Highly-ordered and hierarchical porosity scaffolds for nerve repair
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1 Highly-ordered and hierarchical porosity scaffolds for nerve repair J. SAKAMOTO 1 (PH. D.), D. LYNAM 1 (PH. D.), D. SHAHRIARI 1, K. KOFFLER 2 (PH. D.), P. WALTON (SC. D.) C. CHAN 1 (PH. D.), AND M.H. TUSZYNSKI 2,3 (M.D./PH.D.) 1 MICHIGAN STATE UNIVERSITY, EAST LANSING, MI 2 UNIV. OF CALIFORNIA-SAN DIEGO, LA JOLLA, CA 3 DIRECTOR OF NEURAL REPAIR NIBIB: 1R01EB Bioengineered Scaffolds for Spinal Cord Injury
2 Outline Introduction Nerve guidance scaffold design and fabrication Acellular drug delivery 2
3 Peripheral nerve regeneration Initial trauma/nerve severance 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): ,
4 1.9% OF THE U.S. SUFFERS FROM SOME FORM OF PARALYSIS 1 Condroitin sulfate proteoglycans and myelin-associated inhibitors cause irreversible scarring. 4
5 State-of-the-art scaffold approaches Terry W Hudson, Gregory RD Evans, and Christine E Schmidt. Engineering strategies for peripheral nerve repair. Clinics in plastic surgery, 26(4),
6 State-of-the-art scaffold examples Inverted Umbilical Artery Crouzier et al. J Biomed Mater Res 2009;89A: Braided polylactic co-glyc. acid Tube Bini et al. J Biomed Mater Res 2004;68A: Silicone Tube Lundborg et al. Exp Neur 1982;76: Punched Collagen Tube Moellers et al. Tissue Eng Pt A 2009;15:
7 Our approach: precision, micro-channel scaffolds 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 S Stokols, J Sakamoto, C Breckon, T Holt, J Weiss, and MH Tuszynski. Tissue Eng. 2006;12:
8 Clinically-relevant scaffold fabrication technology Requirements: 1. Discrete linear channels 1-2cm in length mm diameter channels 3. Highly-ordered or uniform wall thickness 4. High channel/lumen volume: 40-80% 5. Compatible with biomaterials 6. Several cm 2 of area 7. Precision superficial geometry (match to MRI scan) 8. Degradable 9. Capable of providing drug delivery 8
9 State-of-the art patterning technology 3D printing J. Friedman et al., Neurosurgery, 51, 3 (2002) (Mayo Clinic, Minnesota) Digital micro-mirror-array device (DMD) Chen et al. Biomed Microdevices (2011) 13:
10 Fiber templating & electrospun scaffolds phema: PCL fiber template Electrospun PLLA fibers L. Flynn, P. Dalton and M. Shoichet, Biomaterials, 24 (2003) (Univ. Toronto) PLGA: sugar fiber template Shea et al. Biomaterials (2013). Gilbert et al. Acta Biomaterialia 6 (2010)
11 Macro-scale self-assembled templates Paradigm Optics Inc. PMMA cladding PS core Lateral dimensions range from: 100nm up to 1mm 11
12 Non-polar Molten Polar Solvent Agarose 12
13 Microchannel Volume Analysis Varying template W/MC ratio allows an areal fraction of open microchannels ranging from Solid Line Dashed Line
14 Scale-up 200um 1cm 14
15 Clinically-relevant scaffold fabrication technology Requirements: 1. Discrete linear channels 1-2cm in length mm diameter channels 3. Highly-ordered or uniform wall thickness 4. High channel/lumen volume: 40-80% 5. Compatible with biomaterials 6. Several cm 2 of area 7. Precision superficial geometry (match to MRI scan)? 8. Degradable? 9. Capable of providing drug delivery? 15
16 In vivo cellular drug delivery Previous work: Bone marrow stromal cells (BMSC) transfected to secrete brain derived neurotrophic factor (BDNF) C4 Dorsal column model Scalebar = 100 mm S Stokols, J Sakamoto, C Breckon, T Holt, J Weiss, and MH Tuszynski. Tissue Eng. 2006;12:
17 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 100nm Assembly in acidic environment Disassembly in neutral ph environment 17
18 Layer-by-Layer Assembly Hydrogel Network 100nm S Mehrotra, D Lynam, R Maloney, KM Pawelec, MH Tuszynski, I Lee, C Chan, JS Sakamoto. Adv Func Mater. 2009;20:
19 Lysozyme Release Per Day (µg/ml) - Log Scale Lysozyme Release 100nm 10 5wt%ag/50wt%sucrose Daily protein release from layer-by-layer 100nm 1 5wt%a/65wt%sucrose 0.1 Optimal BDNF release concentration 50ng/mL Day 19
20 Brain derived neurotrophic factor (BDNF) Release 20
21 Characterizing BDNF bioactivity after release Proliferation Assay NIH 3T3 Fibroblasts 5 Days 21
22 E14 Stem cell graft Cell 25 mm 25 mm 200um 22
23 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) 23
24 Acknowledgements Professor Kris Chan Professor Pat Walton Dr. Daniel Lynam Dena Shahriari National Science Foundation Graduate Fellowship K. Koffler (Ph. D.), M.H. Tuszynski (M.D./Ph.D) NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING: 1R01EB Bioengineered Scaffolds for Spinal Cord Injury 24
25 Brain Derived Neurotrophic Factor Incorporation into LbL Hydrochloric acid effects on BDNF, ph 2 Does layer-by-layer assembly maintain BDNF activity? H+ SH H + SH Cl - Cl - Folded, Denatured, active inactive protein protein S-S SH S-S S-S How to stabilize BDNF for LbL? 1. More neutral assembly ph H+ 2. Acetic acid incorporation 3. 4 C Assembly 4. Incorporation of carrier protein BSA Cl
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