Richelle C. Thomas, Christine E. Schmidt Department of Chemical Engineering

1. Richelle C. Thomas, Christine E. Schmidt Department of Chemical Engineering University of Texas at Austin The In-Vitro Cellular Response to Three-Dimensional Hyaluronic Acid Hydrogels Templated by Self-Assembled Poragen Architecture

2. Motivation: Peripheral Neuropathy (PN) 1B affected patients in U.S., Europe & Japan >20M Americans suffer from some form of peripheral neuropathy >2.5% growth annually Annual cost to Medicare US$3.5 B + US $1B industry by 2012 The Neuropathy Association http://www.neuropathy.org/site/PageServer?pagename=About_Facts “Peripheral Neuropathy Market Approaches US$1B by 2012”, 04/7/2010 /PRNewswire/ http://www.prnewswire.co.uk/cgi/news/release?id=120786

3. Neuropathy Occurs by Disease • Congenital birth defects • Diabetes • Cancerous tumors

4. Neuropathy Occurs by Injury • Motor Vehicle Accidents • Wartime • Cuts/Scrapes

5. Current Treatments Address Symptoms • Eliminating symptoms • Pain • Numbness • Addressing the root cause of the nerve damage http://www.prnewswire.co.uk/cgi/news/release?id=120786 http://saveyourself.ca/articles/sciatica.php

6. Degrees of Peripheral Nerve Injuries Peripheral Nerve Axon Epineurium Perineurium Endoneurium where Schwann cells are Injuries Requiring Treatment Injuries That Autologously Recover First Degree Second Degree Fourth Degree Third Degree Fifth Degree

7. Treatment Options for Peripheral Neuropathy due to Injury Grafting End-to-End Suturing Conduit/Implant Communication between nerve stumps Physical guidance to regenerating axons

8. Biomaterials for PN Require Proper Integration of Stimuli Chemical Electrical Contact Huang et al. (2008) Lee et al. (2002) Gomez et al. (2006)

9. Hyaluronic Acid (HA) Advantageous for PN Applications • Extracellular matrix component • Polyanionic • Hydrophillic • Involved in mediating wound repair • Non-cell adhesive

10. Photocrosslinkable Hydrogels High Swelling HA Hydrogels Advantages Biocompatible Non-cell adhesive Control local chemical properties Optically transparent 20 min 1 min Modulate Swelling Degradation Mechanical Properties 2 min 5 min 10 min UV Exposure Problem: Amorphous gels only offer chemical and no physical cues

11. 25 mm 12 mm Role of Physical vs. Chemical Cues Microchannels vs. Laminin Gomez, Chen, Schmidt (2007). J. R. Soc Interface. 4(13): 223-233. Physical cues are preferred over chemical cues for axon initiation (polarization) 70 %  Physical Cues (microchannels) 30 %  Chemical Cues (NGF or Laminin) Gomez, N., Schmidt, C.E. (2007) Biomaterials. 28 (2): 271-284

12. Techniques to Impose 3D Architecture • Photolithography • Microfluidic patterning • Electrochemical deposition • 3D printing Excellent X-Y control Limited control over z-direction coherence of layers and local chemistry Limited in extent of complexity can achieve Expensive equipment required

13. Goal Template natural polymer hydrogels using self-assembling colloidal crystals for 3D internal architecture

14. Templated Hydrogel Synthesis Methacrylated Hyaluronic Acid 0.1-1% Photoinitiator Poragen of choice

15. Poragen Selection Dendritic Crystalline Structure Spindle Crystalline Structure Potassium Dihydrogen Phosphate Urea Glycine Guanidine

16. Not-templated Hydrogels Lack Architecture 0.5% GMHA, 0.5% I2959, cryoSEM

17. Urea Crystal Growth in 2D HA films Zawko, Schmidt. (2010) Acta Biomaterialia. Zawko, S.

18. Internal Morphology of Urea TemplatedHydrogels

19. 3D Structure of Urea Templated Hydrogels Dextran-FITC 2 mm 1.5 mm Scale Bar: 150 um 1.5 mm • spindle crystalline morphology present • macroscopic porous network apparent throughout z-dimension • complexity not restricted to x-y dimension Scale Bar: 500 um

20. Potassium Dihydrogen Phosphate Crystal Growth in HA Zawko, S. Zawko, Schmidt. (2010) Acta Biomaterialia.

21. Surface Morphology of KH2PO4Templated Hydrogels

22. 3D Structure of KH2PO4Templated Hydrogels Dextran-FITC Scale Bar: 200 um Scale Bar: 300 um • sparse crystalline network • templating traverses the z-dimension • macroscopic porous network apparent throughout z-dimension

23. Hydrogel Synthesis Affects Bulk Properties • Bulk Properties • Swelling • Degradation • Storage Modulus • Effect of templating on hydrogel properties

24. Degradation Protocol 1 W0 dI H2O 2 WS 3 10mM PBS Wt 1 hr, 40˚C 50 U/mLHyaluronidase WS - Wt % Degradation = __________ x 100% WS

26. Not templated hydrogels have ability for degradation to be modulated through PI content and UV exposure

27. Templated hydrogels retain ability for degradation to be modulated through PI content and UV exposure

28. Swelling Protocol WD 1 2 WS WS - WD Swell Ratio = _________ 10mM PBS WD

29. At completion, templated hydrogels swell more than amorphous • Higher PI amounts, reduced swelling

30. Rheological Evaluation • Strain sweep at constant frequency until reaches plateau • Choose strain in plateau region • Conduct frequency sweep at constant strain and measure storage modulus

32. Cytotoxicity Experiment Protocol • Culture cells in 24 well plate for 24 hours • 25,000 cells per well • Put gels in transwells suspended in cell media for 24 hours • Analyze ATP with CellTiterGlo assay

34. Conclusions Both chemical and physical guidance cues are important to achieve optimum scaffold Simple system for tissue engineering applications – able to be tailored for tissues/applications Retain control over hydrogel properties after templating Impose architecture over 1.5mm z-length scales [urea] Impose architecture that sparsely traverses the z-dimension [KH2PO4] Fibroblast cell viability not hindered as result of templating process

35. Acknowledgements • Dr. Christine E. Schmidt • Labmates • Dr. Leandro Forciniti, Sarah Mayes, Dr. John Hardy • Undergraduate Researchers • Shan Modi, Paul Chung, Brittany McGhee

36. Questions?