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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
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