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Using a Novel Transgenic Mouse to Identify Endochondral Ossification Precursor Cells and to Induce Bone Growth
Board Board 1 / Mon 15:25, 13 Apr 2015

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Title: Defining the Role of the Vasculogenic Niche in Trauma-Induced Heterotopic Ossification Using Novel Imaging and Lineage-tracing Mice


Heterotopic ossification (HO), the anomalous growth of bone in extra-skeletal tissues, occurs following musculoskeletal trauma and burns.
Highly vascular environment surrounds growing ectopic bone based on clinical experience.
Studies suggest that endothelial cells differentiate into osteoblasts.


To demonstrate the role of endothelial cells in the formation of heterotopic bone following trauma using our burn/tenotomy mouse model.
To demonstrate that endothelial progenitor cells exhibit pro-osteogenic characteristics in vitro. 


Trauma-Induced Heterotopic Ossification

•30% TBSA partial-thickness burn
•Achilles tenotomy


•Wild type
•ODD-luc (HIF-1 reporters)



Nano-CT: Microfil perfusion 9 weeks after burn/tenotomy

Histology: Immunofluorescent  (IF) staining

In Vitro Confocal: Cdh5-Cre/RFPfx/fxvascular lineage tracing mice.

Flow Cytometry: Mature Endothelium and Endothelial Progenitors

Cell Assays: Alkaline phosphatase

RT-PCR: Osteogenic gene expression

Bioluminescence: HIF-1 expression

Statistics: Two-tailed Student׳s t-test: Burn Tenotomy vs. Control


•Burn/tenotomy results in sustained hypoxia and vascular flow at tendon injury site(Fig. 3A,B).
•Heterotopic ossification forms in close proximity to growing blood vessels (Fig. 4A).
•Lineage-tracing mice demonstrate robust endothelial tissue at HO sites by 3-weeks (Fig. 4B).
•IF demonstrates endothelial colocalization with the chondrogenic marker Sox9 (Fig. 4C).
•Endothelial lineage-cells are absent from mature HO by 6 weeks post-injury (Fig. 4D).
•Mature endothelium and EPCs are enriched with burn tenotomy versus burn alone (Fig. 4E).
•Mature endothelium and EPCs isolated from tenotomy site are highly osteogenic (Fig. 4F,G).
•PX-478 inhibits HIF-1α expression by hypoxic chondrocytes in vitro (Fig. 4H).
•PX-478 diminishes inflammation and HIF-1α expression at the tenotomy site (Fig. 4I).

Burn/tenotomy is followed by robust and sustained hypoxic and vascular responses localizing to sites of developing HO.
Endothelial markers directly co-localize to immature HO.
Endothelial tissues isolated from the site of injury demonstrate increased osteogenic character versus non-endothelial cells from the same site.

PX-478 treatment is effective at decreasing HIF-1 expression in chondrocytes in vitro and diminishes inflammation at tenotomy site.


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