Testing Bone Formation Induction by Calvarial Injection Assay in vivo.

Bone formation occurs during embryogenesis, skeletal growth and during the process of skeletal renewal throughout life. In the process of bone formation, osteoblasts lay down a collagen-containing matrix, termed osteoid, which is gradually hardened by incorporation of mineral crystals. Although osteoblasts can be induced to differentiate and to deposit mineral in culture, this system does not always provide results that reflect the ability of agents to stimulate bone formation in vivo. This protocol describes a rapid and reliable method for testing local administration of agents on bone formation in vivo. In this method, mice are injected with the agent of question for 5 successive days. Fluorochrome labels are injected prior to, and after agents used for testing, and samples are collected and analysed by undecalcified bone histology and histomorphometry. This provides a robust method for assessing the ability of agents to stimulate bone formation, and if a short-term modification is used, can also be used for testing gene responses in bone to the same stimuli.