Examining thein vivofunctionality of the magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI).

Objective. Although neural-enabled prostheses have been used to restore some lost functionality in clinical trials, they have faced difficulty in achieving high degree of freedom, natural use compared to healthy limbs. This study investigated thein vivofunctionality of a flexible and scalable regenerative peripheral-nerve interface suspended within a microchannel-embedded, tissue-engineered hydrogel (the magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI)) as a potential approach to improving current issues in peripheral nerve interfaces.Approach. Assembled MARTEENI devices were implanted in the gaps of severed sciatic nerves in Lewis rats. Both acute and chronic electrophysiology were recorded, and channel-isolated activity was examined. In terminal experiments, evoked activity during paw compression and stimulus response curves generated from proximal nerve stimulation were examined. Electrochemical impedance spectroscopy was performed to assess the complex impedance of recording sites during chronic data collection. Features of the foreign-body response (FBR) in non-functional implants were examined using immunohistological methods.Main results. Channel-isolated activity was observed in acute, chronic, and terminal experiments and showed a typically biphasic morphology with peak-to-peak amplitudes varying between 50 and 500µV. For chronic experiments, electrophysiology was observed for 77 days post-implant. Within the templated hydrogel, regenerating axons formed minifascicles that varied in both size and axon count and were also found to surround device threads. No axons were found to penetrate the FBR. Together these results suggest the MARTEENI is a promising approach for interfacing with peripheral nerves.Significance. Findings demonstrate a high likelihood that observed electrophysiological activity recorded from implanted MARTEENIs originated from neural tissue. The variation in minifascicle size seen histologically suggests that amplitude distributions observed in functional MARTEENIs may be due to a combination of individual axon and mini-compound action potentials. This study provided an assessment of a functional MARTEENI in anin vivoanimal model for the first time.

The Materials Science Foundation Supporting the Microfabrication of Reliable Polyimide-Metal Neuroelectronic Interfaces.

Thin-film polyimide-metal neuroelectronic interfaces hold the potential to alleviate many neurological disorders. However, their long-term reliability is challenged by an aggressive implant environment that causes delamination and degradation of critical materials, resulting in a degradation or complete loss of implant function. Herein, a rigorous and in-depth analysis is presented on the fabrication and modification of critical materials in these thin-film neural interfaces. Special attention is given to improving the interfacial adhesion between thin films and processing modifications to maximize device reliability. Fundamental material analyses are performed on the polyimide substrate and adhesion-promotion candidates, including amorphous silicon carbide (a-SiC:H), amorphous carbon, and silane coupling agents. Basic fabrication rules are identified to markedly improve polyimide self-adhesion, including optimizing the polyimide-cure profile and maximizing high-energy surface activation. In general, oxide-forming materials are identified as poor adhesive aids to polyimide without targeted modifications. Methods are identified to incorporate effective a-SiC:H interfacial layers to improve metal adherence to polyimide, in addition to examples of alloying between adjacent material layers that can impact the trace resistivity and long-term reliability of the thin-film interfaces. The provided rationale and consequences of key decisions made should promote more reproducible science using robust and reliable neuroelectronic technology.

Development of a magnetically aligned regenerative tissue-engineered electronic nerve interface for peripheral nerve applications.

Peripheral nerve injuries can be debilitating to motor and sensory function, with severe cases often resulting in complete limb amputation. Over the past two decades, prosthetic limb technology has rapidly advanced to provide users with crude motor control of up to 20° of freedom; however, the nerve-interfacing technology required to provide high movement selectivity has not progressed at the same rate. The work presented here focuses on the development of a magnetically aligned regenerative tissue-engineered electronic nerve interface (MARTEENI) that combines polyimide "threads" encapsulated within a magnetically aligned hydrogel scaffold. The technology exploits tissue-engineered strategies to address concerns over traditional peripheral nerve interfaces including poor axonal sampling through the nerve and rigid substrates. A magnetically templated hydrogel is used to physically support the polyimide threads while also promoting regeneration in close proximity to the electrode sites on the polyimide. This work demonstrates the utility of magnetic templating for use in tuning the mechanical properties of hydrogel scaffolds to match the stiffness of native nerve tissue while providing an aligned substrate for Schwann cell migration in vitro. MARTEENI devices were fabricated and implanted within a 5-mm-long rat sciatic-nerve transection model to assess regeneration at 6 and 12 weeks. MARTEENI devices do not disrupt tissue remodeling and show axon densities equivalent to fresh tissue controls around the polyimide substrates. Devices are observed to have attenuated foreign-body responses around the polyimide threads. It is expected that future studies with functional MARTEENI devices will be able to record and stimulate single axons with high selectivity and low stimulation regimes.

Anti-biofouling properties of poly(dimethyl siloxane) with RAFT photopolymerized acrylate/methacrylate surface grafts against model marine organisms.

Biofouling of man-made surfaces by marine organisms is a global problem with both financial and environmental consequences. However, the development of non-toxic anti-biofouling coatings is challenged by the diversity of fouling organisms. One possible solution leverages coatings composed of diverse chemical constituents. Reversible addition-fragmentation chain-transfer (RAFT) photopolymerization was used to modify poly(dimethylsiloxane) (PDMSe) surfaces with polymeric grafts composed of three successive combinations of acrylamide, acrylic acid, and hydroxyethyl methacrylate. RAFT limited conflicting variables and allowed for the effect of graft chemistry to be isolated. While all compositions enhanced the anti-biofouling performance compared with the PDMSe control, the ternary, amphiphilic copolymer was the most effective with 98% inhibition of the attachment of zoospores of the green alga Ulva linza, 94% removal of cells of the diatom Navicula incerta, and 62% removal of cells of the bacterium Cellulophaga lytica. However, none of the graft compositions tested were able to mitigate reattachment of adult barnacles, Amphibalanus amphitrite.

Microtopographical patterns promote different responses in fibroblasts and Schwann cells: A possible feature for neural implants.

The chronic reliability of bioelectronic neural interfaces has been challenged by foreign body reactions (FBRs) resulting in fibrotic encapsulation and poor integration with neural tissue. Engineered microtopographies could alleviate these challenges by manipulating cellular responses to the implanted device. Parallel microchannels have been shown to modulate neuronal cell alignment and axonal growth, and Sharklet™ microtopographies of targeted feature sizes can modulate bio-adhesion of an array of bacteria, marine organisms, and epithelial cells due to their unique geometry. We hypothesized that a Sharklet™ micropattern could be identified that inhibited fibroblasts partially responsible for FBR while promoting Schwann cell proliferation and alignment. in vitro cell assays were used to screen the effect of Sharklet™ and channel micropatterns of varying dimensions from 2 to 20 µm on fibroblast and Schwann cell metrics (e.g., morphology/alignment, nuclei count, metabolic activity), and a hierarchical analysis of variance was used to compare treatments. In general, Schwann cells were found to be more metabolically active and aligned than fibroblasts when compared between the same pattern. 20 µm wide channels spaced 2 µm apart were found to promote Schwann cell attachment and alignment while simultaneously inhibiting fibroblasts and warrant further in vivo study on neural interface devices. No statistically significant trends between cellular responses and geometrical parameters were identified because mammalian cells can change their morphology dependent on their environment in a manner dissimilar to bacteria. Our results showed although surface patterning is a strong physical tool for modulating cell behavior, responses to micropatterns are highly dependent on the cell type.

Integration of flexible polyimide arrays into soft extracellular matrix-based hydrogel materials for a tissue-engineered electronic nerve interface (TEENI).

BACKGROUND: Biomimetic hydrogels used in tissue engineering can improve tissue regeneration and enable targeted cellular behavior; there is growing interest in combining hydrogels with microelectronics to create new neural interface platforms to help patient populations. However, effective processes must be developed to integrate flexible but relatively stiff (e.g., 1-10 GPa) microelectronic arrays within soft (e.g., 1-10 kPa) hydrogels. NEW METHOD: Here, a novel method for integrating polyimide microelectrode arrays within a biomimetic hydrogel scaffold is demonstrated for use as a tissue-engineered electronic nerve interface (TEENI). Tygon tubing and a series of 3D printed molds were used to facilitate hydrogel fabrication and device assembly. COMPARISON WITH EXISTING METHODS: Other comparable regenerative peripheral nerve interface technologies do not utilize the flexible microelectrode array design nor the hydrogel scaffold described here. These methods typically use stiff electrode arrays that are affixed to a similarly stiff implantable tube serving as the nerve guidance conduit. RESULTS: Our results indicate that there is a substantial mechanical mismatch between the flexible microelectronic arrays and the soft hydrogel. However, using the methods described here, there is consistent fabrication of these regenerative peripheral nerve interfaces suitable for implantation. CONCLUSIONS: The assembly process that was developed resulted in repeatable and consistent integration of microelectrode arrays within a soft tissue-engineered hydrogel. As reported elsewhere, these devices have been successfully implanted in a rat sciatic nerve model and yielded neural recordings. This process can be adapted for other applications and hydrogels in which flexible electronic materials are combined with soft regenerative scaffolds.

In situ flow cell platform for examining calcium oxalate and calcium phosphate crystallization on films of basement membrane extract in the presence of urinary 'inhibitors'.

A significant portion of the population suffers from idipoathic calcium oxalate (CaOx) kidney stones, and current clinical treatments of stones have limited lasting success with a high rate of patients suffering from reoccurring stones. Understanding the role of physiologically relevant urinary species on the formation, aggregation, and growth of CaOx crystals can allow for better understanding of this complex biomineralization process and lead to more effective clinical treatments. Our prior work has focused on developing a two-stage model system, where the first stage emulates the formation of Randall's plaque, and the second stage examines the influence of the plaque on overgrowth of CaOx into a stone. Herein, we report on the development of an easy-to-use flow-cell platform that utilizes basement membrane extract (BME) as a biologically relevant crystallization substrate to study the influence of urinary 'inhibitors' on the in situ formation and growth of CaOx on BME under flow conditions. Magnesium, citrate, and osteopontin were studied because of their known ability to inhibit CaOx formation, but their influence also led to interesting modifications to the terminal crystal habit. Magnesium had little to no effect on the CaOx crystallization, but both citrate and osteopontin resulted in significant changes to the crystallization kinetics and the terminal crystal habits. Triply inhibited artificial urine solutions resulted in CaOx monohydrate formations that resembled physiological stones, and the in situ platform allowed for morphogenesis to be dynamically monitored. The BME was also used in a two-stage model system to first grow CaP that mimicked Randall's plaques, whereby the impact of the CaP crystallizing surface on CaOx formation could be studied. It was found that the CaP surface did not result in any significant changes in CaOx crystal formation or growth indicating that the urinary inhibitors and the basement membrane substrate were the dominant factors in modulating CaOx crystallization. It was also found that the basement membrane surface promoted the attachment and/or nucleation and growth of both CaOx and CaP crystals compared to bare glass surfaces, thereby enabling easy study of the urinary inhibitors. The work presented here has elucidated the terminal growth habit of different COM structures and has provided an easy to use platform that can be widely adopted by the kidney stone and other crystallization communities.

Engineered Chemical Nanotopographies: Reversible Addition-Fragmentation Chain-Transfer Mediated Grafting of Anisotropic Poly(acrylamide) Patterns on Poly(dimethylsiloxane) To Modulate Marine Biofouling.

Effectively negating the deleterious impact of marine biofouling on the world's maritime fleet in an environmentally conscientious manner presents a difficult challenge due to a variety of factors including the complexity and diversity of fouling species and the differing surface adhesion strategies. Understanding how surface properties relate to biofouling can inform and guide the development of new antibiofouling coatings to address this challenge. Herein, we report on the development of a living photopolymerization strategy used to tailor the surface properties of silicone rubber using controlled anisotropic poly(acrylamide) patterns and the resulting antibiofouling efficacy of these surfaces against zoospores of the model marine fouling organism, Ulva linza. Chemical patterns were fabricated using reversible addition-fragmentation chain-transfer (RAFT) living polymerization in conjunction with photolithography. Pattern geometries were inspired by the physical (i.e., nonchemical) Sharklet engineered microtopography system that has been shown to be effective against the same model organism. Sharklet chemical patterns and analogous parallel channels were fabricated in sizes ranging from 2 to 10 µm in the lateral dimension with tailorable feature heights ranging from tens to hundreds of nanometers. Nonpatterned, chemically grafted poly(acrylamide) silicone surfaces inhibited algal spore attachment density by 59% compared to the silicone control; however, attachment density on chemical nanotopographies was not statistically different from the control. While these results indicate that the chemical nanotopographies chosen do not represent an effective antibiofouling coating, it was found that the Sharklet pattern geometry, when sized below the 5 µm critical attachment size of the spores, significantly reduced the algal spore density compared to the equally sized channel geometry. These results indicate that specific chemical geometry of the proper sizing can impact the behavior of the algal spores and could be used to further study the mechanistic behavior of biofouling organisms.

Fabrication of a Monolithic Implantable Neural Interface from Cubic Silicon Carbide.

One of the main issues with micron-sized intracortical neural interfaces (INIs) is their long-term reliability, with one major factor stemming from the material failure caused by the heterogeneous integration of multiple materials used to realize the implant. Single crystalline cubic silicon carbide (3C-SiC) is a semiconductor material that has been long recognized for its mechanical robustness and chemical inertness. It has the benefit of demonstrated biocompatibility, which makes it a promising candidate for chronically-stable, implantable INIs. Here, we report on the fabrication and initial electrochemical characterization of a nearly monolithic, Michigan-style 3C-SiC microelectrode array (MEA) probe. The probe consists of a single 5 mm-long shank with 16 electrode sites. An ~8 µm-thick p-type 3C-SiC epilayer was grown on a silicon-on-insulator (SOI) wafer, which was followed by a ~2 µm-thick epilayer of heavily n-type (n+) 3C-SiC in order to form conductive traces and the electrode sites. Diodes formed between the p and n+ layers provided substrate isolation between the channels. A thin layer of amorphous silicon carbide (a-SiC) was deposited via plasma-enhanced chemical vapor deposition (PECVD) to insulate the surface of the probe from the external environment. Forming the probes on a SOI wafer supported the ease of probe removal from the handle wafer by simple immersion in HF, thus aiding in the manufacturability of the probes. Free-standing probes and planar single-ended test microelectrodes were fabricated from the same 3C-SiC epiwafers. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed on test microelectrodes with an area of 491 µm2 in phosphate buffered saline (PBS) solution. The measurements showed an impedance magnitude of 165 kΩ ± 14.7 kΩ (mean ± standard deviation) at 1 kHz, anodic charge storage capacity (CSC) of 15.4 ± 1.46 mC/cm2, and a cathodic CSC of 15.2 ± 1.03 mC/cm2. Current-voltage tests were conducted to characterize the p-n diode, n-p-n junction isolation, and leakage currents. The turn-on voltage was determined to be on the order of ~1.4 V and the leakage current was less than 8 µArms. This all-SiC neural probe realizes nearly monolithic integration of device components to provide a likely neurocompatible INI that should mitigate long-term reliability issues associated with chronic implantation.

In Vitro Reactive-Accelerated-Aging Assessment of Anisotropic Conductive Adhesive and Back-End Packaging for Electronic Neural Interfaces.

Bioelectronic neural interfaces can fail in vivo due to water penetration and corrosion of the packaging technology used to protect sensitive portions of the device. Although anisotropic conductive adhesive (ACA) is gaining popularity in the neural interface community to connect fabricated electrode arrays with back-end packages, the durability of ACA in chronic implants is largely unknown. We have designed a platform that uses an aggressive reactive-accelerated aging (RAA) environment to rapidly assess the ability of ACA and silicone-rubber encapsulation to maintain electrical integrity in vitro. All RAA experiments were performed at 77°C, for 24 days, and with 10 to 20 mM H2O2, which approximates a 1 year implantation. Results from these experiments showed that ACA rapidly fails (i.e., 2 to 4 days RAA) due to water absorption through the silicone encapsulant. Electrical impedance spectroscopy (EIS) confirmed water penetration through the package and the resulting corrosion of the sensitive metallic components.

in vitro Reactive-Accelerated-Aging (RAA) Assessment of Tissue-Engineered Electronic Nerve Interfaces (TEENI).

A reactive-accelerated-aging (RAA) soak-test has been employed to challenge microfabricated neural interface devices against an aggressive environment that mimics worstcase chronic physiological inflammation. The RAA tests were able to determine the ability of different materials to increase the adhesive strength of the polyimide and platinum-goldplatinum metallization thin-film interface. It was found that a 3-day RAA soak-test at 87 °C in phosphate buffered saline with 10 to 20 mM hydrogen peroxide resulted in adhesive failure of the metal-polyimide interface when titanium was used as the primary adhesion promotor. The addition of hydrogenated amorphous silicon carbide was able to eliminate the onset of adhesive failure of the metal-polyimide interface during 7-day RAA soak tests. However, sporadic cracking of the silicon carbide layer resulted in a minority of broken metal interconnects that resulted in failed electrodes. These tests have demonstrated the ability of RAA soak tests to provide rapid in vitro assessment of microfabricated neural interfaces and thereby reduce the time needed to develop synthetic methods to fabricate chronically reliable devices.

Marine anti-biofouling efficacy of amphiphilic poly(coacrylate) grafted PDMSe: effect of graft molecular weight.

There is currently strong motivation due to ecological concerns to develop effective anti-biofouling coatings that are environmentally benign, durable, and stable for use by the maritime industry. The antifouling (AF) and fouling-release (FR) efficacy of amphiphilic, charged copolymers composed of ~52% acrylamide, ~34% acrylic acid, and ~14% methyl acrylate grafted to poly(dimethyl siloxane) (PDMSe) surfaces were tested against zoospores of the green alga Ulva linza and the diatom Navicula incerta. The biofouling response to molecular weight variation was analyzed for grafts ranging from ~100 to 1,400 kg mol-1, The amphiphilic coatings showed a marked improvement in the FR response, with a 55% increase in the percentage removal of diatoms and increased AF efficacy, with 92% reduction in initial attachment density of zoospores, compared to PDMSe controls. However, graft molecular weight, in the range tested, was statistically insignificant. Grafting copolymers to PDMSe embossed with the Sharklet™ microtopography did not produce enhanced AF efficacy.