EVERY year, hundreds of thousands of people suffer from paralyzed limbs as a result of peripheral nerve injury. Recently, implantation of artificial nerve grafts has become the method of choice for repairing damaged peripheral nerves. Grafts can lead to some degree of functional recovery when a short segment of nerve is damaged. But they are of little use when it comes to regenerating nerves over distances greater than a few millimeters, and such injuries therefore often lead to permanent paralysis.
Now though, surgeons from Germany have made what could be a significant advance in nerve tissue engineering. They have developed artificial nerve grafts made from hollowed-out pig veins filled with spider silk fibres and, in a series of animal experiments, showed that the grafts can enhance the regeneration of peripheral nerves over distances of up to 6cm. Their findings have just been published in the open access journal PLoS One.
Peripheral nerves have a greater regenerative capacity than those in the central nervous system, but regenerating them properly is challenging. The individual nerve fibres must not only regrow into the damaged area, but also find their proper targets. Furthermore, the regenerated nerve will not function properly unless it is populated by Schwann cells, which produce myelin. This fatty tissue is essential for full recovery, as it wraps itself around the nerve fibres at regular intervals (a process called myelination), facilitating the conductance of nervous impulses along their length.
Conventionally, damaged peripheral nerves are treated either by suturing or by implantation of nerve grafts. The two ends of a severed nerve can be surgically re-attached to each other, as long as the nerve is not stretched in the process. This is not possible for gaps longer than about 5mm, in which case a short length of nerve from elsewhere in the patient’s body can be grafted into the damaged area. But this often causes causes pain in the donor area, and it can be difficult to find a nerve segment that has the same diameter as the damaged nerve. Nerves can be obtained from another person, but they can be rejected by the recipient’s immune system, so drugs that suppress the immune response are usually administered.
An alternative approach, which has emerged in the past ten years or so, is the use of artificial nerve grafts made from silicon or synthetic polymers such as polyethylene. These form scaffolds which bridge the gap in the damaged nerve and serve as conduits through which the nerve fibres can regrow. Artificial grafts can lead to some degree of functional recovery, but they can become toxic with time, or they can constrict the nerve.
These problems can potentially be overcome by using nerve grafts made from biodegradable materials. Five years ago, Peter Vogt and his colleagues in the Department of Plastic, Hand and Reconstructive Surgery at Hannover Medical School reported that Schwann cells readily ensheath spider silk fibres when grown on them, and that nerve grafts made of de-cellularized veins filled with spider silk can be maintained in culture for periods of up to a week. More recently, they showed that spider silk vein grafts can be used to regenerate 20mm gap in the sciatic nerve of rats, either alone or when supplemented with Schwann cells.
In the new study, Vogt’s group dissected 6cm lengths from the small veins in pigs’ legs, washed them and stripped away most of the endothelial cells from their inner walls. They then harvested dragline silk from the golden silk spider Nephila clavipes and pulled the silk through the de-cellularized veins, until it filled about one quarter of their diameter. Using adult sheep, the researchers removed a 6cm length of the tibial nerve in the leg. In one group of animals, the gap was bridged with the spider silk constructs; in another, the section of nerve that had been removed was replaced in reverse orientation.
Defects in the animals’ gait became apparent immediately after the surgery – the hind limb was partially paralyzed and flexed abnormally. But within three weeks there was a significant improvement, with both groups of animals being able to stand properly. By four months, the animals could stand upright on both hind limbs, the hind limbs moved in co-ordination with one another during walking, and there was no obvious difference in strength between the operated and unoperated limbs.
Ten months after surgery, the sheep were killed and their regenerated nerves examined under the microscope. In both groups of animals, the severed nerve fibres had regrown into the nerve grafts to bridge the 6cm gap; Schwann cells had migrated into the grafts and wrapped themselves around the entire length of the regenerated nerves; and the sodium channels required for generating nerve impulses were distributed irregularly along the fibres. This shows that myelination had occurred properly, with the formation of Nodes of Ranvier, the regular gaps in the myelin sheath at which the sodium channels normally cluster. No trace of residual spider silk was detected in the experimental animals, and there was no sign of inflammation at the repair site, indicating that the silk fibres were absorbed subtly without adverse effects.
These findings could have important applications in reconstructive nerve surgery. This is the first time that a large animal model has been used to study nerve regeneration, and the study is the first in which a defect longer than 2cm in length has been successfully repaired. The spider silk constructs enhanced nerve regeneration at least as effectively as the sheeps’ own nerves, and would be advantageous in the clinic, because transplanting large lengths of a patient’s own nerves is unfeasible.
More work will be needed before the technique can be applied to humans. Meanwhile, the regeneration reported here could be further enhanced in a number of ways. The spider silk constructs could, for example, be loaded with substances such as Nerve Growth Factor, or they could be grafted together with Schwann cells, to speed up nerve regrowth. But ultimately, engineering fully functional peripheral nerves will probably require a combination of advanced microsurgery, transplantation of both cells and tissues, advances in materials science and, possibly, gene transfer for the effective delivery of growth factors.
Radtke, C. et al. (2011). Spider Silk Constructs Enhance Axonal Regeneration and Remyelination in Long Nerve Defects in Sheep. PLoS ONE, 6 (2) DOI: 10.1371/journal.pone.0016990.