Figure 3.

Impact of LDN-193189 on ectopic ossification in vivo. (a) Conditional caALK2–expressing mice receiving vehicle after Ad.Cre injection on P7 developed radiographic disease at P13, progressing to fusion of left hindlimb joints by P30–P60, whereas treatment with LDN-193189 diminished ectopic bone formation and preserved joint spaces over the same interval without inducing fractures, osteopenia or skeletal abnormalities (n = 3–5 mice per treatment group; data are representative of six independent experiments). (b,c) Alizarin red and Alcian blue staining of mice at P15 showing ectopic calcifications encasing the left tibia and fibula in vehicle-treated conditional caALK2–expressing mice, but not in LDN-193189–treated mice. Higher magnification images are shown in c. Ectopic bone or cartilage are absent in the wild-type hindlimb. A-P, anterior-posterior; LAT, lateral; P-A, posterior-anterior. (d) μCT imaging showing attenuated ectopic calcification in LDN-193189–treated mice as compared to vehicle-treated mice at P15 and P30. (e) Fixed extension of left hip, knee and ankle joints, as evident in anesthetized and flaccid Ad.Cre-injected conditional caALK2–mutant mice at P30. The extension is attenuated in LDN-193189-treated mice. (f) Passive range of motion impairment score, as assessed by the minimum angle formed by the ankle and tibia with passive dorsoflexion. (g) Impact of vehicle and LDN-193189 treatment upon passive range of motion impairment in Ad.Cre-injected conditional caALK2–mutant mice at P15 and P30 (n as indicated, bars represent mean, *P < 0.001).