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Influence of fibronectin on the adherence of Staphylococcus epidermidis to coated and uncoated intraocular lenses
LABORATORY SCIENCE
Influence of fibronectin on the adherence of Staphylococcus epidermidis to coated and uncoated intraocular lenses Andreas C. Schroeder, MD, Josef M. Schmidbauer, MD, Astrid Sobke, MD, Berthold Seitz, MD, FEBO, Klaus W. Ruprecht, MD, Mathias Herrmann, MD
PURPOSE: To determine the effect of the modification of intraocular lens (IOL) surface properties on the adhesion of Staphylococcus epidermidis caused by fibronectin (FN) as the predominant proadhesive glycoprotein of the eye’s initial foreign body reaction. SETTING: University of Saarland, Homburg/Saar, Germany. METHODS: Eleven IOL types were tested. The IOLs were of poly(methyl methacrylate), acrylate, or silicone. Some were surface modified with heparin or polysaccharide coating. The IOLs, unadsorbed or preadsorbed with fibronectin (FN), were incubated with [3H]-thymidine-labeled S epidermidis Rp62a, and the amount of adherent microorganisms was determined. RESULTS: Attachment of S epidermidis adhesion to various types of IOLs, both unadsorbed and FN precoated, varied significantly. The attachment to highly adhesive IOLs was almost 4-fold greater than that to low-adhesive IOLs. Attachment to FN precoated IOLs was generally enhanced compared with attachment to unadsorbed IOLs. Heparin surface modification resulted in no or a modest reduction in bacterial adhesion compared with unmodified IOLs. Bacterial adhesion was highly statistically significantly less on IOLs with polysaccharide surface modification. CONCLUSIONS: There was significant variability in S epidermidis adhesion to IOLs as a function of design, material, surface modification, and FN preadsorption. Application of the findings may foster new developments to further reduce the major complication in cataract surgery, infective endophthalmitis. J Cataract Refract Surg 2008; 34:497–504 Q 2008 ASCRS and ESCRS
When Harold Ridley implanted the first intraocular lens (IOL) made of poly(methyl methacrylate) (PMMA) into the posterior chamber of a patient’s eye in 1949,1 it was the first step in a development that lead to modern cataract surgery being the most frequently performed operative intervention in the world. However, from Ridley’s time2–4 to now,5–7 postoperative endophthalmitis remains the most feared complication of cataract surgery. Improvements in operative techniques and the introduction of new IOL materials and surface modifications have reduced the incidence of this dreaded complication to a rate of 0.05% to 0.50%.6,7 Although this rate appears to be low, the consequences of prosthetic lens– associated endophthalmitis are potentially grave. As the Endophthalmitis Vitrectomy Study reports,6 47% of patients present with a visual acuity of 0.5 or worse 9 to 12 months after cataract surgery. However, even without frank infection, IOL implantation is accompanied by mild inflammatory tissue reaction.8–10 Q 2008 ASCRS and ESCRS Published by Elsevier Inc.
Moreover, as shown in foreign-body infection models, the presence of prosthetic material triggers a local immune defect with functional impairment of neutrophils derived from the periprosthetic fluid, reduced local levels of the cytokine levels within the first hours after implantation, and an ensuing reduction in complement-mediated opsonic activity.11 It is thought that this compromised innate immunity contributes to the susceptibility of medical implants, including IOLs. Immediately after implantation of a foreign body, the implant’s surface characteristics are profoundly modified by immediate adsorption of a protein layer.12 Vitronectin, fibronectin (FN), laminin, and collagen type IV have been found on explanted IOLs.9,10 In particular, FN has played a crucial role in the promotion of bacterial adhesion to biomaterial surfaces, as seen in in vitro and in vivo studies.11,13–18 Fibronectin, a protein of the extracellular matrix, exists in a soluble form in most fluids of the human body. This multifunctional 0886-3350/08/$dsee front matter doi:10.1016/j.jcrs.2007.10.042
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protein contains binding sites for fibrin, collagen, integrins, heparin, and other cellular and extracellular molecules. Upon adsorption to foreign bodies, FN has been shown to act as a mediator for bacterial adhesion.14,18 A detailed analysis of the promotion of bacterial attachment as a function of IOL design and material in conjunction with the presence or absence of adhesive host factors is not available. To model the clinical situation of IOL implantation, 2 scenarios of bacterial interaction with the IOL surface must be taken into account: (1) bacterial adhesion occurs before IOL implantation (ie, to unadsorbed IOL material) or (2) adhesion occurs during or after IOL implantation (ie, to IOL preadsorbed with host factors). Staphylococcus epidermidis is the most frequently encountered causative pathogen in endophthalmitis after cataract surgery.7 It is believed that the pathogenicity of this organism in foreign-body infections results from its ability to adhere to plain and host factor–adsorbed polymer material and to produce a glycocalix biofilm that interferes with host immune defense and with antibiotic treatment.19,20 Accordingly, the goal of this study was to determine the adhesion characteristics of S epidermidis to IOLs of various designs with and without the presence of FN as a model proadhesive host factor. MATERIALS AND METHODS Intraocular Lenses Eleven types of IOLs were supplied by 3 manufacturers: Acri.Tec, Advanced Medical Optics (AMO), Corneal. The
Accepted for publication October 25, 2007. From the Department and Hospital of Ophthalmology (Schroeder, Schmidbauer, Seitz, Ruprecht) and the Institute of Medical Microbiology and Hygiene (Sobke, Herrmann), University of Saarland Hospital, Homburg/Saar, Germany. No author has a financial or proprietary interest in any material or method mentioned. Supported by a grant from the Medical Faculty, University of Saarland, Homberg/Saar, Germany. Presented at the annual meeting of the XXII Congress of the European Society of Cataract & Refractive Surgeons, Paris, France, September 2004, and in part at the ASCRS Symposium on Cataract, IOL and Refractive Surgery, San Francisco, California, USA, March 2006. K. Hilgert provided technical help. Corresponding author: A.C. Schroeder, MD, Department of Ophthalmology, University of Saarland, Kirrberger Strasse 1, D-66424 Homburg (Saar), Germany. E-mail: [email protected].
IOLs were PMMA, hydrophilic acrylate, hydrophobic acrylate, or silicone (Table 1). Three types of IOLs were surface modified by the manufacturer: The ACR 6D SE IOL, which is heparin surface modified (HSM) hydrophilic acrylate, was compared with the unmodified 600 SE, which is of the same hydrophilic acrylate. The New Six IOL, which is HSM PMMA, was compared with the uncoated Phac 57 T of otherwise identical design. Finally, 2 silicone IOLs were analyzed: a polysaccharide-surface-modified (PSM) 73NAL/B prototype and a 73N-AL uncoated. The various acrylate materials differ in water content and hydrophobicity. Unpublished studies of these materials suggest, however, that contact-angle determinations do not clearly allow one to assign IOL materials to categories defined by water content. Accordingly, the results presented here were obtained based on the primary composition of the IOL material (eg, all acrylate materials were analyzed in 1 category).
Bacterial Strain The strongly biofilm-producing strain S epidermidis RP 62a was used throughout the study. For bacterial labeling, 40 mL of a fresh overnight bacterial suspension were incubated with 50 mL [3H]-thymidine in 1 mL Mueller-Hinton broth for 3 hours in a 37 C shaking water bath. Colony-forming units were counted on Mueller-Hinton plates before every experiment to control for a reproducible inoculum size throughout the study.
In Vitro Adhesion Assay of Staphylococcus Epidermidis to Intraocular Lenses Experimental Settings Bacterial adherence was determined using a modification of a an adhesion assay.15 First, attachment to cover slips of the model material PMMA was tested. Next, to simulate the 2 scenarios of IOL contamination by microorganisms in vivo, 2 experimental settings were established. In experimental setting 1, the IOL was directly removed from its original packing and incubated with the labeled microorganisms in unsupplemented phosphate-buffered saline (PBS) in an adhesion assay as described below. This assay monitors bacterial staphylococcal attachment primarily due to nonspecific physicochemical interactions, particularly surface charge and hydrophobicity. In setting 2, the IOL was precoated with FN and then incubated in PBS supplemented with 0.5% albumin to simulate aqueous humor. This assay reflects adhesion primarily due to specific interactions of bacterial surface components with the adsorbed glycoprotein as nonspecific attachment is blocked. Fibronectin Preadsorption Pilot experiments using plain PMMA slides showed the ideal concentration of FN for the coating to be 2 mg/mL PBS (data not shown). Accordingly, each IOL in the second experimental setting was immersed in 1 mL of this solution for 1 hour at 37 C. Afterward, the solution was decanted; the IOLs were rinsed with PBS and then stored at 4 C for a maximum of 2 hours. Adhesion Assay A parallel examination of 8 IOLs per type and setting were performed during each experiment. For each attachment test, 1 IOL was immersed in 960 mL PBS with 40 mL 3H-labeled bacteria solution added. In experiments in setting 1, unadsorbed IOLs were incubated in unsupplemented PBS. In setting 2, an FN-coated IOL was
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Table 1. Intraocular lenses tested in study. Material
Manufacturer
IOL
Haptics
Optic (mm)
Haptics (mm)
Polymer (Surface Modification)
Acrylate Acrylate Acrylate Acrylate Acrylate PMMA PMMA PMMA Silicone Silicone Silicone
Acri.Tec Acri.Tec AMO Corneal Corneal Acri.Tec Corneal Corneal Acri.Tec Acri.Tec AMO
49S 43C-5 AR 40e 600 SE ACR 6D SE 14C Phac 57 T New Six 73N-AL 73N-AL/B Clariflex B
Plate 1-piece 3-piece 3-piece 1-piece 3-piece 3-piece 3-piece 3-piece 3-piece 3-piece
5.0 6.0 6.0 6.0 6.0 7.0 5.7 6.0 6.0 6.0 6.0
11.00 10.50 13.00 13.00 12.00 10.50 12.50 12.75 12.50 12.50 13.00
HEMA/MMA HEMA/MMA EA/EMA/TFEMA HEMA/MMA HEMA/MMA (heparin) PMMA PMMA PMMA (heparin) PDMS PDMS (polysaccharide) PDMDPS
EA Z ethyl acrylate; EMA Z ethyl methacrylate; HEMA Z 2-hydroxylethyl methacrylate; IOL Z intraocular lens; MMA Z methyl methacrylate; PDMDPS Z poly(dimethyldiphenylsiloxane); PDMS Z poly(dimethylsiloxane); PMMA Z poly(methyl methacrylate); TFEMA Z 2,2,2-trifluoroethyl methacrylate
immersed in PBS supplemented with 0.5% human serum albumin. Subsequently, the IOLs were incubated at 37 C in a shaking water bath for 1 hour. Thereafter, the supernatant was removed; the IOLs were washed twice in PBS to remove the loosely attached bacteria and then transferred into scintillation vials for determination of adherent radioactivity. Bacterial adherence (%) was determined as adherent cpm/ inoculated cpm 100.
Statistical Analysis Data are presented as means and standard error of the mean (SEM). To determine the significance of the results, an analysis of variance (ANOVA) was performed for the results in each experimental setting. If the ANOVA showed significant differences, a Student t test was used to compare adherence values of the various IOLs to a standard IOL (as described in Results) or to compare the effect of FN precoating or of a manufacturer-performed surface modification with the respective uncoated or unmodified IOL material. A P value of 0.05 or less was considered statistically significant.
Staphylococcus Epidermidis Adhesion to Unadsorbed Intraocular Lenses (Setting 1) The results of the adhesion assays to plain IOLs are shown in Figure 2 and Table 2. A significant variation in results was found by ANOVA (F Z 8.88; P!.001). To compare staphylococcal attachment between the groups of acrylate IOLs, the AR40 was used as the standard acrylic IOL (mean adhesion 0.92% G 0.20%). This IOL type was the most frequently used acrylate IOL in clinical use at the authors’ institution at the time of the study. Statistically significant differences in bacterial attachment were found between this standard IOL and the other acrylic IOLs. Bacterial
RESULTS Staphylococcus Epidermidis Adhesion to Poly(methyl methacrylate) Cover Slips A concentration of 0.5% bovine serum albumin (BSA) blocked the adhesion maximally and significantly (P Z .001) (Figure 1). In contrast, precoating of PMMA cover slips with FN resulted in restored bacterial adhesion even in the presence of albumin in the incubation medium. Compared with attachment to uncoated, albumin-free PMMA surfaces, attachment to FN-coated surfaces was significantly elevated. These experiments confirmed the role of FN-promoted attachment of S epidermidis to a PMMA substrate independent of nonspecific interaction forces.
Figure 1. S epidermidis RP62A adhesion to PMMA slides as a function of PBS supplementation with BSA and preadsorption with FN. The PMMA cover slips were incubated with [3H]-thymidine–labeled S epidermidis in the absence of (left bar) or in the presence of BSA (center bar). Alternatively, PMMA cover slips were precoated with FN and then incubated with S epidermidis in BSA PBS (right column) (n Z 3) (** Z P!.05; *** Z P!.01; BSA Z bovine serum albumin; FN Z fibronectin; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
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Figure 2. Results in setting 1: S epidermidis RP62A adherence to various IOLs in noncolloidal medium. Bacteria were incubated with IOLs in unsupplemented PBS analogous as in Figure 1, and relative bacterial adhesion was determined (n Z 8) (** Z P!.05; *** Z P!.01; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
attachment to the 49S IOL was lowest and to the 43C-5 IOL, highest. Of the 2 types of PMMA IOLs tested, the Phac 57 T had statistically significantly lower staphylococcal adhesion than the 14C IOL. In contrast, the 2 silicone IOLs did not have statistically significant differences in S epidermidis adhesion. Overall, analysis of the results of attachment to the different groups of IOL material showed no statistically significant differences. The median bacterial attachment was 0.88%, 0.89%, and 0.90% to acrylate, PMMA, and silicone, respectively. Staphylococcus Epidermidis Adhesion to Fibronectin-Adsorbed Poly(methyl methacrylate) (Setting 2) The results for the FN-coated IOLs in setting 2 showed greater variability than the results in setting
1 (F Z 11.92; P!.001; ANOVA) (Figure 3 and Table 2). Overall, bacterial attachment values were more elevated than values for unadsorbed IOLs (Figure 4). Compared to rates for the standard AR40 IOL, attachment rates to the various acrylate IOLs differed significantly, with the 600 SE IOL having the lowest adhesion and the 43C-5 the highest. In contrast to assays with unadsorbed IOLs, attachment to PMMA IOLs did not significantly differ; the results obtained with the 2 silicone IOLs were comparable. No statistically significant difference was found to be attributable to the IOL materials in general. The median adhesion values were 1.39%, 1.59%, and 1.85% for silicone, PMMA, and acrylate, respectively. Influence of Manufacturer-Provided Intraocular Lens Surface Modifications Poly(Methyl Methacrylate) Versus Heparin-SurfaceModified Poly(methyl methacrylate) In setting 1 and setting 2, comparison of the HSM Phac 57 T IOL and the unmodified New Six IOL showed slightly decreased S epidermidis adherence to the unmodified IOL; however, the difference was not significant (Figure 5). Similarly, although FN preadsorption did not modify this relative adherence result with respect to heparin surface modification, FN significantly promoted bacterial attachment to both IOL types. Unmodified Acrylate Versus Heparin-Surface-Modified Acrylate In contrast to heparin modification of PMMA, heparin surface modification of acrylate IOLs significantly modified bacterial attachment in both experimental settings, albeit in a different manner as a function of FN preadsorption. In setting 1 (unadsorbed), the S epidermidis attachment was significantly reduced by the heparin modification (600 SE versus ACR6D SE, P Z .015). In setting 2 (FN adsorbed),
Table 2. Bacterial adhesion rates. Bacterial Adhesion Rate (%) G SEM Material
IOL
Acrylate 49S Acrylate 43C-5 Acrylate AR 40e Acrylate 600 SE Acrylate ACR 6D SE PMMA 14C PMMA Phac 57 T PMMA New Six Silicone 73N-AL Silicone 73N-AL/B Silicone Clariflex B
Plain IOL 0.40 G 0.07 1.77 G 0.30 0.92 G 0.20 0.44 G 0.05 0.29 G 0.02 1.15 G 0.14 0.64 G 0.08 0.48 G 0.08 0.98 G 0.23 0.18 G 0.02 0.81 G 0.19
FN-Coated IOL P Value 1.26 G 0.21 3.27 G 0.48 1.98 G 0.24 0.87 G 0.10 1.05 G 0.08 1.81 G 0.20 1.36 G 0.15 1.09 G 0.12 1.62 G 0.22 0.54 G 0.06 1.96 G 0.15
!.01 !.05 !.01 !.01 !.01 !.05 !.01 !.01 NS !.01 NS
FN Z fibronectin; IOL Z intraocular lens; NS Z not significant
Figure 3. Staphylococcus epidermidis RP62A adherence to FN-preadsorbed IOLs in a colloidal buffer medium. Bacteria were incubated with IOLs in BSA-supplemented PBS analogous as in Figure 1, and relative bacterial adhesion was determined (n Z 8) (** Z P!.05; *** Z P!.01; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
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Figure 4. Comparison of S epidermidis adhesion to IOLs of acrylate (triangles), PMMA (squares), or silicone (circles) as a function of IOL preadsorption with FN (n Z 8) (BSA Z bovine serum albumin; FN Z fibronectin; IOL Z intraocular lens; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
Figure 5. Staphylococcus epidermidis adherence to PMMA IOLs as a function of heparin surface modification. Unmodified IOLs or HSM-modified IOLs were tested for S epidermidis adhesion as in Figure 2 in plain PBS or after FN preadsorption in BSA–PBS. (n Z 8) (*** Z P!.01; FN Z fibronectin; HSM Z heparin surface modified; IOLZ intraocular lens; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
the HSM IOLs had more elevated attachment values than the unmodified IOLs (P Z .182) (Figure 6). Similar to findings with the PMMA IOLs, bacterial attachment to both acrylate IOLs was increased by FN coating to a highly significant extent.
a difference that was highly statistically significant compared with the values for the uncoated silicone IOLs (73N-AL versus 73N-AL/B; setting I: P Z .004; setting II: P!.001) (Figure. 7). In contrast to other surface modifications and IOL materials, FN-adsorption did not result in a significant adhesion-promoting effect when comparing the unmodified silicone IOLs, whereas FN significantly promoted attachment to the PSM silicone IOL, although the adhesion values of FN-adsorbed PSM silicone IOLs were still lower than those of unmodified silicone IOLs in the absence of FN.
Unmodified Silicone Versus Polysaccharide SurfaceModified Silicone The most significant influence of surface modification was found when comparing silicone IOLs with or without PSM. In both experimental settings, the reduction in the adhesion values was between 5-fold (setting 1) and 3-fold (setting 2),
Figure 6. Staphylococcus epidermidis adherence to acrylate IOLs as a function of HSM. Unmodified IOLs or HSM-modified IOLs were tested for S epidermidis adhesion as in Figure 2 in plain PBS or after FN preadsorption in BSA–PBS (n Z 8) (** Z P!.05; *** Z P!.01; FN Z fibronectin; HSM Z heparin surface modified; IOLZ intraocular lens; n.s. Z not significant; Rel. Bact. Z relative bacterial).
Figure 7. Staphylococcus epidermidis adherence to silicone IOLs as a function of PSM. Unmodified IOLs or PSM-modified IOLs were tested for S epidermidis adhesion as described in Figure 2 in plain PBS or after FN preadsorption in BSA–PBS (n Z 8) (*** Z P!.01; FN Z fibronectin; IOLZ intraocular lens; n.s. Z not significant; PSM Z polysaccharide surface modified; Rel. Bact. Z relative bacterial).
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DISCUSSION In this study, 11 types of IOLs of different materials and with different surface modifications were compared with respect to their adhesive properties for S epidermidis, the most often identified isolate in postsurgical endophthalmitis to the various materials. Our results show that the surface modification an IOL provides has important potential in modulating adherence of S epidermidis in vitro. We found bacterial adherence rates to plain IOLs that are in agreement with the results in other studies performed with IOLs in vitro.20–27 The exact mechanisms resulting in IOL colonization and subsequent infection are not entirely resolved. In particular, although it is likely that most infections occur during the time of insertion, in vitro models reflecting the pathogenic steps must consider the presence of proadhesive host factors in the incisional wound and intraocular fluid composition readily adsorbing the artificial substrate. In vitro models of such pathogenic mechanisms must therefore investigate both scenarios; that is, bacterial interaction with IOL substrates preexposed to proadhesive factors such as FN as well as uncoated IOL substrates. Several factors are important in the initial phase of bacterial adhesion; that is, physicochemical forces, the capsular polysaccharide of the bacteria, and the presence of surface proteins.28 Although physicochemical forces underlie all attachment events and the presence or absence of bacterial capsular polysaccharides is a strain-dependent characteristic, we modeled the presence or absence of adsorbed host factors on the different IOL materials. Upon exposure of artificial lens material to vitreous humor, such almost instant deposition of adhesive host molecules is a passive yet physiological mechanism viewed as an integral part of the foreign-body reaction in the aqueous humor.9,10,29–32 The in vitro adsorption of soluble FN to different types of IOL has been studied,30 and it has been shown that FN adheres significantly better to acrylate IOLs and PMMA IOLs than to silicone IOLs (both P!.01). These findings correspond to the changes in staphylococcal adhesion found in our study, as FN promoted staphylococcal adhesion to a larger extent when preadsorbed to acrylate or PMMA than when preadsorbed to silicone. To our knowledge, bacterial attachment to in vivo IOLs was evaluated in only 1 study.33 In this study, the foreign-body reaction and bacterial attachment to IOLs explanted from pigs 1 week after implantation were evaluated. The study results are in accordance to results in our experiments determining bacterial adhesion after FN preadsorption. Bacterial adhesion to protein-modified IOL substrates was also assessed
in another study using cerebrospinal fluid as an incubation medium (a medium containing multiple adhesive proteins including FN)34; however, as FN-free conditions were not examined in this study, the specific role of adhesive proteins could not be determined. In the development of new IOL generations, surface treatment is often used in an attempt to modify foreign-body biocompatibility. Of the interventions proposed, smoothening the material surface has proven to be effective35,36 and has now become standard in IOL manufacturing. In our study, the 49S and 43C-5 IOL pair demonstrate the potential advantage of design improvement. Both are of the same 2-hydroxylethyl methacrylate/methyl methacrylate copolymer, representing 2 generations of acrylate IOLs. The obsolescent 43C-5 IOL had the highest staphylococcal adhesion, whereas the newer generation 49S had comparably low attachment results. The modifications of the nature of the material between the 2 IOL generations are the intellectual property of the manufacturer and therefore not disclosed. However, modifications of the new acrylate IOL material (proprietary name Acri.Lyc) are not due to any coating or etching process and appear not to confer a significant change in surface hydrophobicity, as we determined by contact-angle measurement (data not shown). Surface hydrophobicity versus hydrophilicity, however, may be a potential adhesion-relevant characteristic aimed at by substrate modification. Manufacturers have attempted to reduce bacterial colonization by introducing more hydrophilic lens surfaces. Such surface modification strategy can be achieved by coating with heparin. Our results with heparin-adsorbed surfaces yielded an effect on staphylococcal adhesion that was unanticipated. Although with PMMA there was nonsignificant tendency toward reduced attachment to the heparin coated surface, with acrylate opposite effects dependent on FN precoating were observed. Heparin bonding resulted in significantly reduced attachment to the unmodified IOL; however, after FN coating the difference was not only eliminated, it was reversed. One possible explanation for this striking finding is that the hydrophilic surface of the heparinized acrylate modifies the presentation and conformation of the FN molecule, enhancing the FN–S epidermidis interaction. Clinical disadvantages of heparin surface modification were presented by Winther-Nielson et al.37 in 1998. The authors found significantly higher rates of posterior capsule opacification (PCO) between HSM PMMA and PMMA. On the other hand, a promising approach shown with the polysaccharide-coated silicone IOL (73N-AL/B), which was a prototype at the time of this study. The clear and significant reduction in staphylococcal adhesion independent of the FN coating was
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not anticipated and may have promising indications in the design of new IOL generations. In essence, when an IOL is implanted, host factor adsorption occurs independent of the nature of the IOL material.38 Such reaction must be considered when examining the interaction between the IOL and the surrounding humor and tissue with respect to bacterial adhesion and development of PCO. To further improve biocompatibility, focusing on modulation of this reaction rather than on developing and using new materials could be beneficial.38 Our results showing the effect of a modification of IOL surface properties on the adhesion of S epidermidis can be considered an important step toward this goal. REFERENCES 1. Ridley H. Intra-ocular acrylic lenses; a recent development in the surgery of cataract. Br J Ophthalmol 1952; 36:113–122 2. Reese WS. Results in 39 bilateral Ridley intra-ocular implants. Br J Ophthalmol 1960; 44:713–717 3. Ridley H. Further experiences of intra-ocular acrylic lens surgery; with a report of more than 100 cases. Br J Ophthalmol 1954; 38:156–162 4. Ridley H. Intra-ocular acrylic lenses; 10 years’ development. Br J Ophthalmol 1960; 44:705–712 5. Barequet IS, Baker AS, Schein OD. Ocular infections. In: Waldvogel FA, Bisno AI, eds, Infections Associated with Indwelling Medical Devices 3rd ed. Washington, DC, American Society for Microbiology, 2000; 287–306 6. Endophthalmitis Vitrectomy Study Group. Results of the Endophthalmitis Vitrectomy Study; a randomized trial of immediate vitrectomy and of intravenous antibiotics for the treatment of postoperative bacterial endophthalmitis; Endophthalmitis Vitrectomy Study Group. Arch Ophthalmol 1995; 113:1479–1496 7. Heaven CJ, Mann PJ, Boase DL. Endophthalmitis following extracapsular cataract surgery: a review of 32 cases. Br J Ophthalmol 1992; 76:419–423 8. Kanagawa R, Saika S, Ohmi S, Tamura M, Nakao T. Presence and distribution of fibronectin on the surface of implanted intraocular lenses in rabbits. Graefes Arch Clin Exp Ophthalmol 1990; 228:398–400 9. Linnola RJ, Werner L, Pandey SK, et al. Adhesion of fibronectin, vitronectin, laminin, and collagen type IV to intraocular lens materials in pseudophakic human autopsy eyes. Part 2: explanted intraocular lenses. J Cataract Refract Surg 2000; 26:1807–1818 10. Linnola RJ, Werner L, Pandey SK, et al. Adhesion of fibronectin, vitronectin, laminin, and collagen type IV to intraocular lens materials in pseudophakic human autopsy eyes. Part 1: histological sections. J Cataract Refract Surg 2000; 26:1792–1806 11. Vaudaux P, Lew DP, Waldvogel FA. Host factors predisposing to and influencing therapy of foreign body infections. In: Bisno Al, Waldvogel FA, eds, Infections Associated with Indwelling Devices 2nd ed. Washington, DC, American Society for Microbiology, 1994; 1–29 12. Ratner BD. Replacing and renewing: synthetic materials, biomimetics, and tissue engineering in implant dentistry. J Dent Edu 2001; 65:1340–1347 13. Vaudaux P, Pittet D, Haeberli A, et al. Host factors selectively increase staphylococcal adherence on inserted catheters: a role for fibronectin and fibrinogen or fibrin. J Infect Dis 1989; 160:865–875
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14. Vaudaux P, Pittet D, Haeberli A, et al. Fibronectin is more active than fibrin or fibrinogen in promoting Staphylococcus aureus adherence to inserted intravascular catheters. J Infect Dis 1993; 167:633–641 15. Vaudaux PE, Waldvogel FA, Morgenthaler JJ, Nydegger UE. Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence. Infect Immun 1984; 45:768–774 16. Foster TJ, McDevitt D. Molecular basis of adherence of Staphylococci to biomaterials. In: Bisno AI, Waldvogel FA, eds, Infections Associated with Indwelling Medical Devices 2nd ed. Washington, DC, American Society for Microbiology, 1994; 31–44 17. Christensen GD, Baldassarri L, Simpson WA. Colonization of medical devices by coagulase-negative Staphylococci. In: Bisno AI, Waldvogel FA, eds, Infections Associated with Indwelling Medical Devices 2nd ed. Washington, DC, American Society for Microbiology, 1994; 45–78 18. Herrmann M, Vaudaux PE, Pittet D, et al. Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J Infect Dis 1988; 158:693–701 19. Griffiths PG, Elliot TSJ, McTaggart L. Adherence of Staphylococcus epidermidis to intraocular lenses. Br J Ophthalmol 1989; 73:402–406 20. Kodjikian L, Burillon C, Roques C, et al. Bacterial adherence of Staphylococcus epidermidis to intraocular lenses: a bioluminescence and scanning electron microscopy study. Invest Ophthalmol Vis Sci 2003; 44:4388–4394. Available at: http:// www.iovs.org/cgi/reprint/44/10/4388. Accessed November 30, 2007 21. Garcı´a-Sa´enz MC, Arias-Puente A, Fresnadillo-Martinez MJ, Matilla-Rodriguez A. In vitro adhesion of Staphylococcus epidermidis to intraocular lenses. J. Cataract Refract Surg 2000; 26:1673–1679 22. Ng EWM, Barrett GD, Bowman R. In vitro bacterial adherence to hydrogel and poly(methyl methacrylate) intraocular lenses. J Cataract Refract Surg 1996; 22:1331–1335 23. Pinna A, Zanetti S, Sechi LA, et al. In vitro adherence of Staphylococcus epidermidis to polymethyl methacrylate and Acrysof intraocular lenses. Ophthalmology 2000; 107:1042–1046 24. Portole´s M, Refojo MF, Leong F-L. Reduced bacterial adhesion to heparin-surface-modified intraocular lenses. J Cataract Refract Surg 1993; 19:755–759 25. Prosdocimo G, Grandesso S, Amici G. Influence of optic and haptic materials on the adherence of Staphylococcus epidermidis to intraocular lenses: a pilot study. Eur J Ophthalmol 1997; 7:241–244 26. Schauersberger J, Amon M, Aichinger D, Georgopoulos A. Bacterial adhesion to rigid and foldable posterior chamber intraocular lenses; in vitro study. J Cataract Refract Surg 2003; 29: 361–366 27. Schlo¨ricke E, Schmidt H, Schulze HA, et al. Adha¨renz von Staphylokokken unterschiedlicher Hydrophobizita¨t; Untersuchung an verschiedenen Intraokularlinsen. [Adherence of staphylococci to various intraocular lenses with different levels of hydrophobicity.]. Ophthalmologe 1997; 94:785–790 28. Kodjikian L, Burillon C, Lina G, et al. Biofilm formation on intraocular lenses by a clinical strain encoding the ica locus: a scanning electron microscopy study. Invest Ophthalmol Vis Sci 2003; 44:4382–4387. Available at: http://www.iovs.org/cgi/reprint/44/ 10/4382. Assessed November 30, 2006 29. Johnston RL, Spalton DJ, Hussain A, Marshall J. In vitro protein adsorption to 2 intraocular lens materials. J Cataract Refract Surg 1999; 25:1109–1115
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30. Linnola RJ, Sund M, Ylo¨nen R, Pihlajaniemi T. Adhesion of soluble fibronectin, vitronectin, and collagen type IV to intraocular lens materials. J Cataract Refract Surg 2003; 29:146–152 31. Saika S, Tanaka S, Ohmi S, et al. Deposition of extracellular matrix on intraocular lenses in rabbits: an immunohistochemical and transmission electron microscopic study. Graefes Arch Clin Exp Ophthalmol 1997; 235:241–247 32. Saika S, Miyamoto T, Yamanaka A, et al. Immunohistochemical evaluation of cellular deposits on posterior chamber intraocular lenses. Graefes Arch Clin Exp Ophthalmol 1998; 236:758–765 33. Kodjikian L, Burillon C, Chanloy C, et al. In vivo study of bacterial adhesion to five types of intraocular lenses. Invest Ophthalmol Vis Sci 2002; 43:3717–3721. Available at: http://www.iovs. org/cgi/reprint/43/12/3717. Accessed November 30, 2007 34. Lundberg F, Gouda I, Larm O, et al. A new model to assess staphylococcal adhesion to intraocular lenses under in vitro flow conditions. Biomaterials 1998; 19:1727–1733 35. Amon M, Menapace R, Radax U, Freyler H. Der Einfluß unterschiedlicher Oberfla¨cheneigenschaften intraokularer Implantate aus PMMA auf deren Biokompatibilita¨t. [Influence of different
surface-properties of IOLs made of PMMA on their biocompatibility.]. Spektrum Augenheilkd 1995; 9:30–35 36. Majima K. An evaluation of the biocompatibility of intraocular lenses. Ophthalmic Surg Lasers 1996; 27:946–951 37. Winther-Nielson A, Johansen J, Pedersen GK, Corydon L. Posterior capsule opacification and neodymium:YAG capsulotomy with heparin-surface-modified intraocular lenses. J Cataract Refract Surg 1998; 24:940–944 38. Ratner BD. Ophthalmologic biocompatibility: anachronism or oxymoron? [guest editorial] J Cataract Refract Surg 1998; 24:288–290. Available at: http://www.ascrs.org/publications/ jcrs/gueditmar8.html. Assessed November 28, 2007
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First author: Andreas C. Schroeder, MD Department of Ophthalmology, University of Saarland, Homburg (Saar), Germany
Influence of fibronectin on the adherence of Staphylococcus epidermidis to coated and uncoated intraocular lenses Andreas C. Schroeder, MD, Josef M. Schmidbauer, MD, Astrid Sobke, MD, Berthold Seitz, MD, FEBO, Klaus W. Ruprecht, MD, Mathias Herrmann, MD
PURPOSE: To determine the effect of the modification of intraocular lens (IOL) surface properties on the adhesion of Staphylococcus epidermidis caused by fibronectin (FN) as the predominant proadhesive glycoprotein of the eye’s initial foreign body reaction. SETTING: University of Saarland, Homburg/Saar, Germany. METHODS: Eleven IOL types were tested. The IOLs were of poly(methyl methacrylate), acrylate, or silicone. Some were surface modified with heparin or polysaccharide coating. The IOLs, unadsorbed or preadsorbed with fibronectin (FN), were incubated with [3H]-thymidine-labeled S epidermidis Rp62a, and the amount of adherent microorganisms was determined. RESULTS: Attachment of S epidermidis adhesion to various types of IOLs, both unadsorbed and FN precoated, varied significantly. The attachment to highly adhesive IOLs was almost 4-fold greater than that to low-adhesive IOLs. Attachment to FN precoated IOLs was generally enhanced compared with attachment to unadsorbed IOLs. Heparin surface modification resulted in no or a modest reduction in bacterial adhesion compared with unmodified IOLs. Bacterial adhesion was highly statistically significantly less on IOLs with polysaccharide surface modification. CONCLUSIONS: There was significant variability in S epidermidis adhesion to IOLs as a function of design, material, surface modification, and FN preadsorption. Application of the findings may foster new developments to further reduce the major complication in cataract surgery, infective endophthalmitis. J Cataract Refract Surg 2008; 34:497–504 Q 2008 ASCRS and ESCRS
When Harold Ridley implanted the first intraocular lens (IOL) made of poly(methyl methacrylate) (PMMA) into the posterior chamber of a patient’s eye in 1949,1 it was the first step in a development that lead to modern cataract surgery being the most frequently performed operative intervention in the world. However, from Ridley’s time2–4 to now,5–7 postoperative endophthalmitis remains the most feared complication of cataract surgery. Improvements in operative techniques and the introduction of new IOL materials and surface modifications have reduced the incidence of this dreaded complication to a rate of 0.05% to 0.50%.6,7 Although this rate appears to be low, the consequences of prosthetic lens– associated endophthalmitis are potentially grave. As the Endophthalmitis Vitrectomy Study reports,6 47% of patients present with a visual acuity of 0.5 or worse 9 to 12 months after cataract surgery. However, even without frank infection, IOL implantation is accompanied by mild inflammatory tissue reaction.8–10 Q 2008 ASCRS and ESCRS Published by Elsevier Inc.
Moreover, as shown in foreign-body infection models, the presence of prosthetic material triggers a local immune defect with functional impairment of neutrophils derived from the periprosthetic fluid, reduced local levels of the cytokine levels within the first hours after implantation, and an ensuing reduction in complement-mediated opsonic activity.11 It is thought that this compromised innate immunity contributes to the susceptibility of medical implants, including IOLs. Immediately after implantation of a foreign body, the implant’s surface characteristics are profoundly modified by immediate adsorption of a protein layer.12 Vitronectin, fibronectin (FN), laminin, and collagen type IV have been found on explanted IOLs.9,10 In particular, FN has played a crucial role in the promotion of bacterial adhesion to biomaterial surfaces, as seen in in vitro and in vivo studies.11,13–18 Fibronectin, a protein of the extracellular matrix, exists in a soluble form in most fluids of the human body. This multifunctional 0886-3350/08/$dsee front matter doi:10.1016/j.jcrs.2007.10.042
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protein contains binding sites for fibrin, collagen, integrins, heparin, and other cellular and extracellular molecules. Upon adsorption to foreign bodies, FN has been shown to act as a mediator for bacterial adhesion.14,18 A detailed analysis of the promotion of bacterial attachment as a function of IOL design and material in conjunction with the presence or absence of adhesive host factors is not available. To model the clinical situation of IOL implantation, 2 scenarios of bacterial interaction with the IOL surface must be taken into account: (1) bacterial adhesion occurs before IOL implantation (ie, to unadsorbed IOL material) or (2) adhesion occurs during or after IOL implantation (ie, to IOL preadsorbed with host factors). Staphylococcus epidermidis is the most frequently encountered causative pathogen in endophthalmitis after cataract surgery.7 It is believed that the pathogenicity of this organism in foreign-body infections results from its ability to adhere to plain and host factor–adsorbed polymer material and to produce a glycocalix biofilm that interferes with host immune defense and with antibiotic treatment.19,20 Accordingly, the goal of this study was to determine the adhesion characteristics of S epidermidis to IOLs of various designs with and without the presence of FN as a model proadhesive host factor. MATERIALS AND METHODS Intraocular Lenses Eleven types of IOLs were supplied by 3 manufacturers: Acri.Tec, Advanced Medical Optics (AMO), Corneal. The
Accepted for publication October 25, 2007. From the Department and Hospital of Ophthalmology (Schroeder, Schmidbauer, Seitz, Ruprecht) and the Institute of Medical Microbiology and Hygiene (Sobke, Herrmann), University of Saarland Hospital, Homburg/Saar, Germany. No author has a financial or proprietary interest in any material or method mentioned. Supported by a grant from the Medical Faculty, University of Saarland, Homberg/Saar, Germany. Presented at the annual meeting of the XXII Congress of the European Society of Cataract & Refractive Surgeons, Paris, France, September 2004, and in part at the ASCRS Symposium on Cataract, IOL and Refractive Surgery, San Francisco, California, USA, March 2006. K. Hilgert provided technical help. Corresponding author: A.C. Schroeder, MD, Department of Ophthalmology, University of Saarland, Kirrberger Strasse 1, D-66424 Homburg (Saar), Germany. E-mail: [email protected].
IOLs were PMMA, hydrophilic acrylate, hydrophobic acrylate, or silicone (Table 1). Three types of IOLs were surface modified by the manufacturer: The ACR 6D SE IOL, which is heparin surface modified (HSM) hydrophilic acrylate, was compared with the unmodified 600 SE, which is of the same hydrophilic acrylate. The New Six IOL, which is HSM PMMA, was compared with the uncoated Phac 57 T of otherwise identical design. Finally, 2 silicone IOLs were analyzed: a polysaccharide-surface-modified (PSM) 73NAL/B prototype and a 73N-AL uncoated. The various acrylate materials differ in water content and hydrophobicity. Unpublished studies of these materials suggest, however, that contact-angle determinations do not clearly allow one to assign IOL materials to categories defined by water content. Accordingly, the results presented here were obtained based on the primary composition of the IOL material (eg, all acrylate materials were analyzed in 1 category).
Bacterial Strain The strongly biofilm-producing strain S epidermidis RP 62a was used throughout the study. For bacterial labeling, 40 mL of a fresh overnight bacterial suspension were incubated with 50 mL [3H]-thymidine in 1 mL Mueller-Hinton broth for 3 hours in a 37 C shaking water bath. Colony-forming units were counted on Mueller-Hinton plates before every experiment to control for a reproducible inoculum size throughout the study.
In Vitro Adhesion Assay of Staphylococcus Epidermidis to Intraocular Lenses Experimental Settings Bacterial adherence was determined using a modification of a an adhesion assay.15 First, attachment to cover slips of the model material PMMA was tested. Next, to simulate the 2 scenarios of IOL contamination by microorganisms in vivo, 2 experimental settings were established. In experimental setting 1, the IOL was directly removed from its original packing and incubated with the labeled microorganisms in unsupplemented phosphate-buffered saline (PBS) in an adhesion assay as described below. This assay monitors bacterial staphylococcal attachment primarily due to nonspecific physicochemical interactions, particularly surface charge and hydrophobicity. In setting 2, the IOL was precoated with FN and then incubated in PBS supplemented with 0.5% albumin to simulate aqueous humor. This assay reflects adhesion primarily due to specific interactions of bacterial surface components with the adsorbed glycoprotein as nonspecific attachment is blocked. Fibronectin Preadsorption Pilot experiments using plain PMMA slides showed the ideal concentration of FN for the coating to be 2 mg/mL PBS (data not shown). Accordingly, each IOL in the second experimental setting was immersed in 1 mL of this solution for 1 hour at 37 C. Afterward, the solution was decanted; the IOLs were rinsed with PBS and then stored at 4 C for a maximum of 2 hours. Adhesion Assay A parallel examination of 8 IOLs per type and setting were performed during each experiment. For each attachment test, 1 IOL was immersed in 960 mL PBS with 40 mL 3H-labeled bacteria solution added. In experiments in setting 1, unadsorbed IOLs were incubated in unsupplemented PBS. In setting 2, an FN-coated IOL was
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Table 1. Intraocular lenses tested in study. Material
Manufacturer
IOL
Haptics
Optic (mm)
Haptics (mm)
Polymer (Surface Modification)
Acrylate Acrylate Acrylate Acrylate Acrylate PMMA PMMA PMMA Silicone Silicone Silicone
Acri.Tec Acri.Tec AMO Corneal Corneal Acri.Tec Corneal Corneal Acri.Tec Acri.Tec AMO
49S 43C-5 AR 40e 600 SE ACR 6D SE 14C Phac 57 T New Six 73N-AL 73N-AL/B Clariflex B
Plate 1-piece 3-piece 3-piece 1-piece 3-piece 3-piece 3-piece 3-piece 3-piece 3-piece
5.0 6.0 6.0 6.0 6.0 7.0 5.7 6.0 6.0 6.0 6.0
11.00 10.50 13.00 13.00 12.00 10.50 12.50 12.75 12.50 12.50 13.00
HEMA/MMA HEMA/MMA EA/EMA/TFEMA HEMA/MMA HEMA/MMA (heparin) PMMA PMMA PMMA (heparin) PDMS PDMS (polysaccharide) PDMDPS
EA Z ethyl acrylate; EMA Z ethyl methacrylate; HEMA Z 2-hydroxylethyl methacrylate; IOL Z intraocular lens; MMA Z methyl methacrylate; PDMDPS Z poly(dimethyldiphenylsiloxane); PDMS Z poly(dimethylsiloxane); PMMA Z poly(methyl methacrylate); TFEMA Z 2,2,2-trifluoroethyl methacrylate
immersed in PBS supplemented with 0.5% human serum albumin. Subsequently, the IOLs were incubated at 37 C in a shaking water bath for 1 hour. Thereafter, the supernatant was removed; the IOLs were washed twice in PBS to remove the loosely attached bacteria and then transferred into scintillation vials for determination of adherent radioactivity. Bacterial adherence (%) was determined as adherent cpm/ inoculated cpm 100.
Statistical Analysis Data are presented as means and standard error of the mean (SEM). To determine the significance of the results, an analysis of variance (ANOVA) was performed for the results in each experimental setting. If the ANOVA showed significant differences, a Student t test was used to compare adherence values of the various IOLs to a standard IOL (as described in Results) or to compare the effect of FN precoating or of a manufacturer-performed surface modification with the respective uncoated or unmodified IOL material. A P value of 0.05 or less was considered statistically significant.
Staphylococcus Epidermidis Adhesion to Unadsorbed Intraocular Lenses (Setting 1) The results of the adhesion assays to plain IOLs are shown in Figure 2 and Table 2. A significant variation in results was found by ANOVA (F Z 8.88; P!.001). To compare staphylococcal attachment between the groups of acrylate IOLs, the AR40 was used as the standard acrylic IOL (mean adhesion 0.92% G 0.20%). This IOL type was the most frequently used acrylate IOL in clinical use at the authors’ institution at the time of the study. Statistically significant differences in bacterial attachment were found between this standard IOL and the other acrylic IOLs. Bacterial
RESULTS Staphylococcus Epidermidis Adhesion to Poly(methyl methacrylate) Cover Slips A concentration of 0.5% bovine serum albumin (BSA) blocked the adhesion maximally and significantly (P Z .001) (Figure 1). In contrast, precoating of PMMA cover slips with FN resulted in restored bacterial adhesion even in the presence of albumin in the incubation medium. Compared with attachment to uncoated, albumin-free PMMA surfaces, attachment to FN-coated surfaces was significantly elevated. These experiments confirmed the role of FN-promoted attachment of S epidermidis to a PMMA substrate independent of nonspecific interaction forces.
Figure 1. S epidermidis RP62A adhesion to PMMA slides as a function of PBS supplementation with BSA and preadsorption with FN. The PMMA cover slips were incubated with [3H]-thymidine–labeled S epidermidis in the absence of (left bar) or in the presence of BSA (center bar). Alternatively, PMMA cover slips were precoated with FN and then incubated with S epidermidis in BSA PBS (right column) (n Z 3) (** Z P!.05; *** Z P!.01; BSA Z bovine serum albumin; FN Z fibronectin; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
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Figure 2. Results in setting 1: S epidermidis RP62A adherence to various IOLs in noncolloidal medium. Bacteria were incubated with IOLs in unsupplemented PBS analogous as in Figure 1, and relative bacterial adhesion was determined (n Z 8) (** Z P!.05; *** Z P!.01; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
attachment to the 49S IOL was lowest and to the 43C-5 IOL, highest. Of the 2 types of PMMA IOLs tested, the Phac 57 T had statistically significantly lower staphylococcal adhesion than the 14C IOL. In contrast, the 2 silicone IOLs did not have statistically significant differences in S epidermidis adhesion. Overall, analysis of the results of attachment to the different groups of IOL material showed no statistically significant differences. The median bacterial attachment was 0.88%, 0.89%, and 0.90% to acrylate, PMMA, and silicone, respectively. Staphylococcus Epidermidis Adhesion to Fibronectin-Adsorbed Poly(methyl methacrylate) (Setting 2) The results for the FN-coated IOLs in setting 2 showed greater variability than the results in setting
1 (F Z 11.92; P!.001; ANOVA) (Figure 3 and Table 2). Overall, bacterial attachment values were more elevated than values for unadsorbed IOLs (Figure 4). Compared to rates for the standard AR40 IOL, attachment rates to the various acrylate IOLs differed significantly, with the 600 SE IOL having the lowest adhesion and the 43C-5 the highest. In contrast to assays with unadsorbed IOLs, attachment to PMMA IOLs did not significantly differ; the results obtained with the 2 silicone IOLs were comparable. No statistically significant difference was found to be attributable to the IOL materials in general. The median adhesion values were 1.39%, 1.59%, and 1.85% for silicone, PMMA, and acrylate, respectively. Influence of Manufacturer-Provided Intraocular Lens Surface Modifications Poly(Methyl Methacrylate) Versus Heparin-SurfaceModified Poly(methyl methacrylate) In setting 1 and setting 2, comparison of the HSM Phac 57 T IOL and the unmodified New Six IOL showed slightly decreased S epidermidis adherence to the unmodified IOL; however, the difference was not significant (Figure 5). Similarly, although FN preadsorption did not modify this relative adherence result with respect to heparin surface modification, FN significantly promoted bacterial attachment to both IOL types. Unmodified Acrylate Versus Heparin-Surface-Modified Acrylate In contrast to heparin modification of PMMA, heparin surface modification of acrylate IOLs significantly modified bacterial attachment in both experimental settings, albeit in a different manner as a function of FN preadsorption. In setting 1 (unadsorbed), the S epidermidis attachment was significantly reduced by the heparin modification (600 SE versus ACR6D SE, P Z .015). In setting 2 (FN adsorbed),
Table 2. Bacterial adhesion rates. Bacterial Adhesion Rate (%) G SEM Material
IOL
Acrylate 49S Acrylate 43C-5 Acrylate AR 40e Acrylate 600 SE Acrylate ACR 6D SE PMMA 14C PMMA Phac 57 T PMMA New Six Silicone 73N-AL Silicone 73N-AL/B Silicone Clariflex B
Plain IOL 0.40 G 0.07 1.77 G 0.30 0.92 G 0.20 0.44 G 0.05 0.29 G 0.02 1.15 G 0.14 0.64 G 0.08 0.48 G 0.08 0.98 G 0.23 0.18 G 0.02 0.81 G 0.19
FN-Coated IOL P Value 1.26 G 0.21 3.27 G 0.48 1.98 G 0.24 0.87 G 0.10 1.05 G 0.08 1.81 G 0.20 1.36 G 0.15 1.09 G 0.12 1.62 G 0.22 0.54 G 0.06 1.96 G 0.15
!.01 !.05 !.01 !.01 !.01 !.05 !.01 !.01 NS !.01 NS
FN Z fibronectin; IOL Z intraocular lens; NS Z not significant
Figure 3. Staphylococcus epidermidis RP62A adherence to FN-preadsorbed IOLs in a colloidal buffer medium. Bacteria were incubated with IOLs in BSA-supplemented PBS analogous as in Figure 1, and relative bacterial adhesion was determined (n Z 8) (** Z P!.05; *** Z P!.01; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
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Figure 4. Comparison of S epidermidis adhesion to IOLs of acrylate (triangles), PMMA (squares), or silicone (circles) as a function of IOL preadsorption with FN (n Z 8) (BSA Z bovine serum albumin; FN Z fibronectin; IOL Z intraocular lens; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
Figure 5. Staphylococcus epidermidis adherence to PMMA IOLs as a function of heparin surface modification. Unmodified IOLs or HSM-modified IOLs were tested for S epidermidis adhesion as in Figure 2 in plain PBS or after FN preadsorption in BSA–PBS. (n Z 8) (*** Z P!.01; FN Z fibronectin; HSM Z heparin surface modified; IOLZ intraocular lens; n.s. Z not significant; PMMA Z poly[methyl methacrylate]; Rel. Bact. Z relative bacterial).
the HSM IOLs had more elevated attachment values than the unmodified IOLs (P Z .182) (Figure 6). Similar to findings with the PMMA IOLs, bacterial attachment to both acrylate IOLs was increased by FN coating to a highly significant extent.
a difference that was highly statistically significant compared with the values for the uncoated silicone IOLs (73N-AL versus 73N-AL/B; setting I: P Z .004; setting II: P!.001) (Figure. 7). In contrast to other surface modifications and IOL materials, FN-adsorption did not result in a significant adhesion-promoting effect when comparing the unmodified silicone IOLs, whereas FN significantly promoted attachment to the PSM silicone IOL, although the adhesion values of FN-adsorbed PSM silicone IOLs were still lower than those of unmodified silicone IOLs in the absence of FN.
Unmodified Silicone Versus Polysaccharide SurfaceModified Silicone The most significant influence of surface modification was found when comparing silicone IOLs with or without PSM. In both experimental settings, the reduction in the adhesion values was between 5-fold (setting 1) and 3-fold (setting 2),
Figure 6. Staphylococcus epidermidis adherence to acrylate IOLs as a function of HSM. Unmodified IOLs or HSM-modified IOLs were tested for S epidermidis adhesion as in Figure 2 in plain PBS or after FN preadsorption in BSA–PBS (n Z 8) (** Z P!.05; *** Z P!.01; FN Z fibronectin; HSM Z heparin surface modified; IOLZ intraocular lens; n.s. Z not significant; Rel. Bact. Z relative bacterial).
Figure 7. Staphylococcus epidermidis adherence to silicone IOLs as a function of PSM. Unmodified IOLs or PSM-modified IOLs were tested for S epidermidis adhesion as described in Figure 2 in plain PBS or after FN preadsorption in BSA–PBS (n Z 8) (*** Z P!.01; FN Z fibronectin; IOLZ intraocular lens; n.s. Z not significant; PSM Z polysaccharide surface modified; Rel. Bact. Z relative bacterial).
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DISCUSSION In this study, 11 types of IOLs of different materials and with different surface modifications were compared with respect to their adhesive properties for S epidermidis, the most often identified isolate in postsurgical endophthalmitis to the various materials. Our results show that the surface modification an IOL provides has important potential in modulating adherence of S epidermidis in vitro. We found bacterial adherence rates to plain IOLs that are in agreement with the results in other studies performed with IOLs in vitro.20–27 The exact mechanisms resulting in IOL colonization and subsequent infection are not entirely resolved. In particular, although it is likely that most infections occur during the time of insertion, in vitro models reflecting the pathogenic steps must consider the presence of proadhesive host factors in the incisional wound and intraocular fluid composition readily adsorbing the artificial substrate. In vitro models of such pathogenic mechanisms must therefore investigate both scenarios; that is, bacterial interaction with IOL substrates preexposed to proadhesive factors such as FN as well as uncoated IOL substrates. Several factors are important in the initial phase of bacterial adhesion; that is, physicochemical forces, the capsular polysaccharide of the bacteria, and the presence of surface proteins.28 Although physicochemical forces underlie all attachment events and the presence or absence of bacterial capsular polysaccharides is a strain-dependent characteristic, we modeled the presence or absence of adsorbed host factors on the different IOL materials. Upon exposure of artificial lens material to vitreous humor, such almost instant deposition of adhesive host molecules is a passive yet physiological mechanism viewed as an integral part of the foreign-body reaction in the aqueous humor.9,10,29–32 The in vitro adsorption of soluble FN to different types of IOL has been studied,30 and it has been shown that FN adheres significantly better to acrylate IOLs and PMMA IOLs than to silicone IOLs (both P!.01). These findings correspond to the changes in staphylococcal adhesion found in our study, as FN promoted staphylococcal adhesion to a larger extent when preadsorbed to acrylate or PMMA than when preadsorbed to silicone. To our knowledge, bacterial attachment to in vivo IOLs was evaluated in only 1 study.33 In this study, the foreign-body reaction and bacterial attachment to IOLs explanted from pigs 1 week after implantation were evaluated. The study results are in accordance to results in our experiments determining bacterial adhesion after FN preadsorption. Bacterial adhesion to protein-modified IOL substrates was also assessed
in another study using cerebrospinal fluid as an incubation medium (a medium containing multiple adhesive proteins including FN)34; however, as FN-free conditions were not examined in this study, the specific role of adhesive proteins could not be determined. In the development of new IOL generations, surface treatment is often used in an attempt to modify foreign-body biocompatibility. Of the interventions proposed, smoothening the material surface has proven to be effective35,36 and has now become standard in IOL manufacturing. In our study, the 49S and 43C-5 IOL pair demonstrate the potential advantage of design improvement. Both are of the same 2-hydroxylethyl methacrylate/methyl methacrylate copolymer, representing 2 generations of acrylate IOLs. The obsolescent 43C-5 IOL had the highest staphylococcal adhesion, whereas the newer generation 49S had comparably low attachment results. The modifications of the nature of the material between the 2 IOL generations are the intellectual property of the manufacturer and therefore not disclosed. However, modifications of the new acrylate IOL material (proprietary name Acri.Lyc) are not due to any coating or etching process and appear not to confer a significant change in surface hydrophobicity, as we determined by contact-angle measurement (data not shown). Surface hydrophobicity versus hydrophilicity, however, may be a potential adhesion-relevant characteristic aimed at by substrate modification. Manufacturers have attempted to reduce bacterial colonization by introducing more hydrophilic lens surfaces. Such surface modification strategy can be achieved by coating with heparin. Our results with heparin-adsorbed surfaces yielded an effect on staphylococcal adhesion that was unanticipated. Although with PMMA there was nonsignificant tendency toward reduced attachment to the heparin coated surface, with acrylate opposite effects dependent on FN precoating were observed. Heparin bonding resulted in significantly reduced attachment to the unmodified IOL; however, after FN coating the difference was not only eliminated, it was reversed. One possible explanation for this striking finding is that the hydrophilic surface of the heparinized acrylate modifies the presentation and conformation of the FN molecule, enhancing the FN–S epidermidis interaction. Clinical disadvantages of heparin surface modification were presented by Winther-Nielson et al.37 in 1998. The authors found significantly higher rates of posterior capsule opacification (PCO) between HSM PMMA and PMMA. On the other hand, a promising approach shown with the polysaccharide-coated silicone IOL (73N-AL/B), which was a prototype at the time of this study. The clear and significant reduction in staphylococcal adhesion independent of the FN coating was
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not anticipated and may have promising indications in the design of new IOL generations. In essence, when an IOL is implanted, host factor adsorption occurs independent of the nature of the IOL material.38 Such reaction must be considered when examining the interaction between the IOL and the surrounding humor and tissue with respect to bacterial adhesion and development of PCO. To further improve biocompatibility, focusing on modulation of this reaction rather than on developing and using new materials could be beneficial.38 Our results showing the effect of a modification of IOL surface properties on the adhesion of S epidermidis can be considered an important step toward this goal. REFERENCES 1. Ridley H. Intra-ocular acrylic lenses; a recent development in the surgery of cataract. Br J Ophthalmol 1952; 36:113–122 2. Reese WS. Results in 39 bilateral Ridley intra-ocular implants. Br J Ophthalmol 1960; 44:713–717 3. Ridley H. Further experiences of intra-ocular acrylic lens surgery; with a report of more than 100 cases. Br J Ophthalmol 1954; 38:156–162 4. Ridley H. Intra-ocular acrylic lenses; 10 years’ development. Br J Ophthalmol 1960; 44:705–712 5. Barequet IS, Baker AS, Schein OD. Ocular infections. In: Waldvogel FA, Bisno AI, eds, Infections Associated with Indwelling Medical Devices 3rd ed. Washington, DC, American Society for Microbiology, 2000; 287–306 6. Endophthalmitis Vitrectomy Study Group. Results of the Endophthalmitis Vitrectomy Study; a randomized trial of immediate vitrectomy and of intravenous antibiotics for the treatment of postoperative bacterial endophthalmitis; Endophthalmitis Vitrectomy Study Group. Arch Ophthalmol 1995; 113:1479–1496 7. Heaven CJ, Mann PJ, Boase DL. Endophthalmitis following extracapsular cataract surgery: a review of 32 cases. Br J Ophthalmol 1992; 76:419–423 8. Kanagawa R, Saika S, Ohmi S, Tamura M, Nakao T. Presence and distribution of fibronectin on the surface of implanted intraocular lenses in rabbits. Graefes Arch Clin Exp Ophthalmol 1990; 228:398–400 9. Linnola RJ, Werner L, Pandey SK, et al. Adhesion of fibronectin, vitronectin, laminin, and collagen type IV to intraocular lens materials in pseudophakic human autopsy eyes. Part 2: explanted intraocular lenses. J Cataract Refract Surg 2000; 26:1807–1818 10. Linnola RJ, Werner L, Pandey SK, et al. Adhesion of fibronectin, vitronectin, laminin, and collagen type IV to intraocular lens materials in pseudophakic human autopsy eyes. Part 1: histological sections. J Cataract Refract Surg 2000; 26:1792–1806 11. Vaudaux P, Lew DP, Waldvogel FA. Host factors predisposing to and influencing therapy of foreign body infections. In: Bisno Al, Waldvogel FA, eds, Infections Associated with Indwelling Devices 2nd ed. Washington, DC, American Society for Microbiology, 1994; 1–29 12. Ratner BD. Replacing and renewing: synthetic materials, biomimetics, and tissue engineering in implant dentistry. J Dent Edu 2001; 65:1340–1347 13. Vaudaux P, Pittet D, Haeberli A, et al. Host factors selectively increase staphylococcal adherence on inserted catheters: a role for fibronectin and fibrinogen or fibrin. J Infect Dis 1989; 160:865–875
503
14. Vaudaux P, Pittet D, Haeberli A, et al. Fibronectin is more active than fibrin or fibrinogen in promoting Staphylococcus aureus adherence to inserted intravascular catheters. J Infect Dis 1993; 167:633–641 15. Vaudaux PE, Waldvogel FA, Morgenthaler JJ, Nydegger UE. Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence. Infect Immun 1984; 45:768–774 16. Foster TJ, McDevitt D. Molecular basis of adherence of Staphylococci to biomaterials. In: Bisno AI, Waldvogel FA, eds, Infections Associated with Indwelling Medical Devices 2nd ed. Washington, DC, American Society for Microbiology, 1994; 31–44 17. Christensen GD, Baldassarri L, Simpson WA. Colonization of medical devices by coagulase-negative Staphylococci. In: Bisno AI, Waldvogel FA, eds, Infections Associated with Indwelling Medical Devices 2nd ed. Washington, DC, American Society for Microbiology, 1994; 45–78 18. Herrmann M, Vaudaux PE, Pittet D, et al. Fibronectin, fibrinogen, and laminin act as mediators of adherence of clinical staphylococcal isolates to foreign material. J Infect Dis 1988; 158:693–701 19. Griffiths PG, Elliot TSJ, McTaggart L. Adherence of Staphylococcus epidermidis to intraocular lenses. Br J Ophthalmol 1989; 73:402–406 20. Kodjikian L, Burillon C, Roques C, et al. Bacterial adherence of Staphylococcus epidermidis to intraocular lenses: a bioluminescence and scanning electron microscopy study. Invest Ophthalmol Vis Sci 2003; 44:4388–4394. Available at: http:// www.iovs.org/cgi/reprint/44/10/4388. Accessed November 30, 2007 21. Garcı´a-Sa´enz MC, Arias-Puente A, Fresnadillo-Martinez MJ, Matilla-Rodriguez A. In vitro adhesion of Staphylococcus epidermidis to intraocular lenses. J. Cataract Refract Surg 2000; 26:1673–1679 22. Ng EWM, Barrett GD, Bowman R. In vitro bacterial adherence to hydrogel and poly(methyl methacrylate) intraocular lenses. J Cataract Refract Surg 1996; 22:1331–1335 23. Pinna A, Zanetti S, Sechi LA, et al. In vitro adherence of Staphylococcus epidermidis to polymethyl methacrylate and Acrysof intraocular lenses. Ophthalmology 2000; 107:1042–1046 24. Portole´s M, Refojo MF, Leong F-L. Reduced bacterial adhesion to heparin-surface-modified intraocular lenses. J Cataract Refract Surg 1993; 19:755–759 25. Prosdocimo G, Grandesso S, Amici G. Influence of optic and haptic materials on the adherence of Staphylococcus epidermidis to intraocular lenses: a pilot study. Eur J Ophthalmol 1997; 7:241–244 26. Schauersberger J, Amon M, Aichinger D, Georgopoulos A. Bacterial adhesion to rigid and foldable posterior chamber intraocular lenses; in vitro study. J Cataract Refract Surg 2003; 29: 361–366 27. Schlo¨ricke E, Schmidt H, Schulze HA, et al. Adha¨renz von Staphylokokken unterschiedlicher Hydrophobizita¨t; Untersuchung an verschiedenen Intraokularlinsen. [Adherence of staphylococci to various intraocular lenses with different levels of hydrophobicity.]. Ophthalmologe 1997; 94:785–790 28. Kodjikian L, Burillon C, Lina G, et al. Biofilm formation on intraocular lenses by a clinical strain encoding the ica locus: a scanning electron microscopy study. Invest Ophthalmol Vis Sci 2003; 44:4382–4387. Available at: http://www.iovs.org/cgi/reprint/44/ 10/4382. Assessed November 30, 2006 29. Johnston RL, Spalton DJ, Hussain A, Marshall J. In vitro protein adsorption to 2 intraocular lens materials. J Cataract Refract Surg 1999; 25:1109–1115
J CATARACT REFRACT SURG - VOL 34, MARCH 2008
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LABORATORY SCIENCE: FIBRONECTIN INFLUENCE ON S EPIDERMIDIS ADHERENCE TO IOLS
30. Linnola RJ, Sund M, Ylo¨nen R, Pihlajaniemi T. Adhesion of soluble fibronectin, vitronectin, and collagen type IV to intraocular lens materials. J Cataract Refract Surg 2003; 29:146–152 31. Saika S, Tanaka S, Ohmi S, et al. Deposition of extracellular matrix on intraocular lenses in rabbits: an immunohistochemical and transmission electron microscopic study. Graefes Arch Clin Exp Ophthalmol 1997; 235:241–247 32. Saika S, Miyamoto T, Yamanaka A, et al. Immunohistochemical evaluation of cellular deposits on posterior chamber intraocular lenses. Graefes Arch Clin Exp Ophthalmol 1998; 236:758–765 33. Kodjikian L, Burillon C, Chanloy C, et al. In vivo study of bacterial adhesion to five types of intraocular lenses. Invest Ophthalmol Vis Sci 2002; 43:3717–3721. Available at: http://www.iovs. org/cgi/reprint/43/12/3717. Accessed November 30, 2007 34. Lundberg F, Gouda I, Larm O, et al. A new model to assess staphylococcal adhesion to intraocular lenses under in vitro flow conditions. Biomaterials 1998; 19:1727–1733 35. Amon M, Menapace R, Radax U, Freyler H. Der Einfluß unterschiedlicher Oberfla¨cheneigenschaften intraokularer Implantate aus PMMA auf deren Biokompatibilita¨t. [Influence of different
surface-properties of IOLs made of PMMA on their biocompatibility.]. Spektrum Augenheilkd 1995; 9:30–35 36. Majima K. An evaluation of the biocompatibility of intraocular lenses. Ophthalmic Surg Lasers 1996; 27:946–951 37. Winther-Nielson A, Johansen J, Pedersen GK, Corydon L. Posterior capsule opacification and neodymium:YAG capsulotomy with heparin-surface-modified intraocular lenses. J Cataract Refract Surg 1998; 24:940–944 38. Ratner BD. Ophthalmologic biocompatibility: anachronism or oxymoron? [guest editorial] J Cataract Refract Surg 1998; 24:288–290. Available at: http://www.ascrs.org/publications/ jcrs/gueditmar8.html. Assessed November 28, 2007
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First author: Andreas C. Schroeder, MD Department of Ophthalmology, University of Saarland, Homburg (Saar), Germany