Jad El-Hoss MSc1,2,*, Kate Sullivan PhD1,2, Tegan Cheng1, Nicole YC Yu BEng(Hons)1, Justin D Bobyn BSc, MBBS2, Lauren Peacock1, Kathy Mikulec1, Paul Baldock PhD3, Ian E Alexander MBBS, FRACP, PhD2, Aaron Schindeler PhD1,2, David G Little MBBS, FRACS(Orth), PhD
Neurofibromatosis type 1 is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1flox/flox and Nf1flox/- mice. The effects of the presence of Nf1null cells were extensively examined. Cultured Nf1null committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with rhBMP-2 treatment. Similarly, Nf1null inducible osteoprogenitors obtained from Nf1 mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1flox/flox and Nf1flox/- mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1flox/flox and Nf1flox/- mice. Histological analyses showed invasion of the Nf1null fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upwards of 10-fold in Nf1null fractures and BrdU staining in closed fractures showed increased numbers of proliferating cells. In Nf1null fractures, TRAP+ cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.
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