E-Poster Presentation 30th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2020

The loss of ephrinB1 in osteoprogenitors delays endochondral ossification during the fracture repair process (#57)

Agnieszka Arthur 1 2 , Sharon Paton 1 2 , Andrew Zannettino 2 3 , Stan Gronthos 1 2
  1. Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health Science, University of Adelaide, Adelaide, SA, Australia
  2. Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia
  3. Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, University of Adelaide, Adelaide, SA, Australia

Background: The process of bone healing comprises mechanical, cellular, and molecular cues. The contact-dependent, membrane-bound Eph receptors and ephrin ligands are implicated in numerous processes required during skeletal development, homeostasis, and bone healing. However, the function of ephrinB1 in fracture repair is unknown.

Aim: To identify the cellular and molecular involvement of ephrinB1 during fracture repair.

Methods: An internal fixation femoral fracture model in conditional male mice lacking ephrinB1 (EfnB1) in the osteo/chondrogenic lineage (EfnB1OBfl/O), driven by the Osterix (Osx) promoter and their Osx:Cre controls was utilised. Micro computed tomography (mCT) and histomorphometric analyses were used to analyse bone parameters, while osteogenic cells were isolated from femora 2 weeks post-fracture (PF) to determine mineral forming potential in vitro. To evaluate ephrinB1 downstream signalling, human bone marrow stem/ stromal cells (hBMSC) were cultured with soluble EphB2-Fc or control IgG-Fc. The phosphorylation status of downstream signalling target TAZ was determined by immunoprecipitation and Western Blot.

Results: In C57Bl/6 wild-type mice ephrinB1 gene expression was elevated 1 week (haematoma) and 2-weeks (soft callus formation/ remodelling) PF. A significant decrease in bone volume/ tissue volume within the fracture callus 2 weeks PF was observed in EfnB1OBfl/O mice. Histomorphometric analysis confirmed a significant increase in chondrogenic tissue and decrease in calcein labelled bone in EfnB1OBfl/O compared to Osx:Cre mice. Mineral synthesis and osteogenic transcription factor Osterix gene expression were significantly reduced in EfnB1OBfl/O cultured BMSC compared to Osx:Cre controls. This study also confirmed a molecular mechanism of TAZ de-phosphorylation in response to ephrinB1 activation in hBMSC, which was consistent with ephrinB1 signalling in mouse stromal cells.

Conclusion: The present study identified that ephrinB1 expressed by the chondrogenic and osteogenic lineages is important for correct soft callus formation and remodelling during the fracture repair process.