Bone Abstracts (2014) 3 PP382 | DOI: 10.1530/boneabs.3.PP382

Osteogenic differentiation of fibroblast derived from patients with fybrodisplasia ossificans progressiva

Nathalie Bravenboer, Dimitra Micha, Huib van Essen, Coen Netelenbos, Marelise Eekhoff & Gerard Pals

VU University Medical Center, Amsterdam, The Netherlands.

Fibrodysplasia ossificans progressiva (FOP) is a rare, extremely disabling genetic disorder characterized by progressive heterotopic ossification preceded by episodic inflammatory soft tissue swellings (flare ups) leading to early death. There is no proven effective treatment yet. We aimed to develop an in vitro system to investigate the working mechanism of flare ups induced ossification.

Skin biopsies were obtained from four patients with FOP. Dermal fibroblasts were cultured in Ham F10 media until passage 3. Fibroblast cell lines from four age-and sex-matched healthy individuals were used as controls. Osteogenic trans-differentiation was induced by culturing for 21 days in osteogenic medium containing beta glycerol phosphate, ascorbic acid and 5% platelate lysate. Osteogenic differentiation was determined by mRNA expression of runx2, alkaline phosphatase, and osteocalcin. Mineralization was detected after 21 days using Alizarin red staining.

In all four cell lines the classical mutation in the activin receptor-like kinase2 (Alk2) receptor was confirmed. Runx2 and alkaline phosphatase mRNA expression started to increase 3 days after addition of osteogenic medium with a maximum increase at day 7, both in control and FOP cell lines. Osteocalcin mRNA expression was not increased until day 14 of culture. After 21 days of culture calcium deposits were detected with alizarin red staining in FOP cell lines, similar to the control cell lines.

We demonstrated osteogenic differentiation in fibroblasts form FOP patients. This in vitro system can be used to test the mechanism of inflammation related flare ups but also enables to test inhibitors of the ossification process.

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