Redox-Responsive Degradable PEG Cryogels as Potential Cell Scaffolds in Tissue Engineering

Article abstract of DOI:10.1002/mabi.201100396

A Michael addition strategy involving the reaction between a maleimide double bond and amine groups is investigated for the synthesis of cryogels at subzero temperature. Low-molecular-weight PEG-based building blocks with amine end groups and disulfide-containing building blocks with maleimide end groups are combined to synthesize redox-responsive PEG cryogels. The cryogels exhibit an interconnected macroporous morphology, a high compressive modulus and gelation yields of around 95%. While the cryogels are stable under physiological conditions, complete dissolution of the cryogels into water-soluble products is obtained in the presence of a reducing agent (glutathione) in the medium. Cell seeding experiments and toxicologic analysis demonstrate their potential as scaffolds in tissue engineering.

Full text of DOI:10.1002/mabi.201100396

Full Paper
Redox-Responsive Degradable PEG Cryogels as
Potential Cell Scaffolds in Tissue Engineering
Tugba Dispinar, Wim Van Camp, Liesbeth J. De Cock, Bruno G. De Geest,
Filip E. Du Prez*
A Michael addition strategy involving the reaction between a maleimide double bond and
amine groups is investigated for the synthesis of cryogels at subzero temperature. Low-
molecular-weight PEG-based building blocks with amine end groups and disulfide-containing
building blocks with maleimide end groups are combined
to synthesize redox-responsive PEG cryogels. The cryogels
exhibit an interconnected macroporous morphology, a high
compressive modulus and gelation yields of around 95%.
While the cryogels are stable under physiological con-
ditions, complete dissolution of the cryogels into water-
soluble products is obtained in the presence of a reducing
agent (glutathione) in the medium. Cell seeding exper-
iments and toxicologic analysis demonstrate their poten-
tial as scaffolds in tissue engineering.
1
. Introduction
sionaldesign. Thescaffoldshouldallowcellularattachment
and provide sufficient space for proliferation inside the
three-dimensional scaffold matrix. A porous scaffold
structure – preferably with interconnected pores – is
required to provide proper oxygen and nutrient transport
within the scaffold matrix. Finally, the scaffold should be
able to degrade into non-toxic water soluble compounds
once it has served its purpose.
The developments in the area of tissue engineering have
highlighted the need for novel degradable scaffolds that are
capable to provide a suitable environment for living cells
[1,2]
and to allow tissue growth.
Since these scaffolds are
used as a support to grow cells and to regenerate tissue
constructs with various shapes and sizes, they have to meet
[3]
several important requirements. Obviously, the scaffold
should consist of a biocompatible material. As it functions
as a support, the material should have a proper mechanical
strength, preferably with an easily tunable three-dimen-
From this point of view, macroporous hydrogels have
received considerable interest in tissue engineering applica-
[4]
tions as hydrophilic, crosslinked networks. The hydro-
philicity of these materials is tuned by the constructive
compound (monomeric unit) and plays a crucial role with
regard to swelling properties of the scaffolds. This hydro-
philic behavior leads to water soluble products during the
degradation process, therefore providing an easy elimina-
tion of the scaffold. The macroporosity of the materials
supplies a relatively high surface area for cell attachment
and also improves the nutrient and oxygen transport inside
the scaffold. Macropores within the hydrogel structure
can be created via various techniques including freeze-
T. Dispinar, Dr. W. Van Camp, Prof. F. E. Du Prez
Department of Organic Chemistry, Polymer Chemistry Research
Group, Ghent University, Krijgslaan 281, S4-bis, B-9000 Ghent,
Belgium
E-mail: filip.duprez@ugent.be
L. J. De Cock, Dr. B. G. De Geest
Laboratory of Pharmaceutical Technology, Department of
Pharmaceutics, Ghent University, Harelbekestraat 72, B-9000
Ghent, Belgium
[5]
[6]
[7]
[8]
drying, porogenation, phaseseparation, gasblowing
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DOI: 10.1002/mabi.201100396
383

T. Dispinar, W. Van Camp, L. J. De Cock, B. G. De Geest, F. E. Du Prez
www.mbs-journal.de
and polymerization of a high-internal-phase emulsion
the use of biodegradable natural compounds such as
dextran, L-lactic acid (LLA), gelatin and chitosan. These
natural compounds have either been used as such or
combined with other natural or synthetic compounds to
prepare degradable cryogels. Examples include HEMA/LLA/
[9,10]
(polyHIPE).
In addition to these techniques, cryotropic
gelation (cryogelation) is considered as a rather new
[11]
approach to prepare macroporous hydrogels.
In this
method, the three-dimensional (3D) gel matrices are
produced in a partially frozen medium in which the
reactants are concentrated in the unfrozen regions and then
polymerize to a dense polymeric matrix in between the
frozen regions (i.e., being ice crystals in the case of a
hydrogel). These ice crystals template continuous inter-
connected pores during the gel formation. Upon completion
of the gelation process, the actual pores appear after melting
of the ice crystals during a defrosting step. Generally,
macroporous hydrogels that are synthesized via cryotropic
gelation are entitled as cryogels. Cryogels usually feature
large interconnected macropores with sizes ranging from 10
[20]
[21]
[22]
dextran-, gelatin-, gelatin/fibrinogen- and gelatin/
[23]
chitosan -based cryogels. These reported cryogel systems
show a hydrolytic degradation behavior under physiologi-
cal conditions. As a result, they exhibit gradual degradation
kinetics with degradation times ranging from days to
months. Degradation rates of these cryogels are adjusted by
variation of the molecular weights of the polymeric
reactants, incorporation of other polymeric segments, or
by adjusting the crosslink density. On the other hand, this
continuous hydrolysis process leads to the gradual weak-
ening of the system during the tissue growth. However, as it
isoftendesirabletokeepthemechanicalsupportthroughout
the whole process of tissue growth, systems with relatively
fast, on-demand degradation kinetics are desirable. An
orthogonal degradation mechanism that allows a rather
quick, on-demand degradation upon completion of the
tissuegrowthwouldbebeneficial.Ideally,thedegradationof
the scaffold should lead to non-toxic, water-soluble com-
pounds as the result of an external trigger. In this respect,
disulfide bondsareofparticularinterestsince theyare stable
against hydrolysis, but can be cleaved orthogonally in the
presence of reducing agents such as dithiothreitol (DTT),
L-cysteine and glutathione through thiol-disulfide exchange
[12]
up to 100 mm.
Furthermore, they show a spongy nature
with fast drying and re-swelling capabilities. These specific
properties make cryogels potential materials for the use in a
wide range of biotechnological applications. (Bio)separa-
[13,14]
[15]
[16]
tion,
(bio)catalysis
and affinity chromatography
are currently the most common application fields of the
cryogels.Moreover,cryogelshavebeenrecentlyinvestigated
as potential cell scaffolds. For this purpose, cryogels were
prepared from biocompatible precursors in aqueous med-
ium. Recently, our group reported on the synthesis of
poly(hydroxyethyl methacrylate) [poly- (HEMA)]-based
cryogels with high potential as cell scaffolds thanks to the
excellent biocompatibility and tunable mechanical proper-
ties of polyHEMA. In addition, the primary alcohol function
of HEMA allows further modification reactions to introduce
desired fuctionalities or even biomolecules such as ther-
apeuticagentsandgrowthfactorsinthefinalcompound.For
instance, alkyne functionalities were introduced via ester-
ificationwith4-pentynoicacidandthenfurtherreactedwith
variousazide-containingmodelcompoundsviathecopper(I)
[24]
reactions.
Therefore, disulfide bonds have been investi-
gatedforavarietyofapplicationsrangingfromdrugdelivery
[25,26]
to soft-tissue engineering.
Especially in soft-tissue
engineering, disulfide containing scaffolds could be more
suitable to culture cells in a 3D environment or to
reconstitute tissues in vitro that might be utilized for the
replacement of diseased tissues in vivo.
This redox-responsive cleavage of disulfide bonds also
allows the design of cryogels with controllable degradation
kinetics, which has already been explored with partial
success. Recently, Andac et al. reported partially degradable
[17]
catalyzed Huisgen ‘click’ cycloaddition reaction.
In
another report, we have tuned the cell adhesion properties
of the cryogels through grafting of stimuli-responsive
poly(N-isopropylacrylamide) from the pore walls by atom-
[27]
disulfide containing polyHEMA-based cryogels.
These
[18]
transfer radical polymerization (ATRP).
Poly(ethylene
cryogels were prepared using a free-radical polymerization
(FRP) process by employing at the same time a disulfide
containing crosslinker as well as a non-degradable cross-
linker in the cryogel synthesis. The second non-degradable
crosslinker was necessary to improve the gel yields and
the stabilityofthe cryogelmatrixsincethecryogel formation
withonly disulfide containing crosslinkerwas notsuccessful.
Until now, the most widely applied synthetic route to
prepare cryogels involves FRP using the ammonium
glycol)(PEG)-basedcryogelshavealsobeenrecentlyreported
[19]
as potential cell scaffolds. The choice for PEG is motivated
by the fact that it is one of the most widely applied synthetic
polymers in hydrogel synthesis for tissue engineering
[4]
because of its biocompatibility. Also in this case, the
microstructure of PEG cryogels could be tuned and it was
shown that the manipulation of the microstructure could be
used as a tool to fine-tune the swelling behavior and
mechanicalproperties ofthe cryogels. Ingeneral, most ofthe
studieshavedealedwithanoptimizationofthephysicaland
structural properties of the cryogels.
0
0
persulfate/N,N,N ,N -tetramethylethylenediamine (APS/TEMED)
initiation system since this redox system can generate
radicals and initiate polymerization, even at low tempera-
[11,28]
Atthesametime,somestudieshavebeenfocusingonthe
design of degradable cryogels. Most of the reports focus on
tures.
Since for specific cases, the FRP process has some
disadvantages, other synthetic strategies have been
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Redox-Responsive Degradable PEG Cryogels as Potential Cell Scaffolds . . .
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explored. Indeed, the disulfide bond might interfere with
the FRP process, causing an undesired radical formation in
another class of nucleophiles that react readily with the
maleimide double bond, however in a slightly slower
[38]
[29,30]
the polymerization medium.
Moreover, chain transfer
fashion compared to the thiols.
reactions might result in additional and permanent cross-
links in the system, and consequently influence the
In 2001, Elbert and Hubbell were the first to use a
conjugate addition strategy reacting an acrylate or vinyl
sulfone end functionalized multi-arm PEG macromono-
[31,32]
degradation ability of the material.
We observed
ourselves that disulfide containing crosslinkers, namely
mers with thiol end functionalized macromonomers to
[39]
0
N,N -bis(acryloyl)cystamine and PEG-based disulfide con-
synthesize PEG-based hydrogels.
Since then, Michael-
taining dimethacrylate, which were employed in the
synthesis of cryogels via FRP, did not result in degradable
type conjugate addition reactions have become a common
[40,41]
reaction to synthesize PEG-based hydrogels.
While
[33]
cryogels.
It should be noted that during cryogel
PEG based step-growth hydrogel materials have been
explored as a new type of biomaterials, up to our
knowledge, step-growth cryogels from PEG-based building
blocks have not been reported. The choice of PEG as a
building block is not only motivated because of its
biocompatible nature but also of its hydrophilic properties,
which is crucial in the design of degradable systems when
water soluble degradation products are targeted.In this
study, the Michael addition strategy between maleimide
double bonds and amine groups has been investigated for
the synthesis of redox-responsive PEG cryogels. Amine
functionalized, low-molecular-weight PEG-based building
blocks and maleimide functionalized disulfide containing
building blocks have been combined in the preparation of
the cryogels. Structural characterization, mechanical prop-
erties and degradation of the cryogels are demonstrated.
The potential use of these cryogels as cell scaffolds in tissue
engineering applications has been verified by a toxicology
analysis of the degradation products, cell viability analysis
and cell seeding experiments.
synthesis, the amountof undesired chain transfer reactions
might be higher as the monomers and crosslinker are
concentrated in the non-frozen regions (cryotropic con-
[11,34]
centration effect).
In addition, the amount of the
crosslinker is generally 10% of the monomer feed to obtain
nice cryogel monoliths. Thus, the rather high concentration
of disulfide groups during the cryogelation process causes
an increased probability of chain transfer reactions, which
give rise to additional, permanent crosslinks in the system.
With regard to the above-mentioned discussion, different
synthetic strategies–not involving any radical species–
should be considered when disulfide containing cryogels
are targeted.
Recently, the formation of networks with step-growth
reactions such as Michael addition chemistry and ‘click’
chemistry has drawn significant attention since these
techniques give more control over the network structure,
and therefore significantly improve the mechanical proper-
ties of the materials compared to the chain-growth
[35,36]
reactions.
Especially, Michael-type conjugate addition
reactionsbecomeattractivetosynthesizenewbiomaterials
to meet the demand of improved materials in the field of
tissue engineering since these reactions may occur under
physiological conditions without the need of any other
reagents. In addition, non free-radical, step-growth reac-
tions do not interfere with disulfide moieties, and are
therefore an interesting alternative to synthesize network
structures that contain disulfide functionalities.
2. Experimental Section
2
.1. Materials
ꢁ1
Glycerol ethoxylate (Mn ¼ 1 000 g ꢀ mol ), triethylamine (99%),
maleic anhydride (99%), methanesulfonyl chloride (99%), cysta-
mine dihydrochloride (98%), glutathione (>98%), phosphate-
buffered saline (PBS, pH ¼ 7.4) in foil pounches, and potassium
hydroxide were purchased from Sigma-Aldrich. The ammonia
solution (25%) and 1,6-hexanediamine (>99.5%) were obtained
from Acros Organics. The other solvents were all HPLC grade and
purchased from Aldrich. All reagents have been used without any
further purification.
From this viewpoint, the Michael-type conjugate addi-
tion reaction between maleimide groups and amine groups
was considered in this research as a suitable way to
synthesize disulfide containing, step-growth cryogels. The
double bonds of maleimide compounds have a low electron
density, making them highly reactive towards Diels-Alder
reactions and 1,3-dipolar addition reactions. In addition,
thesedoublebondsarealsoaccessibletonucleophilicattack
2
.2. Synthesis of the Trifunctional PEG Amine
Trifunctional PEG-amine (PEG-(NH ) has been prepared in two
2 3
)
[
39]
[37]
steps in a manner similar to a previously described method.
via Michael-type conjugate addition reaction.
For
Briefly, hydroxy groups of glycerol ethoxylate were first
mesylated with methanesulfonyl chloride and then converted to
example, the reactions of maleimide double bonds with
thiols or thiolate anions are well-known to proceed
straightforward and form stable thioether linkages. How-
ever, thiol and thiolate nucleophiles might interfere with
disulfide groups when the synthesis of redox-responsive
cryogels is targeted. Alternatively, primary amines are
amine groups in 25% ammonia aqueous solution. Glycerol
ꢁ1
ethoxylate with Mn equal to 1000 g ꢀ mol
(10 g, 11.38 mmol)
was dissolved in 200 mL of dry dichloromethane. After the addition
of triethylamine (5 mL, 35.3 mmol), the solution was cooled to
0 8C. Then, methanesulfonyl chloride (3 mL, 37.55 mmol) was
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added dropwise with stirring. The reaction mixture was stirred
overnight at room temperature. The salt was filtered from the
solution and the solution was washed with a saturated sodium
bicarbonate solution (2 ꢂ 30 mL). The dichloromethane phase
was dried over magnesium sulfate and evaporated to dryness.
Next, mesylated glycerol ethoxylate (10 g, 8 mmol) was dis-
solved in 25% aqueous ammonia solution (150 mL), and the
container was tightly closed. The reaction mixture was vigorously
stirred for 3 d at room temperature. The ammonia was evaporated
and the basic solution was extracted three times with dichlor-
the mixture was heated slowly to reflux with simultaneous
addition of acetic anhyride (13.5 mL, 122.3 mmol). The mixture was
allowed to reflux for an additional 3 h and then acetone was
evaporated. The residue was dissolved in dichloromethane
(100 mL) and washed with
3
a saturated NaHCO solution
(1 ꢂ 50 mL)togetridofthetriethylaminesalt.Thedichloromethane
phase was dried over magnesium sulfate and evaporated. Residual
acetic anhydride was removed via azeotropic distillation with
cyclohexane and the crude compound was purified via column
1
chromatography (40% ethyl acetate-hexane). H NMR (300 MHz,
omethane (100 mL). The presence of terminal amine groups was
CDCl
1.50–1.57 (m, 4 H, ꢁNꢁCH
ꢁNꢁCH ꢁCH ꢁCH ꢁ).
3
, d): 6.66 (s, 4H, ꢁCHꢁCHꢁ), 3.48 (t, 4H, ꢁNꢁCH
2
ꢁCH ꢁ),
2
1
detected by NMR. H NMR (300 MHz, CDCl
3
, d): 3.73 (t, 6H,
ꢁNH ).
2
ꢁCH ꢁCH ꢁ), 1.25–1.29 (m, 4 H,
2
2
ꢁCH
2
ꢁCH
2
ꢁNH
2
), 3.63–3.49 (77H, PEG), 2.84 (t, 6H, ꢁCH
2
2
2
2
2
2
.3. Synthesis of Crosslinker
2.5. Synthesis of Redox-Responsive PEG Cryogels via
Maleimide-Conjugate Addition
Dithiobis(maleimido)ethane (DTME)
To synthesize the disulfide-containing bismaleimide linker DTME,
cystamine dihydrochloride salt was first neutralized to cystamine
using potassium hydroxide (KOH). Cystamine dihydrochloride
Cryogels were prepared in three different total monomer
ꢁ1
concentrations (0.36, 0.18 and 0.12 mmol mL ) at –8 8C in dioxane.
DTME (1.5 equiv.) and PEG-(NH2)3 (1 equiv.) were separately
dissolved in a calculated amount of dioxane. The monomer
solutions were cooled down in an ice bath for a few second and
mixed in open-ended plastic tubes (1 mm in diameter). The plastic
tubes were placed in a cryostate at –8 8C. After 18 h, the tubes were
taken out of the cryostate and put at room temperature. The gel
matrix was first washed with chloroform and then extensively
with ethanol, and finally dried at 60 8C.
(
10 g, 44.4 mmol) was suspended in 100 mL of methanol. After the
addition of KOH (5.5 g, 97.7 mmol), the white suspension was
stirred overnight at room temperature. The next day, a white
precipitate was filtered from solution and methanol was evapo-
rated. The residue was dissolved in dichloromethane (200 mL) and
washed with a saturated NaHCO
3
solution (1 ꢂ 50 mL). The
dichloromethane phase was dried over magnesium sulfate and
pure cystamine was obtained upon evaporation of dichloro-
methane. The rest of the procedure has been adapted from
[42]
previously reported reaction conditions.
Cystamine (4 g,
2.6. Synthesis of the Control Cryogels
2
6.26 mmol) was dissolved in 100 mL of acetone and maleic
Cryogels without disulfide groups have been prepared under the
same conditions as mentioned for the synthesis of disulfide
containing cryogel, using 1,6-bis(maleimido)hexane as the bisma-
leimidecompound.Thecoldsolutionsof1,6-bis(maleimido)hexane
anhydride (5.15 g, 52.53 mmol) was added to the mixture. Sudden
precipitation of the obtained diacid was observed and the mixture
was stirred for one additional hour to complete the reaction. To the
reaction mixture, triethylamine (2 mL, 27.2 mmol) and sodium
acetate (0.03 g) were added. Then the mixture was heated slowly to
reflux with simultaneous addition of acetic anhyride (8 mL,
(
2 3
1.5 equiv.) and PEG-(NH ) (1 equiv.) in dioxane were mixed in
plastic tubes and kept at –8 8C for 18 h. The gel matrix was first
washed with chloroform and then extensively with ethanol, and
finally dried at 60 8C.
7
3
2.5 mmol). The mixture was allowed to reflux for an additional
h and then acetone was evaporated. The residue was dissolved in
dichloromethane (100 mL) and washed with saturated NaHCO
3
solution (1 ꢂ 50 mL) to get rid of the triethylamine salt. The
dichloromethane phase was dried over magnesium sulfate and
evaporated. Residual acetic anhydride was removed via azeotropic
distillation with cyclohexane. The crude compound was then
2
.7. Characterization of Cryogels
2
.7.1. Methods and Instrumentation
purified via column chromatography (40% ethyl acetate/hexane).
1
1
1
1
13
1
D H NMR, 2D H- H correlation (COSY) and 2D H- C hetero-
1
H NMR (300 MHz, CDCl
3
, d): 6.69 (s, 4H, ꢁ–CHꢁCHꢁ), 3.83 (t, 4H,
ꢁCH ꢁS).
nuclear single-quantum correlation (HSQC) spectra were recorded
on a Bruker Avance II 700 spectrometer (700 MHz) using a hr-MAS
NꢁCH
2
ꢁCH ), 2.91 (t, 4H, ꢁCH
2
2
2
1
13 119
probe equipped with a H, C, Sn and gradient channel. Samples
for high-resolution magic angle spinning (hr-MAS) NMR were
prepared as follows: a dry cryogel sample was cut in small pieces
2
.4. Synthesis of 1,6-Bis(maleimido)hexane
1,6-Bis(maleimido)hexane was synthesized from maleic anhyride
7
and put in a 4 mm hr-MAS rotor (50 mL). Next, solvent (DMF-d ) was
andhexanediamineviaadaptingtheprocedureforthesynthesisof
DTME. 1,6-Hexanediamine (5.7 g, 49 mmol) was dissolved in
added to allow the cryogel to swell. Scanning electron microscopy
(SEM) images were recorded with a Quanta 200 FEG FEI scanning
electron microscope operated at an acceleration voltage of 5 kV.
Samples were sputtered with gold prior to SEM analysis.
Compression measurements were performed on swollen cryogels
using a Tinius Olsen-Instrument (H10KT) with a cell load of 100 N.
Samples were compressed between two parallel plates at the
100 mL of acetone and maleic anhydride (9.6 g, 98 mmol) was
added to the mixture. Sudden precipitation of the obtained diacid
wasobservedandthemixturewasstirredforoneadditionalhourto
complete the reaction. To the reaction mixture, triethylamine
(
3.7 mL, 50.7 mmol) and sodium acetate (0.06 g) was added. Then
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ꢁ
3
maximum loading of 20 N with
a
a
compression rate of
dissolubilized in BPS buffer solution containing 5 ꢂ 10
M
ꢁ1
1
mm ꢀ min . Triple experiments have been done for each sample.
glutathione at 37 8C. This solution was further used for cell
Raman spectra were collected using Bruker FRA 106 FT-Raman
spectrometer. Samples were placed on a glass slide to perform the
analysis.
viability analysis.
2
.7.8. Cell Viability
Fibroblasts were seeded in a 96-well plate (450 cells per well) and
cultured in the presence of degradation products. After 24 h, the
cells were quantified using a p-nitrophenyl phosphate (pNPP;
Sigma-Aldrich) cell viability assay. pNPP was hydrolyzed by acid
phosphatase resulting in the release of p-nitrophenol and
phosphate by incubating the cells with a pNPP solution under
2
.7.2. Gelation Yield of Cryogels
Cryogels were first washed with chloroform and then extensively
with ethanol to remove the unreacted gel precursors. Afterwards,
they were dried in an oven at 60 8C until a constant weight was
reached (mdried). The gelation yields of the cryogels were calculated
as (mdried/m
t
) ꢂ 100%, where m
t
is the total mass of the gel
2
acidic condition during 2 h at 37 8C and 5% CO . The reaction was
precursors in the reaction mixture.
stopped with NaOH resulting in the formation of a yellow color,
measuredspectrophotometricallyat 405 nm. All experiments were
performed in triplicate.
2
.7.3. Swelling Ratio Measurement
Afterdryingthecryogelsamplesuntilaconstantweight, theywere
immersed in PBS for 24 h to reach the equilibrium swelling. The
samples were taken out and the excess of water was removed from
the cryogel surface with a tissue paper. The swelling degrees of the
cryogels were calculated from the ratio of weights of swollen
cryogeltodriedcryogel.Tripleexperimentshavebeendoneforeach
sample.
2
.7.9. Cell Seeding on Gel Slab and Immunohistochemistry
The gel slabs were sterilized by 2 h soaking in 70% ethanol followed
by extensive soakingin sterile PBS. Fibroblasts were seeded on a gel
slab and cell adhesion and spreading was evaluated after a 24 h
incubation period. Cells were fixed using 3.7% formaldehyde in PBS
and permeabilized with Triton X-100 (0.1%). After blocking the
samples with Tris buffer containing 5% BSA, actin was stained
fluorescently using Alexa Fluor546 Phalloidin (1/40) (Invitrogen,
Molecular Probes). Cell nuclei were stained with HOECST 33258
2
.7.4. Macropore Volumes
The water inside the swollen cryogel is found in three different
states: polymer bound water, water inside small pores and water
ꢁ1
(
Sigma-Aldrich, 2 mg ꢀ mL
in PBS). Microscopy images were
[43]
recorded on a Nikon EZ-C1 confocal microscope.
inside large pores.
The water present inside large pores is more
free compared to the other types and can be mechanically removed
by simply squeezing the cryogel. This squeezing method can be
used for an approximative estimation of the total volume of
macropores in a cryogel sample. In this study, the volume of
macropores in the cryogel matrix was calculated as follows.
2
.7.10. Cell Seeding in Cryogel
The cryogels were sterilized by 2 h soaking in 70% ethanol followed
by extensive soaking in sterile PBS. 300 000 fibroblasts were seeded
3
on a cryogel (1 ꢂ 1 ꢂ 1 cm ) by drop seeding. After 1 week of
culturing period, fibroblasts were stained using CellTracker Green
(1/5000) (Invitrogen, Molecular Probes) to evaluate the population
of cryogel. Microscopy images were recorded on a Nikon EZ-C1
confocal microscope.
mswollen gel ꢁ msqueezed gel
ꢂ 100%
mswollen gel
2
.7.5. Visual Determination of Residual Primary
2
.7.11. Reduction-Triggered Degradation
Amine Groups
A 6 mm in diameter round shaped cryogel was sliced to obtain a
mm thick sample (20 mg) and put at room temperature in
The 2,4,6-trinitrobenzenesulfonic acid (TNBS) test was used for
visual detection of primary amine groups. A cryogel sample of
1
ꢁ3
aqueous medium (PBS, 10 mL) containing 25 ꢂ 10 M glutathione.
After several time intervals the cryogel was transferred to a glass
cover slip and optical microscopy images were recorded with an
Olympus SZX9 stereomicroscope equipped with a digital camera.
Afterwards, the cryogel was again transferred back to the aqueous
glutathione solution.
1
0 mg was placed in a small glass vial. Three drops of n,n-
diisopropylethylamine (DIPEA) solution (10% in DMF) and three
drops of 1 M aqueous TNBS solution were added successively. The
orange to red color change on the cryogel matrix was visually
inspected after 30 min, which demonstrated qualitatively that
residual primary amine groups are present on the cryogels.
2
.7.6. Cell Culture
Humandermalforeskinfibroblasts(ATCC;CRL-2522)werecultured
in EMEM supplemented with antibiotics (streptomycin/penicillin)
and 20% heat inactivated fetal bovine serum (FBS; Gibco).
Cells were grown at 37 8C in a humidified atmosphere containing
3. Results and Discussion
3
.1. Synthesis of Redox-Responsive Cryogels
5
% CO
2
.
Inafirststep,amineandmaleimidefunctionalizedbuilding
blocks were prepared for the synthesis of cryogels. For the
amine containing building block, an amine terminated
2
.7.7. Degradation of Cryogel for Cell Viability Analysis
The cryogels were sterilized by 2 h soaking in 70% ethanol followed
by extensive soaking in sterile PBS. 4 mg ꢀ mLꢁ1 of the cryogel were
three-arm PEG [PEG-(NH
) ] was synthesized from com-
2 3
mercially available glycerol ethoxylate via mesylation of
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HO
H2N
1
) CH3SO2Cl, Et3N,
o
3
.2. Influence of Polymerization
A)
CH2Cl2, 0
C
Conditions on the Properties of
Redox-Responsive Cryogels
2
) 25 % aqueous
O
O
ammonia solution
O
O
O
O
HO
7
OH
H2N
NH2
7
7
7
PEG-(OH)3
PEG-(NH2)3
In the synthesis of cryogels, both the
crosslink density and the total monomer
O
B)
C)
Et3N, NaO2CCH3,
O
O
(
CH3CO)2O
concentration are key parameters to
[11]
reflux in acetone
S
S
N
S
NH2
O
N
H2N
S
obtain stable monoliths.
In our case,
O
O
O
O
cryogels monoliths are formed via step
growth polymerization of tri-functional
PEG macromonomer and di-functional
DTME (Scheme 1). For the synthesis of
step-growth networks, both the molar
ratios and the molecular weights of
precursors can be considered to adjust
Cystamine
Maleic anydride
DTME
NH
HN
O
O
O
O
O
Dioxane, -8oC, 18h
H
N
O
PEG-(NH2)3
DTME
S
S
N
N
H
N
7
7
O
O
O
[35]
NH
the crosslink density. However, in this
stage of the research, only the molar ratio
of the gel precursors was considered.
HN
2 3
Scheme 1. (A) Synthesis of amine terminated PEG based building block, PEG-(NH ) .
(
B) Synthesis of disulfide-containing bismaleimide crosslinker DTME. (C) Schematic Ideally, a molar ratio of 1.5 [DTME to PEG-
representation of Michael addition reaction in the synthesis of cryogels.
(
2 3
NH ) ] was used to synthesize step-
growth cryogels and gave the most
suitable cryogel monoliths. A further
hydroxyl groups with methanesulfonyl chloride and
subsequent amination reaction with ammonia aqueous
solution (Scheme 1A). Next, a redox-responsive disulfide
bond was incorporated in the structure of the bismaleimide
crosslinker (DTME), which was synthesized by reaction of
cystamine with maleic anhydride (Scheme 1B). The
disulfide containing cryogels were then synthesized via
Michael addition reaction of the electron poor double bond
of the maleimide compound with the amine groups of the
increase (to 1.75) or decrease (to 1.25) in the molar ratio
resulted in a loosely crosslinked system and resulted in
unstable cryogel monoliths. Furthermore, to see the effect
of the total monomer concentration on the properties of the
cryogels such as the degree of swelling, pore sizes and
mechanical properties, cryogels were synthesized starting
from three different total monomer concentrations (0.36,
ꢁ
1
0.18and0.12 mmol ꢀ mL ) at–8 8Cwhile keepingthemolar
ratio of the precursor DTME to PEG-(NH ) at 1.5.
2 3
)
three-arm [PEG-(NH
2
)
3
] moiety (Scheme 1C).
The influence of the total monomer concentration on the
swellingdegreesofthecryogelsisdisplayedinTable1. With
a decreasing initial concentration of the gel precursors, the
cryogels’ degree of swelling increases. Moreover, the
volume of the macropores decreases with increasing initial
total monomer concentration. This result is consistent with
the previously reported assumption that a higher total
monomer concentration causes more dense cryogel struc-
Because of the limited solubility of DTME in water,
dioxane was used as the solvent for the cryogel
synthesis. The conjugate addition between the amine
and maleimide compounds is a straightforward reaction,
which even at subzero temperatures does not require
additional reagents or a catalyst. The reagents are
dissolved separately in dioxane and pre-cooled in an
ice bath for a few seconds before mixing in plastic tubes
[43]
ture with smaller pores and thicker walls.
(
1 mm in diameter). Finally, the mixture is frozen
The differences in the cryogel structure and their
macroporous nature can also be observed by SEM pictures
(Figure 1). The SEM picture of cryogel 1 (Table 1), with a
by immersing the plastic tubes in a cryostate thermostated
at –8 8C.
Table 1. Properties of redox-responsive cryogels produced at –8 8C with different concentrations of gel precursors.
Entry
Concentration of
Yield
[%]
Swelling
degree
Volume of
macropores
[%]
Cryogel
gel precursor
1
[
mmol ꢀ mLꢁ
]
Cryogel 1
Cryogel 2
Cryogel 3
0.36
0.22
0.18
96
94
93
6.5
7.3
8.1
62
66
70
dense
elastic
elastic
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Figure 1. SEM images of redox-responsive degradable cryogels prepared with different monomer concentrations. (A) cryogel
ꢁ1
ꢁ1
ꢁ1
1
(0.36 mmol ꢀ mL ), (B) cryogel 2 (0.18 mmol ꢀ mL ), (C) cryogel 3 (0.12 mmol ꢀ mL ).
relatively high initial monomer concentration, shows a
more oriented structure with smaller pores. As the system
getsmorediluted, thepore sizesofthecryogelsincreaseand
more random pore structures are observed, leading to a
more elastic cryogel structure. On the other hand, it is also
observed that a further decrease in initial precursor
concentration (<0.18 mmol) might have a negative effect
onthestabilityofmonolithscausingthecollapseofthepore
walls.
occur in our system, we performed a detailed analysis to
elucidate the exact chemistry of our approach.
High-resolution magic angle spinning (hr-MAS) NMR
spectroscopy is a valuable tool to characterize the chemical
structure of swollen cryogel samples. This technique was
previously used in our group to monitor and quantify
modification reactions of polyHEMA cryogels using the
copper(I)-catalyzed Huisgen azide/alkyne coupling strat-
[17]
egy.
1
As a specific characteristic related to the cryogelation
procedure, all cryogels show an interconnected macropor-
ous structure (Figure 1). This interconnected nature is
reflected in their extremely fast swelling kinetics.
These cryogels reach their equilibrium swelling ratio
within 1–2 min in PBS (Figure 6a, b).
Figure 2 shows the H hr-MAS NMR spectrum of a redox-
responsive cryogel sample (cryogel 3 in Table 1) that was
swollen in deuterated DMF. This spectrum clearly shows
that the reaction that took place is solely the conjugate
addition between the amine and the maleimide double
bond. The protons of the maleimide doublebond give rise to
a singlet at 6.9 ppm, which completely disappears, demon-
strating the success of the conjugate addition. As a result,
three signals appearing at, d ¼ 2.54 (a), 3.04 (b) and 3.91 (c)
are assigned to the protons of the new succinimide ring
that is conjugated to the PEG-amine. This conjugation is
confirmed by the presence of the signal at d ¼ 2.83 (f), which
Literature studies have demonstrated that the tempera-
ture during cryogel preparation has an important effect on
[11]
the pore size of the cryogels.
Lower synthesis tempera-
tures give rise to a fast freezing process of the solvent,
therefore smaller solvent crystals are formed during the
polymerization and correspondingly, cryogels with smaller
pore sizes are obtained. In our study, cryogels have been
synthesized at –8 8C and further increase in the synthesis
temperature to obtain larger pore sizes has not been
considered since the formation of ordinary gel matrices has
been observed in the system during longer freezing times.
arises from the CH
2
group vicinal to the secondary amine of
conjugated PEG. The signals at d ¼ 3.78 (d) and 2.98 (e)
3
.3. Structural Analysis of Redox-Responsive Cryogels
The structural investigation of cryogels is important since
the maleimide-amine system might react in different ways
depending on the reaction conditions. Other than the
maleimide-amineconjugatereaction,therearetworeactions
that could occur in this system: crosslinking between two
maleimide groups and a ring opening aminolysis reaction
between maleimide and amine groups. The conditions of
[44–47]
these reactions have been studied extensively.
These
studies show that reactions usually require elevated
temperatures (>100 8C) while the solvent choice is also an
important parameter to determine the reaction pathway.
Even though these two reactions are not being expected to
Figure 2. 1H NMR spectrum of
a redox-responsive cryogel
(cryogel 3).
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is surrounded by two other bands at
ꢁ
1
around 694 and 613 cm , which are
assigned to the nC¼O stretching of the
succinimide ring.
3
.5. Mechanical Properties of
Redox-Responsive Cryogels
Compression tests were performed on
swollen cryogel samples to determine
the stress/strain curves and to calculate
the compressive moduli. Samples were
compressed under a maximum loading
of 20 N and the moduli were calculated
from the initial linear region of the
stress/strain curve (0–10% strain). All
1
1
1
13
Figure 3. (A) H- H COSY spectrum and (B) H- C HSQC spectrum of a redox-responsive cryogelsexhibited asimilarcompressive
7
cryogel in DMF-d (cryogel 3).
modulus around 114 kPa (Table 2),
which is likely due to their equal cross-
link density.
belong to the CH groups between the succinimide ring and
Moreover,thestudiesonthemechanicalpropertiesofthe
redox-responsive degradable cryogels also showed that the
initial precursor concentrations have an influence on the
mechanical properties of cryogels. Related to its dense
microstructure, cryogel 1 was not able to unload 20 N and
broke at a maximum loading of 7.6 N at 59% compression
(Figure 5). On the other hand, cryogel 2 and 3 were able to
manage a maximum loading of 20 N at 75% compression
and regained their shape gradually upon removal of the
applied force. This elastic nature of cryogels is further
illustrated in Figure 6. In this figure, it is demonstrated that
a swollen cryogel sample is compressed to a maximum
limit while the cryogel regained its original shape upon
removal of the applied force without any damage on the
cryogel structure. This elastic nature and relatively high
compressive modulus of the cryogels can be attributed to
the step-growth formation of cryogels, leading to a more
ideal network structure in comparison to the radical chain
2
the disulfide group, while the major signal at d ¼ 3.60
2
represents the CH groups of the PEG moeity.
The assignments of these signals are also confirmed via
1
1
1
13
2
D H- H COSY and H- C HSQC NMR spectra recorded
under hr-MAS conditions (Figure 3). In Figure 3A, the
1
1
H- H COSY spectrum also reveals the correlation between
the protons of the new succinimide ring with three signals
appearing at d ¼ 2.54 (a), 3.04 (b) and 3.91 (c).
Apart from NMR analysis, visual analysis on the cryogel
structures by the TNBS test, which is able to detect primary
amine groups, demonstrated that residual amine groups
are present in the cryogel system. In fact, the presence of
primary amine groups on the cryogel structure could be
beneficial for further functionalization studies of the
cryogel matrix.
3.4. Raman Spectroscopy of
Redox-Responsive Cryogels
Raman spectroscopy has been used to
verify the presence of the disulfide
[48]
groups.
The raman spectra of both
blank cryogels (without disulfide groups)
and redox-responsive degradable cryo-
gels (containing disulfide groups) are
shown in Figure 4, traces (a) and (b),
respectively. In the spectrum of the
redox-responsive cryogel (Figure 4b),
the nCS band at 644 and the nSS band at
ꢁ
1
5
08 cm indicate the presence of dis-
ulfides in the system. In the case of the
blankcryogel(Figure4a),thesetwobands
Figure 4. Raman spectra of (a) blank cryogel (without disulfide groups), (b) disulfide
ꢁ
1
arenotdetected. ThenCS bandat644cm
containing cryogel (cryogel 3).
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Redox-Responsive Degradable PEG Cryogels as Potential Cell Scaffolds . . .
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Table 2. Mechanical properties of redox-responsive degradable cryogels.
Entry
Maximum
strain
Compressive
modulus
[kPa]
Maximum
load
Maximum
stress
[
%]
[N]
[MPa]
Cryogel 1
Cryogel 2
Cryogel 3
59
76
75
114
92
7.6
20
20
0.08
0.315
0.399
116
groups are responsible for cryogel degradation.As a nice
demonstration of reduction triggered degradation of
cryogel, a 1 mm thick slice was cut from a cryogel and
incubatedatroomtemperatureinglutathione(25 ꢂ 10ꢁ M)
containing physiological buffer (i.e., PBS; pH ¼ 7.4; 0.150 M
NaCl). After different time intervals the cryogel was
transferred to a glass cover slip and optical microscopy
images were recorded. As shown in Figure 7, the cryogel
gradually swells and decomposes after 12 h of incubation
before turning into a clear solution.
3
3
.7. Potential of the Cryogels as Scaffolds in
Tissue Engineering
In first instance we evaluated whether the degradation
products of the cryogels did affect cell viability. Therefore
we incubated fibroblasts in vitro with solutions ranging
Figure 5. Stress/strain slopes of compression test on redox-
responsive degradable cryogels under the maximum loading of
2
0 N with a displacement control rate of 1 mm ꢀ minꢁ
1
.
ꢁ1
from 0.1 to 1 mg ꢀ mL of the cryogels decomposed in
glutathione containing PBS. None of these solutions did
affect cell viability as shown in Figure 8A. In a second series
of experiments we investigated cell adhesion and spread-
ing on the cryogels as this is essential to serve as tissue
engineering scaffold. For visualization purposes we first
investigated planar gel slabs, which were prepared at room
temperature via film casting. These gels slabs were then
seededwithfibroblasts. AsshowninFigure8B celladhesion
did take place, however the cells exhibited a round
morphologyanddidnotspreadoutasobserved oncommon
tissue culture polystyrene substrates. To improve cellular
behavior, the gel slabs were coated with fibronectin
through simple adsorption, which had a dramatic effect
on cell spreading as shown in the lower row of Figure 8B
where the elongated morphology of the actin filaments –
growth network formation as was observed for hydrogels
[36]
before.
3
.6. Reduction-Triggered Degradation of Cryogels
The redox-responsive degradable cryogels synthesized via
Michael-typeconjugateadditionwere observedto bestable
under physiological conditions (PBS, pH: 7.4). However,
they got solubilized in a few hours in the presence of a
reducing agent in the medium. Blank cryogels that were
prepared from the same PEG-based precursor with a non-
cleavable bismaleimide, namely 1,4-di(maleimido)butane,
did not show any degradation, proving that the disulfide
Figure 6. Redox-responsive cryogel (a) in dry state, (b) in swollen state, (c, d) under compression.
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Figure 7. Optical microscopy images of a round sliced cryogel in dry state after incubation in glutathione during different time intervals.
stained with phalloidin – clearly indicate good cell
spreading.As a final experiment, fibronectin coated cryo-
gels were drop-seeded with fluorescently labeled fibronec-
tin cells. For fluorescent labeling, CellTracker Green
strates the successful population of the cells at different
depths of cryogel matrix. This experiment confirms that the
pore sizes of the cryogel are sufficiently large for cell
penetration when using the cryogels as scaffolds.
[
carboxyfluorescein diacetate succinimidyl ester (CFSE),
green fluorescence] was used to stain the fibronectin cells.
CellTracker Green only stains the living cells, thus it gives
the advantage of tracing living cells inside the cryogel
matrix. After 1 week of culturing period, z-stack confocal
images were taken inside the cryogel matrix at different
depths. In Figure 9, the elongated green structures are
fluorescently labeled living cells, which spread themselves
inside the cryogel matrix, which therefore clearly demon-
4. Conclusion
Disulfide-containing degradable PEG cryogels were suc-
cessfully synthesized thanks to a radical-free conjugate
addition strategy. These step-growth PEG cryogels exhibit
the important requirements to function as potential cell
scaffolds in tissue engineering. For example, they have an
Figure 8. (A) Cell viability of fibroblasts in media containing different concentrations of a decomposed cryogel. Circles: cryogel 1, triangles:
cryogel 2, rectangles: cryogel 3. (B) Confocal microscopy images of fibroblasts seeded onto blank gel slabs and fibronectin coated gel slabs.
Left panels show the overlay of blue and red fluorescence, while the right panels show the DIC channel. Cell nuclei were stained blue with
Hoechst and the actin skeleton was stained red with alexa546-conjugated phalloidin.
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Redox-Responsive Degradable PEG Cryogels as Potential Cell Scaffolds . . .
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Figure 9. Z-stack depicting confocal microscopy sections at different depths of a fibronectin coated cryogel drop-seeded with fibroblasts. For
visualization purposes the fibroblasts were stained green with CellTracker Green before drop-seeding.
interconnected macroporous nature with good mechanical
properties and show good swelling behavior in physiolo-
gical conditions. While these PEG cryogels are stable under
physiological condition, thanks to the presence of disulfide
bridges in the cryogel structure, they are able to degrade to
water soluble components in the presence of an external
stimulus, such as the presence of glutathione as a reducing
agent in this case. Moreover, cell viability experiments
clearly showed that neither the degradation products nor
the gel structure itself are toxic to the cells. Cell seeding
experiments also demonstrated the excellent ability of
the PEG cryogel matrix to serve as scaffolds in tissue
engineering, since the PEG cryogel successfully sustained
the cell population at different depths within the entire
cryogel matrix.
Acknowledgements: T. D. thanks Ghent University for a PhD
scholarship. W. V. C. and B. G. D. G. thank the Research Foundation
Flanders (FWO) for a postdoctoral research fellowship. L. J. D. C.
thanks the IWT for a PhD scholarship. The NMR and Structural
Analysis research group of Ghent University is acknowledged for
the help with the hr-MAS NMR analysis of cryogels.
Received: October 9, 2011; Published online: January 4, 2012;
DOI: 10.1002/mabi.201100396
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T. Dispinar, W. Van Camp, L. J. De Cock, B. G. De Geest, F. E. Du Prez
www.mbs-journal.de
Keywords: cryogels; hydrogels; michael addition; stimuli-sensi-
tive polymers; tissue engineering
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