Presenter NameTitle/Date

Calcium Responsive Composite Hydrogels for Acute Spinal Cord

InjuryMechanical/Chemical Properties and Astrocytic Response

Christopher McKay, Christopher Johnson, Rebecca Pomrenke, Joshua McLane, Nicholas Schaub, Elise DeSimone, Lee Ligon, and Ryan Gilbert

BMES 2014

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SPINAL CORD INJURY AFFECTS OVER 1 MILLION PEOPLE 1

1

1 NCISC 2012

Fewer than 1% Recover

16 300 80

SCI

Lifetime Costs$1-4 Million1

Yearly Costs$10 Billion1

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FEWER THAN 1% EXPERIENCE COMPLETE FUNCTIONAL RECOVERY 1

Force

http://imgarcade.com/1/human-spine-anatomy/

Vertebra

Sp

inal C

ord

2

(1 NSCISC, 2012)

Contusion

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PROGRESSION OF SPINAL CORD INJURY

ACUTE (0-48hr)

SUB-ACUTE (48hr – 2 weeks)

CHRONIC (> 2 weeks )

• Inflammatory Response1

• Reactive Astrocyte Formation1

• Calcium Related Neuronal Death2

Ca++

Ca++

Ca++

Ca++

3 Liverman 2005

• Macroscopic Tissue Damage 1

• Disrupted Blood Brain Barrier

1

1 Tator 1998

• Formation of the Glial Scar3

3

= Reactive Astrocyte

2 Happel, 1981

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DESIGN CRITERIA

Conform to the irregular geometry of the contusion.

An injectable hydrogel is best suited to fill the contusion

The modulus should be near that of native tissue (200-1000Pa).

Hydrogels can be tuned to match the soft SC tissue

Sequester extracellular calcium. Alginate sequesters calcium during hydrogel formation

Facilitate astrocyte integration May improve material integration

4

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ALGINATE

• Biocompatible, low cytotoxicity1

• Crosslinks with Ca2+ ions2,3

• Negatively charged4

CHITOSAN

• Amine Groups shown to enhance cellular adhesion5

GENIPIN

• Natural crosslinking agent• Well suited for spinal cord

environments6-9

1 Wee and Gombotz 1998 2 Braccini and Perez 2001 3 Li et al 2007 4 Rowley et al 1999 5 Zuidema et al 20116 Yamazaki et al 2004 7 Koo et al 2006 8 Moura et al 2011 9 Yamazaki et al 2005

AN INJECTABLE HYDROGEL CAN FILL THE IRREGULAR GEOMETRY OF THE CONTUSION

5

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HYDROGEL STRUCTURE

6

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• Composition varied to control crosslinking behavior

INCREASE THE AMINE GROUPS IN THE SCAFFOLD TO IMPROVE ASTROCYTE

ATTACHMENT

• Internal crosslinking structural influences hydrogel charge

7

Amine Groups

Alginate

?

Chitosan

?

Genipin?

Facilitate astrocyte integration

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High Chitosan

Low Genipin

Low Ca2+

(1.4 mM)

INCREASED CALCIUM FROM IN-SITU CONDITIONS INCREASES THE HYDROGEL

STABILITY

In-situ gelation model

• 500 μL hydrogel injected into chamber slide

• 200 μL neurobasal media (1.4 or 6 mM Ca2+)

• Hydrogel incubated at 37°C. Media replaced daily

• Rheological assessment at 0, 2 and 5 days

High Chitosan

Low Genipin

High Ca2+

(6 mM)

8

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ASTROCYTE ATTACHMENT BEHAVIOR IS NOT DIRECTLY CORRELATED WITH THE AVAILABLE

AMINE GROUPS

Calcein-AM | Hoechst 33342 | 10X Magnification | Scale bar = 300 μm

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 daysNo Chitosan / No

GenipinLow Chitosan / High Genipin High Chitosan / Low

Genipin

0.5

% A

lgin

ate

9

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A A

B

CC

A

• Astrocyte attachment depends on composition

• Altered protein adsorption?

10

Hig

h A

lgin

ate

(0

.5%

)

Increasing NH4+

ASTROCYTE ATTACHMENT BEHAVIOR IS NOT DIRECTLY CORRELATED WITH THE AVAILABLE

AMINE GROUPS

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DESIGN CRITERIA Conform to the irregular geometry of the contusion.

Designed an injectable hydrogel with tunable mechanical properties

The modulus is near that of native tissue (200-1000Pa).

Hydrogels are responsive to calcium and can be tuned to match the soft SC tissue

Sequester extracellular calcium.

Alginate sequesters calcium during hydrogel formation

Facilitate astrocyte integration

Astrocytes attached to hydrogel surface in a composition dependent manner

11

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ACKNOWLEDGEMENTSAdvisor• Dr. Ryan GilbertGilbert Lab Dr. Christopher Rivet• Dr. Jonathan Zuidema• Nicholas SchaubDr. Deanna Thompson Lab• Dr. Linxia Zhang• Dr. Abby Koppes• Dr. 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

12

THANK YOU!THANK YOU!

For more information, see corresponding publication:

McKay C. A., R. D. Pomrenke, J. S. McLane, N. J. Schaub, E. K. DeSimone, L. A. Ligon, and R. J. Gilbert. “An Injectable, Calcium Responsive Composite Hydrogel for the Treatment of Acute Spinal Cord Injury.” ACS Applied Materials & Interfaces 6, no. 3 (February 12, 2014): 1424–38. doi:10.1021/am4027423.