engineering therapies for neural tissue...

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Christine E. Schmidt, Ph.D.

Engineering Therapies for Neural Tissue Regeneration

Biomedical Engineering & Chemical EngineeringThe University of Texas at Austin

Neural Tissue Engineering

PNSRegeneration

CNSRegenerationRegeneration Regeneration

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The Nervous SystemCentral

Gray Matter

Winter, Schmidt (2002). In: Biomimetic Materials and Design. Marcel-Dekker. pp. 375-415.

White Matter

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

Peripheral

Sources of Peripheral Nerve Injury

Traumatic Traumatic

CancerCancer

Auto AccidentsAuto Accidents

InjuriesInjuries

Congenital Congenital DefectsDefects

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Cellular Response to Nerve Injury

PNS CNS

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

PNSRegeneration over long defects (> 3-5 cm)

Key Challenges

g g ( )Appropriate sensory/motor reconnection

CNSGuidance of sufficient axonsCombating glial scar & inhibitory myelinAxon growth past “host-graft” interfaceAppropriate functional reconnections

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Treating Nerve Injury

Goal: Re-establish a continuous pathway for regenerating axons to site of innervation

http://inside.salve.edu/walsh/neuron.jpg

Peripheral Nerve Repair Strategies

Small injuries (< ~1 cm gap): end-to-end surgical repairg p

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

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Larger Defects: Nerve Autograft

Nerve Graft Nerve Graft (e g sural nerve)(e g sural nerve)

Large Nerve Defect (> ~1 cm)Large Nerve Defect (> ~1 cm)(e.g., sural nerve)(e.g., sural nerve)

Injury MuscleNerveCell Body

– Requires two surgeries– “Robs Peter to pay Paul”– Time-consuming/costly procedure

Alternatives: Nerve Guides• Guide regenerating axons• Prevent infiltration of scar tissue• Increase concentration of intraluminal proteins• Regulate diffusion of external macromolecules• Delivery vehicle for proteins, drugs, etc.

NGCproximal

nerve stumpdistal

nerve stumpdirection ofnew axonextension

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Basic Nerve Guide Requirements• Readily formed into conduit• Suturable• Sterilizable• Porous• Low antigenicity• Persists during regeneration

Bi d d bl l t• Biodegradable over long-term• Resists compression or collapse• Pliable (not too rigid)• Contains regeneration stimuli

Graft Reference Autologous Tissue Grafts 1. Nerve grafts (gold standard) (12, 13) 2. Vein grafts (17, 43-45)

3. Muscle grafts (41, 42) 4. Epineurial sheaths (46)

Graft Reference Synthetic Materials 1. Biodegradable synthetic materials Poly(lactic acid) (PLA) (109, 110) Poly(lactic-co-glycolic acid) PLGA (107)

Poly(caprolactone) (111, 113)

Conduit Material

p ( ) 5. Tendon grafts (47) Non-Autologous/Acellular Grafts 1. Immunosuppression w/allografts (373) 2. Acellular allografts and xenografts Thermal decellularization (52, 53, 57) Radiation treatment (54, 58) Chemical decellularization (55, 56) 3. Small intestinal submucosa (SIS) (65, 66, 70) 4. Human amnion (71, 75, 76)

y( p ) ( , ) Poly(urethane) (114) Poly(organo)phosphazene (112) Poly(3-hydroxybutyrate) (116) Poly(ethylene glycol) "glue" (128, 156) Biodegradable glass (117, 118) 2. Electrically active materials Piezoelectric (119) Electrically conducting (120) 3. Non-biodegradable synthetic materials Silicone (122, 127)

Gore-Tex or ePTFE (123-125) Natural-Based Materials 1. ECM protein-based materials Fibronectin (84, 85) Laminin (82, 88) Collagen (90-92) 2. Hyaluronic acid-based materials (95) 3. Fibrin/fibrinogen (96, 97) 4. Other (alginate, agarose...) (99, 100, 102)

Go e e o e ( 3 5)

For cited references, see: Schmidt, C.E., J.B. Leach (2003). Annual Reviews in Biomedical Engineering. 5:293-347.

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What Studies to Date Suggest…

• biocompatible, biodegradable (long-term), Beneficial Properties: Beneficial Properties:

p , g ( g ),nonimmunogenic, suturable, pliable material

• smooth inner lining

• porous outer structure (50 - 100 kDa)

• 3-D, porous, aligned intraluminal matrix

• release of neurotropic and neurotrophic factors

• inherent electrical properties

• support cells or genetically-modified cells

PNS Guidance Conduits in the Clinic• 5 grafts approved for use in humans• All are hollow tubes• Provide simple mechanical support• Use with short defects (< 3-5 cm)

4 mmCol

PLCL

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

PLCL

PGA

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Nerve Structure

www.backpain-guide.com/.../10-1_Nerve_Struct.jpg

Regeneration in a Conduit

Distal Proximal

Day 1: Tube fills with blood proteins

Fibrin cable:Days 2-6: Protein (fibrin) cable formation

Days 7-14: Migration of support cells onto protein cable

Fibrin cable:

• required with empty tube

•cannot form over longer defects

Days 15-28: Axonal elongation (regeneration)Internal matrix

required for longer defects

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Hours

How Nerve Conduits WorkFluid seeps into the void of the conduit.

Days

M th

An hourglass shaped fibrin cable forms.

Cell migration and axonal regeneration occurs, restricted by

Months

Years

the thinnest portion.

Often the resulting tissue is visibly thinner, containing a limited number of regenerated axons.

Increasing Gap Length

Length Limitations of Conduits

At h t l th th fib iAt short gap lengths, the fibrin cable is robust enough to allow regeneration.

Thinning restricts the regenerative space at longer gaps.

Decreasing EEfficacy

The cable does not form when length limits are exceeded. This can result in no regeneration or a neuroma.

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Can We Do Better?


Two Approaches in Our Lab

“Top Down”Modify what exists in

nature

“Bottom Up”Build up from scratch

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“Top-Down” Approach

– Strip native nerve down to basic “skeleton”

– Preserve natural architecture as scaffoldarchitecture as scaffold for new nerve growth

Acellular Nerve Grafts

NerveNerve ScaffoldScaffold

Chemical/Detergent ProcessingChemical/Detergent Processing

GoalsGoals: : -- Remove immunogenic cell componentsRemove immunogenic cell components-- Retain intricate ECM structureRetain intricate ECM structure

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Decell nerve provides 3-D scaffolding to support the body’s own regeneration

Hours

How Acellular Nerve Works

process.

Clean and clear pathways allow cell migration and axonal regeneration.

Axon regeneration is well-

Days

Months distributed throughout the cross-section.

Nerve graft is incorporated into the patient’s own tissue.

Months

Years

Methods of Creating Acellular Grafts

• TemperaturepFragmentation

• RadiationDebris

• Chemical (Sondell Protocol)Selection of Chemicals

Basal Laminae

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Amphoteric and Non-Ionic Detergents Less Destructive to ECM

15) Morphology Basal Laminae Interstitial Endoneurium

5

10

vene

ss S

core

(max

= 1

5 p gy

0Amphoteric Anionic Cationic Non-Ionic

Detergent Category

Effe

ctiv

Cationic and Anionic Detergents Remove More Cellular Material

10

0) Schwann Cell Axon

5

vene

ss S

core

(max

= 1

0 Schwann Cell Axon

0Amphoteric Anionic Cationic Non-Ionic

Detergent Category

Effe

cti

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Optimized Acellular (OA) Graft

- Charge

Anionic Amphoteric

+ Charge

Hudson et al Tissue Engineering 2004

Cell Removal & ECM PreservationNative OA Acellular Graft

100 μm

Cell Removal

100 μm

Structural Preservation

Hudson, Liu, Schmidt (2004). Tissue Eng 10 (9/10), 1346-1358.

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Preservation of Basal Laminae

Native OANerve Graft

10 μm

Freeze Thaw

Sondell(Triton X-100, Na Deoxycholate)

Testing Immunological Response

Isograft (~Autograft) AllograftSame-Strain Cross-Strain

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Immunologically

Accepted

Immunologically

Rejected




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OA Graft: Lack of Immune Response

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ls cells

)4 weeks

2

4

6

8

10

ce o

f Cyt

otox

ic T

-cel

lue

Sta

ined

for

CD8a

+

0Fresh Isograft Fresh Allograft Acellular

IsograftAcellularAllograft

Pre

sen

(% o

f Tis

su

Hudson, Zawko, Deister, Lundy, Hu, Lee, Schmidt (2004). Tissue Eng 10 (11/12): 1641-1651.

Regeneration AssessmentSciaticNerve

Common model: Rat sciatic nerve defect

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Histological Analysis

Immunhistochemistry (stain for axons, Schwann cells, blood vessels, inflammatory cells)

Key Drawback: terminal assay (single data point for an animal)

TEM w/lead citrate and uranyl acetate (visualize myelin, axon counts, fascicle structure)

Proximal Graft Distal

Capacity to Support Regeneration

10mm

RT-97 Sensory Neurofilament Stain

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Regeneration Comparable to Autograft

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14er

atio

n

4

6

8

10

12

o Su

ppor

t Reg

ene

ons

per 1

00 μ

m2 )

0

2

Fresh Graft OptimizedAcelllular Graft

Freeze/ThawAcellular Graft

SondellAcellular GraftC

apac

ity to

(Axo

Hudson, Zawko, Deister, Lundy, Hu, Lee, Schmidt (2004). Tissue Eng 10 (11/12): 1641-1651.

Functional Analysis

Walking track analysis & Sciatic Function Index (SFI)(Bain et al., Plast. Reconstr. Surg., 1989, 83:129-138).

Video tape or ink/paper methods to document foot anatomy

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Sciatic Function Index Calculation

TS Toe SpreadPL Print LengthIT(S) Intermediary Toe SpreadE = ExperimentalN = Normal

SFI 0 (-8.8): normal function

SFI -100: complete impairment

Assessing Functional Recovery

SFI 0: normal function

SFI -100: full impairment

E Experimentalp

N Normal

de Medinaceli et al. Exp Neurol 1982, Bain et al Plastic Reconstr Surg 1989

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Functional Recovery Same as Direct Suture Repair

Functional RecoveryWeek

-60

-50

-40

-30

-20

-10

00 5 10 15 20 25 30 35 40 45

Week

unct

ion

Inde

x (S

FI)

Primary Suture (n=5)OA Graft (n=3)

-100

-90

-80

-70

Scia

tic F

u

Processing Human Nerve

Unprocessed

500 μm 100 μm

Unprocessedhuman nerve

H&E staining reveals structural preservation and cell removal

500 μm 100 μm

Processedhuman nerve

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Clinical Translation

• Acell graft technology Licensed to AxoGen inLicensed to AxoGen in Florida

• Implanted now in 3000+ patients in 250+ centers in US

AxoGen’s AVANCE human nerve graft handles similarly to unprocessed nerve (i.e., the autograft)

We Can Do Better…


BUT ... Longer defects?

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“Bottom-Up” Approach

Biochemical SignalsBiochemical Signals

Nerve Nerve Conduit

Incorporation ofSupport Cells

Biodegradability/Porosity

IntraluminalChannels

Oriented NerveSubstratum

ElectricalActivity

Hyaluronic Acid (HA)

O

H

O

H

HO

H

HOHH

O

COOHO

H

HO

H

H

HNHH

OH

O

O

H

HO

H

OHHO

COOHO

H

HOH

NHH

OH

O

Ubiquitous ECM componentVital role in wound healing (“scar-free” healing)Non-immunogenic FDA-approved

H H H HOCH3

H H H HOCH3 2500-10000

FDA approvedBacterial fermentation production for scale-upCan be easily modified mechanically, chemically,…Non-adhesive “blank slate”Enzymatically degradable

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Photocrosslinked Hyaluronic Acid

Low Swelling HA FilmsHigh Swelling HA Hydrogels

Unswollen Saline Swollen Water Swollen

Modulate:-Swelling-Degradation-Mechanical Properties

Mimicking Nerve Structure

M l ( 10 10 )Macro-scale (~10 μm – 10 mm):Nerve trunk, fascicles

DMD-μSL

Micro-scale (~0.5 - 1 μm):A


AxonsMultiphoton fabrication

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Digital Micromirror Device (DMD)

Lu et al., JBMR, 2006Developed by Yi Lu and Dr. ShaoChen Chen

DMD-μSL-Generated HA Scaffolds

1 mm 1 mm

1 mm 1 mm

In collaboration with Dr. Shaochen Chen, Mechanical Eng., UT Austin

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Gradient Scaffold

1 mm

(unpublished data)

Mimicking Nerve Structure

M l ( 10 10 )Macro-scale (~10 μm – 10 mm):Nerve trunk, fascicles

DMD-μSL

Micro-scale (~0.5 - 1 μm):A


AxonsMultiphoton fabrication

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Role of Physical vs. Chemical Cues

Microchannels vs. Laminin

25 μm 12 μm

Gomez, Chen, Schmidt (2007). J. R. Soc Interface. 4(13): 223-233.

Physical cues are preferred over chemical cues for axon initiation (polarization)

70 % Physical Cues (microchannels)30 % Chemical Cues (NGF or Laminin)

Single Photon Excitation

Multiphoton Excitation (MPE)

Multiphoton Excitation

Brad AmosMRC, CambridgePhotocrosslinkable amino acids:

Tyr, Trp, His, Cys

In collaboration with Dr. Jason Shear, Chemistry, UT Austin

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Multiphoton-Excited Protein Crosslinking

MPEMovie.avi.mov

•Inherently 3D geometries

•Submicron resolution

Protein Microstructure Fabrication

10 µm

•Complex geometries possible

•Chemical AND topographical

10 µm

Kaehr, Shear, PNAS, 2004

Images courtesy of Rex Nielson and Jason Shear

10 µm

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Creating Structures in 3D Hydrogels

Make hydrogels and soakMake hydrogels and soak with protein solution

Crosslink protein structures into hydrogels

Rinse out uncrosslinked solution

3D Protein Structures in HA Gels

Z=40 µm

Z=40 µmZ=20 µm

Z=0 µm

µ

Z=20 µm

50 µm 50 µm

Z=0 µm

50 µm

50 µm 50 µm

50 µm

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Precise 3D Submicron Architecture

20 μm

3D structures inside hydrogels are stable for several months in solution.

NPCs on Protein Structures on HA

Hippocampal progenitors on YIGSR‐modified structures; overlay of phase and immunostaining for ß‐III tubulin

Seidlits, Schmidt, Shear. (2009) Advanced Functional Materials.

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“Bottom-Up” Approach

Biochemical SignalsBiochemical Signals

Nerve Nerve Conduit

Incorporation ofSupport Cells

Biodegradability/Porosity

IntraluminalChannels

Oriented NerveSubstratum

ElectricalActivity

Bottom Line: We Can Do Better…


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Neural Tissue Engineering

PNSRegeneration

CNSRegenerationRegeneration Regeneration

Applications for Spinal Cord Injury

Schwann cell i j ti i t

1 mm

injection into Acellular grafts

“Bypass” graft yp garound injury site and scar tissue

(in collaboration with Dr. Jerry Silver, Case Western)

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Applications for Spinal Cord Injury

Schwann cell i j ti i t

1 mm

injection into Acellular grafts

“Bypass” graft Graftyp garound injury site and scar tissue

(in collaboration with Dr. Jerry Silver, Case Western)

Graft

Lesion

Spinal Cord

Questions?

http://www.bme.utexas.edu/faculty/schmidthttp://www.bme.utexas.edu/faculty/schmidt

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Relevant Review ArticlesBelkas JS, Shoichet MS, Midha R (2004). Peripheral nerve regeneration through guidance tubes.

Neurol Res. 26(2):151-60.

Schmidt CE, JB Leach (2003). Neural Tissue Engineering: Strategies for Repair and Regeneration Annual Reviews in Biomedical Engineering 5:293-347Regeneration. Annual Reviews in Biomedical Engineering. 5:293 347.

Winter JO, CE Schmidt (2002). Biomimetic strategies and applications in the nervous system. In: Dillow, A., Lowman, A., ed. Biomimetic Materials and Design: Biointerfacial Strategies, Tissue Engineering, and Targeted Drug Delivery, Marcel-Dekker. pp. 375-415.

Geller HM, Fawcett JW (2002). Building a bridge: engineering spinal cord repair. Exp Neurol. 174(2):125-36.

Evans GR (2001). Peripheral nerve injury: a review and approach to tissue engineered constructs. Anat Rec. 263(4):396-404.

Tresco PA (2000). Tissue engineering strategies for nervous system repair. Prog Brain Res.128:349-63.

Evans GR (2000). Challenges to nerve regeneration. Semin Surg Oncol. 19(3):312-8.

Bellamkonda, R.V., P. Aebischer (2000). Tissue Engineering in the Nervous System. In: The Biomedical Engineering Handbook. CRC Press, pp. 122-1 to 122-19.

Hudson TW, GRD Evans, CE Schmidt (1999). Engineering strategies for peripheral nerve repair.Clinics in Plastic Surgery. 26(4): 617-628.

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