hydrogels for tissue engineering sarah e. eldred stahl/gellman groups march 6, 2003
TRANSCRIPT
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
Marler, J. L.; Upton, J.; Langer, R.; Vacanti, J. P. Adv. Drug Deliv. Rev., 1998, 33, 165
CellsMatrix
Cell Seeding
ImplantationIncubation
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
O
HOOH
HO2C
O
OHO
NH
HO
O
O
O
HOOH
NaO2C
O
O
O
O
NHO
npoly(hyaluronic acid) poly(sodium alginate)
n
n
poly(ethylene glycol)
n
poly(lactic acid)
n
poly(N-isopropyl acrylamide)
Natural
Synthetic
Preparation of Hydrogels
Hoffman, A. S. Adv. Drug Deliv. Rev., 2002, 43, 3
Monomers Copolymerize
Macromers
Crosslink
Prepolymer
Crosslink
Hydrogel
MonomerHydrogel
Polymerize
Interpenetrating Network (IPN)
Crosslink
Copolymerize
Polymerize
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
Using peptides as labile linkages for enzymatic degradation
O
O N
O
O O
NH
HN
NH
O
O
O
Measuring Biodegradation
Fully Swollen Hydrogel
Buffered Aqueous Solution
Remove Hydrogel From Solution
Complete Dissolution of Hydrogel
Poly(anhydride) Hydrogels
Slower degradation with more hydrophobic monomers
OO
CH2O
O
xn
x = 1, 4, 7
010
20304050
607080
90100
0 10 20 30
Time (days)%
Deg
rad
atio
n
x = 7
x = 4
x = 1
Domb, A. J.; Gallardo, C. F.; Langer, R. Macromolecules, 1989, 22, 3200
Synthesis
Domb, A. J.; Gallardo, C. F.; Langer, R. Macromolecules, 1989, 22, 3200
OHMeO
OBr CH2 COOMe
Ac2O OO
OCH2O
O
OO
OO
CH2O
O
HOOC O CH2 COOH
xn
x = 1, 4, 7
+x
1. MeONa/MeOH
2. NaOH x1
1 + reflux
x2
21. 180 oC
2. vacuum
More Poly(anhydride) Hydrogels
O
O
CH2
O
O
O O
8
methacrylated sebacic anhydride
OO
OO CH2 O
O
OO6
n
n1,6-bis(carboxyphenoxy) hexane
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
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100Time (days)
% M
ass
Lo
ss
100% MSA 50% MSA 40% MSA
25% MSA 0% MSA
Muggli, D. S.; Burkoth, A. K.; Anseth, K. S. J. Biomed. Mater. Res., 1999, 46, 271
O
O
CH2
O
O
O O
MSA
8 n
Poly(ethylene glycol) Hydrogels
Ester bonds added to the backbone using poly(lactide)
Constant mass loss rate
Metters, A. T.; Anseth, K. S.; Bowman, C. N. Polymer, 2000, 41, 3993
O
O
O
OO
O
OO
m n m
Hydrogel Synthesis
Sawhney, A. S.; Pathak, C. P.; Hubbell, J. A. Macromolecules, 1993, 26, 581
O
O
O
OO
O
OO
m n m
HO CH2 CH2 O Hn
+ O
O
O
O
200 oCO
O
OO
O
OH
m n m
H
Acryloyl Chloride
Triethyl Aminephotopolymerization
O
O
O
OO
O
OO
m n m
[Sn]
Controlling the Degradation Rate
Vary the PEG molecular weight
Lower molecular weight monomers, slow degradation due to increased crosslink density
Sawhney, A. S.; Pathak, C. P.; Hubbell, J. A. Macromolecules, 1993, 26, 581
PEG MW Deg. Time
1000 45 days
4000 6 days
6000 5 days
10000 <1 day
Hydrogels with Labile Crosslinkers
Adipic acid dihydrazide to crosslink poly(aldehyde guluronate)
Lee, K. Y.; Bouhadir, K. H.; Mooney, D. J. Macromolecules, 2000, 33, 97
O
O
OHNaO2C
OH
OH
NO N
HO
O
H
O HN
O
NH
O
CO2Na
O
O
N
H
NaO2C
OH
OHNaO2C
OHO
HOCO2Na
OOHNaO2C
OH
O
Controlling the Degradation Rate
Altering the concentration of adipic acid dihydrazide used for crosslinking
100mM <1 equivalent 150mM = 1 equivalent 200mM >1 equivalent
Lee, K. Y.; Bouhadir, K. H.; Mooney, D. J. Macromolecules, 2000, 33, 97
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50
Time (days)
Wei
gh
t L
oss
(%
)
100mM
150mM
200mM
Explanation of the Degradation Rate
Ability of the system to re crosslink with excess crosslinker
Lee, K. Y.; Bouhadir, K. H.; Mooney, D. J. Macromolecules, 2000, 33, 97
Before Degradation After Degradation
100mM
150mM
200mM
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
West, J. L.; Hubbell, J. L. Macromolecules, 1999, 32, 241
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 1 2 3 4 5 6 7
Time (days)W
eig
ht
(g)
2mg/mL 0.2mg/mL Control
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
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
O O
On m
Interpenetrating Networks
Made from poly(N-acryloylglycinamide) (PAG) and poly(acrylic acid) (PAAc)
Hydrogen bonding between the two types of polymer at low temperatures
NH
O NH2
O
HO OPAAcnPAG
n
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
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
H2N NH2
O -20
0
20
40
60
80
100
0 10 20 30 40 50 60
Temp (oC)%
Tra
ns
mit
tan
ce
PAG PAAc 3M urea 2M urea
0.5M urea 0.1M urea 0.01M urea 0M urea
Urea
Other IPNs
From poly(acrylamide), PAAm, and PAAc which form hydrogen bonds at low temperature
Katono, H.; Maruyama, A.; Sanui, K.; Ogata, N.; Okano, T.; Sakurai, Y. J. Controlled Release, 1991, 16, 215
PAAc
PAAm
OO
H
n
HN O
H
n
Explanation of the LCST
LCST = Lower Critical Solution Temperature
The temperature at which a phase transition occurs from a solution to a gel
Taylor, L. T.; Cerankowski, L. D. J. Polym. Sci., Polym. Chem. Ed., 1975, 13, 2551
Gel + Water
Solution
Weight fraction solute
T
Effect of Pendant Groups on LCST
Different monomers to adjust the LCST of a polymeric system
NO
O
RNH2 NH
NHR
O
O
+
Taylor, L. T.; Cerankowski, L. D. J. Polym. Sci., Polym. Chem. Ed., 1975, 13, 2551
R Cloud point of polymer (°C)
Methyl 35
Ethyl 20
Isopropyl 3
Ethyl Methoxy 55
Thermosensitivity of P(NIPAAm) Hydrogels
OHN
P(NIPAAm)
OHO
P(AAc)
n n
Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules, 1999, 32, 7370
Thermosensitivity of P(NIPAAm) Hydrogels
OHN
P(NIPAAm)
OHO
P(AAc)
n n
Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules, 1999, 32, 7370
0
20
40
60
80
100
24 26 28 30 32 34 36 38 40
Temperature (oC)
%T
ran
smit
tan
ce
P(NIPAAm) P(NIPAAm-co-Aac)
Changing the LCST of P(NIPAAm) Hydrogels Copolymers with different amounts of AAm
Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y. J. Biomater. Sci. Polymer Edn., 1994, 6, 585
0
5
10
15
20
25
30
10 15 20 25 30 35 40 45 50
Temperature (oC)
Sw
ellin
g R
atio
30% AAm 20% AAm 10% AAm 5% AAm 0% AAm
Altering the Thermosensitivity
Thermosensitivity of different acrylamide polymers
OHN
P(NIPAAm)
O N
P(DMAAm)
OHN O N
nnn
P(EAAm)n
P(DEAAm)
Okano, T.; Bae, Y. H.; Jacobs, H.; Kim, S. W. J. Controlled Release, 1990, 11, 255
0
2
4
6
8
10
12
14
16
18
20
0 10 20 30 40 50 60
Temperature (oC)
Sw
ellin
g R
atio
Increasing Thermosensitivity
Hydrophilic groups moved further away from the backbone
Aoyagi, T.; Ebara, M.; Sakai, K.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Edn., 2000, 11, 101
OHN
NIPAAm
OHN
OHO
CIPAAm
CIPAAm Synthesis
Aoyagi, T.; Ebara, M.; Sakai, K.; Sakurai, Y.; Okano, T. J. Biomater. Sci., Polym. Edn., 2000, 11, 101
OH
NH2 OHO
O
NH2 OCl
O
CIPAAm
NH
O
O
O
NH
O
OH
O
NEt3/Et2O
1. NaOH
2. HCl/H2O
Changing the LCST?
Ebara, M.; Aoyagi, T.; Sakai, K.; Okano, T. Macromolecules, 2000, 33, 8312
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 10 20 30 40 50
Temperature (oC)
Sw
elli
ng
Rat
io
P(NIPAAm)
P(NIPAAm-co-CIPAAm)
Phase Changes in Acrylamide-Based Hydrogels
Ebara, M.; Aoyagi, T.; Sakai, K.; Okano, T. J. Polym. Sci.: Part A: Polym. Chem., 2001, 39, 335
0 sec
60 sec
80 sec
90 sec
100 sec
120 sec
NIPAAmNIPAAm –
co-AAmNIPAAm –
co- CIPAAm
Another Thermoresponsive Hydrogel
Jeong, B.; Kibbey, M. R.; Birnbaum, J. C.; Won, Y.-Y.; Gutowska, A. Macromolecules, 2000, 33, 8317
HOO
H
n+
OO O
120 oC OO OH
HO
O O
O O
n
OO
O O
mO O O
O O
OHn m
O
O
O
OO
O
O
O
O O O
O O
O
n m
O
O
O
Ox y
130 oC, [Sn]120 oC
Phase Diagram of Graft Copolymer
Jeong, B.; Kibbey, M. R.; Birnbaum, J. C.; Won, Y.-Y.; Gutowska, A. Macromolecules, 2000, 33, 8317
Gel
Sol20
25
30
35
40
45
12 14 16 18 20 22 24 26
Concentration (wt%)
Tem
per
atu
re (
oC
)
Gel
Sol
Phase Diagram of Block Copolymer
Copolymer = PEG-PLGA-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
Lin, H.-H.; Cheng, Y.-L. Macromolecules, 2001, 34, 3710
25 °C 37 °C
In vivo Hydrogel Formation
Elisseeff, J.; Anseth, K.; Sims, D.; McIntosh, W.; Randolph, M.; Yaremchuk, M.; Langer, R. Journal of Plastic and Reconstructive Surgery, 1999, 104, 1014
Isolate and culture cells
Inject polymer/cell solution into
mouse
UV lightHydrogel/cell construct in mouse
O
O On
Cells +
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
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
OHONH
O
P(NIPAAm) P(AAc)
nn
A Proof-of-Principle Experiment
Bae, Y. H.; Vernon, B.; Han, C. K.; Kim, S. W. J. Controlled Release, 1998, 53, 249
Room Temperature
Body Temperature
Solution Phase Cells
Port for Cell Reseeding and Removal
Membrane
Gel Phase
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
(RGD)
Can be attached to synthetic substrates to promote cell attachment
Massia, S. P.; Hubbell, J. A. Cytotechnology, 1992, 10, 189
NH
HN
NH
O
O
O
HN
HN NH2
O
OH
Attaching RGD to Polymers
GRGDY was covalently attached to PAG hydrogels
No cell adhesion assay attempted
Bouhadir, K. H.; Hausman, D. S.; Mooney, D. J. Polymer, 1999, 40, 3575
O
O OH
NaO2COH
OHN
OHNO
NaO2C
OO
O
OH
OHNaO2C
= Peptide
Attaching RGD to Poly(urethane)
Lin, H.-B.; Garcia-Echeverria, C.; Asakura, S.; Sun, W.; Mosher, D. F.; Cooper, S. L. Biomaterials, 1992, 13, 905
NHO
O
NO
O
O OH
NaH
TFA
NO
O
H2N
O
O
NO
O
OHN
NO
O
OHN
NO
O
O ONa+
n
-
n
+
-
n
n
+
n
n= Peptide
Cell Adhesion Assay
Lin, H.-B.; Garcia-Echeverria, C.; Asakura, S.; Sun, W.; Mosher, D. F.; Cooper, S. L. Biomaterials, 1992, 13, 905
0
2000
4000
6000
8000
10000
12000
0 50 100 150 200 250 300
Time (min)
# A
ttac
hed
cel
ls/a
rea
PEU-GRGDVY PEU-GRGDSY PEU-COOH PEU
Attaching RGD to Hydrogels
ON
OO
O
OCH2CH2
O
O
ON
O
O
H2N
NH
O
OCH2CH2
O
NH
O
+
n
+
n
H2N
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
= Peptide
Another Method of RGD Attachment
Random copolymer generated from the following monomers and peptide attached
Capable of trapping cells
Moghaddam, M. J.; Matsuda, T. J. Polym. Sci.: Part A: Polym. Chem., 1993, 31, 1589
O
NO
O
O O O
ON
OO
O
Synthesis and Crosslinking of Monomer Units
Moghaddam, M. J.; Matsuda, T. J. Polym. Sci.: Part A: Polym. Chem., 1993, 31, 1589
O OHOK2CO3
O
O
Br
O O
OO
O
O
O O O
hvCopolymer
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
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