1. Creating and Measuring Variable
Modulus Hydrogels
Luke Boustred

2. What is Regenerative Medicine?
• Branch of medicine that:
“deals with the process of
replacing, engineering or
regenerating human cells,
tissues, etc. to restore or
establish normal function”1
• Multidisciplinary:
– Tissue engineers
– Molecular biologist
• “Translational”
– Bridges research and product
development
– Ultimately, enhances human
health and well-being
1: Regenerative Medicine, 2008, 3(1), 1–5 [47]
Luke!

3. The Story and History: 1900’s–Now
• 1968: First Bone Marrow Transplant
– Siblings, severe combined immunodeficiency
• 1978: Stem cells “discovered”
– Human umbilical cord blood
• 1992: term “regenerative medicine” coined in hospital
administration article
• 2008: first ever tissue engineered trachea transplantation
– Patient’s stem cells  chondrocytes (cartilage)
– Decellularized donor trachea
– Success!
• 2010: Development of “Pixie Dust”
– Decellularized ECM
– Regrow finger!

4. Pixie Dust Testing

5. History: What’s the Point?
• Regenerative Medicine consists of only two
decades of scientific application and
development
• 20th century: “century of physics”
• 21st century: “century of biomedicine”

6. Scaffolds and Regenerative Medicine
• “Pixie Dust”
– ECM, native
– Ideal biocompatibility
• Polymers
– Easy to fabricate
• Quickly
• In large quantities
– Degradable!
– Dynamic application…
• 3D Printing
– Customize polymer:
• Density
• Strength
• Shape
• And more
– Above all: mimic natural
structure and chemistry
• 3D Printing in action:
• https://upload.wikimedia.or
g/wikipedia/commons/5/5d
/Hyperboloid_Print.ogv

7. Hydrogels as Cellular Scaffolds
• Water-insoluble, cross-
linked polymer chains
• Swell when immersed
in water (5-200+ times
dry volume)
• Typically ~90% water by
mass
• Can have single or
multiple polymer
networks

8. VitaGel: Hydrogels in action

9. Motivation for Experimentation
• Explore and Research:
– the fabrication and analysis of multiple network gels has yet to
be extensively explored
– Multiple network gels have greatly increased toughness
compared to single network gels
• Application:
– hydrogels make great cellular scaffolds
– characterize multiple network hydrogels to understand gel
properties
– ultimately, implant scaffolds into body for tissue regeneration
(3D printing)
– Different tissue requires differing toughness–specify hydrogel
toughness with number of polymer networks

10. Variable Modulus Hydrogels
Depiction of Osteochondral Interface: taken from Geistlich Surgery

11. The Polymer Network & Hydrogel
Structure
• The polymer is comprised of many branches and cross-linkers
• Molecular branches are connected through thiol-ene “click
chemistry” bonds
• Molecular lattice formed
• Create many branch-branch linkages, and you get a polymer
network
Schematic of chemical reaction: S. Skaalure, CU Boulder Dept. of Chem and Bio. Eng. Dissertation (2014) [Modified]

12. Methodology
How to Make Multiple Network Gels
Step 1: polymerize precursor
solution (UV exposure)
Step 2: immerse gel into
monomer bath
Step 3: re-expose gel
to UV light
Repeat steps 2-3 as necessary

13. Laboratory Setup
The syringe setup Place Syringes under UV

14. Properties of Interest
• Compressive and Shear
Moduli
– Strength of gel
– mechanical testing
machine
• Swelling Ratio
– Change in mass
– Dry and wet gel mass
• Gel fraction
– % of gel by mass
– Dry and wet gel mass
Strain testing image from Chan,
B et al. in Macromolecular
Biosciences 2012

15. Data Extracted: Mechanical Properties
0
20
40
60
80
100
120
140
160
0 1 2 3 4
CompressiveModulus(kPa)
Diffusion Cycle #
Compressive Modulus vs. Diffusion Cycle
0
10
20
30
40
50
60
0 1 2 3 4
ShearModulus(kPa)
Diffusion Cycle #
Shear Modulus (kPa) vs. Diffusion Cycle

16. Data Extracted: Gel Properties
0.0%
1.0%
2.0%
3.0%
4.0%
5.0%
6.0%
7.0%
8.0%
9.0%
10.0%
0 1 2 3 4
GelFraction
Diffusion Cycle #
Gel Fraction vs. Diffusion Cycle
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
0 1 2 3 4
SwellRatio
Diffusion Cycle #
Swell Ratio vs. Diffusion Cycle #

17. What Do We Learn?
Mechanical Properties
• Compressive and shear
modulus increase by a
factor of 3.5 over 3 diffusion
cycles
– Logarithmic increase
• More networks  greater
force dispersion
• Strength appears to level off
after 4 diffusion cycles
Gel Properties
• Gel fraction increases by
3.1%
– Initially a linear increase
– Minimal change after 2nd
diffusion cycle
• Swelling ratio decreased to
30% of original value
• Less “space” for water

18. Diffusion Cycle Studies
• Extend number of diffusion
cycles
– Max mechanical properties
– Optimize
• Repeat experiment for data
evaluation
0
20
40
60
80
100
120
140
0 2 4 6 8
CompressiveModulus(kPa)
Diffusion Cycle #
Future Direction

19. Thank You!
• A special thanks to Stephanie Bryant and the members of her lab for
donating their time and resources
– Mentors: Callie Fiedler, and Elizabeth Aisenbrey
– Sadhana Sharma
– Marja-Leena Kaariainen
– Luke Amer
– Aaron Aziz
– Stanley Chu
– Margaret Schneider
– Andrea Marks
– Nikki Machalek
– Zachary Reinking
– Anna Iisa
– Conor Messer
• The Faculty of BURST for their guidance and time

20. Interesting Lab features
Incubator Bioreactors (cyclic compression)

21. Questions?
• CU Boulder
• Engineering
• Hydrogels
• The future of regenerative medicine
– Ethics of organ printing and longevity
• What’s it like to be in a research lab?
– I.e. what do you REALLY do with your time?