A Drug-Loaded, Flexible Microelectrode Array to Improve Functional Recovery Following Traumatic Amputation and Peripheral Nerve Injuries

Abstract

Over the last decade, thousands of United States service members have sustained limb injuries from blast exposure, largely attributed to the rising use of improvised explosive devices (IEDs) by terrorist and insurgent groups. These blasts destroy large amounts of tissue causing peripheral nerve injuries and amputations in many cases. The objective of this research was to develop a device to improve functional recovery among these patients by providing in vivo monitoring of neuromuscular regeneration, maintenance of acetylcholine receptors for reinnervation and prevention of muscle atrophy. An implantable, flexible microelectrode array (MEA) was engineered that provides stimulation and recording during acute denervation of muscle distal to nerve injuries and muscle grafts for creation of regenerative peripheral nerve interfaces (RPNIs). One issue that has prevented the development of muscle surface electrodes is the immune-mediated foreign body response resulting in fibrotic capsule formation around the device. To inhibit fibrosis, the electrode was embedded with crystal formulations of a drug called GW2580, a Colony Stimulating Factor 1 (CSF1R) inhibitor allowing for long-term release of the drug from the surface of the MEA. The MEA uses a flexible polyimide elastomer and an array of gold-based microelectrodes featuring Peano curve motifs, which together maintain electrode flexibility. A new surgical technique was developed utilizing a biceps femoris muscle flap along the dorsal surface of the electrode to prevent wound breakdown during implantation. The devices were implanted along the denervated gastrocnemius muscles of rats. These rats underwent therapeutic stimulation using the MEA daily beginning on post-operative day 2. Tissues were harvested on post-operative day 14 and evaluated for quantification acetylcholine receptors and muscle fiber area using immunofluorescence and histological staining. Additionally, the MEAs were implanted along the dorsal surface of free flap grafts to serve as a RPNI for 6 weeks in rats. Using electrophysiological and histological assessments, we compare with unstimulated control grafts. The Young’s modulus was 1.67 GPa, which is comparable to native tendon and muscle. The devices successfully recorded electromyogram data when implanted in rats. When compared to untreated denervated muscles, MEA therapy attenuated atrophy by maintaining larger muscle fiber cross-sectional areas. Furthermore, the acetylcholine receptor areas were markedly larger with MEA treatment. The postoperative course of one animal without the biceps femoris flap (control) was complicated by complete wound dehiscence requiring euthanasia of the animal on postoperative day 4; the remaining control animals showed evidence of ulceration at the implant. The animals that did have the biceps femoris flap did not have ulcerative lesions on postoperative day 7. Expression of specific markers for acute inflammation (TNF-alpha), innate immune cell macrophages (CD68) and fibrosis (alphaSMactin) were also decreased in the flap group by qPCR analysis. Drug-loading with crystalized formulations of GW2580 successfully prevented fibrotic capsule formation and improved therapeutic efficacy of the SMEA. When the MEAs were implanted along the surface of muscle grafts for RPNIs, we found significantly delayed reinnervation and abnormal electromyographic (EMG) signals, with significantly more polyphasia, lower compound muscle action potentials and higher fatigability in stimulated animals. These metrics are suggestive of myopathy in the free flap grafts stimulated with the electrode. Active inflammatory processes and partial necrosis were observed in grafts stimulated with the MEA. This work demonstrates the ability to combine conformability, tensile strength-enhancing metal micropatterning and drug loading for long-term release into a functional implant for both epimysial stimulation and recording during acute denervation injury. However, under the same treatment protocol, implanted epimysial electrodes and electrical stimulation to deinnervated and devascularized flaps during the early recovery phase may be detrimental to regeneration.