Thrombospondin-2 (TSP2) is amatricellular protein with increased expression during growth and
regeneration. TSP2-null mice show accelerated dermal wound healing and enhanced bone formation. We
hypothesized that bone regeneration would be enhanced in the absence of TSP2. Closed, semistabilized
transverse fractures were created in the tibias of wildtype (WT) and TSP2-null mice. The fractures were
examined 5, 10, and 20 days after fracture using mCT, histology, immunohistochemistry, quantitative RT-PCR,
and torsional mechanical testing. Ten days after fracture, TSP2-null mice showed 30%more bone by mCT and
40% less cartilage by histology. Twenty days after fracture, TSP2-null mice showed reduced bone volume
fraction and BMD. Mice were examined 5 days after fracture during the stage of neovascularization and
mesenchymal cell influx to determine a cellular explanation for the phenotype. TSP2-null mice showed increased
cell proliferation with no difference in apoptosis in the highly cellular fracture callus. Although mature
bone and cartilage is minimal 5 days after fracture, TSP2-null mice had reduced expression of collagen IIa and
Sox9 (chondrocyte differentiation markers) but increased expression of osteocalcin and osterix (osteoblast
differentiation markers). Importantly, TSP2-null mice had a 2-fold increase in vessel density that corresponded
with a reduction in vascular endothelial growth factor (VEGF) and Glut-1 (markers of hypoxia inducible factor
[HIF]-regulated transcription). Finally, by expressing TSP2 using adenovirus starting 3 days after fracture,
chondrogenesis was restored in TSP2-null mice. We hypothesize that TSP2 expressed by cells in the fracture
mesenchyme regulates callus vascularization. The increase in vascularity increases tissue oxemia and decreases
HIF; thus, undifferentiated cells in the callus develop into osteoblasts rather than chondrocytes. This leads to an
alternative strategy for achieving fracture healing with reduced endochondral ossification and enhanced
appositional bone formation. Controlling the ratio of cartilage to bone during fracture healing has important
implications for expediting healing or promoting regeneration in nonunions.