Periodontal ligament fibroblasts as a cell model to study osteogenesis and osteoclastogenesis in fibrodysplasia ossificans progressiva.

Abstract

Fibrodysplasia Ossificans Progressiva (FOP) is a progressive disease characterized by periods of heterotopic ossification of soft connective tissues, including ligaments. Though progress has been made in recent years in unraveling the underlying mechanism, patient-derived cell models are necessary to test potential treatment options. Periodontal ligament fibroblasts (PLF) from extracted teeth can be used to study deviant bone modeling processes in vitro since these cells are derived from genuine ligaments. They further provide a tool to study the hitherto unknown role of the bone morphogenesis protein receptor type 1 (BMPR-1) Activin A type 1 receptor ACVR1-R206H mutation in osteoclastogenesis. To further validate this potential model, osteogenesis and osteoclastogenesis was studied in the presence of TGF-β/activin receptor inhibitor GW788388. Control and FOP fibroblasts (n=6 of each) were used in osteogenesis and osteoclastogenesis assays in the absence or presence of TGF-β/activin receptor inhibitor GW788388. For osteogenesis, alkaline phosphatase (ALP) activity, alizarin red staining for mineralization and qPCR for expression of osteogenic markers was assessed. TRACP staining, multinuclearity and expression of osteoclastogenesis markers were used as a measure of osteoclast formation. FOP fibroblasts cultured in osteogenic medium displayed a trend of higher ALP activity at 7days. Gene expression of ALP from FOP fibroblasts was significantly higher at 3days. Mineralization was similar at 21days for both groups. GW788388 did not influence mineral deposition in both groups. Osteoclast formation was inhibited by GW788388 on plastic for both controls and FOP. On cortical bone slices, however, osteoclast formation was significantly lowered by GW788388, only in FOP cultures. qPCR revealed strong expression of RANKL at 7days and a significant decline at 14 and 21days in both FOP and control cultures. In contrast to the osteoclastogenesis results, the RANKL/OPG ratio was higher in the presence of GW788388, only in FOP cultures. TGF-β expression was significantly higher at 14 and 21days compared to 7days, possibly signifying a role in later stages of osteoclast formation. Addition of GW788388 strongly decreased TGF-β expression. Our study shows that periodontal ligament fibroblasts from FOP patients displayed at most slightly enhanced in vitro osteogenesis and osteoclastogenesis. This model could be useful to elucidate molecular mechanisms leading to heterotopic ossification in FOP such as in the presence of specific ACVR1-R206H activators as Activin A.

DOI: 10.1016/j.bone.2017.07.007

Cite this paper

@article{Vries2017PeriodontalLF, title={Periodontal ligament fibroblasts as a cell model to study osteogenesis and osteoclastogenesis in fibrodysplasia ossificans progressiva.}, author={Teun J. de Vries and Ton A M Schoenmaker and Dimitra Micha and Jolanda M A Hogervorst and Siham Bouskla and Tim Forouzanfar and Gerard Pals and Coen Netelenbos and Elisabeth M. W. Eekhoff and N Bravenboer}, journal={Bone}, year={2017} }