A Study of Human Lens Cell Growth in Vitro a Model for Posterior Capsule Opacification

A Study of Human Lens Cell Growth In Vitro A Model for Posterior Capsule Opacification

Chistopher S. C. Lm,*f /. Michael Wormstone,~f George Duncan,^ Julia M. Marcantonio,^ SimF. Webb,-\ and Peter D. Davies*

Purpose. After intraocular lens (IOL) implant surgery for cataract, cell growth on the posterior capsule is responsible for renewed visual impairment in approximately 30% of patients. The authors have, therefore, developed a human lens capsule system to study this growth in vitro. Methods. Sham cataract surgery, including anterior capsulorhexis, nucleus hydroexpression, and aspiration of lens fibers, was performed on donor eyes. In some cases, a polymethylmethacrylate IOL implant was placed in the capsular bag. The capsular bag was dissected free, pinned flat on a plastic culture dish, covered with Eagle's minimum essential medium supple- mented with 10% fetal calf serum and observed by phase-contrast and dark-field microscopy for as long as 100 days. At the end-point, capsules were examined by fluorescence microscopy for actin,. vimentin, and chromatin. Results. Within 24 hours, there was evidence of cell growth in the equatorial region. After 2 to 3 days, cells were normally observed growing from the rhexis onto the posterior capsule and across the anterior surface of the IOL, if present. Growth proceeded rapidly so that the posterior capsule, for example, was totally covered by a confluent monolayer of cells at 5.8 ± 0.6 days and 7.2 ± 0.7 days for capsules aged <40 years and >60 years, respectively. Total cover of the anterior IOL surface generally followed 4 to 5 days behind that of the capsule. Capsular wrinkles became increasingly apparent as time progressed, causing a marked rise in light scatter. An increase in capsular tension also occurred, and the actin filaments became more polarized near the wrinkles. Conclusions. The model presented here for posterior capsule opacification shows many of the changes seen in vivo, including rapid lens cell growth, wrinkling, tensioning, and light scatter in the posterior capsule. It will be possible to develop strategies for inhibiting cell growth with this system. Invest Ophthalmol Vis Sci. 1996; 37:906-914.

An modern extracapsular cataract surgery, to restore cells left behind at surgery.2'3 The resultant cellular the best possible vision, a small artificial lens is im- accumulation and changes to capsular structure give planted into the remaining lens capsular bag from rise to an increase in light scatter, and decentralization which most of the fiber mass has been removed. This of the intraocular lens (IOL) also can occur.3 approach is not without subsequent complications, The most common and indeed successful method however, and within 2 years as many as 35% of patients of treatment is to photodisrupt a section of the poste- experience a significant loss of visual acuity.1 This deg- rior capsule by a high-energy Nd:YAG laser to create radation of the visual image appears to arise from a clear region around the visual axis.3 This treatment colonization of the posterior capsule by lens epithelial is expensive and not without medical complication because it can give rise to an increase in intraocular

From the * Department of Ophthalmology, West Nonirich Hospital, and the f School pressure, retinal detachment, and, in extreme cases, of Biological Sciences, University of East Anglia, Norwich, United Kingdom. pitting of the surface and fracture of the IOL adjacent Presented in part at the annual meeting of the Association for Research in Vision and Of>hthalmology, Fort lxiuderdah, Florida, May 1995. to the cleared region (see refs. 2 and 3 for review). As Supported by The Humane Research Trust and by grant RO1 EY'10558 from the a consequence of these multiple complications, there National Eye Institute. have been strenuous efforts to understand more fully Submitted for publication Sefdember 14, 1995; revised November 14, 1995; accepted December 7, 1995. the underlying mechanism of posterior capsular opac- Proprietary interest categmy: P. ification, and such fundamental investigations either Reprint requests: C Duncan, School of Biological Sciences, University of East Anglia, Nonuich, NR4 7TJ, United Kingdom. have been carried out in vivo in clinical studies or in

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FIGURE l. Low-power, dark-field views of capsular bag preparations, pinned out and immersed in culture medium, pho- tographed within 1 hour of removal from the donor eye. Light-scattering areas arise from the debris left after the operation. In the absence of an intraocular lens (IOL) (a), the disc-shaped opening in the anterior capsule can be seen clearly, revealing the posterior capsule beneath. When an IOL is present (b), the supporting loops (haptics) distend the capsular bag, and two creases (arrows) can be seen on the posterior capsule. These are absent from the capsular bags without an IOL (a). The micrographs represent a field of view of 1.25 X 1.2 cm.

MATERIALS AND METHODS After the removal of corneo-scleral discs for trans- plantation purposes, human donor eyes obtained from East Anglian Eye Bank were used to perform sham cataract surgery, including continuous circular capsulorhexis (5 or 8 mm), hydroexpression of lens fiber mass, and aspiration of residual lens fibers. An IOL could be implanted into the bag if required. The capsular bag was then dissected free from the zonules and secured on a sterile polymethylmethacrylate petri dish. Six to eight entomological pins (Dl; Watkins and Doncaster, Kent, UK) were inserted through the edge of the capsular bag to retain its circular shape (Fig. 1). Cultures were maintained in 1.5 ml of Eagle's minimum essential medium (Sigma, Poole, UK) supple- mented with 10% fetal calf serum and 50 mg/1 genta- micin, and incubation was at 35 °C in a 5% CO2 atmo- sphere. The maintenance medium was replaced every 3 to 4 days. Ongoing observations were performed using phase-contrast and dark-field microscopy. The first group of experiments was conducted without an IOL because this facilitated observation of the earliest stages of cell growth in the capsular bag. Cytoskeletal proteins were visualized by immuno- cytochemistry and epifluorescence microscopy. All re- agents were from Sigma (Poole) unless otherwise stated. Washes were for 3X15 minutes in phosphate- animal model systems45 or have used tissue-cultured 8 buffered saline (PBS)/bovine serum albumin (BSA)- cells largely of nonhuman origin.''" Both approaches Nonidet (0.02% and 0.05%, respectively). The pinned have defects because the details of growth are difficult capsules were fixed for 30 minutes in 4% formalde- 9 to follow in vivo, and the tissue culture approach is hyde in PBS and permeabilized in PBS containing complicated by the fact that the dynamics of cell 0.5% Triton-XlOO, also for 30 minutes. Nonspecific growth are both species and substrate dependent sites were blocked with appropriate serum (1:50 in (compare data in refs. 6, 7, 8, 10). 1% PBS/BSA). Anti-vimentin (Clone V9) was diluted We have, therefore, developed a human lens cap- 1:100 and applied for 60 minutes at 35°C, followed sular bag culture system that not only permits day-to- by washing. Vimentin was visualized with fluorescein day, high-resolution imaging of cell growth on the isothiocyanate-conjugated anti-mouse serum, used at natural substratum but allows a study of the rate of 1:64 for 60 minutes at 35°C. After extensive washing, progression of capsular wrinkling and tensioning, the actin cytoskeleton was stained with Phalloidin- both of which appear to play an important role in tetramethylrhodamine isothiocyanate (TRITC) (2 the postoperative loss of vision in a large number of fjM) for 30 minutes, and cell nuclei with 4,6-Diamid- patients who have undergone cataract surgery.1"3 ino-2-phenylindole (DAPI) at 1 ££g/ml for 10 minutes,

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FIGURE 2. Low-power epifluorescence micrographs of capsular bag preparations at various stages of growth in the absence of an intraocular lens, (a) immediately after pinning out, (b to d) after 3 days, and (e to h) after 8 days. Red micrographs show F-actin stained with TRTTC-phalloidin, green micrographs show vimentin visualized with fluorescein isothiocyanate, and blue micrographs show DAPI staining of cell nuclei. In all cases, micrographs represent a field of view of 565 X 390 //m. (a). In the freshly prepared capsular bag, large patches of cells are present on the anterior capsule, where they remained throughout surgery. However, in some areas (large asterisks) cells were removed; in particular, most cells were removed in the area (small asterisks) immediately adjacent to the rhexis {arron)). Cells are totally absent from the posterior capsule (PC) beyond the capsulorhexis. (b,c) Staining of the dense cytoplasmic network of vimentin filaments in cells after 3 days in culture, (b) Cells growing on the posterior capsule are in focus, whereas the rhexis (between the arrows) is bright but out of focus. With the rhexis in focus (c), it can be seen that cells also grew up onto the outside of the anterior capsule, and out toward the equator, (d) F- actin staining of the same cells growing onto the posterior capsule (the plane of focus is the same as in b). Cells near the rhexis appear radially polarized with parallel stress fibers (see Fig. 4a), whereas cells at the leading edge of the wave of growth have ruffled membranes and actin microspikes (see Fig. 4b). (e). Vimentin staining of original lens cells (in focus) on the anterior capsule, overlaid by new growth (out of focus) on the outside of the anterior capsule. Penetration of the antibody to the interior cells is poor, and they appear lightly stained. Note that cells on the interior of the anterior capsule are small, whereas those growing across the outside surface are much larger, (f to g) Confluent cells on the posterior capsule with one wrinkle, stained to show cell nuclei (f) and corresponding actin (g) and vimentin (h) stain. Note the accumulation of nuclei in die wrinkle (f) and a cell {airoio) undergoing mitosis alongside the wrinkle.

both at room temperature. The stained preparations out, there was evidence of cell growth in the equatorial were again washed extensively, floated onto micro- region, but it was difficult to discern whedier the grow- scope slides, and mounted in Vectashield mounting ing cells were on the anterior or posterior capsular medium (Vector Laboratories, Peterborough, UK). areas because of the disruption in optical images from Images were viewed with a Zeiss Standard R micro- adhering fibers and the overlayering of the two areas. scope (Zeiss, Oberkochen, Germany) and recorded However, once the cells progressed beyond the rhexis on Ektachrome 400 or Tmax 400 pro film (Kodak UK, and were growing toward the center, good optical im- Hemel Hempstead, UK) with an Olympus (London, ages were available, and growth into this region usually UK) OM2 camera. Forty-two capsules were used for occurred within 2 to 3 days after pinning out die cap- all the studies. sule (Fig. 3).

RESULTS The two types of capsular bag preparations used in this study are shown in Figures la and lb. Generally, eight pins were used to preserve the natural circular outline of the bag. The extent of the capsulorhexis can be seen clearly in both cases, and when an IOL was inserted, it slightly distorted the outline of the bag as it would be in vivo. These preparations could be observed by optical microscopy methods for up to 100 days. Immunohistochemical staining for F-actin, performed immediately after pinning out, showed that although the posterior capsule was totally free of cells, large areas of viable cells remained on the anterior FIGURE 3. Phase-contrast micrograph of eel Is growing on the posterior capsule (PC) in the region of the capsulorhexis capsule (Fig. 2a). (in the absence of intraocular lens) 3 days after pinning Growth Studies Without Intraocular Lens out. The rhexis is the phase-contrast bright region and the anterior capsule (*) is not in focus at this level. The vimentin To carry out long-term growth studies, the capsule and actin-stained micrographs corresponding to this stage was observed on a day-to-day basis by phase-contrast of growth are shown in Figures 2b and 2d. This micrograph microscopy. During the first 1 or 2 days after pinning represents a field of view of 0.89 X 0.55 mm.

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stained original cells can be seen in Fig. 2e, overlaid by bright, but out of focus, new growth on the outside of the anterior capsule. Within 6 to 10 days the cells became confluent on the posterior capsule and acquired a regular, cobblestone appearance (Fig. 5). Wrinkles began to form by this stage, and they increased in number and mag- nitude with increasing time in culture (Fig. 6). The wrinkles were regions of light scatter, and this could be seen clearly in the dark-field micrograph (Fig. 6b). Early wrinkles seemed to be initiated beneath individ- ual cells (Fig. 5), but in major wrinkles (Fig. 6), the situation was more complex. DAPI staining of early wrinkles within 2 days of confluency (Fig. 2f) demon- strated that there was already a concentration of nuclei along the wrinkle, with a small area almost devoid of nuclei along each side. The closely packed cells within the wrinkle appeared to have their major actin filaments running in parallel along the length of the wrinkle (Figs. 2g, 7). In regions to each side of the wrinkle, where cell density was comparatively low (Figs. 2f, 2h), major actin filaments were aligned mainly at right angles to the wrinkle (Fig. 7). The net result of the progressive capsular folding was increased tensioning of the capsule, which can clearly be seen as the inwardly bowed region (arrow) between the pins (Fig. 8). FIGURE 4. High-power epifluorescence micrographs showing With the capsular bag systems, it was possible to F-actin in cells growing on the posterior capsule, 3 days after study the effect of donor age and extent of capsulor- pinning out and in the absence of an intraocular lens. In hexis on the rate of growth on the posterior capsule. elongated cells (a) growing off the rhexis, which is out of This was assessed in terms of time for complete cell focus (between the arrows), the actin microfilaments are cover on the posterior capsule. There was a small but highly polarized along the lengtih of the cell, parallel to the significant effect of donor age on the growth charac- direction of growth (right to left). Cells at the leading edge teristics. The capsule was totally covered by a confluent of the growth wave (b) have ruffled membranes and show actin microspikes at right angles to the plasma membrane, both at the leading edge and in regions of cell-cell contact. Some cells also show polygonal arrays of actin filaments. Micrographs represent a field of view of 105 X 68 f/,m.

Vimentin staining clearly showed these cells growing toward the center of the posterior capsule (Fig, 2b), and in another plane of focus, it showed that cells also grew upward from the rhexis and back across the exterior surface of the anterior capsule (Fig. 2c). Staining for fibrous actin (Fig. 2d) of the cells growing on the posterior capsule (see Figs. 2b, 3) showed that they had a well-developed actin cytoskeleton. In cells at the rhexis edge, this appeared to be polarized with the major filaments lying parallel to the direction of FIGURE 5. Formation of a confluent monolayer of cells on growth (Fig. 4a). Cells at the leading edge had fan- the posterior capsule after 9 days in the absence of an intra- shaped, ruffled membranes containing microspikes ocular lens. The phase-contrast micrograph of newly confluent cells at the center of die posterior capsule shows (Fig. 4b). Small spikes appeared to lie in register with their regular cobblestone appearance and reveals that fine those in neighboring cells. Although original epithe- wrinkles are already beginning to form. Because die poste- lial cells stained brightly with TRITG-phalloidin (see rior capsule is not totally flat, only part of the field of view Fig. 2a), the vimentin antibody did not penetrate well is in focus. The micrograph represents a field of view of 0.89 into the region under the anterior capsule. Lightly X 0.58 mm.

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FIGURE 6. (a) Low-power conventional micrograph of severe wrinkling of the posterior capsule approximately 20 days after confluency. The iris diaphragm of the microscope has been closed down to give a graded illumi- nation, which shows the con- tours of the wrinkled areas clearly. Dark-field illumina- tion (b) more clearly shows light scattering associated with the wrinkles. Micro- graphs represent a field of view of 2.4 X 3.6 mm.

cell monolayer at 5.8 ± 0.6 days and 7.2 ± 0.7 days DISCUSSION for capsules aged <40 years and >60 years, respectively. The Student's Rest applied to these data This study (Fig. 1) confirmed data from a number of showed that the means were significantly different at earlier reports where it has been shown that a high P^ 0.01. Increasing the diameter of the capsulorhexis proportion of viable lens epithelial cells are left be- from 5 mm to 8 mm had little effect on the rate of hind after all the manipulations and irrigation that coverage of the posterior capsule (Fig. 9). take place in conventional extracapsular cataract surgery.1"" The exact proportion of the anterior capsule Studies With Intraocular Lens Present covered by viable cells was variable from preparation Inserting an IOL into the bag again had little effect to preparation and ranged from 30% to 80%. This on the growth rate on the posterior capsule, though estimate generally was obtained by observing the ante- in addition there was cell coverage of the anterior rior capsule by phase optics at low magnification surface of the IOL. The pattern and progression of where the non-viable cells were apparent by tfieir in- cell growth on the IOL and on the posterior capsule creased light scatter and prominent nuclei. The cell is shown in Figure 10. The two capsular preparations loss was always greatest nearest the rhexis, but, in spite from the same donor (42 years of age) were found to of the difference in cell mortality, all capsules showed grow at much the same rate. Growth on the posterior a rapid growth of cells across the posterior surface. It capsule was complete after 8 days, and growth on the is a measure of the resilience of human lens cell IOL was complete after 12 or 13 days. growth that, without exception, total and confluent Cell growth onto the IOL proceeded at first in a cell cover of the posterior capsule was obtained. It is manner similar to that on the posterior capsule in that the resilient growth of the epithelial cells left after the rhexis soon fused onto the IOL by cells growing surgery that gives rise to the postoperative opacifica- from the anterior capsule. Once growth was estab- tion of the capsular bag. In vivo cell growth appears lished, the confluent cells had a normal cobblestone to continue for many months and even years, and light appearance (Fig. 11), but cells at the growing edge scattering cell-protein aggregates (Elschnig's pearls) had a much larger and more irregular appearance are formed in dense array.12 In the current model, than the corresponding cells growing on the posterior there was little evidence of such pearl formation, at capsule (see Fig. 3). When the cells on the IOL be- least in capsules cultured for as long as 100 days. came confluent, they increased in density, and most The first evidence of cell growth was observed in of them had an appearance similar to that of conflu- the bow region, where cells were seen moving onto ent cells on the posterior capsule. During the time the posterior capsule. This is in accord with a recent course of our experiments (100 days), there was no study13 of cell growth in the capsular bag, which evidence of cell multilayering or wrinkle formation on showed, using the BrdU technique, that mitotic cells the anterior IOL. are seen first in the bow region. After a few days, cells

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FIGURE 8. Dark-field photograph of capsular bag after 46 days in culture. The increase in tension within the capsule is seen as an inward bowing of the capsular edge between FIGURE 7. High-power epifluorescence micrograph of the ac- die pins (nrrovJ). Light scatter from the wrinkles is present, tin fibers of cells in the region of a wrinkle (see Fig. 2g). In but not very apparent, at this magnification (see Fig. 6b). the numerous cells involved in the wrinkle (see Fig. 2f), Because of the long working distance of the objective lens major actin filaments appear to be aligned along the length used at this magnification, imperfections of the surface of of the fold (between the arrows). In the sparse cells immedi- the plastic culture dish are visible. This micrograph repre- ately alongside the wrinkle (see Fig. 2h), the major actin sents a field of view of 1.1 X 1.15 cm. filaments are aligned mainly at right angles to the fold. This micrograph represents a field of view of 68 x 68 ftm. serum proteins are present at much higher concentra- tions than those found in normal aqueous.'18" It move beyond the rhexis and advance rapidly across should also be noted that the majority of in vitro mod- the posterior capsule. Our findings agree with the els uses lens epithelial cells obtained from noncatarac- movement data of Nagamoto and Miyajima1' obtained tous lenses. There is, however, evidence to suggest from a human capsular bag culture system immobi- that satisfactory growth can be obtained from capsules lized by a ring-supporting system. As expected from a number of tissue culture experiments,1'115 cells on capsules from younger donors did grow at a faster rate than those from older individ- uals, and this agrees with the fact that younger patients show a higher rate of posterior capsule opacification that their older counterparts.' The fact that increasing rhexis size has only a minor delaying effect on cell coverage of the posterior capsule indicates that bow region epithelial cells play a major role in driving cell growth. These cells are the least accessible to mechani- cal disrupting techniques. With our system, the presence of an implant also does not greatly perturb growth on the posterior surface. There is, however, a body of clinical evidence indicating that, in vivo, growth of cells on the posterior capsule is impeded, especially in the case of biconvex optics, where there Time cultured (Days) is a large area of contact between the posterior surface H FIGURE 9. Rate of cell coverage of the posterior capsule be- of the optic and the posterior capsule. Although yond the rhexis with either 5-mm or 8-mm diameter capsu- growth appears rapid in vitro, it should be remem- lorhexis; 100% represents confluency. All donor capsules bered that all in vitro models so far tested actually were >60 years of age, and data were obtained in the ab- use serum proteins to drive growth. In these models, sence of an intraocular lens.

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plays an important role in setting up the elastic tension

CL-201 forces within the capsule because it will obviously act as an anchor zone, limiting the movement of the capsule under tension. The areas free to move in the current model were primarily the posterior and anterior surfaces of the bag, where wrinkling occurs. In vivo the outer edges of the capsule are still attached by the suspensory ligaments, and the tension forces developed are so strong that lateral movement of the IOL (decentralization) and even ligament rupture can ANTERIOR SURFACE OF IOL POSTERIOR CAPSULE occur.3 Wrinkling of the posterior surface is important optically because significant light scattering areas are produced in this way, and the clinical importance of this process has been recognized for some time.2 Some interesting feedback effects must occur that make cells congregate along such stress lines. Immunofluores- cent staining reveals that not only are nuclei lined up within the wrinkle, they are largely absent in areas immediately adjacent. Similarly, actin stress fibers tend to be lined up either along or at right angles to the wrinkle, and the vector effect of such stress forces,

DAY 8 | | DAY 12 small though they may be in a single cell, will be en- hanced considerably when lined up in the same direction in many cells. The net effect of such forces is DAY 9 | I DAY 13 seen clearly in the inward curvature of the capsule

< EXTENT OF CAPSULORHEXIS FIGURE 10, Progression of cell growth on the posterior capsule and the intraocular lens in two capsular bags from the same donor, 42 years of age. Note that, as in Figure 9, only cell growth within the area of the capsulorhexis was moni- tored.

and cells removed from lenses that are both aged and cataractous.14"16 After 2 to 3 days in culture, there was evidence of cell growth in the anterior capsule, and cells began to move across the anterior surface of art implanted IOL just as they do in vivo.9'17 For example, Ibaraki et al9 showed that cellular outgrowths from the capsulorhexis can be seen on the anterior surface of the IOL 1 week after surgery. Again, this is in accord with the observations of Rakic et al13 made on their in situ capsular bag preparation, in which BrdU labeling of anterior epithelial cells followed a day or so after labeling of the bow region cells. The edge of the rhexis rapidly adhered to the surface of the IOL, and this process also occurs in vivo.3 The attachment of the anterior capsule also was apparent in the absence of an IOL, where it adhered to the posterior capsule. In FIGURE ii. Phase-contrast micrograph of cells growing on such preparations, more detail was revealed by immu- the anterior surface of the intraocular lens after 8 days in culture. Note the cobblestone appearance of the confluent nofluorescent studies. These clearly showed the com- cells (right-hand margin; see Figs. 3 and 5), and the irregular plexity of the adherence zone, with cells growing radi- appearance of cells at the growing edge (left-hand margin, ally inward across the posterior capsule, outward arrow). Because of the curved surface of the intraocular lens, across the front surface of the anterior capsule, and only the cells at the growing edge are truly in focus. The upward at the zone itself (Fig. 2c). This zone probably micrograph represents a field of view of 0.69 X 0.76 mm.

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margins in the current model and in the shrinkage G. Pathogenesis of capsular opacification after extra- of the capsule observed in the ring support system capsular cataract extraction: An animal model. Oph- developed by Nagamoto and Bissen-Miyasima.18 thalmology. 1984;91:857-863. It has been suggested from in vivo studies that it 6. McDonnell PJ, Kranse W, Glaser BM. In vitro inhibi- tion of lens epithelial cell proliferation and migration. is not simply the presence of the cell monolayers, ei- Ophthalmic Surg. 1988; 19:25-30. ther on the posterior capsule or on the IOL, that 7. Power WJ, Neylan D, Collum LM. Daunomycin as an causes the major optical problems, but rather the later inhibitor of human lens epithelial cell proliferation pearl formation and wrinkling of the posterior cap- in culture. / Cataract Refract Surg. 1994; 20:287-290. sule.23 Both of these latter changes are important in 8. Kurosaka D, Nagamoto T. Inhibitory effect of TGF- visual degradation, although the relative importance beta 2 in human aqueous humor on bovine lens epi- of either one is a matter for discussion. However, both thelial cell proliferation. Invest Ophthalmol Vis Sci. follow on the resilient growth of human lens cells after 1994; 35:3408-3412. extracapsular surgery. The current model, involving 9. Ibaraki N, Ohara K, Miyamoto T. Membranous out- culture over a relatively short time, reproduces many growth suggesting lens epithelial cell proliferation in pseudophakic eyes. Am J Ophthalmol. 1995; 119:706- of the changes seen in vivo, including resilient cell 711. growth, wrinkling of the posterior capsule, and ten- 10. Olivero DK, Furcht LT. Type IV collagen, laminin and sioning of the capsular bag. It will be possible to ex- fibronectin promote the adhesion and migration of tend the period of study and to investigate whether rabbit lens epithelial cells in vitro Invest Ophthalmol Vis de novo synthesis of lens-specific proteins does occur Sci. 1993; 34:2825-34. late in culture, but it already provides an accurate and 11. Nagamoto T, Bissen-Miyajima H. Postoperative mi- accessible means with which to develop strategies to gration of lens epithelial cells. EurJ Implant Ref Surg. limit cell growth within the capsular bag. 1994; 6:226-227. 12. Budo C, Montanus F, Gofnnet G, Goossens G, Gaill Keywords F. Cytokeratin in lens epithelial cells and its effect on anterior lens capsule opacification. / Cataract Refract cataract, cell growth, epithelial cells, human, lens, posterior Surg. 1993; 19:339-343. capsule opacification 13. Rakic J-M, Galand A, Vrensen G. Lens fibers control human lens epithelial mitotic activity. Vision Res. Acknowledgments 1995;35:S90. The authors thank Mrs. Pam Keeley of the East Anglian Eye 14. Power W, Neylan D, Collum L. Growth characteristics Bank, without whose efforts this study would not have been of human lens epithelial cells in culture: Effect of possible. They also thank Drs. David Spalton, Milind Pande, media and donor age. Doc Ophthalmol. 1993; 84:365- Gijs Vrensen, Jean-Marie Rakic, Albert Galand, and Richard 372. Warn for helpful discussions, Mrs. Diane Alden for technical 15. Bermbach G, Mayer U, Naumann GO. Human lens support, Mrs. Jill Gorton for preparing the manuscript, and epithelial cells in tissue culture. Exp Eye Res. 1991; Pharmacia, IOLAB, and Alcon for providing IOL samples. 52:113-119. 16. Duncan G, Webb SF, Dawson AP, Bootmann MD, El- References liott AJ. Calcium regulation in tissue-cultured human and bovine lens epithelial cells. Invest Ophthalmol Vis 1. Moisseiev J, Bartov E, Schochat A, Blumenthol M. Sci. 1993;34:2835-2842. Long-term study of the prevalence of capsular opaci- 17. Nagamoto T, Hara E. Postoperative membranous pro- fication following extracapsular cataract extraction. / liferation from the anterior capsulotomy margin onto Cataract Refract Surg. 1989; 15:531-533. the intraocular lens optic. / Cataract Report Surg. 2. Ohadi C, Moreira H, McDonnell PJ. Posterior capsule 1991;21:208-211. opacification. Curr Opin Ophthalmol. 1991;2:46-52. 18. Nagamoto T, Bissen-Miyajima H. A ring to support 3. Apple DJ, Solomon KD, Tetz MR, et al. Posterior cap- the capsular bag after continuous curvilinear capsulor- sule opacification. Surv Ophthalmol. 1992; 37:73-116. hexis. / Cataract Refract Surg. 1994; 20:417-420. 4. Odrich MG, Hall SJ, Worgul BV, Trokel SL, Rini FJ. 19. Liu CSC, Duncan G, Wormstone IM, et al. Human Posterior capsule opacification: Experimental analy- lens cell growth on the posterior capsule—a model ses. Ophthalmic Res. 1985; 17:75-84. for aftercataract. Invest Ophthalmol Vis Sci. 1995; 36: 5. Cobo LM, Ohsawa E, Chandler D, Arguello R, George S626.

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