Development of a Regenerative Peripheral Nerve Interface for Control of a Neuroprosthetic Limb

Abstract

Background. The purpose of this experiment was to develop a peripheral nerve interface using cultured myoblasts within a scaffold to provide a biologically stable interface while providing signal amplification for neuroprosthetic control and preventing neuroma formation. Methods. A Regenerative Peripheral Nerve Interface (RPNI) composed of a scaffold and cultured myoblasts was implanted on the end of a divided peroneal nerve in rats (n = 25). The scaffold material consisted of either silicone mesh, acellular muscle, or acellular muscle with chemically polymerized poly(3,4-ethylenedioxythiophene) conductive polymer. Average implantation time was 93 days. Electrophysiological tests were performed at endpoint to determine RPNI viability and ability to transduce neural signals. Tissue samples were examined using both light microscopy and immunohistochemistry. Results. All implanted RPNIs, regardless of scaffold type, remained viable and displayed robust vascularity. Electromyographic activity and stimulated compound muscle action potentials were successfully recorded from all RPNIs. Physiologic efferent motor action potentials were detected from RPNIs in response to sensory foot stimulation. Histology and transmission electron microscopy revealed mature muscle fibers, axonal regeneration without neuroma formation, neovascularization, and synaptogenesis. Desmin staining confirmed the preservation and maturation of myoblasts within the RPNIs. Conclusions. RPNI demonstrates significant myoblast maturation, innervation, and vascularization without neuroma formation.

Figures

Figure 1

Schematic drawing of a Regenerative Peripheral Nerve Interface (RPNI) which is constructed using scaffold material consisting of either silicone mesh, acellular muscle, or acellular muscle with PEDOT conductive polymer. In this example, the end of the peripheral nerve is wrapped by acellular muscle with PEDOT and the construct is populated with cultured myoblasts. (Below) A 2 cm section of the distal common peroneal nerve is removed (A) and the residual nerve (B) is implanted into the RPNI (C) for a minimum of 2 months.

Figure 2

In situ image of Regenerative Peripheral Nerve Interface (RPNI), 4 months after implantation. In this example, 2 × 35 mm plates of myoblasts at culture day 14 were deposited on a one-layer thick sheet (2 cm long) acellular muscle. The common peroneal was transected, a 2 cm length was discarded, and the proximal residual end was tacked to the acellular muscle. The acellular muscle was rolled lengthwise to contain the myoblasts and maintain contact with the transected peroneal nerve. Evoked compound muscle action potential recording with stimulating electrode positioned on the peroneal nerve was 90 μV peak-to-peak.

Figure 3

(a) RPNI constructed with silicone mesh scaffold at postoperative day 111. Note that the myotubes that were implanted within the scaffold have matured and the resultant tissue appears pink and well-vascularized. The silicone mesh remained intact and did not negatively affect viability of the surrounding tissues. (b) RPNI constructed with acellular muscle scaffold at postoperative day 270. (A) implanted electrode; (B) RPNI; (C) peroneal branch of sciatic nerve; and (D) tibial branch of the sciatic nerve.

Figure 4

EMG activity recorded from the RPNI in response to painful foot stimulus during deep and light anesthetic conditions.

Figure 5

Nerve conduction studies from acellular muscle scaffold RPNI (a) and acellular muscle+PEDOT scaffold RPNI (b) showing the generation of an elicited compound muscle action potential.

Figure 6

Histologic localization of acetylcholinesterase (arrows) indicates neuromuscular junctions in an acellular muscle scaffold RPNI (a) and an acellular muscle+PEDOT scaffold RPNI (b). Note the formation of neuromuscular junctions in both specimens which suggests successful nerve-muscle synaptogenesis.

Figure 7

Transmission electron microscopy images of an RPNI created with acellular muscle scaffold at postoperative day 90. Note the presence of mature muscle fibers (A), adjacent regenerating myelinated nerve fibers (B), Schwann cells (C), and the formation of neuromuscular junctions (D).

Figure 8

(a–e) Desmin-positive muscle fibers. Desmin staining with striations confirms the survival of myotubes and maturation into muscle fibers after implantation into the RPNI. (a and b) Normal rat muscle fibers. (c) RPNI muscle fibers after maturing inside acellular muscle coated with PEDOT. (d) RPNI muscle fibers after maturing inside acellular muscle. (e) RPNI muscle fibers after maturing inside silicone mesh. Muscle fibers myoblast-derived muscle fibers were found within all RPNI specimens regardless of scaffold material. (f) Example of peroneal nerve cross section at the entrance to the RPNI.