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Synthesis and characterization of polyurethane-block-poly(2-hydroxyethyl

methacrylate) hydrogels and their surface modification to promote cell
affinity

Article in Journal of Bioactive and Compatible Polymers · March 2011

DOI: 10.1177/0883911511398713

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Article
Journal of Bioactive and
Synthesis and characterization Compatible Polymers
26(2) 114–129

of polyurethane-block- ! The Author(s) 2011

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DOI: 10.1177/0883911511398713
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methacrylate) hydrogels and
their surface modification to
promote cell affinity
Alpesh Patel and Kibret Mequanint

Abstract
Physically crosslinked hydrogels based on poly(2-hydroxyethyl methacrylate) (PHEMA) and
polyurethane macroiniferter (PUMI) were prepared. The synthesis of polyurethane-block-
poly(2-hydroxyethyl methacrylate) (PU-b-PHEMA) was verified by spectroscopic analyses. Due
to the low fibronectin adsorption from culture media, cell attachment on PU-b-PHEMA surface
was poor compared with the PUMI control. To improve the cell affinity of PU-b-PHEMA, fibronectin
was conjugated via surface hydroxyl groups. These biomimetic PU-b-PHEMA hydrogel surfaces
were tested for tissue engineering applications. A short-term cell culture study revealed that,
compared with the unmodified PU-b-PHEMA, fibronectin-conjugated PU-b-PHEMA hydrogel
showed more uniform and dense cell attachment and spreading, indicating a potential use for
tissue engineering applications.

Keywords
biomimetic surfaces, CDI conjugation, cell surface interactions, hydrogels, macroiniferters,
physically crosslinked hydrogels

Introduction
Tissue engineering has emerged as an alternative approach to current treatment modalities
for failed and diseased tissues as well as to improve healthcare standards.1,2 One of the

Department of Chemical and Biochemical Engineering, The University of Western Ontario, London, ON, N6A 5B9,
Canada
Corresponding author:
Kibret Mequanint, Department of Chemical and Biochemical Engineering, The University of Western Ontario, London,
ON, N6A 5B9, Canada
Email: kmequani@eng.uwo.ca
Patel and Mequanint 115

approaches to engineer living tissues involves a polymeric scaffold for cell attachment and
subsequent controlled cell proliferation while maintaining the desired cell function.3,4
To remodel a scaffold by extracellular matrix structure that functions similar to the living
tissue, directing cell function and fate for biomaterials remains a major challenge.
The scaffold platform underlying the cell surface has a significant impact on the behavior
of seeded cells.5 Therefore, any functional biomaterials designed for scaffolding applications
should have biofunctionality for cell attachment, migration, and proliferation. Hydrogels
have many similarities to the physiological properties of living tissues, and have the potential
as one of the most promising scaffold biomaterials.1,6
The properties of hydrogels, such as aqueous surface environment, lower frictional
irritation due to lower surface energy, soft and tissue-like physical properties, nano- and
micro-porous channels for additional nutrition transport, ease of surface modification with
biomolecules, and unique biocompatibility are some of the attractions for tissue engineering
applications.3,7 Naturally occurring hydrogels possess specific cell binding ligands and are
used for scaffolds, but their poor mechanical properties preclude their practical use.1,8
Synthetic crosslinked hydrogels, on the other hand, can be designed to be mechanically
responsive, but their cell adhesion properties are very poor.
The synthetic polymer hydrogel, poly(2-hydroxyethyl methacrylate) (PHEMA), first
reported by Wichterle and Lim,9 is one of the most extensively studied biomaterials for
tissue engineering,10 contact lenses,11 corneal implants,12 cardiovascular implants,13 breast
implants,14 nerve tissue repair,15 drug release devices,16 and tissue repair platform.17
Crosslinking of synthetic hydrogels is imparted by chemical or physical bonding.
However, difficulties in post-synthesis device fabrications and crosslinker toxicity are
drawbacks of chemically crosslinked hydrogels.18 Polyurethanes are known to have
excellent blood compatibility as well as mechanical properties that make them very useful
as biomaterials.19–22 Therefore, to improve the mechanical integrity of PHEMA hydrogels,
polyurethanes could be incorporated as physical crosslinkers during the synthesis stage.
Compared with other physically crosslinked hydrogels, those based on polyurethanes are
attractive for biomedical applications, since they are deemed biocompatible and the presence
of strong hydrogen bonding provides excellent mechanical properties.
Previously, we prepared different physically crosslinked hydrogels using a polyurethane
macroiniferter (PUMI) technique and explored their potential for biomedical
applications.18,23,24 In this study, physically crosslinked polyurethane-block-
poly(2-hydroxyethyl methacrylate) (PU-b-PHEMA) hydrogels were synthesized using the
macroiniferter approach and by conjugating fibronectin to improve cell interaction. The
choice of fibronectin was based on enhanced vascular smooth muscle cell (VSMC)
adhesion that we previously observed when synthetic biomaterials were coated with
fibronectin.25 The potential utility of the current fibronectin-conjugated PU-b-PHEMA
hydrogels for tissue engineering application was also evaluated.

Materials and methods

Materials
All chemicals, unless stated otherwise, were purchased from Sigma–Aldrich (Milwaukee,
WI). Poly(tetramethylene oxide) (PTMO) glycol, Mw ¼ 1000 g/mol (PTMO-1000), was
dried at 90 C under reduced pressure. 2-hydroxyethyl methacrylate (HEMA) was passed
116 Journal of Bioactive and Compatible Polymers 26(2)

through an inhibitor removal column (Sigma–Aldrich, Milwaukee, WI) before use. Methyl
ethyl ketone (MEK) and dimethyl formamide (DMF) were distilled at reduced pressure and
their middle portions were used. All other chemicals and reagents were of the highest purity
available and were used without further purification.

Synthesis of N,N0 -diethyl-N,N0 -bis(2-hydroxyethyl)thiuram disulfide

N,N0 -diethyl-N,N0 -bis(2-hydroxyethyl)thiuram disulfide (DHTD) was synthesized from
2-ethylaminoethanol and carbon disulfide in the presence of iodine as described in a
previous publication.24
1
H-NMR (CDCl3):  (ppm) ¼ 1.10–1.30 (–CH2CH3), 3.50 (–OH), 3.60–4.00 (–CH2CH3,
–S2CNCH2–), and 4.35 (–CH2OH).

Synthesis of PUMI
A mixture of 10.00 g (0.01 mol) of PTMO-1000 and 5.25 g (0.02 mol) of 4,40 -
dicyclohexylmethane diisocyanate (H12MDI) in 20 mL of MEK was heated to 65 C for
3.5 h. The mixture was then cooled to room temperature and a solution of 3.28 g
(0.01 mol) of DHTD in 60 mL of MEK with 0.5 mol.% (based on isocyanate content)
dibutyltin dilaurate catalyst was added. The chain extension reaction was carried out until
the isocyanate peak (2265 cm1) disappeared from the Fourier transform infrared (FTIR)
spectrum. The dithiocarbamate (DC) based PUMI was then precipitated in 10-fold excess
cold methanol and dried at 30 C in a vacuum oven.

Synthesis of (PU-b-PHEMA)
A solution of 0.93 g of PUMI and 4.88 g (0.075 mol) of HEMA in 50 mL of DMF was
purged with nitrogen for 10 min and irradiated with UV light (model B100AP; UVP Inc.,
Upland, CA) at a distance of 6 cm (Scheme 1). After 24 h, the PU-b-PHEMA copolymer was
precipitated by adding a 10-fold excess of cold diethyl ether. The product was recovered by
filtration and dried at 30 C in a vacuum oven. Any PHEMA homopolymer and unreacted
HEMA monomers were then extracted in acetonitrile. The product was then kept in distilled
water for a week in order to leach out any unreacted monomers.

Modification of PU-b-PHEMA copolymer surface with fibronectin

A PU-b-PHEMA film was prepared by a solvent casting using DMF. Circular 0.50-cm discs
were made using a one-hole paper punch. The discs were equilibrated with acetone in
individual vials overnight. The discs were then transferred to vials containing 20 mM of
1,10 -carbonyldiimidazole (CDI) solution in acetone. After 3 h, the unreacted CDI was
removed by rinsing the discs in excess anhydrous acetone three times. The discs were then
sequentially washed with distilled water through increasing ratios of water/acetone solutions
(25/75, 50/50, and 75/25; v/v) and finally with distilled water. The surface-activated discs
were placed into vials containing 600 mL of 50 mg/mL bovine fibronectin (Fn) (TrevigenÕ ,
Gaithersburg, MD, USA) solution in 1% phosphate-buffered saline (PBS). Fibronectin
conjugation was allowed to proceed for 24 h at 4 C. Non-conjugated fibronectin was removed
by washing in 2% sodium dodecyl sulfate (SDS) for 1 h. Finally, the discs were rinsed and
Patel and Mequanint 117

Scheme 1. Schematic diagram of PUMI and PU-b-PHEMA syntheses

equilibrated with PBS overnight. The Fn-modified hydrogel discs (Fn-g-PU-b-PHEMA) were
then stored in sterile PBS (Scheme 2).

Spectroscopic analysis
FTIR spectra were recorded using a Bruker Vector 22 spectrophotometer. Samples were
mounted directly over the sample holder and 32 scans at 4 cm1 resolution were collected for
each sample. Nuclear magnetic resonance (1H-NMR) spectra were recorded on VarianÕ
mercury 400 instrument operating at 400 MHz. The DHTD samples were dissolved in
CDCl3, whereas the PU-b-PHEMA was dissolved in DMSO-d6. The observed peaks were
analyzed using ACD/2D NMR software.

Gel permeation chromatography

The molecular weights were determined using a Waters 2695 separations module equipped
with a Waters 2414 differential refractometer and two PLgel 5 mm mixed-D (300  7.5 mm2)
columns from Polymer Laboratories. The samples, dissolved in DMF with 0.1 M LiBr and
1% (v/v) triethylamine, were injected at 85 C using flow rate of 1 mL/min. The calibration
was performed using polystyrene standards. Empower 2 software was used to analyze the
eluted peaks.
118 Journal of Bioactive and Compatible Polymers 26(2)

OH O O
N N
N NH
N N N
O O
H2N
O
OH O (Fibronectin) O
(CDI)
N NH
N
O 4oC O
Acetone
OH 25oC, 3h O 24h O
N NH
N
O O

PU-b-PHEMA CDI-PU-b-PHEMA Fn-g-PU-b-PHEMA

Scheme 2. Scheme for the modification of PU-b-PHEMA copolymers with bovine fibronectin using CDI
conjugation

Swelling study
Polymer discs (n ¼ 4) were cut into 5 mm diameter discs from cast films. The discs were dried
in a vacuum oven at 50 C, cooled to room temperature, and weighed. They were then
swollen at room temperature in distilled water. Swollen discs were taken out of the water
at regular time intervals and blotted lightly with a filter paper to remove the excess surface
water. The weights of swollen discs were determined and water uptakes of the discs were
calculated using:
Wh  Wd
WU ¼  100 ð1Þ
Wh

where Wh is the weight of the swollen disc at time t and Wd the dry weight of the disc before
swelling.

Thermal analysis
Differential scanning calorimetric (DSC) study and thermogravimetric analysis (TGA) of
PUMI and PU-b-PHEMA were carried out using TA instruments Model Q20 V24.3 Build
115. The samples were dried in a vacuum oven at 50 C for 24 h before use. For
DSC analysis, 5 mg specimens were sealed in a DSC pan and quenched to 110 C. It was
then left to equilibrate for 15 min and heated to 200 C at a rate of 20 C/min. For TGA,
samples were weighed in the range 5–10 mg and heated from 25 C to 700 C at a heating rate
of 20 C/min under nitrogen. TA Instruments Universal Analysis 2000 software was used to
analyze the data.

Protein adsorption study

PUMI and PU-b-PHEMA discs (n ¼ 3) were incubated in 0.10 M PBS solution for 24 h in
24-well plate. The PBS solution (pH 7.4) was replaced by 1 mL of bovine fibronectin solution
(20 mg/mL). The protein adsorption was conducted for 6 h in an incubator at 37 C. The discs
were then rinsed three times with 1 mL of PBS solution, for 10 min each. To extract the
Patel and Mequanint 119

adherent proteins, the discs were treated with 1 mL of aqueous solution of SDS (2%) for 2 h.
The extracted proteins were analyzed using Bicinchoninic acid (BCA) protein assay reagent
kit (Pierce Biotechnology, Rockford, IL) and quantified using a Beckman Coulter DU 520
UV-Vis Spectrophotometer (Mississauga, ON) at 562 nm. Similar experiments were repeated
at higher fibronectin concentration (50 mg/mL). Two fibronectin concentrations were chosen
to simulate the fibronectin content in common serum supplemented culture media.

Preparation of samples for cell culture

Circular 0.5 cm samples were affixed to 1.2 cm diameter glass coverslips using silicone grease,
sterilized with 70% ethanol, and allowed to dry overnight. Coverslips containing polymer
films were placed into individual wells of a 24-well tissue culture plate for cell seeding.

Cell culture study

Primary human coronary artery smooth muscle cells (HCASMCs), purchased from
Cambrex Biosciences (Walkersville, MD, USA), were used. Before cell seeding,
HCASMCs were cultured in smooth muscle growth media with 5% fetal bovine serum
(smooth muscle growth medium (SmGM)-2 with Bullet Kit; Cambrex) at 37 C in a
humidified incubator containing 5% CO2. Growth media was exchanged every 2 days
until approximately 80% confluent cell layer was obtained. The cells were then trypsinized
with 4 mL of 0.05% trypsin–ethylenediaminetetraacetic acid solution, centrifuged at
1100 rpm for 5 min, and resuspended in 3 mL of fresh growth media. The cells (passages
4–6) were seeded onto PUMI and PU-b-PHEMA discs at an initial cell density of
40,000 cells/cm2 and incubated in growth media for specific time intervals. The cells on
the discs were then fixed at room temperature in 2% paraformaldehyde in PBS for 10 min,
and permeabilized in 0.5% Triton X-100 in PBS for another 10 min. After washing three
times with PBS for 5 min, the cell-seeded discs were incubated for 1 h at room temperature in
Alexa-Fluor-568/488 conjugated phalloidin (red/green) in PBS (1 : 50), washed with PBS,
and incubated for 10 min in Hoechst 33342 (20 mg/mL in PBS). The discs were mounted on
glass slides in Trevigen mounting media, sealed, and analyzed with a Leica DM-IRB
fluorescence microscope.

Results and discussion

The availability of a wide range of precursors allows the preparation of polyurethanes with a
wide variety of properties.26,27 However, for physically crosslinked polyurethane hydrogels,
this choice is often lost due to the limited availability of hydrophilic macrodiols. Other than
PEO, available macrodiols are often hydrophobic, making the design of linear polyurethane
hydrogels difficult.28–31 In previous studies, we used macroiniferters to prepare physically
crosslinked polyurethane-based hydrogels which facilitated the inclusion of a family of
hydrophilic free radical polymers onto the polyurethane backbone and expanded the
chemoselectivity of macrodiols to develop linear polyurethane hydrogels.23,24 Moreover,
the use of hydrophilic monomers carrying –OH, –COOH, or –NH2 groups can further
modify the hydrogels with bioactive peptides, growth factors, or biomolecules for tissue
engineering applications.32
120 Journal of Bioactive and Compatible Polymers 26(2)

In this study, H12MDI-based PUMI using DHTD as a chain extender was synthesized.
The PUMI was used as a macroiniferter to synthesize physically crosslinked PU-b-PHEMA
hydrogels (Scheme 1). The prepared hydrogels were soluble in dimethyl sulfoxide, DMF, and
N-methyl-2-pyrrolidone; however, they swelled in the presence of water without dissolving
which confirmed physical crosslinking. To promote the cell interaction, the PU-b-PHEMA
surface was further modified with fibronectin using CDI conjugation (Scheme 2). This
biomimetic surface is able to mediate and enhance the cellular affinity without
compromising the bulk mechanical properties.
The PUMI spectrum in Figure 1 showed H-bonded N–H stretching at around 3330 cm1
and a carbonyl stretching at 1710 cm1 corresponding to the urethane group. A peak at
1226 cm1 was due to the mixed vibrations involving C–N stretching along with N–H
bending of the urethane group. The peak related to the C–O–C stretching of the PTMO
segments was observed at 1080 cm1. All these peaks were also observed in the block
copolymers. The PU-b-PHEMA spectrum showed a broad peak in between 3660 and
3060 cm1 related to H-bonded N–H stretching of urethane linkage as well as O–H
stretching in the HEMA segment. Moreover, peaks related to carbonyl stretching (C ¼ O)
and ester stretching ((CO)–O) of the PHEMA segment were observed at 1660 and
1160 cm1, respectively. The surface-modified Fn-g-PU-b-PHEMA spectrum showed
broad two peaks between 3700 and 3000 cm1 related to the primary amine stretching of
the conjugated fibronectin. Moreover, the ester linkage of the CDI conjugation had been
observed at 1770 cm1.
In the 1H-NMR spectrum of PU-b-PHEMA copolymer (Figure 2), the peaks related to
the PTMO, H12MDI, and PHEMA segments support the success of the copolymer synthesis.
Specifically, the peak related to the urethane proton, observed at 7.95 ppm, supports the
PUMI synthesis, whereas the hydroxyl proton peak observed at 4.81 ppm supports the
presence of PHEMA segment within polyurethane backbone.

F n -g-PU-b-PHEMA
Transmittance

PU-b-PHEMA

PUMI

4000 3000 2000 1000

Wave number, cm–1

Figure 1. FTIR spectra of PUMI, PU-b-PHEMA, and Fn-g-PU-b-PHEMA

Patel and Mequanint 121

Figure 2. 1H-NMR spectrum of PU-b-PHEMA

In Figure 3, the gel permeation chromatography (GPC) traces of PUMI and

PU-b-PHEMA copolymer are shown, both peaks were unimodal and the peak related to
PU-b-PHEMA moved distinctively toward lower elution volume indicating a molecular
weight increase and supports the block copolymer synthesis. In both cases, the
polydispersity index remained below 1.50.

Thermal behavior of macroiniferter and block copolymer

The thermal transitions of PUMI and PU-b-PHEMA are shown in Figure 4. Both PUMI
and PU-b-PHEMA show Tg related to the PTMO soft segment that are higher than the Tg of
pure PTMO (90 C) with molecular weight of 1000 g/mol.33 This could be either due to
strong interaction between soft and hard segments or due to contamination of PTMO with
higher Tg hard segments. The PU-b-PHEMA provides additional Tg at 120 C that is related
to PHEMA segment. Moreover, the Tg related to the PHEMA segment was greater than the
122 Journal of Bioactive and Compatible Polymers 26(2)

PU-b-PHEMA

Mn = 13.64 x 104 g/mol;

PDI = 1.46

PUMI

Mn = 9.54 x 104 g/mol;

PDI = 1.37

10 12 14 16

Elution volume, mL

Figure 3. GPC elution curves of PUMI and PU-b-PHEMA

Tg = –55°C
Endo Heat flow Exo

Tg = –75°C
PUMI

PU–b–PHEMA
Tg = 120°C

–100 –50 0 50 100 150

Temperature, °C

Figure 4. DSC thermographs of PUMI and PU-b-PHEMA

Tg of pure PHEMA (113 C).34 This is probably due to the reduction in the available free
volume and the strong physical interaction between polyurethane and PHEMA segments.
Overall, the present PU-b-PHEMA can be considered as a microphase-separated elastomer.
In both polymers, the absence of peaks related to crystalline phase indicates the amorphous
nature of both polymers.
Patel and Mequanint 123

The TGA data revealed that the PUMI is more thermally stable than the PU-b-PHEMA
(Figure 5). Similar to conventional polyurethanes,35 both polymers followed a two-stage
degradation pathway; in the first stage, weight loss due to hard segment degradation was
followed by weight loss due to soft segment decomposition.

Surface analysis and swelling properties of macroiniferter and block copolymer

The water uptake behavior of PUMI and PU-b-PHEMA at 22 C with respect to immersion
time is shown in Figure 6. The 12.13  1.85% PUMI equilibrium water content (EWC) was
more than that for the MDI-based PUMI systems.23,24 The PU-b-PHEMA hydrogels’
uptake water was rapid in first 10 min; the swelling then slowed to a final EWC of
41.93  0.52%. The incorporation of significant hydrophilic segments within the PUMI
was indicated by an increase in molecular weight (Figure 3) and the expected increase in
EWC observed.

Short-term cell culture. For tissue engineering biomaterials, cell–material interaction is the
first step that provides information regarding their cytocompatibility. The fluorescence
images of cells cultured on PUMI and PU-b-PHEMA hydrogels following 2 days of
culture are shown in Figure 7.
As seen in Figure 7, better cell attachment and spreading were observed for PUMI.
However, the PU-b-PHEMA hydrogels showed poor cell attachment and spreading. The
lack of serum-derived cell-adhesive protein adsorption onto the hydrogel materials could be
the reason for the poor cell adhesive.13,36 To determine this possibility, serum fibronectin
adsorption on both PU and PU-b-PHEMA surfaces was carried out using BCA assay. The
data presented in Figure 8 indicate that fibronectin was adsorbed more readily on PUMI
than that on the PU-b-PHEMA surfaces.
Plasma fibronectin is a known cell adhesive protein due to its cell-binding RGD domains
that are responsible for focal adhesion by binding to integrins which are cell surface
receptors.37 The amount of fibronectin adsorption on synthetic biomaterials regulates cell
interaction. However, other factors such as adsorbed fibronectin distribution, the
conformation, and the integrin binding affinity with the adsorbed fibronectin are also
responsible for other cellular functions, such as differentiation and proliferation.38
As shown in Figure 8, the PU-b-PHEMA hydrogel surfaces adsorbed lower fibronectin
compared with the PUMI surfaces at both fibronectin concentrations. The surface
chemistry, polarity, and topography of the biomaterials may be responsible for
surface fibronectin adsorption. PHEMA is known to undergo dynamic exchange of
its surface groups from methyl to hydroxyl in water during the swelling process
and eventually increases the surface polarity in a hydrated environment.39 Higher
surface polarity due to PHEMA segment ultimately decreases the fibronectin adsorption.
This may be one of the reasons for the lower cell attachment over the PU-b-PHEMA
surfaces.
To improve vascular cell attachment on the polymer surfaces containing hydroxyl groups,
various methods were employed to chemically couple biologically active ligands.40–43 These
methods activate the hydroxyl groups facilitating the coupling of cell-adhesive ligands
mostly via the amino terminus.44 Since CDI is well understood for polypeptide
attachments on PHEMA-based biomaterials,45,46 we used CDI conjugation chemistry to
modify the PU-b-PHEMA surfaces to improve cell adhesion.
124 Journal of Bioactive and Compatible Polymers 26(2)

1.4
100
1.2
80
1.0

Deriv. Wt, %/°C

Weight, %

60 0.8
PUMI
PU- b-PHEMA
0.6
40
0.4
20
0.2

0 0.0
100 200 300 400 500
Temperature, °C

Figure 5. TGA thermographs of PUMI and PU-b-PHEMA

50

40
PU- b -PHEMA

30
Water uptake, %

20

10
PUMI

0
0 100 200 300 400
Swelling time, min

Figure 6. Swelling isotherms of PUMI and PU-b-PHEMA

The fluorescent images of the preliminary 2D cell cultures on PUMI (a and b),
PU-b-PHEMA (c and d), and Fn-g-PU-b-PHEMA (e, f), respectively, are shown in
Figure 9. Similar to Figure 7(c) and (d), the PU-b-PHEMA surfaces had poor cell
attachment and induced cell clustering. When fibronectin was conjugated, the fluorescent
images indicated that cells were uniformly and densely populated throughout the surface.
This suggests that the current PU-b-PHEMA hydrogel could be used for the vascular
Patel and Mequanint 125

Figure 7. Flouroscent microscopic images of VSMC interaction with PUMI (A and B) and PU-b-PHEMA
(C and D) surfaces following 2 days of culture. The nuclei were stained with Hoechst 33342 as blue whereas
the cytoskeleton was stained with Alexa-Fluor-568 conjugated phalloidin for F-actin and is shown as red
(scale bar ¼ 5 mm)

6000
PUMI
PU-b-PHEMA
5000
Fibronectin adsorbed, ng/cm2

4000

3000

2000

1000

0
20 50
Fibronectin concentration, µg/mL

Figure 8. Fibronectin adsorption data of PUMI and PU-b-PHEMA

126 Journal of Bioactive and Compatible Polymers 26(2)

Figure 9. Fluorescent microscopic images of VSMC interaction with PUMI (A and B), PU-b-PHEMA (C and
D), and Fn-g-PU-b-PHEMA (E and F) surfaces.
Note: The cell culture studies were conducted for 2 days (A, C, and E) and 4 days (B, D, and F). The nuclei were
stained with Hoechst 33342 as blue whereas the cytoskeleton was stained with Alexa-Fluor-488 conjugated
phalloidin and is shown as green (scale bar ¼ 5 mm)

tissue engineering applications. To further understand the influence of fibronectin

conjugation over cell migration, proliferation, and phenotype, more detailed cell
interaction studies are needed.

Conclusions
Linear PU-b-PHEMA hydrogels were synthesized using DC-based PUMI and
spectroscopically characterized. The thermal degradation of the polymers followed the
conventional two-stage polyurethane degradation. The water uptake revealed that these
hydrogels had an EWC of 42%. Fibronectin adsorption on PU-b-PHEMA hydrogel was
lower and resulted in poor cell adhesion compared with PUMI. To improve cell attachment,
Patel and Mequanint 127

fibronectin was successfully conjugated onto the PU-b-PHEMA films. Fibronectin-

conjugated PU-b-PHEMA hydrogel promoted more uniform and dense VSMC
attachment and spreading in short-term cultures. These fibronectin-conjugated PU-b-
PHEMA biomaterials are being considered as a potential scaffold material for tissue
engineering applications.

Acknowledgement
This study is supported by the Heart and Stroke Foundation of Canada (HSFC) grant no. NA6345.
Alpesh Patel was funded by a NSERC postgraduate scholarship.

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