Apoptosis-Decellularized Peripheral Nerve Scaffold Allows Regeneration across Nerve Gap.

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.

Lymphatic-to-blood vessel transition in adult microvascular networks: A discovery made possible by a top-down approach to biomimetic model development.

OBJECTIVE: Emerging areas of vascular biology focus on lymphatic/blood vessel mispatterning and the regulation of endothelial cell identity. However, a fundamental question remains unanswered: Can lymphatic vessels become blood vessels in adult tissues? Leveraging a novel tissue culture model, the objective of this study was to track lymphatic endothelial cell fate over the time course of adult microvascular network remodeling. METHODS: Cultured adult Wistar rat mesenteric tissues were labeled with BSI-lectin and time-lapse images were captured over five days of serum-stimulated remodeling. Additionally, rat mesenteric tissues on day 0 and day 3 and 5 post-culture were labeled for PECAM + LYVE-1 or PECAM + podoplanin. RESULTS: Cultured networks were characterized by increases in blood capillary sprouting, lymphatic sprouting, and the number of lymphatic/blood vessel connections. Comparison of images from the same network regions identified incorporation of lymphatic vessels into blood vessels. Mosaic lymphatic/blood vessels contained lymphatic marker positive and negative endothelial cells. CONCLUSIONS: Our results reveal the ability for lymphatic vessels to transition into blood vessels in adult microvascular networks and discover a new paradigm for investigating lymphatic/blood endothelial cell dynamics during microvascular remodeling.

Tissue-Engineered Peripheral Nerve Interfaces.

Research on neural interfaces has historically concentrated on development of systems for the brain; however, there is increasing interest in peripheral nerve interfaces (PNIs) that could provide benefit when peripheral nerve function is compromised, such as for amputees. Efforts focus on designing scalable and high-performance sensory and motor peripheral nervous system interfaces. Current PNIs face several design challenges such as undersampling of signals from the thousands of axons, nerve-fiber selectivity, and device-tissue integration. To improve PNIs, several researchers have turned to tissue engineering. Peripheral nerve tissue engineering has focused on designing regeneration scaffolds that mimic normal nerve extracellular matrix composition, provide advanced microarchitecture to stimulate cell migration, and have mechanical properties like the native nerve. By combining PNIs with tissue engineering, the goal is to promote natural axon regeneration into the devices to facilitate close contact with electrodes; in contrast, traditional PNIs rely on insertion or placement of electrodes into or around existing nerves, or do not utilize materials to actively facilitate axon regeneration. This review presents the state-of-the-art of PNIs and nerve tissue engineering, highlights recent approaches to combine neural-interface technology and tissue engineering, and addresses the remaining challenges with foreign-body response.

Processing-Size Correlations in the Preparation of Magnetic Alginate Microspheres Through Emulsification and Ionic Crosslinking.

Magnetic alginate microspheres are biocompatible due to their alginate matrix, and motion-controllable by applied magnetic fields due to their magnetic character. Therefore, they have the potential of being used as vessels to a broad variety of materials, including drugs and therapeutic agents, facilitating entry to biological systems in a relatively non-invasive manner. Here, magnetic alginate microspheres were prepared through an emulsification and ionic cross-linking process, where a mixture of alginate and iron oxide magnetic nanoparticles was initially dispersed in a continuous phase, followed by gelation of this dispersed phase into microspheres by cross-linking the dispersion with calcium ions. The resulting magnetic alginate microspheres were found to be superparamagnetic and to respond to applied magnetic fields by chain formation. The effect of shear rate, alginate concentration, and magnetic nanoparticle concentration on microsphere size was investigated with the aim to control the size of microspheres with respect to process and formulation parameters. Two of these parameters, shear rate and alginate concentration, were used to correlate experimental results with a theoretical model for the case where the dispersed phase is more viscous than the continuous phase.

Electrical stimuli in the central nervous system microenvironment.

Electrical stimulation to manipulate the central nervous system (CNS) has been applied as early as the 1750s to produce visual sensations of light. Deep brain stimulation (DBS), cochlear implants, visual prosthetics, and functional electrical stimulation (FES) are being applied in the clinic to treat a wide array of neurological diseases, disorders, and injuries. This review describes the history of electrical stimulation of the CNS microenvironment; recent advances in electrical stimulation of the CNS, including DBS to treat essential tremor, Parkinson's disease, and depression; FES for the treatment of spinal cord injuries; and alternative electrical devices to restore vision and hearing via neuroprosthetics (retinal and cochlear implants). It also discusses the role of electrical cues during development and following injury and, importantly, manipulation of these endogenous cues to support regeneration of neural tissue.

Electrical Stimulation of Human Mesenchymal Stem Cells on Conductive Nanofibers Enhances their Differentiation toward Osteogenic Outcomes.

Tissue scaffolds allowing the behavior of the cells that reside within them to be controlled are of particular interest for tissue engineering. Herein, the preparation of conductive fiber-based bone tissue scaffolds (nonwoven mats of electrospun polycaprolactone with an interpenetrating network of polypyrrole and polystyrenesulfonate) is described that enable the electrical stimulation of human mesenchymal stem cells to enhance their differentiation toward osteogenic outcomes.

Macromol. Rapid Commun. 21/2015.

Back Cover: Tissue scaffolds allowing the behavior of the cells that reside within them to be controlled are of particular interest for tissue engineering. Herein, the preparation of conductive nanofiber-based bone tissue scaffolds are described, made from nonwoven mats of electrospun polycaprolactone with an interpenetrating network of polypyrrole and polystyrenesulfonate. These scaffolds enable the electrical stimulation of human mesenchymal stem cells to enhance their differentiation toward osteogenic outcomes. Further details can be found in the article by J. G. Hardy,* M. K. Villancio-Wolter, R. C. Sukhavasi, D. J. Mouser, D. Aguilar Jr., S. A. Geissler, D. L. Kaplan,* and C. E. Schmidt* on page 1884.

Supracolloidal Assemblies as Sacrificial Templates for Porous Silk-Based Biomaterials.

Tissues in the body are hierarchically structured composite materials with tissue-specific properties. Urea self-assembles via hydrogen bonding interactions into crystalline supracolloidal assemblies that can be used to impart macroscopic pores to polymer-based tissue scaffolds. In this communication, we explain the solvent interactions governing the solubility of urea and thereby the scope of compatible polymers. We also highlight the role of solvent interactions on the morphology of the resulting supracolloidal crystals. We elucidate the role of polymer-urea interactions on the morphology of the pores in the resulting biomaterials. Finally, we demonstrate that it is possible to use our urea templating methodology to prepare Bombyx mori silk protein-based biomaterials with pores that human dermal fibroblasts respond to by aligning with the long axis of the pores. This methodology has potential for application in a variety of different tissue engineering niches in which cell alignment is observed, including skin, bone, muscle and nerve.

Decellularized porcine peripheral nerve based injectable hydrogels as a Schwann cell carrier for injured spinal cord regeneration.

Objective.To develop a clinically relevant injectable hydrogel derived from decellularized porcine peripheral nerves and with mechanical properties comparable to native central nervous system (CNS) tissue to be used as a delivery vehicle for Schwann cell transplantation to treat spinal cord injury (SCI).Approach.Porcine peripheral nerves (sciatic and peroneal) were decellularized by chemical decellularization using a sodium deoxycholate and DNase (SDD) method previously developed by our group. The decellularized nerves were delipidated using dichloromethane and ethanol solvent and then digested using pepsin enzyme to form injectable hydrogel formulations. Genipin was used as a crosslinker to enhance mechanical properties. The injectability, mechanical properties, and gelation kinetics of the hydrogels were further analyzed using rheology. Schwann cells encapsulated within the injectable hydrogel formulations were passed through a 25-gauge needle and cell viability was assessed using live/dead staining. The ability of the hydrogel to maintain Schwann cell viability against an inflammatory milieu was assessedin vitrousing inflamed astrocytes co-cultured with Schwann cells.Mainresults. The SDD method effectively removes cells and retains extracellular matrix in decellularized tissues. Using rheological studies, we found that delipidation of decellularized porcine peripheral nerves using dichloromethane and ethanol solvent improves gelation kinetics and mechanical strength of hydrogels. The delipidated and decellularized hydrogels crosslinked using genipin mimicked the mechanical strength of CNS tissue. The hydrogels were found to have shear thinning properties desirable for injectable formulations and they also maintained higher Schwann cell viability during injection compared to saline controls. Usingin vitroco-culture experiments, we found that the genipin-crosslinked hydrogels also protected Schwann cells from astrocyte-mediated inflammation.Significance. Injectable hydrogels developed using delipidated and decellularized porcine peripheral nerves are a potential clinically relevant solution to deliver Schwann cells, and possibly other therapeutic cells, at the SCI site by maintaining higher cellular viability and increasing therapeutic efficacy for SCI treatment.

Development of a SnO2-based 44Ti/44Sc generator for medical applications.

Towards application of 44Sc for diagnostic nuclear medicine, a 44Ti/44Sc generator based on an inorganic resin has been evaluated. Unlike other radionuclide generators used for medical applications, the long-term retention of the parent 44Ti is vital due to its long half life. Herein, tin dioxide (SnO2), a robust inorganic-based resin, has been synthesized and used as the stationary phase for a 44Ti/44Sc generator. The sorption behavior of 44Ti/44Sc was tested on SnO2 with varying acids, concentrations, and times. Preliminary batch study results showed >88 % 44Ti retention to the resin at lower acid concentrations (0.05 M HNO3 and 0.05 M HCl). A pilot generator was evaluated for a year, demonstrating 85.3 ± 2.8 % 44Sc elution yields and 0.71 ± 0.14 % 44Ti breakthrough in 5 M HNO3. Based on capacity studies, a 7.4 MBq (200 µCi) upscaled generator system was constructed for further evaluation of the SnO2 resin stability and the efficacy of the eluted 44Sc for radiolabeling. 44Sc could be regularly eluted from this generator in 5 M HNO3 with an overall average radiochemical yield 84.7 ± 9.5 %. Post-elution processing of the 44Sc with DGA-normal resin removed all 44Ti present and allowed for high 44Sc-DOTA labeling yields of 94.2 ± 0.5 %. Overall, SnO2 has been shown to be a viable material for a 44Ti/44Sc generator.

Current State of 44Ti/44Sc Radionuclide Generator Systems and Separation Chemistry.

In recent years, there has been an increased interest in 44Ti/44Sc generators as an onsite source of 44Sc for medical applications without needing a proximal cyclotron. The relatively short half-life (3.97 hours) and high positron branching ratio (94.3%) of 44Sc make it a viable candidate for positron emission tomography (PET) imaging. This review discusses current 44Ti/44Sc generator designs, focusing on their chemistry, drawbacks, post-elution processing, and relevant preclinical studies of the 44Sc for potential PET radiopharmaceuticals.

Temporal characterization of hyaluronidases after peripheral nerve injury.

Hyaluronic acid (HA) is ubiquitously found in biological tissues and mediates wound healing mechanisms after injury by promoting cell migration and proliferation. With the development of tissue-engineered neural therapeutics, including off-the-shelf grafts for peripheral nerve repair, HA is an attractive material for clinical use because of its various biological roles. HA-based biomaterials have been carefully engineered to elicit specific in vivo host responses, however an important design feature that should be considered in these scaffolds is endogenous degradation. Hyaluronidases (HYALs) are the complementary enzymes that are responsible for HA turnover. Although HYAL expression has been widely characterized in various tissues, including the central nervous system, and for different pathologies, there remains a lack of knowledge of HYAL mediated turnover in peripheral nerve tissue. In this work, gene expression of two hyaluronidases, HYAL1 and HYAL2, and HA-binding receptor, CD44, were studied in two injury models: rat sciatic nerve crush and critical gap transection. HYAL2 and CD44 were shown to be upregulated 3 days after crush injury, whereas HYAL1 was upregulated at 3 weeks, which collectively demonstrate temporal patterning of HA breakdown. Additionally, differences were observed between HYAL and HA expression at 3 weeks when compared for both nerve injury models. The activity of HYAL in peripheral nerve tissue was determined to be approximately 0.11 µmol/min, which could be used to further model HA-based biomaterial breakdown for peripheral nerve applications. Overall, this work provides a landscape of HA turnover in peripheral nerve that can be used for future neural applications.

Injectable neural hydrogel as in vivo therapeutic delivery vehicle.

Purpose: This study demonstrated in vivo delivery of a decellularized, injectable peripheral nerve (iPN) hydrogel and explored options for using iPN in combination with regenerative biomolecular therapies like stem cell secretome. Methods: Rat-derived iPN hydrogel solutions were combined with a dextran-dye before subcutaneous injection into adult Sprague Dawley rats. After injection, an in vivo imaging system (IVIS) was used to visualize hydrogels and quantify dextran-dye release over time. Poly(lactic-co-glycolic) acid (PLGA) was used to encapsulate the dextran-dye to prolong molecular release from the hydrogel scaffolds. Lastly, we investigated use of adipose-derived stem cell (ASC) secretome as a potential future combination strategy with iPN. ASC secretome was assessed for growth factor levels in response to media stimulation and was encapsulated in PLGA to determine loading efficiency. Results: Gelation of iPN hydrogels was successful upon subcutaneous injection. When combined with iPN, a 10 kDa dextran-dye was reduced to 54% its initial signal at 24 hours, while PLGA-encapsulated dextran-dye in iPN was only reduced to 78% by 24 hours. Modified media stimulation resulted in changes in ASC phenotype and dramatic upregulation of VEGF secretion. The PLGA encapsulation protocol was adapted for use with temperature sensitive biomolecules, however, considerations must be made with loading efficiency for cell secretome as the maximum efficiency was 28%. Conclusion: The results of this study demonstrated successful injection and subsequent gelation of our iPN hydrogel formulation in vivo. Biomolecular payloads can be encapsulated in PLGA to help prolong their release from the soft iPN hydrogels in future combination therapies. Lay Summary: We developed an injectable decellularized tissue scaffold from rat peripheral nerve tissue (called iPN), a potential minimally invasive therapeutic meant to fill lesion spaces after injury. This study was the first demonstration of iPN delivery to a living animal. The iPN solution was injected subcutaneously in a rat and properly formed a gelled material upon entering the body. Our results showed that encapsulating biomolecules in an FDA-approved polymer (PLGA) slowed the release of biomolecules from the iPN, which could allow therapeutics more time around the scaffold to help repair native tissue. Lastly, we investigated one potential avenue for combining iPN with other regenerative cues obtained from adipose-derived stem cells. Description of Future Works: Future work must focus on optimal loading conditions and release profiles from the iPN hydrogels. Next steps will be applying iPN in various combination therapies for spinal cord injury. We will focus efforts on developing a pro-regenerative secretome that directly promotes neurite extension and neural cell infiltration into iPN scaffolds upon transplantation in spinal cord.

Development of a bioactive tunable hyaluronic-protein bioconjugate hydrogel for tissue regenerative applications.

Every year, there are approximately 500 000 peripheral nerve injury (PNI) procedures due to trauma in the US alone. Autologous and acellular nerve grafts are among current clinical repair options; however, they are limited largely by the high costs associated with donor nerve tissue harvesting and implant processing, respectively. Therefore, there is a clinical need for an off-the-shelf nerve graft that can recapitulate the native microenvironment of the nerve. In our previous work, we created a hydrogel scaffold that incorporates mechanical and biological cues that mimic the peripheral nerve microenvironment using chemically modified hyaluronic acid (HA). However, with our previous work, the degradation profile and cell adhesivity was not ideal for tissue regeneration, in particular, peripheral nerve regeneration. To improve our previous hydrogel, HA was conjugated with fibrinogen using Michael-addition to assist in cell adhesion and hydrogel degradability. The addition of the fibrinogen linker was found to contribute to faster scaffold degradation via active enzymatic breakdown, compared to HA alone. Additionally, cell count and metabolic activity was significantly higher on HA conjugated fibrinogen compared previous hydrogel formulations. This manuscript discusses the various techniques deployed to characterize our new modified HA fibrinogen chemistry physically, mechanically, and biologically. This work addresses the aforementioned concerns by incorporating controllable degradability and increased cell adhesivity while maintaining incorporation of hyaluronic acid, paving the pathway for use in a variety of applications as a multi-purpose tissue engineering platform.

Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight.

Microphysiological systems provide the opportunity to model accelerated changes at the human tissue level in the extreme space environment. Spaceflight-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting in older adults, known as sarcopenia. These shared attributes provide a rationale for investigating molecular changes in muscle cells exposed to spaceflight that may mimic the underlying pathophysiology of sarcopenia. We report the results from three-dimensional myobundles derived from muscle biopsies from young and older adults, integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analyses comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation. The analyses also revealed downregulated differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were downregulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides an approach to studying the cell autonomous effects of spaceflight on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. We also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLabTM payloads on the ISS.

Neuronal growth promoting sesquiterpene-neolignans; syntheses and biological studies.

The use of small molecules that can promote neuronal growth represents a promising approach to regenerative science. Along these lines we have developed separate short or modular syntheses of the natural products caryolanemagnolol and clovanemagnolol, small molecules previously shown to promote neuronal growth and induce choline acetyltransferase activity. The postulated biosynthetic pathways, potentially leading to the assembly of these molecules in nature, have guided the laboratory syntheses, allowing the preparation of both natural products in as few as two steps. With synthetic access to the compounds as single enantiomers we have examined clovanemagnolol's ability to promote the growth of embryonic hippocampal and cortical neurons. Clovanemagnolol has been shown to be a potent neurotrophic agent, promoting neuronal growth at concentrations of 10 nM.

Towards the translation of electroconductive organic materials for regeneration of neural tissues.

Carbon-based conductive and electroactive materials (e.g., derivatives of graphene, fullerenes, polypyrrole, polythiophene, polyaniline) have been studied since the 1970s for use in a broad range of applications. These materials have electrical properties comparable to those of commonly used metals, while providing other benefits such as flexibility in processing and modification with biologics (e.g., cells, biomolecules), to yield electroactive materials with biomimetic mechanical and chemical properties. In this review, we focus on the uses of these electroconductive materials in the context of the central and peripheral nervous system, specifically recent studies in the peripheral nerve, spinal cord, brain, eye, and ear. We also highlight in vivo studies and clinical trials, as well as a snapshot of emerging classes of electroconductive materials (e.g., biodegradable materials). We believe such specialized electrically conductive biomaterials will clinically impact the field of tissue regeneration in the foreseeable future. STATEMENT OF SIGNIFICANCE: This review addresses the use of conductive and electroactive materials for neural tissue regeneration, which is of significant interest to a broad readership, and of particular relevance to the growing community of scientists, engineers and clinicians in academia and industry who develop novel medical devices for tissue engineering and regenerative medicine. The review covers the materials that may be employed (primarily focusing on derivatives of fullerenes, graphene and conjugated polymers) and techniques used to analyze materials composed thereof, followed by sections on the application of these materials to nervous tissues (i.e., peripheral nerve, spinal cord, brain, optical, and auditory tissues) throughout the body.

Microphysiological system for studying contractile differences in young, active, and old, sedentary adult derived skeletal muscle cells.

Microphysiological systems (MPS), also referred to as tissue chips, incorporating 3D skeletal myobundles are a novel approach for physiological and pharmacological studies to uncover new medical treatments for sarcopenia. We characterize a MPS in which engineered skeletal muscle myobundles derived from donor-specific satellite cells that model aged phenotypes are encapsulated in a perfused tissue chip platform containing platinum electrodes. Our myobundles were derived from CD56+ myogenic cells obtained via percutaneous biopsy of the vastus lateralis from adults phenotyped by age and physical activity. Following 17 days differentiation including 5 days of a 3 V, 2 Hz electrical stimulation regime, the myobundles exhibited fused myotube alignment and upregulation of myogenic, myofiber assembly, signaling and contractile genes as demonstrated by gene array profiling and localization of key components of the sarcomere. Our results demonstrate that myobundles derived from the young, active (YA) group showed high intensity immunofluorescent staining of α-actinin proteins and responded to electrical stimuli with a ~1 µm displacement magnitude compared with non-stimulated myobundles. Myobundles derived from older sedentary group (OS) did not display a synchronous contraction response. Hypertrophic potential is increased in YA-derived myobundles in response to stimulation as shown by upregulation of insulin growth factor (IGF-1), α-actinin (ACTN3, ACTA1) and fast twitch troponin protein (TNNI2) compared with OS-derived myobundles. Our MPS mimics disease states of muscle decline and thus provides an aged system and experimental platform to investigate electrical stimulation mimicking exercise regimes and may be adapted to long duration studies of compound efficacy and toxicity for therapeutic evaluation against sarcopenia.

Decellularized peripheral nerve as an injectable delivery vehicle for neural applications.

Damage to the nervous system can result in loss of sensory and motor function, paralysis, or even death. To facilitate neural regeneration and functional recovery, researchers have employed biomaterials strategies to address both peripheral and central nervous system injuries. Injectable hydrogels that recapitulate native nerve extracellular matrix are especially promising for neural tissue engineering because they offer more flexibility for minimally invasive applications and provide a growth-permissive substrate for neural cell types. Here, we explore the development of injectable hydrogels derived from decellularized rat peripheral nerves (referred to as "injectable peripheral nerve [iPN] hydrogels"), which are processed using a newly developed sodium deoxycholate and DNase (SDD) decellularization method. We assess the gelation kinetics, mechanical properties, cell bioactivity, and drug release kinetics of the iPN hydrogels. The iPN hydrogels thermally gel when exposed to 37°C in under 20 min and have mechanical properties similar to neural tissue. The hydrogels demonstrate in vitro biocompatibility through support of Schwann cell viability and metabolic activity. Additionally, iPN hydrogels promote greater astrocyte spreading compared to collagen I hydrogels. Finally, the iPN is a promising delivery vehicle of drug-loaded microparticles for a combinatorial approach to neural injury therapies.

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.