Direct write of 3d biomolecule microstructures into hydrogel materials
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Direct-Write of 3D Biomolecule Microstructures into Hydrogel Materials. Stephanie Seidlits, Jason Shear, Christine Schmidt University of Texas at Austin. ECM Components. 3D Architecture. Alberts, MBOC, 4th ed. Bochaton-Piallat, Ophtamol Vis Sci, 2000. Engineered in vivo-like Scaffolds.

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Direct-Write of 3D Biomolecule Microstructures into Hydrogel Materials

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Direct write of 3d biomolecule microstructures into hydrogel materials

Direct-Write of 3D Biomolecule Microstructures into Hydrogel Materials

Stephanie Seidlits, Jason Shear, Christine Schmidt

University of Texas at Austin


Direct write of 3d biomolecule microstructures into hydrogel materials

ECM Components

3D Architecture

Alberts, MBOC, 4th ed.

Bochaton-Piallat, Ophtamol Vis Sci, 2000

Engineered in vivo-like Scaffolds

Submicron Features

Spatial Control

Esch, J. Neurosci, 1999

Teixeira, JCS, 2003


Scaffold design

Neuronal Cell Body

Extending Neurite

3D Hydrogel Support

Crosslinked Protein Structures

Scaffold design


Hyaluronic acid

Hyaluronic acid

  • Nonimmunogenic

  • Angiogenesis and --wound healing

  • Enzymatically biodegradable

  • Photocrosslinkable --------------hydrogels (Leach, 2003, Biotech Bioeng)

  • Not cell adhesive

GMHA Reaction

ring opening product

hyaluronic acid

glycidylmethacrylate

transesterification product

Adapted from Derouet, 2002, Eur Poly J


Multiphoton excitation

www.fsu.edu/microscopyprimer

Multiphoton excitation

  • Advantages of MPE photocrosslinking:

  • Inherently 3D

  • Subcellular resolution

  • Complex geometries --possible

  • Heating effects minor ----and localized

  • Can fabricate---------------structures of natural -----materials

www.cellscience.biorad.com


Resolution and biocompatibility of mpe photocrosslinking

Resolution and biocompatibility of MPE photocrosslinking

1 µm

Kaehr, PNAS, 2004


Mpe crosslinking in 3d

Crosslink protein structures directly into saturated GMHA hydrogels using multiphoton excitation

Make GMHA hydrogels and soak in photosensitizer and protein solution

Wash to remove unreacted solution

MPE crosslinking in 3D


Mpe crosslinking in 3d1

MPE crosslinking in 3D


Bsa fitc structures in texas red gmha hydrogels

BSA-FITC structures in Texas Red-GMHA hydrogels

Pyramid base = 50 μm


3d protein structures in ha hydrogels

Z=40 µm

Z=20 µm

50 µm

50 µm

Z=0 µm

50 µm

50 µm

50 µm

50 µm

3D protein structures in HA hydrogels

Z=40 µm

Z=20 µm

Z=0 µm


Scanning electron microscopy

Scanning Electron Microscopy

2 µm

200 nm

2 µm


Avidin biotin functionalization

F

F

F

F

+

+

+

+

+

+

+

B

B

avidin

avidin

bsa

Avidin-biotin functionalization

Strategy 1:

Strategy 2:


Strategy 1 avidin structures

Strategy 1: Avidin structures

Avidin

BSA

150 µm

150 µm

~30 ms exposure time


Strategy 2 bsa structures modified with avidin

50 µm

50 µm

Strategy 2: BSA structures modified with avidin

BSA + Avidin-fluorescein

BSA + unlabeled avidin

~3 ms exposure time


Mpe crosslinking within hydrogels provides

50 µm

MPE crosslinking within hydrogels provides:

  • Flexibility to incorporate multiple bioactive molecules in arbitrary patterns

  • Inherent 3D geometry and biomolecule presentation

  • Submicron sized features


Acknowledgements

Acknowledgements

Principle Investigators:

  • Dr. Christine Schmidt

  • Dr. Jason Shear

    Undergraduates:

  • Rebecca Rosenberger

  • Kat McCarty

  • Jill Heisler

    Lab Members:

  • Curt Deister

  • Bryan Kaehr

  • Rex Nielson

  • Rest of Schmidt and Shear Lab Members

Funding:

NSF-IGERT

NSF BES-0500969 and BES-021744

Welch Foundation


Direct write of 3d biomolecule microstructures into hydrogel materials

IGERT: Cellular and Molecular Imaging for

Diagnostics and Therapeutics

The University of Texas at Austin

Novel Contrast Agent Development

Novel Imaging and Spectroscopy

System Development

Diagnostic and Vital Imaging

Spectroscopy

Imaging to Monitor Therapeutics

Integrative Graduate Education & Research Training Program

Nicholas Peppas, Sc.D., Director

Jennifer Brodbelt, Ph.D., Co-Principal InvestigatorChristine Schmidt, Ph.D., Co-Principal Investigator

http://www.bme.utexas.edu/igert/


Direct write of 3d biomolecule microstructures into hydrogel materials

A)

B)

Figure 4. 3D MPE crosslinked BSA structures within hydrogels. A) Optical transmission images of 200 mg/mL BSA (2% BSA-FITC) photocrosslinked in a 2 wt% GMHA hydrogel with 6 mM FAD using a pulsed TiS laser at 740 nm. Each image was taken in the same xy plane and a different z plane (the first image is focused at the top of the pyramid and the last at the bottom). C) Confocal microscope image of 200 mg/mL BSA (2% BSA-FITC) crosslinked using 6 mM FAD within a 100 mg/mL PEGDA hydrogel. Images are from different planes spanning a total depth of 80 m. Note: concentrations given are those in the uncrosslinked solution. Scale bars = 20 µm.


Crosslinking with fad

Crosslinking with FAD

  • Two Mechanisms:

    • Type I: triplet state abstract hydrogen to create radicals

    • Type II: creates singlet oxygen

      (Spikes 1999 Photochem Photobio)

  • Photocrosslinkable amino acids: Tyr, Trp, His, Cys…


Avidin biotin au np

Avidin-Biotin - Au NP


Advantages of mpe polymerization

Advantages of MPE Polymerization


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