Christopher McKay - Rensselaer Polytechnic Institute - Doctoral Defense Presentation

Hydrogels for Acute Spinal Cord Injury: Physical/Chemical Material Characterization

and Assessment of Astrocytic Response

Christopher A. McKayDoctoral Thesis Defense

April 3rd, 2014

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Societal Impact of Spinal Cord Injury

• Lifetime cost at 25 years2:• Incomplete Motor Function (AIS D)- $1,517,806• High Tetraplegia (C1-C4) - $4,543,182

• Treatment and rehabilitation costs surpass $9.7 billion in the United States alone3

• Less than 1% of patients exhibit complete functional recovery2

1 Christopher and Dana Reeve Foundation 2006,

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2 NSCISC Study 2012, 3 Ackery et al 2004

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Societal Impact of Central Nervous System Injury

•Clinical Treatments:• Spinal Decompression and Fixation2

• Methylprednisilone3

• Clinical Trials• 50+ ongoing trials4

There is a significant need for an efficacious therapy to improve patient outcome following SCI

1 Christopher and Dana Reeve Foundation 2006,

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2 Carlson et al 2003, 3Bracken et al 1997, 4 Unite 2 Fight Paralysis 2014

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There are three distinctly defined periods of spinal cord injury

Acute•Timeframe: 0-48 hours following initial trauma1

• Largely characterized by macroscopic, systemic damage2

•For severe trauma:• Blood vessel hemorrhage • Disruption of blood brain barrier• Infiltration of blood-borne material

SCI is a Dynamic Process:Acute Phase

3 Ronsyn et al 20083 2 Tator 19981 Liverman 2005

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Acute

Sub-Acute

Time frame: 48 hours – 2 weeks following injury1

Changes in the composition of extracellular environment 2,3

Significant inflammatory response1

Induction of secondary injury cascades2,3

These changes lead to the formation of reactive astrocytes

SCI is a Dynamic Process:Sub-Acute Phase

1 Liverman 2005 2 Oyinbo 2011 3 Wingrave et al 2003

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4 Sofroniew 2009

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Postsynaptic Cascade• Activation of glutamatergic receptors

• Increase in intracellular Ca2+

• Activation of calpain and mitochondria failure

• Activation of caspase

Acute

Sub-Acute

Presynaptic Cascade• Damage to presynaptic neuronal membrane

• Accumulation of glutamate in the synaptic cleft

Mechanism of Calcium RelatedSecondary Neuronal Death

1 Syntichaki and Tavernarakis 2003

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Neuronal apoptosis via calcium dependent

glutamate excitotoxicity

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Acute

Sub-Acute

Chronic

1 Liverman 2005

Time frame: 2 weeks following injury – indefinitely1

Characterized by the formation of a glial scar2,3

• Physical and chemical barrier to neuronal regeneration

• Key components: activated astrocytes and chondroitin sulfate proteoglycans (CSPGs)

2 Egn et al 1987 3 Berry et al 1983

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4 Sofroniew 2009

SCI is a Dynamic Process: Chronic Phase

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SCI is a Dynamic Process: Review and Experimental Approach

Acute

Sub-Acute

Chronic

Solution: Prevent early formation of inhibitory cues within the spinal cord lesion

Physical Trauma Structural Damage

Calcium Related Neuronal Death Reactive Astrocyte Formation

Glial Scar Formation Neuronal Regeneration Failure

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Overall Thesis Goal

The overall goal of this research is:

• The development and characterization of an injectable composite hydrogel system that responds to physiological levels of Ca2+

• Assessment of the astrocytic response to hydrogels of varying composition

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Specific Aim 1

Development and Characterization of the Physical, Chemical and Mechanical Behavior of Injectable, Calcium Sensitive Alginate/Chitosan Hydrogels

Specific Aim 1 – Hypothesis

Injectable alginate/chitosan hydrogels can be fabricated to mimic the elastic modulus of native CNS tissue and respond to change in external Ca2+ concentration while exhibiting tunable physical and mechanical properties

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• Spinal cord injuries vary greatly in shape and size

•Contusion injuries are the most common type of injury

•Hydrogels are injectable – conform to the lesion geometry

1 Silver and Miller 2004

Why use a Hydrogel System?

1

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• Homogenous Crosslinking

• Easily Injectable

• Mimic native CNS mechanical properties

• Utilize excess extracellular Ca2+ for in-situ gelation

Hydrogel Design Criteria

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Specific Aim 1 - RationaleAlginate

•Biocompatible, low cytotoxicity

•Crosslinks with Ca2+ ions

•Negatively charged

Chitosan•Positively charged – enhances cellular adhesion

Genipin• Natural crosslinking agent

• Well suited for spinal cord environments

1 Wee and Gombotz 1998 2 Braccini and Perez 2001 3 Li et al 2007 4 Rowley et al 1999 5 Zuidema et al 2011 6 Moura et al 2011 7 Yamazaki et al 2005 8 Yamazaki et al 2004 9 Koo et al 2006

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Specific Aim 1 - Rationale

Chitosan/Genipin• Genipin reacts with positive amine groups on chitosan chains2

Genipin/Genipin•Polymerization of genipin molecules between chitosan chain3

Chitosan/Alginate• Oppositely charged – form polyelectrolyte complexes4,5

Alginate/Ca2+

• Ionic bond formation between Ca2+ ions and guluronic acid residues of alginate6,7

1 Chen et al 2006

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2 Touyama et al 2011 3 Mu et al 2013 4 Sankalia et al 2007 5 Tapia et al 2004 6 Braccini and Perez 2001 7 Li et al 2007

3

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Specific Aim 1 - Results

2% Alginate

5% Alginate

5% Alginate0.5% Chitosan

Can alginate hydrogels be used to facilitate in-situ hydrogel formation?

• Non-homogenous behavior

• Insufficient elastic modulus with CSF Ca2+

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Parameters•Room temperature 37°C

Results•Determines rate of hydrogel formation•Fully gelled: <10% change from final modulus•Linear increase is due to evaporation

Gelation Point

Evaporation

Parameters•Equilibrated at 37°C for gelation time

Results•Determines linear-viscoelastic (LVE) limit•Hydrogels exhibit deformation above LVE•High strain below the LVE limit provides the best output signal

Linear-viscoelastic

limitViable strain values

Deformable Region

Rheological Characterization Protocol

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Gelation Point

Evaporation

Parameters•Equilibrated at 37°C for gelation time

Results•Determines linear-viscoelastic (LVE) limit•Hydrogels exhibit deformation above LVE•High strain below the LVE limit provides the best output signal

Linear-viscoelastic

limitViable strain values

Deformable Region


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Parameters• Strain is chosen based on position of LVE• Equilibrated at 37°C for gelation time

Results• Optimal value: low-frequency plateau

Parameters• Strain is chosen based on position of LVE• Frequency is chosen from low-frequency plateau• Equilibrated at 37°C for gelation time

Results• Determines true ultimate elastic modulus

Low-Frequency

Plateau

Ultimate Elastic Modulus Evaporation


Hydrogel Fabrication Protocol

Two Distinct StepsChitosan/Genipin Crosslinking

Alginate/CaCl2 Crosslinking

Improving Homogeneity: Novel Fabrication Method


6 mL

Two Distinct StepsChitosan/Genipin Crosslinking•Chitosan dissolved in 0.4% acetic acid



~6.8 mL




•Neutralized with 0.5M NaOH



~8.8 mL



•Neutralized with 0.5M NaOH•Addition of Genipin


10 mL




•Neutralized with 0.5M NaOH•Addition of Genipin•Brought to final volume with 0.85% NaCl


10 mL






•Incubated at 37°C for 24 hours

10 mL







•Alginate dissolved in 0.85% NaCl5 mL

15 mL0 mL







•Alginate dissolved in 0.85% NaCl•Chitosan/Genipin solution is added

20 mL0 mL







•Alginate dissolved in 0.85% NaCl•Chitosan/Genipin solution is added•CaCl2 added to solution•Complete solution mixed for 30 minutes

0 mL








20 mL

•Centrifuge for 2 minutes at 2,000 rcf

0 mL








20 mL

•Centrifuge for 2 minutes at 2,000 rcf

0 mL







•Alginate dissolved in 0.85% NaCl•Chitosan/Genipin solution is added•CaCl2 added to solution•Complete solution mixed for 30 minutes•Centrifuge for 2 minutes at 2,000 rcf•Excess liquid aspirated and gel used as needed

~2-5 mL of Usable Gel

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0.25% w/v alginate

Sensitivity to changes in Ca2+ concentration

• Alginate hydrogels demonstrate sensitivity to differences in calcium concentration as low as 1 mM • Fabrication process may allow for in-situ crosslinking at CSF Ca2+ levels

*

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Formulation of alginate chitosan hydrogel blends

• Ca2+ is varied to control elastic modulus

• Hydrogels approximate elastic modulus of spinal cord tissue (300-1000 Pa)


• Composition varied to control crosslinking behavior

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Mechanical response of alginate/chitosan hydrogels to in-situ conditions


In-situ gelation model• 500 μL of hydrogel is injected into a chamber slide• 200 μL neurobasal media is added to chamber slide and replaced daily (1.4 or 6 mM Ca2+)• Hydrogel is incubated at 37°C• Rheological assessment is performed after 2 or 5 days

A5/C0G0/Ca22

Low Ca2+

(1.4 mM)

A5/C25G01/Ca20

Low Ca2+

(1.4 mM)

A5/C25G01/Ca20

High Ca2+

(6 mM)

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Mechanical response of alginate/chitosan hydrogels to in-situ conditions


A

B

A5/C0G0/Ca22

A5/C25G01/Ca20

In-situ gelation model• 500 μL of hydrogel is injected into a chamber slide• 200 μL neurobasal media is added to chamber slide and replaced daily (1.4 or 6 mM Ca2+)• Hydrogel is incubated at 37°C• Rheological assessment is performed after 2 or 5 days

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Degradation Assay• 500 μL of hydrogel is injected into a 24 well plate

• 200 μL of aCSF was added to each well and changed daily (1.4 mM Ca2+)

• Hydrogel removed from well and weighed at designated time points

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Specific Aim 1 - ResultsHydrogel Electrical Charge

• Ninhydrin Assay• Ninhydrin binds to primary amines on chitosan• Absorbance correlates to free amine concentration• Free amine groups are related to hydrogel charge

Alginate

Chitosan

Genipin

Positive Charge

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• Overall similar structure for all hydrogel blends

• Pores size appears concentration dependent

•Hydrogel appearance is composition dependent – related to genipin concentration


A25/C0G0/Ca22

A5/C0G0/Ca22

A25/C125G1/Ca23

A25/C25G05/Ca18

A5/C125G1/Ca24

A5/C25G01/Ca20

Hydrogel Appearance and Internal Structure

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Specific Aim 1 - Conclusions• Alginate/chitosan hydrogels can be fabricated to mimic the elastic

modulus of native CNS tissue• Hydrogels demonstrate sensitivity to mM changes in Ca2+ concentration• Changes in mechanical behavior are observed following incubation in Ca2+

containing media, in a concentration dependent manner – indicative of change in crosslinking behavior

• Degradation, electrical charge and porosity are tunable by altering hydrogel composition


Injectable alginate/chitosan hydrogels can be fabricated to mimic the elastic modulus of native CNS tissue and respond to change in external Ca2+ concentration while exhibiting tunable physical and mechanical properties

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Specific Aim 2

Characterization of Astrocyte Attachment and Activation in Response to Interaction with Alginate/Chitosan Hydrogels


Astrocytes will exhibit greater attachment to hydrogels that demonstrate a higher positive charge while exhibiting no significant increase in reactivity relative to astrocytes cultured on poly-D-lysine coated glass

401 Sofroniew and Vinters 2010

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22 Allen and Barres 2009


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Astrocytes are Active Participants in the CNS Environment

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1Powell et al 1997 2Liesi and Silver 1988 3Tom et al 2004 4Beck et al 2008 5Gris et al 2007 6Christopherson et al 2005 7Hurtado et al 2011 8 Deng et al 2011

Astrocytes Can Direct Neurite Outgrowth

• ECM molecule production1-3

• Inhibitory: CSPGs, versican, keratin sulfate• Beneficial: Laminin, Fibronectin

• Signaling molecules• Axon growth inhibition: ERG-14, TGFβ5, SOX95

• Synapse formation: Thrombospondins6

7 8

Neurons follow migrating astrocytes into biomaterials within the lesion site

GFAP/SMI-31

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Hydrogel Composition Influences Astrocyte Attachment Behavior


A5/C0/G0/Ca22 A5/C125/G1/Ca24 A5/C25/G01/Ca22

Mag – 10XScale bar – 300 μm

Calcein-AMHoechst 33342

Attachment Assay• 500 μL of hydrogel was injected into a chamber slide well• 200 μL of astrocyte media was added• Stained with Calcein-AM and Hoechst 33342 after two days

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Hydrogel Composition Influences Astrocyte Attachment Behavior


• Astrocyte attachment is composition dependent

• Non-homogenous attachment – Clustering behavior is observed

A A

B

CC

A

B

B

AA

AA

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Hydrogel Composition Influences Astrocyte Activation


Astrocyte Activation Assay• 500 μL of hydrogel injected into chamber slide well• Astrocytes cultured for 2 days in astrocyte media. • Protein is isolated and expression was quantified via Western blot

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Specific Aim 2 - Conclusions

• Astrocyte Attachment is Composition Dependent

• Astrocytes Exhibit Differential Attachment Behavior Across Hydrogel Surface

• Hydrogel Composition Significantly Influences Astrocyte Activation


Astrocytes will exhibit greater attachment to hydrogels that demonstrate a higher positive charge while exhibiting no significant increase in reactivity relative to astrocytes cultured on poly-D-lysine coated glass

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Specific Aim 3

Assessment of the Mechanism of Astrocyte Attachment to Alginate/Chitosan Hydrogels and the Influence of Activation on Astrocyte Adhesion


Differential astrocyte adhesion that is observed on hydrogels with different compositions is a consequence of altered astrocyte behavior and the transition of astrocytes to a more reactive state

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Contradictory Attachment Behavior is Observed


Attachment is not correlated with an increased positive charge

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Possible Mechanisms Influencing Attachment

• Increased astrocyte activation

• Increased production of various ECM molecules

• Significant morphological changes hypertrophy and branching

• Increased astrocyte proliferation – Proliferation is correlated with astrocyte reactive state

• Hydrogel crosslinking behavior

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A5/C0/

G0/Ca22

A5/C125

G1/Ca24

A5/C25

G01/Ca22

Artificial Astrocyte Activation• Astrocytes cultured on poly-D-lysine coated well plates or hydrogels for two days• Media was DMEM or DMEM with transforming growth factor β1 (TGF-β1)

Influence of Increased Astrocyte Activation on Astrocyte Attachment

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Specific Aim 3 - ResultsInfluence of Hydrogel Components on Astrocyte Proliferation

Astrocyte Proliferation Assay

• Astrocytes were cultured on poly-D-lysine coated well plates

• 24 hour attachment period

• Following 24 hours, addition of media containing hydrogel components

• After 24 hours, astrocytes stained for Ki-67 and Hoechst 33342

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Specific Aim 3 - ResultsInfluence of Hydrogel Components on Astrocyte Attachment

Astrocyte Attachment Assay

• Astrocytes were cultured on poly-D-lysine coated well plates

• Media containing hydrogel components added simultaneously with astrocytes

• After 48 hours, media was removed and cells were stained with Calcein-AM and Hoechst 33342

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Specific Aim 3 - ResultsInfluence of Hydrogel Components on Astrocyte Morphology

0.5% Alginate

GNP 0.1

24 mM Ca2+

Poly-D-lysine coated glass control

GFAP/Hoechst 33342

0.125% Chitosan/0.1% Genipin 0.25% Chitosan/0.01% Genipin

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Proposed Hydrogel Crosslinking and Attachment Mechanism


Alginate Only

0.125% Chitosan /

0.1% Genipin

0.25% Chitosan / 0.01% Genipin

High genipin/genipin

Low alginate/chitosan

Low genipin/genipin

High alginate/chitosan

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• Increasing astrocyte activation does not influence astrocyte attachment

• Differential attachment is not a consequence of astrocyte proliferation

• Hydrogel components influence astrocyte morphology and attachment in a concentration dependent manner



Differential astrocyte adhesion that is observed on hydrogels with different compositions is a consequence of altered astrocyte behavior and the transition of astrocytes to a more reactive state

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Thesis Summary

• Developed an injectable, calcium sensitive hydrogel material for use in acute SCI

• Demonstrates increased crosslinking in an in situ gelation model

• Variable degradation rate, porosity and charge

• Promotes the attachment and activation of astrocytes in a composition dependent manner

• Activation without proliferation mild activation?

• Attachment is likely controlled by dominant forms of crosslinking within hydrogels

• Varying hydrogel composition can promote/inhibit attachment

Overall, the work in this thesis provide insight into the potential use of a novel calcium sensing biomaterial which may prove beneficial in providing

an environment conducive to neuronal regeneration in a combinatorial treatment for acute spinal cord injury

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Future Directions

• Modifying alginate polymer composition to increase sensitivity to physiological Ca2+ concentrations

• What is the role of ECM production? Do astrocyte produce significant inhibitory ECM molecules?

• Influence on neuronal behavior? Do hydrogels have a significant influence on neuronal excitoxicity in response to increased Ca2+ in media?

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Acknowledgements• Thesis Advisor

• Dr. Ryan Gilbert• Committee Members

• Dr. Deanna Thompson• Dr. Guohao Dai• Dr. Pankaj Karande

• Gilbert Lab Graduate Students• Dr. Christopher Rivet• Jonathan Zuidema• Nicholas Schaub• Christopher Johnson

• Dr. Deanna Thompson Lab• Dr. Linxia Zhang• Dr. Abby Koppes• Courtney Dumont• Chris Bertucci• Kathryn Kearns

This work was funded by support from the National Institutes of Health, National Insititute of Neurological Disorders and Stroke R21NS62392 and NSF CAREER Award 1150125 to R. Gilbert.

• Dr. Lee Ligon Lab• Dr. Lee Ligon• Joshua McLane

• Undergraduate Research Students• Rebecca Pomrenke• Elise DeSimone• Nicholas Zaccor• Greg Desmond

• High School Researchers• Addison Haxo• Austin Kim

• David Frey – Scanning Electron Microscopy

• Cindy, Dennis and Sarah McKay• Jacqueline Zaccor

Science

Christopher McKay - Rensselaer Polytechnic Institute - Doctoral Defense Presentation