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Elsevier Editorial System(tm) for Acta Biomaterialia Manuscript Draft Manuscript Number: Title: Films of dextran-graft-polybutylmethacrylate to enhance endothelialization of materials Article Type: Full Length Article Keywords: Polybutylmethacrylate; Dextran; Copolymer films; Cell proliferation; Endothelial cell migration; Tissue Engineering Corresponding Author: Dr Didier Letourneur, Corresponding Author's Institution: Inserm First Author: Sidi Mohamed Derkaoui, PhD Order of Authors: Sidi Mohamed Derkaoui, PhD; thierry Avramoglou, PhD; Virginie Gueguen, PhD; Christel Barbaud, PhD; Didier Letourneur, PhD Abstract: We have synthesized new structures obtained from amphiphilic copolymers of dextran and polybutylmethacrylate with the aim of endothelialization of biomaterials. Grafting of butylmethacrylate onto dextran has been carried out using ceric ammonium nitrate as initiator. Three copolymers were obtained (11, 30 and 37 wt% dextran) and homogeneous thin films were successfully prepared. In contrast to dextran, the resulting films were stable in water, and copolymers characterized by FTIR, DSC and DMA showed evidence of hybrid properties between the parent homopolymers. Surfaces of films were smooth when analyzed by AFM (roughness 2 +/-1 nm) but greatly differed in their hydrophilicity by increasing the dextran content (water contact angle from 99° to 57°). In contrast to polybutylmethacrylate where the proliferation of vascular smooth muscle cells (VSMCs) was excellent but very low for endothelial cells, the copolymer containing 11% of dextran was excellent for endothelial cells and very limited for VSMCs. An in vitro wound assay demonstrated that copolymer with 11% dextran is even more favorable for EC migration than tissue-culture polystyrene. Increasing the dextran content in the copolymers decreased the proliferation for both vascular cell types. Altogether, these results evidenced that transparent and water-insoluble films made from copolymers of dextran and butylmethacrylate copolymers with an appropriate composition could enhance endothelial cell proliferation and migration. Therefore, a potential benefit of this approach is the availability of surfaces with tunable properties for endothelialization of materials.
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Films of dextran-graft-polybutylmethacrylate to enhance endothelialization of materials
Sidi Mohamed Derkaoui, Thierry Avramoglou, Virgine Gueguen, Christel Barbaud, and
Didier Letourneur*
Inserm, U698, Bio-ingénierie de Polymères Cardiovasculaires
Institut Galilée, Université Paris 13, 99 Av. J.B. Clément, 93430 Villetaneuse (France)
* Corresponding author :
Inserm U698, BPC; Institut Galilée, Université Paris 13, 99 Av. J.B. Clément, 93430
Villetaneuse, France
Tel/Fax : +33 1 4940 3008
Email : [email protected]
Text only
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ABSTRACT
We have synthesized new structures obtained from amphiphilic copolymers of dextran and
polybutylmethacrylate with the aim of endothelialization of biomaterials. Grafting of
butylmethacrylate onto dextran has been carried out using ceric ammonium nitrate as initiator.
Three copolymers were obtained (11, 30 and 37 wt% dextran) and homogeneous thin films
were successfully prepared. In contrast to dextran, the resulting films were stable in water,
and copolymers characterized by FTIR, DSC and DMA showed evidence of hybrid properties
between the parent homopolymers. Surfaces of films were smooth when analyzed by AFM
(roughness 2 +/-1 nm) but greatly differed in their hydrophilicity by increasing the dextran
content (water contact angle from 99° to 57°). In contrast to polybutylmethacrylate where the
proliferation of vascular smooth muscle cells (VSMCs) was excellent but very low for
endothelial cells, the copolymer containing 11% of dextran was excellent for endothelial cells
and very limited for VSMCs. An in vitro wound assay demonstrated that copolymer with 11%
dextran is even more favorable for EC migration than tissue-culture polystyrene. Increasing
the dextran content in the copolymers decreased the proliferation for both vascular cell types.
Altogether, these results evidenced that transparent and water-insoluble films made from
copolymers of dextran and butylmethacrylate copolymers with an appropriate composition
could enhance endothelial cell proliferation and migration. Therefore, a potential benefit of
this approach is the availability of surfaces with tunable properties for endothelialization of
materials.
KEYWORDS : Polybutylmethacrylate; Dextran; Copolymer films; Cell proliferation;
Endothelial cell migration; Tissue Engineering
3
1. Introduction
The development of amphiphilic copolymers based on copolymerization of hydrophilic
and hydrophobic parts is of great importance due to their versatility and potential applications
in regenerative medicine. [1-5]
For instance, methacrylic polymers have been widely studied as
a biocompatible matrix for use in tissue engineering.[6-11]
Some other synthetic polymers
showed physicochemical and mechanical properties comparable to those of biological tissues,
but were not sufficiently biocompatible. In this context, natural biological polymers such as
polysaccharides possess good biocompatibility,[12-15]
but their mechanical properties are often
inadequate. Improvements of the characteristics of synthetic polymers could be attained by
the addition of biological components in the structures. The resulting materials could combine
the appropriate mechanical properties of the synthetic polymer with the biocompatibility of
the natural polymers. The main challenge in achieving this combination relates to the
thermodynamic incompatibility of natural and synthetic polymers that is the result of enthalpy
and entropy barriers caused by the differences in hydrophilicity/hydrophobicity. This
incompatibility is a frequent phenomenon observed for many polymer mixtures and results in
phase separation.[16,17]
In order to overcome this problem, we and others have already
investigated graft copolymerization of a synthetic polymer, polymethylmethacrylate, onto the
natural dextran macromolecule using ceric ammonium nitrate as a redox initiator.[18-22]
This
strategy consisted on the creation of free-radical grafting sites on the dextran backbone by
reaction with cerium IV, the radicals so formed, initiated copolymerization with
methylmethacrylate monomers. We described here new compounds obtained from graft
polymerization of dextran with butylmethacrylate. The materials were prepared as a series of
films, differing in their wettability by varying the concentration of dextran, and we studied
their behaviors with vascular cells[23, 24]
. We found that films with a peculiar composition
4
were able to greatly enhance migration and proliferation of endothelial cells, but not vascular
smooth muscle cells.
2. Materials and methods
2.1 Materials
Dextran 70000 g/mol obtained from SIGMA, was dried under vacuum at 60°C for 24h.
Butylmethacrylate monomers were obtained from Acros, and were purified by washing with
NaOH 5%, NaCl 20%, followed three times by distilled water. Ce(IV) ammonium nitrate and
nitric acid were obtained from Acros.
2.2 Graft copolymerization
Three types of copolymers were synthesized by varying the amount of dextran. The
general experimental procedure was as follow: the reactions were carried out into 1L five-
neck flask equipped with stirrer and condenser then the flask was immersed into thermostated
oil bath at 40°C and purged by nitrogen gas. Three different mass ratios of dextran to BMA
(0.125/2; 0.5/2; 0.75/2 w/w) were used. Dextran was dissolved in 500mL of nitric acid 0.2M
for 10min. Five mL of solution of Ce(IV) dissolved in HNO3 0.2M (2.4 10-4
mol/L) and 2g of
monomers were simultaneously added. The system was continuously stirred for 50 min and
the solution was neutralized to pH 8 by addition of NaOH aqueous solution (10M) and
concentrated with an evaporator. Methanol was used as a non-solvent to precipitate the
polymeric material, that was washed with 50mL EDTA 10-2
M to remove cerium ions. The
three purified copolymers named DB1, DB2 and DB3 were frozen at -20°C and lyophilized
for 24h. To obtain pure graft copolymers, the resulting products were extracted with acetone
in a soxhlet for 24h. Pure copolymers were dried under vacuum and weighted.
2.3 Preparation of films
Preparation of copolymers films was carried out by dissolving 6% (w/v) of copolymers in
mixture THF/H2O 90/10 (v/v). 500µL of the transparent solution obtained was molded in
5
Petri dishes (15mm in diameter) for 24h at room temperature in saturated atmosphere of THF
and in presence of CaCl2 to absorb water. After solvent evaporation, films were released from
the mold upon addition of distilled water, while in the case of PBMA (very fragile), the films
were soaked in distilled water for 2h and then carefully removed from the Petri dishes. The
samples were air dried for 24h to remove the solvent and obtained films are 35µm in
thickness. The films were rinsed several times with PBS. Films of copolymers were cut for
mechanical tests in the shape of 15mm x 5mm, but PBMA film was not prepared because it
was very brittle.
2.4 Infra-red spectral analysis
The infra-red spectra of copolymers films (DB1, DB2 and DB3) were obtained using a
Perkin-Elmer 1600 spectrometer. Each film was mounted on the sample holder. The
atmosphere spectrum was subtracted as background for each scan. Each spectrum was
obtained at a resolution of 1 cm-1
using 32 scans. The studied samples were not gaining
weight by moisture uptake during the acquisition, and similar data were obtained after one
hour. Omnic software was used for data acquisition and analysis. Fourier transform infra-red
(FT-IR) spectroscopy was also used to semi-quantitatively estimate the percentage of dextran
in the copolymers using physical mixtures of dextran and PBMA at different ratios. A
quantity of 15 mg of fine KBr powder was mixed with 1.5mg of physical blend of
dextran/PBMA weight ratio (5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65 and 40/60). We
used the integrated area of (O-H) band at 3400cm-1
with different concentrations of dextran,
PBMA and each synthesized copolymer.
2.5 Differential scanning calorimetric analysis (DSC)
Thermal behavior of homopolymers (dextran and PBMA) and all copolymers (DB1, DB2
and DB3) were performed with a Setaram DSC131 thermal analyzer, operating in
combination with the liquid nitrogen cooling accessory. A mixture of dextran + PBMA (50%,
50% w/w) was also tested. The furnaces were purged with nitrogen and samples were passed
6
in temperature range from -50 to +200°C at heating rate of 10°C/min. In each case, two
consecutive scans were carried out on each sample. The glass transition temperature (Tg) of
the products was taken as the midpoint in the shift of heat flow baseline.
2.6 Dynamic-mechanical analysis (DMA) on copolymer films
DMA test was used to analyze loss modulus, storage modulus and tan of all films of
copolymers (DB1, DB2 and DB3). PBMA was not tested because it was rigid and very fragile
at room temperature. Films of 15 x 5 x 0.035 mm were analyzed on dynamic-mechanical
analyzer, DMA Q800 (TA Instruments). The equipment has operated in a single cantilever
bending mode at 1Hz in frequency, amplitude 5 m and a heating rate of 3°C/min from -100
to +200°C.
2.7 Creep test
To determine the deformation of DB copolymers, a creep test at ambient temperature was
carried out by applying a constant load to DB1, DB2 and DB3 and observing the deformation
with time. Several loads (100g; 125g; 150g, 175g and 200g) were suspended to the extremity
of films with 15mm length, 5mm width and 0.035mm thickness. The expansion of films was
measured from pictures every minute until rupture. Results are obtained as creep curves
according to the following equation: “ε = a[1 − exp(−bt)] + ct”, where (ε) is the distortion and
(t) is the time. Curves were drawn for the percentage of expansion versus time, and initial
deformation velocity obtained with V0 = (d /dt) t 0 = ab + c, is then deduced for all
samples.
2.8 Contact angle measurements
The water contact angles of films were measured at room temperature using a contact
angle system (Krüss, model DSA 10- Mk2). A droplet of deionized water (2µL) was put on
the air-side surface of the films and after 10s the contact angle was measured. The process
was monitored by a video contact angle system. Ten measurements were carried out for each
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simple (PBMA, DB1, DB2 and DB3; dextran gave any film) and the obtained values were
averaged.
2.9 Topography and roughness (AFM)
Atomic force microscopy (AFM) was performed to determine the surface roughness and
the uniformity of coating. All films were imaged with a Nanoscope III Digital instruments
dimension 3100 atomic force microscope (Veeco Metrology Group) and a resonance
frequency range 50-100Hz. Three areas per sample were imaged under Tapping contact mode
and analyzed with WSxM 3.0 beta 10.0 to obtain roughness (Ra) and peak heights of each
film.
2.10 Cell adhesion and proliferation on copolymer films
Endothelial cells (human umbilical vein endothelial cells -HUVEC from ATCC) and
smooth muscle cells (aortic vascular smooth muscle cells -VSMCs, line Rb1[23, 24]
), were used
in this study. The films (15 mm in diameter) were placed on the bottom of 24-well plates, and
the two surfaces were submitted to UV at 254nm for 15min. The ECs and VSMCs cells were
seeded on each film at 5x103 cell/cm
2. Cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM, Gibco) with 4.5 g/L of glucose and 2% L-glutamine supplemented with
10% calf serum and 1% of penicillin and streptomycin. The cells were incubated for 5 days at
37°C in air containing 5% CO2 and in a humidified incubator. The transparency of all films
allowed seeing the cell morphology with a classical inverted phase contrast light microscope
(Zeiss, Axiovert 100). The kinetics of proliferation has been determined daily for 5 days after
starting cell culture. The cell layer adhering to the each disc was washed twice with PBS 1x to
remove remaining materials and loose cells and the cells were detached with trypsin/EDTA
0.25/0.02 (w/v), resuspended and counted with a Coulter (Coulter Counter ZM). The mean
cell number from two wells per tested material was counted. Results are averaged from two
separate experiments. Cell adhesion and proliferation was also observed by fluorescence
microscopy, with cells cultured on films of copolymers for 5 days. Cells were fixed with 10%
8
paraformaldehyde, permeabilized with 0.1% triton X100, and labeled with intracellular F-
actin (phalloïdin FluoProbes547) for cytoskeleton and DAPI for nucleus.
2.11 Endothelial cell migration
In vitro migration assay of ECs was performed as described.[23]
Briefly, ECs at 5x103
cell/cm2
were seeded into 24-well plates, where DB1 film has been placed on the bottom of
wells. Equivalent numbers of cells were also seeded directly into in 24-wells of tissue culture
treated polystyrene surface (TCPS) as a reference surface. After 5 days, EC monolayers were
scraped (denuded) using a 10µL plastic micropipette tip and washed by sterile phosphate
buffered saline solution (PBS, 0.01M, pH = 7.4), afterwards PBS was replaced with DMEM.
Area of migration was observed with optical microscopy and measured at 8, 24, 32, and 48
hours after taken pictures by a digital camera (Pentax istD).
3. Results and discussion
Dextran-graft-polybutylmethacrylate copolymers were synthesized in this work. A redox
initiation was performed with an acidified aqueous solution (pH= 0.32). The decrease in pH
of the reaction mixture, as well as increase of optical density (appearance of tiny white
particles shortly after the cerium and monomer addition) indicated initiation of
copolymerization (Fig. 1). In contrast, this reaction was not obtained in the absence of either
dextran or butylmethacrylate monomer, indicating that the reaction was not the formation of
homopolymers. The resulting copolymers were then purified by extensive extraction with
acetone in a soxhlet to eliminate butylmethacrylate monomer. Three copolymers (DB1, DB2,
DB3) were successfully synthesized by increasing the dextran content in the starting solution.
Interestingly, the resulting copolymers were not soluble in any pure solvents of the parent
homopolymers (water, DMSO, Dichloromethane, Acetone, Chloroform, Dioxane, THF), but
only in a mixture 90/10 of THF/water.
9
The absence of a common solvent between copolymers and the parent homopolymers, did
not allow NMR to be performed. However for semi-quantitative analysis, the comparison of
FT-IR spectra of PBMA and dextran showed for the three copolymers the presence of typical
absorption band of PBMA at 1730 cm−1
corresponding to C=O stretching vibrations, and
characteristic band for dextran corresponding to OH stretching absorption at 3400 cm-1
. The
intensity of O-H band increased with increasing proportion of dextran in copolymers. Semi-
quantitative analysis of the integrated area of the 3400 cm−1
peak as previously described 25
,
gave a calibration curve using physical mixtures of powders made of dextran and PBMA in
known proportions, and then the content of dextran in the copolymers : 11, 30 and 37 weight
% in dextran for DB1, DB2 and DB3, respectively.
The DSC curves of physical mixtures dextran + PBMA evidenced two separate Tg, close
to the Tg of the single homopolymers (28°C for pure PBMA, and 153°C for pure dextran).
Each DB copolymer showed a single glass transition, with an intermediate value compared to
the parent homopolymers (Fig. 2). Tg value of DB copolymers increased with increasing
dextran content (31°C, 38°C, 42°C for DB1, DB2 and DB3, respectively). These values
greatly differed from copolymers when methylmethacrylate was used instead of
butylmethacrylate.22
Thus, the existence of a single glass transition of the copolymers
confirmed the grafting of BMA onto dextran in copolymers.
Copolymers films were then obtained by dissolving copolymers in THF/H2O 90/10 (v/v),
and after controlled solvent evaporation, thin films were obtained. The films from copolymers
were homogenous, transparent and insoluble in water (in contrast to dextran). The dynamic-
mechanical analysis (DMA) gave three parameters: (a) the storage modulus (E’), (b) the loss
modulus (E”), and (c) Tan which is the ratio (E”/E’), for determining the occurrence of
molecular mobility transition. The data revealed a storage modulus E’ of 480 MPa for DB1,
680 MPa for DB2 and 1010 MPa for DB3 at 37 °C, that increased with increasing amount of
dextran. In all cases, E’ decreased with increasing temperature. The elastic modulus, i.e. the
10
stiffness, decreased with increasing amount of dextran in copolymers. On the other hand,
temperature dependence of the Tan showed a maximum peak for DB1 (57°C), DB2 (60°C)
and DB3 (64°C). These values were thus much lower than for pure polymethylmethacrylate
(109°C) or dextran-graft-polymethylmethacrylate (141°C).22
Similar to DSC results, each film
exhibited one glass transition relaxation confirming the graft copolymerization and the
detection of a single peak in Tan is a sufficient criterion to assume the miscibility.
Differences in values with DSC could be attributed to the presence of water tightly associated
to copolymers, which induced changes in mobility of macromolecules.
Creep test was also carried out in order to study differences between DB1, DB2 and DB3
copolymers. Elongation of copolymers under load is high (Fig. 3 left part), with values
reaching 380 %. As shown in figure 3, deformation velocity increases with strain. The
elasticity of films decreases with the increase of dextran in DB copolymer (Fig. 3 right part).
Hydrophilicity of materials was evaluated by water contact angle ( ) measurement, an
indication of surface wettability. PBMA exhibited the highest contact angle ( = 99°) and was
the most hydrophobic material. DB1 ( = 80°) and DB2 ( = 73°) film had moderate level of
wettability, whereas DB3 ( = 57°) film showed the lowest contact angle. Contact angle on
copolymer films exhibited a progressive decrease with increasing dextran content, thus
indicating increased hydrophilicity. All films were further characterized by Atomic Force
Microscope in tapping mode imaging to obtain information on the surface morphology. DB
films exhibited smooth surface, with no notable trends in topographical features upon
increasing of dextran content (Fig. 4). The values for roughness of films were similar (PBMA
Ra = 1.9 ; DB1 Ra =1.6 ; DB2 Ra = 2 ; DB3 Ra = 2.7). Roughness observed on these surfaces
is low, and suitable for comparison between samples for cell adhesion, and potential physical
immobilization of biomolecules.26
11
The copolymers were tested in vitro by seeding ECs and VSMCs on films. After 6 h of
culture, analysis of the VSMC morphology on PBMA film showed clear adhesion with cells
that changed from spherical to fusiform shape and spreaded out of its pseudopodia, but no
adhesion of ECs on hydrophobic PBMA. After 5 days, VSMCs attained confluence on PBMA
(never with ECs), and VSMC proliferation and morphology were comparable to the control
cultures on TCPS (Fig. 5).
VSMCs did not proliferate on DB1 (Fig. 5). Interestingly, analysis of endothelial cell
numbers indicated that on copolymer film DB1 (dextran 11%), ECs proliferated very well and
reached confluence after 5 days (Fig. 6). This marked difference for DB1 film (Fig. 7) was
somehow unexpected, but is likely to be related to a peculiar surface tension of the film and
its subsequent adhesion of serum proteins that could differentially interact with cell receptors
involved in cell adhesion. A detailed study of protein adsorption should be carried out in the
future. When the dextran content in copolymers increases to 30% or 37%, the number of
spreading cells (ECs and VSMCs) decreased considerably, and proliferation for both types of
cells was very limited (Fig. 5-7). This result indicated that EC proliferation is sensitive to the
hydrophobic/hydrophilic properties of films, the chemical components being the same
between the copolymers. In this context, many studies have proved that surface characteristics
of biomaterials such as wettability, greatly affect the cell adhesion and growth on the
materials27,28
with a preferential adsorption of some serum proteins like fibronectin and
vitronectin. In the work of Risbud et al., PMMA substrates were exposed to radio-frequency
argon and nitrogen plasmas to decrease the water contact angle.29
They found that the
untreated PMMA surface did not support endothelial growth and PMMA treated by plasma
was better but exhibited growth rates lower than the TCPS. The chemical approach reported
here by graft polymerization allowed to obtain tunable properties for excellent endothelial cell
coverage of DB1. Surface roughness is another important parameter in biomedical materials,
that may affect cell adhesion30,31
and can compromise hemocompatibility in the case of
12
vascular prostheses32
. According to the AFM results, the roughness was similar among the
different films. Consequently, the proportion of dextran in the copolymer that greatly affected
the hydrophilicity, seems the main parameter to obtain the favorable endothelialization of the
surface of these materials.
The migration assay, created from a wound in a confluent EC monolayer, was used to
assess the effect of DB1 on EC migration compared to TCPS (the other polymer surfaces did
not allowed a confluent EC monolayer). Cell migration was measured as the denuded area in
which ECs invading. The cells on copolymer had extensively migrated from the edge of the
scrape into the denuded space (Fig. 8 top, left panel). They reach confluence at 32h on
copolymer. In contrast, EC migration on TCPS was slower (Fig. 8, right panel), and migration
area remained non-confluent until 48h. This evidenced that surface of DB1 film was
appropriate for both EC migration and proliferation.
4. Conclusion
The aim of this study was to synthesize new copolymers having the biological properties of
polysaccharides and the mechanical properties of polyacrylate. Copolymers of dextran and
butylmethacrylate were successfully synthesized by radical polymerization and characterized.
IR, DSC and DMA revealed that the graft copolymerization has been taken place with the two
different natures and non-miscible polymers. In contrast to dextran, homogenous transparent
and water-insoluble films were prepared from all DB copolymers. The mechanical properties
were enhanced as compared to our previous report with dextran-graft-
polymethylmethacrylate.22
Interestingly, DB1 was able to inhibit VSMC proliferation, and at
the same time to support endothelial cell proliferation and migration. Lastly, cell adhesion and
proliferation can be controlled by varying the content of dextran incorporated into the DB
copolymer. DB1 copolymer exhibited excellent characteristics for applications for
13
endothelialization of materials. Therefore, a potential benefit of this approach is the
availability of bioactive surfaces with tunable properties of interest for biomedical materials.
Acknowledgments
This work was supported by Inserm, University Paris 13, and IFR Paris Nord-Plaine de
France. The authors wish to express their gratitude to Dr. Cédric Lorthioir (CNRS Thiais,
France) for his excellent technical assistance in DMA tests.
14
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16
Legends for Figures
Figure 1. Variation of pH (▲) and optical density (■) during copolymerization. Dextran-
graft-polybutylmethacrylate copolymers were synthesized via redox initiation. The decrease
in pH (left), as well as increase of optical density at 650 nm (right) was plotted as a function
of time. No change was observed in the absence of dextran or butylmethacrylate monomer.
Figure 2. DSC curves showing the glass transition temperature (Tg). Curves for (a) dextran,
(b) PBMA, (c) mixture of dextran + PBMA (50% + 50%), (d) DB1, (e) DB2 and (f) DB3 are
reported in temperature range from -50 to +200°C at heating rate of 10°C/min. In each case,
two consecutive scans were carried out on each sample. The Tg was taken as the midpoint in
the shift of heat flow baseline.
Figure 3. Left) Representative example of elongation of copolymer DB1; Right) Comparison
of initial deformation velocity of DB1 (♦), DB2 (■) and DB3 (▲). A constant load (100g;
125g; 150g, 175g and 200g) was applied to DB1, DB2 and DB3 films (15mm length, 5mm
width and 0.035mm thickness). Elongation of films was measured from pictures every minute
until rupture. Curves were obtained for the percentage of expansion versus the time, and
initial deformation velocity is then deduced for all copolymers. PBMA was too fragile to be
characterized, and dextran gave any film.
Figure 4. Atomic Force Microscopy of PBMA (top left), copolymers DB1 (top right), DB2
(bottom left) and DB3 (bottom right). Scale X, Y = 2 µm/div. All films were imaged under
tapping contact mode with a resonance frequency range of 50-100Hz. Three areas per sample
were imaged and analyzed to obtain roughness (Ra) of each film.
Figure 5. Effect of film copolymers on endothelial cell proliferation. ECs were cultured on
films of PBMA or copolymers DB1, DB2, DB3. Top panel : Pictures were taken after 5 days
in culture (Original magnification 12.5 x). Bottom panel : Endothelial cell growth curve for
PBMA and copolymers DB1, DB2, DB3. Values are means of duplicate wells in two
experiments.
Figure 6. Effect of film copolymers on vascular smooth muscle cell proliferation in vitro.
VSMCs were cultured on films of PBMA or copolymers DB1, DB2, DB3. Top panel :
17
Pictures were taken after 5 days in culture (Original magnification 12.5 x). Bottom panel :
Cell growth curve for PBMA and copolymers DB1, DB2, DB3. Values are means of duplicate
wells in two experiments.
Figure 7. Effect of DB1 film on endothelial cell (left), or vascular smooth muscle cell
proliferation in vitro (right). Cells were cultured on films of copolymer DB1 for 5 days. Cells
were fixed, permeabilized, and labeled with intracellular F-actin (phalloïdin FluoProbes547 in
red) for cytoskeleton and DAPI (in blue) for nucleus. Cells were observed under a fluorescent
microscope. Pictures are representative of three experiments.
Figure 8. EC migration of a denuded EC monolayer on DB1 film (top, left panel) or on TCPS
(top, right panel). Bar is 100µm for each picture. Bottom panel : Quantitative analysis of EC
migration versus time. Area of migration was observed with optical microscopy and measured
at 8, 24, 32, and 48 hours. Values are representative data obtained in two independent
experiments.
Figure 1. Variation of pH (▲) and optical density (■) during copolymerization. Dextran-
graft-polybutylmethacrylate copolymers were synthesized via redox initiation. The decrease
in pH (left), as well as increase of optical density at 650 nm (right) was plotted as a function
of time. No change was observed in the absence of dextran or butylmethacrylate monomer.
Figure(s)
Figure 2. DSC curves showing the glass transition temperature (Tg). Curves for (a) dextran,
(b) PBMA, (c) mixture of dextran + PBMA (50% + 50%), (d) DB1, (e) DB2 and (f) DB3 are
reported in temperature range from -50 to +200°C at heating rate of 10°C/min. In each case,
two consecutive scans were carried out on each sample. The Tg was taken as the midpoint in
the shift of heat flow baseline.
Figure 3. Left) Representative example of elongation of copolymer DB1; Right) Comparison
of initial deformation velocity of DB1 (♦), DB2 (■) and DB3 (▲). A constant load (100g;
125g; 150g, 175g and 200g) was applied to DB1, DB2 and DB3 films (15mm length, 5mm
width and 0.035mm thickness). Elongation of films was measured from pictures every minute
until rupture. Curves were obtained for the percentage of expansion versus the time, and
initial deformation velocity is then deduced for all copolymers. PBMA was too fragile to be
characterized, and dextran gave any film.
Figure 4. Atomic Force Microscopy of PBMA (top left), copolymers DB1 (top right), DB2
(bottom left) and DB3 (bottom right). Scale X, Y = 2 µm/div. All films were imaged under
tapping contact mode with a resonance frequency range of 50-100Hz. Three areas per sample
were imaged and analyzed to obtain roughness (Ra) of each film.
Figure 5. Effect of film copolymers on endothelial cell proliferation. ECs were cultured on
films of PBMA or copolymers DB1, DB2, DB3. Top panel : Pictures were taken after 5 days
in culture (Original magnification 12.5 x). Bottom panel : Endothelial cell growth curve for
PBMA and copolymers DB1, DB2, DB3. Values are means of duplicate wells in two
experiments.
Figure 6. Effect of film copolymers on vascular smooth muscle cell proliferation in vitro.
VSMCs were cultured on films of PBMA or copolymers DB1, DB2, DB3. Top panel :
Pictures were taken after 5 days in culture (Original magnification 12.5 x). Bottom panel :
Cell growth curve for PBMA and copolymers DB1, DB2, DB3. Values are means of duplicate
wells in two experiments.
Figure 7. Effect of DB1 film on endothelial cell (left), or vascular smooth muscle cell
proliferation in vitro (right). Cells were cultured on films of copolymer DB1 for 5 days. Cells
were fixed, permeabilized, and labeled with intracellular F-actin (phalloïdin FluoProbes547 in
red) for cytoskeleton and DAPI (in blue) for nucleus. Cells were observed under a fluorescent
microscope. Pictures are representative of three experiments.
Figure 8. EC migration of a denuded EC monolayer on DB1 film (top, left panel) or on TCPS
(top, right panel). Bar is 100µm for each picture. Bottom panel : Quantitative analysis of EC
migration versus time. Area of migration was observed with optical microscopy and measured
at 8, 24, 32, and 48 hours. Values are representative data obtained in two independent
experiments.
