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Engineering & Biotechnology Strategies for Nerve Repair

Engineering & Biotechnology Strategies for Nerve Repair. Christine E. Schmidt Depts. of Biomedical & Chemical Engineering. Functional Replacement. Regeneration. Cochlear implant. Neural Engineering Goals. Requires two surgeries “Robs Peter to pay Paul” Time-consuming/costly procedure.

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Engineering & Biotechnology Strategies for Nerve Repair

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  1. Engineering & Biotechnology Strategies for Nerve Repair Christine E. Schmidt Depts. of Biomedical & Chemical Engineering

  2. Functional Replacement Regeneration Cochlear implant Neural Engineering Goals

  3. Requires two surgeries “Robs Peter to pay Paul” Time-consuming/costly procedure Nerve Graft (e.g., sural nerve) Large Nerve Defect (> ~1 cm) The Autologous Nerve Graft

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

  5. Role of Electrical Stimuli

  6. Polypyrrole (PPy) • Inherently conductive • Easy & inexpensive to synthesize • Flexibility to alter properties via dopant (X-)

  7. In Vitro Electrical Stimulation 90% increase in median neurite length A = PC-12 cells on PPy B = PC-12 cells on PPy + 100 mV CE Schmidt, VR Shastri, JP Vacanti, R. Langer. PNAS 94: 8948, 1997.

  8. 10 mm 8 weeks regeneration time Distal Nerve Stump Regenerated Nerve Cable Proximal Nerve Stump sciatic nerve defect in rat In Vivo Nerve Regeneration

  9. PPy Modification is Limited  Modification using dopants is restricted to anions of relatively small size

  10. Commercially available phage library with 1012 random peptide inserts Specific binding to surface Incubation with surface PPy PPy 12-mer peptides Repeat interaction pH elution Automated DNA sequencing Binding sequences Amplification Selecting PPy-Binding Peptides

  11. PPy Binding Sequence Isolated for PPyCl “T59 clone” 100 mm 100 mm Random phage (fluorescent tagged anti-phage Ab) T59 phage (fluorescent tagged anti-phage Ab)

  12. T59 Phage Binding Specificity AB Sanghvi, KPH Miller, AM Belcher, CE Schmidt (submitted)

  13. T59 Peptide Binding 100 mm T59 on PPyCl T59 on Polystyrene

  14. T59-RGD Promotes PC12 Cell Adhesion PPyCl + T59-RGD no serum PPyCl no serum 100 mm

  15. Binding Comparison (AFM) n = 30 measurements/sample

  16. Key Amino Acids in T59 T59  THRTSTLDYFVI RT59  IVFYDLTSTRHT BT59  GGGGGGLDYFVI T59HR  TGGTSTLDYFVI T59D  THRTSTLGYFVI FT59  THRTSTGGGGGG 2.50 M) T59 m 2.00 RT59 BT59 1.50 T59HR Surface Coverage ( T59D 1.00 FT59 0.50 0.00 0.00 1 5 15 m Input Conc. ( M)

  17. Acellular Nerve Grafts Scaffold Nerve Processing

  18. Detergent Processing Acellular Graft Native Cell Removal 100 mm Structural Preservation 100 mm TW Hudson, S.Y. Liu, CE Schmidt. Tissue Eng (in press)

  19. Immune Response TW Hudson, … CE Schmidt. Tissue Eng 10 (11/12): 1641-1651, 2004.

  20. Capacity to Support Regeneration 10mm Distal Proximal Graft RT-97 Sensory Neurofilament Stain

  21. Regenerative Capacity TW Hudson, … CE Schmidt. Tissue Eng 10 (11/12): 1641-1651, 2004.

  22. Functional Evaluation

  23. Functional Replacement Regeneration Cochlear implant Neural Engineering Goals

  24. Prosthetics Retinal Implant. Boston Retinal Implant Project http://www.bostonretinalimplant.org/ Therapeutics Microelectrode Arrays. P. Fromherz, et.al. PNAS, 98, p. 10457. Biosensors Electrical Stimulation of Cells Deep Brain Stimulator http://www.medtronic.com

  25. In Vivo labeling ME Akerman et. al. PNAS. 99, 12617, 2002. Unique optical properties Labeling Multi-Labeling Bruchez, Jr., AP Alivisatos, et. al. Science 281, 2013, 1998. Conjugation to NeurotransmittersSJ Rosenthal et. al. JACS 124, 4586, 2002. Quantum Dots 20 Å TEM of CdS quantum dots (D ≈ 30-45 Å) - Size Tunable Fluorescence - Photostability - Narrow Bandwidth

  26. Dipole Moment Y Wang, N Herron. J Phys Chem 95:525, 1991. HEAT Qdot-Cell Properties Light 380 nm Electron Transfer Y Nosaka, et.al. Langmiur 11(4): 1170, 1995. e- + - Cell Quantum Dot Heat Transfer SR Sershen, et. al. JBMR 51: 293, 2000.

  27. Quantum Dot Dipole Moment Hypothesis: Dipole moment strong enough to elicit membrane potential change & open voltage-gated channels CdSe dipole moment ~ 32 Debye Alivisatos, et. al. J Phys Chem 97:730, 1992.

  28. QUANTUM DOT 3 nm C G G A G G D R S RGDS PEPTIDE INTEGRIN RECEPTOR LIPID BILAYER Peptide Attachment to Cells. Quantum Dot-Neuron Interfaces Brightfield Fluorescence (ex 388 nm): Dots -> yellow Cell -> blue B 20 mm Quantum Dot Neuron Interfaces formed with Peptides. JO Winter, TY Liu, BA Korgel, CE Schmidt. Adv Mat 13(22): 1673, 2001.

  29. The Students http://www.bme.utexas.edu/faculty/schmidt

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