Journal: The Journal of Cell Biology
Article Title: Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation
Figure Lengend Snippet: Impairment of periosteal bone formation in truncated BMPR-IB transgenic mice. (A and B) The responsiveness of tg(Col-2.3) homozygous transgenic mice to BMP-2. BMP-2 (20 μg/kg/d, daily for 5 d) in 20 μl PBS containing 0.1% BSA were injected into mice subcutaneously, adjacent to the calvarial bones. Significant amounts of new woven bone, indicated by red arrows and a black arrow head, were formed on the periosteal surface of calvariae in wild-type mice receiving BMP-2, but not in homozygous transgenic mice receiving BMP-2. The existing bones were indicated by a black arrow (A). Similarly, BMP-2 (20 μg/kg/d, ×5d) was administered subcutaneously and Calcein and Alizarin red were injected 7 and 2 d before the animals were killed. The new bone formation during the two fluorochrome injection (5-d period) was observed in wild-type mice (indicated by white arrows and a black arrowhead) but not in tg(Col-2.3) mice (B). (C) Effects of TGFβ on periosteal bone formation. TGFβ (20 μg/kg/d, daily for 5 d) was injected subcutaneously over calvariae. Significant amounts of new woven bone, indicated by red arrows and a black arrowhead, were formed on the periosteal surface of calvariae in wild-type and homozygous transgenic mice. (D and E) In vitro bone formation assay. Calvariae were isolated from 4-d-old wild-type and tg(Col-2.3) mice and cultured for 7 d in BGJ medium. At the end of the incubation, calvariae were fixed and processed for histomorphometric analyses. Osteoblasts on the BS were indicated by red arrows and existing bones were indicated by yellow arrows (D). The thickness of calvariae were measured and presented in (E).
Article Snippet: Tetracycline (15 mg/kg i.p.; Sigma-Aldrich) was administered to 1-mo-old wild-type and transgenic mice, and this was followed by injection of a calcein label (20 mg/kg i.p.; Sigma-Aldrich) 4 d later, mice were sacrificed 48 h after the second label was injected, and bone tissues were removed and fixed in 70% ethanol for 48 h. The specimens were dehydrated through a graded series of ethanol (70–100%) and embedded in methylmethacrylate without prior decalcification.
Techniques: Transgenic Assay, Mouse Assay, Injection, In Vitro, Tube Formation Assay, Isolation, Cell Culture, Incubation