- Email: [email protected]
Capsular tension ring–based in vitro capsule opacification model
LABORATORY SCIENCE
Capsular tension ring–based in vitro capsule opacification model Johannes Burger, MD, Thomas Kreutzer, MD, Claudia S. Alge, MD, Rupert W. Strauss, MD, Kirsten Eibl, MD, Christos Haritoglou, MD, Aljoscha S. Neubauer, MD, Anselm Kampik, MD, Siegfried G. Priglinger, MD
PURPOSE: To evaluate a capsular tension ring (CTR)–supported anterior and posterior capsule opacification (PCO) model in cadaver eyes. The effect of CTR designs on lens capsule shape and lens epithelial cell (LEC) growth were investigated in vitro. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. METHODS: Following open-sky extracapsular cataract extraction, CTR models were implanted in 32 eyes of 16 human donors. The lens capsule expansion by the CTRs was evaluated. The capsular bags supported by the CTRs were excised and maintained at physiological conditions for up to 3 months. The area of LEC coverage over the posterior capsule surface was objectively determined twice a day using a graticule. RESULTS: After CTR implantation, all lens capsules could be safely excised and transferred into organ culture. The CTR designs resulted in different shapes of lens capsule expansion. Complete LEC confluence occurred after a mean of 8.25 days G 2.87 (SD) with the AcriRing KR10 (AcriTec), 6.50 G 1.0 days with the Acrimed, 8.62 G 3.34 days with the InjectoRing (Corneal), 9.00 G 1.87 days with the Morcher 14C, 9.33 G 0.75 days with the Morcher 2A, and 6.25 G 0.5 days with the Ophthalmic Innovation CTR. CONCLUSION: The CTR-supported in vitro PCO model offers a physiological method to support the lens capsule and is a reproducible system for the study of LEC proliferation. J Cataract Refract Surg 2008; 34:1167–1172 Q 2008 ASCRS and ESCRS
Posterior capsule opacification (PCO) remains the most frequent long-term complication after successful cataract extraction and subsequent implantation of an intraocular lens (IOL). Although the incidence of PCO
Accepted for publication March 29, 2008. From the Department of Ophthalmology (Burger, Kreutzer, Alge, Kampik, Priglinger), Ludwig-Maximilians-University, Munich, Germany; and the Department of Ophthalmology (Burger, Alge, Strauss, Eibl, Haritoglou, Neubauer, Priglinger), General Hospital, Linz, Austria. No author has a financial or proprietary interest in any material or method mentioned. Supported by a 2006 research grant from the European Society of Cataract and Refractive Surgeons. Corresponding author: Siegfried Priglinger, MD, Department of Ophthalmology, General Hospital Linz, Linz, Austria. E-mail: [email protected]. Q 2008 ASCRS and ESCRS Published by Elsevier Inc.
development has been significantly reduced over the past few decades, young patients and patients with uveitis or chronic intraocular inflammation show much higher rates of PCO.1–3 Besides IOL material4,5 and design,6–8 major emphasis of PCO prevention focuses on the development of antiproliferative and cytotoxic lens epithelial cell (LEC)-manipulating substances.9–12 Because of possible cell toxicity, it is usually difficult to test new substances and strategies that influence LEC growth in humans. Thus, there is considerable interest in developing in vitro organ culture models for PCO. Currently, 3 in vitro models are available.13–15 Although effectively supporting the lens capsule, all of them exhibit substantial disadvantages, making their reliability debatable. The pin model by Liu et al.13 consists of an isolated capsule secured to the surface of a Petri dish by entomological pins. The lens capsule thereby loses the natural curvature of the lens and capsule folds are induced. The model by Saxby et al.14 consists of a lens capsule containing a huge notched silicone 0886-3350/08/$dsee front matter doi:10.1016/j.jcrs.2008.03.040
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ring that covers a large capsule area and probably interferes with LEC migration. In 2003, El-Osta et al.15 introduced a lens holder system that used cyanoacrylate glue to fixate the lens for surgery and organ culture. This model does not allow separate study of anterior and posterior LEC changes; because of the use of cyanoacrylate, a possible toxic effect on LECs must be calculated. The purpose of the present study was to develop a new in vitro anterior capsule opacification (ACO) and PCO model that would reduce the disadvantages of the existing models. Unlike the existing PCO models, the new in vitro model is based on stabilizing the capsular bag with a conventional capsular tension ring (CTR) that improves visualization of the entire capsule in organ culture and reduces the effect on capsule curvature, thereby more closely resembling in vivo dynamics. MATERIALS AND METHODS Source of Tissue Thirty-two eyes of 16 human donors between 24 years of age and 76 years of age were obtained from the Eye Bank, Ludwig-Maximilians-University, Munich, Germany. Only eyes from donors with specific consent for research purposes and with contraindications for transplantation according to the guidelines of the German Federal Physician Association were included.16 All eyes were preserved in a moist chamber at 4 C within 45 hours postmortem. All experiments were performed in accord with the Declaration of Helsinki.
Surgical Procedure Whole donor eyes were thoroughly cleansed in NaCl 0.9% solution, immersed in polyvinylpyrrolidone-iodine 5%, and rinsed again in the sodium-chloride solution. Corneoscleral disks were excised and prepared for further corneal transplantation. After the iris was removed, open-sky cataract extraction was performed under sterile conditions: After continuous curvilinear capsulorhexis using a forceps, the lens nucleus and epinucleus were extracted by hydroexpression. Residual cortex was removed by irrigation with phosphate-buffered saline (PBS, pH 7.4) and a micro forceps (Kolibri, G-18950, Geuder). The capsular bag was then stabilized by implanting a capsular tension ring (CTR), circularly excised by cutting the zonules with scissors, and safely transferred into organ culture (Figure 1). The CTRs were the AcriRing KR10 (4), Acrimed (4), InjectoRing (8), Morcher 14C (6), Morcher 2A (6), and Ophthalmic Innovation (CTR12) (4). The lens capsule expansion by the CTRs was evaluated. Lens capsular shape was documented immediately after CTR implantation (before excision) and in organ culture.
Organ Culture Storage Capsular tension ring-stabilized lens capsules were stored in 35 mm wells containing 2.5 mL Eagle’s minimum essential culture medium containing 2% fetal calf serum with 100 U/ mL penicillin, 0.1 mg/mL streptomycin, and 0.25 mg/mL amphotericin B (Biochrom) and maintained at 37 C and 5% CO2 in a standard incubator for up to 3 months. The medium
was changed every 5 days.17 The LEC growth (and migration) was monitored every second day using transmission light microscopy (Leica). The area of LEC coverage over the posterior capsule surface was determined objectively 2 times a day using a grid. The ends of the CTRs were used as landmarks, making it possible to repeat observation of the same area of LEC migration.
Immunohistochemistry For staining of fibronectin in vitro, the entire lens capsule with a complete LEC monolayer on the posterior capsule was fixed in paraformaldehyde 4%. After 3 washes in Trisbuffered saline (TBS, pH 7.6) and preincubation with bovine serum albumin 3% (BSA, Roche) for 30 minutes at room temperature to minimize nonspecific staining, specimens were incubated for 15 hours at 4 C with rabbit antifibronectin (Sigma-Aldrich). The antibodies were diluted 1:100 in TBS containing BSA 3%. After 3 washes in TBS, LECs were incubated with swine anti-rabbit IgG Cy-2 (Dianova) diluted 1:100 in blocking buffer. For negative immunostaining control, LECs were incubated with BSA–TBS replacing the primary antibody. A fluorescence microscope (Leica) was used to visualize the immunofluorescent staining.
Statistical Analysis Statistical analysis was based on the Mann-Whitney U test. A P value of 0.05 or less was considered statistically significant.
RESULTS Lens Capsule Expansion The CTR designs caused different shapes of capsule expansion (Figure 1): The Acrimed, AcriRing, and Morcher 14C and 2A CTRs induced circular expansion of the lens capsule with complete closure at the ring ends; the CTR12 and the InjectoRing presented with distinct dehiscence. In addition, the InjectoRing showed irregular oval expansion of the capsule bag, thereby inducing lens capsule streaks. Lens Epithelial Cell Growth Viable LECs were detected in all free-floating CTRexpanded capsules. In the organ culture, the LECs started to migrate and proliferate, similar to the in vivo situation: After approximately 4 days, fibrotic LECs were observed crossing the capsulorhexis margin (Figure 2, A) and about 8 days G 2.34 (SD) later, a homogenous LEC monolayer covered the posterior lens capsule (Figure 2, B). If the tension ring ends did not close completely, initial growth of LEC migration toward the center of the lens capsule was observed at the dehiscence of the CTR (Figure 3). Immunostaining for fibronectin (Figure 2, C) revealed an extracellular network of immunoreactivity. Control cells incubated without the respective primary antibodies were unstained (data not shown). Immunostaining for fibronectin proved that LECs have
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Figure 1. Lens capsules stabilized by the CTRs (transmission light microscopy; original magnification 20).
transdifferentiated into fibroblast-like cell types, as described for PCO. Lens Epithelial Cell Confluence Complete LEC confluence (Figure 4) occurred after a mean of 8.25 G 2.87 days (AcriRing), 6.50 G 1.0 days (Acrimed), 8.62 G 3.34 days (InjectoRing), 9.00
G 1.87 days (Morcher 14C), 9.33 G 0.75 days (Morcher 2A), and 6.25 G 0.5 days (CTR12). Statistical analysis comparing the time until complete LEC confluence of the CTRs showed statistically significant differences (P Z .003 Wilcoxon). Subanalysis of the CTRs showed significantly delayed LEC confluence with the AcriRing, the Morcher CTRs, and the InjectoRing compared with the Acrimed and CTR12 (P!.05 each,
Figure 2. A: Migration of LECs 5 days after transfer to culture medium. B: Confluent LECs on the entire posterior capsule (day 12 of organ culture). C: Immunohistochemical staining for fibronectin (original magnification 100).
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Figure 3. Oval expansion of a lens capsule by an InjectoRing, resulting in a dehiscence at the CTR ends. The initial growth of LECs on the posterior capsule is seen in the gap between the CTR ends.
Mann-Whitney U test. Between the 4 CTRs with delayed LEC confluence, there was no statistically significant difference (PO.05 each, Mann-Whitney U test). DISCUSSION This study showed that a CTR-stabilized lens capsule was an excellent tool for in vitro evaluation of LEC growth. Capsule stabilization with conventional CTRs allowed easy and safe transfer of lens capsules into organ culture. Capsular tension ring stabilization showed capsule expansion with minimal effects on the natural shape of the capsule. This may allow LEC growth that closely resembles the in vivo dynamics of PCO development. The free-floating CTR-expanded capsule provided a physiological setting for LEC growth in organ culture as there was always a layer of culture medium between the outer surface of the posterior capsule and the culture dish, ensuring uninfluenced metabolic supply, which is a major advantage of our model. In addition, the CTR model allowed detailed and close observation of postoperative PCO development; the dynamics of cell growth could be observed since minimal capsule area was concealed by the CTR.
Figure 4. Confluence of LECs with various CTRs.
Several in vitro organ culture models that have different approaches to supporting the lens capsule exist.13–15 Although they allow lens capsule culture, they limit the in vitro evaluation of natural LEC growth because they modify the capsule in various ways postoperatively. Liu et al.13 describe a PCO model in which the isolated capsule is secured to the surface of a Petri dish with 6 to 8 entomological pins. Pinning the specimen to the capsule dish changes the natural curvature of the capsule, resulting in multiple folds of the capsule. We think the nonphysiological deformation of the lens capsule and the contact with the culture dish are major disadvantages because they might influence natural LEC growth. Pinning the specimen to the capsule could also lead to peripheral collapse of the capsule, inducing contact zones between the anterior and posterior capsules. These contact zones allow direct LEC migration from the capsulorhexis edge onto the posterior capsule. Therefore, areas in the peripheral capsule are occluded and investigation of LEC growth in these areas is hindered. However, in IOL implantation, contact between the anterior and posterior capsules is desirable to be able to study the effect of IOL designs on LEC migration. In their model, Liekfeld et al.18 used 6 to 8 entomological pins through the edge of the capsular bag to provide maximum contact between the posterior capsule and the posterior IOL surface. A major advantage of our in vitro PCO model is that using CTRs with different heights, it is possible to provide anterior and posterior capsule contact or noncontact. With a thicker CTR, such as the foldable Morcher 2A, the contact between anterior and posterior capsules can easily be avoided, allowing anterior and posterior capsule phenomena to be studied separately. With a thinner CTR, such as the InjectorRing, contact is achieved in the first week of organ culture, allowing appropriate PCO study after IOL implantation. The endocapsular ring model of Saxby et al.14 separates the anterior and posterior capsules and prevents capsule collapse by supporting the capsule internally with a notched 9.63 mm 1.78 mm (external diameter height) silicone ring. Because of this large dimension, interference with LEC migration must be assumed. Furthermore, the large area covered cannot be adequately monitored by transmission light microscopy. In contrast, the CTRs used in our PCO model cover only a small capsule area, allowing investigation of more peripheral capsule areas. Intraocular lens implantation is possible with Saxby et al.’s model; however, the resultant cell behavior does not reproduce the in vivo situation as no contact between anterior and posterior capsules can be achieved. Furthermore, the endocapsular ring model of Saxby et al. shows continuous contact between the posterior capsule and the
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bottom of the culture dish, which might impede metabolic supply. El-Osta et al.15 introduced a lens holder system with the use of cyanoacrylate glue to fixate the lens for surgery and organ culture. The special lens holders have a central aperture that allows visualization of LEC growth in this area; however, in the equator region, continuous observation of LEC growth with transmission light microscopy is limited because the area of rigid fixation of the capsule to the lens holder is quite large. Furthermore, El-Osta et al.’s lens holder model does not allow separate study of anterior and posterior LEC changes because the capsules are in contact with one another. Therefore, at the capsulorhexis margin, direct migration of LECs from the anterior to the posterior capsule occurs. The major drawback of El-Osta et al.’s model is the use of cyanoacrylate. Cyanoacrylates are adhesive substances that have been widely used in surgical procedures.19,20 However, their specific effect on cell growth and metabolism is not fully understood. In El-Osta et al.’s PCO model, higher concentrations of cyanoacrylate resulted in significant cell death as shown in 3-(4,5 dimethylthiazole-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) and live-dead assays.15 Although vitality of LECs was less affected by lower concentrations (1 mL/mL), a significant number of cells died. Therefore, cell toxicity can not be excluded and a possible negative influence on LEC behavior in this model must be calculated. A major advantage of our in vitro model is the excellent stabilization of the lens capsule by the implanted CTR without changes in the natural capsule shape after cataract extraction. Even in cases that exhibit a significant degree of zonule damage after cornea removal during the eye-banking procedure, CTR implantation leads to excellent stability that allows safe capsule excision and transfer into organ culture. Once in organ culture, it is not necessary to fixate, pin, or glue the capsule on the culture dish as the CTR itself expands and stabilizes the capsule. All the CTRs used in this study differed in the shape of capsule expansion. The overall time to LEC confluence was approximately 8 days, with the Ophthalmic Innovation model having the shortest delay in full coverage of the posterior capsule (6.25 days). We also observed that LECs always started to migrate toward the center of the posterior capsule at the CTR dehiscence. We therefore initially assumed that CTRs with no dehiscence at the ring ends might have a prolonged interval until LEC confluence. Although there was a slight tendency of earlier LEC confluence in CTRs with incomplete tension ring closure, statistical analysis revealed that complete closure of the CTR had no effect on the duration until complete LEC confluence on the posterior capsule. The spring constants
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of CTRs,21 another factor that might influence LEC growth after CTR implantation in vitro, also showed no effect on LEC growth in organ culture. However, the fact that no statistical difference in overall coverage time was found with CTRs with closed or open ends and different spring constants does not imply that the CTR does not affect LEC growth. The CTR-stabilized PCO model offered excellent stabilization of the lens capsule. Depending on the CTR used, contact or noncontact of the anterior and posterior capsules can be achieved. A wide area of the model is accessible for monitoring by transmission light microscopy; it is simple and user friendly and mimics physiological conditions. These properties make this model an ideal tool for studies of ACO and PCO development and enables evaluation of LEC migration and proliferation in vitro. Furthermore, this in vitro model allows testing of new strategies for PCO prevention and for objectively grading IOL performance in vitro. Nevertheless, one has to be aware of the limitation of any model to mirror the dynamics of PCO in vivo. REFERENCES 1. Pandey SK, Apple DJ, Werner L, Maloof AJ, Milverton EJ. Posterior capsule opacification: a review of the aetiopathogenesis, experimental and clinical studies and factors for prevention. Indian J Ophthalmol 2004; 52:99–112 2. Sundelin K, Sjo¨strand J. Posterior capsule opacification 5 years after extracapsular cataract extraction. J Cataract Refract Surg 1999; 25:246–250 3. Bertelmann E, Kojetinsky C. Posterior capsule opacification and anterior capsule opacification. Curr Opin Ophthalmol 2001; 12:35–40 4. Hollick EJ, Spalton DJ, Ursell PG, Meacock WJ, Barman SA, Boyce JF. Posterior capsular opacification with hydrogel, polymethylmethacrylate, and silicone intraocular lenses: two-year results of a randomized prospective trial. Am J Ophthalmol 2000; 129:577–584 5. Ursell PG, Spalton DJ, Pande MV, Hollick EJ, Barman S, Boyce J, Tilling K. Relationship between intraocular lens biomaterials and posterior capsule opacification. J Cataract Refract Surg 1998; 24:352–360 6. Findl O, Buehl W, Menapace R, Sacu S, Georgopoulos M, Rainer G. Long-term effect of sharp optic edges of a polymethyl methacrylate intraocular lens on posterior capsule opacification; a randomized trial. Ophthalmology 2005; 112:2004–2008 7. Kohnen T, Fabian E, Gerl R, Hunold W, Hu¨tz W, Strobel J, Hoyer H, Mester U. Optic edge design as long-term factor for posterior capsular opacification rates. In press, Ophthalmology 2008 8. Auffarth GU, Rabsilber TM, Reuland AJ. Neue Methoden der Nachstarpra¨vention. [New methods for the prevention of posterior capsule opacification.] Ophthalmologe 2005; 102:579–586 9. Rabsilber TM, Auffarth GU. Pharmakologische Ansa¨tze zur Pra¨vention der Cataracta Secondaria. [Pharmacological means to prevent secondary cataract.] Klin Monatsbl Augenheilkd 2006; 223:559–567 10. Power WJ, Neylan D, Collum LMT. Daunomycin as an inhibitor of human lens epithelial cell proliferation in culture. J Cataract Refract Surg 1994; 20:287–290
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11. Tetz MR, Ries MW, Lucas C, Stricker H, Vo¨lcker HE. Inhibition of posterior capsule opacification by an intraocular-lens-bound sustained drug delivery system: an experimental animal study and literature review. J Cataract Refract Surg 1996; 22: 1070–1078 12. Nishi O, Nishi K, Yamada Y, Mizumoto Y. Effect of indomethacin-coated posterior chamber intraocular lenses on postoperative inflammation and posterior capsule opacification. J Cataract Refract Surg 1995; 21:574–578 13. Liu CSC, Wormstone IM, Duncan G, Marcantonio JM, Webb SF, Davies PD. A study of human lens cell growth in vitro; a model for posterior capsule opacification. Invest Ophthalmol Vis Sci 1996; 37:906–914. Available at: http://www.iovs.org/cgi/reprint/37/5/ 906. Accessed April 10, 2008 14. Saxby L, Rosen E, Boulton M. Lens epithelial cell proliferation, migration, and metaplasia following capsulorhexis. Br J Ophthalmol 1998; 82:945–952 15. El-Osta AAR, Spalton DJ, Marshall J. In vitro model for the study of human posterior capsule opacification. J Cataract Refract Surg 2003; 29:1593–1600 16. Bundesa¨rtzekammer. Richtlinien zum Fu¨hren einer Hornhaut¨ rzteblatt. Berlin, Germany, 2000; 97: bank. Deutsches A B-1805–B-1807 17. Priglinger SG, Neubauer AS, May C-A, Alge CS, Wolf AH, Mueller A, Ludwig K, Kampik A, Welge-Luessen U. Optical
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coherence tomography for the detection of laser in situ keratomileusis in donor corneas. Cornea 2003; 22:46–50 Liekfeld A, Pahms N, Torun N, Porstmann AU, Jaroszewski J, Harmann C. Evaluation of a human capsular bag model for secondary cataract determination after intraocular lens implantation. Graefes Arch Clin Exp Ophthalmol 2005; 243: 43–48 Charters A. Wound glue: a comparative study of tissue adhesives. Accid Emerg Nurs 2000; 8:223–237 Yaron M, Erin MH, Huffer W, Cairns C. Efficacy of tissue glue for laceration repair in an animal model. Acad Emerg Med 1995; 2:259–263 Kurz S, Dick HB. Spring constants of capsular tension rings. J Cataract Refract Surg 2004; 30:1993–1997
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First author: Johannes Burger, MD General Hospital Linz, Linz, Austria.
Capsular tension ring–based in vitro capsule opacification model Johannes Burger, MD, Thomas Kreutzer, MD, Claudia S. Alge, MD, Rupert W. Strauss, MD, Kirsten Eibl, MD, Christos Haritoglou, MD, Aljoscha S. Neubauer, MD, Anselm Kampik, MD, Siegfried G. Priglinger, MD
PURPOSE: To evaluate a capsular tension ring (CTR)–supported anterior and posterior capsule opacification (PCO) model in cadaver eyes. The effect of CTR designs on lens capsule shape and lens epithelial cell (LEC) growth were investigated in vitro. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. METHODS: Following open-sky extracapsular cataract extraction, CTR models were implanted in 32 eyes of 16 human donors. The lens capsule expansion by the CTRs was evaluated. The capsular bags supported by the CTRs were excised and maintained at physiological conditions for up to 3 months. The area of LEC coverage over the posterior capsule surface was objectively determined twice a day using a graticule. RESULTS: After CTR implantation, all lens capsules could be safely excised and transferred into organ culture. The CTR designs resulted in different shapes of lens capsule expansion. Complete LEC confluence occurred after a mean of 8.25 days G 2.87 (SD) with the AcriRing KR10 (AcriTec), 6.50 G 1.0 days with the Acrimed, 8.62 G 3.34 days with the InjectoRing (Corneal), 9.00 G 1.87 days with the Morcher 14C, 9.33 G 0.75 days with the Morcher 2A, and 6.25 G 0.5 days with the Ophthalmic Innovation CTR. CONCLUSION: The CTR-supported in vitro PCO model offers a physiological method to support the lens capsule and is a reproducible system for the study of LEC proliferation. J Cataract Refract Surg 2008; 34:1167–1172 Q 2008 ASCRS and ESCRS
Posterior capsule opacification (PCO) remains the most frequent long-term complication after successful cataract extraction and subsequent implantation of an intraocular lens (IOL). Although the incidence of PCO
Accepted for publication March 29, 2008. From the Department of Ophthalmology (Burger, Kreutzer, Alge, Kampik, Priglinger), Ludwig-Maximilians-University, Munich, Germany; and the Department of Ophthalmology (Burger, Alge, Strauss, Eibl, Haritoglou, Neubauer, Priglinger), General Hospital, Linz, Austria. No author has a financial or proprietary interest in any material or method mentioned. Supported by a 2006 research grant from the European Society of Cataract and Refractive Surgeons. Corresponding author: Siegfried Priglinger, MD, Department of Ophthalmology, General Hospital Linz, Linz, Austria. E-mail: [email protected]. Q 2008 ASCRS and ESCRS Published by Elsevier Inc.
development has been significantly reduced over the past few decades, young patients and patients with uveitis or chronic intraocular inflammation show much higher rates of PCO.1–3 Besides IOL material4,5 and design,6–8 major emphasis of PCO prevention focuses on the development of antiproliferative and cytotoxic lens epithelial cell (LEC)-manipulating substances.9–12 Because of possible cell toxicity, it is usually difficult to test new substances and strategies that influence LEC growth in humans. Thus, there is considerable interest in developing in vitro organ culture models for PCO. Currently, 3 in vitro models are available.13–15 Although effectively supporting the lens capsule, all of them exhibit substantial disadvantages, making their reliability debatable. The pin model by Liu et al.13 consists of an isolated capsule secured to the surface of a Petri dish by entomological pins. The lens capsule thereby loses the natural curvature of the lens and capsule folds are induced. The model by Saxby et al.14 consists of a lens capsule containing a huge notched silicone 0886-3350/08/$dsee front matter doi:10.1016/j.jcrs.2008.03.040
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ring that covers a large capsule area and probably interferes with LEC migration. In 2003, El-Osta et al.15 introduced a lens holder system that used cyanoacrylate glue to fixate the lens for surgery and organ culture. This model does not allow separate study of anterior and posterior LEC changes; because of the use of cyanoacrylate, a possible toxic effect on LECs must be calculated. The purpose of the present study was to develop a new in vitro anterior capsule opacification (ACO) and PCO model that would reduce the disadvantages of the existing models. Unlike the existing PCO models, the new in vitro model is based on stabilizing the capsular bag with a conventional capsular tension ring (CTR) that improves visualization of the entire capsule in organ culture and reduces the effect on capsule curvature, thereby more closely resembling in vivo dynamics. MATERIALS AND METHODS Source of Tissue Thirty-two eyes of 16 human donors between 24 years of age and 76 years of age were obtained from the Eye Bank, Ludwig-Maximilians-University, Munich, Germany. Only eyes from donors with specific consent for research purposes and with contraindications for transplantation according to the guidelines of the German Federal Physician Association were included.16 All eyes were preserved in a moist chamber at 4 C within 45 hours postmortem. All experiments were performed in accord with the Declaration of Helsinki.
Surgical Procedure Whole donor eyes were thoroughly cleansed in NaCl 0.9% solution, immersed in polyvinylpyrrolidone-iodine 5%, and rinsed again in the sodium-chloride solution. Corneoscleral disks were excised and prepared for further corneal transplantation. After the iris was removed, open-sky cataract extraction was performed under sterile conditions: After continuous curvilinear capsulorhexis using a forceps, the lens nucleus and epinucleus were extracted by hydroexpression. Residual cortex was removed by irrigation with phosphate-buffered saline (PBS, pH 7.4) and a micro forceps (Kolibri, G-18950, Geuder). The capsular bag was then stabilized by implanting a capsular tension ring (CTR), circularly excised by cutting the zonules with scissors, and safely transferred into organ culture (Figure 1). The CTRs were the AcriRing KR10 (4), Acrimed (4), InjectoRing (8), Morcher 14C (6), Morcher 2A (6), and Ophthalmic Innovation (CTR12) (4). The lens capsule expansion by the CTRs was evaluated. Lens capsular shape was documented immediately after CTR implantation (before excision) and in organ culture.
Organ Culture Storage Capsular tension ring-stabilized lens capsules were stored in 35 mm wells containing 2.5 mL Eagle’s minimum essential culture medium containing 2% fetal calf serum with 100 U/ mL penicillin, 0.1 mg/mL streptomycin, and 0.25 mg/mL amphotericin B (Biochrom) and maintained at 37 C and 5% CO2 in a standard incubator for up to 3 months. The medium
was changed every 5 days.17 The LEC growth (and migration) was monitored every second day using transmission light microscopy (Leica). The area of LEC coverage over the posterior capsule surface was determined objectively 2 times a day using a grid. The ends of the CTRs were used as landmarks, making it possible to repeat observation of the same area of LEC migration.
Immunohistochemistry For staining of fibronectin in vitro, the entire lens capsule with a complete LEC monolayer on the posterior capsule was fixed in paraformaldehyde 4%. After 3 washes in Trisbuffered saline (TBS, pH 7.6) and preincubation with bovine serum albumin 3% (BSA, Roche) for 30 minutes at room temperature to minimize nonspecific staining, specimens were incubated for 15 hours at 4 C with rabbit antifibronectin (Sigma-Aldrich). The antibodies were diluted 1:100 in TBS containing BSA 3%. After 3 washes in TBS, LECs were incubated with swine anti-rabbit IgG Cy-2 (Dianova) diluted 1:100 in blocking buffer. For negative immunostaining control, LECs were incubated with BSA–TBS replacing the primary antibody. A fluorescence microscope (Leica) was used to visualize the immunofluorescent staining.
Statistical Analysis Statistical analysis was based on the Mann-Whitney U test. A P value of 0.05 or less was considered statistically significant.
RESULTS Lens Capsule Expansion The CTR designs caused different shapes of capsule expansion (Figure 1): The Acrimed, AcriRing, and Morcher 14C and 2A CTRs induced circular expansion of the lens capsule with complete closure at the ring ends; the CTR12 and the InjectoRing presented with distinct dehiscence. In addition, the InjectoRing showed irregular oval expansion of the capsule bag, thereby inducing lens capsule streaks. Lens Epithelial Cell Growth Viable LECs were detected in all free-floating CTRexpanded capsules. In the organ culture, the LECs started to migrate and proliferate, similar to the in vivo situation: After approximately 4 days, fibrotic LECs were observed crossing the capsulorhexis margin (Figure 2, A) and about 8 days G 2.34 (SD) later, a homogenous LEC monolayer covered the posterior lens capsule (Figure 2, B). If the tension ring ends did not close completely, initial growth of LEC migration toward the center of the lens capsule was observed at the dehiscence of the CTR (Figure 3). Immunostaining for fibronectin (Figure 2, C) revealed an extracellular network of immunoreactivity. Control cells incubated without the respective primary antibodies were unstained (data not shown). Immunostaining for fibronectin proved that LECs have
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Figure 1. Lens capsules stabilized by the CTRs (transmission light microscopy; original magnification 20).
transdifferentiated into fibroblast-like cell types, as described for PCO. Lens Epithelial Cell Confluence Complete LEC confluence (Figure 4) occurred after a mean of 8.25 G 2.87 days (AcriRing), 6.50 G 1.0 days (Acrimed), 8.62 G 3.34 days (InjectoRing), 9.00
G 1.87 days (Morcher 14C), 9.33 G 0.75 days (Morcher 2A), and 6.25 G 0.5 days (CTR12). Statistical analysis comparing the time until complete LEC confluence of the CTRs showed statistically significant differences (P Z .003 Wilcoxon). Subanalysis of the CTRs showed significantly delayed LEC confluence with the AcriRing, the Morcher CTRs, and the InjectoRing compared with the Acrimed and CTR12 (P!.05 each,
Figure 2. A: Migration of LECs 5 days after transfer to culture medium. B: Confluent LECs on the entire posterior capsule (day 12 of organ culture). C: Immunohistochemical staining for fibronectin (original magnification 100).
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Figure 3. Oval expansion of a lens capsule by an InjectoRing, resulting in a dehiscence at the CTR ends. The initial growth of LECs on the posterior capsule is seen in the gap between the CTR ends.
Mann-Whitney U test. Between the 4 CTRs with delayed LEC confluence, there was no statistically significant difference (PO.05 each, Mann-Whitney U test). DISCUSSION This study showed that a CTR-stabilized lens capsule was an excellent tool for in vitro evaluation of LEC growth. Capsule stabilization with conventional CTRs allowed easy and safe transfer of lens capsules into organ culture. Capsular tension ring stabilization showed capsule expansion with minimal effects on the natural shape of the capsule. This may allow LEC growth that closely resembles the in vivo dynamics of PCO development. The free-floating CTR-expanded capsule provided a physiological setting for LEC growth in organ culture as there was always a layer of culture medium between the outer surface of the posterior capsule and the culture dish, ensuring uninfluenced metabolic supply, which is a major advantage of our model. In addition, the CTR model allowed detailed and close observation of postoperative PCO development; the dynamics of cell growth could be observed since minimal capsule area was concealed by the CTR.
Figure 4. Confluence of LECs with various CTRs.
Several in vitro organ culture models that have different approaches to supporting the lens capsule exist.13–15 Although they allow lens capsule culture, they limit the in vitro evaluation of natural LEC growth because they modify the capsule in various ways postoperatively. Liu et al.13 describe a PCO model in which the isolated capsule is secured to the surface of a Petri dish with 6 to 8 entomological pins. Pinning the specimen to the capsule dish changes the natural curvature of the capsule, resulting in multiple folds of the capsule. We think the nonphysiological deformation of the lens capsule and the contact with the culture dish are major disadvantages because they might influence natural LEC growth. Pinning the specimen to the capsule could also lead to peripheral collapse of the capsule, inducing contact zones between the anterior and posterior capsules. These contact zones allow direct LEC migration from the capsulorhexis edge onto the posterior capsule. Therefore, areas in the peripheral capsule are occluded and investigation of LEC growth in these areas is hindered. However, in IOL implantation, contact between the anterior and posterior capsules is desirable to be able to study the effect of IOL designs on LEC migration. In their model, Liekfeld et al.18 used 6 to 8 entomological pins through the edge of the capsular bag to provide maximum contact between the posterior capsule and the posterior IOL surface. A major advantage of our in vitro PCO model is that using CTRs with different heights, it is possible to provide anterior and posterior capsule contact or noncontact. With a thicker CTR, such as the foldable Morcher 2A, the contact between anterior and posterior capsules can easily be avoided, allowing anterior and posterior capsule phenomena to be studied separately. With a thinner CTR, such as the InjectorRing, contact is achieved in the first week of organ culture, allowing appropriate PCO study after IOL implantation. The endocapsular ring model of Saxby et al.14 separates the anterior and posterior capsules and prevents capsule collapse by supporting the capsule internally with a notched 9.63 mm 1.78 mm (external diameter height) silicone ring. Because of this large dimension, interference with LEC migration must be assumed. Furthermore, the large area covered cannot be adequately monitored by transmission light microscopy. In contrast, the CTRs used in our PCO model cover only a small capsule area, allowing investigation of more peripheral capsule areas. Intraocular lens implantation is possible with Saxby et al.’s model; however, the resultant cell behavior does not reproduce the in vivo situation as no contact between anterior and posterior capsules can be achieved. Furthermore, the endocapsular ring model of Saxby et al. shows continuous contact between the posterior capsule and the
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LABORATORY SCIENCE: TENSION RING–BASED PCO MODEL
bottom of the culture dish, which might impede metabolic supply. El-Osta et al.15 introduced a lens holder system with the use of cyanoacrylate glue to fixate the lens for surgery and organ culture. The special lens holders have a central aperture that allows visualization of LEC growth in this area; however, in the equator region, continuous observation of LEC growth with transmission light microscopy is limited because the area of rigid fixation of the capsule to the lens holder is quite large. Furthermore, El-Osta et al.’s lens holder model does not allow separate study of anterior and posterior LEC changes because the capsules are in contact with one another. Therefore, at the capsulorhexis margin, direct migration of LECs from the anterior to the posterior capsule occurs. The major drawback of El-Osta et al.’s model is the use of cyanoacrylate. Cyanoacrylates are adhesive substances that have been widely used in surgical procedures.19,20 However, their specific effect on cell growth and metabolism is not fully understood. In El-Osta et al.’s PCO model, higher concentrations of cyanoacrylate resulted in significant cell death as shown in 3-(4,5 dimethylthiazole-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) and live-dead assays.15 Although vitality of LECs was less affected by lower concentrations (1 mL/mL), a significant number of cells died. Therefore, cell toxicity can not be excluded and a possible negative influence on LEC behavior in this model must be calculated. A major advantage of our in vitro model is the excellent stabilization of the lens capsule by the implanted CTR without changes in the natural capsule shape after cataract extraction. Even in cases that exhibit a significant degree of zonule damage after cornea removal during the eye-banking procedure, CTR implantation leads to excellent stability that allows safe capsule excision and transfer into organ culture. Once in organ culture, it is not necessary to fixate, pin, or glue the capsule on the culture dish as the CTR itself expands and stabilizes the capsule. All the CTRs used in this study differed in the shape of capsule expansion. The overall time to LEC confluence was approximately 8 days, with the Ophthalmic Innovation model having the shortest delay in full coverage of the posterior capsule (6.25 days). We also observed that LECs always started to migrate toward the center of the posterior capsule at the CTR dehiscence. We therefore initially assumed that CTRs with no dehiscence at the ring ends might have a prolonged interval until LEC confluence. Although there was a slight tendency of earlier LEC confluence in CTRs with incomplete tension ring closure, statistical analysis revealed that complete closure of the CTR had no effect on the duration until complete LEC confluence on the posterior capsule. The spring constants
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of CTRs,21 another factor that might influence LEC growth after CTR implantation in vitro, also showed no effect on LEC growth in organ culture. However, the fact that no statistical difference in overall coverage time was found with CTRs with closed or open ends and different spring constants does not imply that the CTR does not affect LEC growth. The CTR-stabilized PCO model offered excellent stabilization of the lens capsule. Depending on the CTR used, contact or noncontact of the anterior and posterior capsules can be achieved. A wide area of the model is accessible for monitoring by transmission light microscopy; it is simple and user friendly and mimics physiological conditions. These properties make this model an ideal tool for studies of ACO and PCO development and enables evaluation of LEC migration and proliferation in vitro. Furthermore, this in vitro model allows testing of new strategies for PCO prevention and for objectively grading IOL performance in vitro. Nevertheless, one has to be aware of the limitation of any model to mirror the dynamics of PCO in vivo. REFERENCES 1. Pandey SK, Apple DJ, Werner L, Maloof AJ, Milverton EJ. Posterior capsule opacification: a review of the aetiopathogenesis, experimental and clinical studies and factors for prevention. Indian J Ophthalmol 2004; 52:99–112 2. Sundelin K, Sjo¨strand J. Posterior capsule opacification 5 years after extracapsular cataract extraction. J Cataract Refract Surg 1999; 25:246–250 3. Bertelmann E, Kojetinsky C. Posterior capsule opacification and anterior capsule opacification. Curr Opin Ophthalmol 2001; 12:35–40 4. Hollick EJ, Spalton DJ, Ursell PG, Meacock WJ, Barman SA, Boyce JF. Posterior capsular opacification with hydrogel, polymethylmethacrylate, and silicone intraocular lenses: two-year results of a randomized prospective trial. Am J Ophthalmol 2000; 129:577–584 5. Ursell PG, Spalton DJ, Pande MV, Hollick EJ, Barman S, Boyce J, Tilling K. Relationship between intraocular lens biomaterials and posterior capsule opacification. J Cataract Refract Surg 1998; 24:352–360 6. Findl O, Buehl W, Menapace R, Sacu S, Georgopoulos M, Rainer G. Long-term effect of sharp optic edges of a polymethyl methacrylate intraocular lens on posterior capsule opacification; a randomized trial. Ophthalmology 2005; 112:2004–2008 7. Kohnen T, Fabian E, Gerl R, Hunold W, Hu¨tz W, Strobel J, Hoyer H, Mester U. Optic edge design as long-term factor for posterior capsular opacification rates. In press, Ophthalmology 2008 8. Auffarth GU, Rabsilber TM, Reuland AJ. Neue Methoden der Nachstarpra¨vention. [New methods for the prevention of posterior capsule opacification.] Ophthalmologe 2005; 102:579–586 9. Rabsilber TM, Auffarth GU. Pharmakologische Ansa¨tze zur Pra¨vention der Cataracta Secondaria. [Pharmacological means to prevent secondary cataract.] Klin Monatsbl Augenheilkd 2006; 223:559–567 10. Power WJ, Neylan D, Collum LMT. Daunomycin as an inhibitor of human lens epithelial cell proliferation in culture. J Cataract Refract Surg 1994; 20:287–290
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11. Tetz MR, Ries MW, Lucas C, Stricker H, Vo¨lcker HE. Inhibition of posterior capsule opacification by an intraocular-lens-bound sustained drug delivery system: an experimental animal study and literature review. J Cataract Refract Surg 1996; 22: 1070–1078 12. Nishi O, Nishi K, Yamada Y, Mizumoto Y. Effect of indomethacin-coated posterior chamber intraocular lenses on postoperative inflammation and posterior capsule opacification. J Cataract Refract Surg 1995; 21:574–578 13. Liu CSC, Wormstone IM, Duncan G, Marcantonio JM, Webb SF, Davies PD. A study of human lens cell growth in vitro; a model for posterior capsule opacification. Invest Ophthalmol Vis Sci 1996; 37:906–914. Available at: http://www.iovs.org/cgi/reprint/37/5/ 906. Accessed April 10, 2008 14. Saxby L, Rosen E, Boulton M. Lens epithelial cell proliferation, migration, and metaplasia following capsulorhexis. Br J Ophthalmol 1998; 82:945–952 15. El-Osta AAR, Spalton DJ, Marshall J. In vitro model for the study of human posterior capsule opacification. J Cataract Refract Surg 2003; 29:1593–1600 16. Bundesa¨rtzekammer. Richtlinien zum Fu¨hren einer Hornhaut¨ rzteblatt. Berlin, Germany, 2000; 97: bank. Deutsches A B-1805–B-1807 17. Priglinger SG, Neubauer AS, May C-A, Alge CS, Wolf AH, Mueller A, Ludwig K, Kampik A, Welge-Luessen U. Optical
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First author: Johannes Burger, MD General Hospital Linz, Linz, Austria.