Stimulation and Repair of Peripheral Nerves Using Bioadhesive Graft‐Antenna

Abstract An original wireless stimulator for peripheral nerves based on a metal loop (diameter ≈1 mm) that is powered by a transcranial magnetic stimulator (TMS) and does not require circuitry components is reported. The loop can be integrated in a chitosan scaffold that functions as a graft when applied onto transected nerves (graft‐antenna). The graft‐antenna is bonded to rat sciatic nerves by a laser without sutures; it does not migrate after implantation and is able to trigger steady compound muscle action potentials for 12 weeks (CMAP ≈1.3 mV). Eight weeks postoperatively, axon regeneration is facilitated in transected nerves that are repaired with the graft‐antenna and stimulated by the TMS for 1 h per week. The graft‐antenna is an innovative and minimally‐invasive device that functions concurrently as a wireless stimulator and adhesive scaffold for nerve repair.

cm of the sciatic nerve proximal its distal trifurcation. Under an Olympus operating microscope (1-40x), the nerve (diameter ~1 mm) was freed by dissecting surrounding connective tissue using a microscissor; care was taken to minimize nerve handling. The electrical response of the muscle was measured using a purpose-built AC coupled differential amplifier (100x gain, 1 hz High pass filter) and recorded using a digital to analog converter (Model 1401, Cambridge Electronic Design, UK). The recording electrode was placed in the rectus femoris muscle, the reference electrode was positioned to the adjacent tissues while the ground electrode was fixed to the leg skin of the rat. The relative position of electrodes was carefully measured with callipers for further data analysis.

Supplementary 1.2 (Ammeter)
A bespoke ammeter designed around the low noise instrumentation amplifier (INA118, Texas Instruments INC., USA) was used to measure the current flow induced in the loop antenna.
The small current flow is transformed into a small voltage using a sampling resistor (nominal conversion factor: 1 µA = 1.2 mV). This resistor (1.2 Ω) is connected directly between the INA118 inputs and generates a voltage directly proportional to the current flow that is amplified by a factor of 1000 [V/V]. The amplified signal is directly acquired by the Powerlab system that it is used to record the action potential. The reference terminal of the INA118 is directly connected to the general grounding, which includes the rat and the full instrumentation, to avoid creation of ground loops. The bespoke ammeter is calibrated using a precise current source obtained with a 1/3 of REF200 (Texas Instruments INC., USA).
Calibration can be verified by switching the current input of the sampling resistor to the calibrated constant current (100 µA) contained inside the REF200.
Simplified diagram of the bespoke ammeter (see text).

Supplementary 1.3 (Graft-Antenna)
The adhesive film was prepared using the method published earlier by our group [28,29] .
Briefly, medium molecular weight chitosan (598 cps viscosity, 81% deacetylation; Sigma-Aldrich, Sydney, NSW, Australia) was dissolved at a concentration of 1.7% (w/v) in deionized water that contained 2% (v/v) acetic acid and 0.01% (w/v) rose bengal. The viscous solution was stirred for 14 days at room temperature (~25 °C) in the dark to avoid photobleaching of rose bengal. Insoluble matter was removed by centrifuging the rose bengalchitosan solution at 3270×g for an hour. The collected supernatant was spread uniformly (~1.2 ml over ~12 cm 2 ) on a dry and sterile Perspex plate at room temperature. The solution was allowed to dry over 3 weeks which caused ~90% water content loss, forming a thin film which did not dissolve in water [30] . The rose bengal-chitosan film was carefully detached from the plate avoiding damage and small rectangular sections (~5x5 mm) were cut with scissors. An Emitech K550X gold coater (Quorum Emitech, East Sussex, England) sputtered a strip of gold onto the adhesive using a filter paper template. The chitosan adhesive was placed underneath the template and a gold strip was deposited with a width of 0.8 ± 0.1 mm and thickness of 50-80 nm. When this adhesive is placed around the nerve, the gold strip becomes a loop antenna that can receive electromagnetic radiation. The adhesive graftantennas were stored in a sterile plastic box and kept in the dark at room temperature to avoid dye photobleaching.

Supplementary 1.4 (Histology)
Before euthanasia, sciatic nerves were exposed at the site of operation and inspected for neuroma formation, tissue adhesion and uncharacteristic inflammation. The nerves were then harvested in ~15 mm lengths and fixed in 5% paraformaldehyde solution in 0.1 M phosphate buffer for 24 hours at ~4 C. Nerves were serially dehydrated in ethanol and embedded in paraffin. Transverse sections of 5-10 mm thickness were made 5 mm proximal and distal to the adhesive site. Samples were stained with Luxol Fast Blue to visualize myelinated axons and Haematoxylin and Eosin (H&E) to evaluate any adverse effect on nerves due to the TMS stimulation. Histological slides were scanned and analysed using an Aperio XT Slide Scanner (Aperio, Vista, CA, USA). Images were numbered and their identity concealed during 4 analysis, which was carried out on ~55% of the cross-sectional area of the operated and nonoperated (contralateral) nerves. Figure S1. DC stimulation of sciatic nerves. Compound muscle action potentials (CMAPs) triggered in healthy sciatic nerves (controls) by a DC stimulator at different current levels. The signal amplitude decreases sharply from 0.9 to 0.5 mV when the current drops from 18 µA to 15 µA, respectively (n = 3). Nerve Fiber Diameter (µm) 5.9 ± 1.6 5.8 ± 1.7 5.6 ± 1.5 Axon Diameter (µm) 3.6 ± 1.5 3.6 ± 1.6 3.5 ± 1.5