Integrating chemical and topographical cues to enhance Schwann cell migration in 3D

1. Derek Hernandez July 9, 2012 Integrating chemical and topographical cues to enhance Schwann cell migration in 3D

2. Motivation • Chemical • Matrix composition • Growth factors • Designed a method to control topography and chemistry in 3D • Improve scaffold designs for treating nerve injuries • Cell behavior • Migration • Adhesion • Differentiation • Proliferation • Contact • Matrix stiffness • Topography • Compliance • Cellular • Junctions • Paracrine signals Lust, JR. University of Rochester, Institute of Optics. Scale bar = 2 µm

3. Schwann cells • Major glial cell of the peripheral nervous system • Primary function to support and protect neurons • Myelinatingand non-myelinating phenotype

4. Role of Schwann cells Neural Development Post-injury in vivo • Aid in the repair of nerve injury Nave, KA and Schwab, MH. 2005 AxonsSchwann Cells Son, YJ and Thompson, WJ. 1995 Scale bar = 20 µm • Regeneration speed correlates directly with SC migration speed How do we recapitulate the developmental environment to promote regeneration?

5. Desirable properties for nerve guidance channels Li and Hoffman-Kim. Tiss Eng. Part B. 2008

6. Chemical cues • Chemotaxis -directed cellular behavior in response to chemical gradients • Gradients play a major role in neural development Adams, DN. et al. J Neurobio. 2005. Scale bar = 50 µm

7. Topographical cues • Topographical cues in vivo • Topography impacts cell alignment and motility in vitro Mitchell, JA. et. al. PloS ONE. 2011

8. Bridging the gap • Translate research to controllable 3D environments • Decouple the effects of chemical and topographical features • Lay the groundwork for future designs of nerve regeneration scaffolds

9. Research objectives

10. Aim 1: Goals

11. Chemical modification techniques

12. Introduction to multiphoton lithography • Near simultaneous absorption of 2 or more photons Kaehr, B. 2007 Courtesy of Brad Amos MRC, Cambridge

13. Dynamic-mask multiphoton lithography Scan mirror Sample Nielson, R. et al. Small. 2009. Scale bar = 10 µm Ti:S Digital micromirror device nimblegen.com Reproducible and rapid fabrication of 3D protein structures DMD

14. Protein substrate • Bovine serum albumin • 66 kDa protein • pI = 4.7 • Biocompatible • Low immunogenic response • Gelatin, avidin, lysozyme Kaehr, B. et al. PNAS. 2004 Scale bar = 1 µm

15. Protocol to immobilize cues on protein structures • 1) Fabricate protein structure • Concentrated protein solution • Photosensitizer • High laser intensity • 2) Immobilize BP-biotin • 2 mg/mL BP-biotin solution • Reduced laser intensity Remove fabrication solution Protein structure Benzophenone-biotin Neutravidin Biotinylated peptide with PEG linker 3) Bind peptide using neutravidin-biotin chemistry Remove BP-biotin solution

16. Benzophenone immobilization chemistry Benzophenone-DPEG-Biotin First time benzophenone reacted with multiphoton excitation λ = 700-800 nm Reaction occurs at a lower laser intensity than fabrication

17. Controlling the degree of immobilization • Benzophenone concentration dependent on laser fluence Laser power (1 scan/plane) Scan number (17 mW) 4 0 7 1 2 0 [mW] 10 13 16 [scans/plane] 6

18. Continuous gradients using a Pockel’s cell Triangle function Sine function www.microscopyu.com Power Range: 6 - 20 mW Automated and reproducible modulation of laser fluence

19. Immobilized gradients on BSA ramps A Front view Isometric view A B B C C BSA – Blue Scale bars = 10 µm Fluorescent NA - White

20. Assess the impact of immobilization on the structure • Immobilization does not alter the mechanical properties of the substrate Atomic Force Microscopy (AFM) • Surface roughness • Elastic modulus

21. No effect of immobilization on surface roughness All structures identically fabricated Performed immobilization at 85% of fabrication power *Error bars represent the standard deviation (n=5)

22. Force mapping to determine elastic modulus www.azonano.com Hertz model Force (N) F = force (N) Rc = radius of bead (m) E = elastic modulus (Pa) δ = indentation (m) v = poisson’s ratio Extension (µm)

23. Protein structure 2D SC adhesion study Benzophenone Biotin Positive control PLL coated coverslip Negative controls Neutravidin Cues: RGD, IKVAV Cue with PEG linker Medium: DMEM, High glucose, 1% FBS Seed SCs 6 - 8 hrs Fix and Image Count cells/substrate Scrambled Peptide

24. Aim 1 summary • Achieved a range of concentrations without altering substrate roughness • Applied chemistry to functionalize of patterns on 3D substrates • Still need to assess: • Elastic modulus • SC adhesion

25. Aim 2: Goals

26. Previous work • IKVAVfunctionalized BSA structures in hydrogels support DRG cell adhesion and migration • Limitations • Unable to incorporate chemical gradients • Structure height limited to ~30 µm Seidlits, SK. et al. AFM. 2009. Scale bar = 50 µm

27. Hyaluronic Acid • Natural material • Chemically modifiable • Controllable material properties • Biocompatible • Non cell-adhesive • Enzymatically degradable (e.g. hyaluronidase) Leach, JB. et al. Biotech Bioeng. 2004.

28. Fabrication and functionalization in HA gels 1-2% GMHA, 1% I2959 8 hr buffer rinse 2 - 4 min UV exposure 30 min BP-biotin incubation 30 min. in protein solution Buffer wash

29. Fabrication in HA gels • Influenced by basal lamina tubes of native nerve tissue • Fabricated BSA tubes 100 µm long • Fabrication time = 20 minutes Hudson, TW et al. 2004. Scale bar = 10 µm Major gridlines = 10 µm

30. Proposed inner wall topographies BSA tubes on glass • Dimensions are adjustable 4 Ridge 8 Ridge Spiral Ridge dimensions: 1 µm tall 1 µm thick Spiral dimensions: Extends 1 µm from wall 1 full turn in 15 µm Ridge dimensions: 2 µm tall 1 µm thick Scale bar = 10 µm

31. Aim 2 experimental summary Topographical Cues Tube geometry Topographical + Chemical Chemical cues Adhesion Migration distance Migration distance Cell alignment Migration distance Cell alignment

32. Optimizing tube geometry for cell adhesion and migration • Variables • Inner tube diameter (d): 10 – 30 µm • Wall thickness (t): 1 – 10 µm • Interstitial spacing (m): 1 – 5 µm • Cell density: 30,000-100,000 cells/gel • Criteria for success • > 80% of structures with cells d t m

33. Independent cue experimental outline Seed Cell-tracker stained SCs Fix at 4, 12, and 24 hours Assess: Migration speed v. controls Cell alignment (end-end angle) Legend DAPI Stain Protein tube Functionalized protein tube Confocal Microscopy Schwann cell

34. Combined cue experimental outline Take the two best performing cues from each group and combine (4 combinations) Seed Cell-tracker stained SCs Assess: Migration speed v. controls Cell alignment (end-end angle) Compare to individual cue results Fix at 4, 12, and 24 hours Legend DAPI Stain Protein tube Functionalized protein tube Confocal Microscopy Schwann cell

35. Aim 2 summary • Developed a dual-scaffold system to incorporate chemical and topographical cues into hydrogels • Employ scaffolds tothoroughly investigate SC migration and alignment

36. Aim 3: Goals

37. Crosstalk between SCs and neurons • SCs promote neurite extension by secreting diffusible signals • Nerve growth factor, brain derived neurotrophic factor, glial derived neurotrophic factor, neurotrophic factor-3 • SC alignment promotes neurite alignment and extension • SC incorporation into scaffolds to treat nerve injury

38. Dissociated dorsal root ganglia • Contain neurons and glia • Model for peripheral nerve repair • Rapidly extend neurites in vitro www.wikipedia.org

39. Neurite extension protocol • Use best performing tube/gradient combinations from Aim 2 • Quantify neurite extension and alignment • Compare SCs response to Aim 2 Seed dissociated DRGs Fix at time = 12 and 24 hours Legend Stain (DAPI, Neurofilament, S100) Protein tube Functionalized protein tube Confocal Microscopy Schwann cell Neuron

40. Do pre-seeded SC scaffolds further enhance neurite extension • Allow SCs to infiltrate matrix 4 and 24 hours prior to seeding DRGs • Compare neurite extension rates to scaffolds that are not pre-seeded Seed Cell-tracker stained SCs Seed dissociated DRGs Legend Protein tube Functionalized protein tube Schwann cell Neuron Stain (DAPI, Neurofilament, S100)

41. Proposed timeline

42. Acknowledgements • Advisors: • Dr. Christine Schmidt • Dr. Jason Shear • Committee: • Dr. Lydia Contreras • Dr. Chris Ellison • Dr. Wesley Thompson

43. Multiphoton reaction details • Triplet state of photosensitizer produces singlet oxygen • Singlet oxygen is a highly reactive species • Aromatics – tyrosine, tryptophan • Thiols - cysteine • Amines - lysine, arginine • Alkenes

44. Competing multiphoton immobilization chemistries • Mono-acrylated-PEG • PEG-DA hydrogel • N-vinyl pyrrolidone • 2,2-dimethoxy-2-phenylacetophenone • Coumarin-maleimide • Coumarin modified agarose gels • Fluorescein-biotin • Mono-acrylated-PEG modified glass Hoffman, JC et al. Soft Matter. 2010 Wylie, RG. et al. Nature Materials. 2011 Scott, MA. et al. Lab on a Chip. 2012

45. Experimental questions • Topography: • Which topography best promotes migration? • Do topographies dictate cell alignment? • Chemical cue: • Which cue best promotes migration? • Do gradients increase migration speed? • Do gradients contribute to cell alignment? • Combinatorial studies: • Do topographical and chemical cues have a synergistic effect?

46. Crosstalk between SCs and neurons • SCs promote axon extension by secreting diffusible signals • Aligned SCs promote neurite alignment and extension Seggio, AM. et al. Journal of Neural Eng. 2010 Armstrong, SJ. et al. Tissue Eng. 2007

47. Current peripheral repair strategies Size of nerve gap: < 1 mm 5-7 cm > 7 cm Leach, JB. And Schmidt, CE. Ann Rev Biomed Eng. 2003. Need to develop biomaterial scaffolds to improve functional nerve regeneration over larger gap distances