A new rapid radiological procedure for routine teratological use in bone ossification assessment: a supplement for staining methods

BACKGROUND: Presently, bone ossification is assessed by the study of single-stained fetal bones (alizarin red-S) or double-stained bones and cartilaginous structures (alcian blue followed by alizarin red-S). Both methods, especially double-staining, are labor-intensive, time-consuming, and provide qualitative information regarding skeleton ossification. Quantitative evaluation of ossification is more difficult and is usually based on determination of calcium and other minerals in the bone by means of atomic absorption spectrometry. Here we introduce a simple new method that allows quantitative determination of skeleton ossification before routine staining examination. METHODS: Fetuses delivered by laparotomy on the 16th and 21st day of gestation as well as 1-day-old rat pups were examined. The fetuses and pups were prenatally subcutaneously exposed to sodium valproate or to physiological saline. Lateral, prone, and supine digital radiograms of each fetus were taken using the Digora-Soredex digital radiography system and the Planmeca Intra intraoral X-ray machine. According to the best visualization, the data concerning vertebra were analyzed. All the fetuses were then routinely double-stained using alcian blue and alizarin red-S. RESULTS: Malformations of axial skeleton (rib, sternum, and thoracic and sacral vertebra) were found in valproate-treated groups. Unlike cartilage malformations, the bone changes were detected in similar frequency in radiological and staining methods. Differences in densities according to the degree of ossification in the vertebral arches and bodies at different levels of the vertebral column, between drug-treated and negative control groups were noted. CONCLUSIONS: The preliminary results suggest that digital radiography examination is a useful method in determining delaying of skeleton ossification not detectable by other methods. It balances qualitative and quantitative aspects of the presently used methods and is also simple, objective, fast, and relatively inexpensive.