1. Engineering Multi-Functional Three- Dimensional Nervous Tissue Constructs Advisor: Dr. Bryan J. Pfister Presenter: Mevan Siriwardane, C 2 PRISM Research Fellow Advisor: Dr. Bryan J. Pfister Presenter: Mevan Siriwardane, C 2 PRISM Research Fellow

2. Development and Growth  Axon Stretch Growth  Mechanical tension in nerves is natural during development in most species  Axon Stretch Growth  Mechanical tension in nerves is natural during development in most species

3. Background  Nerve Guidance Conduits for Treating Nerve Injury  Hollow tubes used in support of nerve regeneration  Nerve Guidance Conduits for Treating Nerve Injury  Hollow tubes used in support of nerve regeneration Questions: What is tissue engineering?

4. 2D Axon Stretch-Growth  Building a Nerve Tissue Transplant: “Axon Stretch-Growth” 2-D Stretch-Growth

5. 2D Axon Stretch-Growth

7. 3D Axon Stretch-Growth  Building a Nerve Tissue Transplant: “Axon Stretch-Growth” 3-D Stretch-Growth

8. 3D Axon Stretch-Growth  Design Considerations  Sustaining stretch-grown axons in a gel  Design Considerations  Sustaining stretch-grown axons in a gel

9. 3D Axon Stretch-Growth  Axons crossing through mesh!

10.  Axons also grow along the fibers of the nylon mesh!!! 3D Axon Stretch-Growth

11. Axon Outgrowth on 2D and 3D Collagen Substrates  Evaluation of Candidate Materials for Nerve Tissue Construct  Rat Tail Collagen Type I  Bovine Tendon Collagen Type I  Study Axon Growth Behavior  2D coating on dishes (Collagen)  3D Hydrogels (Collagen)  Evaluation of Candidate Materials for Nerve Tissue Construct  Rat Tail Collagen Type I  Bovine Tendon Collagen Type I  Study Axon Growth Behavior  2D coating on dishes (Collagen)  3D Hydrogels (Collagen)

12. Axon Outgrowth on 2D Rat Tail and Bovine Tendon Collagen

13.  Morphology Differences b) DRG on Rat Tail Collagen Type I a) DRG on Bovine Tendon Collagen Type I

14. 2D vs. 3D Axonal Outgrowth

15. Hypothesis  The incorporation of uni-axial fibers to soft 3D collagen gels will provided a rigid framework for guiding directional outgrowth for regenerating axons

16. Axon Growth on Fibers a) DRG on Collagen Fibers b) DRG on PLLA Fibers

17. Axon Growth on Fibers PLLA Fiber Collagen Fiber

18. Axon Outgrowth Rate on Collagen Fibers

19. Collagen Bulk Fibers vs. Collagen-Coated PLLA Fibers

20. Future Directions a) DRG on Thick PLLA Fiber b) DRG on Thick Collagen Fiber c) DRG on Thin PLLA Fiber d) DRG on Thin Collagen Fiber Fiber Diameter and Molecular Orientation

21. Future Directions a) DRG on Thick PLLA vs. Collagen Fiber b) DRG on Thin PLLA vs. Collagen Fiber Preferential Growth

22. Future Directions a) DRG on Multi-filament Thin PLLA Fibers b) DRG on Multi-filament Thin Collagen Fibers c) DRG on Multi-filament Thin PLLA Fibers (Spaced) d) DRG on Multi-filament Thin Collagen Fibers (Spaced) Multi-filament and Spacing

23. Future Directions  Protein coatings on collagen fibers  Comparison of rat tail and bovine tendon collagen hydrogels on 3D axon outgrowth  Integration of living axon stretch-grown cultures into hydrogel matrix with fiber-reinforced composites  Clinical evaluation of construct using in vivo rat models  Protein coatings on collagen fibers  Comparison of rat tail and bovine tendon collagen hydrogels on 3D axon outgrowth  Integration of living axon stretch-grown cultures into hydrogel matrix with fiber-reinforced composites  Clinical evaluation of construct using in vivo rat models

24. Questions? 3D Nervous Tissue Construct Fibers