In vitro organotypic culture method to evaluate the biocompatibility of heparin-surface-modified intraocular lenses

In vitro organotypic culture method to evaluate the biocompatibility of heparin-surface-modified intraocular lenses S. Milazzo, M.D., M.F. Sigot-Luizard, M.D., M. Borhan, M.D., G. Montefiore, M.D., P. Turut, M.D., H. Saraux, M.D.

ABSTRACT Using an organotypic culture method, we evaluated the biocompatibility of two kinds of intraocular lenses: conventional poly(methyl methacrylate) (PMMA) and heparin-surface-modified (HSM) PMMA. Chicken corneal endothelium from embryos incubated for 14 days was placed on an agar medium and covered with one of three types of materials: PMMA, HSM PMMA, and a control, Thermanox®. Experiments at five different times measured cell migration, cell multiplication, and cell adhesion. Scanning electron microscopy showed a low level of corneal endothelial adhesion on the HSM IOL surface. There was a significant difference between the HSM and untreated lenses in migration surface and cell density, with the HSM lenses having better biocompatibility.

Key Words: biocompatibility, corneal endothelium, heparin surface modification, organotypic culture, poly(methyl methacrylate)

Several recent studies have explored ways to increase the biocompatibility of intraocular lenses (10Ls). 1- 3 Although these studies have shown that modification of the IOL's surface can increase biocompatibility, postoperative reactions are still observed when these modified lenses are implanted. We used an in vitro organotypic culture model to evaluate the biocompatibility of two kinds of intraocular lenses: conventional PMMA and heparin-surface-modified (HSM) PMMA. We compare our results with those of other studies.

MATERIALS AND METHODS Under strict asepsis, corneas from chicken embryos that had been incubated for 14 days at 37 degrees Celsius were removed under a microscope using microsurgical instruments on a gelose support. The corneas were placed in sterilized saline solution to prevent drying. Four I mm 2 sections were removed from each cornea. The explants were placed so that the endothelial face was in contact with one of two materials: PMMA or HSM PMMA. As a control, a group of explants was

covered with 1 cm2 of Thermanox® (NUNC Inc., Naperville, IL), an inert plastic treated with a cell culture. The culture medium comprised 50% mixture of MEM Iscove 74%, fetal veal serum (SVF) 20%, tricine 4%, glutamine 2%, and 50% gelose at I% agar. Ten dishes of 12 controls, four dishes of I2 HSM PMMA lenses (Kabi Pharmacia, Stockholm), and four dishes of I2 PMMA lenses (Kabi) were examined at five different times. This method provides a quantitative measurement of cell multiplication, migration, and adhesion.

Surface Measurement

At the end of the incubation period, the dishes were opened. The samples were turned over and covered with a drop of isotonic solution of neuter red (2% of lsoton II® filtered at 0.45 Jlm), which permits visualization of cells and prevents cell deterioration during cell counting. 4 •5 After a few minutes of coloration, the samples were rinsed and covered with a drop of Isoton II to prevent drying during measurement. The cells were examined with a scanning electron microscope. Using a device developed by Duval and

From the Clinique Ophtalmologique, Centre Saint- Victor, A miens, France (Milazzo, Borhan, Turut); the Hopi tal Saint Antoine, Paris (Montejiore, Saraux); and the Centre de Recherche Royalieu, Compiegne, France (Sigot-Luizard). Reprint requests to S. Milazzo, M.D., Clinique Ophtalmologique, Centre Saint- Victor, 354 boulevard de Beauvil/e, 80054 Amiens Cedex, France. 638

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DENSITY

(cells/mm2) 20000

...

instilled in each petri dish and then removed with a syringe after 30 seconds. The process was repeated at five, ten, 30, and 60 minutes. After 60 minutes, 4 ml of nondiluted solution was added and removed after 15 minutes. At each time period, the detached cells were counted with· a Coulter® counter. These values were entered into a computer and used to calculate cell density (number of cells/surface units) using the average migration value. These values yielded a cell disconnecting curve (adhesion curve).

PMMA

HSM

...

PMMA

10000

Statistical Analysis

...

T(-)

0+-~--r-~~--~----~--~~

0

Fig. 1.

DENSITY

40000

2

4

8

6

SURFACE

10 (mm2)

(Milazzo) Cellular density as a function of surface; T(-) = Thermanox.

.r------------------------------, HSM PMMA

30000

PMMA

We assigned a key to each culture and entered the key into a computer reference file. Using standard statistical parameters (mean, root-mean-square, variance), we took the results from each time frame and calculated the cell migration, number of cells per explant, and cell density. These results yielded a cell density diagram as a function of migration veil surface and a cell disconnecting curve (percentage of disconnected cells as a function of time) and the abscissas-axis (time in minutes), which shows the cell adhesion properties of each sample. Adhesion was ranked according to a system used in previous studies: 5 •6 strong adhesion, fewer than 3,000 cells per minute; medium adhesion, 3,000 to 4,500 cells per minute; weak adhesion, more than 4,500 cells per minute. This technique uncovers characteristics of the tissue-material interface that probably would be masked in an in vivo process.

RESULTS One dish of control samples became infected and was eliminated from the study. Tables 1 and 2 summarize the findings of the study. SURFACE 2 The chicken embryo corneal endothelium showed 1,1 9,5 strong cell migration in the control sample (Figure 1). Fig. 2. (Milazzo) Histogram of cellular density as a function Thermanox was found in a zone of strong surface with of the surface; T(-) = Thermanox. low density. The HSM PMMA and untreated PMMA, however, were vertically grouped in a zone of thin surface with high density, indicating that cell migration was coauthors, 4 the ex plants' migration surfaces and migra- weaker or more inhibited than in the control samples tion veils (i.e., difference between total surface and ex- (Figure 2). The HSM samples had the highest rate of cell plant surface) were scanned by digital computer and multiplication (cell density). We used the nonparametric Kruskal-Wallis test to stored in an Apple computer. compare our results. There was a significant difference between HSM PMMA and untreated PMMA in migraAdhesion tion surfaces (P = .020) and cell density (P = .042). of solution enzyme an with treated The explants were Scheffe's parametric test showed a significant difference was trypsin-EDTA diluted in Isoton II. This solution Table 1. Cytocompatibility of the three materials. Material

Surface (mm 2 )

Thermanox PMMA HSMPMMA

9.54 ± 3.65 2.05 ± 1.57 1.07 ± 1.02

Density (mm 2 ) 2,604.44 ± 1,450.11 8,715.50 ± 6,240.05 17,494.75 ± 13,836.69

Area (cell ± 3,856.11 ± 3,802.50 ± 4,151.00 ±

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minute) 220.53 280.54 508.45

Adhesion Medium Medium Medium 639

other materials. The PMMA response was closer to that ofthe control (Figure 3). Scanning electron microscopy confirmed the statistical findings (Figures 4 to 6). There was much less cell 80 adhesion to the HSM PMMA than to the untreated PMMA. As time progressed, the PMMA reaction was 60 similar to that of the control. The disconnecting curve values became statistically significant only after the tenminute mark (P = .06). The difference was insignificant 40 T(-) (P = .42) when measured by the Kruskal-Wallis test. PHSM PMMA We used the average of all samples of a material to 20 PMMA determine its cell adhesion, which was calculated from the area of the curve. Values ranged from 3,000 to 4,500 cells per minute. The weakest adhesion, shown by the 5' 10' 20' 30' 60' highest area, was found in the HSM PMMA samples (Figure 7), although the values for the HSM PMMA Fig. 3. (Milazzo) Cell disconnecting curve as a function of (4,151.0 ± 508.4) and untreated PMMA (3,802.5 ± time; T(-) = Thermanox. 280.5) were very close. Disconnection (%)

100

DISCUSSION

AREA

cell/om

5000

4000 3000 2000 1000 0 T(-) Fig. 4.

HSMPMMA

PMMA

(Milazzo) Cell adhesion; T(-) = Thermanox.

(P = .004) between the surface curve/density of the

Thermanox control and the HSM PMMA. The cell dis~ connection curve showed that HSM PMMA had a quicker and more intense response to the cells than the

The principle of in vitro cultures is to keep an organ, tissue, or individual cell alive outside the organism. The terminology committee of the American Association of Tissue Cultures7 distinguishes between a culture in which the organ preserves its function and structure, a tissue culture that has a specific function, and a cell culture, in which the cells or unorganized cells are allowed to grow freely, ensuring precise function with a specific metabolism. The cell culture techniques proposed by national and international standards organizations remain the most commonly used. They are reproducible, allow the indepth study of human cells, and provide fast, quantitative evaluation of the cytotoxicity of specific materials. Yet these techniques are of limited value in a clinical ophthalmology setting because they evaluate only one type of cell and occur in a strictly regimented setting. Thus, we chose an explant, or organotypic, culture technique in which a freshly removed biopsy is cultivated in an upright, exposed position. This system maintains the functions of and interactions between different cells. The principle of this technique is to remove in vivo tissue and maintain it in vitro. The explanted tissue or organ is cultivated on the surface of a semisolid medium, which preserves the interaction between the different cell types that make up a tissue or organ. Under these conditions, the tissue or organ survives without metabolic

Table 2. Cellular disconnecting by time with epithelium in direct contact with material. Minutes Material Thermanox PMMA HSMPMMA 640

5 11.4 ± 9.3 14.7 ± 4.9 21.0 ± 6.2

20

30

28.3 ± 9.7

60.7 ± 12.0

83.3 ± 6.2

95.8 ± 3.0

31.5 ± 6.1

48.2 ± 6.2

76.5 ± 16.3

94.0 ± 1.4

45.0 ± 12.7

62.2 ± 19.9

83.0 ± 12.1

96.5 ± 1.0

lO

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60

Fig. 5.

(Milazzo) Scanning electron microscopy ofThermanox cells.

Fig. 6.

(Milazzo) Scanning electron microscopy of PMMA cells.

modifications. With this primary culture method, the untreated PMMA used as a substrate. Our in vitro study cells do not adapt to the culture medium, as is the case indicates that surface-modified material also inhibits cell growth more than untreated PMMA does. with cell colony cultures. Our results lead to two possibilities: ( 1) the modified In a study of the behavior of organotypic explants in a semisolid medium, Wolff and Marin 8 found that cell surface is toxic and the cells die as they leave the explant; migration of an explant in contact with a material de- (2) the HSM PMMA surface is unfavorable for cell mipends on the synthesis of the extracellular matrix. The gration and adhesion. There was a significant difference matrix spreads on a support, such as Plexiglas or glass, (P = .020) between HSM and Thermanox migration but it cannot accumulate around the explants without surfaces. Scanning electron microscopy confirmed our statisa biocompatible medium. Simple contact with the findings, which agree with in vitro and in vivo tical explant; the outside migrate to cells allows support in the literature. studies histioa into therefore the organotypic explant evolves in vitro study, 1 the development of fibroblasts one In is descendants typic culture, in which a group of cell allowed to grow, ensuring precise function with specific showed that surface modification greatly reduces the initiation of in vitro development. A comparative study metabolism. endothelium contact with freshly excised rabbit corof Later studies confirmed that the level of cell adhesion 11 found that tissue lesions are more severe with an neas is proportional to cytocompatibility. Sigot-Luizard et 10 9 6 5 PMMA lens than with an HSM PMMA IOL. untreated al. • • • used this concept to evaluate quantitatively the experiments by Larsson and coauthors 12•13 vitro In cytocompatibility of biomaterials. The studies focused on the migration veil generated by organ fragments in found very high platelet adhesion with PMMA IOLs; adhesion was practically nonexistent with the surfacecontact with materials. The choice of culture medium depends on the tissue to be cultivated. In the Iscove culture medium, theserum is replaced by purified active ingredients. Very rich in glucose (4.5 g/1), this medium is especially useful in organ typification. Poured into 60 mm diameter petri dishes, it allows cultivation of explants in an exposed, upright position. The work of Sigot-Luizard et al. 10 showed the importance of cell adhesion and celi density in determining the cytocompatibility of a material. Their study was based on the quantitative and reproducible measure of three biological properties of tissue-material interface: cell migration, multiplication, and attachment (adhesion) to the support. Our study found that cell density was much greater in the HSM IOL samples than in the untreated PMMA. All preclinical studies have demonstrated that various cell Fig. 7. (Milazzo) Scanning electron microscopy of HSM PMMA cells. types cultured on HSM PMMA adhere less than on TM

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modified lenses. There was also a weaker in vitro attachment of microphages to the surface of HSM lens than to that of the untreated IOL. An in vivo study ofiOL implantation in the posterior chamber of rabbit eyes found that 24 hours after IOL implantation, the number of leukocytes in the aqueous was three times higher in eyes with a PMMA IOL than in those with an HSM lens. 13 Yet, no significant difference was found after three, seven, and 15 days. A new method based on a mixture of the monoclonal antibody anti-C3a has underscored that modified-surface PMMA does not significantly increase C3a as compared with a control serum but that PMMA does. 14 After the implementation of IOLs in the posterior chamber of monkeys, 2 the inflammatory reaction of the anterior chamber was greatly reduced in the eyes with an HSM IOL at four, eight, and 18 weeks. The results were the same with anterior chamber IOLs. 3 In another in vivo study, 68 patients received posterior chamber HSM IOLs implanted in the lens capsule with no unusual complications or reactions. 15 Another recent trial series 16 demonstrated that HSM IOLs cause much less secondary inflammation than PMMA IOLs, especially in high-risk patients. 14 In conclusion, our study found that biocompatibility is improved when the PMMA surface is modified with immobilized heparin. Trials are currently in progress to evaluate the clinical value of heparin surface modification of IOLs.

5. 6.

7. 8. 9.

10.

11.

12.

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