gms | German Medical Science

Objectives: Blood supply is of critical importance for bone remodeling and adequate fracture healing. Osteoporosis as well as delayed union or nonunion of fractures occur more frequently in patients with vascular diseases. A previous study showed that CXC-Ligand 9 (Cxcl9), a chemokine secreted by osteoblasts, negatively regulates angiogenesis and limits blood supply to the intact bone, ultimately resulting in impaired bone formation in vitro [1]. However, it remained unclear whether 1) Cxcl9 is also of relevance in bone remodeling in vivo and 2) plays a role in bone regeneration after fracture.

Methods: Spine and Tibia of female WT and Cxcl9-deficient (Cxcl9^-/-) mice at the age of 4 and 12 weeks were harvested for non-decalcified histomorphometric analysis. Female WT and Cxcl9^-/- mice received a femoral osteotomy stabilized by an external fixator (Fx) at the age of 12 weeks. Femurs were harvested 7, 14 and 21 days post-surgery for micro-CT and histological analyses, corresponding with early, intermediate, and late stages of bone, respectively. Gene expression analysis of the fracture callus at day 3, 7, and 14 days post-surgery was performed using qRT-PCR.

Results and conclusion: While at the age of 12 weeks no alteration in the static and cellular parameters of bone remodeling was observed, 4-week-old Cxcl9-deficient mice showed reduced trabecular bone volume and a decrease in both osteoblast and osteoclast numbers in the spine. Following femoral osteotomy, 12-week-old Cxcl9-deficient mice showed impaired bone regeneration 14 and 21 days after fracture. In particular, µCT evaluation revealed decreased bone volume, tissue volume and bone surface in the callus of Cxcl9-deficient mice. Histomorphometric quantification of non-decalcified callus sections demonstrated a reduced percentage of mineralized bone in the total callus area 21 days following osteotomy in mutant animals. This was accompanied by a high rate of incomplete bridging of fracture ends in mice lacking Cxcl9, resulting in nonunion. On a molecular level, decreased expression of the osteoblast transcription factor Runx2 at day 7 and the cartilage marker Sox9 at all time points was observed in callus tissue, which was accompanied by reduced mRNA levels of the angiogenesis marker Vegf during the early stage of bone healing. In contrast to a previous report [1], our findings indicate that Cxcl9 does not function as an inhibitor of bone formation and is rather required for adequate bone remodeling in young, growing mice. Moreover, our data demonstrate that Cxcl9 is crucial for bone regeneration and may thus represent a promising therapeutic target to treat patients with impaired fracture healing.