Effects of experimental conditions on the release of 45calcium from prelabeled fetal mouse long bones

Embryonic/neonatal bones in culture are commonly used for the study of osteoclastic resorption in vitro. For this purpose, the release of 45calcium (45Ca) from prelabeled bones is measured as an index of resorption. We studied 45Ca release from two types of long bone explants after different preparation methods: 17-day-old fetal mouse radii/ulnae with and without cartilage ends (intact radii/ulnae and shafts, respectively), and intact 18-day old metacarpals/metatarsals. In addition, we examined the effect of different culture conditions, such as cultures performed under the surface of the medium or at the interphase of medium and air, on 45Ca release and histology. When intact radii/ulnae were cultured under the surface of the medium, there was always a significant amount (10%) of net basal 45Ca release (corrected for physicochemical exchange) that was not due to osteoclastic resorption, as it could not be suppressed by inhibitors of resorption even at high concentrations. Moreover, histologically TRAcP-positive cells were almost absent after culture and the bone marrow/stromal cells in the center of the bone appeared necrotic, possibly due to a lack of oxygen. Under these culture conditions, osteoclasts could survive in shafts as well as in PTH-stimulated intact radii/ulnae, but a constant amount of 10% 45Ca, not due to resorption, was still released in the medium. When these explants were cultured at the interphase of medium and air, basal and stimulated 45Ca release originated from osteoclastic resorption. In contrast, in 18-day-old fetal mouse metacarpals/metatarsals, the experimental conditions applied did not affect 45Ca release, which was always due to resorption of the explants by osteoclasts. In these intact bones, in which at time of explantation the stage of osteoclast development is the same as in 17-day radii/ulnae, osteoclasts survive due to the smaller size of the explants, which allows sufficient diffusion of oxygen even at a lower tension when cultures are performed under the surface of the medium. In conclusion, experimental conditions of prelabeled 17-day-old fetal mouse radii/ulnae can affect results considerably. Therefore, if 45Ca release from these bones is to be used as a reliable index of resorption, the system should be validated with stimulators, as well as inhibitors of osteoclastic resorption. The alternative model we describe, namely, cultures of intact 18-day-old fetal mouse metacarpals/metatarsals, offer the practical advantage that meaningful results about resorption can be obtained independently of the position of these bones in the culture medium, which implies that they are less sensitive to variations in factors such as oxygen tension. Moreover, per embryo, 12 similar sized metacarpaWmetatarsals instead of four radii/ulnae are available for culture, which may reduce experimental variability and can lead to a considerable reduction in the number of experimental animals.