Interaction of micropatterned topographical and biochemical cues to direct neurite growth from spiral ganglion neurons.

Functional outcomes with neural prosthetic devices, such as cochlear implants, are limited in part due to physical separation between the stimulating elements and the neurons they stimulate. One strategy to close this gap aims to precisely guide neurite regeneration to position the neurites in closer proximity to electrode arrays. Here, we explore the ability of micropatterned biochemical and topographic guidance cues, singly and in combination, to direct the growth of spiral ganglion neuron (SGN) neurites, the neurons targeted by cochlear implants. Photopolymerization of methacrylate monomers was used to form unidirectional topographical features of ridges and grooves in addition to multidirectional patterns with 90o angle turns. Microcontact printing was also used to create similar uni- and multi-directional patterns of peptides on polymer surfaces. Biochemical cues included peptides that facilitate (laminin, LN) or repel (EphA4-Fc) neurite growth. On flat surfaces, SGN neurites preferentially grew on LN-coated stripes and avoided EphA4-Fc-coated stripes. LN or EphA4-Fc was selectively adsorbed onto the ridges or grooves to test the neurite response to a combination of topographical and biochemical cues. Coating the ridges with EphA4-Fc and grooves with LN lead to enhanced SGN alignment to topographical patterns. Conversely, EphA4-Fc coating on the grooves or LN coating on the ridges tended to disrupt alignment to topographical patterns. SGN neurites respond to combinations of topographical and biochemical cues and surface patterning that leverages both cues enhance guided neurite growth.

Photograftable Zwitterionic Coatings Prevent Staphylococcus aureus and Staphylococcus epidermidis Adhesion to PDMS Surfaces.

Due to its attractive mechanical properties and biocompatibility, poly(dimethyl)siloxane (PDMS) is widely used in the fabrication of biomedical materials. On the other hand, PDMS is also prone to adsorption of both proteins and bacteria, making PDMS implants susceptible to infection. Herein, we examine the use of durably cross-linked zwitterionic coatings for PDMS surfaces to mitigate bacterial adhesion. Using a single-step photografting technique, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA) thin films were covalently attached to PDMS substrates. The abilities of these coatings to resist the adhesion of Staphylococcus aureus and Staphylococcus epidermidis were tested in vitro under both wet and droplet conditions, as well as in subcutaneous and transcutaneous implantation models using Sprague-Dawley rats. Zwitterionic thin films effectively reduced bacterial adhesion in both in vitro and in vivo conditions. This was particularly true for pCBMA-coated materials, which exhibited significant reduction in bacterial adhesion and growth with respect to S. aureus and S. epidermidis for all in vitro conditions as well as the ability to resist bacterial growth on PDMS implants. The results of this study suggest that a simple and durable photografting process can be used to produce polymer thin films capable of preventing infection of implantable medical devices.

Antifouling Photograftable Zwitterionic Coatings on PDMS Substrates.

The foreign body response (FBR) to implantable materials can negatively impact performance of medical devices such as the cochlear implant. Engineering surfaces that resist the FBR could lead to enhanced functionality including potentially improving outcomes for cochlear implant recipients through reduction in fibrosis. In this work, we coat poly(dimethylsiloxane) (PDMS) surfaces with two zwitterionic polymers, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA), using a simultaneous photografting/photo-cross-linking process to produce a robust grafted zwitterionic hydrogel. reduce nonspecific protein adsorption, the first step of the FBR. The coating process uses benzophenone, a photografting agent and type II photoinitiator, to covalently link the cross-linked zwitterionic thin film to the PDMS surface. As the concentration of benzophenone on the surface increases, the adhesive strength of the zwitterionic thin films to PDMS surfaces increases as determined by shear adhesion. Additionally, with increased concentration of the adsorbed benzophenone, failure of the system changes from adhesive delamination to cohesive failure within the hydrogel, demonstrating that durable adhesive bonds are formed from the photografting process. Interestingly, antifouling properties of the zwitterionic polymers are preserved with significantly lower levels of nonspecific protein adsorption on zwitterion hydrogel-coated samples compared to uncoated controls. Fibroblast adhesion is also dramatically reduced on coated substrates. These results show that cross-linked pSBMA and pCBMA hydrogels can be readily photografted to PDMS substrates and show promise in potentially changing the fibrotic response to implanted biomaterials.

Photopolymerized micropatterns with high feature frequencies overcome chemorepulsive borders to direct neurite growth.

Developing and regenerating neurites respond to a variety of biophysical and biochemical cues in their micro-environment to reach target cells and establish appropriate synapses. Defining the hierarchal relationship of both types of cues to direct neurite growth carries broad significance for neural development, regeneration, and, in particular, engineering of neural prostheses that improve tissue integration with native neural networks. In this work, chemorepulsive biochemical borders are established on substrates with a range of surface microfeatures to determine the potential of physical cues to overcome conflicting biochemical cues. Physical micropatterns are fabricated using photomasking techniques to spatially control photoinitiation events of the polymerization. Temporal control of the reaction allows for generation of microfeatures with the same amplitude across a range of feature frequencies or periodicities. The micropatterned substrates are then modified with repulsive chemical borders between laminin and either EphA4-Fc or tenascin C that compete with the surface microfeatures to direct neurite growth. Behaviour of neurites from spiral ganglion and trigeminal neurons is characterized at biochemical borders as cross, turn, stop, or repel events. Both the chemical borders and physical patterns significantly influence neurite pathfinding. On unpatterned surfaces, most neurites that originate on laminin are deterred by the border with tenascin C or EphA4-Fc. Importantly, substrates with frequent micropattern features overcome the influence of the chemorepulsive border to dominate neurite trajectory. Designing prosthesis interfaces with appropriate surface features may allow for spatially organized neurite outgrowth in vivo even in the presence of conflicting biochemical cues in native target tissues.

Photopolymerized Microfeatures Guide Adult Spiral Ganglion and Dorsal Root Ganglion Neurite Growth.

HYPOTHESIS: Microtopographical patterns generated by photopolymerization of methacrylate polymer systems will direct growth of neurites from adult neurons, including spiral ganglion neurons (SGNs). BACKGROUND: Cochlear implants (CIs) provide hearing perception to patients with severe to profound hearing loss. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by spread of the electrical currents in the inner ear. Directing the regrowth of SGN peripheral processes towards stimulating electrodes could help reduce current spread and improve spatial resolution provided by the CI. Previous work has demonstrated that micro- and nano-scale patterned surfaces precisely guide the growth of neurites from a variety of neonatal neurons including SGNs. Here, we sought to determine the extent to which adult neurons likewise respond to these topographical surface features. METHODS: Photopolymerization was used to fabricate methacrylate polymer substrates with micropatterned surfaces of varying amplitudes and periodicities. Dissociated adult dorsal root ganglion neurons (DRGNs) and SGNs were cultured on these surfaces and the alignment of the neurite processes to the micropatterns was determined. RESULTS: Neurites from both adult DRGNs and SGNs significantly aligned to the patterned surfaces similar to their neonatal counterparts. Further DRGN and SGN neurite alignment increased as the amplitude of the microfeatures increased. Decreased pattern periodicity also improved neurite alignment. CONCLUSION: Microscale surface topographic features direct the growth of adult SGN neurites. Topographical features could prove useful for guiding growth of SGN peripheral axons towards a CI electrode array.

Tuning Surface and Topographical Features to Investigate Competitive Guidance of Spiral Ganglion Neurons.

Cochlear Implants (CIs) suffer from limited tonal resolution due, in large part, to spatial separation between stimulating electrode arrays and primary neural receptors. In this work, a combination of physical and chemical micropatterns, formed on acrylate polymers, are used to direct the growth of primary spiral ganglion neurons (SGNs), the inner ear neurons. Utilizing the inherent temporal and spatial control of photopolymerization, physical microgrooves are fabricated using a photomask in a single step process. Biochemical patterns are generated by adsorbing laminin, a cell adhesion protein, to acrylate polymer surfaces followed by irradiation through a photomask with UV light to deactivate protein in exposed areas and generate parallel biochemical patterns. Laminin deactivation was shown increase as a function of UV light exposure while remaining adsorbed to the polymer surface. SGN neurites show alignment to both biochemical and physical patterns when evaluated individually. Competing biochemical and physical patterns were also examined. The relative guiding strength of physical cues was varied by independently changing both the amplitude and the band spacing of the microgrooves, with higher amplitudes and shorter band spacing providing cues that more effective guide neurite growth. SGN neurites aligned to laminin patterns with lower physical pattern amplitude and thus weaker physical cues. Alignment of SGNs shifted toward the physical pattern with higher amplitude and lower periodicity patterns which represent stronger cues. These results demonstrate the ability of photopolymerized microfeatures to modulate alignment of inner ear neurites even in the presence of conflicting physical and biochemical cues laying the groundwork for next generation cochlear implants and neural prosthetic devices.

Photopolymerizable Zwitterionic Polymer Patterns Control Cell Adhesion and Guide Neural Growth.

Developing materials that reduce or eliminate fibrosis encapsulation of neural prosthetic implants could significantly enhance implant fidelity by improving the tissue/electrode array interface. Here, we report on the photografting and patterning of two zwitterionic materials, sulfobetaine methacrylate (SBMA) and carboxybetaine methacrylate (CBMA), for controlling the adhesion and directionality of cells relevant to neural prosthetics. CBMA and SBMA polymers were photopolymerized and grafted on glass surfaces then characterized by X-ray photoelectron spectroscopy, water contact angle, and protein adsorption. Micropatterned surfaces were fabricated with alternating zwitterionic and uncoated bands. Fibroblasts, cells prevalent in fibrotic tissue, almost exclusively migrate and grow on uncoated bands with little to no cells present on zwitterionic bands, especially for CBMA-coated surfaces. Astrocytes and Schwann cells showed similarly low levels of cell adhesion and morphology changes when cultured on zwitterionic surfaces. Additionally, Schwann cells and inner ear spiral ganglion neuron neurites aligned well to zwitterionic patterns.

Quantifying Spiral Ganglion Neurite and Schwann Behavior on Micropatterned Polymer Substrates.

The first successful in vitro experiments on the cochlea were conducted in 1928 by Honor Fell (Fell, Arch Exp Zellforsch 7(1):69-81, 1928). Since then, techniques for culture of this tissue have been refined, and dissociated primary culture of the spiral ganglion has become a widely accepted in vitro model for studying nerve damage and regeneration in the cochlea. Additionally, patterned substrates have been developed that facilitate and direct neural outgrowth. A number of automated and semi-automated methods for quantifying this neurite outgrowth have been utilized in recent years (Zhang et al., J Neurosci Methods 160(1):149-162, 2007; Tapias et al., Neurobiol Dis 54:158-168, 2013). Here, we describe a method to study the effect of topographical cues on spiral ganglion neurite and Schwann cell alignment. We discuss our microfabrication process, characterization of pattern features, cell culture techniques for both spiral ganglion neurons and spiral ganglion Schwann cells. In addition, we describe protocols for reducing fibroblast count, immunocytochemistry, and methods for quantifying neurite and Schwann cell alignment.

Material stiffness effects on neurite alignment to photopolymerized micropatterns.

The ability to direct neurite growth into a close proximity of stimulating elements of a neural prosthesis, such as a retinal or cochlear implant (CI), may enhance device performance and overcome current spatial signal resolution barriers. In this work, spiral ganglion neurons (SGNs), which are the target neurons to be stimulated by CIs, were cultured on photopolymerized micropatterns with varied matrix stiffnesses to determine the effect of rigidity on neurite alignment to physical cues. Micropatterns were generated on methacrylate thin film surfaces in a simple, rapid photopolymerization step by photomasking the prepolymer formulation with parallel line-space gratings. Two methacrylate series, a nonpolar HMA-co-HDDMA series and a polar PEGDMA-co-EGDMA series, with significantly different surface wetting properties were evaluated. Equivalent pattern periodicity was maintained across each methacrylate series based on photomask band spacing, and the feature amplitude was tuned to a depth of 2 µm amplitude for all compositions using the temporal control afforded by the UV curing methodology. The surface morphology was characterized by scanning electron microscopy and white light interferometry. All micropatterned films adsorb similar amounts of laminin from solution, and no significant difference in SGN survival was observed when the substrate compositions were compared. SGN neurite alignment significantly increases with increasing material modulus for both methacrylate series. Interestingly, SGN neurites respond to material stiffness cues that are orders of magnitude higher (GPa) than what is typically ascribed to neural environments (kPa). The ability to understand neurite response to engineered physical cues and mechanical properties such as matrix stiffness will allow the development of advanced biomaterials that direct de novo neurite growth to address the spatial signal resolution limitations of current neural prosthetics.