Inverse high internal phase emulsion polymerization (i-HIPE) of GMMA, HEMA and GDMA for the preparation of superporous hydrogels as a tissue engineering scaffold

Article abstract of DOI:10.1039/c5tb01873k

A series of novel superporous hydrogels for regenerative medicine were prepared by oil-in-water (o/w) or inverse high internal phase emulsion (i-HIPE) copolymerization of glycerol monomethacrylate (GMMA), 2-hydroxy ethyl methacrylate (HEMA) and glycerol dimethacrylate (GDMA) as a cross-linker using a non toxic solvent and a redox initiator system at the physiological temperature (37 °C). The monomer GMMA was synthesized from glycidyl methacrylate (GMA) by an alternative facile method using Amberlyst-15. The described i-HIPEs showed a significantly wider stability window. The polyHIPE hydrogels were characterized by FTIR, BET method for surface area, mercury porosimetry, SEM, DSC, TGA, XRD, compressive strain and strain recovery. The swelling ratio of the hydrogels and their degradation in 0.007 M NaOH and lipase B (Candida antarctica) solutions were determined gravimetrically and the rate of degradation was explained in terms of the molecular structure of the hydrogels. The morphological studies showed that the pore diameter varied between 20 and 30 μm and the pore throats (interconnecting windows) diameter was in the range of 4-8 μm. The described polyHIPE hydrogels were found to have an open cell morphology and interconnected pore architecture, which are important characteristics for scaffold applications. The initial cytotoxicity study performed according to ISO-10993-5 indicated cytocompatibility (97% cell viability) and the subsequent cell seeding and proliferation study exhibited 55-88% cell viability (increased monotonously from GHG-1 to GHG-5), which could be attributed to modulation of the physical and chemical properties of the hydrogels. The described super porous hydrogels are considered as potential candidates for scaffold materials in tissue engineering applications.

Full text of DOI:10.1039/c5tb01873k

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ARTICLE
Inverse high internal phase emulsion polymerization (i-HIPE) of GMMA, HEMA and GDMA for
preparation of superporous hydrogels as tissue engineering scaffold.
a
c
d
a,b
Archana C. Nalawade , R. V. Ghorpade , Sadiqua Shadbar , Mohammed Shadbar Qureshi* , N.N.
c
e
c
Chavan , Ayesha A. Khan and S. Ponrathnam .
Received 00th January 20xx,
Accepted 00th January 20xx
A series of novel superporous hydrogels for regenerative medicine were prepared by oil-in-water (o/w) or inverse high
internal phase emulsion (i-HIPE) copolymerization of glycerol monomethacrylate (GMMA), 2-hydroxy ethyl methacrylate
DOI: 10.1039/x0xx00000x
(HEMA) and glycerol dimethacrylate (GDMA) as cross-linker using a non toxic solvent and a redox initiator system at the
www.rsc.org/
physiological temperature (37 °C). The monomer GMMA was synthesized from glycidyl methacrylate (GMA) by alternative
facile method using Amberlyst-15. The described i-HIPEs had shown significantly wider stability window. The polyHIPE
hydrogels were characterized by FTIR, BET method for surface area, mercury porosimetry, SEM, DSC, TGA, XRD,
compressive strain and strain recovery. The swelling ratio of the hydrogels and their degradation in 0.007 M NaOH and
lipase B (Candida antarctica) solutions were determined gravimetrically and the rate of degradation was explained in
terms of molecular structure of the hydrogels. The morphological studies showed that the pore diameter varied between
2
0-30 µm and the pore throats (interconnecting windows) diameter in the range of 4-8 µm. The described polyHIPE
hydrogels were found to have open cell morphology and interconnected pore architecture – the important characteristics
for scaffold applications. The initial cytotoxicity study performed according to ISO-10993-5 indicated cytocompatibility
(97% cell viability) and the subsequent cell seeding and proliferation study exhibited 55 - 88 % cell viability (increased
monotonously from GHG-1 to GHG-5), which could be attributed to modulation of the physical and chemical properties of
the hydrogels. The described super porous hydrogels are considered as potential candidate for scaffold materials in tissue
engineering applications.
adhesion, extracellular matrix regeneration and it must allow
spatial cell distribution throughout the scaffold to facilitate
6
-9
homogeneous tissue formation.
Hydrogels are three dimensional physically or chemically cross-
linked polymer networks that can absorb and retain large
amounts of water or physiological fluids while maintaining the
Introduction
The emerging field of tissue engineering attempts to repair
damaged tissues with the help of porous scaffold materials
having the required properties which act as a template for
1
0, 11
structural integrity.
The porous hydrogels are promising
materials as scaffolds for tissue engineering because of their
inherent hydrophilic nature; favorable mass transport
1
tissue regeneration. Polymer based porous materials, as
1
2-14
properties
for molecules like drugs, nutrients, oxygen, cell
scaffolds, have attracted the attention of researchers due to
1
5
2
wastes within swollen hydrogel, structural similarity to the
extracellular matrix of tissues; besides being amenable to
relatively mild processing conditions. High water content of
,4 hydrogels contributes to the biocompatibility and their soft
their versatile properties and applications in this field. The
material to be used as a scaffold should permit cells adhesion
and promote their growth, and allow the retention of
3
differentiated cell functions besides being biocompatible.
1
6
rubbery property resembles the living tissue. Swollen porous
hydrogels can offer native tissue mimicking
The micro structural parameters such as porosity, pore size,
pore interconnectivity influence the cell attachment, cell
5
growth and their proliferation. Therefore, a scaffold should
a
– a
1
7
microenviourment. A variety of synthetic and naturally
derived materials have been used to prepare porous hydrogels
have high enough porosity to provide sufficient space for cell
1
8
for tissue engineering scaffolds and their physical, chemical,
morphological and biological properties could be tailored for a
particular application.
The methods available for synthesis of hierarchically porous
1
9
20
materials include gas foaming, phase separation, solvent
2
1
casting and particle leaching
etc. These methods provide
good control over the porous architecture but involve
multistep complex procedures, which are time consuming. The
polymerized high internal phase emulsions (PolyHIPEs) are
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2
2-24
more recently explored materials as scaffolds.
PolyHIPEs paraffin oil (light, SF 3S630244) were purchased from Merck
have been synthesized using high internal phase emulsions (India). The culture of cells NIH3T3 was purchased from
2
5-27
(HIPEs) as templates.
HIPEs are emulsions that have an National Center for Cell Science (NCCS), Pune (India).
internal phase volume greater than 74% dispersed in
continuous (external) phase. Polymerization of continuous
phase of HIPEs gives rise to porous polymers (polyHIPEs) with
open cells and interconnected pores after removal of
Methods
Synthesis of Glycerol Monomethacrylate
2
8
dispersed internal phase
2
that impart excellent mass Glycerol monomethacrylate (GMMA) monomer is usually
9
transport property for oxygen, nutrients, waste metabolite obtained by the reaction of a protected vicinal ketal form of
which is necessary for uniform cell migration, cell survival and glycerol with methacryloyl chloride followed by acid hydrolysis
44
proliferation.
of the protected ketal group. We have achieved an
In recent times, advances in HIPE methodologies have been economical and environment friendly synthesis of GMMA
2
3
made to produce porous polymeric materials and the most (section 1, ESI) by direct acid-catalyzed hydrolysis of glycidyl
common being polyHIPE systems synthesized by water-in-oil methacrylate (GMA) where solid catalyst Amberlyst-15 – a
emulsions route, where the oil phase consists of polymerizable macro-reticular anion exchange resin – was used. The
4
5
monomers. Several groups have demonstrated the advantages of macro-reticular resin are: its pore size is
3
0-36
applicability of polyHIPE as scaffold for tissue engineering
considerably larger permitting diffusion of reactants freely
+
H )
of which the styrene–DVB polyHIPE is the most studied system throughout the resin that makes the functional group (-SO
3
synthesized by water-in-oil emulsions route. However, these available for the catalytic activity, and it is amenable to
polyHIPEs are intrinsically limited by matrix hydrophobicity and recycling. The hydrolysis of the epoxy ring of GMA gives mainly
the difficulty in introducing surface functionality to make them of mixture of 2, 3-diol and varying amounts of 1, 3-diol isomers
hydrophilic by post polymerization modification which is (Fig. S1, ESI) which finally attains an equilibrium concentration
4
4, 46
hampered due to the inert nature of polymer and harsh of 90:10 respectively on standing for about two weeks.
3
7
reaction conditions. To overcome the intrinsic hydrophobicity
of polyHIPEs synthesized via water-in-oil route, synthesis of
hydrophilic polyHIPE was suggested following oil-in-water
methodology by using hydrophilic monomers in aqueous
PolyHIPE preparation
In a typical experiment, the liquid paraffin oil (dispersed phase,
80 vol. % on total volume) was added drop wise to the
continuous (aqueous) phase consisting of Triton X-305 (10 %
by weight), GMMA and HEMA in various molar concentrations,
fixed amounts of cross-linker GDMA (25 mol % of GMMA and
HEMA) and initiator APS (2 mol% of monomers and cross-
linker) in deionized water (Table 1), while constant stirring at
3
8
(continuous) phase. However, hydrophilic polyHIPEs remain
less explored systems for biomedical applications mainly for
poor emulsion stability, relatively limited synthesis windows,
2
3, 39
low porosity and/or poor interconnectivity
toxic organic solvents.
besides use of
In this study we report the synthesis of superporous polyHIPE
hydrogels for tissue engineering applications by
copolymerization of glycerol monomethacrylate (GMMA),
-hydroxy ethyl methacrylate (HEMA) and glycerol
1
000 rpm. After complete addition of the oil phase, the HIPE
was further stirred for five min. to improve emulsion
uniformity (similar to mayonnaise consistency) and then the
reducing agent TEMED (2 mol% of monomers and cross-linker)
was added. The HIPE was covered and kept at 37 °C in a water
bath for polymerization for 6 h during which cross-linking of
polymer chains locked-in the emulsion geometry of HIPE with
the suspended oil droplets as a pore template to form the
polyHIPE hydrogel. After the polymerization was over, the
polyHIPE was washed thoroughly with deionized water and
Soxhlet extracted for 12 h with acetone to remove the oil
phase. Finally, the resulting polyHIPE was dried overnight to
constant weight under vacuum at 65 °C to produce white fluffy
hydrogel having physical strength similar to popcorn, and a
feel somewhat similar to ethylene-vinyl acetate (EVA)
copolymer, but to a degree lighter. The mass balance for dried
polyHIPEs and co-monomers including cross-linker matched
2
dimethacrylate (GDMA) as a cross-linker following inverse (i.e.
oil-in-water) HIPE polymerization methodology using non toxic
oil phase. So far no study has been reported on the
preparation of porous hydrogels based on inverse HIPE
polymerization of HEMA, GMMA and GDMA. The co-monomer
HEMA was selected for its wide applicability in the biomedical
4
0-43
fields.
In addition, we have also described an alternative
facile method for the synthesis of GMMA co-monomer using
Amberlyst-15 as a recyclable catalyst.
Experimental Section
Materials
Glycidyl methacrylate (GMA), 2-hydroxy ethyl methacrylate with in the experimental limits.
(
HEMA), glycerol dimethacrylate (GDMA), Amberlyst-15
+
(H form) and Triton X-305 (HLB-17.3) were purchased from Characterization
Sigma Aldrich Chemical Co. (USA) and used as such without
further purification. Amberlyst-15 was activated at 120 °C
before use. Ammonium persulphate (APS), reducing agent
N, N, N’, N’-tetra- methylethylenediamine (TEMED) and liquid
Mechanical Testing
The static compression test was carried out in a simplified
manner on the swollen polyHIPE hydrogels to determine their
2
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-
1
-1
mechanical property. The polyHIPE hydrogels test samples at 1220 cm , 1740 cm , 2954 cm , and a broad band centered
-1
-1
-1
were carefully cut into flat rectangular test pieces of about at 3430 cm (range 3149 - 3660 cm ) are attributed to the
×10×5 mm (length × width × thickness) and allowed to swell presence of C-O, C=O, C-H and OH groups respectively. The
8
-1
to the equilibrium state in deionized water for 6 h. The swollen absence of the band at 1630 cm in the FTIR spectrum of
test specimens were kept on a clean glass plate and subjected polymer networks indicated that there was no C=C bond
to a compressive force (F = mg) by gently placing a calibrated unsaturation in the polyHIPE hydrogels. These hydrogels were
mass (m), ensuring that it exerts pressure evenly, and the characterized and found to be promising materials as a
change in thickness was noted. The thickness of compressed scaffold in tissue engineering.
specimens was carefully measured after 5 min. under stress Effect of Surfactant
with the help of callipers. The magnitude of compressive stress
In general, the choice of surfactant and its quantity determines
7
the emulsion stability and thus HIPE formation. However, for
-2
was calculated (force per unit area, Nm ) and the resultant
compressive strain was determined as Δt/t , where Δt is the
change in thickness and is original thickness. The
compressive strain was measured under the static load of
0.32 kPa and the strain recovery was determined by
0
inverse HIPE, the choice of surfactant is critical because of
23
their poor stability. In case of HIPE, surfactant(s) are selected
t
0
mostly on trial and error basis, besides on the hydrophilic-
lipophilic balance (HLB) values; surfactants with the HLB values
1
measuring the thickness when the load was removed and the
polyHIPE hydrogels samples rested for 5 min. The new sample
thickness was divided by the original thickness to calculate the
strain recovery. The subsequent strain recoveries were also
determined as discussed for three consecutive compressive
loading-unloading cycles after resting the samples for 5 min.
The new recovered sample thickness after 5 min. was divided
by the thickness of the previous cycle to calculate the strain
recovery.
2
-6 are used for water-in-oil emulsions and 12-16 for
oil-in-water emulsions. Therefore, in this work, various high
HLB surfactants (HLB>15) such as Tween-40 (HLB, 15.6),
Tween-20 (HLB, 16.6), and Triton X-305 (HLB, 17.3) were tried
for preparing the inverse HIPE. However, in spite of the
comparable HLB values, the most effective surfactant for
obtaining stable emulsions turned out to be Triton X-305. This
was attributed to the fact that the hydrogen bonding plays a
7
critical role in the stabilization of the HIPE by the surfactant.
Degradation study
The surfactants Tween-20 and Tween-40 have more hydrogen
In vitro degradation study was conducted by measuring the bond donor/acceptor sites in the polar head as compared to
-1
percent weight loss in 0.007 M NaOH and 1.0 mg mL lipase Triton X-305. Since the monomers and cross-linker also have
4
7
solutions as described.
the hydrogen bond donor/acceptor sites, there was a stronger
interaction (H-bonding) between the monomers and the cross-
linker and the polar head of the surfactants Tween-20 as well
as Tween-40, as compared to Triton X-305. This perhaps
prevented the polar head of Tween-20 and Tween-40 from
interacting with the aqueous phase of the emulsion and
impaired their ability to stabilize the organic/water interface.
Cross-linker concentration
Details about the degradation study and the other physico-
chemical characterization techniques, instrumentation, and
biological studies are provided under the ESI, section 2.
Results and Discussion
In order to study the effect of cross-linker concentration in the
continuous phase for obtaining stable emulsion with desired
Synthesis of polyHIPE Hydrogels
In order to synthesize polyHIPE hydrogels as tissue scaffold, morphology, the polyHIPE hydrogels were synthesized with 1,
one has to carefully select the right methodology, monomers, 5, 10 and 25 mole percent concentration of GDMA while
disperse phase and optimize the parameters. A series of novel keeping the rest of the composition including the monomers
superporous polyHIPE hydrogels were synthesized by concentration fixed (GMMA:HEMA, 100:0). It was observed
copolymerizing GMMA, HEMA and GDMA on the inverse that the polyHIPE morphology progressively improved from
(oil-in-water) HIPE template stabilized by Triton X-305 almost nonporous to highly porous as the cross-linker
(Scheme 1) with APS/TEMED redox initiation at the concentration increased. At 1 mol% concentration of GDMA
physiological temperature. After the polymerization, removal almost no porosity developed (Fig. 2A) and only rudimentary
of the oil phase yielded the highly porous materials (GHG-1 to porosity (with very irregular pore shape and without pore
GHG-5) having interconnected open porous architecture with interconnectivity) started emerging at 5 mol% concentration
bimodal pore size distribution. The monomers HEMA and (Fig. 2B). As the concentration of the cross-linker was
GMMA were selected as they have been used for neutral increased to 10 mol%, the porosity of the polyHIPE improved
polymeric hydrogels which have found wide applications in further (the pore shape became better) but with thicker pore
biomedical field and GDMA was selected for being hydrophilic walls and fewer interconnects (Fig. 2C). At 25 mol% GDMA
and flexible cross-linker. Among many disperse phases e.g. concentration a polyHIPE hydrogel with desired porous
liquid paraffin oil, toluene, xylene, and cyclohexane, the liquid structure and having highly interconnected open-pore
paraffin oil was chosen for its biocompatibility.
architecture resulted (Fig. 2D). Therefore, all the polyHIPE
The FTIR spectra (Fig. 1) of the polyHIPE hydrogels are in hydrogels, reported in this work, were synthesized keeping the
agreement with the polymer structure as expected. The bands crosslink density at 25 mol%.
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Concentration of cross-linker can impact the emulsion stability Porous structure and mechanical properties
and affect the morphology of polyHIPE by altering the cross-
The concentration of HEMA and GMMA were varied to
examine the effect of composition on the morphology and the
properties of the polyHIPE hydrogels. The SEM images (Fig. 3)
show that all the hydrogels exhibited open porous bimodal
linking kinetics, which depends on the number of cross-linking
48
sites available besides the reactivity of the double bond. In
our case, the gradual increase in GDMA concentration resulted
in larger number of the cross-linking sites (double bonds)
available which in turn enhanced the rate of cross-linking and
hence produced the stable emulsion leading to a polyHIPE
hydrogel with the progressively improved and finally the
desired morphology.
morphologies with high porosity ranging from 92.3% to 96.6%
49
(Table 2) that is typical of the polyHIPE porous structures.
The surface area of the polyHIPE hydrogels varied between
2
.89 and 23.1 m g (Table 3). With the increased proportion
-1
2
of HEMA up to 60 mol% in the copolymer, the porosity of
hydrogels increased (consequently, the surface area and the
Effect of molar ratio of monomers
Initially, we tried to synthesize the superporous hydrogel of pore volume, too), whereas the pore size, pore throat size and
HEMA by the inverse HIPE methodology using GDMA as a pore wall thickness decreased (Table 2). This led to the
cross-linker but the emulsion was not stable. The co-monomer formation of relatively stiffer (GHG-1) to softer (GHG-5)
GMMA was incorporated in the formulation along with HEMA hydrogels as indicated by the decreasing trend in the glass
g
in different mole ratios (Table 1) and it was noted that the transition temperature (T ) and the increasing trend in the
addition of GMMA vastly improved the emulsion stability. % compression (Table 4). The pore and pore throat sizes as
Further, we observed that when the molar ratio of well as the pore wall thickness of these hydrogels were
GMA: HEMA was increased beyond 40:60, the emulsions were determined from the SEM images using Image J software
not stable. The change in the emulsion stability was confirmed (Image J 1.46). The field of the SEM images was divided into
by the optical microscopy (Axioscope-A1, Carl Zeiss, Germany) equal quadrants and more than 30 numbers of pores, pore
by gently casting the emulsion on silanized glass plate. When throats and pore walls each were selected randomly from
the emulsions were stable the optical microscopic images every quadrant for calculating the average and the standard
showed that the emulsion droplets were uniformly distributed deviation.
(Fig. S2A, ESI) but when the emulsion stability started The pore throats or pore interconnect formation takes place
decreasing there was an uneven distribution or deformation of due to the shrinkage of the thin polymer film (separating
the emulsion droplets (Fig. S2B, ESI). Since GMMA played a droplets) during the polymerization as well as during the post
very important role in stabilizing the emulsions, the polyHIPE polymerization process of Soxhlet extraction and drying. As a
hydrogels were synthesized with the composition of GMA: result, the pore throat size was expected to decrease with the
HEMA by restricting the range from 100:0 to 40:60 while the increase in the pore wall thickness. However, contrary to the
molar concentration of the GDMA cross-linker was kept expectation, the pore throat size increased with the increase in
constant at 25 mol % as discussed above.
the pore wall thickness. Similar results have been reported for
5
0
Initiator
the polyHIPEs obtained by the inverse HIPE methodology. All
the polyHIPE hydrogels were amenable to repeated complete
drying and full recovery of their water content on
re-immersion in water indicated that they have an open
The phase separation of the emulsion occurred either when
the polymerization was initiated at the locus of the aqueous
phase thermally with ammonium persulphate (APS) at the
elevated temperatures (>50°C) or when benzoyl peroxide was
employed to initiate the polymerization at the locus of organic
phase. Subsequently, we tried the redox initiated
5
1
interconnected pore structure.
The HIPE emulsions underwent some coalescence (droplet
coarsening) indicated by the presence of the larger secondary
pores compared to the basic or primary pores in the polyHIPE
hydrogels. The basic or primary pores formed during the
emulsification stage morphed into the larger secondary pores
o
polymerization at the physiological temperature (37 C) at the
locus of aqueous phase by using water soluble initiator APS as
an oxidizing agent and TEMED as an accelerator. The redox
initiated polymerization produced highly porous polyHIPE
hydrogels having interconnected open-pore architecture. It
may be noted that the open pore interconnectivity of the
polyHIPE was achieved only with the organic phase initiation
48
through the coalescence during the polymerization process.
The size and frequency of coalesced pores is inversely
proportional to the stability of HIPE. For 60 mol% HEMA in the
co-polymer (GHG-5) the size and the number of coalesced
pores increased (Fig. 3E) and beyond this composition
7
as reported by Robinson et al. and when they used the
(
60 mol% HEMA) the HIPEs were unstable.
aqueous phase initiation the closed pore morphology was
observed. However, this study, with the aqueous phase
initiation, demonstrated that the organic phase initiation was
not a necessity for producing open pore interconnected
polyHIPE hydrogels. In addition, the APS-TEMED redox
initiation allowed the increased cure rate at the physiological
temperature that makes this system potentially viable for in
situ (in vivo) polymerization.
The water swelling ratio (Table 3) and the pore volume
increased with the decrease in the pore size (i.e. increase in
the porosity) and showed a quadratic fit (Fig. 4A), whereas the
pore volume and the mercury intrusion volume showed a
linear relationship (Fig. 4B), as expected.
The polyHIPE hydrogels studied, exhibited high porosity
(>92%) and the bimodal pore morphology comprised of
1
9.6-30.3 µm pores and 3.7-7.6 µm interconnecting pore
4
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throats distributed uniformly (which is necessary to facilitate polyHIPE hydrogels took place very rapidly and reached the
the diffusion of nutrient and waste besides uniform equilibrium swollen state in around 10 min. However, the
distribution of cells by promoting their migration), can be used swelling ratio (Q) given in Table 3 were determined after 4 h.
for soft tissue engineering applications. Many attempts have The highest value of Q (13.6 ± 0.05) was observed for GHG-5
been made to determine the ideal pore size of scaffold for (GMMA: HEMA, 40:60), whereas GHG-1 (100% GMMA)
tissue engineering applications but without any clear showed the lowest values of Q (8.1 ± 0.05).
consensus; often the results are contradictory or may be As was expected, the swelling ratio increased as the
5
specific to cell type. In general, the pore sizes >100 µm are hydrophilicity (total no of –OH groups in the polymer,
2
5
3
considered suitable for this purpose. However, for tissue calculated assuming complete polymerization indicated by
engineering scaffolds, pore sizes 1.4-7.3 µm have also been mass balance for the dried polyHIPEs and comonomers
5
described and recent studies indicate that <40 µm pores including the cross-linker) and the porosity of polyHIPE
0
5
3
improve tissue regeneration.
increased (Table 3). In addition, the swelling parameters (Q)
None of the polyHIPE hydrogels shrunk and crumpled or also increased with increase in the surface area. This could be
collapsed during the drying step. This was partly because of due to the higher percentage of –OH groups became
relatively high degree of cross-linking (25 mol% GDMA) and accessible to the surrounding water molecules for the
partly due the lower glass transition temperature that reduced interaction through the hydrogen bonding. Indeed, the
the stiffness and decreased the resistance to the stress correlation (Fig. 4C) of Q with the hydrophilicity (total no of –
generated during drying, as also observed by Gitlli and OH groups in the polymer) improved vastly (Fig. 4D) when the
5
1
Silvestein. The compressive strain and the initial strain surface area was applied as the weight factor by multiplying it
recovery increased with decreasing GMMA and increasing with the total no. of -OH groups, to determine the corrected
HEMA contents for all the PolyHIPE hydrogels. The hydrogel hydrophilicity.
having 100% GMMA (GHG-1) was the stiffest composition
tested, with a compressive strain of 25.0 when 10.32 KPa load Thermal properties
was applied and showed an initial strain recovery of 80.7% on
The thermal characteristics of the polyHIPE hydrogels were
the load removal. These values increased steadily to 41.9 and
studied with the help of DSC and TGA (Table 4). In the DSC
9
2.2% as GMMA concentration decreased to 40% and HEMA
thermograms for the polyHIPE hydrogels (Fig. 6) both the T
and the melting endotherm peak (T ) are observed. With the
increased HEMA and the decreased GMMA contents, the T
decreased and the T shifted towards the lower temperature
g
increased to 60%. The mechanical properties and the
structural integrity of the scaffolds were mainly determined by
the porous morphology (e.g. porosity, pore and pore throat
size, pore wall thickness and pore volume); crystallinity,
discussed under thermal properties, contributed to a very
small extent.
m
g
m
o
o
initially slowly for GHG-1 (131.9 C) to GHG-3 (130.6 C) and
o
rapidly for GHG-4 (122.8 C) to no melting endotherm for
GHG-5. This endotherm indicated the existence of a small
amount of crystallites which was also substantiated by X-Ray
diffraction patterns (neither very sharp nor completely
diffused, Fig. S4, ESI), in GHG-1 to GHG-4. Similar observation
on small amount of the crystallites was made for other
With the increased porosity, the polyHIPE hydrogels became
increasingly softer as indicated by the increase in the
compressive strain. The compressive strain and the initial
strain recovery are inversely related to the pore size (as well as
the pore throat size, and the pore wall thickness) and these
data showed a quadratic fit (Fig. 5A and 5B). The compressive
strain and the initial strain recovery data for GHG-1 to GHG-5
showed a linear relationship (Fig. 5C) indicating the softer
hydrogels recovered better.
polyHIPE having 22 mol% ethylene glycol dimethacrylate
55
(EGDMA) as a cross-linker, which is flexible and resembles to
GDMA used in this work. When flexible cross-linker e.g. GDMA
or EGDMA) is employed, chain segments with relatively low
(
cross-link densities and, consequently, relatively unrestricted
molecular mobility, can organize themselves into the more
ordered crystalline domains that results into the melting
The initial strain recovery increased with increased HEMA
content, and approached 92% upon subsequent loading which
indicated the ability of these scaffolds to recover from the
strain. The 100% GMMA (GHG-1) exhibited the lowest initial
recovery (80.7%), and the highest recovery was shown by
GHG-5 (92.2%).
55
endotherm. In addition, the formation of these crystalline
domains is assisted by the intra molecular (intra chain)
hydrogen bonding between –OH groups as evidenced by a
-1
broad O-H stretching band centered around 3430 cm (range
Swelling kinetics in water
-1
149 - 3660 cm ) in the FTIR spectrum (Fig. 1). Based on the
3
A study of the swelling behaviour of the polyHIPE hydrogels above data and the discussion, a representative amorphous-
was carried out in water since the wettability and the swelling crystalline schematic model (Fig. 7) is proposed. The model
are very important properties of a scaffold for tissue shows that the polyHIPE hydrogels (GHG-1-GHG-4) are
engineering. The scaffold’s wettability determines the cell predominantly amorphous with the little crystallinity.
5
0
seeding and proliferation whereas the swelling is responsible However, GHG-5 did not show any discernible crystallinity.
5
4
for the nutrient transport
metabolites from) the cells growing on the polymer matrix. It crystallinity in GHG-1 to GHG-4 and for GHG-5 there was no
was observed (Fig. S3, ESI) that the swelling of all the five discernible crystalline region since no T was observed. In
m
to (and removal of toxic The decrease in the T indicated reduction in the degree of
m
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ARTICLE
Journal Name
m g
addition, concomitant to the T , the T also decreased with the The higher rate of degradation of GHG-1, though marginal but
reduction in the degree of crystallinity. As a consequence of discernible (Fig. 9), can be rationalized by hypothysing that
the decreased degree of crystallinity, the density (d) of the the hydrolysis of the ester bonds in the hydrogels takes place
polymer is expected to decrease for less compact packing of via the base catalyzed bimolecular acyl cleavage (
the molecular chains (mass) in a given volume. Indeed, the T mechanism which is the most common under strong basic
decreased with the decrease in density (d) of the polymer condition where the OH nucleophile directly attacks the
determined after grinding the polyHIPE hydrogels to fine electrophilic carbon (>C=O) of the ester and breaks the π bond
AC
B 2)
g
-
(
2
powder to remove the porosity) and showed a perfect (r =1) to form the tetrahedral intermediate leading to organic acid
rd
56
degree polynomial (cubic) correlation (Fig. 8A). The and alcohol formation. Further, the nucleophilic attack of
3
-
was also consistent with the reduced OH , which is the rate determining step (RDS), will be
lowering of the T
g
stiffness (increased compression) of GHG-1 to GHG-5 (Table 4) facilitated if the electrophilic carbon (>C=O) is attached with an
which reduced the resistance to the stress generated during electron withdrawing group(s) and the overall rate of reaction
the drying and thus helped in maintaining the dimensional will increase. There is only one electron withdrawing –OH
5
1
integrity.
group in the polymer chains due to HEMA and GDMA whereas
The TGA and DTG curves (Fig. S5, ESI) show the thermal chain due to GMMA has two –OH groups (scheme 1).
degradation of the polyHIPE hydrogels. All the samples except Furthermore, GMMA has two positional isomers (Scheme S1,
GHG-1 degraded in the three stages viz., the small shoulders at ESI) of which 1,3-hydroxy GMMA (13%) could be more
o
o
around 200 C (corresponding weight loss, 2-4 %) and 275 C susceptible to hydrolysis as both the electron withdrawing –
corresponding weight loss, 8-10 %) and the main (dominant) OH groups are closer to the electrophilic carbon (>C=O) than
(
o
degradation peak at 411.2 ± 5.0 C (corresponding weight loss, 2,3-hydroxy GMMA (87%).
o
69-72 %) and a residual mass of 7-8 % at about 450 C. In Since the polyHIPE hydrogel GHG-1 had 100% GMMA, it was
addition to three DTG peaks, the main degradation peak of more susceptible to hydrolysis and showed the higher rate of
o
GHG-1 showed an additional shoulder at around 350
C
degradation. It may be noted that the higher degradation rate
(
corresponding weight loss, ~30 %) and the degradation peak of GHG-1 became noticeable only after 5-days which further
o
th
at 411.2 C (corresponding weight loss, ~40 %). The weight loss enhanced (differentiated) on 10 day. The other hydrogels
onset temperature of the main peak decreased with the particularly GHG-2 and GHG-3 with relatively higher GMMA
increased HEMA and the decreased GMMA contents. Further, content (80 and 60%, respectively) were also expected to show
o
it was noted that the peak (before 100 C) due to the loss of the enhanced rate of degradation than GHG-4 and GHG-5 in
moisture, shifted towards the lower temperature (Table 4 and the order GHG-1 > GHG-2 > GHG-3 but did not show any
Fig. S5, inset, ESI) from GHG-1 to GHG-5. This was attributed to discernible change on the experimental timescale of 10 days.
the increased porosity (surface area) which facilitated In principle, it would be expected and interesting to see if
relatively easy removal of water from the hydrogels; GHG-2 and GHG-3 show the enhanced rate of degradation in
consequently, the moisture peak shifted to the lower the expected order on the longer timescales.
temperature. The weight loss onset temperature showed a In order to examine the degradability in biological
similar cubic correlation (Fig. 12B) with the density (d) as that environment, degradation of GHG-5, as a representative case,
2
, albeit less than perfect (r =0.9873). Therefore, the T
-1
in 1.0 mg L lipase B (Candida antarctica) solution was studied
of the T
g
g
and the weight loss onset temperature showed a linear in a similar ways as that of the degradation in 0.007 M NaOH.
2
correlation (r =0.9886), which was expected (Fig. 8C). As the The mass loss for the hydrogel, unlike in 0.007 M NaOH, was
polyHIPE hydrogels became denser, the onset degradation not only much lower i.e. 1.0% (2-days), 7.0% (5-days) and 8.4%
temperature, T
g
and T
m
increased. However, the variation in T
m
(10-days), but also did not vary linearly with time. This may be
due to the diffusion of the enzyme lipase, into the bulk of the
was not pronounced but fairly discernible.
4
7
Degradation
hydrogel, is limited by the size of the enzyme.
It was observed that all the hydrogels disintegrated in 0.007 M
The synthesized polyHIPE hydrogels were subjected to the
accelerated degradation at room temperature in 0.007 M
sodium hydroxide solution to test that the hydrolytically labile
ester bonds are accessible by monitoring the % mass loss on
-1
NaOH after 10 days whereas in 1.0 mg L lipase B solution
there was no noticeable change in the integrity of the material.
This showed that the relatively extensive surface as well as the
bulk degradation of the hydrogels took place in the NaOH
solution as compared to the lipase solution.
nd
2
th
th
, 5 and 10 day. All the hydrogels showed the linearly
2
increasing degradation (slope, 1.8727 ± 0.0599 and r > 0.997)
Fig. 9) with an average % mass loss of 7.2 ± 0.6 (2-days), 12.0
0.3 (5-days) and 22.0 ± 0.3 (10-days), except GHG-1. The
average % mass loss for GHG-1 after 2 days was same
7.2 ± 0.6) as those of rest of the four hydrogels but increased
Biocompatibility
(
Cytotoxicity
±
In vitro cytotoxicity evaluation is the first step towards the
biocompatibility of materials. In order to investigate the
potential of the polyHIPE hydrogels as a scaffold for tissue
engineering, assessment of their biocompatibility was carried
out using a well characterized NIH3T3 cell line. The cell viability
was assessed for 5 days relative to TCPS as cytocompatible
control. The assessment indicated that the hydrogels exhibited
(
to 14.1± 0.6 (5-days) and 26.0 ± 0.3 (10-days). Since all the
hydrogels had fixed cross-linking degree at 25% they were
expected to degrade with the same rate including GHG-1.
6
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ARTICLE
greater than 97 % cell viability after 5 days (Fig. 10). The were stable enough and form the interconnected porous
2
images of NIH3T3 cells in the presence of hydrogels and the architecture as compared to the i-HIPE studied earlier.
3,39
control after 5 days are shown (Figure S6, ESI). It was noted Therefore, the polyHIPE hydrogels prepared via inverse HIPE
that there was no significant difference in the cell viability as route in this work are considered promising materials as tissue
compared to TCPS. The cytotoxicity study indicates that these scaffold.
hydrogels are not toxic and hence taken up for cell adhesion
and proliferation study.
Conclusion
Cell adhesion and proliferation
The cell adhesion and proliferation study was carried out as In this work, a series of superporous polyHIPE hydrogels were
discussed under ESI, section 2. The assessment revealed that successfully synthesized by inverse high internal phase
the polyHIPE hydrogel samples exhibited greater than 55 % cell
viability after 7 days (Fig. 11). In all the polyHIPE hydrogels
there was a homogeneous distribution of the pores and
interconnecting windows (pore throats) necessary to facilitate
the diffusion of nutrients and waste metabolites which led to a
growth and migration of the cells from the site of seeding.
Though the hydrogels did not show any significant toxicity
emulsion polymerization (i-HIPE) of the water soluble
monomers, cross-linker at the physiological temperature using
a non toxic organic solvent as dispersed phase. The
morphology and the properties of the materials could be
altered by changing the monomer ratio. The described
materials showed sufficiently high swelling ratio, porosity,
hydrophilicity and thereby increased degradability and
improved biocompatibility. The open porous morphology - a
(97% cell viability), but, in comparison, the cell proliferation
data showed much less cells viability (55-88%) in cell adhesion
and proliferation study. This could be because of modulation key design criterion for tissue scaffold - of the reported
of the physical and chemical properties of these hydrogels polyHIPE hydrogels permitted fast enough convective mass
with the cell growth.
transport of nutrients and oxygen which resulted in the cell
growth and proliferation. In addition, these polyHIPE hydrogels
exhibited desired mechanical properties viz., compressive
strain, strain recovery and dimensional integrity on drying at
After day one, the average % viable cells on all the PolyHIPE
hydrogels was 95.6 ± 2.9 as compared to TCPS (100 ± 1.8 %),
while after seventh day, GHG-1 and GHG-5 showed
56.6 ± 1.7% and 88.4 ± 4.2 % cell viability respectively relative
6
5 °C - a temperature higher than the human body
to TCPS (100 ± 3.8 %). The % cell viability on the polyHIPE
hydrogels increased monotonously from GHG-1 to GHG-5 and
correlated well with their physical and chemical properties as
expected. The increase in % cell viability could be attributed to
the increase in porosity (i.e. decrease in pore and pore throat
temperature. Assessment of in vitro cell adhesion and
proliferation has shown that these materials supported the cell
adhesion, their penetration and proliferation. Overall, the
described polyHIPE hydrogels resembled to many of soft
size), pore volume, surface area, area to volume ratio and tissues of the human body, and therefore can be considered as
hydrophilicity (Fig. 12). The increasing hydrophilicity (increased promising materials for tissue scaffolding. In addition,
–OH groups and increased surface area) of the polyHIPE APS-TEMED redox initiation allowed increased cure rate and
hydrogels promoted the cell adhesion, migration and
quicker development of porous interconnect network at
physiological temperature and thus could be potentially viable
for in situ (in vivo) polymerization, as well.
3
8
proliferation of the seeded cells. Klouda et al. reported that
cell viability greater than 75% is necessary for a material to be
biocompatible. However, materials having cell viability up to
5
7
5
8
about 40% is also reported as biocompatible. The phase
contrast microscopic images (Fig. S7, ESI) show the continual
increasing trend in the cell viability from GHG-1 to GHG-5;
Acknowledgements
whereas, the florescence microscopy (Fig. 13) clearly show We are thankful to the Director, National Chemical Laboratory
that the number of live cells (green) increased and the dead for providing necessary facilities and to the Council of Scientific
cells (red) decreased. The cells were spread with elongated
morphology retaining their nuclear integrity which is a sign of
healthy and proliferating cells. These results confirm the
cytocompatibility of the polyHIPE hydrogels and their ability to
support cell proliferation.
and Industrial Research (CSIR), New Delhi, India for providing
senior research fellowship to one of us (ACN). Thanks are also
due to Ms Sonali M. Bhosle for the help in synthesizing GMMA
monomer used in this work. Thanks are due to Dr. M. S.
Shashidhar for the critical review of the manuscript.
For tissue engineering applications porous scaffold with pore
size ranging from 4 μm to 100 μm have been used depending
5
0 53
on the scaffold function and types of cells to be repaired. ,
Recently, Krajnc et al. have reported the porous monoliths
Notes and references
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4
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Table 4.Thermal and mechanical properties of polyHIPE hydrogels
[
58] D. M. Dragusin, S. V. Vlieberghe, P. Dubruel, M. Dierick, L.
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Sample
T
g
T
m
Onset
temp.
Water
peak
Bulk
Comp.
Strain
recov.
(%)
8
a
b
(
°C)
(°C)
density
strain
(%)
-
1
(°c)
temp.
(g cc )
(°c)
GHG-1
GHG-2
GHG-3
7.43
131.9
131.4
446
412
402
394
390
52.7
48.0
53.0
46.0
43.0
0.114
0.104
0.095
0.094
0.064
25.0
29.4
32.0
35.8
41.9
80.7
83.0
85.5
86.0
92.2
- 5.35
- 10 46 130.6
GHG-4 - 10.93 122.8
GHG-5 - 13.51
Table 1.Composition of inverse HIPE formulations
-
a
Bulk density determined after crushing hydrogels to fine powder.
Under 10.32 kPa load.
Sample
GMMA
mM)
HEMA
(mM)
0
GDMA
(mM)
2.081
2.081
2.427
2.913
3.121
APS
TEMED
(mM)
0.332
0.332
0.388
0.466
0.499
b
(
(mM)
0.166
0.166
0.194
0.233
0.249
GHG-1
GHG-2
GHG-3
GHG-4
GHG-5
8.324
6.658
5.827
5.827
4.994
1.664
3.885
5.826
7.492
Table 2.Characteristics of polyHIPE hydrogels
Sample
Porosi
ty
Pore
Pore
Pore
Hg
intr.
vol.
Pore
vol.
a
a
size
throat
wall
a
-1
(
%)
(µm)
size
(µm)
(cc g )
-
1
(
µm)
(cc g )
GHG-1
GHG-2
GHG-3
GHG-4
GHG-5
92.3
95.3
95.2
94.9
96.6
30.3 ± 7.2
25.4 ± 8.5
21.3 ± 6.5
20.7 ± 6.0
19.6 ± 5.8
7.7 ± 2.0
6.2 ± 1.7
4.6 ± 1.2
4.0 ± 0.9
3.7 ± 0.5
1.8 ± 0.3
1.2 ± 0.4
0.8 ± 0.1
0.7 ± 0.2
0.5 ± 0.6
7.0
8.2
5.0
5.2
5.9
6.0
7.5
9.8
10.9
12.8
a
Calculated from SEM images with the help of Image J 1.46 s/w by randomly
selecting more than 125 pores(refer text).
Table 3.Physico-chemical properties of polyHIPE hydrogels
Sample
Surface
area
No. of
–OH
Surface
area
Swelling
ratio
Cell
viability
(%)
b
2
-1
a
(m g )
groups
to
(Q)
-21
×
10
volume
ratio
-
1
(
m )
GHG-1
GHG-2
GHG-3
GHG-4
GHG-5
2.89
3.43
4.67
10.0
23.1
11.28
10.25
10.82
12.28
12.32
41.3
41.8
47.7
91.7
180.5
8.1 ± 0.05
9.1 ± 0.1
56.6 ±1.7
58.2 ± 3.3
65.2 ± 4.4
77.3 ± 2.2
88.4 ± 4.2
10.7 ± 0.1
11.7 ± 0.05
13.6 ± 0.05
Scheme 1. Copolymerization of GMMA, HEMA and GDMA.
a
See text for calculation of number of –OH group.
Number of viable cells as determined by MTT assay after 7 days.
b
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Fig. 1 FTIR spectra of polyHIPE hydrogels: a) GHG-1, b) GHG-2, c) GHG-3, d) GHG-4,
e) GHG-5; and monomers: f) HEMA and g) GMMA. (It may be noted that the -OH
-
1
stretching peak (3149 – 3660 cm ) is relatively broad in polyHIPE hydrogels as
compared to the monomers indicating increased H-bonding).
Fig. 3 SEM microscopic images of polyHIPE hydrogels: A) GHG-1, B) GHG-2, C) GHG-3,
D)GHG-4 and E) GHG-5.
Fig. 2 SEM microscopic images showing the effect of cross-linker concentration on
polyHIPE hydrogel morphology; (GMMA:HEMA,100:0) and GDMA: A)1 mol %,
B)5 mol %, C)10mol % and D) 25 mol %.
Fig.4 Variation of swelling ratio of polyHIPE hydrogels with A) pore size,
C) hydrophilicity, D) corrected hydrophilicity; and B) correlation between Hg intrusion
volume and pore volume.
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Fig. 7 A schematic amorphous-crystalline model of GMMA-HEMA-GDMA polyHIPE
hydrogels (GHG-1to GHG-4); crystallinity decreases from GHG-1 to GHG-4 (not shown).
Fig. 5 Variation of mechanical properties of polyHIPE hydrogels with pore size:
A) compressive strain, B) strain recovery and C) correlation between compressive strain
and strain recovery.
Fig. 8 Variation of glass transition temperature (T
g
) and decomposition onset
temperature with density A) and B); C) correlation of T
temperature.
g
and decomposition onset
Fig. 6 DSC thermograms of polyHIPE hydrogels: a) GHG-1, b) GHG-2, c) GHG-3,
d) GHG-4 and e) GHG-5.
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Fig. 9 Degradation of polyHIPE hydrogels in 0.007M NaOH with time at ambient
temperature: a) GHG-1, b) GHG-2, c) GHG-3, d) GHG-4 and e) GHG-5; inset: % mass loss
vs. % GMMA after 10 days.
Fig.12 Variation of cell viability with A) swelling ratio, B) pore throat size, C) surface
area to volume ratio and D) corrected hydrophilicity.
Fig.13 Fluorescence microscopic images of NIH3T3 cells seeded on polyHIPE hydrogels:
a) control, b) GHG-1, c) GHG-2, d) GHG-3, e) GHG-4 and f) GHG-5 after 7 days. (It may
be noted that the live cells (green) continually increase and the dead cells (red)
decrease from GHG-1 to GHG-5, see the text also).
Fig. 10 Cytotoxicity test on NIH3T3 cells in presence of polyHIPE hydrogels by MTT
assay.
Fig.11 Cell proliferation test on polyHIPE hydrogels relative to TCPS as control by MTT
assay.
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TOC – Synthesis of superporous hydrogels as tissue engineering scaffold via inverse
high internal phase emulsion (i-HIPE) polymerization.