Direct-Write of 3D Protein Structures into GMHA Hydrogels

1. Direct-Write of 3D Protein Structures into GMHA Hydrogels Stephanie Seidlits April 12, 2006

2. "A tissue engineered construct that is has a well-controlled microstructure will better maintain cell morphology,differentiation, and functionality over long periods of time. A key feature of these constructs will be the replication of in vivo geometry and dimensional size scale that will aid in the maintenance of an in vivo-like cell phenotype.” T.A. Desai, 2000, Medical Engineering and Physics

3. Bochaton-Piallat, Ophtamol Vis Sci, 2000 Alberts, MBOC, 4th ed. ECM Components 3D Architecture Engineered in vivo-like Scaffolds Submicron Features Spatial Control Esch, J. Neurosci, 1999 Teixeira, JCS, 2003

4. 3D scaffolds induce in vivo phenotypes • 3D scaffolds have prominent effects on cell: • Migration • Adhesion • Interactions with matrix and neighboring cells • Cytoskeletal structure • Morphology • Gene and protein expression Cukierman, Science, 2001

5. Nanoscale topography induces in vivo phenotypes Feature size affects cell: • Cytoskeletal organization • Adhesion • Migration • Morphology • ? Teixeira, JCS, 2003

6. Luo, Nature Materials, 2004 Spatial Control of Cells • Spatially-patterned chemical or topographical cues are important for cell: • Guidance • Interactions with supporting cell types • Behavior • Orientation/Morphology Thompson, Tissue Engineering, 2001

7. Shear, Anal. Chem., 1997 Mauro, J. Micromech. Sys., 1998. Multiphoton Excitation • Femtosecond pulsed TiS laser in near-IR • MPE scales nonlinearly with intensity • Peak intensity only affects small area of focus (<1µm3)

8. Cover Glass crossliked protein concentrated protein solution with photosensitizer z y x MPE Crosslinking of Proteins • Use to crosslink proteins into hydrated structures of 250-500 nm diameter • Laminin, BSA (Kaehr, PNAS, 2004), collagen (Basu, Biomacromol, 2005), fibrinogen, and fibronectin (Basu, JBMR, 2004) easily crosslinked while retaining bioactivity

9. Advantages of MPE Polymerization

10. Laminin Yamada, JBC, 1991 Extracellular Matrix • Extracellular matrix molecules influence a wide range of cellular functions: • Cell adhesion • Cell migration • Tissue organization • Cell differentiation • Morphology • Mediation of soluble signals

11. Hyaluronic Acid • Nonimmunogenic • Nonadhesive • Important for wound healing • Bioactive properties - angiogenesis • Enzymatically biodegradable • Can be made into UV-crosslinked porous hydrogels when modified with methacrylate groups (Leach, 2003, Biotech Bioeng) May be able to exploit both physical and biological effects of HA However, HA hydrogels are non-adhesive to cells

12. Adapted from Derouet, 2002, Eur Poly J Methods 1) Make GMHA hydrogels 2) Swell hydrogels in solution of photosensitizer and protein Flavin Adenine Dinucleotide

13. Methods 3) MPE crosslink protein structures inside of GMHA hydrogels with pulsed TiS laser 100 mg/mL BSA, 3mg/mL laminin crosslinked with 3 mM FAD with the TiS at 740 nm in 2% GMHA hydrogel;165 mW power and 50 µm/s scan speed. 4) Rinse solution protein out of hydrogels

16. Avidin Structures

18. Avidin-Biotin - Au NP

19. Laminin/BSA Structures

20. Laminin/BSA Structures 100 mg/mL BSA crosslinked with 3 mM FAD with the TiS at 740 nm in 2% GMHA gel. Top have 3 mg/mL laminin as well.

21. Cells • PC-12 cell would not easily adhere to laminin structures on gels • NG-108s will adhere if allowed to settle for ~2-3 hr after seeding • NG108’s will adhere preferentially to laminin lines (preliminary) • Problems with GMHA sterilization

22. Thanks! • Christine Schmidt • Jason Shear • All the students in the labs • Curt Deister • Rex Nielson