Nerve tissue engineering and regeneration

James Phillips
Professor of Regenerative Medicine, UCL

MECH0031
January 2023
Overview

• Introduction to nerve anatomy

• Nerve damage & degeneration
• Regeneration & surgical repair
• Conduit design: biomaterials & tissue engineering
opportunities
• Issues and alternative strategies
Nervous system anatomy
 Peripheral neurons
• Cell bodies located in CNS or
ganglia (e.g. DRGs)
• Very long axons: >1m
• Very thin axons: <1 – 20 µm
• Post-mitotic
• Need to bend and stretch
• Need structural and metabolic
support

Peripheral nerve structure provides the environment required to support and

protect these long thin delicate cells
Fascicle

Epineurium
Perineurium

Endoneurium
Perineurial
cells
Collagen architecture
 Force - strain curves for the nerve core
and the nerve sheath respectively.
Biomechanical anatomy
4.5 Nerve Core
Nerve Sheath
4

3.5

3
Force - Newton

2.5

1.5

0.5

0
0 20 40 60 80 100

Strain - %

Tillett RL, Afoke A, Hall SM, Brown RA & Phillips JB. (2004). Investigating mechanical behaviour at a core-sheath interface
in peripheral nerves. Journal of the Peripheral Nervous System 9, 255-262.

Georgeu GA, Walbeehm ET, Tillett R, Brown RA, Afoke A & Phillips JB. (2004). Nerve retraction: Contributions of
peripheral nerve components and their response to tension. Cell & Tissue Research 320, 229-234.
 Mechanical considerations: Longitudinal tensile heterogeneity

40 30

15
% strain (in situ)

% strain (ex vivo)

30 proximal
20 joint
10 joint

% strain
20 distal

10
5
10

0 0 0
Joint Non Joint Joint Non Joint A B C D
Region of sciatic nerve
Region of median nerve Region of median nerve

Phillips JB, Smit X, De Zoysa N, Afoke A, & Brown RA. (2004). Peripheral nerves in the rat exhibit localised heterogeneity of
tensile properties during limb movement. Journal of Physiology 557, 879-887
 Tensile testing to measure stiffness

1.00
(joint/non-joint)
Stiffness ratio

0.75

0.50

0.25

0.00
Sciatic Median

Conclusion – less stiffness (greater compliance) at joints

 Mean fibril diameter (nm)
Epineurium Perineurium Endoneurium
Median nerve
Joint 64.9 ± 2.9 *** 34.8 ± 1.1 ** 37.3 ± 0.6 ***
Non joint 74.9 ± 2.2 40.7 ± 1.2 42.9 ± 1.0
Sciatic nerve
Joint 77.0 ± 1.2 40.0 ± 1.4 42.5 ± 1.1
Non joint 77.8 ± 0.8 39.1 ± 1.3 42.1 ± 1.1

Rat median nerve: Thinner collagen fibrils at joints

Mason & Phillips (2011) Journal of the Peripheral Nervous System, 16: 261-269
Nerve biomechanics is important

• Critical to repair choices and normal

function (tension causes fibrosis,
adhesion restricts movement)
• Repair differently depending on
anatomical location?
• Artificial replacement tissues need to
match and restore mechanical
integrity
• Poorly understood

DOI: http://dx.doi.org/10.1016/j.cub.2017.01.007
Nerve damage

• Autoimmune diseases
• Cancer
• Compression/trauma
• Diabetes
• Drugs/toxins
• Motor neuron disease
• Nutritional deficiencies
• Infectious disease
 https://doi.org/10.1007/978-3-030-06217-0_1-2
Nerve Tissue Engineering and Regeneration, Reference Series in Biomedical Engineering,
The History of Nerve Repair, Susan Standring (2020) in J. Phillips et al. (eds.), Peripheral
Overview of Wallerian degeneration. (a–e) events occurring within a myelinated peripheral nerve fiber after crush. (a) normal myelinated axon associated
with Schwann cells and enclosed within a continuous basal lamina; (b, c) within 7 days of injury, the axon sprouts from its proximal stump, Schwann cells
distal to the injury proliferate, and the axon and all of its associated myelin sheaths distal to the crush site are degraded and phagocytosed by
macrophages that have penetrated the basal lamina. The neuronal cell body undergoes significant changes, including up-regulation of immediate early
genes (e.g., c-Jun) and regeneration-associated genes (RAGs) that are sometimes recognisable microscopically as chromatolysis. The daughter
Schwann cells in the distal segment line up within the basal lamina forming a Schwann tube (band of Büngner); (d, e) ingrowing axonal sprouts establish
a relationship with the acutely denervated Schwann cells, are myelinated and may connect with the original end organ, depending on the distance from
injury site to target organ. Myelin sheaths are thinner and internodal lengths are shorter than those along the proximal, undamaged axon.
(f–k) events occurring within myelinated axons after transection and subsequent retraction of proximal and distal stumps. (f) three normal
myelinated axons; (g, h) within a week of transection, axon sprouts and co-migrating Schwann cells grow into the interstump gap. Wallerian
degeneration occurs throughout the distal stump; (i) axon sprouts and co-migrating Schwann cells cross the inter-stump gap and the axons enter
Schwann tubes in the distal stump. The possibility that axonal misrouting will occur at this point is almost inevitable; (j, k) axon sprouts that do not
cross the inter-stump gap may form a neuroma associated with the proximal stump. Almost all chronically denervated Schwann cells in the distal
stump ultimately disappear.

The History of Nerve Repair, Susan Standring (2020) in J. Phillips et al. (eds.), Peripheral Nerve Tissue Engineering
and Regeneration, Reference Series in Biomedical Engineering, https://doi.org/10.1007/978-3-030-06217-0_1-2
 Aberrant sprouting at the proximal stump: Neuroma

Deumens et al., Prog Neurobiol 92 (2010) 245-276

• Suggest the kinds of trauma injury that might
cause peripheral nerve damage.
• What other tissues might be injured
simultaneously?
• How would you be able to detect a nerve injury in
a limb?
Peripheral nerve injury statistics (…market
opportunity)

• Main causes: accidents, fractures, lacerations,

wounding, surgery, compression syndromes.
• 3-5% of trauma cases include PNI
• Incidence: 1M in US & Europe, 1.2M in India, 2M
in China
• Noble 1998: 46% of PNI from car crash, mean age 34

• Rosberg 2005: forearm PNI patients 273 sick days

• Magellan 2013: $1.32 – $1.92 Billion market size

• WiseGuy/PBIGP London 2018: $0.5 Billion market size

Primary repair

• If not too much tension, primary end-to-end

repair is best approach
www.jamestgoodrich.net/peripheral.htm

www.wheelessonline.com/ortho/nerve_repair

Epineurial repair
Autograft

• If there would be too much tension (and

therefore fibrosis) with a primary repair, an
autograft can be used.
www.jamestgoodrich.net/peripheral.htm

www.aboutnerve.net
Autograft

• Clinical ‘gold standard’ approach

• Donor site morbidity
• Limited availability
Provides: Schwann cells (autologous, denervated), tissue architecture,
biomechanical properties,
What does an autograftguidance cues
actually provide?
Does not provide: Neurons
Tube

Deumens et al., Prog Neurobiol 92 (2010) 245-276

US Food and Drug Administration /Conformit Europe- approved absorbable nerve

conduits for clinical repair of peripheral and cranial nerves.
Meek MF, Coert JH. Ann Plast Surg. 2008 Apr;60(4):466-72

FDA approved guidance conduits and wraps for peripheral nerve injury:
A review of materials and efficacy.
Kehoe S, Zhang XF, Boyd D. Injury 2012; 43, 553-572
Decellularised allograft
Peripheral nerve repair options
Suture as primary repair Hollow tube/Connector

www.wheelessonline.com/ortho/nerve_repair

Breakdown of nerve repair interventions by gap length levels. Data based on Magellan Medical Technology
Consultants Inc. analysis of the peripheral nerve repair market in the US, 2013

Processed nerve allograft Autograft

How successful are current approaches?
“Despite more than 100 years of intense laboratory and clinical
investigations, results of nerve repairs are somewhat discouraging, with only
50% of patients regaining useful function.”

Lee, Steve K. MD; Wolfe, Scott W. MD. Peripheral Nerve Injury and Repair. Journal of the American
Academy of Orthopaedic Surgeons: July 2000 - Volume 8 p 243-252

“(1) young patients generally do better than old patients; (2) early repairs
do better than late repairs; (3) repairs of single-function nerves do better
than mixednerve repairs; (4) distal repairs do better than proximal
repairs; and (5) short nerve grafts do better than long nerve grafts.”

Sunderland S: Nerve Injuries and Their Repair: A Critical Appraisal. New York: Churchill
Livingstone, 1991.
Conduits as alternatives to autografts:
Properties? Wide range of natural
and synthetic
polymers available.

•Biocompatible & bioresorbable Physical guidance (micro-

scale fibres/channels),
•Cell guidance substrate (core) containment, alignment.
•Schwann cells (or alternatives)
Autologous? Stem cell-
•Doesn’t adhere to surrounding tissue (sheath) derived, cell-free factor
delivery.
•Mechanical support/protection (sheath)
Surface texture & chemistry,
coatings, gels.

Suture compatible,
stiffness/compliance matched to
repair site.
Biomaterials & Tissue Engineering to replace
the autograft?

W. Daly et al. J. R. Soc. Interface

2012;9:202-221

Faroni et al. Advanced Drug Delivery

Reviews, Volumes 82–83, 2015, 160–167
Anisotropic cellular substrates to
support nerve regeneration in experimental
nerve injury models

• Schwann cell-seeded aligned fibres made from micro-structured synthetic

polymers (PGA) Lietz et al., 2006 Biomaterials 27, 1425-1436
• Schwann cells seeded within longitudinally porous cross linked collagen
scaffolds Bozkurt et al. (2009) Biomaterials 30, 169-179
• Differentiated adipose-derived stem cells seeded within decellularised
nerve tissue Zhang et al. (2010) Biomaterials 31, 5312-5324
• Neural stem cells aligned on the luminal surface of patterned polylactide
tubes Hsu et al. (2009) Artificial Organs 33, 26-35
• Strips of Schwann cell-seeded poly-3-hydroxybutyrate
In all these cases the anisotropic structure was achieved through the
use of a pre-aligned scaffold into which cells were seeded
Collagen gel contraction
Engineered neural tissue (EngNT) Patent: Phillips & Georgiou 2013

Schwann cells in
tethered rectangular
collagen gels self-align

Cell and matrix

alignment is stabilised

31
Georgiou et al. (2013) Biomaterials 34, 7335-7343
 Engineered neural tissue
tested in vitro

S100 S100 S100

EngNT-Schwann cell sheets can support
& direct neuronal growth

SCHWANN CELLS NEURONS NUCLEI

Schwann cell line

Adult rat DRG neurons
Nerve repair devices assembled for in vivo tests

rolling

Bundle together Pack into NeuraWrapTM

§ Pre-clinical model: 15 mm gap in GFP rat sciatic nerve, 8 weeks

§ Established to be ‘critical length’
§ Other assembly approaches tested in short gap experiments
§ Athymic ‘nude’ rats allow human cells to be tested
 Engineered neural tissue used to promote
regeneration in rat peripheral nerve

How to move this tissue engineering technology towards the clinic?

• Sources of cells and materials
• Engagement with commercial sector partners and clinicians
• Development of production technology suitable for scale-up

Georgiou et al. (2013) Biomaterials 34, 7335-7343

EngNT can be made using rat adipose tissue-
derived stem cells
(Collaboration with Dr Paul Kingham, Umea, Sweden)
S100
βIII-Tub
S100

Number of neurites in
distal part of device

Georgiou, Golding, Loughlin, Kingham & Phillips (2014) Biomaterials 37, 242-251
EngNT can be made using human dental
pulp-derived stem cells
(Collaboration with Hasselt University, Belgium)

S100
βIII-Tubulin

In vitro model of peripheral

nerve myelination

Martens, Sanen, Georgiou, Struys, Bronckaers, Ameloot, Phillips & Lambrichts (2014) FASEB J 28, 1634-1643.
Translational development of EngNT
M. Georgiou, S.C.J. Bunting, H.A. Davies, A.J. Loughlin, J.P. Golding and J.B. Phillips (2013). Engineered
neural tissue for peripheral nerve repair. Biomaterials, 34, 7335–7343
M. Georgiou, J.P. Golding, A.J. Loughlin, P.J. Kingham and J.B. Phillips (2014) Engineered neural tissue with
aligned, differentiated adipose-derived stem cells promotes peripheral nerve regeneration across a critical
sized defect in rat sciatic nerve. Biomaterials 37, 242-251
W. Martens, K. Sanen, M. Georgiou, T. Struys, A. Bronckaers, M. Ameloot, J.B. Phillips and I. Lambrichts
(2014). Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite
outgrowth in an aligned tissue-engineered collagen construct in vitro. FASEB J, 28, 1634–1643
K. Sanen, W. Martens, M. Georgiou, M. Ameloot, I. Lambrichts & J.B. Phillips (2017) Engineered neural
tissue with Schwann cell differentiated human dental pulp stem cells: potential for peripheral nerve repair?
Journal of Tissue Engineering and Regenerative Medicine doi: 10.1002/term.2249
F. Gonzalez-Perez, J. Hernández, C. Heimann, J.B. Phillips, E. Udina, X. Navarro (2018) Schwann cells and
Mesenchymal Stem cells on laminin or fibronectin aligned matrices support regeneration across a critical size
defect of 15 mm in the rat sciatic nerve. Journal of Neurosurgery: Spine 28:109-118
C. Schuh, A.G.E. Day, H. Reidl & J.B. Phillips (2018) An optimized collagen-fibrin blend enhances the ability
of engineered neural tissue to promote peripheral nerve repair. Tissue Engineering Part A (in press)
A.G.E. Day, K.Singh-Bhangra, C. Murray-Dunning, L. Stevanato & J.B. Phillips (2017) The Effect of
Hypothermic and Cryogenic Preservation on Engineered Neural Tissue. Tissue Engineering Part C 23, 575-
582
C. O’Rourke, A.G.E. Day, C. Murray-Dunning, L. Thanabalasundaram, J. Cowan, L. Stevanato, N. Grace, G.
Cameron, R.A.L. Drake, J. Sinden & J.B. Phillips (2018) An allogeneic ‘off the shelf’ therapeutic strategy for
peripheral nerve tissue engineering using clinical grade human neural stem cells. Scientific Reports 8, 2951
M.L.D. Rayner, A.G.E. Day, K.S. Bhangra, J. Sinden & J.B. Phillips (2021). Engineered neural tissue made
using clinical-grade human neural stem cells supports regeneration in a long gap peripheral nerve injury
model. Acta Biomaterialia 135, 203-213
 EngNT made using ‘off the shelf’ allogeneic
stem cells?
• Clinical grade human neural stem cells
• Advanced Therapy Medicinal
Product (ATMP)
• Good Manufacturing Practice
(GMP) materials and
processes
• Optimal product design and
appropriate preclinical testing
• Regulatory approval and first in
human trial
Autologous v Allogeneic?
• Autologous
– Non-immunogenic
– Possibly lower regulatory and ethical barriers
– Less risk of disease transmission
– Delay between harvesting and treatment
– Variability of efficacy between donors
– Commercially challenging
• Allogeneic
– Off-the-shelf availability for immediate use
– Bulk manufacture of consistent QC product
– Obvious commercial opportunities
– Immunogenicity
– Safety/ethical concerns with some sources (hESC etc)

• Next generation cells?

– Ex vivo gene therapy / engineered cells (safety, phenotype, tracking)
– iPSCs (phenotype, safety, regulatory)
Engineered Neural Tissue (EngNT)
 EngNT Manufacturing development

Cost summary
Cost of Materials £3,360
Cost of Facilities £3,000
Cost of Product Release £12,400
Number of constructs per batch 80
(12 removed for testing and
product failures)
Grand Total (Cost per Construct) £235

Georgiou, Bunting, Davies, Loughlin, Golding and Phillips (2013). Muangsanit, Day, Dimiou, Ataç, Kayal, Park, Nazhat & Phillips (2020). Rapidly
Engineered neural tissue for peripheral nerve repair. formed stable and aligned dense collagen gels seeded with Schwann cells
Biomaterials, 34, 7335–7343 support peripheral nerve regeneration. J Neural Engineering 17, 046036
 Mathematical modelling to optimise and
organise nerve repair constructs

Random 3D neurite
extension with spatially
dependent probability
distribution

CoMPLEX: Centre for Mathematics, Physics and Engineering in the Life Sciences and Experimental Biology
Optimal distribution of therapeutic cells
seeded within constructs?

• Cell seeding induces spatial variations in O2, VEGF and cell density
• O2 diffuses from nearest vascular source and is metabolised by cells, which in
turn proliferate, die and produce angiogenic factors in proportion to local O2
• Cannot currently predict link between seeded cell density, O2 and VEGF, or
exploit this to propose optimal cell seeding
Modelling the impact of different initial cell seeding density
Modelling the impact of
different initial cell
distribution patterns
 Remaining challenges and other approaches
beyond the tube…
• Optimising source and phenotype of therapeutic cells
– autologous v allogeneic?
– temporary trophic support or long term survival/myelination etc?
– cell biology of human nerve injury?
– gene therapy
– macrophages, endothelial cells, fibroblasts…
• Optimal device design - combining tissue engineering &
mathematical modelling
• Development of new in vivo imaging technology
• Measuring outcomes in patients?
Rayner MLD, Brown HL, Wilcox M, Phillips JB & Quick TJ (2019). Quantifying
regeneration in patients following peripheral nerve injury. JPRAS 73, 201-208
Cattin et al. Cell. 2015 Aug 27;162(5):1127-39
Measuring clinical outcomes in patients?
Volumetric MRI

Rayner MLD, Brown HL, Wilcox M, Phillips JB & Quick TJ (2019). Quantifying regeneration in patients following peripheral nerve
injury. JPRAS 73, 201-208
M Wilcox, L Dos Santos Canas, R Hargunani, T Tidswell, H Brown, M Modat, J Phillips, S Ourselin & T Quick (2021). Volumetric MRI is a
promising outcome measure of muscle reinnervation. Sci Rep 11, 22433
Drugs to improve nerve repair
• PPARɣ agonists
• Growth factors
• PI3K pathway activators
1. Following nerve
injury, downstream • Tacrolimus (FK506)
2. Axons in repaired
axons degenerate
nerves can
and muscle function 4. Without treatment,
regenerate slowly
is lost axon regeneration is
(<1mm per day)
slow and limited.
Functional recovery
of muscles is poor

Nerve
injury (cut, Local Delivery
crush
stretch etc)

Nerve
injury Slow axon
5. After treatment
repaired regrowth
regeneration is faster.
Axons This minimises duration
degenerate Dr of muscle denervation,
ug reducing atrophy and
? improving functional
recovery

3. Muscles without
nerve input undergo
atrophy. This can be
irreversible where
there is a prolonged
delay
Problems in nerve repair

• Neuroma formation
• Muscle atrophy
• Damage due to sensory loss
• Fibrosis
• Cortical remodelling
• Poor functional outcome
Alternative approach?
Micro-channel electrode
arrays

Lacour et al., (2009) IEEE Transactions 17

 https://www.youtube.com/watch?v=Y6fug4pzU4Q

https://www.youtube.com/watch?v=WV0bJkk86pw
Gold standard?

• Full restoration of function is unlikely

• Differences in outcome
• Even the perfect repair won’t overcome the
problems of neuronal apoptosis, the delay in
regeneration of more proximal damage, and the
resulting atrophy, damage and cortical
remodelling required.
• Clinical assessment of nerve repair technology?
Summary
• Peripheral nerve structure facilitates neuronal function (support
provided by Schwann cells and cellular/ECM structure,
biomechanical protection)
• Peripheral nerve damage is followed by Wallerian degeneration
(clears debris and encourages regeneration).
• Functional outcome generally poor
• Primary repair, graft, conduit (tube) used clinically.
• Engineered neural tissue has been developed to prototype ATMP
stage using clinically relevant materials and cells
• Key remaining challenge to optimise cells and materials
• Multidisciplinary approach including mathematical modelling, drug
and gene therapies, biomechanics.
Further reading
S. Hall (2005) The response to injury in the peripheral nervous system.
J Bone Joint Surg (Br) 87: 1309-19
Deumens et al. (2010) Repairing injured peripheral nerves: Bridging
the gap. Prog Neurobiol 92: 245-276
Standring S. (2020) The History of Nerve Repair. In: Phillips J.,
Hercher D., Hausner T. (eds) Peripheral Nerve Tissue Engineering
and Regeneration. https://doi.org/10.1007/978-3-030-06217-0_1-2
Taylor & Haycock (2020) Biomaterials & Scaffolds for Repair of the
Peripheral Nervous System. In: Phillips J., Hercher D., Hausner T.
(eds) Peripheral Nerve Tissue Engineering and Regeneration.
https://doi.org/10.1007/978-3-030-06217-0_3-1
The Nervous System, Basic Science and Clinical Conditions (2007).
Michael-Titus, Revest and Shortland. Churchill Livingstone
Phillips (2021) ‘EngNT’ — Engineering live neural tissue for nerve
replacement. Emerg Top Life Sci 5 (5): 699–703.
www.jamesphillips.org