Nerve Bioengineering - University of Manchester



Nerve Bioengineering and peripheral nerve repair

Peripheral Nervous System

• Cranial nerves

• Spinal nerves

• Peripheral nerves

• Peripheral components of the autonomic nervous system

Peripheral nerves contain a variable mix of fibres both myelinated and unmyelinated of 3 broad types:

• Motor fibres - supply end plates in skeletal muscle

• Sensory fibres - receive information from viscera, skin, muscle, tendon, joints

• Autonomic fibres - both sympathetic and parasympathetic, subserve the blood vessels, viscera, sweat glands, arrector pilae muscles

PN structure

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nerve trunk cut away to expose a single fasciculus from which 3 fibres are shown. 2 myelinated axons, one on each side of a group of non-myelinated axons enclosed within a Schwann cell sheath. The myelinated fibre of the left has been cut away to demonstrate the relationship between the axon the SC and its sheath of myelin.

• Individual nerve fibres are supported by a collagen rich endoneurium

• Fibres are grouped in bundles or fascicles which are contained within the perineurium.

• Perineurium is a specialised tissue composed of flattened perineurial cells, alternating with layers of collagen.

o Maintains the homeostasis of the endoneurial fluid surrounding myelinated and unmyelinated fibres and provides a barrier to diffusion.

• Fasicles are embedded in connective tissue, which in its outer layers condenses to form the epineurium or nerve sheath.

• Each PN may contain one or multiple fascicles.

• Each PN has an extensive blood supply composed of interconnecting epineurial, perineurial and endoneurial plexuses which link with extrinsic regional vessels.

Neuronal cells

• Major cells are neurons and Schwann cells (SCs)

• Axons are closely associated with SCs

• Each axon-SC unit or nerve fibre is contained within a basal lamina

• SCs are a stable non-proliferating population

The major cellular components of the PNS are neurons and Schwann cells.

Neuronal cell body is usually sited in a ganglion where numerous neuronal cells are grouped and from where its processes, dendrites and axons originate.

Schwann cells

In the PNS axons are closely associated with SCs. These wrap along the entire length of the larger axons juxtaposing one another at the nodes of Ranvier and laying down spiral layers of myelin sheath. Each axon-SC unit or nerve fibre is contained within a basal lamina.

SCs are a stable non proliferating population.

ECM

PNS ECM comprises an SC basal lamina and surrounding extracellular space.

Macromolecules in the ECM include:

Laminin-2

Collagen IV, VI

P200

Tenascin-C

F-spondin

Proteoglycans Heparan sulphate and Chondroitin sulphate

Fibronectin

Entactin

Diseases of the PN

PN is susceptible to the same wide range of categories of disease as other tissues – inflammatory, traumatic, metabolic, toxic, genetic, neoplastic.

• Inflammatory neuropathies characterised by

o inflammatory cell infiltrates. In some, an

o infectious agents elicits the inflammatory responses but in others

o immune mechanisms are presumed to be the primary cause.

• Infectious polyneuropathies – many infectious processes affect PN.

• Hereditary neuropathies

• Acquired metabolic and toxic neuropathies – endogenous disorders (eg adult onset diabetes mellitus) or exogenous agents.

• Traumatic neuropathies

o Lacerations from cutting injuries or bone fragments.

o Avulsions, when tension is applied to a PN often as a force applied to one of the limbs.

o Compression neuropathy when a PN is compressed e.g carpal tunnel syndrome (median nerve compression at the level of the wrist within the compartment deliminated by the transverse carpal ligament).

o Benign and malignant tumours can be derived from elements of the nerve sheath.

Injury and Regeneration

• During PN development, SCs synthesise and assemble basal lamina ECM and fibril-forming collagens.

• This synthesis is dependent on axonal contact.

• Following injury ECM molecules promote axonal growth and regeneration.

• Close coordination and interactions between cellular and ECM components.

So PN integrity is maintained by the close coordination and complex interactions of both its cellular and extracellular components.

It is this complexity that makes PN bioengineering such a challenge.

• Regeneration of PN axons is slow.

Regrowth is complicated by discontinuity between the proximal and distal portions of the nerve sheath as well as by the misalignment of individual fascicles.

Wallerian degeneration

• Following transection of a peripheral nerve, in the distal nerve stump Wallerian degeneration occurs

• Characterised by

o Macrophage recruitment

o Degradation of both the axonal and myelin components

o Proliferation of endoneurial fibroblasts and resident SC population which align with the original basal lamina to form bands of Bungner

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a) Nerve injury provokes recruitment and activation of immune cells at the site of a nerve lesion, in the DRG, and in the ventral and dorsal horns of the spinal cord. (b) Top, macrophages, T lymphocytes and mast cells invade the lesion site and spread around the distal stumps of injured nerve fibers. Schwann cells begin to proliferate, dedifferentiate and form bands of Büngner, which serve as guiding tubes for regenerating axons. Middle, macrophages and a few T lymphocytes reside in the DRG before injury. Their numbers increase sharply after injury. Macrophages also move within the sheath that satellite cells form around the cell bodies of primary sensory neurons. Satellite cells begin to proliferate and increase the expression of glial fibrillary acidic protein. Bottom, one week after nerve injury, dense clusters of microglial cells occur in the ventral horn of the spinal cord, surrounding the cell bodies of motor neurons. Massive microglial activation is also found in the dorsal horn, in the projection territories of the central terminals of injured primary afferent fibers.

From the following article

The neuropathic pain triad: neurons, immune cells and glia. Scholz & Woolf. Nature Neuroscience 10, 1361 - 1368 (2007) doi:10.1038/nn1992

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a) Macrophages and Schwann cells produce matrix metalloproteases that interrupt the blood-nerve barrier. CGRP, substance P, bradykinin and nitric oxide released from the proximal stumps of injured nerve fibers induce hyperemia and swelling, promoting the invasion of further monocytes and T lymphocytes. The chemokines CCL2 and CCL3 attract and guide monocytes to the lesion site. Macrophages and mast cells release prostaglandins and the cytokines IL-1[pic], IL-6, IL-18, TNF and LIF. TNF has an autocrine effect on macrophages that is mediated through TNFR1 activation and enhances cytokine synthesis and release. TNF also promotes further macrophage infiltration. (b) Within minutes of the injury, neuregulin, a growth factor constitutively expressed on the axonal membrane, binds to a heteromeric receptor composed of ERBB2 and presumably ERBB3 on Schwann cells. Early ERBB2 activation is involved in demyelination, whereas late signaling through ERBB2 and ERBB3 supports Schwann cell proliferation. In the reverse direction, Schwann cells release the neurotrophic factors NGF and GDNF, prostaglandins, and cytokines; these sensitize nociceptors and modulate sensory neuron gene expression.

Peripheral Nerve Repair

• Unpredictable results.

• Often get impaired function, pain, leading to disability and decreased quality of life.

• In some cases direct end to end repair not feasible due to the gap between the transacted nerve ends.

Peripheral Nerve Repair: Autologous nerve graft

Repair of gap injury using autologous nerve graft was

• Accepted in the 1970s and remains the gold standard

• Commonly harvested from:

o Sural nerve in the leg or

o Medial cutaneous nerve in the forearm of the patient.

• Provides a guidance channel to the regrowing axons as it contains a basal lamina scaffold and endogenous Schwann cells.

Is a natural non-immunogenic ready to use graft and has clear advantages but functional outcome is very variable.

Nerve allograft can be done where autograft not possible but need immune suppression.

Nerve bioengineering for repair and regeneration

Alternatives to nerve autograft have been researched over the last few years, generally in the form of a conduit.

Ideal conduit: inert, immunologically compatible, biodegradable, able to support axonal growth and result in functional recuperation at least comparable to or superior to that achieved with a nerve graft.

Attempts date back to last century: decalcified bone, rubber tubes, fat and fascial sheaths, blood vessels, tubes made out of parchment, metal, but much more recently success reported with both biological and common synthetic materials.

|Non-resorbable polymers |Resorbable polymers |Biological materials |

|Ethylene-vinyl acetate copolymer |PGA |Artery/vein |

|PTFE (some porous) |PLLA |Muscle |

|PE |PGA/PLLA blends |Decalcified bone |

|Silicone elastomers |PCL |Collagen/gelatin |

|PVC |Polyhydroxybutyrate (PHB) |Hyaluronic acid derivatives |

|Polyacrylonitrile (PAN) (semi permeable) |Polyhydroxyethyl methacrylate |Alginate |

|PAN/PVC (semipermeable) | | |

|Polysulphone (PS) | | |

Biological conduits

From biol tissue include

• Acellular muscle grafts – can freeze thaw or heat treat to destroy cellular component, leaving behind the basal lamina

o Muscle grafts have been shown to support regeneration comparable to nerve grafts over a 2cm rat sciatic nerve gap.

o In vivo the graft was penetrated by Schwann cells fibroblasts, perineural and endothelial cells, axon regenerated within 3 weeks.

• Vein grafts to bridge sensory nerve lesions in the hand but inferior to muscle.

• Small intestinal mucosa, stripped of its mucosal and serosal layers leaves an acellular collagen matrix which can be fashioned into a roll to bridge a nerve gap. Results were poor but when seeded with SCells, it promotes significan regen approaching autograft success.

• Collagen can be shaped into a conduit – tubes have been prepared from rat tail tendon and shown to support moderate nerve rege across a 1cm gap.

Synthetic conduits

• Silicone has been one of the most extensively used materials both in experimental models and clinically.

o Non absorbable inert conduit

o Acts as a biological chamber allowing accumulation of GFs, ECM molecules and SCs

which promote nerve regen across short nerve gaps but a clinical trial shown can cause symptoms of irritation and nerve compression occasionally necessitating removal.

More recent interest in biodegradable…

• Polyhydroxybutyrate

o Can be processed into resorbable mats that are strong, flexible and easy to handle making it suitable to use as a wrap around in direct nerve repair.

o Conduits made from these mats promote axonal regeneration

o Can orientate the fibres providing directional and contact guidance to the regrowing axons.

o Vascularisation has been shown

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Fig. 1. A schematic illustration of the components of ‘grafts’ that influence peripheral nerve regeneration. The components include Scaffolds (hydrogel or fibers), ECM proteins, glial or other cells and neurotrophic factors. The spatial distribution of one or more of these components determines the degree of anisotropy of the graft.

From: Bellamkonda, Biomaterials Volume 27, Issue 19, 2006, Pages 3515-3518

doi:10.1016/j.biomaterials.2006.02.030

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Fig. 2. Light micrograph of DRG neurites growing along laminin coated nylon fibers in 3D in 1% agarose gels. Cell body is to the right (out of the picture). (A) Lower magnification (40×). Neurites prefer growing on fibers, however, they continue into the gel when the fibers end. (B) Higher magnification (200×) of the square region in (A).

From: Bellamkonda, Biomaterials Volume 27, Issue 19, 2006, Pages 3515-3518

doi:10.1016/j.biomaterials.2006.02.030

Mimicking the nerve environment

major development is the development of conduits that more closely mimic the nerve environment. – 3Dmatrices that support the regrowing axons and proliferating SCs.

PLGA foamed scaffold:

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Necessity for Neurotrophic Factors and Schwann cells

Evidence that both nat and synth mats can act as nerve conduits, often fail when used alone.

Need for GFs and SCs

Ideally have Controlled release

Gradient concentration

Difficult tho due to multitude of highly coordinated neurotrophic cues

Intro of SCs better as essential for nerve regen

• SCs can align themselves and provide directional cues to regrowing axons.

• SC can synthesise and secrete neurotrophic factors eg NGF, BDNF, CNTF and of course necessary ECM molecules

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Stem cells

• Neural stem cell isolation and culture in 1992

• Have capacity to diff into mature neurons in vitro and in vivo

• Accessibility an issue

• Bone marrow stromal cells more easily available

• MSCs express markers of neural progenitors including

o Neurofilament proteins

o Neuron specific nuclear protein

o (-tubulin III

o Glial fibrillary acidic protein

• Can diff into neurons and glia

Specific substrate cues

Neurite promoting laminin epitope IKVAV

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