1 / 44

Hyaluronic Acid Hydrogel Biomaterials for Soft Tissue Engineering Applications

Hyaluronic Acid Hydrogel Biomaterials for Soft Tissue Engineering Applications. Jennie Baier Leach Supervisor: Christine E. Schmidt. “Biomaterials that heal” Ratner BD . (2002) J Controlled Release . 78:211-8. Aim : To facilitate natural wound healing biology. Biological design :

gyala
Télécharger la présentation

Hyaluronic Acid Hydrogel Biomaterials for Soft Tissue Engineering Applications

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Hyaluronic Acid Hydrogel Biomaterials for Soft Tissue Engineering Applications Jennie Baier Leach Supervisor: Christine E. Schmidt

  2. “Biomaterials that heal” Ratner BD. (2002) J Controlled Release. 78:211-8 Aim: To facilitate natural wound healing biology Biological design: Biomimetic molecules like those present in a wound Limited nonspecific protein adsorption Nonimmunogenic Materials design: Enzymatic degradation Versatile modification strategies Mechanical properties match tissue www.organogenesis.com Hubbell JA. (1999) Curr Opin Biotech. 10:123-9; Stocum DL. (1998) Wound Repair Regen. 6:276-90

  3. Tissue Engineering Scaffolds: State of the Art Proteins Fibrin, Collagen “Sugars” Agarose, Alginate Chitosan, Dextran, Hyaluronic acid Synthetic polymers Polyethylene glycol (PEG) Polylactic-co-glycolic acid (PLGA) Polyhydroxyethyl methacrylate (pHEMA) Inherent biological activity Nonimmunogenic Multiple modification sites Tunable material properties

  4. Hyaluronic Acid (HA) glucuronic acid acetylglucosamine • Natural ECM component • Easily produced in large quantities • Non-immunogenic • Multiple sites available for modification • Enzymatically degradable

  5. HA’s role in wound healing Chen WY & Abatangelo G. (1999) Wound Repair Regen. 7:79-89.

  6. Overall Goal: To develop and characterize hyaluronic acid hydrogel scaffolds for soft tissue engineering applications

  7. Aim 1: To create and characterize HA hydrogels Aim 2: To develop methods for controlling cell-HA hydrogel interactions Peptide-conjugated HA hydrogels Protein-releasing HA hydrogels

  8. Aim 1: To create and characterize HA hydrogels Aim 2: To develop methods for controlling cell-GMHA hydrogel interactions Peptide-conjugated GMHA hydrogels Protein-releasing GMHA hydrogels

  9. glycidyl methacrylate (GM) + Photoinitiator + UV light Crosslinked GMHA GMHA HA GM modification HA Methacrylation and Crosslinking Baier Leach J, Bivens KA, Patrick CW, Jr. & Schmidt CE. (2003) Biotech Bioeng 82:578-89 Crosslinking Variables: GMHA concentration (0.5-2.0%) UV exposure (1-4 min, ~22 mW/cm2) Photoinitiator conc. (0.03-3% Irgacure 2959) % methacrylation (NMR: ~5-11%)

  10. GMHA Solution +UV Remove mold Equilibrate in buffer overnight, weigh Dry completely, weigh Determining the Degree of Crosslinking (Swelling Ratio) Goal: To obtain a relative measure of crosslinking for the GMHA gels

  11. Flory polymer-solvent interaction theory: Estimated pore size: 644 nm 619 nm 539 nm •  % methacrylation • Swelling ratio  Cross-linking • Pore size Effect of Methacrylation on Crosslinking n=3 for each bar

  12. 5% 7% 11% Increasing % methacrylation n>4 for each point Effect of Methacrylation on Degradation Rate GMHA Solution +UV Incubate in hyase Measure weight loss of gel over time Remove mold

  13. Media Hydrogel Crosslinked GMHA 1% GMHA 0.1% Irgacure 2959 0.03% N-vinyl pyrrolidinone 1 minute UV Crosslinked GMHA GMHA in solution Media HA n>6 for each bar Effect of Methacrylation on Cytocompatibility HAEC monolayer www.corning.com

  14. 3. Harvest tissue at 2 weeks (side view without TEC) 1. Fill TEC with hydrogel 4 hydrogels per rat + Control: Fibrin -Control: Agarose 2 HA gels 2. Suture to muscle 4. EC immunostain (CD31) In Vivo Analysis of Endothelial Cell (EC) Infiltration

  15. In Vivo Analysis of EC Infiltration Fibrin (+) GMHA hydrogel 6.63 ± 1.10, n = 9 (% area CD31-positive cells)7.06 ± 0.14, n = 4 2 Week Implant Scale, 200 mm Agarose (-)

  16. Aim 1: To create and characterize HA hydrogels Aim 2: To develop methods for controlling cell-GMHA hydrogel interactions Peptide-conjugated GMHA hydrogels Protein-releasing GMHA hydrogels

  17. Aim 1: To create and characterize HA hydrogels Aim 2: To develop methods for controlling cell-GMHA hydrogel interactions Peptide-conjugated GMHA hydrogels Protein-releasing GMHA hydrogels

  18. Peptide-Conjugated GMHA Hydrogels Baier Leach J, Bivens KA, Collins CN, & Schmidt CE. (submitted) J Biomed Mater Res * Peptides of interest: Cell adhesive fibronectin peptide (GRGDSG) Model peptide, hexaglycine (GGGGGG) * Based on work with PEG hydrogels in J. West and J. Hubbell’s laboratories

  19. Conjugation Efficiency and Yield  Hexaglycine input  Conjugation efficiency  Hexaglycine input  Conjugation yield Targeted yield * * Based on work with PEG hydrogels in J. West and J. Hubbell’s laboratories

  20. Negative control (no hyase) Effect of [PEG] on Degradation 5 u/ml hyase n>3 for each point Incubate in hyase Measure weight loss

  21. Human dermal fibroblasts 3 days Rinse Count number of adherent cells n=3 hydrogels for each bar Fibroblast Adhesion on GMHA-PEG-GRGDSG hydrogels

  22. Aim 1: To create and characterize HA hydrogels Aim 2: To develop methods for controlling cell-GMHA hydrogel interactions Peptide-conjugated GMHA hydrogels Protein-releasing GMHA hydrogels

  23. Growth factors: ~5-20 nm BSA: ~30 nm 4-arm PEG  Density of polymer or crosslinks  Protein diffusion ~50 nm Protein-Releasing GMHA hydrogels Baier Leach J & Schmidt CE. (in preparation) Biomaterials ~500 nm

  24. Fractional release of BSA (Mt/M) n=3 for each point BSA Release From GMHA-Based Hydrogels At each time-point: remove 1 ml, measure protein concentration 15ml

  25. At each time-point: remove 1 ml, measure protein concentration 15ml BSA Release From GMHA-Based Hydrogels Fractional release of BSA (Mt/M) Fractional release of BSA (Mt/M) n=3 for each point

  26. Crosslink/Polymer Density Affects More Than Diffusion ~50 nm  Density of polymer or crosslinks  Protein diffusion  Degradation rate  Stiffness

  27. Hydrogel-Microsphere Composites BSA-PLGA microspheres (SEM) Dv,10 = 8 mm Dv,50 = 16 mm Dv,90 = 34 mm Hydrogel-microsphere composite (brightfield) 100 mm

  28. Extended BSA Release from Hydrogel-Microsphere Composites Fractional release of BSA (Mt/M) 2 weeks n=3 for each point

  29. Aim 1: To create and characterize HA hydrogels Aim 2: To develop methods for controlling cell-GMHA hydrogel interactions Peptide-conjugated GMHA hydrogels Protein-releasing GMHA hydrogels

  30. Overall Goal: To develop and characterize hyaluronic acid hydrogel scaffolds for soft tissue engineering applications

  31. Acknowledgements Dr. Charles W. Patrick, Jr, MD Anderson Cancer Center, Houston, TX Dr. C.P. Pathak, Sulzer Biologics, Austin, TX Dr. Brent Iverson, UT-Austin Dr. Nicholas Peppas, UT-Austin Dr. Donald Paul, Timothy Fornes, UT-Austin Drs. Keith Johnston, Robert O. Williams III, W. Thomas Leach, UT-Austin Kathryn Bivens, Scott Zawko, Schmidt group members Eric Brey, Cindy Frye, Carol Johnston, Patrick group members Chelsea Collins, Kate Lee, Gwang-Yi Hwang, Erik Askenasy, Nabilla Porbandarwalla, undergraduate research assistants

  32. Cross-linked HA hyase hyase Crosslinked HA Hydrogels Native HA hyase hyase

  33. Previous Work: HA Composite Materials Collier J, Hudson TW, Schmidt CE. (2000) JBMR 50:574 Polypyrrole without HA Polypyrrole with HA ~2 fold increase in blood vessels with HA

  34. Effect of Methacrylation on HAEC Proliferation Set-up: “starve” 12h; expose to 1.5 mg/ml fragments for 48h n>9 for each bar

  35. Flory Polymer-Solvent Theory Qv determined from Qm: x: where Mc from simplified Flory-Rehner equation: therefore, ue: • Qv volumetric swelling u specific volume of the dry polymer n # of monomer repeats • Qm mass swelling Mc average MW between crosslinks x mesh size • rp density of dry polymer V1 molar volume of solvent (water) • rs density of solvent (water) ue effective crosslink density • Flory polymer-solvent interaction parameter (assumed to be 0.473 for HA) • (ro2)1/2root mean square distance between crosslinks

  36. Results of Flory Polymer-Solvent Theory Calculations

  37. 1H-NMR spectroscopy

  38. In vivo degradation Muscle or cellular ingrowth 4 weeks 8 weeks Scale, 100 mm H&E stain GMHA hydrogel Gels degrade slowly due to low subcutaneous concentration of hyase

  39. Efficiency and Yield  Hexaglycine / gel reactive sites  Conjugation efficiency  Hexaglycine / gel reactive sites  Conjugation yield

  40. Effect of Peptide Conjugation on Crosslinking • Measure swelling ratio • Swelling ratio  Crosslinking n>2 for each point

  41. Release Data Fits Fickian Model of Diffusion for Mt/M∞ < 0.6, n=3 for each point; n=9 for De calculations

  42. Diffusion Coefficient of BSA n=9 for each bar

  43. Protein stability Collect BSA released from hydrogels Run SEC/HPLC to determine relative amounts of monomeric BSA and oligomeric BSA aggregates n=4 gels, sampled 3 times each

More Related