Development of a standardized in vitro model to reproduce hydrophilic acrylic intraocular lens calcification

Abstract

Opacification through calcification of hydrophilic acrylic intraocular lenses (IOL) is a severe complication after cataract surgery. Causing symptoms that range from glare through to severe vision loss, the only effective therapy is explantation of the opacified IOL so far. Although IOL calcification is a well-described phenomenon, its pathogenesis is not fully understood yet. The purpose of the current study was to develop a laboratory model to replicate IOL calcification. Calcification could be reproduced using a horizontal electrophoresis and aqueous solutions of calcium chloride and disodium hydrogen phosphate. The analysis of the in vitro calcified IOLs was performed using light microscopy, Alizarin Red and Von Kossa staining, scanning electron microscopy, energy dispersive x-ray spectroscopy and electron crystallography using transmission electron microscopy and electron diffraction. The presented laboratory model could be used to identify hydrophilic IOLs that are at risk to develop calcification and to assess the influence of associated risk factors. In addition, it can serve as a research tool to further understand this pathology.

Figure 1

Schematic illustration of experimental setup (a) and hydroxyapatite formation within the intraocular lens (IOL) polymer (b). (a) The IOLs were placed in the double-walled holder having a circular opening exposing the IOL’s surfaces. The holder was then placed in the electrophoresis tank with a rubber seal to avoid leakage of the solutions. (b) The disodium phosphate aqueous solution was set at the cathode side and the calcium chloride aqueous solution at the anode side. Calcium cations and phosphate anions migrate toward the corresponding electrode side—passing through the IOL polymer. Hydroxyapatite was formed as the opposing ions met in the gel.

Figure 2

Analysis methods. After electrophoresis, light microscopy (LM) images were taken of the complete IOLs. Then the IOLs were bisected into X and Y halves. The X half was analyzed using the Alizarin Red and the Von Kossa methods. The anterior and posterior surfaces of the Y half and vertical sections (VS) of these Y halves were investigated using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and electron diffraction (ED).

Figure 3

Light microscopy investigation of IOL 3a. Light microscopy (a) shows flat and granular deposits on the IOL’s surface. Alizarin Red (b) and Von Kossa (c) staining reveal the deposits had formed on the surface (b) and within the IOL polymer (c) and consist of calcium phosphate.

Figure 4

Analysis of surface deposits of IOL 3a. Scanning electron microscopy (a, b) shows deposits formed on the IOL’s surface. Energy dispersive X-ray spectroscopy (c) investigation shows high peaks of 6,5 cps/eV at 2 keV characteristic for phosphorus (P) and 9,5 cps/eV at 3,7 keV characteristic for calcium (Ca). These findings confirm the deposits to be calcium phosphates. Since the sample was placed on a silicon waver for analysis, a silicon peak (Si) can be found in the EDX result. Carbon (C) and oxygen (O) peaks originate from the IOL polymer.

Figure 5

Chemical analysis of the crystals formed within the polymer of IOL 3a. Scanning electron microscopy investigation of a vertical section (a) overview, (b, c) higher magnification) with energy dispersive X-ray spectroscopy (d) confirms the crystals to consist of calcium (Ca) and phosphorus (P). The silicon peak (Si) originates from the silicon waver, carbon (C) and oxygen (O) peaks from the IOL polymer.

Figure 6

Electron crystallography of crystals within the polymer of IOL 3c. Transmission electron microscopy (b) provides the electron diffraction (ED) pattern of the crystals (a). Comparison (c) of the crystals’ ED pattern within IOL 3c to a reference ED of hydroxyapatite (HAP) shows a very high accordance: The ED of IOL 3c shows peaks at a scattering vector of 16, 18.5, 20, 22.5, 32, 34 and 36.5, characteristic for hydroxyapatite.

Figure 7

Distinction between crystals formed within the IOL polymer and hydroxyapatite (HAP) to octacalcium phosphate (OCP). Due to the OCP’s asymmetrical crystal structure, the reference electron diffraction (ED) pattern of OCP shows various diffraction reflections. These reflections are missing in the ED pattern of crystals in IOL 3c. Characteristic reflections to clearly distinguish OCP and HAP are marked (arrows).