MATERIALS FOR PERIPHERALNERVE REGENERATION

By,

Sriram Sankar

CONTENTS

INTRODUCTION

GUIDANCE THERAPIES

BIOMOLECULAR THERAPIES

CELLULAR THERAPIES

ADVANCED THERAPIES

FUTURE & ROAD AHEAD

REFERENCES

NERVOUS SYSTEM Conducts and

interprets signals as well as provides excitatory stimuli to the PNS

Innervate muscle tissue, transmitting sensory and excitatory input to and from the Spinal

column.

CELLULAR COMPONENTS OF NERVOUS SYSTEMS

Typical Neuron:

I. Cell Body(Soma)

II. Dendrites

III. Axons

IV. Nissl Bodies

V. Synaptic terminals

VI. Post synaptic cleft

CELLULAR COMPONENTS OF NERVOUS SYSTEMS Neuroglial Cells: Supporting cells for

Neurons.

Nerve Injury and Regeneration PNS Nerve Injuries(Fig. a)o Wallerian Degeneration

Given an injury achieving total transection of a nerve:

a) Cytoskeletal breakdown

b) Cell membrane dissolution

c) Swelling of proximal stump

d) Shedding of myelin sheath

e) Cleanup by phagocytotic cells

CNS Nerve Injuries (Fig. b)

a) Glycoproteins based inhibition

b) Slower macrophage infiltration

c) Reactive astrocytes forming Glial

scars. Schmidt, Leach (2003). Annual Reviews inBiomedical Engineering 5:293-347.

Nerve Injury Treatment

Group Fascicular SutureEpineural Suture

+ Single surgery - small defects

+ large gaps

End-to-End reconnection

Medial Antebrachial cutaneous nerve

SuperficialradialsensoryMedial nerve

Sural nerve

- donor site func. loss

Autologous Nerve Grafts

NEED FOR GUIDANCE THERAPIES

Physical Guidance of Axons –important for nerve repair1. In 1960’s, Millesi pioneered to accurately align nerve

fascicles in the direct resection of nerve ends, with improved Outcomes & also showed nerve grafts reduced tension and enhanced recovery

2. Later research then proved that physical guidance along with biochemical signals are critical for nerve repair.

Currently autologous nerve graft is the gold standard for nerve repair.

Usage for “Nerve guides or Conduits”:a) Direct axons sprouting from the proximal end

b) Provide a conduit for diffusion of growth factors secreted by nerve end

c) Prevent scar tissue infiltration

NERVE CONDUIT MATERIALSNATURAL MATERIALS AND GRAFTS SYNTHETIC MATERIALS

Autologous tissue grafts Nerve grafts (gold standard) Vein grafts Muscle grafts Epineurial sheaths Tendon grafts

Nonautologous grafts 1. Immunosuppression with allografts 2. Acellular allografts and xenografts 3. Small intestinal submucosa

Human amnion ECM protein-based materials

Fibronectin Laminin Collagen Hyaluronic acid-based materials Fibrin/fibrinogen

Other materials (alginate, agarose )

Biodegradable synthetic materials

Poly(lactic acid) (PLA) Poly(lactic-co-glycolic acid) PLGA Poly(caprolactone) Poly(urethane) Poly(organo)phosphazene Poly(3-hydroxybutyrate) Poly(ethylene glycol) “glue” Biodegradable glass

Electrically active materials Piezoelectric Electrically conducting

Non biodegradable synthetic materials

Silicone Gore-Tex or ePTFE

NERVE CONDUIT DESIGN

Design RequirementsSuturable Sterilizable Low antigenicity Persists during regeneration

Biodegradable long-term Resists compression/collapse Pliable

PNS CONDUITS IN CLINIC 5 grafts approved for use in humans All are hollow tubes Provide simple mechanical support Use with short defects (< 3-5 cm)

Col

PLCL

PGASchlosshauer et al. (2006). Neurosurgery 59;740-

748.

Improved Results obtained using Natural Materials

Silicone tubes+laminin+fibronectin+collagen improved results compared to bare silicon tubes(control) 10mm sciatic rat nerve.

Oriented Fibronectin mats for 10 mm nerve defects in rats, results close to that of the nerve autograft.

Collagen tubes containing a porous collagen-glycosaminoglycan matrix suitable for Schwann cell and neurite migration & enhances nerve repair.

Current studies:a. Modulation of fibrin gels either using magnetic fields

to align the polymer fibers & provide appropriate biomolecules to enhance neurite extension.

b. Laser guided orientation on natural polymer fibres.

Improved Results obtained using Natural Materials

PGA,PLA,PLGA –processed into foams and seeded

with Schwann cells. PEG used to “fuse”membranes of severed nerve ends cross-linked PEG hydrogels + growth factors to mimic

the ECM are under development.

Poly(L-lactic acid) foam nerve guidance channels.

PC12 cells extending neurites on a PEG hydrogel + RGDS covalently incorporated into the material.

SYNTHETIC NERVE CONDUITS

FIBRIN Scaffold Orientation ( ROBERT LANGER LAB)

PLGA Conduits

PLGA Conduits

Talac, Friedman, Moore, Lu, Jabbari, Windebank,Currier, Yaszemski (2004). Biomaterials. 25:1505-10.

PLGA-NGF Conduits

Reinforced PHEMA ConduitsKatayama…Shoichet (2006). Biomaterials 27:505–518.

Yang, De Laporte, Rives, (2005). J Control Release. 104:433-46.

ADVANCES IN CONDUITS Mimicking the Spinal Cord Architecture

Moore … Yaszemski, (2006). Biomaterials 27:419–429.

Chitin Tubes with PLGA Microspheres

PHEMA Tubeswith PLGAMicrospheres

Shoichet (2006). Journal of Controlled Release 110:400– 407.

Piotrowicz, Shoichet, Biomaterials (2006).

27:2018–2027.

BIOMOLECULAR THERAPIESThe Neurotrophins include :

a. Nerve growth factor (NGF), b. brain derived Neurotrophic factor (BDNF),c. neurotrophin-3 (NT-3), and neurotrophin-

4/5 (NT-4/5). Other factors of importance are Ciliary

Neurotrophic factor (CNTF), Glial cell line-derived growth factor

(GDNF), Acidic and Basic fibroblast growth factor

(aFGF, bFGF).

ROLE OF NEURONAL FACTORS Neural response

1. M.N survival

2. M.N outgrowth

3. S. N survival

4. S.N outgrowth

5. S.C regeneration

6. PNS regeneration

7. Sensory nerve growth across the PNS & CINS

Neurotrophic factors

1. BDNF, NT-3, NT-4/5, CNTF, GDNF.

2. Same as above

3. NGF,NT-4/5,GNDF

4. NGF,BNDF,NT-3

5. NGF, NT-3, CNTF, FGFs

6. NGF, NT-3, NT-4/5,

CNTF, GDNF, FGFs

7. NGF, NT-3,GDNF,

BIOMOLECULE DELIVERY Earlier– osmotic pumps & Silicon

reservoirsToday- Polymer Matrices,

Microspheres & Gene therapy.Finite reservoirs Gene

Therapy( Viral & Non-Viral)[ issue of inflammation& safeness]

Cell transfection of DNA using gene guns.

Lipoplexes and cationic lipidsCell specific targetted ligands &

GAMS allow repeated transfection throughout neural outgrowth

INTRINSIC NEURONAL FACTORS Manipulation of RAG’s, neuronal cytoskeletal

elements and anti-apoptosis. RAG’s( GAP-43 & CAP-23) reappear during

regeneration(overexpression+ coordination) Actin polymerisation and rearrangement(migration

of fibroblasts & extension). Actin accesory proteins(gelsolin) important role. Anti-apoptosis factor bcl-2(axonal growth) Adhesion molecules( L1,NCAM,N-cadherin) Axon guidance and path finders

(semaphorins, slits, netrins and ephrins) Synaptogenic factors- Agrin, S-laminin & ARIA

CELLULAR THERAPIES Glial cells, OEC’s, Stem cells & G.M cells. Schwann cells- ECM, cell adhesion molecules,

integrins & neurotrophins+ synapse formation. Schwann cell+ demylination= regeneration.

Challenges- axons fail to leave Schwann cells. Remylination beyond injury Exacerbate chondroitin sulphate( scar formation)

Macrophages= angiogenesis + Schwann cell infiltration + axonal regeneration.

Conflicting report discussion. OEC ~ Schwann, astrocytes migrate along with

axons and produce neurotrophins and CAM’s.a. Provide permissive substrate axonal growth through scars

b. Coexist with astrocytes, however loss of smell for specimen.

STEM CELLS & G.M CELLSNeural stem cells Neurons, Astrocytes & oligodendrocyes (2-5 weeks in rat spinal cord)

Control over stem cell diff & environment control.

Glial progenitor cells not thoroughly explored.

GM Fibroblasts for delivery of neurotrophins.

Molecular “on switches”control NGF gene expression via tetracyline-responsive promoters.

Coloured markers for tracing OEC’s or use transgenic animals expressing a modified xenogenic proteins which acts as a source of modified OEC’s

ADVANCED THERAPIES Complex nerve guides- magentic polymer

alignment, injection moulding, phase separation, solid free form fabrication & ink-jet polymer printing

Foams-amorphous & irregular- not good for guidance, so longitudinal aligned matrices conduits.

One approach sub chambers and lamina to mimic fascicular structure Li,

Liu, Hoffman-Kim (2007)

anti-CS anti-LN

NEURONAL INGROWTH

Gomez, Li, Chen, Schmidt (2007). Biomaterials 28: 271-284.

Gomez., Chen, Schmidt (in press). J. Royal Society inteface

ADVANCED THERAPIESPLGA- Collagen conduits filled with

collagen fibres showed 80mm nerve defects to be treated in dogs.

Not well controlled because of handmade fabrication techniques and no scale up possible.

Seidlits, Shear, Schmidt (2006). BSA & LN LINES CREATED BY 2- PHOTON LASER ON PEG & HA.

ADVANCED THERAPIES Magentic aligned Fibrin and collagen fibres better off

compared to random fibres. PLGA-Injection moulded thermally induced phase

transition process longitudinal aligned channels channels

“Two-phase” conduit for spinal cord repair.a. Inner portion emulated the gray matter and was

composed of a porous polymer seeded with neural stem cells, which provided trophic support.

b. outer portion mimicked the white matter with long, axially oriented pores for axonal guidance and radial porosity to allow for fluid transport while inhibiting the in growth of scar tissue.

c. inner-oriented pores were created from poly(lactico-glycolic acid) using a solid-liquid phase separation technique.

SOLID FREEFORM FABRICATION Produce features as small as 6 microns. 3DP employs powder processing in the

construction of devices in a layer wise manner 3DP, capable of fabricating from a computer

model and can handle complex features, such as internal walls, porosity gradients, tortuous channels, and multiple material regions.

Printing or scaffold preparation can be done. Scaffolds created using SFF can contain

biomimetic internal architectures that prove valuable for tissue engineering.

high cost and inability to incorporate biological components under processing conditions.

INK JET PRINTING OF POLYMERSPrecise delivery of polymer solutions.3D structures with desired thickness,

dimensions, incorporation of biomolecules possible.

Micro Fab. have generated bifurcated degradable polymer tubes that have ridges

With permission-MicroFab Corporation Schmidt, Leach (2003). Annual Reviews in Biomedical Engineering 5:293-347.

NANO STRUCTURES THE ANSWER ?

Self-assembledpeptide amphiphilenanofiber scaffolds

Silva (2006). Nature ReviewsNeuroscience, 7: 65-74

NEURAL NEO-TISSUES ??

Mahoney, Saltzman (2001). Nat Biotechnol. 19:934-9.

ISSUES & ROAD AHEAD ….Combination approachesNanometer-scale materialsNeural progenitor/stem cellsModulation of internal cellular factors

(cAMP levels, GTPases, GAP-43)

Systematic studies on cue combinations

Consistency with regeneration models

TRANSLATION

REFERENCESMahoney, Saltzman (2001). Nat Biotechnol.

19:934-9.Silva (2006). Nature Reviews Neuroscience, 7:

65-74Gomez, Li, Chen, Schmidt (2007). Biomaterials

28: 271-284.Gomez., Chen, Schmidt (in press). J. Royal

Society interfaceYaszemski, (2006). Biomaterials 27:419–429Shoichet (2006). Journal of Controlled Release

110:400– 407Piotrowicz, Shoichet, Biomaterials (2006)

27:2018–2027.Talac, Friedman, Moore, Lu, Jabbari, Windebank,

Currier, Yaszemski (2004). Biomaterials. 25:1505-10.

Yang, De Laporte, Rives, (2005). J Control Release. 104:433-46.

Schlosshauer et al. (2006). Neurosurgery 59;740-748.

Schmidt, Leach (2003). Annual Reviews in Biomedical Engineering 5:293-347

THANK YOU