Ординатура / Офтальмология / Английские материалы / Biomaterials and regenerative medicine in ophthalmology_Chirila_2010

Vitreous

13.1 Ocular anatomy (Swindle and Ravi, 2007).

In recent years, research into vitreous substitutes has focused on polymeric hydrogels, and these have been reviewed extensively (Chan et al., 1984; Chirila et al., 1998; Soman and Banerjee, 2003 Swindle and Ravi, 2007). However, these preformed equilibrium-swollen hydrogels disintegrate when injected and sheared through a small gauge needle (Chirila and Hong, 1998). We have previously designed, synthesized, and characterized water-soluble thiol-containing copolymers that gel in the presence of oxygen (Foster et al., 2006). Thus, prior to injection, they exist in polymeric form. The use of a reversible disulfide crosslinker in the initial formation of the hydrogel enables chemical reduction to a substantially pure thiol-containing copolymer that can undergo subsequent exhaustive purification and can regel under physiological conditions in the eye. In this work, the young porcine vitreous was characterized and used as an animal model for the development of the specification for vitreous substitutes. Then, potential vitreous substitutes that are in vivo-forming hydrogels were developed and characterized to meet the criteria established via testing of the natural vitreous humor.

13.2 Biomechanics of the vitreous humor

13.2.1 Background

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(G′) than loss modulus (G≤), which indicates its viscoelastic solid behavior. G′ represents the elastic or recoverable component, whereas G≤ represents the viscous component or dissipated energy. Its viscosity is highest in the posterior and decreases toward the anterior segment (Lee et al., 1992).

The mechanical properties of the vitreous humor have been studied by several groups. Beginning in 1976, Bettelheim and Wang tested the viscoelastic properties of bovine eyes by inserting compression chucks in the vitreous cavity. A dynamic viscoelastometer applied compressional sinusoidal strain via electromagnetic transducers. In bovine vitreous, the storage and loss moduli were found to be 4.2–4.7 Pa and 1.9–3.7 Pa, respectively. They hypothesized that hyaluronic acid contributed to the viscosity and collagen contributed to the elasticity. Their results showed that the elastic and viscous components were of the same magnitude, but the elasticity was slightly higher (Bettelheim and Wang, 1976). The two biopolymers in the vitreous interact to form a stable hydrogel without syneresis or mechanical collapse when subjected to conditions that would normally destroy collagen networks (Chirila et al., 1998).

In 1980, Zimmerman measured the viscoelasticity of the human vitreous in vivo by light scattering. He reported an elastic shear modulus of 0.05 Pa (Zimmerman, 1980). Tokita et al. used a torsional pendulum to measure the complex shear modulus of bovine vitreous at low frequencies, giving a shear modulus value of 0.5 Pa (Tokita et al., 1984).

In the early 1990s, a magnetic microrheometer was developed by Lee et al. because typical rotational rheometers may destroy the vitreous structure.

The fluid was stressed by moving a microscopic iron sphere in a horizontal direction under the influence of magnetic force magnets. They then used an empirical four-parameter viscoelastic model to calculate the creep compliance of human, bovine, and porcine vitreous. The model is mathematically equivalent to an ideal Burgers model, but the parameters are valid for their data from the microrheometric creep test. The Maxwell viscosity represents the unrecoverable viscosity, while the Kelvin viscosity represents the internal viscosity (Lee et al., 1994). Several conclusions could be drawn from their work. The human vitreous has lower retardation times than the bovine or porcine vitreous, indicating faster recovery in the human eye. The vitreous humor is most viscous at the posterior in order to protect the retina and is less viscous at the anterior in order to allow rapid accommodation. The mechanical properties of the human vitreous are more similar to the porcine than to the bovine vitreous, and the human vitreous most closely resembles that of the central region of the porcine vitreous. Their results indicate that the porcine vitreous would serve as a suitable animal model for the human vitreous humor (Lee et al., 1994).

A novel cleat geometry was recently developed to overcome wall slip in shear rheometry (Nickerson et al., 2005). Initial G′ and G≤ values were 30

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collapse (Balazs, 1973). It is evident that a vitreous substitute should mimic the natural vitreous by being viscoelastic and transparent.

13.2.2 Experimental methods

Porcine eyes (N = 90) were used for analysis of the physical and mechanical properties of the natural vitreous humor. The eyes were obtained from 6-month-old pigs from a local abattoir (Weyhaupt Brothers Packing Company, Belleville, IL, USA). All eyes were tested within 12 hours of death to ensure retention of vitreous structure. Rheological analysis of the porcine vitreous is challenging because of the nature of the vitreous humor. The natural vitreous is a heterogeneous gel with solid gel components and liquid components that flow easily.

The sclera was cut around the exterior of the cornea and lens. The anterior segment of the eye was removed with tweezers, while the vitreous body remained attached to the lens. Finally, the vitreous was placed in a Petri dish and the lens was gently removed from the vitreous sample. When initially removed from the eye, the vitreous is a viscoelastic solid, but with time the structure degrades and the gel becomes fluidic and slowly starts to creep.

Viscoelastic properties were determined using the Vilastic-3 oscillatory capillary rheometer (Vilastic, Austin, TX, USA). The capillary rheometer was used for rheological evaluation because it enables testing of small samples and the central section of the porcine vitreous rather than the encapsulated vitreous body, which showed slippage effects on a parallel plate rheometer (Nickerson et al., 2005). The capillary tube had an inner diameter of 0.149 cm. The anterior segment was removed from fresh porcine eyes, the vitreous was removed from the vitreous cavity, and approximately 0.5 cm3 of the intact vitreous was aspirated for testing.

Samples were tested at 25 °C rather than at physiological temperature because Tokita et al. showed that the mechanical properties of the vitreous were temperature invariant from approximately 10 to 40 °C (Tokita et al., 1984). The porcine vitreous was analyzed by frequency scans from 0.05 to 20 Hz at 0.05 strain, increasing shear rate from 0.05 to 30 s–1 at 2 Hz, or for 30 minutes at 2 Hz constant frequency and 0.5 s–1 shear rate. A frequency of 2 Hz was chosen for the constant frequency experiments because it was found to be the region most sensitive to changes in moduli (Buchsbaum et al., 1984), and it was the frequency used by other groups that analyzed the vitreous humor (Nickerson et al., 2005).

13.2.3 Results and disscussion

Modulus (Pa)

10

G′ G≤

1

Modulus (Pa)

10

G′ G≤

1

moduli compare well with those obtained by other groups that analyzed the porcine vitreous humor by other methods. The viscoelastic properties obtained were used as the animal model for targeting the ideal vitreous substitute.

13.3 Vitreous substitutes

13.3.1 Background

A vitrectomy is a surgical procedure where the vitreous is cut and aspirated, and this is normally followed by replacement with an artificial substitute.

A vitrectomy is usually performed for relief of traction and removal of blood from the ocular cavity. Currently, gases (air, sulfur hexafluoride, or perfluoropropane), perfluorocarbon liquids, fluorosilicone oil, or silicone oil (polydimethylsiloxane) are used as temporary vitreous substitutes to tamponade the detached retina against the posterior of the eye. These substitutes are not satisfactory for several reasons. For example, depending on the location of the retinal tear, these substitutes may require the patients to position themselves face down for days (Colthurst et al., 2000). Silicone oil can be difficult to remove, has shown toxicity to intraocular structures, is capable of emulsification, and has been associated with glaucoma and corneal decompensation, both of which can lead to blindness (Leaver and Billington, 1989; Giordano and Refojo, 1998; Jonas et al., 2001). Silicone oil is only 70% effective in retinal reattachment (Jonas et al., 2001), and often

the patient has to undergo subsequent cataract surgery after use of silicone oil as a tamponade (Leaver and Billington, 1989). Most importantly, none of these clinically available substitutes can be left in the eye safely for more than a few months (Giordano and Refojo, 1998). While silicone oil has been successful in retinal reattachment in some severe cases, it is evident that a better long-term vitreous substitute is needed. Silicone oils, perfluorocarbon liquids, and gases as vitreous substitutes have been extensively reviewed (Leaver and Billington, 1989; Sparrow et al., 1990; Giordano and Refojo, 1998; Colthurst et al., 2000; Jonas et al., 2001; Versura et al., 2001; Wolf et al., 2003).

Initial research on vitreous substitutes focused on replacing the vitreous humor with vitreous from animals. However, because of the degradation of the vitreous outside the eye, these failed as vitreous substitutes (Deutschmann, 1906; Cutler, 1947). Next, researchers focused on developing vitreous substitutes from the natural components. However, the failure of collagen (Pruett et al., 1972; Pruett et al., 1974) Nakagawa et al., 1997; Nayak, 1999; Liang et al., 1998 and hyaluronic acid (Pruett et al., 1979; Nakagawa et al., 1997) to mimic the natural vitreous led to research into synthetic polymers. These synthetic polymeric vitreous substitutes, uncrosslinked and crosslinked hydrogels, are discussed below. Work with synthetic polymers began in the 1950s after poly(methyl methacrylate) was used in lens and cornea prostheses (Refojo, 1971).

In recent years, research of vitreous substitutes has focused on polymeric hydrogels, and these have been reviewed extensively (Chan et al., 1984; Chirila et al., 1998; Soman and Banerjee, 2003; Swindle and Ravi, 2007). However, these preformed equilibrium-swollen hydrogels disintegrate when injected and sheared through a small-gauge needle (Chirila and Hong, 1998). We have developed reversible hydrogels from acrylamide and N, N′-bis(acryloyl) cystamine (BAC), a disulfide crosslinker. The disulfide bonds were reduced to thiol groups, enabling purification and removal of all unreacted toxic monomer and low molecular weight polymers (Aliyar et al., 2004). Gel elasticity was maintained after injection in human cadaver eyes (Foster et al., 2006) and porcine eyes (Swindle et al., 2006) ex vivo. The patient would not have to remain face-down for extended periods of time as is required for vitrectomy with traditional materials (Aliyar et al., 2004). This work confirmed that these gels may be formed in the eye and that it is possible for a hydrogel to produce osmotic pressure in the vitreous cavity (Foster et al., 2006). The addition of a hydrophobic monomer, N-phenylacrylamide (NPA), to the acrylamide and BAC greatly improved biocompatibility. Toxicity tests on Chinese Hamster Ovary (CHO) cells showed a viability of approximately 100% after 5 days at a concentration of 15 mg/mL. Additionally, rheological testing showed that the storage and loss moduli of this hydrogel formulation matched those of the natural porcine vitreous (Swindle et al., 2008).

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The future of vitreous substitutes is to find a formulation that can be left in the eye in the long term. Additionally, it would be preferable to mimic the mechanical properties, water content, and light transmittance of the natural vitreous humor. Silicone oil, currently the most commonly employed vitreous substitute, accomplishes none of these things. Polymeric hydrogel vitreous substitutes developed and tested experimentally have proven capable of matching these properties. Rheological testing can help match the mechanical properties of the polymeric substitutes to those of the natural vitreous. Furthermore, the process of in situ gelation is the key to viable vitreous substitutes because polymers that are injected as gels rather than as liquids fragment due to shear, lose their elasticity, and can cause inflammatory reactions in ocular tissues. In the future, there will be further research conducted on in situ gelling polymeric vitreous substitutes. Polymers can be tailored to alter their mechanical properties to match those of the natural vitreous humor. The goal is to match those properties, have a transparent hydrogel that is 99% water, and to find a substitute that will not cause cytotoxic reactions and will be retained in the eye.

13.3.2 Experimental methods

Statistical experimental design

Statistical experimental design is a useful tool for rapid screening of candidates and for determining important factors. Scheffe simplex-lattice designs were developed specifically to analyze mixture designs. Mixture design of experiments is used to determine the optimum concentration of chemical constituents that elicit a particular response while minimizing the number of experiments to be run. In a mixture experiment, the independent factors are proportions of different components of a blend and must sum to 100%. The purpose of the mixture design is to be able to predict empirically the response of a mixture for any combination of ingredients, or measure the influence of each component singly and in combination with others on the response (Anderson and Whitcomb, 2000). A range of polymeric hydrogel formulations may be analyzed using a mixture design with the monomer components along the backbone treated as mixture variables and the polymer concentration in the final hydrogel as a process factor.

StatEase Design-Expert 7.1 software (Minneapolis, MN, USA) enabled rapid selection of experimental parameters and analysis of data. The fundamentals are rooted in statistical experimental design, in which the test formulations chosen have monomer feed ratios and polymer concentrations within the ranges specified, as well as midpoints to determine non-linearity and error.

The assumptions used in factorial design apply to mixture design. After the experimental data were obtained, analysis of variance (ANOVA) was used

Reductive liquefaction and purification

After exhaustive washing with double distilled water, hydrogel formulations were reduced from the S—S crosslinks to linear polymers with S—H groups with DTT. In order to improve the biocompatibility and decrease polydispersity, the reduced polymer solutions were dialyzed with Spectra/Por tubing with a 25 kDa molecular mass cut-off, in nitrogen-bubbled water at pH 4 to prevent oxidation. The dialysis washing solution was changed three times over 72 hours. After dialysis, the polymers were precipitated in either acetone or a 25:75 mixture of methanol and diethyl ether. Precipitation in acetone was advantageous when large batches were produced because the solvent was more readily available than the diethyl ether. However, precipitation in diethyl ether produced a white powder that did not aggregate as it had a tendency to do in acetone. The precipitate was freeze-dried in a lyophilizer and was stored under vacuum to prevent oxidation.

Regelation

The reduced polymers were regelled by air oxidation at physiological pH of

7.4 in Dulbecco’s phosphate buffered saline (DPBS). Figure 13.6 shows the complete reaction scheme of the AA copolymer, and the reaction mechanism for oxidative regelation of the polymers with air is shown in Fig. 13.7.

Table 13.1 Poly(acrylic acid) hydrogel compositions tested