lecture 22: tissue engineering of peripheral nerves · 2020. 2. 4. · properties and structure of...

Martin Luther University Halle-Wittenberg

Lecture 22:Tissue Engineering of

Peripheral Nerves

Prof. Thomas Groth

Biomedizinische Materialien



Content

• Structure and function of nerves

• Nerve pulse emission and propagation

• Established therapies for surgical repair of nerve defects

• Nerve conduits as biomaterial-based scaffolds

• Bioactive Hydrogels

• Application of cells


Structure of Nervous System

Spinal cord

Brain

Peripheralnerve systemPNS

Centralnerve systemCNS


Task of CNS


Autonomous (ANS) and (Somatic) NervousSystem


Supportive Cells (Glia)

2 types in peripheral (PNS), 4 types in CNS

• Tasks: Nutrition of neurons/ mechanicalsupport/ electrical insulation/ lead structureduring growth

• Schwann cells surround larger nerve fibres, generate myelin sheets, essential forregeneration of peripheral nerves

• Satellite cells envelop neurones in ganglia, function unknown


Glia in CNS - Astrocytes

J Cell Biol. 2005 Dec 19;171(6):1001-12.

There are differences between glia cells and neurons:- neurons can generate action potentials glial cells cannot- neurons have chemical synapses; glial cells do not have synapses- there are more glial cells than neurons


Schwann & Satellite Cells


Schwann Cells

Fig. 1 (a) A myelinated axon in theperipheral nervous system and (b) itsdevelopment. Each Schwann cellmyelinates a single axon, to which it isdirectly apposed. During development (anticlockwise) Schwann cells loosely ensheath axonsand the myelin sheath grows aroundthe axon to form concentric layers, which become tightly apposed


Structure of Motoneurones


Neurons

Glia cells (astrocytes red)Neurons (green)

http://www.greenspine.ca/en/jigsaw_puzzles.html


Axones can be pretty long– Example Motoneurones Cell body in spinal cord and synapsis on muscle cells in foot


Properties and Structure of Nerve Cells

• Transmission of information by means of electrical impulses

• Extremely long life-time - Maximum. > 100 years

• Amitotic – no replacement after cell death (exception olfactoric neurons and certain neurons in hippocampus)

• Extremely active metabolism Necessity of permanent supply of glucose and oxygen Without supply cell death after a few minutes

• Relatively large, branched cells with partly one meter long extensions (axons)

• Structure normally dendrits as „entrance“, main cell body „integration“ and axons „exit“


Nerve Conduction

The semipermeable membrane of a cell has differentconcentrations of ions inside and out. Diffusion movesthe K+ and Cl− ions in the direction shown, until theCoulomb force halts further transfer.This results in a layer of positive charge on the outside,a layer of negative charge on the inside, and thus avoltage across the cell membrane. The membrane isnormally impermeable to Na+.

An action potential is the pulse of voltage inside a nerve cell graphed here. It is caused by movements of ions across the cell membrane as shown. Depolarization occurs when a stimulus makes the membrane permeable to Na+

ions. Repolarization follows as the membrane again becomes impermeable to Na+, and K+ moves from high to low concentration.


Axons

• Normally only one axon per neuronal cell; can be very long and highly branched (axon collaterales)

• Origin at axon „hill“ (also origin of action potential)

• Terminates in upto 10.000 axon „buttons‘

• Supply with nutrients, etc. from nuclear region (active axonal transport) with replacement of membrane components, mitochondria, enzymes, etc.


Myelin Sheets

•Articularly found in thick axones

•white lipoproteins for electricalinsulation

•Acceleration of nerve pulse up tofactor 100 150 m/s

•Formed by Schwann cells (rollingaround axon)

•Myelin-free areas are found in regulardistances (1 mm)

Rings of Ranvier‘ (there are foundalso collaterals)

•Nerve fibres, which are enveloped bySchwann cells without rolling aroundthem „non-myelinated“, (up to 15 Axons/Schwann cells)

Transmission electron micrograph of a myelinated axon. Generated at the Electron Microscopy Facility at Trinity College, Hartford, CT.


Saltatoric Nerve Pulse

• Membrane potential of resting axon about -70 mV• Opening of sodium channels along axon leads depolarization and progression of

action potential along axon• Non-myelinated axons slow progression from sodium channel to sodium

channel (about 1-3 m/sec)• Myelinated axons Progression of action potential from node of Ranvier to

node of Ranvier (about 100 m/sec)


Motivation for Tissue Engineering/ Regeneration of Nerves

• Multitude of diseases of nerve system – more than 100 neuropathies

• Examples CNS: Morbus Parkinson (paralysis agitans) death ofdopamine-producing cells of substantia nigra in mid brain

• Example PNS: Polyneuropaties due diabetis Destruction of myelinsheet of nerves

• Traumata of CNS and PNS e.g. spinal cord injury


Regeneration of PNS

• PNS complex + many long range connections Healing only by regeneration

• Formation of scar tissue (fibrosis) no reformation of electrical connection

• Diseases like diabetis, other neuropathies and large nerve defectsnegligible regeneration

• CNS with brain und spinal cord limited capacity for healing andregeneration


Structure of Peripheral Nerves

www.histology-world.com/keyfeatures/pnerve1.htm

Epineurum/Endoneurum/Perineurum – composed ofconnective tissue withepithlioid myofibroblasts andpartly fat cells and bloodvessels.

http://www.histology-world.com/keyfeatures/pnerve1.htm


Histology Peripheral Nerves

http://www.octc.kctcs.edu/gcaplan/anat/Histology/API%20histo%20nervous.htm


Injury of Peripheral Nerves


Surgical Repair of Peripheral Nerves

• Without surgical intervention seldomlyregeneration of function after seriousnerve defects Reasons:

- Death of neurons after injury Lack ofneurotropic factors

- Big gaps between nerve stumpsGrowing axon cannot find distal end

- Formation of scar tissue in defective region Fibrous tissue forms physical barrier forgrowing axon

• Neuroma bundle of growing axonswithout orientation and connection todistal regions (can be painful)

Neuroma formation after nerve injury


Surgical Techniques

• Neurolysis Removal ofinterfascicular fibrosis allows growthof axons

• Nerve transplantation Removal ofextended obstacles (fibrosis) andbridging of defects byautotransplants

• Autotransplantats Skin nerves forbridging of defects

• Disadvantages Quantity of usefuldonor nerves limited, formation ofpainful neuroms at donor sitepossible

Sural nerve graft harvesting – most commondonor neve utilized for grafting


Autologous Nerve Transplantation


Surgical Reconstruction with Biomaterials

• Application of surgical glue to connect nerve ends e.g. fibrin glue

• Employment of tubular lead structures (nerve conduits) Connection of proximal und distal ends of nerve stumps

• Advantages:

- Use of autologous nerves not necessary no risk of donor morbidity

- Controlled distance between nerve stumps

- Prevention/reduction of fibrotic processes due to separation from connective tissue

- Tubes as reservoir for biopharmaceuticals Tissue Engineering of nerves.


„Nerve Guidance Channel“ – „Nerve Conduits“

http://www.bio-pro.de/en/region/stern/magazin/02152/index.html

Implanted tube shall be replaced by patient´s tissuesupports regeneration of nerves and promotesvascularization (at best)

Dissected nerves, and connected by biomaterial implanttube


Nerve Conduits

Rinsing of tube lumen with salt solution toremove blood clots Diminishes inflammatoryreaction

Porous wall allows exchange of solutesSupply of oxygen and nutrients

Lohme


Regeneration in a Conduit

Distal Proximal

Day 1: Tube filled with blood proteins

Day 2-6: Protein (fibrin) cable formation

Distal Proximal

Distal Proximal

Day 7-14Migration of support cells along fibrin cables

Distal Proximal

Day 15-28 Axonal elongation (regeration )

C.E. Schmidt, University of Texas, Austin


How Nerve Conduits Work

Tissue fluid (plasma) enters voidof tube

An hourglass shaped fibrin cableis formed

Cell migration and axonalregeneration occurs resitrictedby thinnest portion

Resulting tissue often visiblythinner contains limited numberof neurons

Hours

Days

Month

Years



Length Limitations of Conduits

Short gap length fibrincable robust enough toallow regeneration

Thinning restrictsregenerative space at longergaps

No fibrin cable formationwhen lenght limit isexceeded no regenerationbut fibrosis & neuromaformation

Increasing gap length

Decreasin

gefficacy



Clinical Application of Nerve Conduits

Surgical procedure of primary reconstruction of ulnar nervus digitalis with nerve conduit


Healing Response Nerve Conduits (10 mm)

• Within a few hours Filling of tubes with blood plasma fromsurrounding blood vessels contains fibrin and neurotropic factors

• After one week Filling of lumen with longitudinal oriented fibrin fibres Formation of bridge between both nerve ends

• Subsequently Fibrin matrix serves as guiding structure for migrationof cells from proximal to distal end of nerve (Fibroblasts organizing at the periphery, Schwann cells, macrophages, endothelial cells)

• After two weeks first axons growing from proximal end with envelopof Schwann cells

• After four weeks first myelinated axons reach distal end (depends on distance)


Advantages of Nerve Conduits

• Easier surgical procedure

• Reduced mechanical tension of nerves at anastomosis, which is importantfor healing

• Prevention/reduction of formation of scar tissue

• Guidance for growing nerve tissue

• Accumulation of endogous, neuroactive molecules

• Exclusion of inhibitory molecules


Biomaterials for Nerve Conduits


Problems with Nerve Tubes

• Success strongly dependent of material and its permeability

• Disadvantage of biostable materials Risk of compression of nervesand soft tissue; Trigger for inflammatory response

• Advantage of degradable materials dissolve during regeneration, however:

• Disadvantage Damage of nerves due to swelling of material anddegradation products, difficulties to control degradation inductionscar tissue


Tissue Engineering PNS

RV Bellamkonda, Biomaterials19 (2006) 3515-3518


Options to Place Growth Factors forNerve Regeneration

• Upper example growth factor deliveryfrom solution or hydrogel as filler oflumen

• Middle: Application of cells that form guiding structure and release growthfactors & produce ECM proteins like laminins

• Lower: Transfection of cells with genes that promote release of neurotropicgrowth factors


Growth Factors Important for Nerve Regeneration

Neuro-specific growth factors Effect/Function

Nerve growth factor (NGF) Survival Neuron, Axon-Schwann cell interact.

Brain-derived neurotrophic factor (BDNF) Survival Neuron

Ciliary neurotrophic factor (CNTF) Survival Neuron

Glia-derived neurotrophic factor (GDNF) Survival Neuron

Glia growth factor (GGF) Mitogen for Schwann cells

Neurotrophin-3 (NT) Survival Neuron

Neurotrophin-4/5 (NT-4/5) Survival Neuron

General factors

Insulin-like growth factor 1 (IGF 1) Axonal growth, Schwann cell migration

Insulin-like growth factor - 2 Motoneurone, growth, Reinnervation muscle

Platelet-derived growth factor (PDGF) Cell growth, neuronal survival

acidic fibroblast growth factor (aFGF) Neurite regeneration, cell proliferation

basic fibroblast growth factor (bFGF) Neurite regeneration, neovascularisation


Survey on GF Delivery Strategies for Nerve Regeneration


Gels for Filling of Nerve Conduits

• Acceleration of healing and crossing of larger gaps possible

• Application of matrix factors Laminin, Collagen, Matrigel, different glycosaminoglycanes, fibrin

• Use of neurotropic growth factors dosage, dose regime ????

• Viscositity of gels very important for healing high viscosity is followed bypoorer healing than with saline solution


Bioactive Hydrogels

• Filling of nerve tubes with bioactive hydrogels based on fibrin polymers, collagen, but also synthetic polymers like polyethylene glycol cross-linkedwith peptides or GAGs

• Modifcation with peptide sequences from matrix proteins and celladhesion receptors

• Collagen (RGD, DGEA), Fibronectin (RGD), Laminin (SIKVAV, YIGSR, RGD)

• Neural Adhesion Molecule (N-CAM) N-Cadherin

• Immobilisation of neurotropic growth factors

• Also direct application of hydrogels in defective areas of peripheral nerves


Functionalized Hydrogels

• Cross-linking of fibrin polymers by linker through Factor XIIIa (Transglutaminase)

• Electrostatic or covalent bindung of heparin to fibrin, Heparin binds specifically NGF andother growth factors

• Growing axons split fibrin through plasmin paves the way


Degradable PEG-PLA Hydrogels

•Cultivation of neuronal cells in hydrogel in comparison to monolayer culture

•Improved survival and growth in hydrogel

Source: Manoney and Anseth, Biomaterials 27 (2006) 2265-2274


• Cultivation ofneuronal cells in hydrogel

• (a) + (b) 10 und 12 days Growth ofneurites withinculture well

• (c) + (d) Growth ofneurites in surrounding gel


In situ Repair Through Hydrogels

• Regeneration of nerve defects through hydrogels in injured area

www.biomed.metu.edu.tr/courses/term_papers/NE...


Cells for Regeneration

• Schwann cells surround axons produce myelin sheets and basal lamina

• Isolation of cells from biopsies + proliferation in vitro

• In vitro culture of Schwann cells difficult Application of growth factorsnecessary

• Immobilisation of autologous Schwann cells in nerve tubes before surgery improved healing response in animal models

• Other cells: CNS Glia cells (astrozytes),

• Ectomesenchymal stem cells (EMSC) Precursor cells for Schwann cells


Role of Schwann Cells During Regeneration

• Formation of cellular columns after injury

• Secretion of basal lamina with neurite promoting substances (laminin. collagen IV; etc.)

• Secretion von Nerve growth factors and other neurotropic molecules

• Expression ofNGF receptors sequestration of NGF at injurypresentation of NGF to stimulate neurons

• Transfection of Schwann cells Promoting secretion of neurotrophicfactors


Use of Cell Sheets for Nerve Regeneration

A flat sheet of Engineered Neural Tissue contains aligned glial cells (green) that support and guide neuronal regeneration


Use of Cell Sheets for Nerve Regeneration

Regenerating neurons (green) grow through the implanted repair device (left), supportedand guided by aligned Schwann cells (red, centre) and penetrate the distal stump (right).


Comparison Autograft, PLGA Nerve Tubesand Cell-Based Therapies I

• Use of EMSC for colonization ofnerve tubes

• EMSC comparable to autografts

• Poor results for nerve tubesmade of PLGA only

Nie et al. Int. J. Oral Maxillofacial Surg. 36 (2007) 36-48


Comparison Autograft, PLGA Nerve Tubesand Cell-Based Therapies II

Nerve regeneration cell-based therapies after 4 month – (A) Methylene bue staining(400x), (B) 4000 x with many axons (C) Axon surrounded by Schwann cells, (D) PLGA only a few axons formed during the same time


Summary

• Conventionall surgical techniques to treat defects of peripheral nerves limited success due to shortage of autografts

• Biomaterial-based therapies with application of nerve tubes onlybridging of short defects (max 10 mm) possible.

• Combination degradable scaffolds with bioactive hydrogels and cells(Tissue Engineering) Results comparable to application of autografts


Literature

• G. Penkert, Chirurgie der Nervenverletzungen Teil I, Der Chirurg 70 (1999) 959-967.

• J. Lohmeyer u.a.. Überbrückung peripherer Nervendefekte durch Einsatz von Nervenröhrchen, Der Chirurg 78 (2007) 142-147.

• EG Fine et al. Nerve Regeneration In Principles of Tissue Engineering, 785-798

lecture 22: tissue engineering of peripheral nerves · 2020. 2. 4. · properties and structure of...

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