Download Nerve Bioengineering

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
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
(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
(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 , 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
Ethylene-vinyl
acetate
copolymer
PTFE (some porous)
PE
Silicone elastomers
PVC
Resorbable polymers
PGA
PLLA
PGA/PLLA blends
PCL
Polyhydroxybutyrate
(PHB)
Polyacrylonitrile
(PAN) Polyhydroxyethyl
(semi permeable)
methacrylate
PAN/PVC (semipermeable)
Polysulphone (PS)
Biological materials
Artery/vein
Muscle
Decalcified bone
Collagen/gelatin
Hyaluronic acid derivatives
Alginate
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
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
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:
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
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