1. Richelle C. Thomas Department of Chemical Engineering University of Texas at Austin April 20, 2011 Data Blitz

2. Motivation: Peripheral Neuropathy • Soft tissue scaffold • Focus on injury • Supports wound healing • Reduces inflammation • Promotes tissue reorganization

3. Motivation • Statement: Hyaluronic Acid gels beneficial for wound healing applications • Problem: Hydrogels are amorphous and do not provide any significant physical contact guidance to infiltrating cells beyond their inherent porosity

4. Goal Develop natural polymer hydrogel with 3D internal architecture Three-Dimensional Hydrogel with Internal Architecture Colloidal Crystal Amorphous Hydrogel

5. Specific Aim 1 Specific Aim 2 Specific Aim 3 Synthesize and characterize photocrosslinkable three-dimensionally patterned natural polymer Hyaluronic Acid hydrogels Optimize colloidal crystal templating protocol for three-dimensional construct. Evaluate degradation profile, degree of swelling and void area of templated gels relative to virgin scaffolds. Assess viscoelastic differences between amorphous and templated gels. Acquire confocal images of poragen structure within bulk hydrogel.

6. HA-Urea Film Porous Structure Scale bar: 10 lm. TRITC- labeled Green fluorescent albumin–FITC • connectivity among the pores of the scaffold • protein diffusion was restricted to the pores • tightly crosslinked, low-permeability hydrogel Zawko, S.A. , et al. (2010. “Crystal templating dendritic pore networks and fibrillar microstructure into hydrogels.” ActaBiomaterialia 6(7): 2415-2421

7. HA-Urea Hydrogel Porous Structure Pore size 50-100 μ m FITC labeled, Scale bar: 150 μm Scale Bar: 250 μm

8. HA-Urea Hydrogel Porous Structure FITC labeled, scale bar = 200 μm

9. Specific Aim 1 Specific Aim 2 Specific Aim 3 • Cell studies • - Peripheral nerve regeneration (urea) • Vascularized systems (less dense, highly branched) • KH2PO4 • Guanidine • Glycine • CHAPS

10. Guanidine TRITC labeled, 4X Magnification

11. KH2PO4 FITC labeled, 4X Magnification, 3D Projection

12. KH2PO4 * Scale bar 200 um, (* 150 um)

13. Cytotoxicity Astrocytes 50 mg/ml GMHA 4X Control 10 X 4X Experiment 10 X Redo with sterile HA, DAPI stain

14. Thiol films • Form covalent bonds chemically • Form thiol-acrylate (ester) bonds • Hydrolyzable at physiological pH • Acrylate:thiol mixed in 7:3 ratio by mass • Rydholm, A.E., Anseth, K.S. and Bowman, C.N. “Effects of neighboring sulfides and pH on ester hydrolysis in thiol–acrylate photopolymers” ActaBiomaterialia, Volume 3, Issue 4, (2007) Pages 449-455

15. Thiol-Capsaicin Films 30 mg/ml GMHA, 10 ug/ml Thiol 7.2 mg/ml Capsaicin 80% MeOH 4.5mg/ml Capsaicin 80% MeOH 4.5 mg/ml Capsaicin 50% MeOH 2.78mg/ml Capsaicin 50% EtOH 4X 10X 20X

16. Thiol-Capsaicin Data How to identify/separate different phases within film? 30mg/ml GMHA | 2.78mg/ml Capsacin| 50% EtOH | 10ug/ml thiol Both images at 10X Magnification

17. Thiol-capsacindata Empty domains within film 30mg/ml GMHA | 2.78mg/ml Capsaicin | 50% EtOH | 10ug/ml thiol 4X 10X

18. Thiol-Capsaicin Data 30 mg/ml GMHA, 7.2 mg/ml Capsaicin, 80% MeOH, 10mg/ml thiol Ordered domains within film… 4X

19. Future Work • Peripheral Nerve Regeneration • Cytoxicity study • Evaluate cell neurite extension within hydrogel • Screen remaining vascular poragen candidates • Thiol films • Characterize • Evaluate ability of thiol-capsaicin films to provide sustained drug release over time

20. Questions?