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Using a Novel Transgenic Mouse to Identify Endochondral Ossification Precursor Cells and to Induce Bone Growth
Shailesh Agarwal, MD, Shawn J. Loder, BS, Cameron Brownley, BS, Eboda Oluwatobi, BS, Jonathan Peterson, BS, Yuji Mishina, PhD, Steven R. Buchman, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE Patients with fibrodysplasia osseous progressive (FOP) develop heterotopic bone secondary to mutations resulting in BMP type I receptor Alk2 constitutive activity (caAlk2). We hypothesize that introduction of caAlk2 in cells driven by the cardiac developmental promoter Mef2c[AHF] induce robust chondrogenesis and osteogenesis.
METHODS Transgenic Mef2c[AHF]-Cre+/- mice were crossed with caALK2flox/flox mice yielding caALK2flox/flox/Mef2c[AHF]-Cre+. Heterotopic cartilage and bone were followed by MicroCT, histology, whole mount skeletal preparations and immunohistochemistry for SOX9 (chondrogenesis) and pSMAD1/5 (osteogenesis). ROSALacZ/Mef2c[AHF]-Cre+ mice were stained with X-gal to identify recombined cells. Primary cells were isolated from the heterotopic bone and native bone from mutant and control mice.
RESULTS Transgenic caALK2flox/flox/ Mef2c[AHF]-Cre+ mice survived beyond birth and developed robust heterotopic cartilage and bone at the ankle compared to littermate controls by Alcian blue staining(A), microCT(B), and pentachrome stain(C). ROSA reporter mice had X-gal staining within the Achilles’ tendon and along cartilage borders; osteoblasts were negative for X-gal(D). Cells harvested from mutant heterotopic bone were more osteogenic compared to skeletal bone and bone from wild type mice by alizarin red and qRT PCR (p<0.05).
CONCLUSIONS We describe a novel transgenic mouse with heterotopic cartilage and bone after introduction of the FOP mutation in a subset of cells. These non-osteoblastic cells induce robust chondrogenesis and osteogenesis. Cartilage precursor cells harvested from these mice remain more osteogenic ex vivo holding potential for bone tissue engineering.


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