DOI: 10.5301/IJAO.2011.6398
Int J Artif Organs 2011 ; 34 ( 2): 198- 209
ORIGINAL ARTICLE
Polyurethanes as supports for human retinal pigment
epithelium cell growth
Gisele R. da Silva1, Armando da S. C. Junior2, Juliana B. Saliba2, Marianne Berdugo3, Brigitte T.
Goldenberg3, Marie C. Naud3, Eliane Ayres4, Rodrigo L. Oréfice4, Francine B. Cohen3,5,6
School of Pharmacy, Federal University of São João Del Rei, Chanadour, Divinópolis - Brazil
School of Pharmacy, Federal University of Minas Gerais, Belo Horizonte - Brazil
3
INSERM UMR872, Physiopathology of Ocular Diseases: Therapeutic Innovations, Institut des Cordeliers, Paris - France
4
Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais, Belo Horizonte - Brazil
5
Fondation Ophtalmologique Adolphe de Rothschild, Paris - France
6
Université René Descartes, Hotel Dieu University Hospital, Paris - France
1
2
ABSTRACT
Purpose: The transplant of retinal pigment epithelium (RPE) cells on supports may well be an effective therapeutic approach to improve the visual results of patients with age-related macular degeneration. In this
study, two biodegradable polyurethanes were investigated as supports for human RPE cells (ARPE-19).
Methods: Polyurethane aqueous dispersions based on poly(caprolactone) and/or poly(ethylene glycol)
as soft segments, and isophorone diisocyanate and hydrazine as hard segments were prepared. Polyurethane films were produced by casting the dispersions and allowing them to dry at room temperature
for one week. The ARPE-19 cells were seeded onto the polyurethane films and they were investigated
as supports for in vitro adhesion, proliferation, and uniform distribution of differentiated ARPE-19 cells.
Additionally, the in vivo ocular biocompatibility of the polyurethane films was evaluated.
Results: The RPE adhered to and proliferated onto the polyurethane supports, thus establishing cellPUD surface interactions. Upon confluence, the cells formed an organized monolayer, exhibited a
polygonal appearance, and displayed actin filaments which ran along the upper cytoplasm. At 15 days
of seeding, the occluding expression was confirmed between adjacent cells, representing the barrier
functionality of epithelial cells on polymeric surfaces and the establishment of cell-cell interactions.
Results from the in vivo study indicated that polyurethanes exhibited a high degree of short-term intraocular biocompatibility.
Conclusions: Biodegradable polyurethane films display the proper mechanical properties for an easy
transscleral-driven subretinal implantation and can be considered as biocompatible supports for a
functional ARPE-19 monolayer.
Key words: Retinal pigment epithelium (RPE), Biodegradable polyurethane, Polyurethane aqueous
dispersion, In vivo short-term biocompatibility, Age-related macular degeneration, RPE graft
Accepted: December 2, 2010
INTRODUCTION
The retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that lies in close association with the neurosensory retina (1). The apical membrane of the RPE faces the
198
photoreceptor’s outer segments, whereas the basolateral
membrane faces Bruch’s membrane, which separates the
RPE from fenestrated endothelium of the choriocapillaris
(2). RPE cells are connected to each other by four types
of junctions situated at the lateral membrane of the cells:
© 2011 Wichtig Editore - ISSN 0391-3988
da Silva et al
tight junctions, adherent junctions, desmosomes, and gap
junctions. RPE tight junctions are part of blood-eye barrier,
as they regulate the diffusion of blood components to the
retina (3)(4).
The RPE cells have crucial multiple functions as they
aid the absorption of scattering light that enters the eye
via the pupil (2), constitute the outer retinal barrier, and
recycle the visual pigments through rod outer segment
specific phagocytosis (5). RPE cells also control the
delivery of nutrients to the photoreceptors as well as
the bidirectional transport of metabolic products, ions,
and water between the subretinal space and blood (6).
Furthermore, RPE cells secrete the vascular endothelial
growth factor (VEGF), a multifunctional cytokine strongly
implicated in angiogenesis (7), and the pigment epithelial derived factor (PEDF), a potent inhibitor of angiogenesis (8). The extracellular matrix, which can have an
antiangiogenic function, is also secreted by RPE. This
potential for modulation of the extracellular milieu and
modulation of disease processes means that RPE can
aid in restoring the normal anatomy and support function for photoreceptor needs (9).
A failure of any one of these different RPE functions can
lead to the degeneration of the retina, the loss of visual
functions, and blindness. The most widespread disease
associated with RPE dysfunction is age-related macular
degeneration (ARMD) (3). The ARMD is classified into
two subgroups: atrophic (dry form) and exudative (wet
form). The dry form is characterized by a progressive
degeneration of RPE and photoreceptors. The exudative
form is linked to choroidal neovascularization directed
toward the subretinal macular region, with subsequent
bleeding and/or fluid leakage, which may result in a sudden loss of central vision. In the two types of ARMD,
the presence of hypo- and/or hyperpigmentation of the
RPE (10) is common. ARMD most commonly affects patients of over 60 years of age in the Western world (11).
It has led to blindness and visual disability in 14.4% of
those between 55 and 64 years of age, in 19.4% of those between 65 and 74 years of age, and in 36.8% of
those over 75 years of age. This prevalence is expected
to increase in the West as life expectancy continues to
increase (12).
One therapeutic strategy to curb visual deterioration in
ARMD patients is RPE transplants. Many studies have
been carried out to identify a substrate to support and
maintain an organized monolayer of healthy RPE cells (13),
which can subsequently be transplanted. The healthy transplanted RPE cells can repopulate the Bruch’s membrane,
as they are protected by support from the diseased tissue.
It has been suggested that a diseased Bruch’s membrane prevents cells growth and limits the attachment of RPE
transplants (14, 15).
Biological materials have been applied experimentally as
supports for RPE growth, including collagen films (16), Descemet’s membrane (17), the anterior lens capsule (18), the
amniotic membrane (19); synthetic biostable polymer films,
such as hydrogels (20), commercially available polyurethanes (Pellethane®, Tecoflex®, Zytar®) (21), polydimethylsiloxane (13); as well as synthetic temporary polymers, such
as poly-L-lactic (PLLA) and poly-DL-lactic-co-glycolic acid
(PLGA) (3, 22-24). These natural and synthetic materials
are suitable for the adhesion and proliferation of RPE cells and have the potential of delivering RPE cells, but they
have not been tried at clinical levels.
Biodegradable polyurethanes, derived from poly (caprolactone) and poly(ethylene glycol), as a soft segment, and
isophorone diisocyanate and hydrazine, as hard segments,
have recently been developed by producing a water dispersion of the polymers, followed by a drying step (25).
This procedure of synthesis avoids the use of organic and
toxic solvents usually employed in the formation of PLGA
and PLA films. The biodegradation of the polyurethanes
was characterized by hydrolysis of the ester bonds of the
poly(caprolactone) incorporated into soft segments (26).
These soft thermoplastic polyurethanes displayed high levels of elasticity and resistance, a controllable degradation
rate, and good in vivo biocompatibility in the subcutaneous
tissue (27, 28). Furthermore, they can be manufactured with
a controllable thickness. There are advantages in using thin
but semi-rigid films as RPE supports, including the safety
of the surgical procedure, the prevention of distortion of
the retina adjacent to the implant, a greater potential for reestablishing the RPE-Bruch’s membrane interaction, and a
greater diffusion of nutrients (23, 24).
In this study, these polymer films derived from biodegradable aqueous polyurethane dispersions were investigated as
supports for in vitro adhesion, proliferation, and uniform distribution of a differentiated monolayer of human RPE cells
(ARPE-19). Additionally, the in vivo biocompatibility of the
polymers was evaluated through their implantation on the
eyes of rats. It was hypothesized that these polyurethanes
could be experimentally evaluated as temporary devices
for cell therapy in the treatment of ocular diseases.
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Polyurethanes for human retinal pigment epithelium
MATERIALS AND METHODS
TABLE I - COMPOSITION (WT.%) OF THE AQUEOUS POLyURETHANE DISPERSIONS*
Synthesis of biodegradable aqueous polyurethane dispersion (PUD)
Aqueous polyurethane dispersions (PUD) were prepared
by the prepolymer mixing process, using a 250 mL threeneck glass flask equipped with a heating mantel, a mechanical stirrer, and a thermometer in a nitrogen atmosphere.
The macrodiol components poly(ethylene glycol) (PEG,
Mn=1500 g.mol-1; Sigma-Aldrich, St. Louis, MO, USA), polycaprolactone-diol (PCL 1000, Tone Polyol 2221, Mn=1000
g.mol-1, Dow Chemical Company, Midland, MI, USA) and
polycaprolactone-diol (PCL 2000, Tone Polyol 0249, Mn =
2000 g.mol-1, Dow Chemical Company, Midland, MI, USA),
2.2-bis(hydroxymethyl) propionic acid (DMPA, 98.3%; Fluka, Sigma-Aldrich, St. Louis, MO, USA), and isophorone diisocyanate (IPDI, Bayer, San Paulo, Brazil) (NCO/OH ratio of
2.3) were added to the reactor in the presence of dibutyl tin
dilaurate (DBDLT, Miracema Nuodex, Brazil). The reaction
was carried out at 70ºC to 75ºC in a nitrogen atmosphere
for 4 hours. The number of free NCO groups was determined on a percentage basis by applying the standard di-butylamine back titration method. After titration, the prepolymer
temperature was allowed to drop to 40ºC. The carboxylic
acid groups were neutralized by the addition of trethylamine (TEA, 98%; VETEC Química Fina Ltda, Rio de Janeiro,
Brazil). The mixture was stirred for another 40 minutes to
ensure that the reaction had been completed. All samples
were dispersed by adding deionized water to the neutralized
prepolymer, which was stirred vigorously. After the dispersion, the amount of hydrazine (HZ solution 64%; Miracema
Nuodex, Brazil), enough to react with free NCO groups, was
added to the reactor, together with a small amount of water,
and stirred for another 30 minutes. This chemical procedure
proved to be successful in producing polyurethane dispersions with a solid content of approximately 25% (PUD). The
compositions of the prepared samples are shown in Table I.
Films were produced by casting the dispersions in a Teflon
mold and allowing them to dry at room temperature for one
week. After, the films were placed in an oven at 60ºC for 24
hours to dry. Two types of polyurethanes were produced: (1)
PUD5, which contains only PCL as the soft segment; and (2)
PUD6, which contains both PEG and PCL as soft segments.
Thicknesses of the films were between 50 µm to 100 µm
and 200 µm.
200
Reagents
PUD5
PUD6
Isophorone diisocyanate (IPDI)
8.58
8.58
Polycaprolactone-diol (PCL 1000)
4.85
4.85
Polycaprolactone-diol (PCL 2000)
9.09
8.36
Poly(ethylene glycol) (PEG 1500)
2.2-bis(hydroxymethyl) propionic acid
(DMPA)
-
0.73
0.97
0.97
Trethylamine (TEA)
0.73
0.73
Water
74.70
74.70
Hydrazine (HZ)
1.08
1.08
* 0.01 % of Dibutyl tin dilaurate (DBDLT) based on the amounts of IPDI, PEG,
and DMPA.
ARPE-19 cell culture
ARPE-19 cells, an established but non-immortalized human
RPE cell line (29), were graciously provided by Dr. Hjelmeland (University of California, Davis, CA, USA) and were
grown in a Dulbecco’s Modified Eagles Medium and Ham’s
F12 medium (DMEM/F12; Gibco BRL, Grand Island, Ny,
USA) with 10% fetal bovine serum (FBS Gibco BRL, Grand
Island, Ny) in a 37ºC humidified atmosphere of 5% CO2 and
95% air. The culture medium was refreshed every 2 days.
Upon confluence, cells were rinsed with 2 mL of a 0.05%
trypsin-EDTA (Gibco BRL, Grand Island, Ny, USA) solution
and incubated with 5 mL of trypsin-EDTA at 37ºC in a humidified atmosphere of 5% CO2 and 95% air. Next, within
5 to 15 minutes, the trypsin enzyme activity was stopped
by the addition of 5 mL of complete growth medium and
centrifuged for 5 minutes at 1500 rpm. The supernatant was
discarded, while the cells were resuspended in 13 mL of
fresh complete DMEM/F12 and seeded onto culture flasks
for further propagation and subsequent passages.
ARPE-19 cell culture on PUD films
The PUD films were cut into round pieces (4.5 mm in diameter), and disinfected by exposure to UV light for 90 minutes on each side prior to cell culture. ARPE-19 cells were
plated on top of each polymer and polyester tissue culture
polystyrene (TCPS) (Costar, Cambridge, MA, USA), as a
control at a density of 4 x 103 cells/well.
© 2011 Wichtig Editore - ISSN 0391-3988
da Silva et al
Number of adherent cells on PUD films
Fluorescence and immunostaining
After 8 hours in the culture, the medium was aspirated, and
the cells on PUD films and control TCPS were rinsed with
phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (Merck Eurolab, Fontelay Sous-Bois, France)
for 15 minutes. Next, fixed cells were rinsed again with
PBS for 5 min and immersed in PBS containing 0.3% Triton
X-100 (Sigma- Aldrich) for 15 minutes. After rising in PBS
for 5 minutes, the nuclei were stained with Propidium Iodide (Sigma-Aldrich, St. Louis, MO, USA) in PBS (1:100) for
10 minutes at room temperature. Finally, the cells were washed five times at 5 minute intervals with PBS and one time
with water, mounted in Gel Mount (Biomeda, Burlingame,
CA, USA) and viewed using an Olympus IX70 fluorescent
microscope attached to a digital camera. Five fields were
photographed per PUD film and control TCPS (total of 15
fields per surface per time-point). The nuclei were counted
for each field of view (0.59 mm2). The average number of
nuclei on the control surface was set as 100%, while the
average number of nuclei ± 1 standard deviation (SD) on
each polymer surface was obtained as a percentage of the
control. Data were presented as a histogram.
At 7 and 15 days of culture, the cells on PUD films were
submitted for the same procedure described for the attachment study. After nuclei staining with Propidium Iodide, F-actin fibers were labeled with Phalloidin FITC (SigmaAldrich, St. Louis, MO, USA) in PBS (1:250) for 30 minutes
at room temperature. Cells were then rinsed, mounted, and
viewed using a fluorescent microscope.
At 15 days of culture, for the labeling of occludin tight
junctions, the cells grown on PUD films were fixed with
a 4% p-formaldehyde for 30 minutes at room temperature. The fixed cells were incubated with PBS containing 0.1% Triton X-100 for 30 minutes. This was followed by incubation with the rabbit anti-Occludin (Zymed
Laboratories, South San Francisco, CA, USA) in PBS
containing 0.1% Triton X-100 (1:100) for 60 minutes.
The cells were rinsed twice with PBS for 10 minutes
and incubated with an Alexa Fluor 488 goat anti-rabbit
secondary antibody (Molecular Probes, Eugene, OR,
USA) in PBS (1:250) for 60 minutes in the dark. Finally,
cells were rinsed, mounted, and viewed using a fluorescent microscope.
Cell proliferation on PUD films (nuclear count)
Transmission electron microscopy of the cells on
PUD films (TEM)
At set time intervals of 1, 2, 7, and 15 days, the cells on
PUD films and control TCPS were submitted for the same
procedure described for the attachment study. The average number of nuclei ± 1 standard deviation (SD) per surface per time-point was presented as a histogram.
Scanning electron microscopy (SEM) of the cells
on PUD films
At 1 day of culture, SEM was performed on PUD films for
cell morphology analysis. Cells were fixed with 2% glutaraldehyde in PBS for 60 minutes at room temperature,
rinsed three times at 10-minute intervals with PBS, and
post-fixed with 2% osmium tetroxide in PBS (1:1) for 30
minutes at room temperature. Cells were dehydrated in
a series of graded ethanol (50-100%). Afterwards, the
cells were dried using hexamethyldisilazane (Sigma-Aldrich, St. Louis, MO, USA), coated with gold (Balzer MD
010; Bal Tec AG, Fürstentum, Lichtenstein), and examined by an electron microscope at 10 kV (JEOL 840A;
Tokyo, Japan).
At 15 days of culture, TEM was performed on PUD films
for cell morphology analysis. The cells grown on PUD films
were washed in PBS, fixed with 2% glutaraldehyde for 30
minutes at room temperature, washed in 0.1 M of a pH
7.4 cacodylate buffer, and post-fixed with 1% osmium tetroxyde for 15 minutes at room temperature. Cells were
dehydrated in a graded series of ethanol (70-100%) and
flat-embedded in Epon. Ultra-thin sections (70-80 nm)
were obtained using an ultra-microtome (Reichert OM, UZ;
Reichert Co., Vienna, Austria), counterstained with uranyl
acetate and lead citrate and examined using an electron
microscope (Philips CM10).
Statistical analysis
Results were expressed as mean ± SD. Data were tested
for normality and investigated for statistical significance
using the Student´s t test and a one-way analysis of variance (ANOVA), where appropriate. A p-value of less than
0.05 was considered significant.
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Polyurethanes for human retinal pigment epithelium
conjunctiva were sutured using 8-0 vicryl suture (Ethicon,
Piscataway, NJ, USA).
Animals were sacrificed using a lethal dose of pentobarbital (100 mg/kg – intraperitoneal injection) at 15 days
post-implant. The eyes were carefully collected and fixed
in formalin (10% in isotonic saline) for 48 hours. The fixed
eyes were embedded in paraffin and 5 µm-thick sections
were obtained. The sections were stained with toluidine
blue and visualized using an Olympus IX70 microscope
(Olympus, Shinjuku, Japan) attached to a digital camera
(Olympus, Shinjuku, Japan).
Fig. 1 - ARPE-19 cells attached to PUD5 and PUD6 surfaces after 8 h
of seeding. Data are expressed as a percentage of the control TCPS
± SD (n = 15 for each PUD, n = 10 for control) (p < 0.05).
In vivo short-term biocompatibility of PUD films
Six- to eight-week-old female Brown Norway rats (Harlan
Laboratories, Horst, The Netherlands) were maintained in
individual cages with food and water ad libitum and with a
controlled temperature and humidity. Animals were housed and cared for according to the guidelines set forth by
the Association for Research in Vision and Ophthalmology
(ARVO) regarding the use of animals in ophthalmic and vision research. Experiments were also performed in accordance with stipulations set forth by eye Ethics Committee
in Animal Experimentation of the Université Paris Descartes, which gave approval to the protocol. Only one eye of
each animal was used.
PUD5 and PUD6 films of 2.0 mm in side were disinfected using ultra-violet light at 254 nm (45 W). Each site of
the film was irradiated during 45 minutes before the implant surgery. Before implantations, the films were cut in
an arrow-shaped manner of 1 mm x 2.5 mm (Figs. 7A and
B). The animals were anesthetized with an intraperitoneal
injection of xylazine (20 mg/kg) and ketamine (80 mg/kg).
The left pupil was dilated with tropicamide eye drops (Johnson & Johnson Vision Care, Jacksonville, FL, USA). To
implant the PUD films into the subretinal space (n=3 for
each polymer), the conjunctiva was dissected at the limbus
in the temporo-superior quadrant and a 1 mm sclerotomy
was performed at 2 mm posterior to the limbus (Fig. 7C).
The PUD films were introduced into the subretinal space
through a trans-choroidal approach. The sclerotomy and
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RESULTS
Number of adherent cells on PUD films
The adhesion of ARPE-19 cells on the surfaces of PUD5,
PUD6, and control TCPS was expressed as the mean
number of cell nuclei (%) ± SD after 8 hours of incubation
(Fig. 1). The RPE attachment rate on PUD5 and PUD6 surfaces was 86.57 ± 6.33% and 81.59 ± 7.54%, respectively.
It was observed that the percentage of attached RPE cells
on the PUD5 surface (with only PCL as a soft segment)
is slightly higher than PUD6 surface (with both PCL and
PEG as soft segments). However, the statistical analysis
(Student’s t-test) showed that there was no significant difference in the mean number of cells attached to polymeric
supports (p<0.05) after 8 hours of in vitro culture.
Cell proliferation on PUD films (nuclear count)
The proliferation of ARPE-19 cells on the surfaces of PUD5,
PUD6 and control TCPS was expressed as percentage of
cells in comparison with day 1. On day 2, the percentage
of RPE cells on the control TCPS, PUD5 and PUD6 was
73.1%, 72.4% and 43.5%, respectively. On day 7, the percentage of RPE cells increased significantly on the surfaces
of the control TCPS, PUD5 and PUD6 (143.7, 108.62 and
66.09%, respectively). On day 15, the percentage of RPE
cells on the control TCPS, PUD5 and PUD6 was 341.2%,
246.7% and 222.6%, respectively, demonstrating cell proliferation in both polyurethane substrates. Over the period
of culture, the percentage of cells indicated the following
order of ARPE-19 proliferation on the supports: TCPS >
PUD5 > PUD6. Although the number of cells was greater
© 2011 Wichtig Editore - ISSN 0391-3988
da Silva et al
Fig. 2 - Proliferation kinetics of ARPE-19 cells cultured in vitro on
the control TCPS, PUD5, and PUD6 surfaces. Data are expressed
as mean number of nuclei ± SD for each time-point (n = 15 per each
PUD, n = 10 for control, per each day) (p < 0.05).
in the control TCPS for all time intervals (Fig. 2), the statistical analysis (one-way ANOVA) showed that there was
no significant differences in cell proliferation on polymeric
supports and the control TCPS (p<0.05) after 1, 2, 7, and
15 days of in vitro culture. It was also observed that the
PUD5 and PUD6 surfaces provided the establishment of
important cell-polymeric surface interactions, since RPE
cells were able to attach, migrate, and proliferate on the
given substrates.
Scanning electron microscopy (SEM)
SEM was employed to evaluate the ARPE-19 morphology
attached on PUD5 and PUD6 on the first day of seeding. Cell
morphology was similar on PUD5 and PUD6 surfaces. After
settling down, one group of cells showed a rounded shape, organized as small well-attached clusters (Figs. 3A, B,
and C). These RPE cells could be seen in areas where there
was a high cell density. Another group of cells exhibited an
elongated and flattened shape, with projections rising out of
them (Figs. 3D and E). These irregular shaped cells were observed in areas with low RPE density, suggesting that they
displayed a mobility to cover the substrates and were not
extensively connected to the polymeric surfaces.
Fluorescent staining and immunostaining
After 7 days in culture, the ARPE-19 cells reached a confluent level on PUD5 and PUD6 and covered the entire polymeric surfaces as an organized monolayer. As the number
of cells increased on substrates, the cell size progressively
decreased and acquired a polygonal appearance. It was ob-
Fig. 3 - Scanning electron micrographs of small clusters of rounded
ARPE-19 cells on PUD5 (A) (×50) and PUD6 (C) (×100) surfaces after
1 day of culture. Higher magnification views of cells with apical microvilli on PUD5 (B) (×20) and elongated cells on PUD6 (D) (×20) with
projections of PUD6 (E) (×10).
served that cell shape was directly influenced by cell density
(22). Phalloidin staining revealed that actin fibers were intensely concentrated around the entire perimeter of the cells,
representing large surface attachments (30). The actin filaments could also be viewed running parallel to one another
through the upper part of the cytoplasm, and being inserted
into the intercellular membrane of adjacent cells, thus providing a connection between them. Propidium iodide staining
of cell nuclei demonstrated that they were located centrally
and did not appear to overlap, suggesting a monolayer formation. Binucleated cells could also be observed, resulting
in cell division. This morphological assessment of confluent
ARPE-19 cells on PUD5 and PUD6 is illustrated in Figure
4.A and 4.B, respectively. The areas shown are representative of the entire cell population on polymers.
Zonula occludens are formed by transmembrane adhesive
molecules, such as occludin (31). They act as a barrier to
the diffusion of solutes through the intracellular space (32).
In this study, ARPE-19 cells on PUD5 and PUD6 substrates presented the ability of expressing occludin, which demonstrated not only the functionality of epithelial cells, but
also the establishment of cell-cell interactions (Fig. 5).
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Polyurethanes for human retinal pigment epithelium
Fig. 5 - Intercellular tight junctions between adjacent ARPE-19 cells
on the control TCPS (A), PUD5 (B), and PUD6 (C) surfaces stained
with occludin after 15 days of culture (×40).
Fig. 4 - Micrographs of ARPE-19 cells on the control TCPS (A),
PUD5 (C), and PUD6 (E) surfaces, demonstrating F-actin stained
with Phalloidin FITC after 7 days of culture. Nuclei were stained with
Propidium Iodide. Micrographs (B), (D), and (F) represent a merging
of stained F-actin and nuclei of cells on both polymers (×40).
Transmission electron microscopy (TEM) of the
cells on PUD films
Electron photomicrographs showed that organelles, cytoplasmatic structures, and membranes of the RPE cells on
the PUD supports remained intact. Golgi apparatus, mitochondria, nucleus, rugosus endoplasmic reticulum, vesicles, and gap junction structures showed no sign of morphological compromise (Fig. 6).
In vivo short-term biocompatibility of PUD films
PUD5 and PUD6 films implanted in vivo under the retina
or in the supra choroidal space were well tolerated, considering that no clinical evidence of immediate or delayed
intraocular inflammation could be seen. Particularly, the
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Fig. 6 - Retinal pigment epithelial cells electronic photomicrograph
showing that organelles and cytoplasmatic structures and membranes remained intact. G – Golgi apparatus; M – mitochondria;
N-nucleus; R-rugosus endoplasmic reticulum; V- vesicles. Arrows
indicate gap junction structures between PUD support and cells.
polymer under the retina could be clearly observed (Fig.
7D) without any vitreous reaction at 7 and 15 days after
surgery. Histological examination of the ocular tissues in
the implants region showed a preserved architecture of
the retina. No infiltrating cells were observed on any of the
sections (Fig. 7E). The retinal pigment epithelial cells just
above the polymer displayed normal structure (Fig. 7E at
higher magnification).
DISCUSSION
Age-related macular degeneration (ARMD) is a disease of
the macula, the central portion of the retina responsible
for high resolution vision. The macular degeneration can
lead to severe loss of vision and even blindness in the elderly population. There are two different types of ARMD:
atrophic (dry form) and exudative (wet form). The dry form
© 2011 Wichtig Editore - ISSN 0391-3988
da Silva et al
Fig. 7 - (A) Macroscopic view of
PUD film. (B) The PUD film presented a triangular shape and
white color. (C) Implantation of
the PUD film in the choroid of a
Lewis mouse eye. (D) The PUD
film implanted within the choroid
of a Lewis mouse eye. (E) Toluidine blue stained histological
section on postoperative day 15
with PUD implanted within the
choroid of an eye. The polyurethane degraded due to the fixation process of the eye. Square
indicated the retina on the polymeric matrix (×40). (F) The architecture of the retina on the PUD
was preserved (×60).
F
is characterized by the presence of drusen and RPE atrophy. The loss of RPE functions leads to the degeneration
of photoreceptors and, consequently, a progressive loss of
vision. The wet form is defined as the development of choroidal neovascularization. The new abnormal blood vessels
are weak, and have a propensity to leak, thus damaging
the RPE and the photoreceptors. The consequence of bleeding is a rapid and severe decline in vision (10, 33, 34).
Over the last decades, there have been significant advances in the management of exudative ARMD and geographic atrophy (35). However, patients are still losing vision,
especially when the available therapeutic approaches do
not allow the effective treatment of causal disease. The
possibility of replacing dysfunctional RPE represents the
initial attempt for macular reconstitution. Currently, the
transplant of a suspension of RPE cells is applied to ARMD
treatment. In a technique of this sort, however, the cells
may fail to form an ordered monolayer (36). Subretinal fibrosis, invasion of the retina by RPE cells, and proliferative
vitreoretinopathy are other complications associated with
this technique (37). For RPE cell transplants to be successful, an organized epithelial monolayer should be transferred onto biodegradable substrates.
Biological materials have been experimentally applied as
supports for RPE growth, including Descemet’s membrane
(17), the microcontact printed human lens capsule (38), the
porcine anterior lens capsule (ALC) (20), and the amniotic
membrane (39). These materials allowed for the attachment and expansion of a monolayer of RPE cells, which
showed typical epithelial morphology and a ZO-1 tight junction. Porcine ALC, which was transplanted to the subretinal space of pig eyes, was well-tolerated and caused no
inflammation when Bruch’s membrane was undamaged.
However, the stickiness of the ALC and its tendency to curl
are problems to be overcome (18). Natural materials have
also been studied as substrates for RPE cells, including
collagen (40) and fibrinogen (41). However, these natural
materials are associated with disadvantages, such as poor
processing properties, inhibition of cell proliferation, and
variation in enzymatic biodegradation rates (3). Additionally, synthetic materials have been used as substrates for
RPE proliferation, such as polydimethylsiloxane (PDMS)
(13) and polyether urethanes (PU) (21). These biostable
materials were surface modified by air plasma treatment to
optimize the RPE cell-substrate interaction, thus allowing
for the formation of an intact functional monolayer of RPE
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Polyurethanes for human retinal pigment epithelium
cells that are able to phagocytose photoreceptor outer segments. Although PDMS and PU have been effective in the
establishment of RPE monolayers, it is necessary to carefully manipulate the hydrophilicity of their polymeric surfaces
using the air plasma treatment. Synthetic biodegradable
materials have been evaluated as substrates for RPE cells, such as poly(DL-lactic-co-glycolic acid) (PLGA) and/or
poly(lactic acid) (PLA) (3, 22, 23). These acted as suitable
substrates for RPE the attachment, proliferation, and maintenance of the differentiated function, and may therefore be
designed to act as temporary supports for the transplanted
RPE cells, but the gap between the performed studies and
clinical applications is still far from being overcome.
In this study, two biodegradable polyurethanes were used
as substrates for differentiated human RPE cell growth. The
cell attachment on PUD5 and PUD6 polymers was evaluated
so as to establish the correlation among polymeric surface
properties and cell adhesion. The macromolecular structure
of the polymer’s surface and the surface charge are important factors which determine the affinity of adsorbed proteins and subsequent cellular-substrate interactions.
PUD5 and PUD6 displayed a complex structure consisting of hydrophobic and hydrophilic domains created by
the microphase separation derived from the low compatibility between soft and hard segments. The PCL alone in
the PUD5 soft segment led to a more defined two-phase
polyurethane. The PUD6 exhibited a larger degree of phase
mixing, possibly due to the interaction between polar hard
segments and hydrophilic PEG segments, in turn leading to
a less heterogeneous structure (28). The number of attached
RPE cells on the PUD5 surface is slightly higher than on
the PUD6 after 8 hours of incubation, suggesting that there
was a dependence of cell adhesion and PUD hydrophobicity. The PUD5 backbone showed an increased hydrophobic
character, in turn enhancing the adsorption of serum adhesion proteins as well as cellular interactions. The presence
of PEG on the PUD6 structure decreased its hydrophobic
character, preventing protein absorption and/or forming
weak interactions, which resulted in a decreased RPE attachment. It is well-documented that PEG can prevent protein absorption as well as inhibit cell attachment (42, 43).
However, the statistical analysis (Student’s t-test) showed
that there was no significant difference in the mean number
of attached cells on polymeric supports (p<0.05). Since the
statistical difference could not be viewed, it was suggested
that the incorporation of just 3% of PEG in the PUD6 soft
segment was not enough to form a PEG rich phase on the
206
surface, which would promote the repulsion of proteins and
cells that approached the surface.
The surface charge of an artificial material represents
another factor to influence protein absorption. PUD5
and PUD6 films displayed negatively charged carboxylic
groups (COO-) at 7.4 pH. This ionic center was formed due
to the incorporation of dimethylol propionic acid (DMPA)
within the hard segment during the synthesis process. The
negatively charged surfaces of the polymers most likely
promoted electrostatic interactions with serum adhesion
proteins, resulting in their adsorption and subsequent RPE
adhesion. In summary, the protein absorption phenomenon on PUD5 and PUD6 surfaces was generated by different interactions, including hydrophobic and electrostatic
interactions.
The ARPE-19 proliferation on polymeric supports and control TCPS was evaluated over a 15-day period, and the
number of proliferated RPE cells on PUD5 surfaces was
higher than on PUD6 surfaces. However, the statistical
analysis (one-way ANOVA) showed that there were no significant differences in the cell proliferation on polymeric
supports and the control TCPS (p<0.05). The differences
in the cell proliferation rate on PUD5 and PUD6 substrates
can be attributed to the presence of PEG in the soft segment. PEG can orient their hydrophilic chains to the hydrated environment and tends to repel membrane proteins of
the cells, thus preventing their establishment. On the other
hand, the ratio of PEG incorporated in the PUD6 soft segment was not enough to prevent an intense modification in
the RPE proliferation rate.
The morphological analysis of RPE cells on PUD5 and
PUD6 surfaces was assessed by scanning electron microscopy and immunocytochemistry. After 1 day of seeding,
small clusters of RPE cells showed a rounded shape, although individual cells presented a non-uniform morphology. Upon attaining the confluence level and extended
period, RPE cells achieved and maintained a polygonal
appearance. It was suggested that the epithelial cell shape
became more homogeneous not only due to the higher cell
density on the surfaces, but also due to the establishment
of interactions of actin filaments within two different sites:
1. with an intercellular membrane of adjacent cells (a site
of cell-cell attachment); and 2. around the perimeter of
the cells, representing large surface attachments (a site of
cell-substrate attachment). Although the epithelial cells on
PUD5 and PUD6 surfaces achieved a suitable level of organization, their shape is not as uniform as that of epithelial
© 2011 Wichtig Editore - ISSN 0391-3988
da Silva et al
cells within the tissue. This fact can be attributed to the
differences between the artificial culture system and the
natural environment (44).
After a 15-day period, it was observed that ARPE-19 cells on the PUD5 and PUD6 surfaces expressed occludin,
suggesting the presence of tight junctions among them.
This result indicated that cell growth on the polymeric supports provided the formation of cell-cell interactions, which
is essential for the formation of a functional epithelial monolayer. The RPE must adopt a tight epithelial monolayer
phenotype, which acts as a blood-retinal barrier (16). Electron photomicrographs also showed the establishment of
cell-PUD support interactions, represented by junction
structures lining the surface of the polymers.
This study also investigated the short-term tolerance of
the polymers in vivo in pigmented rats. The histological
and clinical results from the in vivo study indicated good
tolerance of PUD5 and PUD6 films. There were no signs
of acute and chronic inflammation or necrosis. The cells
involved in the inflammatory reaction, such as macrophages and foreign body giant cells, were not observed in
the ocular tissues 15 days after implantation. Additionally,
the architecture of the retina and other ocular tissues was
preserved. Our results were significantly better than those described by Foulds and coworkers (45) after having
implanted the urethane-based hydrophilic polymer within
the suprachoroidal space. In this study, the following was
observed: 1. mononuclear macrophagic response to the
polymer; 2. adjacent fibrosis in the choroid; 3. chorioretinal
fusion (45). According to Einmahl and coworkers (46), in
some specimens, choroidal fibrosis led to total retinal atrophy, whereas in others a reactionary proliferation of atrophic RPE cells could be observed. Moreover, the obtained
results were as interesting as those described by Heller
and coworkers (47) after injecting a viscous poly(ortho
ester) into the suprachoroidal space of the rabbit eyes. In
these particular cases, histopathological studies indicated
good biocompatibility of the polymer. Furthermore, the polyurethanes presented suitable mechanical properties for
an easy transscleral-driven subretinal implantation.
Finally, it is important to emphasize that the reagents used
as precursors of these polyurethanes were carefully selected, so that the hydrolytic biodegradation process was favorable and the degradation products were non-cytotoxic
to RPE cells and soluble in water. Thus the biodegradation
of the polyurethanes indicated that the ester bonds of the
poly(caprolactone) incorporated into the soft segments
were hydrolytic broken. The hydrolysis of poly(caprolactone)
ester bonds enhanced the mobility of the lower molar
mass chains, increasing their ability to pack in a crystalline
structure and resulting in the re-organization of the initially
amorphous phases of the polyurethanes. Furthermore, the
degradation products from the polyurethanes were noncytotoxic to the RPE cells (26). Therefore, the polyurethanes were designed to act as temporary supports for the
transplanted RPE cells, providing a greater potential for reestablishing the RPE-Bruch’s membrane interaction, and
a greater diffusion of nutrients. Further studies have now
been performed in order to reduce the lifetime of the polymer by controlling their thickness.
In summary, PUD5 and PUD6 provided a suitable base support for better adhesion, growth and functionality of ARPE-19
cells in culture. Moreover, the in vivo study revealed a promising short-term biocompatibility of biodegradable synthetic
polyurethanes. Further experiments will be performed to evaluate the long-term biocompatibility and the ophthalmologic
applicability of these biomaterials. It is hypothesized that the
PUD5 and PUD6 could be applied as temporary RPE support
as a means for aiding therapies for age-related macular degeneration (ARMD). One therapeutic strategy to arrest visual
deterioration in ARMD patients is RPE transplants by means
of a substrate to support and maintain an organized monolayer of healthy and functional RPE cells.
CONCLUSIONS
We were able to verify that PUD5 and PUD6 surfaces were
able to establish important interactions with ARPE-19 cells,
thus allowing for their adhesion, migration, and proliferation, which are essential in forming an RPE monolayer. The
presence of a polygonal RPE monolayer was demonstrated
by the staining of actin filaments, which were connected to
intercellular membranes of the cells and associated with
stress fibers. The identification of the occludin expression
represented the functionality of confluent epithelial cells on
PUD5 and PUD6 supports. Additionally, PUD5 and PUD6
demonstrated intraocular biocompatibility, since there
were no signs of inflammatory response or toxic reactions
in the ocular tissues. They also exhibit proper mechanical
properties for subretinal implantation. The results obtained
in this study showed the possibility of using synthetic biodegradable and biocompatible polyurethanes as supports
for retinal epithelial cells.
© 2011 Wichtig Editore - ISSN 0391-3988
207
Polyurethanes for human retinal pigment epithelium
Financial support: The authors would like to acknowledge financial
support from the following institutions: CAPES/MEC (Brazil), CNPq/
MCT (Brazil) and FAPEMIG (Minas Gerais - Brazil).
Conflict of interest statement: The authors confirm that there are no
known conflicts of interest associated with this publication and there
has been no significant financial support for this work that could have
influenced its outcome.
Address for correspondence:
Prof. Francine Behar-Cohen
INSERM UMR872
Institut des Cordeliers
15, rue de l’Ecole de Médecine
75006, Paris, France
e-mail: francine.behar@gmail.com
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