transfer) with specificity against 1 and 2 basepair mismatches in DNA detection, molecular separations capability in solutions of ∼10-10 M, and permits simultaneous or parallel, multiplexed separation of protein and DNA. Thus, this versatile platform enables self-assembly-based rapid, sensitive, and specific detection and separation of biomolecules, simultaneously and with real-time visualization. This technology demonstrates potential for nanoscale assembly, biosensing, and bioseparations.

Effect of Electrospun Fiber Mat Thickness and Support Method on Cell Morphology.

Electrospun fiber mats (EFMs) are highly versatile biomaterials used in a myriad of biomedical applications. Whereas some facets of EFMs are well studied and can be highly tuned (e.g., pore size, fiber diameter, etc.), other features are under characterized. For example, although substrate mechanics have been explored by several groups, most studies rely on Young's modulus alone as a characterization variable. The influence of fiber mat thickness and the effect of supports are variables that are often not considered when evaluating cell-mechanical response. To assay the role of these features in EFM scaffold design and to improve understanding of scaffold mechanical properties, we designed EFM scaffolds with varying thickness (50-200 µm) and supporting methodologies. EFM scaffolds were comprised of polycaprolactone and were either electrospun directly onto a support, suspended across an annulus (3 or 10 mm inner diameter), or "tension-released" and then suspended across an annulus. Then, single cell spreading (i.e., Feret diameter) was measured in the presence of these different features. Cells were sensitive to EFM thickness and suspended gap diameter. Overall, cell spreading was greatest for 50 µm thick EFMs suspended over a 3 mm gap, which was the smallest thickness and gap investigated. These results are counterintuitive to conventional understanding in mechanobiology, which suggests that stiffer materials, such as thicker, supported EFMs, should elicit greater cell polarization. Additional experiments with 50 µm thick EFMs on polystyrene and polydimethylsiloxane (PDMS) supports demonstrated that cells can "feel" the support underlying the EFM if it is rigid, similar to previous results in hydrogels. These results also suggest that EFM curvature may play a role in cell response, separate from Young's modulus, possibly because of internal tension generated. These parameters are not often considered in EFM design and could improve scaffold performance and ultimately patient outcomes.

Micelle-templated, poly(lactic-co-glycolic acid) nanoparticles for hydrophobic drug delivery.

PURPOSE: Poly(lactic-co-glycolic acid) (PLGA) is widely used for drug delivery because of its biocompatibility, ability to solubilize a wide variety of drugs, and tunable degradation. However, achieving sub-100 nm nanoparticles (NPs), as might be desired for delivery via the enhanced permeability and retention effect, is extremely difficult via typical top-down emulsion approaches. METHODS: Here, we present a bottom-up synthesis method yielding PLGA/block copolymer hybrids (ie, "PolyDots"), consisting of hydrophobic PLGA chains entrapped within self-assembling poly(styrene-b-ethylene oxide) (PS-b-PEO) micelles. RESULTS: PolyDots exhibit average diameters <50 nm and lower polydispersity than conventional PLGA NPs. Drug encapsulation efficiencies of PolyDots match conventional PLGA NPs (ie, ~30%) and are greater than those obtained from PS-b-PEO micelles (ie, ~7%). Increasing the PLGA:PS-b-PEO weight ratio alters the drug release mechanism from chain relaxation to erosion controlled. PolyDots are taken up by model glioma cells via endocytotic mechanisms within 24 hours, providing a potential means for delivery to cytoplasm. PolyDots can be lyophilized with minimal change in morphology and encapsulant functionality, and can be produced at scale using electrospray. CONCLUSION: Encapsulation of PLGA within micelles provides a bottom-up route for the synthesis of sub-100 nm PLGA-based nanocarriers with enhanced stability and drug-loading capacity, and tunable drug release, suitable for potential clinical applications.

Hydrogels That Allow and Facilitate Bone Repair, Remodeling, and Regeneration.

Bone defects can originate from a variety of causes, including trauma, cancer, congenital deformity, and surgical reconstruction. Success of the current "gold standard" treatment (i.e., autologous bone grafts) is greatly influenced by insufficient or inappropriate bone stock. There is thus a critical need for the development of new, engineered materials for bone repair. This review describes the use of natural and synthetic hydrogels as scaffolds for bone tissue engineering. We discuss many of the advantages that hydrogels offer as bone repair materials, including their potential for osteoconductivity, biodegradability, controlled growth factor release, and cell encapsulation. We also discuss the use of hydrogels in composite devices with metals, ceramics, or polymers. These composites are useful because of the low mechanical moduli of hydrogels. Finally, the potential for thermosetting and photo-cross-linked hydrogels as three-dimensionally (3D) printed, patient-specific devices is highlighted. Three-dimensional printing enables controlled spatial distribution of scaffold materials, cells, and growth factors. Hydrogels, especially natural hydrogels present in bone matrix, have great potential to augment existing bone tissue engineering devices for the treatment of critical size bone defects.

Glioma-astrocyte interactions on white matter tract-mimetic aligned electrospun nanofibers.

Gliomas are highly invasive forms of brain cancer comprising more than 50% of brain tumor cases in adults, and astrocytomas account for ∼60-70% of all gliomas. As a result of multiple factors, including enhanced migratory properties and extracellular matrix remodeling, even with current standards of care, mean survival time for patients is only ∼12 months. Because glioblastoma multiforme (GBM) cells arise from astrocytes, there is great interest in elucidating the interactions of these two cell types in vivo. Previous work performed on two-dimensional assays (i.e., tissue culture plastic and Boyden chamber assays) utilizes substrates that lack the complexities of the natural microenvironment. Here, we employed a three-dimensional, electrospun poly-(caprolactone) (PCL) nanofiber system (NFS) to mimic some features of topographical properties evidenced in vivo. Co-cultures of human GBM cells and rat astrocytes, as performed on the NFS, showed a significant increase in astrocyte GFAP expression, particularly in the presence of extracellular matrix (ECM) deposited by GBM cells. In addition, GBM migration increased in the presence of astrocytes or soluble factors (i.e., conditioned media). However, the presence of fixed astrocytes acted as an antagonist, lowering GBM migration rates. This data suggests that astrocytes and GBM cells interact through a multitude of pathways, including soluble factors and direct contact. This work demonstrates the potential of the NFS to duplicate some topographical features of the GBM tumor microenvironment, permitting analysis of topographical effects in GBM migration.

Toward 3D biomimetic models to understand the behavior of glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) tumors are one of the most deadly forms of human cancer and despite improved treatments, median survival time for the majority of patients is a dismal 12-15 months. A hallmark of these aggressive tumors is their unique ability to diffusively infiltrate normal brain tissue. To understand this behavior and successfully target the mechanisms underlying tumor progression, it is crucial to develop robust experimental ex vivo disease models. This review discusses current two-dimensional (2D) experimental models, as well as animal-based models used to examine GBM cell migration, including their advantages and disadvantages. Recent attempts to develop three-dimensional (3D) tissue engineering-inspired models and their utility in unraveling the role of microenvironment on tumor cell behaviors are also highlighted. Further, the use of 3D models to bridge the gap between 2D and animal models is explored. Finally, the broad utility of such models in the context of brain cancer research is examined.

Glioblastoma behaviors in three-dimensional collagen-hyaluronan composite hydrogels.

Glioblastoma multiforme (GBM) tumors, which arise from glia in the central nervous system (CNS), are one of the most deadly forms of human cancer with a median survival time of ∼1 year. Their high infiltrative capacity makes them extremely difficult to treat, and even with aggressive multimodal clinical therapies, outcomes are dismal. To improve understanding of cell migration in these tumors, three-dimensional (3D) multicomponent composite hydrogels consisting of collagen and hyaluronic acid, or hyaluronan (HA), were developed. Collagen is a component of blood vessels known to be associated with GBM migration; whereas, HA is one of the major components of the native brain extracellular matrix (ECM). We characterized hydrogel microstructural features and utilized these materials to investigate patient tumor-derived, single cell morphology, spreading, and migration in 3D culture. GBM morphology was influenced by collagen type with cells adopting a rounded morphology in collagen-IV versus a spindle-shaped morphology in collagen-I/III. GBM spreading and migration were inversely dependent on HA concentration; with higher concentrations promoting little or no migration. Further, noncancerous astrocytes primarily displayed rounded morphologies at lower concentrations of HA; in contrast to the spindle-shaped (spread) morphologies of GBMs. These results suggest that GBM behaviors are sensitive to ECM mimetic materials in 3D and that these composite hydrogels could be used to develop 3D brain mimetic models for studying migration processes.

Mimicking white matter tract topography using core-shell electrospun nanofibers to examine migration of malignant brain tumors.

Glioblastoma multiforme (GBM), one of the deadliest forms of human cancer, is characterized by its high infiltration capacity, partially regulated by the neural extracellular matrix (ECM). A major limitation in developing effective treatments is the lack of in vitro models that mimic features of GBM migration highways. Ideally, these models would permit tunable control of mechanics and chemistry to allow the unique role of each of these components to be examined. To address this need, we developed aligned nanofiber biomaterials via core-shell electrospinning that permit systematic study of mechanical and chemical influences on cell adhesion and migration. These models mimic the topography of white matter tracts, a major GBM migration 'highway'. To independently investigate the influence of chemistry and mechanics on GBM behaviors, nanofiber mechanics were modulated by using different polymers (i.e., gelatin, poly(ethersulfone), poly(dimethylsiloxane)) in the 'core' while employing a common poly(ε-caprolactone) (PCL) 'shell' to conserve surface chemistry. These materials revealed GBM sensitivity to nanofiber mechanics, with single cell morphology (Feret diameter), migration speed, focal adhesion kinase (FAK) and myosin light chain 2 (MLC2) expression all showing a strong dependence on nanofiber modulus. Similarly, modulating nanofiber chemistry using extracellular matrix molecules (i.e., hyaluronic acid (HA), collagen, and Matrigel) in the 'shell' material with a common PCL 'core' to conserve mechanical properties revealed GBM sensitivity to HA; specifically, a negative effect on migration. This system, which mimics the topographical features of white matter tracts, should allow further examination of the complex interplay of mechanics, chemistry, and topography in regulating brain tumor behaviors.

Inherent interfacial mechanical gradients in 3D hydrogels influence tumor cell behaviors.

Cells sense and respond to the rigidity of their microenvironment by altering their morphology and migration behavior. To examine this response, hydrogels with a range of moduli or mechanical gradients have been developed. Here, we show that edge effects inherent in hydrogels supported on rigid substrates also influence cell behavior. A Matrigel hydrogel was supported on a rigid glass substrate, an interface which computational techniques revealed to yield relative stiffening close to the rigid substrate support. To explore the influence of these gradients in 3D, hydrogels of varying Matrigel content were synthesized and the morphology, spreading, actin organization, and migration of glioblastoma multiforme (GBM) tumor cells were examined at the lowest (<50 µm) and highest (>500 µm) gel positions. GBMs adopted bipolar morphologies, displayed actin stress fiber formation, and evidenced fast, mesenchymal migration close to the substrate, whereas away from the interface, they adopted more rounded or ellipsoid morphologies, displayed poor actin architecture, and evidenced slow migration with some amoeboid characteristics. Mechanical gradients produced via edge effects could be observed with other hydrogels and substrates and permit observation of responses to multiple mechanical environments in a single hydrogel. Thus, hydrogel-support edge effects could be used to explore mechanosensitivity in a single 3D hydrogel system and should be considered in 3D hydrogel cell culture systems.

Hydrogel-electrospun fiber composite materials for hydrophilic protein release.

Although hydrogels are widely used in controlled-release systems, obtaining extended, uniform drug release with little initial burst has been challenging. However, recently researchers have shown that combining hydrogels with another drug delivery material can dramatically improve release kinetics. Here we describe a novel hydrogel-based composite material that exhibits stable, near-linear, sustained release of a model hydrophilic protein (e.g., bovine albumin serum, BSA) for over two months with a significant reduction in initial burst release (7% vs. 20%). The composite is comprised of poly(ε-caprolactone) (PCL) electrospun fiber mats coupled with poly(ethylene glycol)-poly(ε-caprolactone) diacrylate (PEGPCL) hydrogels through photo-polymerization. It is believed that the additional diffusion barrier provided by hydrophobic electrospun fiber mats reduces hydrogel swelling and water penetration rates and increases the diffusion path length, resulting in delayed, more uniform drug release. Further, released proteins remain bioactive as demonstrated by PC12 cell neurite extension in response to released nerve growth factor (NGF). The use of electrospun fiber mats to modulate hydrogel drug release provides a new method to control release kinetics of hydrophilic proteins, reducing burst release and extending the release duration.

Polylysine-modified PEG-based hydrogels to enhance the neuro-electrode interface.

Neural prostheses are a promising technology in the treatment of lost neural function. However, poor biocompatibility of these devices inhibits the formation of a robust neuro-electrode interface. Several factors including mechanical mismatch between the device and tissue, inflammation at the implantation site, and possible electrical damage contribute to this response. Many researchers are investigating polymeric brain mimetic coatings as a means to improve integration with nervous tissue. Specifically, hydrogels, constructs also employed in tissue engineering, have been explored because of their structural and mechanical similarity to native tissue. However, many hydrogel materials (e.g., poly(ethylene glycol) (PEG)) do not support cell adhesion. In this work, we report a technique to enhance the interface between polymeric brain mimetic coatings and neural tissue using adhesion molecules. In particular, polylysine-modified PEG-based hydrogels were synthesized, characterized and shown to promote neural adhesion using a PC12 cell line. In addition, we examined adhesion behavior of a PEG-co-polymer and found that these materials adhere to electrodes for at least 4 weeks. These results suggest that polylysine-PEG hydrogel biomaterials are biocompatible and can enhance stability of chronic neural interfaces.

pH sensitive CdS-iron oxide fluorescent-magnetic nanocomposites.

There has been great interest in the use of nanoparticles for imaging, particularly in multimodal applications (e.g., combination of MRI and fluorescence). Yet creating particles with multiple functionalities has been challenging. Here, we report the synthesis of pH sensitive, fluorescent-magnetic, nanocomposites created through a simple aqueous procedure. Separately synthesized superparamagnetic iron oxide nanoparticles and mercaptopropionic acid (MPA)-coated CdS quantum dots were crosslinked using 3-mercaptopropyl trimethoxysilane (MPS) as a bifunctional linker to yield CdS-iron oxide conjugates. Conjugates formed clusters of 0.1-1.0 microm diameter, with the smallest observed particle diameter approximately 50 nm. Particle solubility and photoluminescent (PL) intensity were sensitive to solution pH, with the highest PL intensity and stability obtained at pH values < 3.0 and MPS:Cd:Fe ratios of 1:10:1. pH sensitivity is believed to result from changes in nanoparticle solubility within the silica-based matrix. Given these unique properties, this material might find application in separation, pH sensitive detection (e.g., endosomal tracking) and biosensing.

Retinal prostheses: current challenges and future outlook.

Blindness from retinal diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP), usually causes a significant decline in quality of life for affected patients. Currently there is no cure for these conditions. However, over the last decade, several groups have been developing retinal prostheses which hopefully will provide some degree of improved visual function to these patients. Several such devices are now in clinical trials. Unfortunately, the possibility of electrode or tissue damage limits excitation schemes to those that may be employed with electrodes that have relatively low charge densities. Further, the excitation thresholds that have been required to achieve vision to date, in general, are relatively high. This may result in part from poor apposition between neurons and the stimulating electrodes and is confounded by the effects of the photoreceptor loss, which initiates other pathology in the surviving retinal tissue. The combination of these and other factors imposes a restriction on the pixel density that can be used for devices that actively deliver electrical stimulation to the retina. The resultant use of devices with relatively low pixel densities presumably will limit the degree of visual resolution that can be obtained with these devices. Further increases in pixel density, and therefore increased visual acuity, will necessitate either improved electrode-tissue biocompatibility or lower stimulation thresholds. To meet this challenge, innovations in materials and devices have been proposed. Here, we review the types of retinal prostheses investigated, the extent of their current biocompatibility and future improvements designed to surmount these limitations.

Neurotrophin-eluting hydrogel coatings for neural stimulating electrodes.

Improved sensory and motor prostheses for the central nervous system will require large numbers of electrodes with low electrical thresholds for neural excitation. With the eventual goal of reducing stimulation thresholds, we have investigated the use of biodegradable, neurotrophin-eluting hydrogels (i.e., poly(ethylene glycol)-poly(lactic acid), PEGPLA) as a means of attracting neurites to the surface of stimulating electrodes. PEGPLA hydrogels with release rates ranging from 1.5 to 3 weeks were synthesized. These hydrogels were applied to multielectrode arrays with sputtered iridium oxide charge-injection sites. The coatings had little impact on the iridium oxide electrochemical properties, including charge storage capacity, impedance, and voltage transients during current pulsing. Additionally, we quantitatively examined the ability of neurotrophin-eluting, PEGPLA hydrogels to promote neurite extension in vitro using a PC12 cell culture model. Hydrogels released neurotrophin (nerve growth factor, NGF) for at least 1 week, with neurite extension near that of an NGF positive control and much higher than extension seen from sham, bovine serum albumin-releasing boluses, and a negative control. These results show that neurotrophin-eluting hydrogels can be applied to multielectrode arrays, and suggest a method to improve neuron-electrode proximity, which could result in lowered electrical stimulation thresholds. Reduced thresholds support the creation of smaller electrode structures and high density electrode prostheses, greatly enhancing prosthesis control and function.