Capsular Bag Model Research Articles

Stereolithographic Rapid Prototyping of Clear, Foldable, Non-Refractive Intraocular Lens Designs: A Proof-of-Concept Study

Purpose A cataract is a cloudy area in the crystalline lens. Cataracts are the leading cause of blindness and the second cause of severe vision impairment worldwide. During cataract surgery, the clouded lens is extracted and replaced with an artificial intraocular lens, which restores the optical power. The fabrication of intraocular lenses using existing molding and lathing techniques is a complex and time-consuming process that limits the development of novel materials and designs. To overcome these limitations, we have developed a stereolithography-based process for producing models of clear lens designs without refractive function, serving as a proof of concept. This process has the potential to contribute toward new lens development, allowing for unlimited design iterations and an expanded range of materials for scientists to explore. Methods Lens-like 3D objects without refractive function were fabricated by using stereolithography. A photopolymerizable resin containing 2-phenoxyethyl acrylate, poly (ethylene glycol) dimethacrylate, and a suitable photoinitiator was developed for the production of lens-like 3D object prototypes. The morphology of the printed devices was characterized by scanning electron microscopy. The transparency and thermal properties were analyzed using spectrophotometry and differential scanning calorimetry, respectively. The biocompatibility of the devices was investigated in a cultured human lens cell line (FHL-124), using a standard lactate dehydrogenase assay, and the lenses were folded and implanted in the human capsular bag model. Results One-piece lens-like 3D objects without refractive function and with loop-haptic design were successfully fabricated using Stereolithography (SLA) technique. The resulting 3D objects were transparent, as determined by UV spectroscopy. The lactate dehydrogenase test demonstrated the tolerance of lens cells to the prototyping material, and apparent foldability and shape recovery was observed during direct injection into a human capsular bag model in vitro. Conclusions This proof-of-principle study demonstrated the potential and significance of the rapid prototyping process for research and development of lens-like 3D object prototypes, such as intraocular lenses.

Conditions for modifying intraocular lenses as drug carriers for methotrexate using poly (lactic-co-glycolic acid).

The intraocular lens (IOL) can be used as a slow-release drug carrier in cataract surgery to alleviate posterior capsular opacification (PCO). The following is a systematic development of an IOL using methotrexate and the solvent casting process with poly (lactic-co-glycolic acid) (PLGA) as a carrier polymer. Different solvents for PLGA and methotrexate were tested for dissolution properties and possible damage to the IOL. The required biological concentration of methotrexate was determined in human capsular bags implanted with an IOL. To detect fibrosis, α-SMA, f-actin, and fibronectin were labelled by immunofluorescence staining. Cell proliferation and extracellular matrix contraction were observed in a lens epithelial cell line (FHL-124). Finally, the IOL was designed, and an ocular pharmacokinetic model was used to measure drug release. Solvent mixtures were found to allow coating of the IOL with drug and PLGA without damaging it. PCO in the capsular bag model was inhibited above 1 μM methotrexate (p = 0.02). Proliferation in FHL-124 was significantly reduced above a concentration of 10 nM (p = 0.04) and matrix contraction at 100 nM (p = 0.02). The release profile showed a steady state within therapeutic range. After determination of the required physicochemical manufacturing conditions, a drug releasing IOL was designed. A favourable release profile in an ocular pharmacokinetics model could be shown.

The human capsular bag model as a pre‐clinical evaluation tool for IOLs

Cataract affects the vision and well‐being of millions. At present the only means of treating cataract is through surgical intervention. Modern cataract surgery typically retains a ring of anterior lens capsule and the entire posterior capsule. This is known as a capsular bag, which can house an intraocular lens (IOL) implant. Initial surgical outcomes are excellent, but with time lens cells that remain following surgery undergo a wound‐healing response. In essence the trauma of surgery provokes cells to proliferate and migrate across the acellular capsular surfaces and in some case the IOL surface. Most importantly, cells ultimately grow within the visual axis, on the central posterior capsule, and undergo fibrotic events that are associated with transdifferentiation to myofibroblasts, matrix deposition and matrix contraction that can lead to a secondary loss of vision termed posterior capsule opacification (PCO). PCO affects a large proportion of patients and is one of the most common fibrotic conditions in the world. It is therefore of great importance to better understand this condition and thus strong experimental systems are required for this purpose. To this end cataract surgery can be performed on human donor eyes within a laboratory setting to provide a biological product that is configured in the same manner as in the patient. This is known as the human capsular bag model. Using this clinically relevant model it has been possible to tease our key regulatory molecules driving human lens growth and fibrosis. While the human capsular bag model has been a useful tool to identify therapeutic targets or test putative therapeutics no pharmacological treatment is currently in clinical use. As a result, the intraocular lens is currently the major tool employed to manage PCO progression. Characteristics required for an IOL include strong optical performance, stability within the lens capsular bag and anti‐PCO properties. To meet international standards for IOL manufacture a number of tests are performed to predict IOL behaviour when implanted. However, ISO guided tests take place in a non‐biological setting and are limited to assessing purely physical characteristics. Therefore, understanding how the IOL interacts with the capsular bag and how wound‐healing responses could influence IOL position and stability is an area of great importance. The human capsular bag model can address this shortfall in the IOL development pipeline to relate design features to IOL stability and anti‐PCO performance in a clinically relevant setting. The presentation will highlight the evolution of the model as a tool for IOL testing and development.

Extracellular HSP90 promotes differentiation of lens epithelial cells to fiber cells by activating LRP1-YAP-PROX1 axis.

Capsular residual lens epithelial cells (CRLEC) undergo differentiation to fiber cells for lens regeneration or tansdifferentiation to myofibroblasts leading to posterior capsular opacification (PCO) after cataract surgery. The underlying regulatory mechanism remains unclear. Using human lens epithelial cell lines and the ex vivo cultured rat lens capsular bag model, we found that the lens epithelial cells secrete HSP90α extracellularly (eHSP90) through an autophagy-associated pathway. Administration of recombinant GST-HSP90α protein or its M-domain induces the elongation of rat CRLEC cells with concomitant upregulation of the crucial fiber cell transcriptional factor PROX1and its downstream targets, β- and γ-crystallins and structure proteins. This regulation is abolished by PROX1 siRNA. GST-HSP90α upregulates PROX1 by binding to LRP1 and activating LRP1-AKT mediated YAP degradation. The upregulation of GST-HSP90α on PROX1 expression and CRLEC cell elongation is inhibited by LRP1 and AKT inhibitors, but activated by YAP-1 inhibitor (VP). These data demonstrated that the capsular residue epithelial cells upregulate and secrete eHSP90α, which in turn drive the differentiation of lens epithelial cell to fiber cells. The recombinant HSP90α protein is a potential novel differentiation regulator during lens regeneration.

Nicotinamide improves in vitro lens regeneration in a mouse capsular bag model

BackgroundMammalian lens regeneration holds great potential as a cataract therapy. However, the mechanism of mammalian lens regeneration is unclear, and the methods for optimization remain in question.MethodsWe developed an in vitro lens regeneration model using mouse capsular bag culture and improved the transparency of the regenerated lens using nicotinamide (NAM). We used D4476 and SSTC3 as a casein kinase 1A inhibitor and agonist, respectively. The expression of lens-specific markers was examined by real-time PCR, immunostaining, and western blotting. The structure of the in vitro regenerated lens was investigated using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) and methylene blue staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and transmission electron microscopy.ResultsThe in vitro lens regeneration model was developed to mimic the process of in vivo mammalian lens regeneration in a mouse capsular bag culture. In the early stage, the remanent lens epithelial cells proliferated across the posterior capsule and differentiated into lens fiber cells (LFCs). The regenerated lenses appeared opaque after 28 days; however, NAM treatment effectively maintained the transparency of the regenerated lens. We demonstrated that NAM maintained lens epithelial cell survival, promoted the differentiation and regular cellular arrangement of LFCs, and reduced lens-related cell apoptosis. Mechanistically, NAM enhanced the differentiation and transparency of regenerative lenses partly by inhibiting casein kinase 1A activity.ConclusionThis study provides a new in vitro model for regeneration study and demonstrates the potential of NAM in in vitro mammalian lens regeneration.

Assessment of intraocular lens/capsular bag biomechanical interactions following cataract surgery in a human in vitro graded culture capsular bag model

Intraocular lenses (IOLs) are implanted during cataract surgery. For optimum results, stable positioning of the IOL in the capsular bag is important. Wound-healing events following cataract surgery lead to modification of the capsular bag and secondary visual loss due to posterior capsule opacification. At present, it is unclear how these biological events can affect stability of the IOL within the capsular bag. In the present study, a human in vitro graded culture capsular bag model was the experimental system. Capsulorhexis and lens extraction performed on human donor eyes generated suspended capsular bags (5 match-paired experiments). Preparations were secured by pinning the ciliary body to a silicone ring and maintained in 6 mL of medium for 84 days using a graded culture system: days 1–3, 5% human serum and 10 ng/mL transforming growth factor β (TGFβ2); days 4–7, 2% human serum and 1 ng/mL TGFβ2; days 8–14, 1% human serum and 0.1 ng/mL TGFβ2; days 15–84, serum-free Eagle's minimum essential medium (EMEM). A CT LUCIA 611PY IOL was implanted in all preparations. Quantitative measures were determined from whole bag images captured weekly. Images were registered using FIJI and analysed in ImageJ to determine capsular bag area; distortion; angle of contact; haptic stability; capsulorhexis area; and a fusion footprint associated with connection between the anterior and posterior capsules. Cell coverage and light scatter were quantified at end-point. The transdifferentiation marker, α-SMA was assessed by immunocytochemistry. Immediately following surgery, distortion of the capsular bag was evident, such that a long axis is generated between haptics relative to the non-haptic regions (short axis). The angle of contact between the haptics and the peripheral bag appeared inversely correlated to capsular bag area. Growth on the peripheral posterior capsule was observed 1 week after surgery and beneath the IOL within 1 month. As coverage of the posterior capsule progressed this was associated with matrix contraction/wrinkles of both the central posterior capsule and peripheral capsular bag. Cells on the central posterior capsule expressed αSMA. Fusion footprints formed in non-haptic regions of the peripheral bag and progressively increased over the culture period. Within and at the edge of the fusion footprint, refractive structures resembling lens fibre cells and Elschnig's pearls were observed. Cell attachment to the IOL was limited. An impression in the posterior capsule associated with the CT LUCIA 611PY optic edge was evident; cell density was much greater peripheral to this indent. Wound-healing events following surgery reduced capsular bag area. This was associated with the long/short axis ratio and angle of contact increasing with time. In summary, we have developed a human capsular bag model that exhibits features of fibrotic and regenerative PCO. The model permits biomechanical information to be obtained that enables better understanding of IOL characteristics in a clinically relevant biological system. Throughout culture the CT LUCIA 611PY appeared stable in its position and capsular bag modifications did not change this. We propose that the CT LUCIA 611PY optic edge shows an enhanced barrier function, which is likely to provide better PCO management in patients.

Supercritical fluid technology for the development of innovative ophthalmic medical devices: Drug loaded intraocular lenses to mitigate posterior capsule opacification

Supercritical impregnation technology was applied to load acrylic intraocular lenses (IOLs) with methotrexate to produce a sustained drug delivery device to mitigate posterior capsule opacification. Drug release kinetics were studied in vitro and used to determine the drug loading. Loaded IOLs and control IOLs treated under the same operating conditions, but without drug, were implanted ex vivo in human donor capsular bags. The typical cell growth was observed and immunofluorescence staining of three common fibrosis markers, fibronectin, F-actin and α-smooth muscle actin was carried out. Transparent IOLs presenting a sustained release of methotrexate for more than 80 days were produced. Drug loading varying between 0.43 and 0.75 ± 0.03 µgdrug·mg−1IOL were obtained when varying the supercritical impregnation pressure (8 and 25 MPa) and duration (30 and 240 min) at 308 K. The use of ethanol (5 mol%) as a co-solvent did not influence the impregnation efficiency and was even unfavorable at certain conditions. Even if the implantation of methotrexate loaded IOLs did not lead to a statistically significant variation in the duration required for a full cell coverage of the posterior capsule in the human capsular bag model, it was shown to reduce fibrosis by inhibiting epithelial-mesenchymal transformation. The innovative application presented has the potential to gain clinical relevance.

The human capsular bag model of posterior capsule opacification.

Posterior capsule opacification (PCO) is the most common complication following cataract surgery and affects millions of patients. PCO is a consequence of surgical injury promoting a wound-healing response. Following surgery, residual lens epithelial cells grow on acellular regions of the lens capsule, including the central posterior capsule. These cells can undergo fibrotic changes, such that cell transdifferentiation to myofibroblasts, matrix deposition and matrix contraction can occur, which contribute to light scatter and the need for further corrective Nd:YAG laser capsulotomy in many patients. It is therefore of great importance to better understand how PCO develops and determine better approaches to manage the condition. To achieve this, experimental systems are required, and many are available to study PCO. While there may be a number of common features associated with PCO in different species, the mechanisms governing the condition can differ. Consequently, where possible, human systems should be employed. The human capsular bag model was established in a laboratory setting on donor eyes. A capsulorhexis is performed to create an opening in the anterior capsule followed by removal of the lens fibre mass. Residual fibre cells can be removed by irrigation/aspiration and if required, an intraocular lens can be implanted. The capsular bag is isolated from the eye and transferred to a dish for culture. The human capsular bag model has played an important role in understanding the biological processes driving PCO and enables evaluation of surgical approaches, IOLs and putative therapeutic agents to better manage PCO.

HSP90 as a novel therapeutic target for posterior capsule opacification

Posterior capsule opacification (PCO) is a common complication of cataract surgery, resulting from a combination of proliferation, migration, epithelial-mesenchymal transition (EMT) of residual capsular epithelial cells and fibrosis of myofibroblasts. HSP90 is known to regulate the proteostasis of cells under pathophysiological conditions. The role of HSP90 in PCO formation, however, is not clear. To do this, the lens epithelial cell lines and an ex vivo cultured rat capsular bag model were used to study the role of HSP90 in PCO formation. The expression of protein and mRNA was measured by immunoblotting and quantitative RT-PCR, and cell apoptosis was measured by TUNEL(TdT-mediated dUTP nick-end labeling). The cell proliferation was measured by cell viability assays. The results showed that 17-AAG (Tanespimycin), an inhibitor of HSP90, suppresses the proliferation of immortalized lens epithelial cell lines HLE-B3, SRA01/04, and mLEC, with IC50 values of 0.27, 0.27, and 0.49 μM, respectively. In an ex vivo cultured rat capsular model, the capsular residual epithelial cells resisted the stress of the capsulorhexis surgery and took 3–6 days to completely overlay the capsular posterior wall. During this process, heat shock factor 1 and its downstream targets HSP90, HSP25, αB-crystallin, and HSP40 were upregulated. Treatment with 17-AAG inhibited the viability of capsular residual epithelial cells and induced the cells apoptosis, characterized by increases in ROS levels, apoptotic DNA injury, and the activation of caspases 9 and 3. HSP90 participated in regulating both EGF receptor (EGFR) and TGF receptor (TGFR) signaling pathways. HSP90 was found to interact with the EGFR, such that inhibition of HSP90 by 17-AAG destabilized the EGFR protein and suppressed p-ERK1/2 and p-AKT levels. 17-AAG also inhibited the TGF-β-induced phosphorylation of SMAD2/3 and ERK1/2 and the decrease in E-cadherin and ZO-1 expression. Accordingly, these data suggest that the induction of HSP90 protects capsular residual epithelial cells against capsulorhexis-induced stress and participates in regulating the processes of proliferation, EMT and migration of rat capsular residual epithelial cells, at least partly, through the EGFR and TGFR signaling pathways. Treatment with 17-AAG suppresses PCO formation and is therefore a potential therapeutic candidate for PCO prevention.

Resveratrol Inhibits Wound Healing and Lens Fibrosis: A Putative Candidate for Posterior Capsule Opacification Prevention.

Posterior capsule opacification (PCO) is a common complication of cataract surgery. In addition to improved surgical methods and IOL designs, it is likely additional agents will be needed to improve patient outcomes. Presently no pharmacological agent is in clinical use to prevent PCO. Here we investigate the putative ability of resveratrol (RESV), a naturally occurring polyphenol, as a therapeutic agent. The human lens epithelial cell line FHL124, a human lens capsular bag model, and central anterior epithelium were used as experimental systems. Standard culture was in 5% fetal calf serum Eagle's minimum essential medium; 10 ng/mL transforming growth factor-β2 (TGFβ2) was used to induce fibrotic changes. A scratch wound assay was used to measure cell migration and the patch assay was used to assess matrix contraction by FHL124 cells. Protein expression was assessed by immunocytochemistry and Western blot and gene expression by quantitative RT-PCR. In capsular bags, cell growth across the posterior lens capsule, capsular wrinkling, and epithelial-to-mesenchymal transition were determined by image analysis. In FHL124 cells, addition of 30 μM RESV significantly impeded cell migration in a wound-healing assay. RESV significantly inhibited TGFβ2-induced expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) at both the message and protein levels, as well as significantly inhibiting matrix contraction induced by TGFβ2. In human capsular bags, 30 μM RESV significantly inhibited cell growth. TGFβ2-induced α-SMA expression and capsular wrinkling were also significantly inhibited by RESV treatment. RESV significantly suppressed expression of TGFβ2-induced genes associated with fibrotic disease, including matrix metalloproteinase-2 in FHL124 cells, capsular bags, and central anterior epithelium. RESV can counter PCO-related physiological events in two human lens model systems. RESV therefore has the potential to be used as a candidate agent for the prevention of PCO, which in turn could benefit millions of cataract patients.

Gremlin is a potential target for posterior capsular opacification

ABSTRACTObjective: The present study was conducted to determine the role of gremlin during the development of posterior capsular opacification (PCO) via in vitro and in vivo experiments.Methods: The activation, roles and relationships of the BMPs/Smad1/5, MAPK, FAK and AKT signaling pathways in human lens epithelial cells (HLECs) after gremlin induction were detected by western blotting and real-time PCR. Wound-healing, transwell, capsular bag models and rat PCO models assays were used to test the effects of gremlin on HLECs’ migration, proliferation, EMT-specific protein α-smooth muscle actin（α-SMA）and development of PCO in rats.Results: Our data showed that knockdown of the gremlin inhibited the development of PCO and reduced expression of α-SMA in rats. While gremlin did not alter the migration of HLECs, it increased the expression of p-ERK and p-AKT. Knockout of Smad2 or Smad3 inhibited the expression of p-ERK and p-AKT proteins induced by gremlin. Gremlin also reduced BMP4-induced expression of the p-Smad1/5 protein. Finally, knockout of Smad1/5 increased gremlin-induced expression of α-SMA, fibronectin and type I collagen (COL-1) in HLECs.Conclusion: These results suggested that gremlin contributed to the development of PCO by promoting LEC proliferation, activation of TGF-β/Smad, ERK and AKT signaling and inhibition of BMPs/Smad1/5 signaling. Furthermore, inhibiting gremlin effectively impaired both PCO development in rats and EMT in the lens capsule. Thus, our data suggest that gremlin might be a potential target for PCO.

An In Vitro Human Lens Capsular Bag Model Adopting a Graded Culture Regime to Assess Putative Impact of IOLs on PCO Formation.

To develop a culture regime for the in vitro human lens capsular bag model that better reflects clinical events following cataract surgery and to use this refined model to evaluate the putative impact of IOLs on PCO formation. Capsulorhexis and lens extraction were performed on human donor eyes to generate capsular bags attached to the ciliary body by the zonules. Preparations were secured by pinning the ciliary body to a silicone ring and maintaining in 6 mL serum-free EMEM for 28 days or in a graded culture system (days 1-3, 5% human serum and 10 ng/mL TGFβ2; days 4-7, 2% human serum and 1 ng/mL TGFβ2; days 8-14, 1% human serum and 0.1 ng/mL TGFβ2; days 15-28, serum-free EMEM), which better mimics clinical changes. Preparations were monitored with phase-contrast and modified-dark-field microscopy. Cell coverage and light scatter were quantified using image analysis software. The transdifferentiation marker, α-SMA and matrix component, fibronectin were assessed by immunocytochemistry. To assess IOLs in the model, Alcon Acrysof or Hoya Vivinex IOLs were implanted in match-paired capsular bags. Match-paired experiments showed that graded culture enhanced growth, facilitated matrix contraction, increased transdifferentiation, and promoted matrix deposition relative to serum-free culture. The graded culture protocol was applied to match-paired bags implanted with a Hoya Vivinex or an Alcon Acrysof IOL. The Vivinex demonstrated a lag in growth across the posterior capsule. However, by day 28, coverage was similar, but light-scatter was greater with Acrysof implanted. Cell growth on the Acrysof IOL anterior surface was significantly greater than Vivinex. The graded culture human capsular bag model serves as an excellent system to evaluate and develop intraocular lenses. The Hoya Vivinex IOL showed an overall better level of performance against postsurgical wound healing and PCO than the Alcon Acrysof using this model.

The intraocular lens as a drug delivery device for an epidermal growth factor-Receptor inhibitor for prophylaxis of posterior capsule opacification.

Posterior capsule opacification (PCO) occurs as a common complication after cataract surgery. Erlotinib is an inhibitor of the epidermal growth factor-Receptor and reduces critical cellular events leading to PCO. In this invitro study, Erlotinib-modified intraocular lenses (IOLs) employed as a drug delivery device have been evaluated for PCO prevention. The IC50 concentration of Erlotinib was determined by using FHL-124 cells. For the human capsular bag model, 40 cadaver eyes underwent sham cataract surgery. Sixteen capsular bags were exposed to the IC50 of Erlotinib. Intraocular lens (IOL) of three different materials was pharmacologically modified and tested in the anterior segment model and implanted into 24 capsular bags. To test for corneal toxicity, pairs of human cornea were exposed to high concentrations of Erlotinib and corneal endothelial cells (CEC) were exposed to the modified IOL. Release kinetics of Erlotinib from the IOL was measured. IC50 of Erlotinib was determined to be 10μm. Erlotinib alone (p=0.002) and when soaked into IOLs (p<0.001) significantly increased the number of days needed until total cell coverage of the capsular bags in comparison with the control. Modified IOLs mitigated cell growth in the anterior segment model (p<0.001). No short-term corneal toxicity was observed up to a concentration of 100μm, and IOLs did not show toxicity on CEC. Erlotinib was released constantly from IOL. Erlotinib might be of clinical relevance in PCO prophylaxis, as its short-term application induces a long-term deceleration of cellular growth. Erlotinib can be introduced into the eye via soaked IOLs.

Poly(lactic-co-glycolic) Acid as a Slow-Release Drug-Carrying Matrix for Methotrexate Coated onto Intraocular Lenses to Conquer Posterior Capsule Opacification

ABSTRACTPurpose: Posterior capsule opacification (PCO) still represents the main long-term complication of cataract surgery. Research into pharmacologic PCO prophylaxis is extensive. One promising candidate drug is methotrexate (MTX). Our aim is to determine the in vitro feasibility of MTX-loaded poly(lactic-co-glycolic) (PLGA) biomatrices sprayed on intraocular lenses (IOLs) as a drug-delivery implant.Methods: Hydrophilic and hydrophobic acrylic IOLs were spray-coated with MTX-loaded PLGA. Unsprayed, solvent only, and solvent-PLGA-sprayed IOLs served as controls. All IOLs were evaluated for their growth-inhibiting properties in an in vitro anterior segment model and the ex vivo human capsular bag. The release kinetics of MTX from the IOLs was determined. The toxicity of MTX on corneal endothelial cells was evaluated by using a dye reduction colorimetric assay. MTX was also used in a scratch assay.Results: MTX-PLGA-IOL showed a significant difference in cell proliferation and migration compared with all controls in the anterior segment model (p < 0.001) and in the human capsular bag model (p = 0.04). No difference in viability was observed on corneal endothelial cells (p = 0.43; p = 0.61). MTX significantly inhibited cells in the scratch assay (p = 0.02). At all measured points, the released MTX dose remained above EC50 and below the toxic dose for the endothelium.Conclusions: In view of the strong inhibition of PCO in vitro with the lack of toxic effects on a corneal cell line, MTX encapsulating microspheres seem to be a promising method for modifying IOL.

The Intraocular Lens as a Drug Delivery Device: In Vitro Screening of Pharmacologic Substances for the Prophylaxis of Posterior Capsule Opacification.

Numerous pharmacologic substances have been proposed for preventing posterior capsule opacification (PCO). The following trial was to compare those drugs to find more suitable options. IOL should then be modified by the pharmaceuticals as a drug-delivery device. A systematic literature search was performed to identify published substances. FHL-124 was used to determine cell proliferation and toxicity using a dye reduction test (XTT). Prescreened substances showing a reduction on cell growth without being toxic were soaked into an IOL. Those IOL were tested for their effect on PCO in an anterior-segment model and the human ex vivo capsular bag model. Toxicity on a corneal endothelial cell line (CEC-SV40) was determined. Release kinetics of methotrexate from the IOL was measured. Toxicity testing in both cell lines was done in serum-free conditions. All growth assays were exposed to 10% fetal calf serum (FCS)-supplemented medium. The substances inhibited cell growth at the following EC50: caffeic acid phenethyl ester 1.6 ± 0.9 nM, disulfiram 359 ± 33 nM, methotrexate 98.0 ± 29.7 nM, rapamycin 70.2 ± 14.0 pM, and retinoic acid 1.1 ± 0.12 nM. All but disulfiram showed an effect in the anterior segment model when soaked into an IOL. Long-term inhibitory effects in the human capsular bag model were observed for caffeic acid phenethyl ester and methotrexate IOLs. Only methotrexate and disulfiram did not show any toxicity on endothelial cells. Methotrexate was released constantly from the hydrophilic IOL for 2 weeks. We could identify caffeic acid phenethyl ester and methotrexate in vitro as potential candidates for IOL modification for PCO prophylaxis.

Inhibition effects of sulforaphane-induced autophagy flux to cell proliferation on ex vivo human capsular bags

Background Sulforaphane (SFN) is an effective chemopreventive agent and can regulate the biological molecular mechanisms to inhibit the overgrowth of cells.Autophagy is a biological process of maintaining cellular internal environment.Understanding the affection of SFN to biological behavior of human lens epithelial cells (LECs) and the association of SFN with autophagy is helpful for the prevention and target treatment of posterior capsule opacification (PCO). Objective This study was to investigate the eradication effeccts of SFN on residual lens cell population in vitro posterior capsule opacification (PCO) model and evaluate the mechanism of SFN-induced cell death. Methods In vitro human capsular bag models were generated from fresh donor eyes by phacoemulsification and were cultured in EMEM containing 2% fetal bovine serum (FBS). Different concentrations of SFN (0, 1, 10 and 100 μmol) were added in the medium for 30 days respectively according to grouping, and the growth of LECs was observed by optical microscope and immunofluorescence technique.FHL124, a human LEC line, was cultured with EMEM containing 5%FBS and divided into 0, 1, 10, 30 and 100 μmol SFN groups.Lactate dehydrogenase (LDH) release rate in the medium was detected to evaluate cell damage/death.The migration of the cells on capsular bags was assessed by scratch test.The ultrastructure and number of autophagosomes were examined under the transmission electron microscope.The expression of LC3 in the cells were detected using Western blot in the presence or absence of autophagy inhibitors. Results The cell coverage rates on the capsular bags were significantly lower in the 10 and 100 μmol/L SFN groups than those in the 0 and 1 μmol/L SFN groups, with a statistically significant difference among the groups (F=48.57, P<0.01). Immunofluorescence showed that the density of F-actin-and Vimentin-positive cells was evidently decreased in the 10 and 100 μmol/L SFN groups compared with 0 and 1 μmol/L SFN groups.The releasing levels of LDH (absorbancy) were 0.19±0.03, 0.39±0.06, 0.56±0.07, 0.68±0.08 and 0.89±0.09 in the 0, 1, 10, 30 and 100 μmol/L SFN groups, respectively, and the releasing level of LDH was gradually increased in the 10 and 100 μmol/L SFN groups in comparison with the 1 μmol/L SFN group (all at P<0.01). With the increase of SFN concentration, the reduction rate of scratched area decreased with the increase of SFN concentration, and the decrease of scratch area was significantly lower than that of adjacent low mass concentration group and the differences were statistically significant (P<0.05). The relative expressions of LC3-Ⅱ protein were 0.423±0.003, 14.543±0.024, 0.668±0.024 and 0.576±0.056 in the blank control group, SFN group, SFN+ 3-MA group and 3-MA group, respectively, and the relative expressions of LC3-Ⅱ protein were significantly lower in the SFN+ 3-MA group and 3-MA group than those in the SFN group (all at P<0.01). The number of autophagosomes was 4.07±0.32, 4.13±0.34, 9.21±0.53 and 21.02±1.34 in the blank control group, and 1, 10, 100 μmol/L SFN groups, and the number of autophagosomes in the 10 and 100 μmol/L SFN groups was significantly higher than that in the blank control group and 1 μmol/L SFN group (all at P<0.01). Conclusions SFN mediates LECs death by promoting autophagy in ex vivo capsular bags, and SFN may be a novel agent of potential chemopreventive and target treatment for PCO. Key words: Lens capsule, crystalline; Isothiocyanates; Thiocyanates/therapeutic use; Cell line; Lens epithelial cells; Models, biological; Autophagy/drug effects; Posterior capsule opacification

Sulforaphane promotes ER stress, autophagy, and cell death: implications for cataract surgery

Posterior capsule opacification (PCO) commonly develops following cataract surgery and is a wound-healing response that can ultimately lead to secondary visual loss. Improved management of this problem is required. The isothiocyanate, sulforaphane (SFN), is reported to exert cytoprotective and cytotoxic actions, and the latter may be exploited to treat/prevent PCO. SFN concentrations of 10 μM and above significantly impaired wound-healing in a human lens capsular bag model. A similar pattern of response was also seen with a human lens cell line, FHL124. SFN treatment promoted increased expression of endoplasmic reticulum (ER) stress genes, which also corresponded with protein expression. Evidence of autophagy was observed in response to SFN as determined by increased microtubule-associated protein 1A/1B-light chain 3 (LC3)-II levels and detection of autophagic vesicles. This response was disrupted by established autophagy inhibitors chloroquine and 3-MA. SFN was found to promote MAPK signaling, and inhibition of ERK activation using U0126 prevented SFN-induced LC3-II elevation and vesicle formation. SFN also significantly increased levels of reactive oxygen species. Taken together, our findings suggest that SFN is capable of reducing lens cell growth and viability and thus could serve as a putative therapeutic agent for PCO.Key messageSFN reduces lens epithelial cell growth, migration, and viability.SFN can promote ER stress and autophagy in lens cells.SFN promotes MAPK signaling, and inhibition of MEK can suppress SFN-induced autophagy.ER stress and autophagy in lens cells are likely promoted by ROS production.SFN may help prevent posterior capsule opacification after cataract surgery.

Effect of femtosecond laser‐assisted lens surgery on posterior capsule opacification in the human capsular bag in vitro

To compare posterior capsule opacification (PCO) by observing lens epithelial cell growth in the human capsular bag invitro between conventional lens surgery using phacoemulsification (Phaco) technique and femtosecond laser-assisted lens surgery (FLACS). For the invitro human capsular bag model, 18 cadaver eyes from nine human donors underwent three types of lens surgery. Three groups consisting of six capsular bags were established, that is FLACS, Phaco and extracapsular lens extraction (ECCE). The capsular bag was transferred into equal cell culture conditions after using one of the defined surgical approaches. Cellular growth of lens epithelial cells was observed and photo-documented. The time until full cell-coverage of the capsular bag was measured. The human capsular bag model can be successfully prepared using FLACS. There was no statistically significant difference in time until cell-coverage of the human donor capsular bag invitro in all three surgical settings (ECCE versus Phaco p=0.6; ECCE versus FLACS p=1.0; Phaco versus FLACS p=1.0). In our invitro human capsular bag model, we could not observe a statistically significant difference in PCO formation using different surgical approaches of lens extraction. Therefore, PCO formation might not be attributed to the type of surgery. Furthermore, this study shows that FLACS can be used for the human capsular bag model preparation and validates the human capsular bag model for future research.

Effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab) on lens epithelial cells.

The molecular and cellular effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab) on lens epithelial cells (LECs) were examined using both an immortalized human lens epithelial cell line and a porcine capsular bag model. After treatment with various concentrations of bevacizumab, cell viability and proliferation patterns were evaluated using the water-soluble tetrazolium salt assay and 5-bromo-2′-deoxyuridine enzyme-linked immunosorbent assay, respectively. The scratch assay and Western blot analysis were employed to validate the cell migration pattern and altered expression levels of signaling molecules related to the epithelial–mesenchymal transition (EMT). Application of bevacizumab induced a range of altered cellular events in a concentration-dependent manner. A 0.1–2 mg/mL concentration demonstrated dose-dependent increase in proliferation and viability of LECs. However, 4 mg/mL decreased cell proliferation and viability. Cell migrations displayed dose-dependent retardation from 0.1 mg/mL bevacizumab treatment. Transforming growth factor-β2 expression was markedly increased in a dose-dependent manner, and α-smooth muscle actin, matrix metalloproteinase-9, and vimentin expression levels showed dose-dependent changes in a B3 cell line. Microscopic observation of porcine capsular bag revealed changes in cellular morphology and a decline in cell density compared to the control after 2 mg/mL treatment. The central aspect of posterior capsule showed delayed confluence, and the factors related to EMT revealed similar expression patterns to those identified in the cell line. Based on these results, bevacizumab modulates the proliferation and viability of LECs and induces morphological alterations through the modulation of expression patterns of specific factors related to the EMT.

Growth factor restriction impedes progression of wound healing following cataract surgery: identification of VEGF as a putative therapeutic target.

Secondary visual loss occurs in millions of patients due to a wound-healing response, known as posterior capsule opacification (PCO), following cataract surgery. An intraocular lens (IOL) is implanted into residual lens tissue, known as the capsular bag, following cataract removal. Standard IOLs allow the anterior and posterior capsules to become physically connected. This places pressure on the IOL and improves contact with the underlying posterior capsule. New open bag IOL designs separate the anterior capsule and posterior capsules and further reduce PCO incidence. It is hypothesised that this results from reduced cytokine availability due to greater irrigation of the bag. We therefore explored the role of growth factor restriction on PCO using human lens cell and tissue culture models. We demonstrate that cytokine dilution, by increasing medium volume, significantly reduced cell coverage in both closed and open capsular bag models. This coincided with reduced cell density and myofibroblast formation. A screen of 27 cytokines identified nine candidates whose expression profile correlated with growth. In particular, VEGF was found to regulate cell survival, growth and myofibroblast formation. VEGF provides a therapeutic target to further manage PCO development and will yield best results when used in conjunction with open bag IOL designs.