Download Internal Osmotic Pressure as a Mechanism of Retinal Attachment in

Internal Osmotic Pressure as a
Mechanism of Retinal Attachment
in a Vitreous Substitute
WILLIAM J. FOSTER, HYDER A. ALIYAR, PAUL HAMILTON AND NATHAN RAVI*
Surgical Services, St. Louis VA Medical Center, St. Louis, MO, 63106, USA
and Department of Ophthalmology and Visual Sciences, Washington
University, St. Louis, MO 63110, USA
ABSTRACT: In this study, the possibility of using the internal osmotic pressure of intraocular polymeric hydrogel materials to attach the retina in the
repair of a retinal tear or hole was investigated. This is in contrast to the conventional methods of retinal detachment repair (intraocular gas, polydimethylsiloxane, or n-perfluorooctane), which rely on surface tension and have
recognized limits. The system selected for implementation of this scheme was
based on an acrylamide copolymer that was crosslinked in an aqueous solution
to provide a transparent hydrogel which allowed control of the swelling pressure. Synthetic hydrogels, such as those selected here, provide an alternative
to materials currently used as vitreous prostheses.
KEY WORDS: vitreous substitute, osmotic pressure, retinal detachment,
retinal tamponade, hydrogel, vitreous prothesis, macular holes, retinal tears,
retinal detachments, vitreous hemorrhage.
INTRODUCTION
Vitreous Biochemistry and Physiology
he eye is conventionally divided into two anatomic regions, the
anterior segment and the posterior segment. The anterior segment
includes the cornea, iris, and lens (Figure 1). The posterior segment is
composed of the vitreous body (also called the vitreous), the ciliary
T
*Author to whom correspondence should be addressed.
E-mail: [email protected]
Figures 1, 5, 6, and 7 appear in color online: http://ibe.sagepub.com
Journal of BIOACTIVE AND COMPATIBLE POLYMERS, Vol. 21 — May 2006
0883-9115/06/03 0221–15 $10.00/0
DOI: 10.1177/0883911506064368
© 2006 SAGE Publications
221
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W. J. FOSTER ET AL.
Figure 1. Schematic diagram of the eye.
body, retina, choroid, and sclera. The anterior segment structures are
designed to admit light into the eye and to focus that light on the
retina. The posterior segment structures are designed to detect light
and transmit the resulting signal to the brain.
The vitreous body is simply the clear “jelly-like” substance in the
middle of the eye, posterior to the lens. It is a paucicellular natural
hydrogel that is made up primarily of non-crosslinked Type II collagen
fibrils and hyaluronic acid [1] and is approximately 99% water by
weight. The non-crosslinked collagen forms a random polymer
network that runs from one end of the vitreous cavity to the other and
is responsible for the gelatinous vitreous. Intermeshed in the collagen
polymer network, and also present in the region of liquid vitreous, is
hyaluronic acid. This glycosaminoglycan, made up of two alternating
monosaccharides (N-acetylglucosamine and glucuronic acid linked by
glycoside bonds), forms a 1,000–10,000 Mw unbranched, coiled polyanion that has a high hydrated specific volume (2,000–3,000 cc/g). The
concentration of the hyaluronic acid varies from 0.03–0.10% in
humans [1].
The vitreous is non-uniform in density, with a 100–200 m-thick
layer of solid cortical gel adjacent to the retina, ciliary body, and lens
[1]. In addition, Cloquet’s canal, the central portion of the vitreous
anterior to the optic nerve, frequently contains thin, multilayered, fenestrated sheaths of basal lamina tissue [1].
The vitreous is firmly attached to the anterior retina, where collagen fibers penetrate the retina and attach to the basement membrane
of the ciliary epithelium and peripheral retinal glial cells [2]. The
retina, the tissue that detects light, lines the inside of the eye and is in
contact with the vitreous. The choroid is a highly vascular structure
Internal Osmotic Pressure
223
that surrounds the retina (i.e., lies outside the retina) and provides
nutrients to the retina. The sclera, which surrounds the choroid, is
made of collagen fibrils with varying cross-sectional diameters and a
haphazard arrangement [3]. This results in a lack of transparency for
this tough outer coat of the eye.
In young children, the vitreous, which is secreted by hyalocytes, is
firm and helps to maintain the retina and keep it pressed against the
back of the eye. It acts as a viscoelastic damper, thought to be due to
the presence of hyaluronic acid [1] and the uniform distribution of the
osmotic pressure ensures retinal attachment to choroid. As a person
ages, the vitreous develops liquefied pockets, characterized by a
paucity of collagen [1], and eventually separates from the retina completely, except for the most anterior retina. This has been ascribed to
aggregation of the collagen fibrils into thicker cables [4–7] as well as
changes and thickening of the basement membrane of the retina that
result in loss of collagen fibril adherence [1] except in the anterior eye,
where collagen fibrils remain firmly attached.
During the acute phase of this separation (known as posterior vitreous detachment or PVD), the vitreous may be abnormally attached to
the retina at some focal point. The vitreous will naturally move within
the eye during normal eye movement and in the process can create a
tear or hole in the retina at the point of adhesion. This retinal tear
should be repaired as it can lead to retinal detachment (fluid under the
retina) with the risk of blindness.
Retinal Detachments and Treatments
The vitreous body undergoes a nonuniform transition from a gellike substance in a young child to a more fluid-like substance in an
older adult. Associated with this transition are a number of visionthreatening phenomena such as macular holes, retinal tears, retinal
detachments, and vitreous hemorrhage. The mechanism for these phenomena is considered [8] to involve traction of the liquefying vitreous
gel on the retina when currents are created in the vitreous by eye
movements.
The creation of a hole or tear in the retina by vitreous traction is a
surgical emergency. Treatment of this hole or tear involves relieving
the traction and blocking egress of fluid through the hole by reapproximation of the retina to the underlying tissues. Details of treatment
options for this condition have been thoroughly reviewed by Wilkinson
and Rice [8]. An increasingly common technique to achieve these
results is vitrectomy surgery. When a patient undergoes a vitrectomy,
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much of the vitreous is removed by using aspiration and cutting techniques to relieve the traction on the retina. Frequently a gas (air,
sulfur hexafluoride, or perfluoropropane) is introduced into the eye to
hold the retina against the back of the eye (the retinal pigment epithelial layer) until a scar is formed between the retina and the underlying
tissue. This process can take weeks, and during this time, the installed
compound must remain in contact with the retinal hole. For this
reason, patients who have received intraocular gas are usually positioned for a week or more in a face-down position that many find difficult to maintain. Obviously, holes located in the inferior retina are not
easily amenable to closure by intraocular gas.
Silicone oil (polydimethylsiloxane) has been instilled in the eye for
complex retinal tears, for inferior tears, or in patients unable to position themselves. Given its lower surface tension at the water interface
(30 dynes/cm as opposed to 70 dynes/cm at the gas/water interface),
the success rate for hole closure is frequently less [9]. In addition, the
silicone oil must be removed later, which is difficult to remove completely, since it is toxic to intraocular structures and is associated with
glaucoma and corneal decompensation [10] (both of which can lead to
blindness).
Vitreous Substitutes, Perfluoron and Silicone Oils
Over the years, attempts to find vitreous substitutes has been
mostly trial-and-error [11]. The materials currently used include the
following:
•
•
•
•
air;
perfluorocarbon gasses (especially n-perfluoropropane);
sulfur hexafluoride;
saturated perfluoronated oils (particularly n-perfluorooctane [12]
and n-perfluorodecalin [13]);
• polydimethylsiloxane [14].
Materials that have been experimentally investigated include the
following:
•
•
•
•
•
•
perfluorophenanthrene [15];
perfluorotri-n-propylamine [16];
polymethyl-3,3,3-trifluoropropylsiloxane-co-dimethylsiloxane [17];
poly(2-hydroxyethyl acrylate) [18];
semifluorinated alkanes [19];
silicone gel [20];
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Internal Osmotic Pressure
•
•
•
•
•
•
methylated collagen [21];
collagen/hyaluronic acid mixtures [22];
hydroxypropylmethyl cellulose [23];
crosslinked poly(vynyl) alcohol [24];
polymethylacrylamidoglycolate methyl ester [25];
crosslinked poly(1-vinyl-2-pyrrolidinone) [26].
All of these compounds either depend on surface tension [27] or are
structurally similar to silicone oils and presumably depend on surface
tension and the (as yet incompletely determined) other mechanisms
through which silicone oils may act.
In this paper, our efforts to develop alternatives to silicone oil and
perfluorocarbon liquids for repair of complex retinal detachments are
described. Unique to our system is the ability to form a hydrogel under
physiologic conditions and then generate internal osmotic pressure,
thus closing the retinal tear. We are trying to create an artificial
version of the of the juvenile vitreous by developing new polymeric solgel systems. This would avoid issues related to patient positioning and
known toxicity of current materials as well as provide better treatment
of inferior tears. By selecting such systems, we also can avoid exposure
of the intraocular tissues to toxic monomers and the thermal energy
present in exothermic in vivo polymerization reactions.
Injection, using a small-gauge needle, is the only practical procedure
for the delivery of a vitreous substitute. The injection process induces
some loss of elasticity when preformed hydrogel materials were
injected [26] due to breakage of some of the crosslinks in the gel
network. Such loss of elasticity does not occur in the hydrogel system
investigated in this work because gelation occurs only in the vitreous
cavity after the polymer is injected.
For retinal tamponade with osmotic swelling to be achieved, the
swelling pressure of the polymer in the eye should be positive but tailored in such a manner as to avoid excessive pressure on the retinal
vasculature (possibly causing vascular occlusion) and retina. According
to the Flory–Rehner theory [28], the swelling pressure can be
expressed as:
[ln(1 ) w ] G
RT
2
3
(1)
where is the molar volume of the solvent, is the volume fraction of
the polymer, and w are the second- and third-order interaction parameters, and G is the elastic modulus of the swollen network. This relationship can be used to select an appropriate polymer composition.
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W. J. FOSTER ET AL.
METHODS
The chemicals were purchased from Aldrich-Sigma Chemical Co. St.
Louis, MO, USA and used without further purification. All reagents
used were analytical grade.
Preparation of poly(acrylamide-co-2-thiol ethyl acrylamide)
hydrogel
Acrylamide and bisacryloylcystamine (BAC) were copolymerized at
acrylic mole ratios of 96/4 at 5% (w/w) monomer concentration in 25%
ethanol (25:75 ethanol:water v/v). The hydrogel was then reductively
liquefied followed by precipitation in methanol to obtain water-soluble
thiol-containing polymer. These procedures have been previously
reported [29,30]. The resulting soluble polymer obtained was labeled
“the copolymer”.
Characterization of the Copolymer
The thiol (—SH) content present in the copolymer was determined
by Ellman’s analysis [31]. The molecular weight of the reduced copolymer was determined with an HPLC-GPC system equipped with a triple
detector (refractive index, light scattering, and viscosity; Viscotek,
Houston, TX, USA), with a G4000PWXL column (Tosoh Biosep, Montgomeryville, PA, USA). The mobile phase was 20 mM Bis-Tris buffer
(pH 6.0, 0.1% sodium azide). Samples were prepared in water (pH 4
and N2 saturated) at a concentration of 0.5% (w/v). Poly(ethylene
glycol) standards (Viscotek, Houston, TX, USA) of molecular weight
(Mw) 1,000 to 950,000 were used for calibration.
Regelation in the Vitreous Cavity
A stock solution of the copolymer (20 mL, 8% w/v, pH 5 in water)
was prepared. Four milliliter aliquots with a final copolymer concentration of 7% w/v were prepared from this stock solution. The pH of
each 4 mL aliquot was adjusted to 7.0 with a small amount of 1 M
NaOH. This thiol copolymer solution was regelled in situ in the vitreous cavity by two methods: first, an equivalent amount of dithiodipropionic acid (DTDP) was added to a 1:1 molar ratio of the —SH content
contained in the copolymer. The DTDP copolymer mixture was well
stirred just before injection into the vitreous cavity. The copolymer
rapidly regelled within minutes. A second method involved bubbling
Internal Osmotic Pressure
227
air through the dissolved 8% polymer solution at pH 4 for 30 min
before raising the pH to 7.0. The regellation process usually took
several hours.
Toxicity Testing of Materials
Viability and toxicity testing were carried out on Chinese hamster
ovary (CHO) cells in tissue culture using the methylthiazole tetrazolium (MTT) assay [32]. In this assay, 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (thiazolyl blue) is converted to
formazan, which forms a water-insoluble precipitate by an enzymatic
reaction that occurs only in living cells. The experiment was performed
as follows: CHO cells were plated at a density of 10,000 cells per a 16mm well in 24 well clusters. The media used was 1 mL per well of
minimal essential medium (MEM). All tissue culture reagents were
from Sigma Chemical Co (St. Louis, MO) with 10% fetal calf serum
with gentamycin and penicillin/streptomycin. Twenty-four hours after
plating, the media was aspirated, and fresh media containing the
material to be tested was added. Cells were then incubated for 72 h.
After incubation, 0.5 mL of M199 medium without phenol red, containing thiazolyl blue (MTT) (Sigma/Aldrich) (0.65 mg/mL) and 10% fetal
calf serum was added. The cells were then incubated an additional 4 h.
The medium was aspirated, and the precipitated formazan in the cell
monolayer was dissolved in 200 L of DMSO. Samples were transferred to a microtiter plate and the absorbance at 540 nm was measured. Samples were performed in quadruplicate.
Results obtained from the cells treated with the test compounds
were expressed as a percent of results obtained from control, or
untreated, cells. The IC50 was derived from a plot of the data as the
concentration where the value obtained would equal 50% of control
and was used as a measure of toxicity.
To test for toxicity, a specific amount of the methanol precipitated,
lyophilized copolymer (300 mg) was added to acidified (pH 6.0) MEM
media (9.7 mL). This copolymer solution was allowed to dissolve for
24 h. The pH was then adjusted to pH 7.4 and the solution mixed
with 10 mL of MEM with 20% fetal calf serum to give a final concentration of 15 mg/mL polymer in MEM with 10% fetal calf serum. The
material was allowed to mix in the presence of air to allow the formation of disulfide bonds. The resultant media was added to the CHO
cells for testing. In addition to the copolymer, linear acrylamide,
acrylamide monomer, and DTDP were also tested under identical
conditions.
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W. J. FOSTER ET AL.
Surgery
Fresh human cadaver globes were obtained from the hospital
morgue. A pneumatic vitrectomy was performed with a modified
version of the standard technique [33]. The cadaver eyes were tilted so
that the visual axis was horizontal. In 1 mL increments, air was
injected into the vitreous cavity of the eyes, in 10 min intervals,
through a pars plana approach (4 mm posterior to the limbus) with a
30-gauge needle. A 27-gauge needle was then placed through the pars
plana inferiorly to allow vitreous fluid to drain. A total of 5 mL of air
was injected. Ten minutes after the final injection of air, 4 mL of the
copolymer solution was injected into the vitreous cavity of each eye
through the pars plana with a 27-gauge needle. The eyes were
wrapped in saline-saturated gauze and were kept overnight at room
temperature. The globes were then formalin fixed, sectioned with a
Surgitron cutting device (Ellman International Inc. Oceanside, NY),
and photographed.
RESULTS
Preparation and Characterization of the Copolymers
Copolymerization of acrylamide and BAC provided a unique hydrogel. The critical step in obtaining the desired water-soluble copolymers
from crosslinked gels involves the complete reduction of all the disulfide bonds (—S—S—) in the gels into —SH groups, as shown in Figure
2. More details on the preparation and characterization of a similar
thiol polymer can be obtained from previous reports [29,30].
The disulfide bonds of the gels were almost completely reduced by
dithiothreitol (DTT). Ellman’s analysis shows a —SH content of
(CH2
Ch2 CH CONH2
A
AAm
CH CO NH CH2CH2S)2
BAC
s
s
s s
s
s
s
SH
s
B
C
HS
s
s
s
s
s
s
s
s s
s
s
SH
HS
s
Figure 2. Schematic representation for preparation, liquefaction of the crosslinked gel
to give the copolymer. (a) Copolymerization with APS and TEMED. (b) Swelling and
washing of the gel to remove unreacted acrylamide () and BAC () monomers. (c)
Reductive liquefaction of the gel with DTT followed by precipitation in methanol, filtration, and vacuum drying to again get the copolymer.
229
Internal Osmotic Pressure
5.1 104 moles/g. The predicted value was 5.4 104 moles/g. The molecular weight distribution analysis of the copolymer showed a broad
distribution with a polydispersity of 3.4 and weight average molecular
weight (Mw) of 3.8 105.
In Situ Regelation of the Copolymer Solution
Regelation of copolymer can be achieved by air oxidation of the thiol
groups or by a thiol-disulfide exchange reaction. The air oxidation took
several hours to form gels at pH 7.5, but the exchange reaction yielded
gelation in 5 min at pH 7.0. The gels obtained were optically transparent. The suitability of the water-soluble copolymer solution for in vivo
regelation was established in the human cadaver vitreous cavities in
vitro.
Regelation was performed with 7% w/v (final concentration) solution
of the copolymer. The thiol-disulfide exchange reaction, leading to the
formation of gel, is schematically represented in Figure 3. As the
copolymer was prepared monomer free and injected at the desired concentration, monomer toxicity was avoided.
The swelling pressure of the polymer in the eye should be positive
but tailored in such a manner as to avoid excessive pressure on the
retinal vasculature (possibly causing vascular occlusion) and retina.
The hydrated gel formed from the 7% w/v copolymer was noted to have
a slightly higher elastic modulus compared with the consistency of
natural vitreous. Gels of similar composition, formed in vitro, have an
elastic modulus of 100 to 150 Pa and contain more than 95% water.
The mechanical properties and swelling characteristics of the regelled
hydrogel can be tuned through appropriate modification of the properties
SH
HS
S
SH
S
HS
S
SH
HS
S
SH
HS
S
S
S
S
Figure 3. Schematic representation of regelation of copolymer solution through thioldisulfide exchange reaction with DTDP at pH 7.0 in the vitreous cavity.
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W. J. FOSTER ET AL.
Linear polyacrylamide
AB4
MTT Abs, % of cont
100.0
50.0
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
MG/ML
Figure 4. Toxicity of linear polyacrylamide, polymerized in the absence of a crosslinker
compared with polyacrylamide polymerized with BAC thiol as a crosslinker (labeled
AB4) (both polymers were methanol precipitated and the lyophilized material was used
for toxicity testing as described).
of the thiol polymer, such as molecular weight, thiol content, and
hydrophobicity.
Intravitreal Gel
This protocol in cadaveric eyes was successfully implemented as
depicted in Figures 5 to 7. The hydrated gels were consistently formed
and were observed to have vitreous-like consistency. In performing
this series of experiments, we have developed critical experience that
will guide us in animal studies and trials of other sol-gel systems.
Toxicity Results
From the toxicity curves (Figure 4), linear polyacrylamide has an
IC50 of 3.2 mg/mL, whereas the copolymer has an IC50 of 7.25 mg/mL.
Toxicity can be reduced by further purification of the copolymer by
Soxhlet extraction and dialysis, or multiple methanol reprecipitations
[34]. Consistent with other observed work [35], when the acrylamide
monomer was tested under identical conditions to those of the copolymer, it showed a high level of toxicity in CHO cells, with an IC50 of
Internal Osmotic Pressure
231
Figure 5. The formed polymeric gel is visible after the sclera has been cut away.
Figure 6. The eye, with gel in situ, has been coarsely cut to show the anatomically
correct position of the gel within the globe.
0.02 mg/mL. Based on a weight/weight ratio, linear polyacrylamide
showed a decrease in toxicity of 160-fold, and the thiol polyacrylamide
polymer showed a decrease of 360-fold when compared with acrylamide monomer. DTDP was assayed and an IC50 of 7.8 mg/mL or
37 mM was obtained. The final concentration of DTDP needed to regel
the copolymer at 7% w/w was 32 mM.
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W. J. FOSTER ET AL.
Figure 7. The removal of the polymer from the globe shows that the polymer is transparent.
CONCLUSION
Spontaneously forming reversible sol-gel systems are a reasonable
alternative to current vitreous substitutes. By changing the paradigm
from a liquid or gas to an artificial vitreous gel that approximates the
retina against the RPE, we may avoid the limitations of current substitutes, such as, the need for face-down positioning and the need for a
second operation to remove oil to prevent glaucoma and toxicity.
Further work in vivo is needed to evaluate the long-term toxicity and
the risk of complications.
For the first time, the use of internal osmotic pressure to reapproximate the retina has been observed. In a series of proof-of-principle
experiments, a commonly available monomer, acrylamide, was used to
form hydrogels. Poly(acrylamide-co-BAC) hydrogels were prepared and
reduced to obtain a water-soluble copolymer with pendant thiol (—SH)
groups. An aqueous solution of the thiol polymers was then regelled to
form a hydrogel, through a thiol-disulfide exchange reaction. In vitro
regelation of this polymer system inside the vitreous cavity of human
cadaver eyes indicates that this is a reasonable and potentially advantageous alternative to current vitreous substitutes. By changing the
paradigm from a liquid or gas to a formed artificial vitreous that reapproximates the retina against the RPE through a mechanism of
internal osmotic pressure, the limitations of current substitutes such
as the need for face-down positioning are potentially avoided. Prelimi-
Internal Osmotic Pressure
233
nary toxicity testing shows that the scrupulous removal of the acrylamide monomers by multiple methanol reprecipitation greatly
reduced the toxicity of the material. Also, the introduction of thiol
groups in the polymer matrix appears to have decreased the toxicity by
two-fold over linear polyacrylamide. Further work is in progress to
evaluate toxicity, the risk of complications of this material, and synthesis of new and better gel formulations.
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