Ex vivo biophysical characterization of a hydrogel-based artificial vitreous substitute

Abstract

Purpose: To characterize the biophysical properties of an artificial vitreous body substitute (VBS), which consists of a biocompatible, cross-linked, hyaluronic acid (HA)-based hydrogel, by analysing the VBS's influence on intraocular pressure (IOP) and retinal integrity in distinct ex vivo eye models in order to evaluate the its potential for in vivo biocompatibility testing.

Methods: Pig eyes were obtained immediately postmortem, and VBS was injected after core-vitrectomy. IOP was followed for 24 h (n = 5). VBS influence on retinal integrity was investigated using isolated bovine retinas superfused with an oxygen saturated nutrient solution. An electroretinogram (ERG) was recorded on explanted bovine retinae using silver/silver chloride electrodes; after application of VBS for 2 min, a washout period of 70 min was employed. The percentage of a-and b-wave reduction at the end of the washout phase was compared to baseline values (n = 5). Data were calculated throughout as the mean and the standard deviation. qRT-PCR (Bax/Bcl-2-ratio, GFAP- and PGP9.5-levels) or western blot analysis was used to test for toxicity of Princess Volume after 24 h (and β-3 tubulin with GAPDH as a control gene). Significance was estimated by Student´s t-test; p ≤0.05 was considered to be statistically significant.

Results: The IOP increased non-significantly by 10% after 24 h. Short-term biocompatibility testing using isolated superfused bovine retinas showed neither significant reductions of the b-wave nor the a-wave amplitudes (b-wave reduction 14.2%, p>0.05; a-wave reduction 23.9%, p>0.05). qRT-PCR and western blot analysis did not reveal significant toxicity after 24 h.

Conclusions: The manufactured HA-based hydrogel showed highly favourable biophysical characteristics in the explored ex vivo models, justifying in vivo studies enabling the assessment of biocompatibility.

Fig 1. Overview of physicochemical characteristics of VBS.

The blue, red and green points represent the storage moduli, the cubes represent the loss moduli (20-gauge needle) and the triangles the elasticity.

Fig 2. Effects of VBS on the IOP in the porcine vitrectomy model.

Average of series (n = 5). Representative standard deviations for each measurement are given.

Fig 3. Effects of BSS serving as control on the IOP in the porcine vitrectomy model.

Average of series (n = 5). Representative standard deviations for each measurement are given.

Fig 4. Effects of VBS on the a- and b-wave amplitude of the ERG taken from isolated superperfused bovine retinae.

Average of series (n = 5). The blue line represents the a-wave amplitudes, the red line represents the b-wave amplitudes. Representative standard deviations for each drug series are given.