Fig. 4

Silencing H19 obstructs endochondral osteogenesis leading to delayed bone remodeling. (A) The same proportion of AdBMP2, AdBMP2+simH19, AdGFP, AdBMP2+H19-infected cartilage fragments were subcutaneously implanted into the lateral abdomen of nude mice without thymus (nu/nu) for 3 weeks. In appearance, the tumor size of AdGFP group was the smallest. The masses in AdGFP and AdBMP2+simH19 groups showed a more distinct cartilage phenotype than those in AdBMP2 and AdBMP2+H19 groups. (B) Micro-CT quantitative analysis showed that compared with AdGFP, bone mass and trabecular mass parameters in AdBMP2 group were higher, while bone volume fraction and trabecular mass parameters in AdBMP2+simH19 group were lower than AdBMP2 group. “*” means p < 0.01 as compared with AdBMP2 group; “#” means p < 0.01 as compared with AdBMP2+simH19 group. BV, bone tissue volume; BV/TV, bone volume fraction; SMI, structure model index; Tb.N, trabecular number; Tb.Th, trabecular thickness; Conn.Dn, Connectivity Density. (C) The proportion analysis of cartilage hypertrophy, calcification and mineral deposition determined by saffranine O staining of cartilage mass showed that compared with AdGFP, chondrocytes in AdBMP2 group were significantly hypertrophic; Compared with AdBMP2, hypertrophic chondrocytes decreased in AdBMP2+simH19 group. Compared with AdBMP2+simH19, hypertrophic chondrocytes increased in AdBMP2+H19 group. (D) Immunofluorescence (IF) detection of CD31 in cartilage mass showed that compared with AdBMP2, the expression of CD31 (red) in cartilage ossification area in AdBMP2+simH19 group was significantly reduced. (E) Immunofluorescence (IF) detection of CD31 in mouse fracture sections showed that in the PO-2 W and PO-4 W, the expression of CD31 (red) of AdsimH19 groups was significantly lower than that in the AdGFP group in the cartilage ossification region, and the silence of H19 hindered vascularization, resulting in delayed endochondral osteogenesis.