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.

Three-Dimensional Bioprinted Hyaluronic Acid Hydrogel Test Beds for Assessing Neural Cell Responses to Competitive Growth Stimuli.

Hyaluronic acid (HA) is an abundant extracellular matrix (ECM) component in soft tissues throughout the body and has found wide adoption in tissue engineering. This study focuses on the optimization of methacrylated HA (MeHA) for three-dimensional (3D) bioprinting to create in vitro test beds that incorporate regeneration-promoting growth factors in neural repair processes. To evaluate MeHA as a potential bioink, rheological studies were performed with PC-12 cells to demonstrate shear thinning properties maintained when printing with and without cells. Next, an extrusion-based Cellink BIO X 3D printer was used to bioprint various MeHA solutions combined with collagen-I to determine which formulation was the most optimal for creating 3D features. Results indicated that MeHA (10 mg/mL) with collagen-I (3 mg/mL) was most suitable. As Schwann cells (SCs) are a critical component of neural repair and regeneration, SC adhesion assessment via integrin ß1 immunostaining indicated that the bioink candidate adequately supported SC adhesion and migration when compared to Col-I, a highly cell-adhesive ECM component. MeHA/collagen-I bioink was adapted for neural specific applications by printing with the neural growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). These test beds were conducive for SC infiltration and presented differential migration responses. Finally, a two-chamber in vitro test bed design was created to study competitive biochemical cues. Dorsal root ganglia were seeded in test beds and demonstrated directional neurite extension (measured by ß-III tubulin and GAP43 immunostaining) in response to NGF and GDNF. Overall, the selected MeHA/collagen-I bioink was bioprintable, improved cell viability compared to molded controls, and was conducive for cell adhesion, growth factor sequestration, and neural cell infiltration. MeHA is a suitable bioink candidate for extrusion-based bioprinting and will be useful in future development of spatially complex test beds to advance in vitro models as an alternative to common in vivo tests for neural repair applications.

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.

Tunable methacrylated hyaluronic acid-based hydrogels as scaffolds for soft tissue engineering applications.

Hyaluronic acid (HA)-based biomaterials have been explored for a number of applications in biomedical engineering, particularly as tissue regeneration scaffolds. Crosslinked forms of HA are more robust and provide tunable mechanical properties and degradation rates that are critical in regenerative medicine; however, crosslinking modalities reported in the literature vary and there are few comparisons of different scaffold properties for various crosslinking approaches. In this study, we offer direct comparison of two methacrylation techniques for HA (glycidyl methacrylate HA [GMHA] or methacrylic anhydride HA [MAHA]). The two methods for methacrylating HA provide degrees of methacrylation ranging from 2.4 to 86%, reflecting a wider range of properties than is possible using only a single methacrylation technique. We have also characterized mechanical properties for nine different tissues isolated from rat (ranging from lung at the softest to muscle at the stiffest) using indentation techniques and show that we can match the full range of mechanical properties (0.35-6.13 kPa) using either GMHA or MAHA. To illustrate utility for neural tissue engineering applications, functional hydrogels with adhesive proteins (either GMHA or MAHA base hydrogels with collagen I and laminin) were designed with effective moduli mechanically matched to rat sciatic nerve (2.47 ± 0.31 kPa). We demonstrated ability of these hydrogels to support three-dimensional axonal elongation from dorsal root ganglia cultures. Overall, we have shown that methacrylated HA provides a tunable platform with a wide range of properties for use in soft tissue engineering.

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.

Conductive interpenetrating networks of polypyrrole and polycaprolactone encourage electrophysiological development of cardiac cells.

Conductive and electroactive polymers have the potential to enhance engineered cardiac tissue function. In this study, an interpenetrating network of the electrically-conductive polymer polypyrrole (PPy) was grown within a matrix of flexible polycaprolactone (PCL) and evaluated as a platform for directing the formation of functional cardiac cell sheets. PCL films were either treated with sodium hydroxide to render them more hydrophilic and enhance cell adhesion or rendered electroactive with PPy grown via chemical polymerization yielding PPy-PCL that had a resistivity of 1.0 ± 0.4 kΩ cm, which is similar to native cardiac tissue. Both PCL and PPy-PCL films supported cardiomyocyte attachment; increasing the duration of PCL pre-treatment with NaOH resulted in higher numbers of adherent cardiomyocytes per unit area, generating cell densities which were more similar to those on PPy-PCL films (1568 ± 126 cells mm(-2), 2880 ± 439 cells mm(-2), 3623 ± 456 cells mm(-2) for PCL with 0, 24, 48 h of NaOH pretreatment, respectively; 2434 ± 166 cells mm(-2) for PPy-PCL). When cardiomyocytes were cultured on the electrically-conductive PPy-PCL, more cells were observed to have peripheral localization of the gap junction protein connexin-43 (Cx43) as compared to cells on NaOH-treated PCL (60.3 ± 4.3% vs. 46.6 ± 5.7%). Cx43 gene expression remained unchanged between materials. Importantly, the velocity of calcium wave propagation was faster and calcium transient duration was shorter for cardiomyocyte monolayers on PPy-PCL (1612 ± 143 µm/s, 910 ± 63 ms) relative to cells on PCL (1129 ± 247 µm/s, 1130 ± 20 ms). In summary, PPy-PCL has demonstrated suitability as an electrically-conductive substrate for culture of cardiomyocytes, yielding enhanced functional properties; results encourage further development of conductive substrates for use in differentiation of stem cell-derived cardiomyocytes and cardiac tissue engineering applications. STATEMENT OF SIGNIFICANCE: Current conductive materials for use in cardiac regeneration are limited by cytotoxicity or cost in implementation. In this manuscript, we demonstrate for the first time the application of a biocompatible, conductive polypyrrole-polycaprolactone film as a platform for culturing cardiomyocytes for cardiac regeneration. This study shows that the novel conductive film is capable of enhancing cell-cell communication through the formation of connexin-43, leading to higher velocities for calcium wave propagation and reduced calcium transient durations among cultured cardiomyocyte monolayers. Furthermore, it was demonstrated that chemical modification of polycaprolactone through alkaline-mediated hydrolysis increased overall cardiomyocyte adhesion. The results of this study provide insight into how cardiomyocytes interact with conductive substrates and will inform future research efforts to enhance the functional properties of cardiomyocytes, which is critical for their use in pharmaceutical testing and cell therapy.