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Guiding Neuronal Cells in 3- dimensions

Guiding Neuronal Cells in 3- dimensions. Derek Hernandez University of Texas at Austin. Motivation. Hundreds of thousands Americans affected by PNS and CNS nerve damage Nearly half result in irreversible paraplegia due to deficient treatment strategies

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Guiding Neuronal Cells in 3- dimensions

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  1. Guiding Neuronal Cells in 3- dimensions Derek Hernandez University of Texas at Austin

  2. Motivation • Hundreds of thousands Americans affected by PNS and CNS nerve damage • Nearly half result in irreversible paraplegia due to deficient treatment strategies • Current in vitro models are unable to comprehensively study nerve regeneration

  3. Current treatment strategies • Reattachment of the nerve • Autologous nerve graft • Decellularized nerve • Synthetic nerve tubes Leach, JB. And Schmidt, CE. Ann Rev Biomed Eng. 2003. • Problems • Degradation • Swelling • Poor axonal targeting or dispersion • Lack microarchitectural supports De Ruiter, GCW. Biomat. 2010

  4. Background • Durotaxis Chemotaxis Engler, AJ. et al. Cell. 2006. Jeon, NL. et al. Nature Biotech. 2002.

  5. What we have achieved Seidlits, SK. et al. AFM. 2009.

  6. Multiphoton chemistry Kaehr, B. 2007 Kaehr, B. PNAS. 2008 Nielson, R. et al. Small. 2009

  7. Research strategy • Aim 1: Create tunable gradients of neural cues (e.g. Laminin derived IKVAV peptide) on planar and 3D surfaces • Aim 2: Create gradients of mechanical cues (e.g. stiffness) on planar and 3D surfaces • Aim 3: Optimize schwann cell migration and DRG patterning rates using chemical and mechanical cues

  8. Aim 1 • Aim 1: Create tunable gradients of neural cues (e.g. Laminin derived IKVAV peptide) on planar and 3D surfaces Growth cones migrate in direction of higher immobilized IKVAV concentration Adams, DN. et al. J Neurobio. 2005.

  9. Objective λ = 345 Benzophenone *Dorman, G. and Prestwich, GD. Biochemistry. 1994. Hypolite, CL. Bioconj. Chem. 1997.

  10. Experimental methods Fabricate microstructures of BSA on glass (400 mg/ml BSA and 4-10 mMphotosensitizer) Functionalize with benzophenone-biotin using a secondary scan Incubate in Neutravidin-fluorophore overnight Courtesy of Stephanie Seidlits Kaehr, B. 2007

  11. Immobilized chemical gradients 15 65 10 mW step gradient 15 mW step gradient 15 60 15 60

  12. Gradients continued 20 110 80 20 • We can immobilize chemicals in arbitrary geometries and a variety of slopes using a collection of variables

  13. Assessing structural deformation

  14. More AFM

  15. 3-Dimensional functionalization *Courtesy of Stephanie Seidlits

  16. Conclusions • We can immobilize chemical gradients of arbitrary geometries with sub-micron 3D spatial resolution • Chemical functionalization does not alter mechanical features of the matrix (*preliminary results)

  17. Future directions • 3D immobilization • Complete more analysis using the AFM • Force mapping

  18. Acknowledgements • Dr. Christine Schmidt • Dr. Jason Shear • Dr. Stephanie Seidlits • Dr. Eric Ritchsdorff • Eric Spivey

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