Low Loading of Collagen in Electrospun Polyester Nerve Conduits for Repairing Segmental Nerve Defect: An Experimental Study Using the Tibial Nerve in Rats with Multiple Measurements

Abstract

The present study provides in vivo trials of electrospun poly(l-lactide-co-ε-caprolactone), PLCL, copolymer 67:33 mol %, and electrospun PLCL blend with a low loading of collagen (0.5% w/v), PLCL-Col, as a connecting porous biodegradable nerve conduit to repair 7 mm long segmentary tibial nerve lesions in rats compared with the standard autograft technique. The electrospun PLCL scaffolds reveal a matrix of fibers with a mean diameter of 476 ± 60 nm and an average pore size of 253 ± 5 nm. Blending collagen with the PLCL results in a comparatively denser matrix of fibers with a mean diameter of 417 ± 42 nm and a pore size of 244 ± 3 nm. For in vivo testing, a total of 30 male Wistar rats were divided into 3 groups of 10 and each group was subjected to a different nerve repair procedure for evaluation of nerve regeneration after reconstruction. Evaluation of nerve regeneration was compared in terms of the tibial functional index (TFI), nerve conduction velocity (NCV), gastrocnemius muscle weight (%GMW), and a histomorphometric study. After 12 weeks of implantation, there was evidence of nerve regeneration across the gap from the histomorphologic study. All parameters of nerve regeneration were observed in every animal of the study groups. Our results clearly showed that there are reinnervation and return of function in all groups, similarly to the autograft group. PLCL-Col showed better results than PLCL and autograft, which suggested that PLCL-Col porous conduits may serve as a scaffold for peripheral nerve regeneration.

Figure 1

SEM images of tubular electrospun scaffolds: (a) PLCL and (b) PLCL-Col (scale bars = 500 μm; magnification = ×35; n = 10). (Bottom left) Insets show macroscopic images of the electrospun scaffolds. (Lower right) Insets display the previously reported SEM morphology of PLCL and PLCL-Col fibers [reprinted (adapted or reprinted in part) with permission from Daranarong, Copyright 2013 Society of Chemical Industry], illustrating the nanofibrous diameter (scale bars = 10 μm; magnification = ×1500; n = 5).

Figure 2

Mean %NCV of PLCL-Col, PLCL, and autograft (n = 10).

Figure 3

% Gastrocnemius muscle weight for animals implanted with control autografts, PLCL, and PLCL-Col conduits, and harvested at 12 weeks (n = 10).

Figure 4

Foreign body multinucleated giant cells (FBGC) and Langhans type giant cells (LGC) adjacent to the inner surface of the nerve conduit in light microscopic H&E staining (scale bars, 100 μm, n = 10).

Figure 5

Schwann cells (SCs), myelinated nerve fibers (MNFs), and macrophage (Mac) of the tibial nerve distal to the nerve conduit in H&E staining (scale bars, 50 μm, n = 10).

Figure 6

Monoclonal antibody TUJ1 immunohistochemistry was used to stain the tibial nerve (PLCL group). (A) Proximal (P) part of the nerve conduit (NT). (B) Middle part of the nerve conduit. (C) Distal (D) part of the nerve conduit (scale bars, 100 μm). (D) Myelinated nerve fiber (MNFs) stained by TUJ1 (brown color) and Schwann cells (SCs) in high-power magnification (scale bars, 50 μm, n = 10).

Figure 7

Semithin cross section (A–D) and transmission electron microscopy (TEM) (E–F) of the tibial nerve of the experimental side sample of the PLCL-Col group. Round shape of the nerve and regular axon distribution in the proximal portion of the tibial nerve on low (A) and moderate (B) power magnification. Irregular shape and axon distribution in the distal portion of the tibial nerve on low (C) and moderate (D) power magnification (scale bars, 100 μm). Mast cells (arrow) were found in the distal portion as the regeneration process of the axon. TEM shows degeneration of the axon in some part ((E), arrow) (scale bars, 3000 nm) and myelinated nerve fibers ((F), MNFs) (scale bars, 2000 m, n = 10).

Figure 8

Semithin cross section (A, B) and transmission electron microscopy (TEM) (C, D) of the tibial nerve of the experimental side sample of the PLCL group. Irregular shape and axon distribution in the distal portion of the tibial nerve on low (A) (Scale bars, 100 μm) and moderate (B) power magnification (scale bars, 100 μm). Mast cells (arrow) were found in the distal portion as the regeneration process of the axon. TEM shows degeneration of the axon in some part ((C), arrow), myelinated nerve fibers (C, MNFs) (scale bars, 3000 nm), and (D) Schwann cells (SCs) surrounding the axon (Ax) (scale bars, 600 nm, n = 10).