A novel ex vivo organotypic culture model of alkaptonuria-ochronosis

Alkaptonuria (AKU) is an orphan disease that has an estimated prevalence of 0.3/100,000. The disease is caused by the lack of activity of homogentisic acid oxidase (HGO), an enzyme involved in tyrosine and phenylalanine metabolism. To date, there is only one drug, the nitisinone, with orphan designation authorised by both Food and Drug Administration (FDA) and European Medical Agency (EMA) for AKU. A clinical trial on AKU patients using nitisinone has recently been completed but it needs further investigation for long-term therapy. In recent years our group has developed a series of AKU in vitro models using cell lines, primary chondrocytes and human plasma in order to test the efficacy of new substances, mainly antioxidant compounds, for AKU therapy. Herein, we report the optimisation of an ex vivo reproducible culture method exploiting cartilage slices in order to investigate the deposition of ochronotic pigment in this kind of connective tissue. Human normal cartilage slices, obtained after surgery for prosthesis replacement, were cultured for several days in the presence of a sublethal concentration of homogentisic acid (HGA). After two months of incubation with HGA, the peculiar melanin-like ochronotic pigmentation can be observed into the cartilage tissue. This novel organo-typic ex vivo model could be extremely useful to investigate the efficacy of substances able to ameliorate the conditions of AKU patients. Moreover, it could be used for genetic and proteomic investigations to better define AKU pathophysiology.