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Polymer International 41 (1996) 419-425
Influence of Hard Segments of
Polyurethane on Cell Growth
P. C. Lee,' L. W. Chen,2* J. R . Lin,' K. H. Hsieh2& L. L. H. Huang3
Institute of Materials Science and Engineering, Department of Chemical Engineering, Center for Biomedical Engineering,
National Taiwan University, Taipei, Taiwan
(Received 14 May 1996; accepted 4 July 1996)
Abstract: Polyurethanes (PU) with suitable soft segments have been found to be
good blood-compatible polymers and have attracted much attention recently. In
this study, various molar amounts of 4,4'-methylene bisphenyl isocyanate reacted
with poly(tetramethy1eneoxide) were synthesized to explore the optimal ratio of
hard/soft segments for cell attachment and proliferation in in uitro systems. Differential scanning calorimetry and dynamic mechanical analysis were used to
determine the physical properties, hydrogen bonding index (HBI) and transmission electron microscopy to observe the phase-separation phenomena in the
materials, and 3T3 fibroblast to evaluate the dependence of the cell proliferation
at 37°C on the material properties. Our results show that cell attachment and
proliferation are closely related to the cell growth surface, which in turn is controlled by (1) the ratio of hard to total segment concentration and (2) the recrystallization temperature (T,)of PU. To obtain a good cell growth surface, the
ratio of hard to total segment concentration is found to be between 0.4 and 0.6,
and HBI is between 1.5 and 2.1. Furthermore, when the T, of PU is near the
physiology temperature, a stable surface for cell growth can be provided. The
shorter molecules in the soft segment region can rearrange the molecular chain at
37°C.
K e y words: polyurethane, cell growth, phase separation, hydrogen bonding
index, cytotoxicity.
INTRO D UCTlON
c o - ~ o r k e r s . They
~ , ~ observed selective calcification by
the 1000 molecular weight species, in contrast to the
higher and lower molecular weight macroglycols, which
reduced this behaviour. Thoma et aL6 hypothesized that
the PTMO 1000 was capable of forming ring or crown
structures, which can effectively allow passage of
specific-sized molecules and hence play an important
role in the biostability of polyurethane. Based on these
results, PTMO 1000 was used as the soft segment in the
polyurethane series in this study.
A composition ratio of diisocyanate : polyol : chain
extender = 2 : 1 : 1 has often been used to assess blood
compatibility and cell ~ompatibility.~-'~
However, since
we were particularly interested in the weight fraction of
hard segment and its effect on cell growth, we changed
Among polymers, polyurethane has been considered to
have great potential for application in medical devices.
Polyurethane is adopted for implant application
because of such advantages as its high tensile strength,
lubricity, good abrasion resistance, ease of handling and
extruding and good 'bi~compatibility'.l-~
In this study, we focus on using poly(tetramethy1ene
oxide) (PTMO 1000) to synthesize the polyurethane. In
relation to the higher and lower molecular weight polyether macroglycols in polyurethanes, the behaviour of
PTMO 1000 has been reported by Phillips, Thoma and
* To whom all correspondence should be addressed.
419
Polymer ZnternationalO959-8103/96/$09.00
0 1996 SCI. Printed in Great Britain
P. C. Lee et al.
420
the molar ratio of diisocyanate 4,4'-methylene bisphenyl
diisocyanate, MOI) as a hard segment, and polyol
(PTMO 1000) as a soft segment in this polyurethane
series. The correlation of physical properties with cell
growth was investigated.
EXPERIMENTAL
reaction was complete, the polymer solution was put
under vacuum for 5min to degas the polymer solution,
cast in moulds; cured at 70°C for 24 h, and post-cured
at 110°C for 24h. The samples were placed under
vacuum at room temperature to remove the residual
solvent. All the specimens were conditioned at room
temperature and 50% humidity for at least 2 weeks
prior to testing.
Materials
Preparation of polymer film
The materials used and their vendors were as follows.
4,4'-Methylene
diphenyl
diisocyanate
(MDI),
poly(tetramethy1ene oxide) with average molecular
weight 1000 (PTMO lOOO), dimethylformamide (DMF)
and 1,Cbutanediol were purchased from Aldrich
Chemical Company, Inc. (USA). Round glass coverslips,
with diameter of 15mm, were purchased from Matsunami Glass Industries, Ltd (Japan). Dimethylsulphoxide
(DMSO), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) and RuO, were purchased
from Sigma Chemical Company (USA). DMEM, RPMI
medium 1640, fetal bovine serum (FBS), trypsin-EDTA
and streptomycin/penicillin were all cell culture grade
and were purchased from Gibco BRL Life Technologies, Inc. (USA).
The polymer membranes were supported on a 24-well
tissue culture to detect cell attachment and proliferation
on the polymer surface. Films were cast onto optical
coverslips using a 3% solution of polyurethane in
DMF. The glass coverslips were first cleaned thoroughly with chromic acid, then doubly distilled water
and detergents under sonication, and washed with
ethanol and dried under vacuum at 50°C. After coating
with polymer solution, the coverslips were dried in air
at 70°C for 24 h and kept under vacuum at room temperature for 48 h. They were stored in a desiccator until
used.
Synthesis of polyurethanes with various molar ratios
of hard segments
This series of polyurethanes was prepared in degassed
DMF solution using a two-stage solution polymerization method at 65°C under dry nitrogen. The
molar ratios of MDI : PTMO 1000 : chain extender and
their codes are shown in Table 1. A dry three-necked
flask was used to prepare the prepolymer. 1,4-Butanediol, as chain extender, and 50wt% degassed DMF
were added to promote the polymerization. No catalyst
was added. The reaction was continued until the isocyanate functional group had disappeared from the
Fourier transform infrared (FTIR) spectra. When the
TABLE 1. Symbols of PUS synthesized with various
MDI ratios ( M D I = 4.4'-methylene
bisphenyldiisocyanate; polyol = PTMO 1000; chain extender =
1.4-butanediol)
Symbol
MDI : polyol : chain extender
CE
1 :0:1
1 :I :o
2 : l :I
4:l :3
6:l :5
8:l :7
1-1 000
2-1 000
4-1 000
6-1 000
817 or 8-1 000
1 019 or 10-1 000
12111 or 12-1 000
110 or
211 or
41 3 or
615 or
10:1:9
1 2 : l :I1
Surface hydrophilicity analysis
Analysis of the dynamic contact angle was performed
on the polymer-coated film at 37°C by the Wilhelmy
plate technique. Dynamic advancing and receding
contact angles were calculated from the force-depth
curve, based on measurements at advancing and receding rates of 25 mm min-l. Surface tension was measured
for fresh films as well as for films soaked in octane at
room temperature for 24 h. The instrument was calibrated using a Pt plate. All glass slides used in the
experiment were cleaned with chromic acid and then
washed several times with doubly distilled water. The
size of sample used for testing was 2 cm x 3 cm. Measurements of five films were taken and the results averaged. The samples used as control glass surfaces were as
f01lows.~(1) Hydrophilic glass surface: the glass was
cleaned with cold chromium sulphuric acid for 24 h and
rinsed thoroughly with distilled water. Thereafter the
slides were put into 2% hydrofluoric acid for 2min and
rinsed with distilled water. The cleaned glasses were
stored in doubly distilled water to retain hydrophilicity.
(2) Hydrophobic glass surface: dry glass slides were
stored in dimethyloctadecylsilane (2% v/v) and washed
in chloroform for 24 h. After soaking, the glasses were
rinsed with chloroform three times.
Dynamic mechanical analysis ( DM A ) and differential
scanning calorimetry (DSC) measurements
DMA and DSC were performed with a thermomechanical analyser (DuPont 100). Rectangular samples
POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996
Injuence of hard segments of PU
2.5in x 0.5in x 0.125in were torqued at 1Hz for
DMA measurements.
Under a 20mlmin-' dry nitrogen purge, the DSC
samples (10 mg) were heated in sealed aluminium pans
at 10"Cmin-' from room temperature to 200"C, kept
for 3 min, cooled to - 150°C at the same rate, and then
reheated to 250°C to obtain the second scan data.
Attenuated total reflection (ATR)-FTIR analysis of
polyurethane surface
A Nicolet FTIR system with a resolution of 4cm-' was
used to collect the ATR-FTIR spectra. The absorbance
intensity at 17OOcm-' (bonded C=O) ratioed to that at
1730cm-' (free bonded C=O) was used to define the
hydrogen bonding index (HBI).
Transmission electron microscopy (TEM)
observations
To prepare specimens for TEM observation, 0.3%
polymer solutions were placed on a 200 mesh coated
slot grid. Specimens were stained using vaporized RuO,
for 30 min and observations, as well as photographs,
were taken using a transmission electron microscope
(JEM- 1200EXII).
Evaluation of cytotoxicity of polyurethane extracts
To examine the extracts of polymers from the cell
culture, we used the ASTM F 624-93 method. The
extracts were first completely immersed in sterilized
DMEM at 37°C and then processed by shaking at
1000rpm for 120h in a borosilicate glass tube. After
shaking, the extracts were immediately diluted with
culture medium at a ratio of nine times its volume, and
seeded with the fibroblast 3T3 cell line at a cell density
of 5 x lo4 per well in the 24-well plate.
The determination of fibroblast activation after
contact with the extracts was assessed by the tetrazolium salt test (MTT). The details of MTT are reported
in Refs 17 and 18; at the end of the incubation time, the
fibroblasts were detached by trypsin and the trypsinization was terminated by DMEM with 10% serum.
Then, 180pl/well of trypsinization solution and 20 pl/well
of 0.5% MTT in RPMI medium were added to the
96-well plates. After incubation at 37°C for 3h, the
culture plates were centrifuged, the supernatant was discarded, and the intracellular formazan crystals were
solubilized with 100pl/well of DMSO. The absorbance
of samples in each plate was determined at 570nm to
obtain the optical density (OD) value.
Direct contact cell to polyurethane to evaluate cell
attachment and proliferation
Polymer-coated glass coverslips, placed in a 24 multiwell cell culture plate, were sterilized with 70% alcohol
POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996
42 1
and 30min UV radiation. The 3T3 fibroblast cell line,
with a 20th-pass approach was used in this study. Supplemented with 10% fetal calf serum (FCS), streptomycin (50mg1-') and penicillin (5 x lo4 units 1-I) in
DMEM as medium, the cells were harvested with
0.15% trypsin/0.02% EDTA in PBS (pH 7.4). The cells
were resuspended in the medium at a cell density of
5 x lo4 cells ml-'. The 1ml cell suspension was seeded
on the polymer-coated coverslips placed on a 24-well
tissue culture plate. After culture for a predetermined
time, the cells were washed with PBS twice, with gentle
shaking of the plate to remove the serum in the well.
The cells were detached with 2 0 0 4 of 0.2% EDTA/
0.15% trypsin in PBS by incubating at 37°C for 3 min.
Then, 6 0 0 ~ 1DMEM/10% FCS was added to stop the
trypsization. The cells were pipetted out thoroughly and
the viability in suspension was measured by MTT
assay. The cell growth rate was compared with that
from PS tissue culture wells. Four samples were measured and the standard deviation was calculated.
RESULTS AND DISCUSSION
Polyurethane characterization
In this study, the hard segment denotes the MDI chain
extender region from the reaction of isocyanate and the
chain extender. The soft segment denotes the major
chain of PTMO. The calculated theoretical value of
hard segment, HBI, recrystallization exotherm temperature (T,) and glass transition temperature (T,) of
hard segment are shown in Table 2.
Hydrogen bonding index in polyurethanes. In this study,
the HBI was used to identify the interaction between
the hard segment of N-H and the C=O group in
forming hydrogen bonds in the polymer chain. As
shown in Table 2, HBI increases with increase in the
ratio of hard segment in PU. Compared with the PU of
lower hard segment ratio, the PU of higher hard
segment ratio has some intermolecules which exert a
TABLE 2. Hard segment weight fraction, T,, Toand
HBI in P T M O 1 OOO series PUS
Sample
Calculated hard
segment weight
fraction
HBI
T,("C)
T,("C)
110
21 1
41 3
615
817
1019
12111
0.227
0.392
0.575
0.673
0.734
0.776
0.806
1.03
1.54
2.14
2.52
2.93
3.42
3.98
-50
-25
-10
0
2
6
10
-
30
46
75
100
86
97
P . C. Lee et al.
422
stronger interaction. Therefore, a separation is developed between the hard and soft segments. In other
words, this higher ratio of ordered chains of hard segments will exhibit more phase separation.
Morphology of polyurethanes observed by T E M . To
observe the hard segment distribution in the structure,
the vapour-stained KuO, materials were examined by
TEM. The results are shown in Fig. 1. Since RuO, is a
good staining agent for benzene rings, the hard segments are observed in the black or grey region of the
TEM, while the soft segments are in the white domain.
Figure 1A shows the TEM picture of sample 110.
Because the weight fraction of hard segments is less in
this system, they are dispersed in the structure. The
stained area is increased with increase in value of the
hard segments. The 211, 413 and 615 polyurethanes
possess hard segments in the dispersed phase, but the
( A ) 110
(B)2I1
(IT) 817
817 polyurethane has a continuous phase of hard segments. The 817 material possesses a calculated hard
segment weight fraction of 0.73 and HBI of 2.93. Continuous phases of hard segments were also observed in
the 1019 and 12111 composites which possess of HBIs
3.42 and 3.98, respectively.
Mechanical properties of polyurethanes. The dynamic
mechanical spectra of flexural storage moduli ( E ) of
samples based on PTMO 1000 are shown in Fig. 2. It
can be seen that increasing the MDI content of the
polyurethane significantly increases E' through the
rubbery plateau region. Furthermore, the onset of the
rubbery plateau region is in a lower temperature region
for the lower ratios of MDI. These trends indicate that
the samples with lower MDI weight fractions have
higher mobility to rearrange the hard-segment chains in
the bulk at the physiological temperature of 37°C. This
assumption is reasonable, because our cell culture
system at 37°C is close to the annealing condition such
that molecular chains can be easily rearranged as the
MDI ratio is lowered. On the other hand, the polyurethanes with high hard segment ratios cannot move
freely, especially samples 615, 817, 1019 and 12 111. At
the cell culture incubation temperature, 37"C, only 110
and 211 have soft segments which move rather easily,
resulting in a surface suitable for the attachment and
proliferation of cells.
DSC was used to investigate the melting temperature
(T,) of crystallites in these polymers. The results are
shown in Fig. 3. The endotherms show that AH and
melting temperature increased as the hard segment
content increased. The 615, 817, 1019 and 12 111 polyurethanes have an obvious T, at about 200°C. Also
observed in Fig. 3 are the recrystallization exotherm
temperatures (K), which are also related to the weight
fraction of hard segments in the samples. In 211 polyurethane, the T, is 40°C which is near to the physiology
temperature. The 615 and polyurethanes with higher
10
(C) 413
Amplitude (P-ol-O.&O mm
(F) 1019
IIG ) I 2 1 I I
(D)615
Fig. 1. TEM pictures of PU samples: (A) 110; (B) 211; (C)
413;(D) 615;(E) 817;(F)1019;(G)12 111.
7 !
-200
-100
0
100
Temperature YC)
Fig. 2. Flexural storage moduli (E') versus temperature for
various MDI ratios.
POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996
Influence of hard segments of PU
423
of higher mobility of the molecular chains in the 211
component.
In vitro evaluation of cell growth on polyurethanes
-=a&
-0.5-
I
'
.
0 -1.0-
g
.
-1.5-
-z.s!
-150
.
,
.
.
-lw
,
-60
.
0
sb
100
Temperature ('c)
160
260
z
Fig. 3. DSC scans for various MDI ratios at a heating rate of
10°C min-
'.
hard segment ratio possess T, higher than 75°C. Since
there is a molecular weight distribution of the polymer
chains, the lower T, of materials with lower weight fractions of MDI is due to thermally induced recrystallization of hard segments for the shorter molecular
chains existing in the soft segments.
Surface tension of polyurethanes. The results of surface
tension measurements of specimens immersed in octane
for 24h at 37°C are shown in Fig. 4. Compared with
the control specimens (hydrophilic and hydrophobic
glasses), all the polyurethanes possess intermediate
behaviour between hydrophilic and hydrophobic,
implying the existence of both hydrophilic and hydrophobic molecules in the molecular structure. The 211
polyurethane possesses higher surface tension and contains more hydrophilic groups on the surface than other
polyurethanes. It is suggested that this is a consequence
Cytotoxicity of extract. Several methods have been
reported for detecting the cytotoxicity of materials. In
the present study, we used (1) extract dilution assay and
(2) direct contact assay on cell cultures to evaluate the
in uitro cytotoxicity and biocompatibility of materials.
In addition, we used MTT assay to determine the cell
viability in examining cell attachment and proliferation.
It is a rapid and reproducible colorimetric method,
which is based on the cleavage of a yellow tetrazolium
salt from purple formazan crystals by mitochondria1
enzymes of metabolically active cells. As shown in Fig.
5, no oytotoxicity was detected in the extracts of the
polyurethanes since the cell viability results were similar
to the control value of the tissue culture well. Therefore,
we can rule out extract toxicity in these materials and
conclude that the properties of the material surface have
a dominant influence on the experimental results.
Cell attachment and proliferation. To compare cellular
growth on these materials, the 3T3 fibroblast cell line
was directly seeded on the PU series. In Fig. 6, the
results show that all the materials can allow attachment
(i.e. seeding after 2 h) of the 3T3 fibroblast. It can also
be seen that the polyurethanes have less cell attachment
than the tissue culture well and hydrophilic glass.
Among the polyurethanes, the 211 and 413 have better
surfaces supporting cell attachment. Furthermore, cell
attachment decreases with increasing MDI ratio and
110 polyurethane, i.e. 12 111 PU has the least number of
cell attachments. The results of cell proliferation of the
PU series at 37°C after 72 h and 144h are shown in Fig.
40
0.40
I
35
6
-
h
30
a
5
I 1
25
-
E
1
0.25
o
v)
5
2 20
g
15
-m
I
10
rn
e
0.20
-1-1000
3
>
-2-1000
-4-1000
0.15
x
0 0.10
x
t
t
1
2
4
6
8
10
12
Hi
Hb
Molar ratio of MDI per PTMO 1000
Fig. 4. Surface tension of PTMO 1000 series PU, where Hi
denotes a hydrophilic glass surface and Hb denotes a hydrophobic glass surface.
POLYMER INTERNATIONAL VOL. 41, NO. 4, 1996
0.00
6-1000
-8-1000
- A - 10-1000
5
0
T.C. Well
-CE
c
5
0
CI
0.30
12-1000
'
24h
48h
72h
96h
120h
144h
Growth Time ( hours)
Fig. 5. Cell proliferation in extracts of PTMO 1OOO series PU
by MTT assay.
P . C. Lee et al.
424
0.035
0.030
0.025
0
b
m
T
0.020
0,
4
0.015
0
0.010
>
0
0.005
0.000
2
1
a
6
4
H.G. T.C.
well
12
10
Molar ratio of MDI per PTMO 1000
Fig. 6. Cell attachment of PTMO 1000 series PU after 2 h.
7. The molecular chains on the surface may achieve an
equilibrium state during a longer-term incubation at
37°C. After 144 h proliferation, the cells almost achieve
confluence on each of the PUS,except for 110 PU. With
a low ratio of hard segments, 110 PU has the worst cell
growth behaviour after 72 and 144h of cell proliferation, implying that the softest material may not be a
good supporting matrix for cell growth owing to the
lack of hard segments in the structure. It seems that
suitable hard segments are a necessary element for cell
growth. On the other hand, 211 PU has the best cell
growth among this polyurethane series. The same trend
in cell attachment and cell proliferation on the various
PUS is that increasing the MDI ratio causes a decrease
in cell numbers on the surface. Based on the results of
TEM and cell proliferation for this polyurethane series,
it appears that the cells can grow perfectly on the disperse phase of hard segments, but not on the continuous hard segment surface. However, the requirement
0.40
i
OlMN
0.35
E
0.30
E
0
[;;
c
m
0.25
Q,
0.20
>
0.15
-m3
6
0 0.10
0.05
0.00
Glass T.C.
C.E.
I
2
4
6
8
10
12
well
Molar ratio of MDI per PTMO 1000
Fig. 7. Cell proliferation of PTMO 1000 series PU after 12
and 144 h.
for cell growth is not only a continuous soft segment
domain, but also a ratio of hard/total segments, which
is between 0.4 and 0.6. The 211 and 413 PUS fall into
this range.
The general concept of enhanced interaction with
hydrophilic materials has been fostered by experiments,
demonstrating that cells usually attach much more
readily to glass than to hydrophobic surfaces such as
Teflon,’ siliconized glass,” polystyrene” or parafilm.” Although the importance of substrate interfacial
tension has often been cited in cell adhesion, the surface
tension values we have measured are only minimally
affected. The reason for this is that the measurement of
surface tension is an averaged value of the macrophase
on the surface, but cell attachment and proliferation is a
microphase factor of the specimen surface. Hence, the
microphase, observed by TEM, is a direct and significant factor.
In view of the above results it is reasonable to expect
that physical and chemical properties are equally
important in yielding a suitable environment for cell
growth. In this context, two factors are considered to be
crucial in determining the cell attachment and cell proliferation properties of the surface. The first factor is the
surface morphology, which has been demonstrated from
TEM, and the existence of an optimally dispersed hard
segment phase in the PUS. The second factor is the
hydrophilic property, which in principle can be induced
readily when the polymer chains have high mobility at
37°C. Hence the high weight fraction of hard segments
has a lower cell growth than 211 and 413 PUS.
CONCLUSION
In this study, the bulk properties of a series of hard
segment polyurethanes and cell interaction on these
materials were studied by DSC, DMA and TEM. The
results show that cell proliferation is affected by mechanical factors of the materials such as T,, T, and rigidity.
A lower Tg for the lower weight fraction of hard segments gives a higher mobility to the soft segments in the
molecular structure. With T, close to 37”C, the hard
segments of 21 1 and 413 PUS can rearrange the molecular surface to provide a stable morphology for cell
growth. The softest material, 110 PU, and the hardest
material, 12 111 PU, are not suitable for cell attachment
and proliferation. The results also indicate the absence
of a cytotoxicity response for extracts from the polyurethanes studied. Furthermore, there was not a direct
relation between surface tension and cell proliferation,
since surface tension is measured for the macrostructure
of the surface, while cell growth depends on the microphase structure of the surface, which can only be
detected by TEM. The domain of hard-soft segment
distribution is found to affect cell attachment or proliferation. The cells tended to grow on the optimally disPOLYMER INTERNATIONAL VOL. 41, NO. 4, 1996
InJIuence of hard segments of PU
persed hard segments of PUS, especially on the surface
with weight fraction of hard segments between 0.4 and
0.6. The corresponding HBI is between 1.5 and 2.1.
These PUS may possess suitable chemical structures
and physical properties for cell growth. The phaseinverse region is at the hard segment ratio 0.7 (817 PU);
however, a continuous phase of hard segments is not
suitable for cell attachment and proliferation.
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425
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