Visible Light Activation of Nucleophilic Thiol-X Addition via Thioether Bimane Photocleavage for Polymer Cross-Linking.

On-demand photo-uncaging of reactive thiols have been employed in engineering biomaterial scaffolds for regulation of cellular activities. A drawback of the current photo-uncaging chemistry is the utilization of high energy UV light or 2-photon laser light, which may be harmful to cells and cause undesired side reactions within the biological environment. We introduce an effective approach for the caging of thiol using monobromobimane, which can be removed under irradiation of light at λ = 420 nm and in the presence of electrophiles, such as acrylate, propiolate and maleimide, for trapping of the newly release thiol. This chemical approach can be used in visible light-induced polymer coupling and cross-linking for the preparation of cell-laden hydrogels.

Nonswelling Click-Cross-Linked Gelatin and PEG Hydrogels with Tunable Properties Using Pluronic Linkers.

Swelling of hydrogels leads to a decrease in mechanical performance coupled with complications in solute diffusion. In addition, hydrogel swelling affects patient safety in biomedical applications such as compression of tissue and fluid blockage. A conventional strategy for suppressing swelling is to introduce a thermoresponsive polymer with a lower critical solution temperature (LCST) within the network structure to counter the water uptake at elevated temperature. However, altering the gel's mechanical strength via modification of the network structure often affects the water uptake behavior and thus a nonswelling platform with tunable mechanical properties suitable for various biomedical applications is desirable. In this study we applied the commercially available triblock PEG-PPG-PEG (Pluronic) as a cross-linker for the preparation of nucleophilic thiol-yne click cross-linked hydrogels with suppressed swelling at physiologically relevant temperature. The mechanical properties and degradation rate of these nonswelling hydrogels can be tuned by judicious combinations of the available linkers. The Pluronic linkers can be applied to prepare biologically relevant gelatin based hydrogels with suppressed swelling under physiological conditions that support attachment of fibroblast cells in 2D culture and controlled release of albumin, paving the way for the development of reliable and better performing soft biomaterials.

Low Fouling Electrospun Scaffolds with Clicked Bioactive Peptides for Specific Cell Attachment.

While electrospun fibers are of interest as scaffolds for tissue engineering applications, nonspecific surface interactions such as protein adsorption often prevent researchers from controlling the exact interactions between cells and the underlying material. In this study we prepared electrospun fibers from a polystyrene-based macroinitiator, which were then grafted with polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). These brush coatings incorporated a trimethylsilyl-protected PEG-alkyne monomer, allowing azide functional molecules to be covalently attached, while simultaneously reducing nonspecific protein adsorption on the fibers. Cells were able to attach and spread on fibrous substrates functionalized with a pendant RGD-containing peptide, while spreading was significantly reduced on nonfunctionalized fibers and those with the equivalent RGE control peptide. This effect was observed both in the presence and absence of serum in the culture media, indicating that protein adsorption on the fibers was minimal and cell adhesion within the fibrous scaffold was mediated almost entirely through the cell-adhesive RGD-containing peptide.

3D Electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes.

Astrocytes are a target for regenerative neurobiology because in brain injury their phenotype arbitrates brain integrity, neuronal death and subsequent repair and reconstruction. We explored the ability of 3D scaffolds to direct astrocytes into phenotypes with the potential to support neuronal survival. Poly-ε-caprolactone scaffolds were electrospun with random and aligned fibre orientations on which murine astrocytes were sub-cultured and analysed at 4 and 12 DIV. Astrocytes survived, proliferated and migrated into scaffolds adopting 3D morphologies, mimicking in vivo stellated phenotypes. Cells on random poly-ε-caprolactone scaffolds grew as circular colonies extending processes deep within sub-micron fibres, whereas astrocytes on aligned scaffolds exhibited rectangular colonies with processes following not only the direction of fibre alignment but also penetrating the scaffold. Cell viability was maintained over 12 DIV, and cytochemistry for F-/G-actin showed fewer stress fibres on bioscaffolds relative to 2D astrocytes. Reduced cytoskeletal stress was confirmed by the decreased expression of glial fibrillary acidic protein. PCR demonstrated up-regulation of genes (excitatory amino acid transporter 2, brain-derived neurotrophic factor and anti-oxidant) reflecting healthy biologies of mature astrocytes in our extended culture protocol. This study illustrates the therapeutic potential of bioengineering strategies using 3D electrospun scaffolds which direct astrocytes into phenotypes supporting brain repair. Astrocytes exist in phenotypes with pro-survival and destructive components, and their biology can be modulated by changing phenotype. Our findings demonstrate murine astrocytes adopt a healthy phenotype when cultured in 3D. Astrocytes proliferate and extend into poly-ε-caprolactone scaffolds displaying 3D stellated morphologies with reduced GFAP expression and actin stress fibres, plus a cytotrophic gene profile. Bioengineered 3D scaffolds have potential to direct inflammation to aid regenerative neurobiology.

Nanotopographic surfaces with defined surface chemistries from amyloid fibril networks can control cell attachment.

We show for the first time the possibility of using networks of amyloid fibrils, adsorbed to solid supports and with plasma polymer coatings, for the fabrication of chemically homogeneous surfaces with well-defined nanoscale surface features reminiscent of the topography of the extracellular matrix. The robust nature of the fibrils allows them to withstand the plasma polymer deposition conditions used with no obvious deleterious effect, thus enabling the underlying fibril topography to be replicated at the polymer surface. This effect was seen despite the polymer coating thickness being an order of magnitude greater than the fibril network. The in vitro culture of fibroblast cells on these surfaces resulted in increased attachment and spreading compared to flat plasma polymer films with the same chemical composition. The demonstrated technique allows for the rapid and reproducible fabrication of substrates with nanoscale fibrous topography that we believe will have applications in the development of new biomaterials allowing, for example, the investigation of the effect of extracellular matrix mimicking nanoscale morphology on cellular phenotype.

3D Functional Neuronal Networks in Free-Standing Bioprinted Hydrogel Constructs.

The composition, elasticity, and organization of the extracellular matrix within the central nervous system contribute to the architecture and function of the brain. From an in vitro modeling perspective, soft biomaterials are needed to mimic the 3D neural microenvironments. While many studies have investigated 3D culture and neural network formation in bulk hydrogel systems, these approaches have limited ability to position cells to mimic sophisticated brain architectures. In this study, cortical neurons and astrocytes acutely isolated from the brains of rats are bioprinted in a hydrogel to form 3D neuronal constructs. Successful bioprinting of cellular and acellular strands in a multi-bioink approach allows the subsequent formation of gray- and white-matter tracts reminiscent of cortical structures. Immunohistochemistry shows the formation of dense, 3D axon networks. Calcium signaling and extracellular electrophysiology in these 3D neuronal networks confirm spontaneous activity in addition to evoked activities under pharmacological and electrical stimulation. The system and bioprinting approaches are capable of fabricating soft, free-standing neuronal structures of different bioink and cell types with high resolution and throughput, which provide a promising platform for understanding fundamental questions of neural networks, engineering neuromorphic circuits, and for in vitro drug screening.

Cell Microencapsulation within Gelatin-PEG Microgels Using a Simple Pipet Tip-Based Device.

Microgels are microscale particles of hydrogel that can be laden with cells and used to create macroporous tissue constructs. Their ability to support cell-ECM and cell-cell interactions, along with the high levels of nutrient and metabolite exchange facilitated by their high surface area-to-volume ratio, means that they are attracting increasing attention for a variety of tissue regeneration applications. Here, we present methods for fabricating and modifying the structure of microfluidic devices using commonly available laboratory consumables including pipet tips and PTFE and silicon tubing to produce microgels. Different microfluidic devices realized the controlled generation of a wide size range (130-800 µm) of microgels for cell encapsulation. Subsequently, we describe the process of encapsulating mesenchymal stromal cells in microgels formed by photo-cross-linking of gelatin-norbornene and PEG dithiol. The introduced pipet-based chip offers simplicity, tunability, and versatility, making it easily assembled in most laboratories to effectively produce cell-laden microgels for various applications in tissue engineering.

Cell-laden injectable microgels: Current status and future prospects for cartilage regeneration.

Injectable hydrogels have been employed extensively as versatile materials for cartilage regeneration due to their excellent biocompatibility, tunable structure, and ability to accommodate bioactive factors, as well as their ability to be locally delivered via minimally invasive injection to fill irregular defects. More recently, in vitro and in vivo studies have revealed that processing these materials to produce cell-laden microgels can enhance cell-cell and cell-matrix interactions and boost nutrient and metabolite exchange. Moreover, these studies have demonstrated gene expression profiles and matrix regeneration that are superior compared to conventional injectable bulk hydrogels. As cell-laden microgels and their application in cartilage repair are moving closer to clinical translation, this review aims to present an overview of the recent developments in this field. Here we focus on the currently used biomaterials and crosslinking strategies, the innovative fabrication techniques being used for the production of microgels, the cell sources used, the signals used for induction of chondrogenic differentiation and the resultant biological responses, and the ability to create three-dimensional, functional cartilage tissues. In addition, this review also covers the current clinical approaches for repairing cartilage as well as specific challenges faced when attempting the regeneration of damaged cartilage tissue. New findings related to the macroporous nature of the structures formed by the assembled microgel building blocks and the novel use of microgels in 3D printing for cartilage tissue engineering are also highlighted. Finally, we outline the challenges and future opportunities for employing cell-laden microgels in clinical applications.

Biomaterial Strategies for Restorative Therapies in Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurological disorder, in which dopaminergic midbrain neurons degenerate, leading to dopamine depletion that is associated with neuronal death. In this Review, we initially describe the pathogenesis of PD and established therapies that unfortunately only delay progression of the disease. With a rapidly escalating incidence in PD, there is an urgent need to develop new therapies that not only halt progression but even reverse degeneration. Biomaterials are playing critical roles in these new therapies which include controlled and site-specific delivery of neurotrophins, increased engraftment of implanted neural stem cells, and redirection of endogenous stem cell populations away from their niche to encourage reparative mechanisms. This Review will therefore cover important design features of biomaterials used in regenerative medicine and tissue engineering strategies targeted at PD.

One-step method for generating PEG-like plasma polymer gradients: chemical characterization and analysis of protein interactions.

In this work we report a one-step method for the fabrication of poly(ethylene glycol) PEG-like chemical gradients, which were deposited via continuous wave radio frequency glow discharge plasma polymerization of diethylene glycol dimethyl ether (DG). A knife edge top electrode was used to produce the gradient coatings at plasma load powers of 5 and 30 W. The chemistry across the gradients was analyzed using a number of complementary techniques including spatially resolved synchrotron source grazing incidence FTIR microspectroscopy, X-ray photoelectron spectroscopy (XPS) and synchrotron source near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Gradients deposited at lower load power retained a higher degree of monomer like functionality as did the central region directly underneath the knife edge electrode of each gradient film. Surface derivatization experiments were employed to investigate the concentration of residual ether units in the films. In addition, surface derivatization was used to investigate the reactivity of the gradient films toward primary amine groups in a graft copolymer of poly (L-lysine) and poly(ethylene glycol) (PLL-g-PEG copolymer) which was correlated to residual aldehyde, ketone and carboxylic acid functionalities within the films. The protein adsorption characteristics of the gradients were analyzed using three proteins of varying size and charge. Protein adsorption varied and was dependent on the chemistry and the physical properties (such as size and charge) of the proteins. A correlation between the concentration of ether functionality and the protein fouling characteristics along the gradient films was observed. The gradient coating technique developed in this work allows for the efficient and high-throughput study of biomaterial gradient coating interactions.

Gelatin-Based 3D Microgels for In Vitro T Lineage Cell Generation.

T cells are predominantly produced by the thymus and play a significant role in maintaining our adaptive immune system. Physiological involution of the thymus occurs gradually with age, compromising naive T cell output, which can have severe clinical complications. Also, T cells are utilized as therapeutic agents in cancer immunotherapies. Therefore, there is an increasing need for strategies aimed at generating naive T cells. The majority of in vitro T cell generation studies are performed in two-dimensional (2D) cultures, which ignore the physiological thymic microenvironment and are not scalable; therefore, we applied a new three-dimensional (3D) approach. Here, we use a gelatin-based 3D microgel system for T lineage induction by co-culturing OP9-DL4 cells and mouse fetal-liver-derived hematopoietic stem cells (HSCs). Flow cytometric analysis revealed that microgel co-cultures supported T lineage induction similar to 2D cultures while providing a 3D environment. We also encapsulated mouse embryonic thymic epithelial cells (TECs) within the microgels to provide a defined 3D culture platform. The microgel system supported TEC maintenance and retained their phenotype. Together, these data show that our microgel system has the capacity for TEC maintenance and induction of in vitro T lineage differentiation with potential for scalability.

Electrochemical and mechanical performance of reduced graphene oxide, conductive hydrogel, and electrodeposited Pt-Ir coated electrodes: an active in vitro study.

OBJECTIVE: To systematically compare the in vitro electrochemical and mechanical properties of several electrode coatings that have been reported to increase the efficacy of medical bionics devices by increasing the amount of charge that can be delivered safely to the target neural tissue. APPROACH: Smooth platinum (Pt) ring and disc electrodes were coated with reduced graphene oxide, conductive hydrogel, or electrodeposited Pt-Ir. Electrodes with coatings were compared with uncoated smooth Pt electrodes before and after an in vitro accelerated aging protocol. The various coatings were compared mechanically using the adhesion-by-tape test. Electrodes were stimulated in saline for 24 hours/day 7 days/week for 21 d at 85 °C (1.6-year equivalence) at a constant charge density of 200 µC/cm2/phase. Electrodes were graded on surface corrosion and trace analysis of Pt in the electrolyte after aging. Electrochemical measurements performed before, during, and after aging included electrochemical impedance spectroscopy, cyclic voltammetry, and charge injection limit and impedance from voltage transient recordings. MAIN RESULTS: All three coatings adhered well to smooth Pt and exhibited electrochemical advantage over smooth Pt electrodes prior to aging. After aging, graphene coated electrodes displayed a stimulation-induced increase in impedance and reduction in the charge injection limit (p  < 0.001), alongside extensive corrosion and release of Pt into the electrolyte. In contrast, both conductive hydrogel and Pt-Ir coated electrodes had smaller impedances and larger charge injection limits than smooth Pt electrodes (p  < 0.001) following aging regardless of the stimulus level and with little evidence of corrosion or Pt dissolution. SIGNIFICANCE: This study rigorously tested the mechanical and electrochemical performance of electrode coatings in vitro and provided suitable candidates for future in vivo testing.

Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells.

Current clinical approaches to treat articular cartilage degeneration provide only a limited ability to regenerate tissue with long-term durability and functionality. In this application, injectable bulk hydrogels and microgels containing stem cells can provide a suitable environment for tissue regeneration. However insufficient cell-cell interactions, low differentiation efficiency and poor tissue adhesion hinder the formation of high-quality hyaline type cartilage. Here, we have designed a higher order tissue-like structure using injectable cell-laden microgels as the building blocks to achieve human bone marrow-derived mesenchymal stem cell (hBMSC) long-term maintenance and chondrogenesis. We have demonstrated that a 4-arm poly(ethylene glycol)-N-hydroxysuccinimide (NHS) crosslinker induces covalent bonding between the microgel building blocks as well as the surrounding tissue mimic. The crosslinking process assembles the microgels into a 3D construct and preserves the viability and cellular functions of the encapsulated hBMSCs. This assembled microgel construct encourages upregulation of chondrogenic markers in both gene and glycosaminoglycan (GAG) expression levels. In addition, the regenerated tissue in the assembled microgels stained positively with Alcian blue and Safranin O exhibiting unique hyaline-like cartilage features. Furthermore, the immunostaining showed a favourable distribution and significantly higher content of type II collagen in the assembled microgels when compared to both the bulk hydrogel and pellet cultures. Collectively, this tissue adhesive hBMSC-laden microgel construct provides potential clinical opportunities for articular cartilage repair and other applications in regenerative medicine. STATEMENT OF SIGNIFICANCE: A reliable approach to reconstruct durable and fully functional articular cartilage tissue is required for effective clinical therapies. Here, injectable hydrogels together with cell-based therapies offer new treatment strategies in cartilage repair. For effective cartilage regeneration, the injectable hydrogel system needs to be bonded to the surrounding tissue and at the same time needs to be sufficiently stable for prolonged chondrogenesis. In this work, we utilised injectable hBMSC-laden microgels as the building blocks to create an assembled construct via N-hydroxysuccinimide-amine coupling. This crosslinking process also allows for rapid bonding between the assembled microgels and a surrounding tissue mimic. The resultant assembled microgel-construct provides both a physically stable and biologically dynamic environment for hBMSC chondrogenesis, leading to the production of a mature hyaline type cartilage structure.

Mouse embryonic stem cell colonisation of carbonated apatite surfaces.

Apatites play a crucial role in the body and have been used extensively in biomedical implants. The influence on stem cell behaviour is not known and so this study will explore whether sintered carbonated apatites are favourable for propagation of stem cells. Different weight substitutions of carbonated apatite, specifically 2.5 wt% (2.5 wt%CAP) and 5 wt% (5 wt%CAP), were sintered and characterised prior to the investigation of their potential as a matrix for the support of mouse embryonic stem (ES) cells. Characterisation of the apatites included elemental analysis, X-ray diffraction, surface roughness, specific surface area, density, and solubility. The ability of carbonated apatite to support mouse ES cell colonisation and maintenance in the presence of leukaemia inhibitory factor was determined by an enumeration of live versus dead cells within a population, and immunoreactivity to Oct4, a transcription factor and stem cell marker, following growth on each matrix. It was found that while both compositions allowed for the colonisation of mouse ES cells, the cells were not maintained in an undifferentiated state, as evidenced by a reduction in the number of cells staining positive for Oct4 expression. This study shows that an increase in carbonate content within sintered apatites leads to a higher cell number, a desired aspect for stem cells to populate scaffolds intended for tissue engineering. This study presents carbonated apatites as a suitable matrix for the initial colonisation and differentiation of ES cells for tissue engineering applications.

In situ-forming click-crosslinked gelatin based hydrogels for 3D culture of thymic epithelial cells.

Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications.

Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain.

Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration.

Cell infiltration into a 3D electrospun fiber and hydrogel hybrid scaffold implanted in the brain.

Tissue engineering scaffolds are often designed without appropriate consideration for the translational potential of the material. Solid scaffolds implanted into central nervous system (CNS) tissue to promote regeneration may require tissue resection to accommodate implantation. Or alternatively, the solid scaffold may be cut or shaped to better fit an irregular injury geometry, but some features of the augmented scaffold may fail to integreate with surrounding tissue reducing regeneration potential. To create a biomaterial able to completely fill the irregular geometry of CNS injury and yet still provide sufficient cell migratory cues, an injectable, hybrid scaffold was created to present the physical architecture of electrospun fibers in an agarose/methylcellulose hydrogel. When injected into the rat striatum, infiltrating macrophages/microglia and resident astrocytes are able to locate the fibers and utilize their cues for migration into the hybrid matrix. Thus, hydrogels containing electrospun fibers may be an appropriate platform to encourage regeneration of the injured brain.

Optimization of Aqueous SI-ATRP Grafting of Poly(Oligo(Ethylene Glycol) Methacrylate) Brushes from Benzyl Chloride Macroinitiator Surfaces.

Poly(oligo(ethylene glycol) methacrylate) (pOEGMA) brushes were grafted via surface-initiated atom transfer radical polymerization (SI-ATRP) from a poly(styrene-co-vinylbenzyl chloride) macroinitiator. While bromoisobutyryl initiator groups are most commonly used for this purpose, benzyl chloride initiators may be advantageous for some applications due to superior stability. Water-only graft solutions produced thicker brush coatings with superior low fouling properties (low protein adsorption and cell adhesion) versus mixed water/alcohol solutions. Coatings produced using 475 Da OEGMA (methyl ether terminated) further reduced non-specific interactions compared to 360 Da OEGMA (hydroxyl terminated). Initiator density had minimal effect on low fouling properties.

3D presentation of a neurotrophic factor for the regulation of neural progenitor cells.

BACKGROUND: Adequate cell-scaffold interactions and neurotrophin support are essential factors for neural regeneration. AIM: To provide insight into the biofunctionalization of complex 3D scaffolds with nanoscale precision, as well as the effect of spatial distribution of brain-derived neurotrophic factor (BDNF) and its prolonged stimulation in combination with enhanced cell affinity of nanofibrous scaffolds on the survival/proliferation and neurite outgrowth. METHODS & MATERIALS: We developed a versatile approach using layer-by-layer self-assembly to incorporate cell adhesion and spatial representation of neurotrophic factors into complex nanofibrous scaffolds. RESULTS: Heparin/poly-L-lysine (PLL) polyelectrolyte multilayers (PEMs) were deposited on electrospun poly-ε-caprolatone nanofibers. Well-controlled amounts of BDNF were immobilized on the PEM-modified nanofibers. In addition, longer neurite outgrowth was observed in neural progenitor cells cultured on PLL-terminating PEM scaffolds. The immobilized BDNF on PLL-terminated PEM scaffolds resulted in significantly longer neurites and higher cell numbers (p < 0.01) compared with BDNF-free and BDNF-adsorbed PLL-terminating scaffolds. Interestingly, there was no upregulation of TrkB-FL, TrkB-T1 or GAP-43 mRNAs with immobilized BDNF in day 5 cultures. DISCUSSION & CONCLUSION: This work reinforces the importance of the combinatorial effects of biomaterial scaffold nanostructure and spatial presentation of neurotrophins in directing neural progenitor cell fates.

Neuronal electrophysiological function and control of neurite outgrowth on electrospun polymer nanofibers are cell type dependent.

Modeling of cellular environments with nanofabricated biomaterial scaffolds has the potential to improve the growth and functional development of cultured cellular models, as well as assist in tissue engineering efforts. An understanding of how such substrates may alter cellular function is critical. Highly plastic central nervous system hippocampal cells and non-network forming peripheral nervous system dorsal root ganglion (DRG) cells from embryonic rats were cultured upon laminin-coated degradable polycaprolactone (PCL) and nondegradable polystyrene (PS) electrospun nanofibrous scaffolds with fiber diameters similar to those of neuronal processes. The two cell types displayed intrinsically different growth patterns on the nanofibrous scaffolds. Hippocampal neurites grew both parallel and perpendicular to the nanofibers, a property that would increase neurite-to-neurite contacts and maximize potential synapse development, essential for extensive network formation in a highly plastic cell type. In contrast, non-network-forming DRG neurons grew neurites exclusively along fibers, recapitulating the simple direct unbranching pathway between sensory ending and synapse in the spinal cord that occurs in vivo. In addition, the two primary neuronal types showed different functional capacities under patch clamp testing. The substrate composition did not alter the neuronal functional development, supporting electrospun PCL and PS as candidate materials for controlled cellular environments in culture and electrospun PCL for directed neurite outgrowth in tissue engineering applications.