Ultrastructure of cells cultured onto various intraocular lens materials

Ultrastructure of cells cultured onto various intraocular lens materials Piera Versura, B.S.D., Roberto Caramazza, M.D.

... . .. .. ' ABSTRACT Therespouse .of three cell types (human fibroblasts, mGDOcytes, and platelets),. cultured Gr seeded onto. different intraGcular lens m~terials~ was analyzed by .ultrastructural· examination. The mate.' rhuscollsidere
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Although poly(methyl methacrylate) (PMMA) is the material used for the manufacture of the majority of implanted intraocular lenses (IOLs), several clinical concerns about its use have arisen. These issues have been partially addressed by a chemical and/or physical modification of the basic PMMA to change its surface characteristics or by the use of soft hydrogel IOL material as an alternative to the PMMA.l In this study we considered the following: (1) heparin surface modified PMMA (HSM-PMMA), a material derived from the covalent end point attachment of heparin molecules which achieves a complete and continuous coverage of the PMMA 2,3; this modification has already been applied to the surfaces of vascular prostheses 4 ,5; (2) plasma-treated PMMA (PT-PMMA), a material obtained by a physical treatment that breaks the hydrophobic bonds through partial oxidation and introduction of the hydrophilic group -OH; (3) hydrogel, a poly-HEMA with a 38% water content. The in vitro tests ofbiocompatibility used highly reproducible cell cultures which were analyzed by

standardized techniques to assess their health and behavior. The in situ correlative ultrastructural studies provide information on those specific cells closely adherent to the material under investigation. These are the cells that playa crucial role in the recruitment of all the other competent elements onto the implant surfaces in vivo. From an ultrastructural standpoint the present study analyzes various cell types cultured onto different IOL materials. The behavior of human fibroblasts was analyzed to evaluate the in vitro attachment of mesenchymal cells. This reaction occurs quite commonly in pseudophakic eyes and often leads to synechial formation. Monocytes from peripheral normal human blood were also considered because of the interrelationship between the macrophages and the multinucleated giant cells present on the IOL surfaces, a feature indicating the onset of a foreignbody reaction against the prosthesis. In addition, the degree of adhesion of normal human platelet onto the various materials was evaluated.

Reprint requests to Dr, Piera Versura, Institute of Ophthalmology, University of Bologna, Via Massarenti, 9, 40138 Bologna, Italy, 58

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MATERIALS AND METHODS Small discs (1 cm diameter) of PMMA were obtained from a blank; they were carefully smoothed at both sides, polished and cleaned, washed several times in phosphate-buffered saline (PBS, pH 7.4), and air dried. Some were sterilized and then used in the tissue cultures as control specimens; others were surface modified as described. The HSM-PMMA was prepared according to the method described by Olsson et al. 3 The plasma treatment consisted of PMMA discs set in an oxygen rarefied atmosphere, then treated for two minutes at room temperature with an electromagnetic field at 13 MHz frequency and 100 W/cm 2 power. As reported for other physical processes based on similar procedures of surface modification, 6 this method also renders the molecular layers at the surface more highly oriented and therefore reduces the concentration of the surface defects. The hydrogel we studied was a poly-2-hydroxyethyl-methacrylate (poly-HEMA) containing 38% water, named PT64, from Ciba Vision, Italy, a material proven to be biocompatible for the manufacturing of contact lenses. Human fibroblasts, monocytes, and platelets were cultured or seeded onto the PMMA, HSMPMMA, PT-PMMA, and the hydrogel, as follows. The human fibroblast cell line IMR-90 was used in the experiments. We seeded 20,000 cells in 20 III Dulbecco's modified Eagle medium, pH 7.3, supplemented with 20% Ham's F-12, 10% newborn calf serum (NCS), and 1 % antibiotic antimycotic solution (AAS) (all reagents purchased from Gibco) on each disc. The discs were set in a 24-well tissue culture plate. After adherence for two hours, 1 ml of the culture medium was gently added to each disc-containing well. The cells were incubated for four days at 5% CO 2 in air at 37°C. Cells from the 15th to the 23rd passages were used. Monocytes from human leukocyte-rich normal plasma were isolated by hyperosmotic density gradient centrifugation on hycodeny R monocytes (from Nyegaard). We seeded 20,000 cells in 20 III RPMI 1640, supplemented with 20% NCS and 1 % AAS, on the discs and the tissue culture plate was placed in a humid chamber at 5% CO 2 in air at 37°C. After adherence for three hours, 1 ml of the culture medium with 10% NCS was gently added to each disc-containing well. The cells were incubated up to five days. Normal human EDT A blood was centrifuged at 200 g for ten minutes to obtain plasma. We spun 10 ml plasma at 1,000 g for ten minutes and the platelet pellet was resuspended in 5 ml of the culture

medium used for the fibroblasts. We then placed 20 III of the obtained cell suspension on each disc. The cells were allowed to adhere for two hours in a humid chamber at 5% CO 2 in air at 37°C; 1 mlof the culture medium was then added. Four different discs of the same material were used at the same time for each experiment. The experiments were repeated five times. After the specified time, the discs were removed from the culture medium and fixed in 2.5% glutaraldehyde dissolved in 0.1 M phosphate buffer for one hour. After an overnight washing in 0.15 M phosphate buffer, the specimens were fixed in 1 % OsO 4 in Veronal buffer and dehydrated through an ascending series of ethanol. They were divided in half to obtain information from the same disc by transmission (TEM) and scanning (SEM) electron microscopy. For TEM the specimens were embedded in Epon, ultrathin sections were cut with a diamond knife, collected on grids, counterstained with uranyle acetate and lead citrate, and observed with a Philips 400 T. For SEM, the specimens underwent criticalpoint drying, were mounted on aluminum stubs with silver conducting paint, and sputtered with a thin layer of pure gold to limit the charging artefacts during observation. They were examined with a Philips 505 SEM operating in a range of 5 to 10 kV. RESULTS Fibroblasts After four hours the cells cultured onto PMMA and PT-PMMA flattened and large areas of the discs appeared to be covered by them. After the same hours, the fibroblasts on the HSM-PMMA and the hydrogel were still rounded and no flattening had occurred. After one day, the PMMA discs were completely covered by spindle-shaped and confluent fibroblasts, which subsequently formed three to four layers. The same process was observed on the PT-PMMA, but this culture formed a monolayer. The cells on the HSM-PMMA were dramatically reduced in number; they appeared to be rounded and stuck to each other, with no tendency to flatten and spread. Some residual cell debris was observed on the hydrogel surfaces. This situation remained unaltered to the time of disc removal (Figures 1 to 4). The submicroscopic cytology of the cells cultured onto PMMA, as shown by TEM, was characterized by large nuclei with prominent nucleoli and several profiles of rough endoplasmic reticulum . The cytoskeletal elements were regularly arranged

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Fig. 1.

(Versura) Fibroblasts cultured onto PMMA. The cells (f) are elongated, confluent, multilayered, and cover all the material surface (SEM 420 x).

Fig. 3.

(Versura) Fibroblasts cultured onto HSM-PMMA. The cells (f) appear to be round, stuck to each other, and to the material (arrows), but do not show any evidence of flattening and elongation (SEM 200 x).

along the cortical area of the cytoplasm, and the microtubuli were located at the cell front edge. Numerous points of focal contact with the underlying disc, resembling the so-called "podosomes," were observed at the interface (Figure 5). The fibroblasts on the PT-PMMA surface ap-

peared to have reduced the podosomes and lost the normal arrangement of the cytoskeletal elements. Although nuclei and nucleoli were prominent, the cytoplasm of the cells more closely adherent to the discs were filled up with large lysosomes (Figure 6). The few cells adherent to the HSM-PMMA sur-

Fig. 2.

Fig. 4.

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(Versura) Fibroblasts cultured onto PT-PMMA. The cells (f) are elongated but not always confluent, and large areas of the substrate are seen (*). In some points the cells are two to three layered (arrows) (SEM 360 x).

(Versura) Fibroblasts cultured onto hydrogel. Only rare cellular debris (arrows) are seen on the material surface (SEM 740 x).

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Fig. 5.

(Versura) Fibroblasts cultured onto PMMA. The cells appear to be healthy and actively synthesizing, as demonstrated by the prominent, rough endoplasmic reticulum (rer). Points of focal contacts with the substrate are numerous (po do somes = p) (TEM 28,000 x).

face appeared to be normal in cell organelle content, but they were devoid of the podosomes and the cytoskeletal elements were completely disorganized (Figure 7). We cannot document any ultrastructural feature of the inner organization of the fibroblasts cultured

Fig. 6.

(Versura) Fibroblasts cultured onto PT-PMMA. The cytoplasm of the cells appears to be filled up with large lysosomes (arrows) (TEM 10,200 x).

Fig. 7.

(Versura) Fibroblasts cultured onto HSM-PMMA. The front edge of the cell is devoid of microtubules and the micro filaments arranged along the cortical cytoplasm appear to be interrupted (arrows) (TEM 26,000 x).

onto the hydrogel. Because of the paucity of their presence on this material, we could not observe any grid containing a complete cell section. Monocytes The monocytes cultured onto the PMMA were intermingled with platelets and polymerized fibrin filaments, all of which almost covered the disc surface (Figure 8). These cells and serum proteins, although still contemporaneously present, appeared to be greatly reduced in number on the HSMPMMA discs. The surface morphology, as observed by SEM, showed ruffled and flattened monocytes on the PMMA, while these same cells on HSM-PMMA did not display any tendency to spread and adhere more closely to the disc in an attempt to phagocytose it. No differences between these two materials were observed in cell organelle content and ultrastructural morphology; the cells appeared to be normal in both cases, but of course a correct conclusion about the state of resting or activation of a cell when in contact with a given substrate cannot rely solely on its morphological analysis. Ruffled and flattened monocytes were also seen on the PT-PMMA, intermingled with granulocytes, platelets, and a few fibrin filaments (Figure 9); by TEM examination the cells appeared to be normally constituted. Very rare cell debris and no cells at all were seen on the hydrogel surfaces (Figure 10).

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Fig. 8.

(Versura) Monocytes cultured onto PMMA. The cells (m) are intermingled with platelets (p) and polymerized fibrin filaments (arrows) . Small areas of the material are uncovered (.) (SEM 390 x).

Fig. 10.

(Versura) Monocytes cultured onto hydrogel. Only rare cellular debris (arrows) are observed (SEM 2,260 x) .

Platelets The platelets seeded onto the PMMA discs displayed all the forms that indicate a state of activation; that is, the intermediate and fully spread forms. Transmission electron microscopy confirmed the peculiar arrangement of the cytoplasmic organelles. The platelets on the PT-PMMA

were mainly the dendritic form (Figure 11) while those on the HSM-PMMA showed only the round and abortive forms, suggestive of a resting state. The hydrogel surfaces had very few round cells (Figure 12). The results obtained for all three cell types are summarized in Tables 1, 2, and 3.

Fig. 9.

Fig. II.

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(Versura) Monocytes cultured onto PT-PMMA. The cells (m) appear to be rumed and flattened. A close interrelationship with granulocytes (g) and platelets (p) is demonstrated (SEM 2,100 x).

(Versura) Platelets cultured onto PT-PMMA. The cells (p) show mainly the dendritic form (SEM 3,000 x) .

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Table 2. Summary of the results from the monocyte cultures. The monocytes cultured onto -PMMA appear to be in a phase of activation -PT-PMMA are mixed with granulocytes and platelets -HSM-PMMA are present to a lesser extent and do not display any sign of activation -Hydrogel are extremely few ; only cell debris is present

The majority of the synthetic materials used to replace human body structures have been developed to enhance their full integration with the surrounding tissues . They were selected for chemical or physical characteristics that would facilitate colonization and adherence of the relevant cells in vivo. Intraocular lens materials are somewhat different. They should theoretically display surface features that inhibit the deposition of cells, primarily from the aqueous humor, since the optical function of the IOL could be compromised. Various materials have recently been produced to modify the surface of the most popular IOL

material, PMMA, or to develop soft materials such as hydrogels with varying water content. These new biomaterials have reflected the well-recognized fact that cell adhesion and proliferation tend to decrease on negatively charged surfaces. 7 This probably occurs because at physiological pH values the cells also carry a negative surface charge; therefore a barrier of electrostatic nature could account for the prevention of cell attachment. In addition, the degree of cell attachment decreases with increasing surface tension 8 and increases with wettable materialsY The cells under investigation showed a differential behavior when cultured or seeded onto the materials we tested. In a general way, the adhesion of fibroblasts, monocytes, and platelets occurred to the greatest degree on PMMA and was progressively less on PT-PMMA, HSM-PMMA, and polyHEMA. Fibroblasts appeared to loose their adherence capability as a consequence of disarrangement of the cytoskeletal filaments. It is recognized that a contact cell substrate occurs early, mediated by specific ligand-receptor-like interactions; this results in a reorganization of the microfilaments and microtubules, which then flattens the cell. On the basis of our morphological data, we believe that specific receptors localized on the plasma membranes of cultured fibroblasts fail to recognize

Table 1. Summary of the results from the fibroblast cultures.

Table 3. Summary of the results obtained from the platelet seeding.

The fibroblasts cultured onto

The platelets seeded onto

-PMMA are 3 to 4 layers thick and confluent; display podosomes and a normal cytoskeleton

-PMMA display the basic form of resting and activated state

-PT-PMMA are monolayered, spread but not confluent, do not show podosomes and a normal cytoskeleton; the cytoplasms are degenerated

-PT-PMMA display a dendritic form

-HSM-PMMA are scattered and unspread, lack focal contacts with the material; the cortical cytoskeletal filaments appear to be disorganized

-Hydrogels are extre mely few and in 0/+ round/abortive forms On the right, the number of cells/square area of the surface material: + = presence of cells, 0 = absence of cells

Fig. 12.

(Versura) Platelets cultured onto hydrogel. The rare cells (p) only display the round form , suggestive of a resting state (SEM 2,300 x).

DISCUSSION

-Hydrogel are similar to those cultured onto HSM-PMMA

-HSM-PMMA display only the round and abortive forms

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+++ ++/+++ +

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HSM-PMMA and hydrogel surfaces. We are currently testing this hypothesis. Culturing fibroblasts onto the PT-PMMA, in which evidence of degeneration was shown, especially at the level of those cells directly adhering to the material, appears to be controversial. This observation could account for a cytotoxic effect occurring only through a direct contact of cell and material, but specific studies are needed to resolve the question. The monocytes cultured onto HSM-PMMA and hydrogel do not show any tendency to flatten as is the case when PMMA and PT-PMMA are used as substrates. For the first two materials only rare and isolated cells were seen, while for the second two a close relationship with granulocytes and platelets was observed. This latter finding is not entirely understood. It is well recognized that no morphological feature is considered a reliable target for the expression of a resting or an activated cell; therefore the results from our study on the monocytes represent a point to begin an analysis of this problem. Interpretation of the results for the platelets appears to be less intriguing in respect to the others. The activation of these cells is visually observed by their progressive change in shape from the round to the fully spread form, avoiding an artefactual activation of the platelets during the procedures of isolation from the peripheral blood. Our results demonstrate a progressive activation from the HSM-PMMA to the PT-PMMA up to the PMMA, in which all the cells appear to be in an intermediate or fully spread form. The hydrogel is completely

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devoid of adherent platelets. A conclusive interpretation cannot be drawn on the basis of these data. Further investigations are required to deal with several of the issues arising from the present study of the behavior of these three cell types. However, in a preliminary way we can say that HSM-PMMA and the hydrogel have been demonstrated to prevent cell adhesion successfully while preserving cellular physiology. REFERENCES 1. Pedley DC, Skelly pJ, Tighe BJ. Hydrogels in biomedical applications. Br Polymer J 1980; 12:99-110 2. Larm 0, Larsson R, Olsson PA. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue. Biomater Med Devices Artif Organs 1983; 11: 161-173 3. Olsson P, Larm 0, Larsson R, et al. Requirements for thromboresistance for surface-heparinized materials. Ann N Y Acad Sci 1983; 416:525 4. Cholakis CH, Zingg W, Sefton MV. Effect of heparin-PVA hydrogel on platelets in a chronic canine arterio-venous shunt. J Biomed Mater Res 1989; 23:417-441 5. Salzman EW, Merrill EW, Binder A, et al. Protein-platelet interaction on heparinized surfaces. J Biomed Mater Res 1969; 3:69-81 6. Hansma PK, Elings VB, Marti 0, Bracker CEo Scanning tunneling microscopy and atomic force microscopy: application to biology and technology. Science 1989; 242:209-216 7. Hattori S, Andrade JD, Hibbs JB, et al. Fibroblast cell proliferation on hydroxyethyl methacrylate copolymers. J Coli Interface Sci 1985; 104:72-78 8. Absolom DR, Thomson C, Hawthorn LA, et al. Kinetics of cell adhesion to polymer surfaces. J Biomed Mater Res 1988; 22:215-229 9. Ratner BD, Horbett T, Hoffman AS, Hauschka SD. Cell adhesion to polymeric materials: implications with respect to biocompatibility. J Biomed Mater Res 1975; 9:407-422

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