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

13.6 Acrylic acid copolymer regelation procedure: A, initial polymerization and crosslinking; B, reduction, C, regelation in situ.

AB—SH + –OH AB—S– + H2O

AB—S– + O2 AB—S• + O2

2 AB—S• AB—SS—AB

13.7 Free radical regelation procedure. AB, acrylamide or acrylic acid; B, BAC crosslinker; SS, crosslinked disulfide bond; SH, thiol group when not cross linked.

Hydrogel analysis

An Abbe refractometer was used to measure the refractive index of each tested sample (ATAGO Abbe Refractometer NAR-1T, Kirkland, WA, USA). The testing was done at a visible light wavelength of 552 nm at 37 °C. Storage and loss moduli of the hydrogels were determined using the Vilastic-3 oscillatory capillary rheometer. All samples were evaluated at 37 oC at 2 Hz, with the shear rate increasing from 0.1 to 100 s–1. Additionally, the samples were analyzed at increasing frequency at a constant shear rate. Several samples were too stiff to be analyzed on the capillary rheometer, so they were analyzed on the cone and plate rheometer by frequency scan at 5% strain or strain scan at 2 Hz frequency.

Toxicity testing was carried out following the tetrazolium-based colorimetric (MTT) assay described by Bruining et al. (2000), in which 3-(4,5-dimethylthiol-2-yl)-2,5-diphenyl tetrazolium bromide (thiazolyl blue) is converted to formazan, an insoluble precipitate, by an enzymatic reaction performed only by living cells. Human retinal pigment epithelial (RPE) cells (ARPE-19, American Type Culture Collection (ATCC), Manassas, VA, USA) were used for cytotoxicity evaluation because they are the cells that would actually come into contact with the vitreous substitute in vivo.

RPE cells were plated at a density of 15 000 cells per 16-mm well in 1 mL of 10% calf serum (CS) minimum essential medium (MEM) with

352 Biomaterials and regenerative medicine in ophthalmology

antibiotics. Media were aspirated after 24 hours and replaced with fresh media containing polymeric materials. Cells were incubated 72 hours. Media containing polymer were replaced by 0.5 mL 10% CS M199 medium without phenol red with 0.65 mg/mL thiazolyl blue. After 4 hours, the media were aspirated and formazan precipitate was dissolved in 200 μL dimethylsulfoxide. Absorbance was measured at 540 nm in a microtiter plate. All samples were tested in quadruplicate. To test for toxicity, polymer solutions of varying concentrations were added to acidified (pH 6) MEM media, dissolved, and gelled by air oxidation at pH 7.4 at final concentration of 15 mg/mL in 10%

CS MEM. The viability was calculated as a percentage of absorbance of the test sample with respect to the control sample (Bruining et al., 2000).

13.3.3 Results and discussion

The adhesive property of the polymer was one of the reasons why PAA was selected as a potential vitreous substitute (Park and Robinson, 1987). The natural vitreous humor attaches to the retina at several points, and it would be desirable to replace the vitreous with a hydrogel that could adhere to the retina and could mimic those attachment points.

Table 13.2 summarizes the PAA formulations tested, and the results for storage modulus, loss modulus, and refractive index. More midpoints, axial points, and replicates were chosen for the PAA in an attempt to gather information about the non-linear factors that may contribute to the modulus of the hydrogel vitreous substitutes.

Figure 13.8 plots the refractive indices of the hydrogel formulations and compares them to the natural vitreous humor. The noticeable trend is that the refractive index is dependent primarily upon polymer concentration in the hydrogel, as expected. It is also important to note that hydrogel formulations near 1% polymer concentration match the refractive index of the vitreous humor. By comparison, the refractive index of silicone oil is 1.4.

Figures 13.9 and 13.10 show storage modulus and loss modulus, respectively, versus frequency at 5% strain for all PAA hydrogels analyzed. Even at 1% polymer concentration, these hydrogels were stronger than the natural vitreous humor. All formulations have a higher storage modulus than loss modulus and act as viscoelastic solids, like the natural vitreous humor.

For analysis of the storage and loss moduli, the data underwent a log transformation. The retractive index results required no data transformation. A quadratic Scheffe model was used to correlate the data. The models for storage modulus, loss modulus, and retractive index were all significant, with p values of 0.0001 for storage modulus and retractive index, and 0.0004 for loss modulus. The p, R2, and F values are summarized in Table 13.3.

The resultant equations produced to predict the values of storage modulus, loss modulus, and refractive index are shown below. In the equations below,

AA, BAC, NPA, and Gel refer to the percentage concentrations. For example, 1% AAB5N5 would have the input values of 1 for Gel, 90 for AA, 5 for BAC, and 5 for NPA.

log(G′) = 0.618 ∞ BAC + 0.514 ∞ NPA – 0.016 ∞ AA – 0.128 ∞ BAC ∞ NPA – 0.002 ∞ BAC ∞ Gel – 0.025

Statistical analysis of the results using the targeted values for the refractive index and moduli shown in Table 13.4 produced an optimal hydrogel formulation of 1.20% AAB3N4, or a hydrogel containing 1.20% polymer by weight with 3% crosslinker and 4% NPA along the backbone. The predicted

1% AAB3N3

1% AAB3N5

1% AAB4N4

1% AAB5N3

1% AAB5N4

1% AAB5N5

1.5% AAB4N3

1.5% AAB4N4

1.5% AAB4N5

2% AAB3N3

2% AAB3N5

2% AAB4N4

2% AAB5N3

2% AAB4N5

2.5% AAB4N5

3% AAB3N5

3% AAB4N4

3% AAB4N4

3% AAB5N3

3% AAB5N4

3% AAB5N5 Porcine vitreous

13.10 Poly(acrylic acid) loss modulus versus frequency at 5% strain.

1% AAB3N3

1% AAB5N5

1% AAB4N4

1% AAB5N3

1% AAB5N4

1% AAB5N5

1.5% AAB4N3

1.5% AAB4N4

1.5% AAB4N5

2% AAB5N3

2% AAB3N5

2% AAB4N4

2% AAB5N3

2% AAB5N5

2.5% AAB4N5

3% AAB3N3

3% AAB3N5

3% AAB4N4

3% AAV5N3

3% AAB5N4

3% AAB5N5 Porcine vitreous

surgery ophthalmic in substitutes vitreous as Hydrogels

and experimental values for this formulation are shown in Table 13.5. The storage and loss moduli of 1.2% AAB3N4 are plotted against frequency in Fig. 13.11.

The statistical model results are represented graphically in several plots. The darker shaded portions of the figures illustrate the regions that have storage modulus values between 5 and 15 Pa, loss modulus values between 0.5 and 5 Pa, and refractive index values between 1.334 and 1.336. Figures 13.12 to 13.14 show the targeted areas for 0.75%, 1.00%, and 1.25% hydrogels, respectively. A 1% hydrogel is a desirable formulation to owing due to its similar water content to the natural vitreous humor and lower viscosity than formulations containing higher polymer content.

The following plots represent graphically the full range of possibilities for PAA formulations that could behave as biomimetic vitreous substitutes. Figures 13.15 to 13.17 show storage modulus, loss modulus, and refractive index, respectively. Finally, the darker shaded region of Fig. 13.18 shows all possible PAA formulations that fall within the targeted values of the optomechanical properties of the ideal vitreous substitute.

Microindentation of several formulations of PAA hydrogels at 7.5% polymer concentration revealed a trend in elastic modulus. The results are summarized in Table 13.6. As expected, an increase in crosslinker

G′

2.00

20%

1.50

30.0863

Gel (%)

20.1583

1.00

15.0558

10.0275

0.50

13.15 Model predictions for poly(acrylic acid) storage modulus.

concentration increased the modulus of the hydrogel. However, it is also noticeable that an increase in the NPA content decreased the modulus. This is due to ineffective crosslinking in the hydrogels containing more NPA. These hydrogels often form more intramolecular crosslinks because of the clustering of the hydrophobic groups along the polymer backbone.

In addition to the determination of the refractive index, storage modulus, and loss modulus, the in vitro toxicity of PAA was determined using RPE cells. The results of four formulations are shown in Fig. 13.19. Upon first glance, the PAA hydrogel vitreous substitutes appear to be more toxic than the polyacrylamide vitreous substitutes (Swindle et al., 2008). There are a few confounding factors. The polyacrylamide formulations were tested using CHO cells, which are less sensitive than RPE cells. In addition, the PAA formulations are far more viscous and adhere to the cells, so some error resulted when the cells stuck to the polymer samples and therefore did not remain in the culture dish, resulting in an artificially diminished number due to aspiration rather than cell death. These results are not discouraging because the toxicity shown corresponds to the linear polymer chains and

not the less toxic crosslinked hydrogels. An important trend to note is that the higher the concentration of NPA along the backbone, the higher the in vitro biocompatibility. This was also noted in preliminary toxicity studies with CHO cells and polyacrylamide hydrogels.

The refractive index and viscoelasticity of the natural vitreous humor can be mimicked by a number of different formulations of PAA copolymeric hydrogels. The optimal formulation determined by the statistical design models had refractive index, storage modulus, and loss modulus values very close to ose of the natural vitreous humor.

13.4 Osmotic pressure

13.4.1 Background