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Using a Novel Transgenic Mouse to Identify Endochondral Ossification Precursor Cells and to Induce Bone Growth
Shawn Loder, BS, Shailesh Agarwal, MD, Cameron Brownley, BS, John Li, MD, Michael Sorkin, MD, Shuli Li, PhD, Jonathan Peterson, BA, Yuji Mishina, PhD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
PURPOSE: Bone morphogenetic protein (BMP) signaling mediated by Activin A receptor, type I (ACVR1/ALK2) plays a critical role for development of cardiovascular and skeletal systems. Patients with fibrodysplasia osseous progressiva (FOP) develop heterotopic bone secondary to mutations resulting in BMP type I receptor Alk2 constitutive activity (caAlk2). We describe a novel mouse model of heterotopic ossification in which caAlk2 expression is driven by the Nfatc1 gene. We hypothesized that this mutation would enhance heterotopic ossification and would stimulate enrichment of bone progenitor cells at sites of endochondral ossification.
METHODS:Transgenic Nfatc1-Cre+/- mice were crossed with caALK2flox/WT mice yielding Nfatc1-Cre+/caALK2flox/WT (mutant) and Cre- or Nfatc1-Cre+/caALK2WT/WT (littermate controls) mice.
Heterotopic cartilage and bone were followed by MicroCT, nanoCT, histology, whole mount skeletal preparations and immunohistochemistry for SOX9 (chondrogenesis) and osteocalcin (osteogenesis). R26RlacZ/WT/ Nfatc1-Cre+ mice stained with X-gal and R26RlacZ/WT/ Nfatc1-Cre+ mice were imaged to identify Nfatc1-Cre cells. Cells present in the heterotopic bone were analyzed by flow cytometry and compared to normal bone from the same mice and from wild type controls. Primary cells from the heterotopic bone and native bone from mutant and control mice were isolated and differentiated.
RESULTS:Transgenic Nfatc1-Cre+/caALK2flox/WT mice survived beyond birth unlike mice expressing caALK2 globally. Mutants developed robust heterotopic cartilage and bone at sites of endochondral ossification (growth plates) compared to littermate controls by MicroCT, histology and Alcian blue staining (Fig. 1A-C). Mutant mice also exhibited decreased ankle range of motion (Fig. 1D). Cells analyzed from the region of heterotopic ossification had over a 600% enrichment of bone progenitor cells (CD105+/63C-/Alpha5+/Thy1-) by flow cytometry (Fig. 2A,B). Interestingly, this phenotype was not present ubiquitously, as the tibia appeared normal in mutants and littermate controls away from the ankle. Additionally, more proximal joints (knee and elbow) developed less heterotopic bone. Reporter mice showed Nfatc1-Cre expressing cells within the Achilles’ tendon and along cartilage borders while osteoblasts were negative for X-gal. Osteoblasts harvested from mutant heterotopic bone remained significantly more osteogenic compared to skeletal osteoblasts from mutant and wild type mice mice by alkaline phosphatase, alizarin red and qRT PCR in vitro (p<0.05).
CONCLUSION:We describe a novel transgenic mouse which develops heterotopic cartilage and bone after introduction of the FOP mutation only at sites of endochondral bone formation. These non-osteoblastic cells induce robust chondrogenesis and osteogenesis. These findings suggest that a limited population of cells which express ca-ALK2 are capable of causing ectopic bone formation at sites of endochondral ossification, and may serve as a target for patients with fibrodysplasia ossificans progressiva and trauma induced heterotopic ossification. The finding of more HO at distal joints suggests that force may potentiate this effect. Finally, cartilage precursor cells harvested from these mice remain more osteogenic ex vivo holding potential for bone tissue engineering.
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