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Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep.

Radtke C, Allmeling C, Waldmann KH, Reimers K, Thies K, Schenk HC, Hillmer A, Guggenheim M, Brandes G, Vogt PM - PLoS ONE (2011)

Bottom Line: The nerve constructs were compared to autologous nerve grafts.Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons.This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic, Hand and Reconstructive Surgery, Hannover Medical School, Hannover, Germany. radtke.christine@mh-hannover.de

ABSTRACT

Background: Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/principal findings: The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/significance: This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.

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Histological analysis of regenerated fibers following spider silk construct implantation for nerve defect bridging.Immunostaining of regenerated nerve sections with neurofilament (NF, A–D in green) for regenerated axons and S100 for identification Schwann cells (A–D in red) demonstrating that axons regenerated throughout the construct and endogenous Schwann cells migrated into the construct (A and B, red). Cell nuclei are stained with DAPI (blue). Cross sectioning of regenerated nerve fibers by implantation of spider silk construct showed immunopositive staining for S100 and co-localization of neurofilament revealed ensheathment of regenerated axons by endogenous Schwann indicative for remyelination of regenerated nerve fibers in the construct (C and D). Scale bar in C = 10 µm, scale bar in D = 8 µm.
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pone-0016990-g002: Histological analysis of regenerated fibers following spider silk construct implantation for nerve defect bridging.Immunostaining of regenerated nerve sections with neurofilament (NF, A–D in green) for regenerated axons and S100 for identification Schwann cells (A–D in red) demonstrating that axons regenerated throughout the construct and endogenous Schwann cells migrated into the construct (A and B, red). Cell nuclei are stained with DAPI (blue). Cross sectioning of regenerated nerve fibers by implantation of spider silk construct showed immunopositive staining for S100 and co-localization of neurofilament revealed ensheathment of regenerated axons by endogenous Schwann indicative for remyelination of regenerated nerve fibers in the construct (C and D). Scale bar in C = 10 µm, scale bar in D = 8 µm.

Mentions: Ten months following surgery, the animals were euthanized and the nerves removed and frozen sections were prepared for immunostaining procedures. In both, the control group (autologous nerve transplantation, data not shown) and the spider silk construct transplantation group, neurofilament (NF) immunostaining for axons indicated that axons regenerated across the suture repair site and into the nerve grafts bridging the entire length of the defect. Axons were longitudinally aligned throughout the entire length of the regenerated lesion site. Note that even fascicle structure of nerves was reestablished in regenerated nerve segments and that endogenous Schwann cells (SCs) are present within regenerated nerves in the spider silk construct group (Fig. 2A–D) as identified in the nerve by S100 staining (Fig. 2A–D, red). The SCs in the nerve construct group arose from migration as the grafts were deficient in SCs. In longitudinal sections of the regenerated nerve, SCs alignment was in close approximation with the regenerated axons and cross sections of the nerves showed that SCs were wrapped around regenerated axons indicating that endogenous migrated SCs not only migrated and survived in the construct, but also functionally ensheathed regenerated axons. The cells were positioned on the NF labeled axons (green) in the typical position of myelinating Schwann cells [19].


Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep.

Radtke C, Allmeling C, Waldmann KH, Reimers K, Thies K, Schenk HC, Hillmer A, Guggenheim M, Brandes G, Vogt PM - PLoS ONE (2011)

Histological analysis of regenerated fibers following spider silk construct implantation for nerve defect bridging.Immunostaining of regenerated nerve sections with neurofilament (NF, A–D in green) for regenerated axons and S100 for identification Schwann cells (A–D in red) demonstrating that axons regenerated throughout the construct and endogenous Schwann cells migrated into the construct (A and B, red). Cell nuclei are stained with DAPI (blue). Cross sectioning of regenerated nerve fibers by implantation of spider silk construct showed immunopositive staining for S100 and co-localization of neurofilament revealed ensheathment of regenerated axons by endogenous Schwann indicative for remyelination of regenerated nerve fibers in the construct (C and D). Scale bar in C = 10 µm, scale bar in D = 8 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045382&req=5

pone-0016990-g002: Histological analysis of regenerated fibers following spider silk construct implantation for nerve defect bridging.Immunostaining of regenerated nerve sections with neurofilament (NF, A–D in green) for regenerated axons and S100 for identification Schwann cells (A–D in red) demonstrating that axons regenerated throughout the construct and endogenous Schwann cells migrated into the construct (A and B, red). Cell nuclei are stained with DAPI (blue). Cross sectioning of regenerated nerve fibers by implantation of spider silk construct showed immunopositive staining for S100 and co-localization of neurofilament revealed ensheathment of regenerated axons by endogenous Schwann indicative for remyelination of regenerated nerve fibers in the construct (C and D). Scale bar in C = 10 µm, scale bar in D = 8 µm.
Mentions: Ten months following surgery, the animals were euthanized and the nerves removed and frozen sections were prepared for immunostaining procedures. In both, the control group (autologous nerve transplantation, data not shown) and the spider silk construct transplantation group, neurofilament (NF) immunostaining for axons indicated that axons regenerated across the suture repair site and into the nerve grafts bridging the entire length of the defect. Axons were longitudinally aligned throughout the entire length of the regenerated lesion site. Note that even fascicle structure of nerves was reestablished in regenerated nerve segments and that endogenous Schwann cells (SCs) are present within regenerated nerves in the spider silk construct group (Fig. 2A–D) as identified in the nerve by S100 staining (Fig. 2A–D, red). The SCs in the nerve construct group arose from migration as the grafts were deficient in SCs. In longitudinal sections of the regenerated nerve, SCs alignment was in close approximation with the regenerated axons and cross sections of the nerves showed that SCs were wrapped around regenerated axons indicating that endogenous migrated SCs not only migrated and survived in the construct, but also functionally ensheathed regenerated axons. The cells were positioned on the NF labeled axons (green) in the typical position of myelinating Schwann cells [19].

Bottom Line: The nerve constructs were compared to autologous nerve grafts.Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons.This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.

View Article: PubMed Central - PubMed

Affiliation: Department of Plastic, Hand and Reconstructive Surgery, Hannover Medical School, Hannover, Germany. radtke.christine@mh-hannover.de

ABSTRACT

Background: Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/principal findings: The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/significance: This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.

Show MeSH
Related in: MedlinePlus