Abstract
Purpose :
Optic disc drusen are acellular calcified deposits in the anterior, unmyelinated optic nerve and occur in up to 2.4% of the population. The majority of patients develop vison loss due to optic neuropathy. These ectopic calcifications have been shown to consist of calcified mitochondria, which result from a combination of narrow scleral canal, impaired axoplasmic flow, and local metabolic stress, ischemia and inflammation. Both intra-and extracellular calcium concentrations are highly regulated by biological process, and mitochondria is known to be effective calcium buffering organelle intracellularly. We aimed to assess different conditions that promote calcification in human skin-derived primary human fibroblast cultures.
Methods :
We cultured human fibroblasts and treated with different conditions for 21 days: 1-4 mM potassium phosphate, 2-6 mM tri-calcium phosphate, 2-3.2 mM sodium phosphate, 0.4-2 mM ibotenic acid, 1-10 mM 3-nitro-propionic acid, and osteogenic differentiation media (0.01 mM dexamethasone, 10 mM B-glycerophosphate, and 50 mM ascorbic acid). Calcification was evaluated by staining with Alizarin-red S and quantified by measuring optical density at 560nm using a plate reader. To assess cell viability, we performed MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and absorbance measurement at 570 nm. We performed statistical analysis using one-way ANOVA and unpaired T test, and p <0.05 was considered as statistically significant.
Results :
Calcification in normal human fibroblast cultures, indicated by Alizarin-red S staining, was significantly induced by culturing for 21 days in elevated extracellular concentrations of calcium using potassium phosphate, tri-calcium phosphate, and sodium phosphate. This avid calcification did not impact cell viability per MTT assay. Excitotoxic toxins using ibotenic acid and 3-nitro-propionic acid did not induce calcification but did lead to lower cell viability. The highest level of calcification was induced with 4mM potassium phosphate (5.69±1) and 3.2 mM sodium phosphate (5.21±0.4).
Conclusions :
Our study determined the optimal concentration of extracellular potassium and sodium phosphate that an induce calcification in primary human fibroblasts after 7 and 21 days in culture. This finding will help us design a novel in vitro assay for assessing increased risk of calcification in patients with optic disc drusen.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.