A novel internal fixator device for peripheral nerve regeneration

Abstract

Recovery from peripheral nerve damage, especially for a transected nerve, is rarely complete, resulting in impaired motor function, sensory loss, and chronic pain with inappropriate autonomic responses that seriously impair quality of life. In consequence, strategies for enhancing peripheral nerve repair are of high clinical importance. Tension is a key determinant of neuronal growth and function. In vitro and in vivo experiments have shown that moderate levels of imposed tension (strain) can encourage axonal outgrowth; however, few strategies of peripheral nerve repair emphasize the mechanical environment of the injured nerve. Toward the development of more effective nerve regeneration strategies, we demonstrate the design, fabrication, and implementation of a novel, modular nerve-lengthening device, which allows the imposition of moderate tensile loads in parallel with existing scaffold-based tissue engineering strategies for nerve repair. This concept would enable nerve regeneration in two superposed regimes of nerve extension--traditional extension through axonal outgrowth into a scaffold and extension in intact regions of the proximal nerve, such as that occurring during growth or limb-lengthening. Self-sizing silicone nerve cuffs were fabricated to grip nerve stumps without slippage, and nerves were deformed by actuating a telescoping internal fixator. Poly(lactic co-glycolic) acid (PLGA) constructs mounted on the telescoping rods were apposed to the nerve stumps to guide axonal outgrowth. Neuronal cells were exposed to PLGA using direct contact and extract methods, and they exhibited no signs of cytotoxic effects in terms of cell morphology and viability. We confirmed the feasibility of implanting and actuating our device within a sciatic nerve gap and observed axonal outgrowth following device implantation. The successful fabrication and implementation of our device provides a novel method for examining mechanical influences on nerve regeneration.

FIG. 1.

Device design. The nerve stretching device is composed of spiral nerve cuffs, poly(lactic co-glycolic) acid (PLGA) nerve guidance channel (NGC), and stainless steel backbone (a) Two regions of regeneration, A: Enhanced axonal outgrowth into a tissue engineered nerve guide/scaffold; B: Lengthening of intact regions of nerve stumps–cf. limb lengthening. (b) Dimensions of the device. Color images available online at www.liebertpub.com/tec

FIG. 2.

Fabrication and characterization of spiral nerve cuff. (a) Two layers of silicone sheets (with one being stretched) are glued together; (c) the microgroove pattern on the slab is transferred to the cuff; (b) the resultant curled nerve cuff after curing; (d) inner diameter decreases with increasing % strain of prestretched silicone sheet; (e) sample ex vivo testing of trypan blue-labeled nerve in cuff revealed no appreciable compression or tethering. Color images available online at www.liebertpub.com/tec

FIG. 3.

Fabrication of PLGA NGC. (a) Alginate rod was immersed in PLGA/Pluronic F-127 solution for 10 min. Tubular PLGA layer is formed due to phase separation when water diffuses out the alginate rod; coated alginate rod was thoroughly washed in water before alginate rod is removed (b) PLGA tube is cut into short segments to serve as NGC. (c) Cross section scanning electron microscopy picture of PLGA tube.

FIG. 4.

PLGA cytotoxicity. (a–d) Cells were fed with PLGA-incubated culture medium to test whether leachable substances from a PLGA scaffold would have cytotoxic effects on the cells. (a, b) No signs of PLGA cytotoxicity was observed in terms of cell morphology; (c, d) Live/Dead^® cell viability assay indicated very few dead cells; (e) No statistical difference was found between control and PLGA group, p=0.05. (f) Visualization of morphology of cells grown on PLGA by fluorescent wheat germ agglutinin staining of cell membranes. SH-SY5Y cells attached, spread, and proliferated on PLGA. Color images available online at www.liebertpub.com/tec

FIG. 5.

Demonstration of slip-free nerve deformation. (a, b) Device fully extended and maximally actuated, ex vivo. (c, d) Fully extended device and actuated device in a rat sciatic nerve defect. Note that implanted device can stretch the nerve stumps 6 mm without slippage. Color images available online at www.liebertpub.com/tec

FIG. 6.

Response to 2-week implantation of device. (a) A nerve gap was created by removal of 10 mm segment from the rat sciatic nerve. (b) Following 2 weeks of implantation, minor fibrosis was observed at the implantation site. (c) The stainless steel backbone was cleanly extracted at 2 weeks. (d) The proximal stump was still securely held by the nerve cuff (red arrow), and the regenerating tip extended beyond the cuff ∼6 mm (blue arrow). (e, f) In the absence of device implantation the two stumps remained disconnected and misaligned. A bulge was observed at the proximal stump and the degenerating distal stump appeared fused with surrounding fatty/connective tissue. Color images available online at www.liebertpub.com/tec

FIG. 7.

Evidence of regenerative neural outgrowth. (a) The regenerating nerve extended ∼6 mm beyond the cuff, and was stained with anti-SMI-31 (a–c) and anti-S100 (e, f) antibodies, which labeled phosphorylated neurofilaments and Schwann cells, respectively. (d, g) The contralateral sciatic nerve was stained with the same two markers and served as a control. Color images available online at www.liebertpub.com/tec