In Vitro Study On Relationship Between Posterior Capsular Opacification And Materials Of Intraocular Lenses

BackgroundCataract remains the leading cause of the blindness in the world. Surgical intervention is the unique treatment of the cataracts and phacoemusification combined with intraocular lenses (IOLs) implantation is the major method, It, however, is associated with possible complications, for example, endophthalmitis, cystoid macular edema(CME), and posterior capsule opacification(PCO) so on. PCO is the most frequent long-time complications which may suffer patients second visual loss and even blind.The PCO is consequence of proliferation, migration, epithelial-mesenchymal transition (EMT) and lens fiber regeneration of the residual lens epithelial cells(LECs) in the posterior capsule after cataract extraction and may stretch the lens luxation and dystopia due to capsule contraction. Many factors affect the IOLs biocompatibility, such as the patients profiles, the surgeons’s technique, the surgical technology and the IOLs, within which the IOLs itself is the determining factor. The elements of IOLs include the hepatic design and the lens material which contains itself properties and its surface qualities. The commonly use IOLs materials inculde hydrophobic acrylic, hydrophilic acrylic, silicone and PMMA. Many studies demonstrated the effects of the square-edge IOLs on preventing the PCO, however, there are disputs about the conclusion that PCO is affected by the materials of IOLs, and so is it that the IOLs materials type may cause the least PCO.The formation of PCO is relative to multifactorial, many cytokines and different signal pathways. The researches of cytokines, IOLs and PCO were the detections of cytokines in aqueous humor from animals eyes which were implanted IOLs for some time. However it is unclear that the cytokines are secreted from lens epithelial cells or from the blood circulation due to the damage of aqueous fluid circulation. The studies about the expressions of cytokines of human lens epithelial cells in vitro and the connection between cytokines and the IOLs materials were barely found domestic and abroad. Tissue growth factor beta 2 (TGF-P2) is an important factor of the lens epithelial mesenchymal transformation (EMT) signaling cascade that promotes PCO, which is consensus in ophthalmology field. IL-6, MMP-2 and MMP-9 relative to the process of PCO have already been demonstrated by many researches. However there are no relevant reports about relationships among the TGF-β2, IL-6, MMP-2 and MMP-9 and the IOL materials.Part I In vitro research on the capsule biocompatibility of intraocular lenses of differential materials[OBJECTIVE]Types of intraocular lenses of differential materials are for ophthalmologists and cataracts. This study is aiming to differentiate the capsule biocompatibility of differential intraocular lenses using human lens epithelial cells culture system: analyzing the cell adhesion, proliferation and epithelial mesenchymal transition.[METHODS]1. Cells sources Human lens epithelial cells were HLEC-B3 line purchased from American Type Culture Collection(ATCC). Good quality and quantity of HLEC-B3 were gotten by recovering, passaging and freezing repeatedly.2. Cell adhesion test Human lens epithelial cells (HLEC-B3) were grown on hydrophobic acrylic IOLs, hydrophilic acrylic IOLs, silicone IOLs, PMMA IOLs. They are grouped by A((PC156C55, PMMA), B(Crystalens HD, silicone), C(Akreos Advance Optics Aspheric Lens, hydrophilic acrylic), D1(Envista, hydrophobic acrylic), D2(ZCB00 Lens, hydrophobic acrylic), D3(Acrysof, hydrophobic acrylic). After 6 hours and 24 hours incubation, cellular adhesion and morphology were assessed and imaged under microscope.3. Cell proliferation assay The proliferation of HLEC-B3 grown on IOLs was measured with a CCK-8 cell counting kit.4. EMT assay After next 48 hours incubation, immunofluorescence on alpha smooth muscle actin(a-SMA) as the cytoplasm biomarker was performed to estimate the EMT and the rate of EMT of IOLs groups were counted.5. HE assay HE stained IOLs were detected to assessed the morphology and proliferation of cells on IOLs after 72 hours incubation.6. Statistical analysis The results were expressed as mean±standard deviation(x±SD), Data analysis was made by one-way analysis of variance and further pairwise comparison by least significant differences test using SPSS 20.0 software(IBM SPSS, Chicago, USA), value of α=0.05 was as significant level and a value of P<0.05 was considered to be statistical significance.[RESULTS]1. Normol cell morphology The HLECs-B3 cultured on flask showed a typical epithelial-like morphology, displaying small round, ellipse and polygon-shaped.2. Cell adhesion results(1) At 6 hours after cells seeding on IOLs, the overwhelming majority of cultured cells grew against the wall of IOLs. The cells counts adhesion on hydrophobic acrylic IOLs groups and silicone IOLs groups were the most, and followed by PMMA groups, while the hydrophilic acrylic IOLs groups were the least; the morphology of HLECs on hydrophobic acrylic groups, hydrophilic acrylic IOLs groups and silicone IOLs groups exhibited mall round, scalene triangle and oval-shape, displaying an epithelial-like morphology, however, isolated spindle-shaped cells were observed. The connects between cells were obvious; while cells on PMMA IOLs groups aggregated to mass, and the extension cells were fused completely and the cellular morphology were unclear; more, the shape of fibroblast were seen.(2)At 24 hours after cells seeding on IOLs, all cells were expanding, the cells on hydrophobic acrylic IOLs groups showed more ellipse-shape and less myofibroblast-like; while the HLECs on the hydrophilic acrylic IOLs groups almost came off, and there were floating round cells on the culture mediums; HLECs on PMMA IOLs were less and less, most cells clumped together and spindle-shaped LECs were found beside by cell mass.3. Cell proliferation results The proliferation results of HLECs on IOLs detected by CCK-8 Cell Counting were shown as:D3(0.2384±0.007)>D2(0.2338± 0.002)>D 1 (0.2260±0.005)>B(0.1781±0.007)>A(0.1580±0.007)>C(0.1242±0.002); and there was no distinct difference between A and E(P=0.155), however, there were remarkable contrast among othere groups (P<0.01).4. EMT results Due to almost all cells on hydrophilic acrylic IOLs fell off, the C groups had no immunofluorescence results; the figures of immunofluorescence shown the bule-fluorescence nucleus and green-fluorescence a-SMA on A, B, D1, D2 and D3 groups. The ration of cells with green-fluorescence a-SMA was counted under fluorescence microscope and resulted in A(84.16±10.48)%>B(17.33±5.71)%> D2(14.22±4.64)%>D1(13.99±9.11)%>D3(9.98±3.80)%. There were significant difference between A and B, D1, D2 and D3 respectively, however, there were no statistical significant among B, Dl, D2, D3 groups.5.HE results The staining of HLECs on IOLs after 48 hours cultured displayed blue cell nucleus and red dish-stained cytoplasm on A, B, C, D1, D2 and D3 groups. For a field of vision, the cell counts of B, Dl, D2 and D3 groups were larger than of A groups, and the cell structures were integrated but not unclear, the nucleus were round-shaped and unequal-sized, Karyorrhexis, Pyknosis and undergoing meiosis nucleus were visible occasionally; For C group, though the cells fell off the IOLs in the forward part, the HE results dispalyed blue cell nucleus and red dish-stained cytoplasm. However, the cellular structure were not integrity and some cells were not with nucleus; big vacuoles were found in some cells and filled with the whole cytoplasm; the most cellular nucleus were inhomogeneity and various shapes, which shown quadrangle, triangle, pengaton, small round and elliptic type.while on the A groups, the cells counts were significantly smaller and gathered to mass; the nucleus of cells were darker and closer together, Pyknosis were more and nuclear fragments were found in cytoplasm.[CONCLUSION]In vitro experiment, the capsule biocompatibility of IOLs may affected by the IOLs materials. And the hydrophobic acrylic IOLs may have the best capsule biocompatibility, then followed by silicone IOLs and PMMA IOLs, the hydrophilic acrylic IOLs may show the worst capsule biocompatibility taking the adhesion, proliferation and epithelial mesenchymal transition into all considerations. The morphology of cells adhesion on IOLs and the counts of cell proliferation may be relative to the epithelial mesenchymal transition, and a certain proportion of momolayer cells may prevent PCO.Part II The cytokines expressions of human lens epithelial cells[OBJECTIVE]To study cytokines expressions of human lens epithelial cells cultured with intraocular lenses and the possible connection between the cytokines expression and the intraocular lenses materials.[METHODS]1. At 24 hours after the HLECs seeding on the different IOLs, the culture mediums were collected into tubes and stored in-80℃ refrigerator;2. The culture mediums were procedural thawing and sub-packed to four tubes; Enzym-linked immunosorbent assay (ELISA) detected the level of TGF-β2, IL-6, MMP-2 and MMP-9 in cell culture medium secreted by HLECs;3.The results were expressed as mean±standard deviation (x±SD), Data analysis was made by one-way analysis of variance and further pairwise comparison by least significant differences test using SPSS 20.0 software.[RESULTS]The culture mediums were detected the secretions of TGF-β2, IL-6, MMP-2 and MMP-9; and the secretions lever of IL-6 and MMP-9 were almost consistent; the culture mediums in the PMMA IOLs group had the highest lever of IL-6 and MMP-9, and then hydrophobic acrylic IOLs groups, followed by silicone IOLs groups, the hydrophilic acrylic IOLs groups displayed the lowest lever. For the TGF-β2 and MMP-2, the levers were relatively lower, and the hydrophobic acrylic IOLs groups were detected the highest lever, then silicone IOLs groups and then PMMA IOLs groups and hydrophilic acrylic IOLs groups. However there were no statistical significant for TGF-β2, IL-6, MMP-2 and MMP-9 among all groups.[CONCLUSION]The human lens epithelial cells can express the TGF-β2, IL-6, MMP-2 and MMP-9; The intraocular lenses may induce the secretions of TGF-β2, IL-6, MMP-2 and MMP-9 of human lens epithelial cells; The expression of TGF-β2, IL-6, MMP-2 and MMP-9 of human lens epithelial cells may be not affected by the materials of intraocular lens.