Targeting The Signaling Pathway Of A Multi-potent Connective Tissue Stem Cell To Treat Heterotopic Ossification And Muscle Fibrosis
Shailesh Agarwal, MD, Shawn Loder, MD, James Drake, BS, Christopher Breuler, BS, John Li, MD, David Cholok, BS, Shuli Li, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
PURPOSE: Heterotopic ossification (HO) occurs with severe trauma causing significant functional limitations. Identification of progenitor cells and signaling mechanisms which cause these processes is critical for identifying therapeutic options.
METHODS: C57Bl/6 mice underwent Achilles’ tenotomy and 30% TBSA burn to induce HO. Immunostaining and mRNA quantification was performed for the tendon lineage marker, Scleraxis. Separately, mice expressing hyperactive BMP receptor (Scx-creERT/caACVR1fl/fl) were generated and received cardiotoxin to induce intramuscular HO followed by histologic analysis after 3 weeks. Given its known effects on tendon, ciprofloxacin was used to treat mesenchymal cells in vitro followed by mRNA quantification of Scx and osteogenic differentiation assays. Finally, mice treated with control or ciprofloxacin(10mg/kg) received burn/tenotomy followed by microCT after 9 weeks (n=4/group).
RESULTS: Burn/tenotomy increased Scleraxis expression at the tenotomy site (A). Scleraxis-lineage cells with hyperactive BMP receptor activity were sufficient to form intramuscular HO after trauma (B). Ciprofloxacin significantly reduced Scleraxis gene expression in vitro, with a corresponding decrease in osteogenic differentiation (C). Treatment of burn/tenotomy mice with ciprofloxacin significantly reduced HO after 9 weeks (8.50 v. 3.67mm^3, p<0.05) (D).
CONCLUSIONS: Muscle- and tendon-resident progenitor cells marked by expression of Scleraxis, a tendon lineage marker, are capable of forming cartilage and osseous lesions. Targeted inhibition of Scleraxis gene expression using ciprofloxacin, a readily-available therapeutic is a powerful a treatment for patients at risk for muscle fibrosis and HO.
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