Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration

Abstract

Objective: In our study, we describe the use of spider silk fibres as a new material in nerve tissue engineering, in a 20-mm sciatic nerve defect in rats.

Materials and methods: We compared isogenic nerve grafts to vein grafts with spider silk fibres, either alone or supplemented with Schwann cells, or Schwann cells and matrigel. Controls, consisting of veins and matrigel, were transplanted. After 6 months, regeneration was evaluated for clinical outcome, as well as for histological and morphometrical performance.

Results: Nerve regeneration was achieved with isogenic nerve grafts as well as with all constructs, but not in the control group. Effective regeneration by isogenic nerve grafts and grafts containing spider silk was corroborated by diminished degeneration of the gastrocnemius muscle and by good histological evaluation results. Nerves stained for S-100 and neurofilament indicated existence of Schwann cells and axonal re-growth. Axons were aligned regularly and had a healthy appearance on ultrastructural examination. Interestingly, in contrast to recently published studies, we found that bridging an extensive gap by cell-free constructs based on vein and spider silk was highly effective in nerve regeneration.

Conclusion: We conclude that spider silk is a viable guiding material for Schwann cell migration and proliferation as well as for axonal re-growth in a long-distance model for peripheral nerve regeneration.

Figure 1

Macroscopic examination six months after nerve grafting. (a) Anatomic microdissection of the regenerated sciatic nerve. A 20 mm gap in the nerve had been interposed with an isogenic vein filled with spider silk fibres. The graft has maintained its original volume, is well integrated into the host tissue and well vascularized. (b) Comparison between the ipsilateral and contralateral gastrocnemius muscles in an animal grafted with veins and matrigel. On the left, the control is shown, compared to which the muscle on the right has atrophied following denervation. (c) Comparison between the ipsilateral and contralateral gastrocnemius in an animal grafted with veins and spider silk. On the left, the control is displayed, compared to which the muscle on the right has completely retained its mass. (d) Muscle to weight ratio of the ipsilateral and contralateral gastrocnemius muscles. Experimental groups are indicated according to Table 1. Data represent means ± SEM (n= 5). Statistical analysis was performed with paired student's t‐test and adjusted according to Bonferroni. Stars indicate significant differences (P < 0.05).

Figure 2

Comparative examination of the five experimental groups. Hamatoxylin‐eosin staining of paraffin‐embedded distal parts of the analyzed nerve grafts, harvested six months after implantation. Longitudinal sections were analyzed under a light microscope. The original magnification was 100×. (a) Isogenic nerve graft. Neural tissue is regularly patterned along the longitudinal axis. (b) Artificial nerve conduit consisting of vein and spider silk. The neural tissue is regularly patterned. Note the epineural and perineural sheath at the margin of the construct. (c) Artificial nerve conduit consisting of vein, spider silk and Schwann cells. (d) Artificial nerve conduit consisting of vein, spider silk, Schwann cells and matrigel. Axons are aligned along the longitudinal axis and epineural and perineural sheaths are visible. (e) Artificial nerve conduit consisting of vein and matrigel. Axonal regeneration is not visible, and no cells can be detected inside the conduit.

Figure 3

Remyelinisation of the artificial nerve conduits. The distal part of the nerve graft was paraffin‐embedded and processed for immunostaining with anti‐S100 followed by primary antibody detection with Alexa Fluor 488. Photomicrographs were taken at an original magnification of 400×. (a) Longitudinal section through a conduit consisting of vein, spider silk, Schwann cells and matrigel. Schwann cells stained positive alongside the neurites. (b) Longitudinal section through a conduit consisting of vein and spider silk. Schwann cells stained positive alongside the neurites, although the section was slightly misaligned with the longitudinal axis. Note that the cell‐free conduit was repopulated with Schwann cells until the distal end. (c) Negative control without primary antibody shows only a slight level of autofluorescence.

Figure 4

Toluidine‐blue‐stained cross‐section. (a–e) A distal part of the conduit was embedded in Epon and processed for Toluidine blue staining. Photomicrographs were taken at an original magnification of 400×. (a) Cross section through an isogenic nerve. This group showed a high number of axons and good remyelination. (b) Cross section through a conduit consisting of vein and spider silk. This group showed remyelinated axons with proper myelin sheaths and a fascicular structure. (c) Cross section through a conduit consisting of vein, spider silk and Schwann cells. The axons are evenly distributed throughout the conduit and encircled by myelin sheaths. (d) Cross section through a conduit consisting of vein, spider silk, Schwann cells and matrigel. An irregular organisation with somewhat lower numbers of axons was observed in the distal part of the regenerated nerve. (e) Cross section through a conduit originally consisting of vein and matrigel without Schwann cells and spider silk. This group showed no remyelinated axons. (f) Analysis of fibre counts. The images were assessed for the presence of axons/myelin complexes in 200 µm² cross sectional areas. Data represent the mean + SEM (n= 5). Statistical analysis was performed with paired student's t‐test and adjusted according to Bonferroni. Stars indicate significant differences (P < 0.05).

Figure 5

Axonal regeneration in a conduit consisting of vein, spider silk, Schwann cells and matrigel. (a–c) The distal part of the nerve graft was paraffin‐embedded and processed for immunostaining with anti‐neurofilament followed by primary antibody labelling with Alexa Fluor 488. Photomicrographs were taken at an original magnification of 400×. (a) Longitudinal section. Regenerated axons stained positive for neurofilament. The axons traverse the conduit in a regular pattern. (b) Cross section. The axons are arranged in a regular bundle. (c) Negative control without primary antibody shows only slight autofluorescence. Nuclei were stained with DAPI a nuclear labelling agent emitting blue fluorescence upon binding to AT regions of DNA. (d) A distal part of the conduit was embedded in Epon and processed for transmission electron microscopy. Photomicrograph was taken at an original magnification of 16000×. The image shows a cross section of a regenerated axon with bundles of interfilaments, mitochondria and vesicles following the microtubules. The axon is encircled by a myelin sheath.