Highly-ordered and hierarchical porosity scaffolds for nerve repair

J. SAKAMOTO1 (PH. D.) , D. LYNAM1 (PH. D.), D. SHAHRIARI1, K. KOFFLER2 (PH. D.), P. WALTON (SC. D.) C. CHAN1 (PH. D.),

AND M.H. TUSZYNSKI2,3 (M.D./PH.D.)

1MICHIGAN STATE UNIVERSITY, EAST LANSING, MI 2UNIV. OF CALIFORNIA-SAN DIEGO, LA JOLLA, CA

3DIRECTOR OF NEURAL REPAIR

NIBIB: 1R01EB014986-01 Bioengineered Scaffolds for Spinal Cord Injury

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Outline

•Introduction

•Nerve guidance scaffold design and fabrication

•Acellular drug delivery

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Initial trauma/nerve severance

Peripheral nerve regeneration

Metabolic function: changes from neuro transmittance to wound repair. Wallerian degeneration after 2-4 days clears fragments.

Remaining Schwann cells provide environment to promote axon extension.

Steve K Lee and ScottWWolfe. Peripheral nerve injury and repair. Journal of the American Academy of Orthopaedic Surgeons, 8(4):243–252, 2000.

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1.9% OF THE U.S. SUFFERS FROM SOME FORM OF PARALYSIS1

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Condroitin sulfate proteoglycans and myelin-associated inhibitors cause irreversible scarring.

5 Terry W Hudson, Gregory RD Evans, and Christine E Schmidt. Engineering strategies for peripheral nerve repair. Clinics in plastic surgery, 26(4), 1999.

State-of-the-art scaffold approaches

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Inverted Umbilical Artery Crouzier et al. J Biomed Mater Res 2009;89A:818-28

Braided polylactic co-glyc. acid Tube Bini et al. J Biomed Mater Res 2004;68A:286-95

Silicone Tube Lundborg et al. Exp Neur 1982;76:361-75

Punched Collagen Tube Moellers et al. Tissue Eng Pt A 2009;15:461-72

State-of-the-art scaffold examples

7 S Stokols, J Sakamoto, C Breckon, T Holt, J Weiss, and MH Tuszynski. Tissue Eng. 2006;12:2777-2787

•Continuous linear channels to provide physical guidance, “bridge-the-gap”

•Uniform wall thickness to enable high channel volume/scaffold volume

•Scaffold material: soft for biocompatibility, stiff enough to guide, and eventually degrade

•Timed and sustained delivery of drugs

Our approach: precision, micro-channel scaffolds

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Clinically-relevant scaffold fabrication technology

Requirements: 1. Discrete linear channels 1-2cm in length

2. 100-200mm diameter channels

3. Highly-ordered or uniform wall thickness

4. High channel/lumen volume: 40-80%

5. Compatible with biomaterials

6. Several cm2 of area

7. Precision superficial geometry (match to MRI scan)

8. Degradable

9. Capable of providing drug delivery

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J. Friedman et al., Neurosurgery, 51, 3 (2002) 742-752. (Mayo Clinic, Minnesota)

State-of-the art patterning technology

3D printing

Chen et al. Biomed Microdevices (2011) 13:983–993

Digital micro-mirror-array device (DMD)

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Gilbert et al. Acta Biomaterialia 6 (2010) 2970–2978

L. Flynn, P. Dalton and M. Shoichet, Biomaterials, 24 (2003) 4265-4272. (Univ. Toronto)

Shea et al. Biomaterials (2013).

PLGA: sugar fiber template

pHEMA: PCL fiber template Electrospun PLLA fibers

Fiber templating & electrospun scaffolds

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Macro-scale self-assembled templates

PMMA cladding

PS core

Paradigm Optics Inc.

Lateral dimensions range from: 100nm up to 1mm

Non-polar Solvent Molten Agarose Polar Solvent

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Microchannel Volume Analysis

• Varying template W/MC ratio allows an areal fraction of open microchannels ranging from 0.4-0.79

Solid Line

Dashed Line

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1cm

200um

Scale-up

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Clinically-relevant scaffold fabrication technology

Requirements: 1. Discrete linear channels 1-2cm in length

2. 100-200mm diameter channels

3. Highly-ordered or uniform wall thickness

4. High channel/lumen volume: 40-80%

5. Compatible with biomaterials

6. Several cm2 of area

7. Precision superficial geometry (match to MRI scan)

8. Degradable

9. Capable of providing drug delivery

? ?

?

In vivo cellular drug delivery

S Stokols, J Sakamoto, C Breckon, T Holt, J Weiss, and MH Tuszynski. Tissue Eng. 2006;12:2777-2787

Previous work: Bone marrow stromal cells (BMSC) transfected to

secrete brain derived neurotrophic factor (BDNF)

C4 Dorsal

column model

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Scalebar = 100 mm

Acellular Drug Delivery

• Alternating layers of hydrogen-bonded polymers to deliver drugs or proteins – Layer-by-Layer – Hydrogen bond donor and hydrogen bond acceptor polymers with

protein in-between – In acidic environment, polymer layers assemble. At neutral pH,

polymers slowly disassemble – Can augment release by increasing surface area

Assembly in acidic environment Disassembly in neutral pH environment 17

100nm

Layer-by-Layer Assembly

100nm

Hydrogel Network

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S Mehrotra, D Lynam, R Maloney, KM Pawelec, MH Tuszynski, I Lee, C Chan, JS Sakamoto. Adv Func Mater. 2009;20:247–258

Lysozyme Release

5wt%ag/50wt%sucrose

100nm

5wt%a/65wt%sucrose

100nm

0.01

0.1

1

10

0 1 2 3 4 5 6 7 8 9 10

Lyso

zym

e R

ele

ase

Pe

r D

ay

(µ

g/m

L) -

Lo

g Sc

ale

Day

Optimal BDNF release concentration

50ng/mL

Daily protein release from layer-by-layer

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Brain derived neurotrophic factor (BDNF) Release

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Proliferation AssayNIH 3T3 Fibroblasts

5 Days

Characterizing BDNF bioactivity after release

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E14 Stem cell graft

25 mm 25 mm

200um

Cell

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Summary

Developing scaffold patterning technology High aspect ratio channels, with clinically relevant dimensions

Compatible with hydrogels (agarose and alginate) Demonstrated high channel volume (80%) ? Fabricate biodegradable scaffolds (lasting months)

Developing and integrated drug delivery technology Layer-by-layer technology provides timed and relevant dose response

Combined Layer-by-layer with hydrogels to augment release ? Demonstrate clinically-relevant, bioactive BDNF release

On-going/future work

• Myelination-recruiting support cells • Peripheral nerve repair (closer to clinical relevance)

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Acknowledgements

NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING: 1R01EB014986-01 Bioengineered Scaffolds for Spinal Cord Injury

Dr. Daniel Lynam Dena Shahriari National Science Foundation Graduate Fellowship

K. Koffler (Ph. D.), M.H. Tuszynski (M.D./Ph.D)

Professor Kris Chan Professor Pat Walton

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Brain Derived Neurotrophic Factor Incorporation into LbL

• Hydrochloric acid effects on BDNF, pH 2

S-S

S-S S-S

H+

H+

H+

Cl-

Cl-

Cl-

SH

SH

SH

Folded, active protein Denatured, inactive protein

❌ Does layer-by-layer assembly maintain BDNF activity?

How to stabilize BDNF for LbL? 1. More neutral assembly pH 2. Acetic acid incorporation 3. 4°C Assembly 4. Incorporation of carrier protein BSA 25