Osteoblast-specific Krm2 overexpression and Lrp5 deficiency have different effects on fracture healing in mice.

The canonical Wnt/ß-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5-/-) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5-/- mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5-/- mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active ß-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis.

Midkine-deficiency increases the anabolic response of cortical bone to mechanical loading.

The adaptive response of bone to load is dependent on molecular factors, including growth factor signaling, which is involved in the regulation of proliferation, differentiation and function of osteoblasts and osteoclasts. Based on a recent study, which has shown that the deficiency of growth factor midkine (Mdk) in mice at 12 and 18 months of age resulted in increased trabecular bone formation, we hypothesized that mechanically-induced bone remodeling may, at least in part, be dependent on Mdk expression. To investigate this, we loaded the ulnae of Mdk-deficient mice and appropriate wild-type mice at the age of 12 months using the in vivo ulna loading model. Histomorphometric quantification of the periosteal bone demonstrated an increased mineralizing surface, mineral apposition rate, and bone formation rate in ulnae of Mdk-deficient mice compared to wild-type mice in response to loading. Because Mdk has been shown to bind to a complex of receptor-type protein tyrosine phosphatase zeta (Ptprz) and low density lipoprotein receptor-related protein-6 (Lrp-6) together with the α4ß1- and α6ß1-integrins, we performed in vitro studies using osteoblastic cells, transiently over-expressing Mdk, Wnt-3a, and Ptprz to evaluate whether Mdk has a role in regulating bone formation by modulating Wnt signaling. We observed a negative effect of Mdk on Wnt signaling, the extent of which appeared to be dependent on Ptprz expression. Moreover, we performed in vitro loading studies with osteoblasts treated with recombinant Mdk and observed a negative effect on the expression of Wnt target genes, which play a critical role in osteoblast proliferation. In summary, our data demonstrate that Mdk-deficiency in mice has an anabolic effect on mechanically induced cortical bone formation. This could be due to an improved osteoblast function based on an enhancement of ß-catenin-dependent Wnt signaling by both Mdk-deficiency and mechanical loading.

Fracture healing in mice under controlled rigid and flexible conditions using an adjustable external fixator.

Mice are increasingly used to investigate mechanobiology in fracture healing. The need exists for standardized models allowing for adjustment of the mechanical conditions in the fracture gap. We introduced such a model using rigid and flexible external fixators with considerably different stiffness (axial stiffnesses of 18.1 and 0.82 N/mm, respectively). Both fixators were used to stabilize a 0.5 mm osteotomy gap in the femur of C57BL/6 mice (each n = 8). Three-point bending tests, µCT, and histomorphometry demonstrated a different healing pattern after 21 days. Both fixations induced callus formation with a mixture of intramembranous and enchondral ossification. Under flexible conditions, the bending stiffness of the callus was significantly reduced, and a larger but qualitatively inferior callus with a significantly lower fraction of bone but a higher fraction of cartilage and soft tissue was formed. Monitoring of the animal movement and the ground reaction forces demonstrated physiological loading with no significant differences between the groups, suggesting that the differences in healing were not based on a different loading behavior. In summary, flexible external fracture fixation of the mouse femur led to delayed fracture healing in comparison to a more rigid situation.