Human Lens Capsule Research Articles

Adhesion study of cultured human lens capsule cells on hydrophilic intraocular lenses coated with polyethylene glycol

To evaluate the adhesion of human lens capsule cells on hydrophilic acrylic intraocular lenses (IOLs) coated with polyethylene glycol (PEG). Department of Ophthalmology, Faculty of Medicine, Universidade Estadual Paulista-Botucatu, São Paulo, Brazil. Experimental study. Human anterior lens capsules obtained during cataract surgery were cultured and seeded (200 cells/IOLs) in triplicates on the surface of a copolymer comprising hydroxyethyl methacrylate, ethyl methacrylate, and methyl methacrylate IOLs (Loflex) treated or not treated with PEG. After 26 hours, the number of viable adherent cells was estimated by counting in a hemocytometer. The coating of hydrophilic acrylic IOLs with PEG was effective in inhibiting cell adhesion (P < .05). Cells showing 2 distinct morphologic patterns-epithelial and dendritic-like-were observed during the in vitro establishment of the cultures. A tendency toward greater adhesion of dendritic-like cells was observed in untreated IOLs compared with treated IOLs (P = .095). Coating hydrophilic acrylic IOLs with PEG was effective in inhibiting cell adhesion. This treatment might play a role in posterior capsule opacification prevention. No author has a financial or proprietary interest in any material or method mentioned.

Finite element implementation of a multiscale model of the human lens capsule.

An axisymmetric finite element implementation of a previously described structural constitutive model for the human lens capsule (Burd in Biomech Model Mechanobiol 8(3):217-231, 2009) is presented. This constitutive model is based on a hyperelastic approach in which the network of collagen IV within the capsule is represented by an irregular hexagonal planar network of hyperelastic bars, embedded in a hyperelastic matrix. The paper gives a detailed specification of the model and the periodic boundary conditions adopted for the network component. Momentum balance equations for the network are derived in variational form. These balance equations are used to develop a nonlinear solution scheme to enable the equilibrium configuration of the network to be computed. The constitutive model is implemented within a macroscopic finite element framework to give a multiscale model of the lens capsule. The possibility of capsule wrinkling is included in the formulation. To achieve this implementation, values of the first and second derivatives of the strain energy density with respect to the in-plane stretch ratios need to be computed at the local, constitutive model, level. Procedures to determine these strain energy derivatives at equilibrium configurations of the network are described. The multiscale model is calibrated against previously published experimental data on isolated inflation and uniaxial stretching of ex vivo human capsule samples. Two independent example lens capsule inflation analyses are presented.

Mutations in the ABCA3 gene are associated with cataract-microcornea syndrome.

Cataract-microcornea syndrome (CCMC) is an autosomal dominant inherited disease characterized by the association of congenital cataract and microcornea without any other systemic anomaly or dysmorphism. Although mutations of several genes have been shown to cause dominant CCMC, in many patients the causative gene has not yet been identified. Our aim was to identify the disease-associated gene in Chinese patients with CCMC. The CCMC patients from two unrelated Chinese families and 26 sporadic patients were enrolled. All the patients were screened by Sanger sequencing with no identified mutations. Genetic variations were screened by whole-exome sequencing and then validated using Sanger sequencing. By sequencing the whole exome of three patients in a Chinese four-generation dominant CCMC family (Family A), three heterozygous missense mutation (c.115C>G, c.277G>A, and c.4393G>A) were identified in ATP-binding cassette protein A3 (ABCA3). At highly conserved positions, changes (c.115C>G and c.4393G>A) were predicted to have functional impacts and completely cosegregated with the phenotype. We further confirmed our finding by identifying another heterozygous missense mutation, c.2408C>T, in ABCA3 in an additional dominant CCMC family (Family B), which also cosegregated with the phenotype. Moreover, four heterozygous mutations, two missense mutations (c.4253A>T, c.2069A>T) and two splice site mutations (c.4053+2T>C, c.2765-1G>T) were identified from the sporadic patients. The ABCA3 protein was expressed in human lens capsule, choroid-retinal pigment epithelium and retinal pigment epithelial cells. Mutations in the human ABCA3 gene were associated with lethal respiratory distress. Our study showed, for the first time to our knowledge, that mutations in ABCA3 were associated with CCMC, warranting further investigations on the pathogenesis of this disorder.

Mesothelial cells: a cellular surrogate for tissue engineering of corneal endothelium.

To evaluate whether mouse adipose tissue mesothelial cells (ATMCs) share morphologic and biochemical characteristics with mouse corneal endothelial cells (CECs) and to evaluate their capacity to adhere to the decellularized basal membrane of human anterior lens capsules (HALCs) as a potential tissue-engineered surrogate for corneal endothelium replacement. Adipose tissue mesothelial cells were isolated from the visceral adipose tissue of adult mice, and their expression of several corneal endothelium markers was determined with quantitative RT-PCR, immunofluorescence, and Western blotting. Adipose tissue mesothelial cells were cultured in a mesothelial retaining phenotype medium (MRPM) and further seeded and cultured on top of the decellularized basal membrane of HALCs. ATMC-HALC composites were evaluated by optical microscopy, immunofluorescence, and transmission electron microscopy. Mesothelial retaining phenotype medium-cultured ATMCs express the corneal endothelium markers COL4A2, COL8A2, SLC4A4, CAR2, sodium- and potassium-dependent adenosine triphosphatase (Na(+)/K(+)-ATPase), β-catenin, zona occludens-1, and N-cadherin in a pattern similar to that in mouse CECs. Furthermore, ATMCs displayed strong adhesion capacity onto the basal membrane of HALCs and formed a confluent monolayer within 72 hours of culture in MRPM. Ultrastructural morphologic and marker characteristics displayed by ATMC monolayer on HALCs clearly indicated that ATMCs retained their original phenotype of squamous epithelial-like cells. Corneal endothelial cells and ATMCs share morphologic (structural) and marker (functional) similarities [corrected]. The ATMCs adhered and formed structures mimicking focal adhesion complexes with the HALC basal membrane. Monolayer structure and achieved density of ATMCs support the proposal to use adult human mesothelial cells (MCs) as a possible surrogate for damaged corneal endothelium.

Meta-analysis of genome-wide association studies in multiethnic Asians identifies two loci for age-related nuclear cataract.

Age-related cataract is a leading cause of blindness worldwide, especially in developing countries where access to cataract surgery remains limited. Previous linkage and candidate gene studies suggested genetic influences on age-related nuclear cataract but few genetic markers have been identified thus far. We conducted genome-wide association studies on 4569 Asians (including 2369 Malays and 2200 Indians), and replicated our analysis in 2481 Chinese from two independent cohorts (1768 Chinese in Singapore and 803 Chinese in Beijing). We confirmed two genome-wide significant loci for nuclear cataract in the combined meta-analysis of four cohorts (n = 7140). The first locus was at chromosome 3q25.31 in KCNAB1 (rs7615568, fixed-effect Pmeta = 2.30 × 10(-8); random-effect Pmeta = 1.08 × 10(-8)). The second locus was at chromosome 21 in the proximity of CRYAA (rs11911275, fixed-effect Pmeta = 2.77 × 10(-8); random-effect Pmeta = 1.98 × 10(-9)), a major protein component of eye lens. The findings were further supported by up-regulation and down-regulation of KCNAB1 and CRYAA in human lens capsule, respectively, as the severity of nuclear cataract increases. The results offer additional insights into the pathogenesis of nuclear cataract in Asians.

Sensitized gelatin as a versatile biomaterial with tailored mechanical and optical properties

We have found that gelatin doped with tot'hema (medication used for curing anemia) and eosin becomes elastic, while retaining good optical properties. The mechanical properties of tot'hema–eosin-sensitized gelatin films (TESG) have been investigated for various concentrations of tot'hema (ranging from 5 to 30% v/v). TESG specimens were prepared according to the ASTM standards for elastic materials. Tensile strength, strain at break and Young's modulus were measured. The results show that the extensibility of TESG film increases with increasing tot'hema concentration, while the Young's modulus and stress at break exponentially decrease. Mechanical properties can be tailored to suit various biomedical applications such as blood vessels, human lens capsules and biosensors based on microlenses. Tunable (strain responsive) TESG microlenses were produced and a mechanical model of blood vessels was prepared.

Measurements of elastic modulus for human anterior lens capsule with atomic force microscopy: the effect of loading force

The purpose of the study was to appraise the effect of loading force magnitude on the determination of the elastic modulus of the anterior lens capsule through atomic force microscopy. Four human anterior lens capsules taken during phacoemulsification cataract surgery were studied, free of epithelial cells, with atomic force microscopy. For the experiment, five different indentation loading forces were applied to near areas of the specimen. Experimental data was exported and analyzed according to the Hertz model to obtain the Young's modulus with regards to the elastic behavior of the material. Force-distance curves were acquired by applying a load of 2, 5, 10, 20 and 30 nN. When examining the results it was evident that determination of Young's modulus of the anterior lens capsule is dependent on the loading force concerning the examined range. Loading forces of 10 and 20 nN led to results without significant difference (p > 0.05) and more reproducible (coefficients of variation 12.4 and 11.7 %, respectively).

Increase in lens capsule stiffness caused by vital dyes

To assess potential changes in lens capsule mechanical properties after staining with brilliant blue, indocyanine green (ICG), and trypan blue. Department of Ophthalmology and Applied Physics and Center for NanoScience, Ludwig-Maximilians-University, Munich, Germany. Experimental study. Fifteen unstained lens capsules were dissected into 7 wedge-shaped parts. Three fragments were stained with brilliant blue 0.025%, ICG 0.05%, and trypan blue 0.06%, respectively, for 1 minute. Another 3 specimens were additionally illuminated using a standard light source. The seventh part served as an untreated control. All specimens were analyzed using atomic force microscopy (AFM) in contact mode with a scan rate of 0.6 Hz. Two scan regions of 10 μm × 10 μm were chosen, and stiffness was determined using AFM in a force spectroscopy mode. The force curves were performed with a data rate of 5000 Hz. Staining of the samples resulted in an increase in tissue stiffness (brilliant blue: P<.001; ICG: P<.01; trypan blue: P<.05). Additional illumination after staining further increased tissue stiffness, but not significantly. Mean increase in the relative elasticity values were 1.61 ± 0.15 (SD) for brilliant blue, 2.04 ± 0.21 for brilliant blue with illumination, 1.63 ± 0.22 for ICG, 2.01 ± 0.22 for ICG with illumination, 1.23 ± 0.11 for trypan blue, and 1.39 ± 0.11 for trypan blue with illumination. In relation to unstained tissue, the relative elasticity of the stained tissue increased 1.2-fold after illumination. Staining significantly increased the mechanical properties of the human lens capsule. No author has a financial or proprietary interest in any material or method mentioned.

Morphological and proliferative studies on ex vivo cultured human anterior capsule lens epithelial cells

AbstractPurpose The proliferation and migration of lens epithelial cells (LECs) that remain in the capsular bag after cataract surgery can lead to development of posterior capsule opacification (PCO) ‐ a major cause of post‐operative visual loss. The multipotency of anterior lens capsule (LC) LECs is still controversial. We aimed to test the proliferating and migrating potential and to characterize structurally the ex vivo cultured LECs growing out off human anterior LC.Methods The explants of LCs were obtained from uneventful cataract surgery and consisted of basal membrane and monolayer of LECs that were cultivated under adherent conditions. The size and shape of the outgrowing cells were recorded by scanning electron microscopy (SEM) and their migration and proliferation potential were followed on a daily basis throughout their continued growth using light microscope.Results The migration and proliferation of anterior LC LECs filling up the denuded region of the LC could be followed by light microscopy. These cells could also migrate to the opposite side of the capsule as shown by both, light microscopy and SEM. The migration and proliferation of LECs on glass or plastic culture surfaces could be shown by both techniques, while the distribution of the LECs and their morphology could be analyzed in details by SEM.Conclusion Classic light microscopy and SEM studies can be used to show that human anterior LC harbors LECs that can migrate and proliferate, suggesting their multipotency and putative stem cell nature. The anterior LC explants can be used to study PCO and the potential of different pharmaceutical or physical treatments against PCO development.

Permeability of the Anterior Lens Capsule for Large Molecules and Small Drugs

Purpose: For developing injectable lenses the retention properties of the capsular bag are important. Therefore the apparent permeability coefficients of sodium fluorescein and fluorescent dextrans of different sizes were determined for the human anterior lens capsule to calculate a molecular weight cutoff from these data. In addition, permeability coefficients of drugs helpful for the suppression of secondary cataract were determined.Materials and Methods: Capsulorhexis specimens were fixed in a specially designed two compartment diffusion chamber to investigate the permeation of sodium fluorescein and fluorescent dextrans of different sizes (10, 40, 70 and 150 kDa) for 24 h (n ≥ 3) and of the antiproliferative drugs actinomycin D and methotrexate for 0.5, 24, 48 and 72 h (n ≥ 3).Results: The molecular weight cutoff of the anterior lens capsule was found to be 166 ± 82 kDa. After 0.5 h, no passage of actinomycin D and methotrexate was detectable through the lens capsule. The apparent permeability coefficients for actinomycin D and methotrexate were calculated to 0.71 ± 0.02 µm/s and to 0.80 ± 0.13 µm/s, respectively.Conclusions: The capsular bag retains fluorescent dextrans with a molecular weight of >166 kDa. Hence, prepolymers are required to polymerize rapidly to be retained inside of the capsular bag. In addition, low-molecular substances intended as antiproliferative drugs for secondary cataract prevention should be applied within a time frame of five minutes in such a way that cells adjacent to the capsular bag will not be damaged.

MALDI MS imaging analysis of apolipoprotein E and lysyl oxidase-like 1 in human lens capsules affected by pseudoexfoliation syndrome

Pseudoexfoliation (PEX) syndrome is an age-related systemic disease of the extracellular matrix, characterized by the presence of amyloid-like fibrillar deposits on the anterior lens capsule. The pathological deposits (PEX material) can obstruct aqueous outflow leading to increased intraocular pressure that in turn can result in glaucoma. PEX syndrome is the most common risk factor for glaucoma. In our previous work, we reported a protocol for the analysis of human lens capsules by MALDI MS imaging. Here, we extend our previous work applying the developed protocol to the analysis of human lens capsules affected by PEX syndrome. We focus our investigation on known components of the PEX material, namely lysyl oxidase-like 1 (LOXL1) and apolipoprotein E (APOE). Our results show that LOXL1 is more abundant in the deposits in the iris region and, alternatively APOE is concentrated in the PEX material accumulated in the pupillary area of the anterior lens capsule. Furthermore, we identify potentially relevant post-translational modifications which may have an important role in promoting the cross-linking processes in PEX syndrome and stabilize aggregate structures within the proteinaceous PEX material. This paper is about the identification and localization of apolipoprotein E and lysyl oxidase-like 1 in human lens capsules affected by PEX syndrome by MALDI MS imaging. With this study we expand the clinical application of MALDI MSI toward the use of non-sectioned tissue samples analyzed after in situ enzymatic digestion and advance the knowledge regarding a common pathology like PEX syndrome.

The Preparations Used to Study Calcium in Lens Epithelial Cells and Its Role in Cataract Formation

This review summarizes relevant publications and our recent work related to the structure and the function of the lens epithelium and its role in the formation of cataract as well as its correlation with changes in intracellular calcium, as studied on different types of preparations. It is emphasized that the human lens capsule preparation isolated during the cataract surgery is an adequate source for the studies of lens epithelial cells and we highlight the possibilities for studying the intracellular calcium homeostasis and the cataract.

Cultivation and Characterization of Cornea Limbal Epithelial Stem Cells on Lens Capsule in Animal Material-Free Medium

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2×2×0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.

Cultivation and characterization of corneal limbal epithelial stem cells on lens capsule in animal material‐free medium

AbstractPurpose To develop simple, reproducible and animal‐materials free method for cultivating and differentiating human limbal stem cells (LSCs) into corneal epithelium on human lens capsule (LC) for clinical transplantation.Methods Limbal tissues (2x2x0,25 mm) were harvested from cadavers (44 males and 33 females, age 70,5+9,3 years) within 12 hours of biologic death. All tissue collection complied with the guidelines of the Helsinki Declaration and was approved by the Regional Ethical Committee. Cell viability was measured with the MTT and annexin‐FITC/propidium iodide assays. Characterization of the growing cells was performed by RT‐qPCR and immunfluorescence stainings accordingly.Results Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (&gt;97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were tested for positivity for markers of stemness (P63, ABCG2, CK19, ITGA9, VIM), proliferation (Ki‐67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non‐hematopoietic, ‐endothelial and ‐mesenchymal stem cell phenotype, while the cell adhesion molecules, integrins and lectin‐based surface carbohydrate profiling showed a specific pattern on the LESCs.Conclusion We report a method for isolating and expanding cornea LESCs from cadavers or alternatively from autologous donors capable of producing viable cell outgrowth on LC for possible treatment of LESC deficiency.

Gaps between lens epithelial cells due to contractions

AbstractPurpose The purpose of the study was to characterize the contractions of human anterior lens capsule epithelial cells that create gaps between cells, and to assess the physiological mechanisms and a possible association of the contractions with cataract formation.Methods Lens capsules obtained during cataract surgery were stained with fluorescent dye Fura‐2. Its fluorescence, upon excitation at 360 and 380 nm, was imaged to monitor changes in cell morphology and cytosolic free Ca2+ concentrations ([Ca2+]i) in response to pharmacological stimulation by acetylcholine and to mechanical stimulation. Contractions were also studied by scanning electron microscope.Results Epithelial cells contracted in about a third of preparations after stimulation. Contractions started either before or at best simultaneously with the rise in [Ca2+]i. They also occurred when there was hardly any change in [Ca2+]i upon application of physiological saline alone. The probability of contractions occurring did not differ significantly among cortical, nuclear and combined cortical+nuclear cataract.Conclusion The contractions of the anterior lens epithelial cells occur in significant part of human lens anterior capsule postoperative preparations. They can be mechanically induced, are localized and reversible, have a fast response and did not differ among different types of cataract. They are at least partly independent of changes in [Ca2+]i. This mechanism could represent physiological basis of cataract formation in phakic intraocular lenses that touch the crystalline lens.

Human anterior lens capsule serving as a substrate for human trabecular meshwork cells cultivation

To investigate if human anterior lens capsule is a suitable substrate for the culture of primary human trabecular meshwork (HTM) cells. Trabecular meshwork cells derived from four human donors were seeded on anterior lens capsules that were prepared from the lenses of donor eyes. Cell morphology and viability were examined at 1, 3, 5 and 7 days. Cell viability was measured based on a two-colour fluorescence assay (membrane-impermeable propidium iodide and membrane permeable Hoechst 33342). Immunocytochemistry studied Zonula occludens-1 (ZO-1), vimentin, tissue transglutaminase (tTgase) and Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase). Morphology of the cultivated cells followed a typical model while their viability was > 95% in all cases. ZO-1 was found at the cell boundaries of the HTM-AC complex. Vimentin was located at the lateral membranes of the HTM cells. Na(+)/K(+)-ATPase was found at the basolateral membrane of the HTM cells. tTgase was also identified. Anterior lens capsule can be considered as a suitable alternative substrate for cultivation of HTM cells and assist the expansion of existing knowledge about glaucoma pathophysiology and therapy.

Rabbit models for continuous curvilinear capsulorhexis instruction

To develop a rabbit model for continuous curvilinear capsulorhexis (CCC) instruction. University of California San Francisco, San Francisco, California, USA. Experimental study. Isolated rabbit lenses were immersed in 2% to 8% paraformaldehyde (PFA) fixative from 15 minutes to 6 hours. Rabbit eyes were treated by substituting aqueous with 2% to 4%PFA for 30 minutes to 6 hours, followed by washes with a balanced salt solution. Treated lenses and eyes were held in purpose-designed holders using vacuum. A panel of 6 cataract surgeons with 5 to 15 years of experience performed CCC on treated lenses and eyes and responded to a questionnaire regarding the utility of these models for resident teaching using a 5-item Likert scale. The expert panel found that rabbit lenses treated with increasing amounts of fixative simulated CCC on human lens capsules from the third to the seventh decade of life. The panel also found fixative-treated rabbit eyes to simulate some of the experience of CCC within the human anterior chamber but noted a shallower anterior chamber depth, variation in pupil size, and corneal clouding under some treatment conditions. Experienced cataract surgeons who performed CCC on these rabbit models strongly agreed that isolated rabbit lenses treated with fixative provide a realistic simulation of CCC in human patients and that both models were useful tools for capsulorhexis instruction. Results indicate that rabbit lenses treated with 8% PFA for 15 minutes is a model with good fidelity for CCC training. No author has a financial or proprietary interest in any material or method mentioned.

Descemet's membrane substrate from human donor lens anterior capsule

To study the potential use of human donor anterior lens capsule as a Descemet's membrane substrate. Anterior lens capsules were recovered from the lenses of 30 cornea donors. Human corneal endothelial cells were recovered from the remaining corneal sclera rims of 15 donor corneas used for penetrating keratoplasty. Samples were sorted into three groups. Group 1 consisted of 10 samples in which the endothelial cells were allowed to grow on anterior lens capsules. In Group 2 human corneal endothelial cells grew on a collagen membrane and in Group 3 on polystyrene culture plates. Cell density, morphology and adherence of the cell-capsule complex were evaluated at 1, 4, 7 and 14days with a phase-contrast microscope, a scanning electron microscope and by histology. Cell viability was quantified by a microscopic live-dead assay. Expression of zonula occludens-1, Na(+) /K(+) -adenosine triphosphatase, tissue transglutaminase and vimentin were investigated by immunohistochemistry. A mean diameter of 10.05±0.13mm of anterior capsule was obtained as a substrate for cell culture. Endothelial cell density of Group 1 was measured at 2455.4±283.8cells/mm(2) , which was also comparable with the cell density of the control group. Cell viability was 95% or superior in all groups and multiple cellular interconnections developed between growing cells. Immunohistochemical analysis demonstrated strongly positive staining for all investigated proteins. Electron microscopy confirmed the adherence and monolayer growth of the endothelial cells. Human donor anterior lens capsule might therefore be a potential scaffold for the ex vivo expansion of human corneal endothelial cells.

Synthesis, staining properties, and biocompatibility of a new cyanine dye for ILM peeling

The aim of this work is to investigate the biocompatibility and staining properties of DSS: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS). Dye concentrations of 0.5, 0.25, and 0.1% were evaluated (290 and 295 mOsm). Toxicity was assessed using a colorimetric test measuring the inhibition of ARPE 19 cell, human primary RPE cell, and human Müller cell proliferation. Exposure time was 30, 60, 120, and 300 s. Indocyanine green (ICG) (0.5, 0.25, and 0.1%) served as a control. Cells were also illuminated with plain white light (750 mW/cm(2)) for 10 min to assess phototoxic effects. Besides staining of porcine and human lens capsule, internal limiting membrane (ILM)-staining was assessed by applying 0.25 and 0.5% DSS over the macula in two human post-mortem eyes. DSS of 0.25 and 0.1% showed no toxic effect on primary RPE cells and MIO-M1cells, and 0.5, 0.25, and 0.1% for ARPE-19 cells. In MIO-M1cells, 0.5% dye showed a significant reduction of mitochondrial dehydrogenase activity only following an exposure time of 300 s. Following illumination, ICG showed a significantly more pronounced effect on cell viability in primary RPE cells and MIO-M1cells compared to DSS. The absorption maximum is found at 591 nm; the even more bathochromic fluorescence proceeds with a common Stokes' shift where maxima at 620 and 660 nm with a quantum yield of 32% were found. The fluorescence is sufficiently hypsochromic and the fluorescence quantum yield high enough for an easy visual detection. The contrast and staining properties at the ILM were excellent and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted. Our results indicate that this new cyanine dye DSS may represent an alternative for ILM staining due to its matched absorption concerning visibility and fluorescence qualities as well as its good biocompatibility.

AFM Study for Morphological Characteristics and Biomechanical Properties of Human Cataract Anterior Lens Capsules

The aim of this study was to quantitatively investigate the morphologies (surface roughness) and biomechanical properties (Young's modulus) of human anterior lens capsules (ALCs) for noncataract and cataract groups using atomic force microscopy. Eight human ALCs obtained during phacoemulsification from patients with senile cataracts (72 ± 13 years) were investigated in both the hydrated and dehydrated conditions. The cataract group showed clearly the proliferated lens epithelial cells (LECs) with a monomorphic cell structure, a diameter of 12.54 ± 4.31 μm, and a height of 0.23 ± 0.04 μm, whereas the control group showed no LECs. A substantial amount of false-positive calcification was observed caused by the deposition of remnants of dried salt solution. Cataract group showed significantly higher surface roughness (382.06 nm, p ≤ 0.001) than control group in the anterior side of ALCs, whereas cataract group showed significantly lower surface roughness (353.79 nm, p ≤ 0.001) than control group in their posterior side. Cataract group showed significantly higher Young's modulus (69.52 kPa, p ≤ 0.001) compared to the control group, regardless of the ALC side. Therefore, it is significant that this study provides a new method to examine the nanostructural characteristic and biomechanical property of human ALCs through a nanometer-scale resolution microscopy technique.