Three-dimensional imaging of trabecular bone using the computer numerically controlled milling technique.

Although various techniques exist for high-resolution, three-dimensional imaging of trabecular bone, a common limitation is that resolution depends on specimen size. Most techniques also have limited availability due to their expense and complexity. We therefore developed a simple, accurate technique that has a resolution that is independent of specimen size. Thin layers are serially removed from an embedded bone specimen using a computer numerically controlled (CNC) milling machine, and each exposed cross section is imaged using a low-magnification digital camera. Precise positioning of the specimen under the camera is achieved using the programmable feature of the CNC milling machine. Large specimens are imaged without loss of resolution by moving the specimen under the camera such that an array of field-of-views spans the full cross section. The images from each field-of-view are easily assembled and registered in the postprocessing. High-contrast sections are achieved by staining the bone black with silver nitrate and embedding it in whitened methylmethacrylate. Due to the high contrast nature and high resolution of the images, thresholding at a single value yielded excellent predictions of morphological parameters such as bone volume fraction (mean +/- SD percent error = 0.70 +/- 4.28%). The main limitations of this fully automated "CNC milling technique" are that the specimen is destroyed and the process is relatively slow. However, because of its accuracy, independence of image resolution from specimen size, and ease of implementation, this new technique is an excellent method for ex situ imaging of trabecular architecture, particularly when high resolution is required.