derek hernandez july 9, 2012. designed a method to control topography and chemistry in 3d improve...
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Integrating chemical and topographical cues to enhance Schwann cell migration in 3DDerek HernandezJuly 9, 2012
Motivation
Designed a method to control topography and chemistry in 3D
Improve scaffold designs for treating nerve injuries
Lust, JR. University of Rochester, Institute of Optics. Scale bar = 2 µm
Chemical• Matrix composition• Growth factorsContact• Matrix stiffness• Topography• Compliance
Cell behavior• Migration• Adhesion• Differentiation• Proliferation
Cellular• Junctions• Paracrine signals
Schwann cells
Major glial cell of the peripheral nervous system
Primary function to support and protect neurons
Myelinating and non-myelinating phenotype
Role of Schwann cells
Son, YJ and Thompson, WJ. 1995Scale bar = 20 µm
Nave, KA and Schwab, MH. 2005
Neural Development
Post-injury in vivo
Axons Schwann Cells
• Regeneration speed correlates directly with SC migration speed
How do we recapitulate the developmental environment to promote regeneration?
• Aid in the repair of nerve injury
Desirable properties for nerve guidance channels
Li and Hoffman-Kim. Tiss Eng. Part B. 2008
Adams, DN. et al. J Neurobio. 2005.Scale bar = 50 µm
Chemical cues
Chemotaxis -directed cellular behavior in response to chemical gradients
Gradients play a major role in neural development
Topographical cues in vivo
Topography impacts cell alignment and motility in vitro
Topographical cues
Mitchell, JA. et. al. PloS ONE. 2011
Bridging the gap
Translate research to controllable 3D environments
Decouple the effects of chemical and topographical features
Lay the groundwork for future designs of nerve regeneration scaffolds
Research objectives
Aim 1:Develop a benzophenone-based multiphoton immobilization chemistry
Aim 2:Create a 3D construct to investigate the effect of gradients and topographies on SC migration
Aim 3:Explore the relationship between SCs and neurons
Aim 1: Goals
Develop a multiphoton immobilization chemistry to generate gradients of bound chemical cues in user-defined, 3-dimensional patterns
Characterize and optimize benzophenone-biotin chemistry
Assess the mechanical changes due to benzophenone-biotin immobilization
Render protein microstructures biofunctional for SC culture
Chemical modification techniques
Method Advantages Disadvantages
Soft lithography low cost, simple, 10 nm resolution, rapid 2D patterns
Microfluidics (absorption) Cheap, reproducible
diffusion limited patterns, large
solution volumes required
Photolithography Parallel processing, 100 nm resolution
expensive, 2D surfaces, unable to
control surface chemistry
3D printing 3Dlow resolution, limited materials, large shear
forces
Multiphoton lithography
sub-micron resolution, 3D
time intensive, expensive
Near simultaneous absorption of 2 or more photons
Introduction to multiphoton lithography
Kaehr, B. 2007 Courtesy of Brad Amos MRC, Cambridge
Dynamic-mask multiphoton lithography
Nielson, R. et al. Small. 2009.Scale bar = 10 µm
Reproducible and rapid fabrication of 3D protein structures
Digital micromirror device
nimblegen.com
Ti:S
Scan mirror
Sample
DMD
Protein substrate
Bovine serum albumin 66 kDa protein pI = 4.7 Biocompatible Low immunogenic response
Gelatin, avidin, lysozyme
Kaehr, B. et al. PNAS. 2004Scale bar = 1 µm
Protocol to immobilize cues on protein structures
Benzophenone-biotin
Neutravidin
Biotinylated peptide with PEG linker
Protein structure
1) Fabricate protein structure
• Concentrated protein solution
• Photosensitizer• High laser intensity
2) Immobilize BP-biotin
• 2 mg/mL BP-biotin solution
• Reduced laser intensity
Remove fabrication
solution
3) Bind peptide using neutravidin-biotin
chemistryRemove BP-biotin solution
Benzophenone immobilization chemistry
Benzophenone-DPEG-Biotin
λ = 700-800 nm
First time benzophenone reacted with multiphoton excitation
Reaction occurs at a lower laser intensity than fabrication
Controlling the degree of immobilization
Benzophenone concentration dependent on laser fluence
7 10 13 16 1 2 4 60 0[mW]
[scans/plane]
0 4 8 12 16 20 24 28 32 36 40
Distance (µm)
0 3 6 9 1215182124273033360
2000
4000
6000
8000
10000
Distance (µm)
Avera
ge P
ixel In
-te
nsit
y
Laser power (1 scan/plane)
Scan number (17 mW)
Continuous gradients using a Pockel’s cell
Power Range: 6 - 20 mWwww.microscopyu.com
Automated and reproducible modulation of laser fluence
Triangle function
Sine function
Immobilized gradients on BSA ramps
A
B
C
BSA – Blue Scale bars = 10 µmFluorescent NA - White
Front view Isometric viewA
B
C
Assess the impact of immobilization on the structure
Immobilization does not alter the mechanical properties of the substrate
Atomic Force Microscopy (AFM)
Surface roughness
Elastic modulus
No effect of immobilization on surface roughness
0 2 4 6565860626466687072
Functionalization Scans
RM
S (
nm
)
0 2 4 60
500
1000
1500
2000
2500
3000
3500
Functionalization Scans
Ave
rag
e I
nte
nsit
y
All structures identically fabricated
Performed immobilization at 85% of fabrication power
*Error bars represent the standard deviation (n=5)
Force mapping to determine elastic modulus
Hertz model
F = force (N)Rc = radius of bead (m)E = elastic modulus (Pa)δ = indentation (m)v = poisson’s ratio
www.azonano.com
Extension (µm)
Forc
e
(N)
2D SC adhesion study
BenzophenoneBiotin
Neutravidin
Cue with PEG linker
Protein structure
Scrambled Peptide
Negative controlsPositive control
PLL coated coverslip
Fix and Image
6 - 8 hrs
Cues: RGD, IKVAV
Count cells/substrate
Seed SCsMedium: DMEM, High glucose, 1% FBS
Aim 1 summary
Achieved a range of concentrations without altering substrate roughness
Applied chemistry to functionalize of patterns on 3D substrates
Still need to assess: Elastic modulus SC adhesion
Aim 2: Goals
Develop a 3D construct to study the effects of immobilized chemical gradients and topographies on SC adhesion and migration
Incorporate topographical cues and chemical gradients in HA based hydrogels
Optimize SC adhesion to protein structures by controlling geometry
Investigate SC migration speed and alignment in response to various chemical and topographical cues
IKVAV functionalized BSA structures in hydrogels support DRG cell adhesion and migration
Limitations Unable to incorporate chemical gradients Structure height limited to ~30 µm
Previous work
Seidlits, SK. et al. AFM. 2009. Scale bar = 50 µm
Hyaluronic Acid
Natural material
Chemically modifiable
Controllable material properties
Biocompatible
Non cell-adhesive
Enzymatically degradable (e.g. hyaluronidase)
Leach, JB. et al. Biotech Bioeng. 2004.
Fabrication and functionalization in HA gels
8 hr buffer rinse
30 min. in protein solution
30 min BP-biotin incubation
Buffer wash
2 - 4 minUV exposure
1-2% GMHA, 1% I2959
Fabrication in HA gels
Influenced by basal lamina tubes of native nerve tissue
Fabricated BSA tubes 100 µm long Fabrication time = 20 minutes
Hudson, TW et al. 2004.Scale bar = 10 µm
Major gridlines = 10 µm
Proposed inner wall topographies
4 Ridge
8 Ridge
Spiral
Ridge dimensions:2 µm tall1 µm thick
Ridge dimensions:1 µm tall1 µm thick
Spiral dimensions:Extends 1 µm from wall1 full turn in 15 µm
Scale bar = 10 µm
Dimensions are adjustable
BSA tubes on glass
Aim 2 experimental summary
Tube geometry
Adhesion
Migration distance
Topographical
Cues
Chemical cues
Migration distance
Cell alignment
Topographical +
Chemical
Migration distance
Cell alignment
Optimizing tube geometry for cell adhesion and migration
Variables Inner tube diameter (d): 10 – 30 µm Wall thickness (t): 1 – 10 µm Interstitial spacing (m): 1 – 5 µm Cell density: 30,000-100,000 cells/gel
Criteria for success > 80% of structures with cells
td
m
Independent cue experimental outline
Seed Cell-tracker stained SCs
Fix at 4, 12, and 24 hours
Confocal Microscopy
DAPI Stain
Variable Characteristics (n)
Topography (non-functionalized)
none (8), 4 ridge (8), 8 ridge (8), spiral (8)
Chemical cue IKVAV or RGD
Gradient slope (no topography)
constant(8), low(8), steep(8)
Assess:1) Migration speed v.
controls2) Cell alignment (end-
end angle)
Protein tube
Functionalized protein tubeSchwann cell
Legend
Combined cue experimental outline
Seed Cell-tracker stained SCs
Fix at 4, 12, and 24 hours
Confocal Microscopy
DAPI Stain
Assess:1)Migration speed v. controls2)Cell alignment (end-end
angle)3)Compare to individual cue
results
Take the two best performing cues from each group and combine (4 combinations)
Protein tube
Functionalized protein tubeSchwann cell
Legend
Aim 2 summary
Developed a dual-scaffold system to incorporate chemical and topographical cues into hydrogels
Employ scaffolds to thoroughly investigate SC migration and alignment
Aim 3: Goals
Study the relationship between SC migration and neurite extension by seeding dissociated DRGs onto scaffolds
Determine if SC migration speed directly correlates to neurite extension
Determine if scaffolds pre-seeded with SCs improve neurite extension rates
Crosstalk between SCs and neurons
SCs promote neurite extension by secreting diffusible signals Nerve growth factor, brain derived neurotrophic factor,
glial derived neurotrophic factor, neurotrophic factor-3
SC alignment promotes neurite alignment and extension
SC incorporation into scaffolds to treat nerve injury
Dissociated dorsal root ganglia
Contain neurons and glia
Model for peripheral nerve repair
Rapidly extend neurites in vitro
www.wikipedia.org
Neurite extension protocol
Seed dissociated DRGs
Fix at time = 12 and 24 hours
Confocal Microscopy
Stain (DAPI, Neurofilament, S100) Protein tube
Functionalized protein tubeSchwann cell
Legend
Neuron
Use best performing tube/gradient combinations from Aim 2
Quantify neurite extension and alignment
Compare SCs response to Aim 2
Do pre-seeded SC scaffolds further enhance neurite extension
Seed Cell-tracker stained SCs
Seed dissociated DRGs
Stain (DAPI, Neurofilament, S100)
Allow SCs to infiltrate matrix 4 and 24 hours prior to seeding DRGs
Compare neurite extension rates to scaffolds that are not pre-seeded
Protein tube
Functionalized protein tubeSchwann cell
Legend
Neuron
Proposed timeline
Acknowledgements
Advisors: Dr. Christine Schmidt Dr. Jason Shear
Committee: Dr. Lydia Contreras Dr. Chris Ellison Dr. Wesley Thompson
Multiphoton reaction details
Triplet state of photosensitizer produces singlet oxygen
Singlet oxygen is a highly reactive species Aromatics – tyrosine, tryptophan Thiols - cysteine Amines - lysine, arginine Alkenes
Competing multiphoton immobilization chemistries
Mono-acrylated-PEG PEG-DA hydrogel N-vinyl pyrrolidone 2,2-dimethoxy-2-phenylacetophenone
Coumarin-maleimide Coumarin modified agarose gels
Fluorescein-biotin Mono-acrylated-PEG modified glass
Hoffman, JC et al. Soft Matter. 2010
Wylie, RG. et al. Nature Materials. 2011
Scott, MA. et al. Lab on a Chip. 2012
Experimental questions
Topography: Which topography best promotes migration? Do topographies dictate cell alignment?
Chemical cue: Which cue best promotes migration? Do gradients increase migration speed? Do gradients contribute to cell alignment?
Combinatorial studies: Do topographical and chemical cues have a
synergistic effect?
Crosstalk between SCs and neurons
SCs promote axon extension by secreting diffusible signals
Aligned SCs promote neurite alignment and extension
Armstrong, SJ. et al. Tissue Eng. 2007
Seggio, AM. et al. Journal of Neural Eng. 2010
Current peripheral repair strategies
Leach, JB. And Schmidt, CE. Ann Rev Biomed Eng. 2003.
Size of nerve gap:
< 1 mm 5-7 cm > 7 cm
Need to develop biomaterial scaffolds to improve functional nerve regeneration over larger gap distances