Derek Hernandez July 9, 2012. Designed a method to control topography and chemistry in 3D Improve scaffold designs for treating nerve injuries Lust,

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