Deterioration of fracture healing in the mouse model of NF1 long bone dysplasia.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 manifests as a heritable susceptibility to tumours of neural tissue mainly located in the skin (neurofibromas) and pigmented skin lesions. Besides these more common clinical manifestations, many NF1 patients (50%) have abnormalities of the skeleton. Long bones are often affected (usually the tibia) and the clinical signs range from bowing to spontaneous fractures and non-unions. Here we present the analysis of bone fracture healing in the Nf1(Prx1)-knock-out mouse, a model of NF1 long bone dysplasia. In line with previously reported cortical bone injury results, fracture healing was impaired in Nf1(Prx1) mice. We showed that the defective fracture healing in Nf1(Prx1) mice is characterized by diminished cartilaginous callus formation and a thickening of the periosteal bone. These changes are paralleled by fibrous tissue accumulation within the fracture site. We identify a population of fibrous tissue cells within the Nf1 deficient fracture as alpha-smooth muscle actin positive myofibroblasts. Additionally, histological and in-situ hybridization analysis reveal a direct contact of the fracture site with muscle fascia, suggesting a possible involvement of muscle derived cells in the fracture deterioration.

Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite.

It has been shown for developing enamel and zebrafish fin that hydroxyapatite (HA) is preceded by an amorphous precursor, motivating us to examine the mineral development in mammalian bone, particularly femur and tibia of fetal and young mice. Mineral particle thickness and arrangement were characterized by (synchrotron) small-angle X-ray scattering (SAXS) combined with wide-angle X-ray diffraction (WAXD) and X-ray fluorescence (XRF) analysis. Simultaneous measurements of the local calcium content and the HA content via XRF and WAXD, respectively, revealed the total calcium contained in HA crystals. Interestingly, bones of fetal as well as newborn mice contained a certain fraction of calcium which is not part of the HA crystals. Mineral deposition could be first detected in fetal tibia at day 16.5 by environmental scanning electron microscopy (ESEM). SAXS revealed a complete lack of orientation in the mineral particles at this stage, whereas 1day after birth particles were predominantly aligned parallel to the longitudinal bone axis, with the highest degree of alignment in the midshaft. Moreover, we found that mineral particle length increased with age as well as the thickness, while fetal particles were thicker but much shorter. In summary, this study revealed strong differences in size and orientation of the mineral particles between fetal and postnatal bone, with bulkier, randomly oriented particles at the fetal stage, and highly aligned, much longer particles after birth. Moreover, a part of the calcium seems to be present in other form than HA at all stages of development.