Models of tibial fracture healing in normal and Nf1‐deficient mice

Abstract

Delayed union and nonunion are common complications associated with tibial fractures, particularly in the distal tibia. Existing mouse tibial fracture models are typically closed and middiaphyseal, and thus poorly recapitulate the prevailing conditions following surgery on a human open distal tibial fracture. This report describes our development of two open tibial fracture models in the mouse, where the bone is broken either in the tibial midshaft (mid-diaphysis) or in the distal tibia. Fractures in the distal tibial model showed delayed repair compared to fractures in the tibial midshaft. These tibial fracture models were applied to both wild-type and Nf1-deficient (Nf1 þ/À ) mice. Bone repair has been reported to be exceptionally problematic in human NF1 patients, and these patients can also spontaneously develop tibial nonunions (known as congenital pseudarthrosis of the tibia), which are recalcitrant to even vigorous intervention. pQCT analysis confirmed no fundamental differences in cortical or cancellous bone in Nf1-deficient mouse tibiae compared to wild-type mice. Although no difference in bone healing was seen in the tibial midshaft fracture model, the healing of distal tibial fractures was found to be impaired in Nf1 þ/À mice. The histological features associated with nonunited Nf1 þ/À fractures were variable, but included delayed cartilage removal, disproportionate fibrous invasion, insufficient new bone anabolism, and excessive catabolism. These findings imply that the pathology of tibial pseudarthrosis in human NF1 is complex and likely to be multifactorial. ß

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast-like cells

“…(10) Heterozygous Nf1 þ/À deficient mice exhibited an impaired response in BMP-7 in an ectopic bone formation assay (11) as well as reduced bone healing in a distal tibial fracture model. (12) More advanced models have been subsequently generated in an attempt to model the double inactivation of NF1 seen in some human bone defects. Nf1 À=À Prx1 mice lacking Nf1 in the calvaria and limb mesenchyme showed runting, congenitally bowed tibias, and impaired bone healing in a drill hole defect.…”

“…This mouse midshaft fracture model has been previously described. (12) In summary, anesthesia was induced with ketamine (35 mg/kg) and xylazine (4.5 mg/kg) intraperitoneally (i.p.) and maintained using inhaled isoflurane.…”

“…This was previously used to show reduced fracture healing in Nf1 þ/À mice. (12) A lack of union at 3 weeks does not necessarily imply that fractures would proceed to non-union, only that normal healing was delayed. In this study, fractures were assessed by up to two blinded assessors, and statistical significance was determined using a Fisher's exact test.…”

“…Whereas it has been previously reported that Nf1 þ/À mice show delayed healing in the distal tibia, (12) we generated a novel model of AdCre-mediated deletion of both Nf1 alleles. To confirm local recombination in mice, AdCre was injected directly into the fracture site at the time of fracture.…”

Skeletal abnormalities in neurofibromatosis type 1: Approaches to therapeutic options

“…Initial preclinical studies using these mice reported delayed bone healing in Nf1 þ/À mice upon distal tibia fracture [Schindeler et al, 2008a]. While Nf1 þ/À mice do not replicate the bowing and spontaneous fractures seen in patients with NF1 and tibial dysplasia, they indicate that Nf1 haploinsufficiency is sufficient to model defective NF1 bone repair.…”