Transcript Hydrogels for Tissue Engineering

Hydrogels for Tissue
Engineering
Sarah E. Eldred
Stahl/Gellman Groups
March 6, 2003
Organ Failure





Transplantation
Over 79,000 people in the United States on
organ waitlist in 2002
Over 6,000 waitlist deaths in 2002
15% average fatality rate within one year of
transplant
Lifelong immunosuppressant therapy
http://www.ustransplant.org
Outline

Background and Introduction

The Use of Hydrogels in Tissue Engineering




Implant Persistence – Biodegradability
Surgical Issues – Injectable Hydrogels
Cell Attachment – Peptide Enhanced Hydrogels
Outlook
Treatments for Organ Failure

Surgical Reconstruction



Can result in long-term
problems
Ineffective
Use of mechanical
organ substitutes


Cannot replace all
functions of the diseased
organ
Usually cannot halt
patient deterioration
Langer, R. P.; Vacanti, J. P. Science, 1993, 260, 920
Tissue Engineering

A multidisciplinary field aimed at “develop[ing]
biological substitutes that restore, maintain,
or improve tissue function”

Can involve transplantation of cells in artificial
matrices

Could lead to new therapies
Langer, R. P.; Vacanti, J. P. Science, 1993, 260, 920
Matrix Based Cell Transplantation

Matrix purposes




Maintain structural
integrity of the implant
Guide the growth of new
tissue
Allow for the invasion of
blood vessels
Provide necessary
mechanical forces to
cells
Cells
Matrix
Cell
Seeding
Incubation
Implantation
Marler, J. L.; Upton, J.; Langer, R.; Vacanti, J. P. Adv. Drug Deliv. Rev., 1998, 33, 165
Materials Used for Cell Matrices



Ceramics (bone)
Steel (arteries)
Polymers

Natural



Collagen
Gelatin
Synthetic



Poly(ethylene oxide)
Poly(acrylic acid)
Poly(vinyl alcohol)
Peppas, N. A.; Langer, R. Science, 1994, 263, 1715
Lee, K. Y.; Mooney, D. J. Chem. Rev., 2001, 101, 1869
Why Polymers?




Less likely than metals to harm surrounding
tissue
Useful for more varied types of tissue
Easier to seed cells into polymers than into
other types of materials
More chemical diversity
Peppas, N. A.; Langer, R. Science, 1994, 263, 1715
Hydrogels

Hydrophilic polymeric networks that can
absorb water without dissolving

Can be composed of natural or synthetic
polymers

First suggested for use in biomedical
applications in 1960
Hoffman, A. S. Adv. Drug Deliv. Rev., 2002, 43, 3
Wichterle, O.; Lim, D. Nature, 1960, 185, 117
Natural vs. Synthetic Hydrogels

Natural





Most closely resemble
the tissues they are
meant to replace
Almost always
biocompatible
Biodegradable
Difficult to isolate from
biological tissues
Restricted versatility

Synthetic





Can be reliably produced
Greater control over
polymer structure
May not be
biocompatible
Not always
biodegradable
Use of toxic reagents a
problem
Lee, K. Y.; Mooney, D. J. Chem. Rev., 2001, 101, 1869
Hydrogels as Tissue Engineering
Matrices

Advantages


Aqueous environment for
cells
Porous to allow for
nutrient transport

Easily modified

Usually biocompatible

Disadvantages

Hard to handle

Physically weak

Difficult to sterilize
Hoffman, A. S. Adv. Drug Deliv. Rev., 2002, 43, 3
Some Hydrogel Forming Polymers
Natural
HO2C
HO
O HO
O
OH
HO
poly(hyaluronic acid)
NaO2C
O
NH
O
O
HO
O
OH
n
n
O
poly(sodium alginate)
Synthetic
O
n
O
n
poly(lactic acid)
O
NH
poly(N-isopropyl acrylamide)
O
n
poly(ethylene glycol)
Preparation of Hydrogels
Monomers
Copolymerize
Copolymerize
Crosslink
Hydrogel
Macromers
Crosslink
Crosslink
Prepolymer
Polymerize
Polymerize
Monomer
Hydrogel
Interpenetrating
Network (IPN)
Hoffman, A. S. Adv. Drug Deliv. Rev., 2002, 43, 3
Outline

Background and Introduction

The Use of Hydrogels in Tissue Engineering




Implant Persistence – Biodegradability
Surgical Issues – Injectable Hydrogels
Cell Attachment – Peptide Enhanced Hydrogels
Outlook
Implant Persistence

Problems with non-biodegradable cell
matrices





Immunoresponse
Weakening of surrounding tissues
Lack of integration into body
Possibility of additional surgery
Ideal degradation of implants over time
Incorporating Biodegradability

Using labile bonds in the polymer backbone
and/or crosslinkers
O
O
N
O
O

O
Using peptides as labile linkages for
enzymatic degradation
O
N
H
H
N
O
O
N
H
Measuring Biodegradation
Fully Swollen Hydrogel
Buffered Aqueous
Solution
Remove Hydrogel
From Solution
Complete Dissolution of Hydrogel
Poly(anhydride) Hydrogels
O
O
CH2
x
O
n
x = 1, 4, 7

Slower degradation
with more hydrophobic
monomers
% Degradation
O
100
90
80
70
60
50
40
30
20
10
0
x=7
x=4
x=1
0
10
20
30
Time (days)
Domb, A. J.; Gallardo, C. F.; Langer, R. Macromolecules, 1989, 22, 3200
Synthesis
O
OH + Br
MeO
CH2
x
COOMe
1. MeONa/MeOH
2. NaOH
HOOC
O
CH2
COOH
x
1
1 + Ac2O
reflux
O
O
O
O
CH2
O
x
O
O
2
2
1. 180 oC
2. vacuum
O
O
O
CH2
x
O
n
x = 1, 4, 7
Domb, A. J.; Gallardo, C. F.; Langer, R. Macromolecules, 1989, 22, 3200
More Poly(anhydride) Hydrogels
O
O
O
O
CH2
O
O
8
n
methacrylated sebacic anhydride
O
O
O
O
O
CH2
O
O
6
1,6-bis(carboxyphenoxy) hexane
nO

Degradation from the surface of the hydrogel inward

Acrylate functionalities for crosslinking
Muggli, D. S.; Burkoth, A. K.; Anseth, K. S. J. Biomed. Mater. Res., 1999, 46, 271
Degradation Rates
Degradation rate
controlled by the ratios
of anhydride monomers
in the polymerization
feed
O
O
O
O
100
90
80
70
% Mass Loss

60
50
40
30
20
10
0
0
20
40
60
80
Time (days)
O
CH2
O
8
n
100% MSA
50% MSA
25% MSA
0% MSA
40% MSA
MSA
Muggli, D. S.; Burkoth, A. K.; Anseth, K. S. J. Biomed. Mater. Res., 1999, 46, 271
100
Poly(ethylene glycol) Hydrogels

Ester bonds added to the backbone using
poly(lactide)
O
O
O
O
O
Om

O
n
O
m
Constant mass loss rate
Metters, A. T.; Anseth, K. S.; Bowman, C. N. Polymer, 2000, 41, 3993
Hydrogel Synthesis
O
O
HO
CH2 CH2 O
n
O
H +
O
[Sn]
O
H O
200 oC
O
O
O
Acryloyl Chloride
n
H
m
O
O
Triethyl Amine
m
O
O
O
O
O
Om
n
O
O
photopolymerization
m
O
O
O
Om
O
O
n
O
m
Sawhney, A. S.; Pathak, C. P.; Hubbell, J. A. Macromolecules, 1993, 26, 581
Controlling the Degradation Rate


Vary the PEG
molecular weight
Lower molecular weight
monomers, slow
degradation due to
increased crosslink
density
PEG MW
Deg. Time
1000
45 days
4000
6 days
6000
5 days
10000
<1 day
Sawhney, A. S.; Pathak, C. P.; Hubbell, J. A. Macromolecules, 1993, 26, 581
Hydrogels with Labile Crosslinkers

Adipic acid dihydrazide to crosslink poly(aldehyde
guluronate)
O
O
O
OH
NaO2C
OH
O
H
NaO2C
N N
O
H
O
H
OH
OH
N
HO
H
N N
CO2Na
O
O
O
NaO2C
OH
H
O
O
O
OH
NaO2C
HO
HO
O
CO2Na
Lee, K. Y.; Bouhadir, K. H.; Mooney, D. J. Macromolecules, 2000, 33, 97
Controlling the Degradation Rate



Altering the
concentration of adipic
acid dihydrazide used
for crosslinking
100mM <1 equivalent
150mM = 1 equivalent
200mM >1 equivalent
100
90
80
Weight Loss (%)

70
60
100mM
50
150mM
40
200mM
30
20
10
0
0
10
20
30
40
50
Tim e (days)
Lee, K. Y.; Bouhadir, K. H.; Mooney, D. J. Macromolecules, 2000, 33, 97
Explanation of the Degradation Rate

Ability of the system to re crosslink with
excess crosslinker
Before Degradation
After Degradation
100mM
150mM
200mM
Lee, K. Y.; Bouhadir, K. H.; Mooney, D. J. Macromolecules, 2000, 33, 97
Hydrogels Degraded by Enzymes
PEG hydrogel with an
Ala-Pro-Glu-Leu
tetrapeptide as a
copolymer block


Susceptible to
collagenase enzymes
Collagenase
concentration
dependent degradation
rate
0.45
0.4
0.35
0.3
Weight (g)

0.25
0.2
0.15
0.1
0.05
0
0
1
2
3
4
5
Time (days)
2mg/mL
0.2mg/mL
West, J. L.; Hubbell, J. L. Macromolecules, 1999, 32, 241
Control
6
7
Outline

Background and Introduction

The Use of Hydrogels in Tissue Engineering




Implant Persistence – Biodegradability
Surgical Issues – Injectable Hydrogels
Cell Attachment – Peptide Enhanced Hydrogels
Outlook
Surgical Issues

Large incisions necessary for implantation of
tissue engineering hydrogels

Difficult to fill irregularly shaped spaces
(cartilage, bone)

Implantation without major surgery is
desirable
Fabrication of Injectable Hydrogels

Exploitation of the sol-gel phase transition
upon cooling

Adjustment of the lower critical solution
temperature (LCST) to be below body
temperature

Crosslinking the polymer in vivo
Measuring the Phase Change

% Transmittance – solutions are transparent,
gels are opaque

Swelling Ratio = Swollen Weight – Dry Weight
Dry Weight
Manipulating the Sol-Gel Transition
Temperature

Temperature dependent gel-sol phase
transition in PEG-PLA block copolymers
O
O
n
m
O


Gelation from packing of PLA segments
Injectable at 45°C and would gel upon
cooling to body temperature (~37°C)
Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature, 1997, 388, 860
Interpenetrating Networks


Made from poly(N-acryloylglycinamide) (PAG) and
poly(acrylic acid) (PAAc)
Hydrogen bonding between the two types of
polymer at low temperatures
PAG
PAAc
n
O
N
H
NH2
HO
O
n
O
Sasase, H.; Aoki, T.;Katono, H.;Sanui, K.; Ogata, N.; Ohta, R.; Kondo, T.; Okano, T.;
Sakurai, Y. Makromol. Chem., Rapid Commun., 1992, 13, 577
Addition of Urea
Temperature
dependent gel-sol
phase transition that
can be altered by the
addition of urea
100
80
% Transmittance

60
40
20
0
0
20
30
40
50
-20
O
H2N
10
Temp (oC)
NH2
PAG
0.5M urea
PAAc
0.1M urea
3M urea
0.01M urea
2M urea
0M urea
Urea
Sasase, H.; Aoki, T.;Katono, H.;Sanui, K.; Ogata, N.; Ohta, R.; Kondo, T.; Okano, T.;
Sakurai, Y. Makromol. Chem., Rapid Commun., 1992, 13, 577
60
Other IPNs

From poly(acrylamide),
PAAm, and PAAc which
form hydrogen bonds at
low temperature
n
PAAc
O
H
HN
O
H
O
PAAm
n
Katono, H.; Maruyama, A.; Sanui, K.; Ogata, N.; Okano, T.; Sakurai, Y. J. Controlled
Release, 1991, 16, 215
Explanation of the LCST


LCST = Lower Critical
Solution Temperature
The temperature at
which a phase
transition occurs from a
solution to a gel
T
Gel + Water
Solution
Weight fraction solute
Taylor, L. T.; Cerankowski, L. D. J. Polym. Sci., Polym. Chem. Ed., 1975, 13, 2551
Effect of Pendant Groups on LCST

Different monomers to adjust the LCST of a
polymeric system
O
N
R
O
O
+
RNH2
N
H
NHR
O
Cloud point of polymer (°C)
Methyl
35
Ethyl
20
Isopropyl
3
Ethyl Methoxy
55
Taylor, L. T.; Cerankowski, L. D. J. Polym. Sci., Polym. Chem. Ed., 1975, 13, 2551
Thermosensitivity of P(NIPAAm)
P(AAc)
Hydrogels P(NIPAAm)
n
HN
O
n
HO
O
Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules, 1999, 32, 7370
Thermosensitivity of P(NIPAAm)
P(AAc)
Hydrogels P(NIPAAm)
n
HN
n
O
HO
O
%Transmittance
100
80
60
40
20
0
24
26
28
30
32
34
36
38
40
Temperature (o C)
P(NIPAAm)
P(NIPAAm-co-Aac)
Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules, 1999, 32, 7370
Changing the LCST of P(NIPAAm)
Hydrogels

Copolymers with different amounts of AAm
30
Swelling Ratio
25
20
15
10
5
0
10
15
20
25
30
35
40
45
50
Temperature (oC)
30% AAm
20% AAm
10% AAm
5% AAm
0% AAm
Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y. J. Biomater. Sci. Polymer Edn., 1994, 6,
585

Thermosensitivity of
different acrylamide
polymers
Swelling Ratio
Altering the Thermosensitivity
20
18
16
14
12
10
8
6
4
2
0
0
10
20
30
40
50
Temperature (oC)
O
H
N
n
P(EAAm)
O
N
n
P(DMAAm)
O
H
N
n
P(NIPAAm)
O
N
n
P(DEAAm)
Okano, T.; Bae, Y. H.; Jacobs, H.; Kim, S. W. J. Controlled Release, 1990, 11, 255
60
Increasing Thermosensitivity

Hydrophilic groups moved further away from
the backbone
NIPAAm
HN
O
CIPAAm
HN
O
HO
O
Aoyagi, T.; Ebara, M.; Sakai, K.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Edn.,
2000, 11, 101
CIPAAm Synthesis
O
HO
NH2 O
NH2 O
OH
O
O
O
N
H
Cl
1. NaOH
O
2. HCl/H2O
NEt3/Et2O
O
O
N
OH
H
CIPAAm
Aoyagi, T.; Ebara, M.; Sakai, K.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Edn.,
2000, 11, 101
Changing the LCST?
4.5
Swelling Ratio
4
3.5
3
P(NIPAAm)
2.5
2
P(NIPAAm-coCIPAAm)
1.5
1
0.5
0
0
10
20
30
40
50
Temperature (o C)
Ebara, M.; Aoyagi, T.; Sakai, K.; Okano, T. Macromolecules, 2000, 33, 8312
Phase Changes in Acrylamide-Based
NIPAAm – NIPAAm –
Hydrogels NIPAAm co-AAm co- CIPAAm
0 sec
60 sec
80 sec
90 sec
100 sec
120 sec
Ebara, M.; Aoyagi, T.; Sakai, K.; Okano, T. J. Polym. Sci.: Part A: Polym. Chem., 2001,
39, 335
Another Thermoresponsive Hydrogel
H
HO
O n
O
o
120 C
+
O
O
HO
O
O
O
O
O
O
O
O
O
O
m
120 oC
O
O
n
O
n
O
O
O
O
O
O
O
130 oC, [Sn]
m
OH
O
OH
n
O
O
O
O
O
O
O
m
O
O
O
O
O
x
y
Jeong, B.; Kibbey, M. R.; Birnbaum, J. C.; Won, Y.-Y.; Gutowska, A. Macromolecules,
2000, 33, 8317
Phase Diagram of Graft Copolymer
Temperature ( o C)
45
40
35
Gel
30
Sol
25
Gel
20
Sol
12
14
16
18
20
22
24
26
Concentration (wt%)
Jeong, B.; Kibbey, M. R.; Birnbaum, J. C.; Won, Y.-Y.; Gutowska, A. Macromolecules,
2000, 33, 8317
Phase Diagram of Block Copolymer

Copolymer = PEGPLGA-PEG

Same shape as that of
graft copolymer

More hydrophobic –
more gelation
Jeong, B.; Bae, Y. H.; Kim, S. W. Macromolecules, 1999, 32, 7064
Thermoresponsive Hydrogels

Image of a hydrogel on either side of its LCST
25 °C
37 °C
Lin, H.-H.; Cheng, Y.-L. Macromolecules, 2001, 34, 3710
In vivo Hydrogel Formation
Cells +
Isolate and
culture cells
O
n
O
O
Inject polymer/cell
solution into
mouse
Hydrogel/cell construct in
mouse
UV light
Elisseeff, J.; Anseth, K.; Sims, D.; McIntosh, W.; Randolph, M.; Yaremchuk, M.; Langer,
R. Journal of Plastic and Reconstructive Surgery, 1999, 104, 1014
Use of Thermoresponsive Hydrogels
to Create an Artificial Organ



Injectable tissue engineering matrix to
implant Islets of Langerhans
Clear solutions in water at 25°C and immobile
gels at 35°C
Continued to produce insulin for several
weeks
P(NIPAAm)
P(AAc)
n
O
N
H
n
O
OH
Gutowska, A.; Kim, S. W.; Bae, Y. H. Macromol. Symp., 1996, 109, 155
Bae, Y. H.; Vernon, B.; Han, C. K.; Kim, S. W. J. Controlled Release, 1998, 53, 249
A Proof-of-Principle Experiment
Port for Cell Reseeding and Removal
Membrane
Solution
Phase
Gel Phase
Cells
Room
Temperature
Body
Temperature
Bae, Y. H.; Vernon, B.; Han, C. K.; Kim, S. W. J. Controlled Release, 1998, 53, 249
Outline

Background and Introduction

The Use of Hydrogels in Tissue Engineering




Implant Persistence – Biodegradability
Surgical Issues – Injectable Hydrogels
Cell Attachment – Peptide Enhanced Hydrogels
Outlook
Peptide Enhanced Hydrogels

Hydrogels in tissue engineering applications
= extracellular matrices

PEG’s lack of cell adhesiveness

Cell adhesion peptides on hydrogels
Proteins for Cell Attachment


Integrins – membrane bound receptors in
cells that bind to cell adhesion proteins
Bind to the peptide sequence Arg-Gly-Asp
HN
NH2
(RGD)
HN
O
O
N
H

H
N
O
O
OH
N
H
Can be attached to synthetic substrates to
promote cell attachment
Massia, S. P.; Hubbell, J. A. Cytotechnology, 1992, 10, 189
Attaching RGD to Polymers
O


GRGDY was covalently
attached to PAG
hydrogels
No cell adhesion assay
attempted
OH
OH
NaO2C
O
O
NaO2C
HN
O
HN
O
O
OH
O
NaO2C
OH
O
= Peptide
Bouhadir, K. H.; Hausman, D. S.; Mooney, D. J. Polymer, 1999, 40, 3575
Attaching RGD to Poly(urethane)
O
O
N
NaH
NH
O
O
O
O
+
O
N
O
O
O
n
n
n
OH
O
O
+
O Na
H
N
+ H2N
O
N
N
O
O
n
n
O
H
N
TFA
O
N
= Peptide
O
n
Lin, H.-B.; Garcia-Echeverria, C.; Asakura, S.; Sun, W.; Mosher, D. F.; Cooper, S. L.
Biomaterials, 1992, 13, 905
Cell Adhesion Assay
# Attached cells/area
12000
10000
8000
6000
4000
2000
0
0
50
100
150
200
250
300
Time (min)
PEU-GRGDVY
PEU-GRGDSY
PEU-COOH
PEU
Lin, H.-B.; Garcia-Echeverria, C.; Asakura, S.; Sun, W.; Mosher, D. F.; Cooper, S. L.
Biomaterials, 1992, 13, 905
Attaching RGD to Hydrogels
O
O
O
O
+ H2N
N
N
H
O
O
O
OCH2CH2
n
O
O
N
+ H2N
O
O
O
OCH2CH2
n
N
H
= Peptide

The hydrogels with the PEG spacer exhibited
superior cell adhesion to those without it
Hern, D. L.; Hubbell, J. A. J. Biomed. Mater. Res., 1998, 39, 266
Another Method of RGD Attachment

Random copolymer generated from the
following monomers and peptide attached
O
O
N

O
O
O
O
N
O
O
O
O
Capable of trapping cells
Moghaddam, M. J.; Matsuda, T. J. Polym. Sci.: Part A: Polym. Chem., 1993, 31, 1589
Synthesis and Crosslinking of
Monomer Units
O
HO
O
O
K2CO3
O
O
O
O
O
O
Br
O
Copolymer
O
hv
O
O
Moghaddam, M. J.; Matsuda, T. J. Polym. Sci.: Part A: Polym. Chem., 1993, 31, 1589
Outline

Background and Introduction

The Use of Hydrogels in Tissue Engineering




Implant Persistence – Biodegradability
Surgical Issues – Injectable Hydrogels
Cell Attachment – Peptide Enhanced Hydrogels
Outlook
The Future of Tissue Engineering



Current use of tissue engineered materials
Far cry from whole organs
Key issues for future work

Materials Science and Chemistry


Better scaffolds
Biology and Medicine


Cell differentiation
Surgical techniques
Zandonella, C. Nature, 2003, 421, 884
Acknowledgements
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
Shannon Stahl and Sam Gellman
The Gellman Group and the Stahl Group
Greg Hanson
Neil Strotman
Reagan Miller
Sharon Beetner
Nate Bowling
Erin Sabath
Matt Bowman
Stephen Seitz
Jeff Johnson
Will Lee