HALF of all women born in this country will suffer a bone fracture because of osteoporosis. In osteoporosis, the bones become so fragile that they can break almost spontaneously. It is also true that more women die each year as a consequence of osteoporotic fracture than die of breast cancer. With numbers like these, the need to find a cure for osteoporosis is an urgent one.
Scientists at Lawrence Livermore National Laboratory are actively involved in this cause using the x-ray tomographic microscope (XTM) to produce three-dimensional images of bone. We are using these images to detect microscopic changes in bone structure of small laboratory animals and to study bone loss as well as increases in bone volume after treatment.
The only other method for producing accurate images of the microstructure of bone is sectioning, a time-consuming process that requires slicing the bone very thinly. This method destroys the sample and often introduces tiny pieces of debris, called artifacts, that can obscure important information. Furthermore, sectioning only produces two-dimensional images, which can be used to depict three-dimensional bone structure but not always with complete accuracy. XTM is the only method currently available for studying bone three-dimensionally without destroying it. This means that studies can even be made in vivo.

The XTM at Work
The XTM was developed in 1991 as a spin-off of work on x-ray lasers for the Strategic Defense Initiative, and its inventors at LLNL and Sandia National Laboratories, Livermore, won an R&D 100 Award for the efforts. (See the October 1991 Energy&Technology Review for a detailed description of the XTM.) The XTM is a form of computed tomography, or CT, which was developed in the 1970s as a medical diagnostic tool. (The commonly used term "CAT scan" is a vestige of the earlier name "computerized axial tomography.") The LLNL configuration of the XTM is shown above.
The XTM's spatial resolution is about 2 micrometers, shown at right. Using monochromatic (single-energy) synchrotron radiation at Stanford University's Synchrotron Radiation Laboratory (a part of the Stanford Linear Accelerator), the XTM can obtain spatial resolutions better than that of the best medical CT scanners. Monochromatic synchrotron radiation is used rather than conventional x rays; the former produces less distortion and, hence, better resolution because of its high brightness and the nearly parallel quality of its beam, known as collimation. The XTM is also superior to magnetic resonance imaging (MRI) because MRI cannot be used on metallic materials and because the resolution of the XTM is many times greater.
The XTM is excellent for nondestructive evaluation of a wide variety of industrial and military materials, but the radiation dose required to produce the XTM's high-resolution images currently limits its use in medical studies to laboratory animals or cadavers. Work continues to reduce the radiation exposure levels.






Searching for a Cure
Researchers from the Laboratory and the University of California, San Francisco, are studying osteoporosis, looking at bone loss due to estrogen depletion and at potential treatments. The hope is to understand critical clinical time points in the development of osteoporosis to establish more effective interventions.
As with many studies of osteoporosis, our studies focus on trabecular bone, the sponge-like, connecting bone tissue that forms an internal supporting network mostly near joints where it fills the interior of the cortex (the hard, outer shell of bone tissue). The wrist bones and the neck of the femur where the femur goes into the hip joint have considerable trabecular bone; the vertebrae are almost entirely trabecular bone with very little cortex. Most osteoporotic fractures occur at these three sites.
Female laboratory rats are being used as subjects, half of which have had their ovaries removed to induce estrogen depletion. The non-ovariectomized rats serve as controls. Rats are excellent subjects for osteoporosis studies because estrogen depletion affects the bones of rats and humans in similar ways but much more quickly in rats than in humans.
In the first study, we took XTM images of the rats' proximal tibias before their ovaries were removed, and again five weeks later to determine bone loss. (See images below.) Trabecular bone volume decreased by approximately 60% in the estrogen-depleted animals compared to the control group. In addition, there was a significant change from an interconnected plate- and strut-like structure to one that was mostly disconnected struts. Dangling trabecular elements, supported only by marrow, were also seen in the ovariectomized animals. While these dangling elements contribute to total bone mass, they do not contribute to the stiffness or strength of the bone. We found that the number of trabecular interconnections decreased by 90% in the rats without ovaries compared to the control group. Combinations of broken trabecular struts and dangling elements most likely contribute to fracture risk.





In our most recent study of a potential treatment for osteoporosis, ovariectomized rats were given various intermittent doses of human parathyroid hormone (hPTH) because it appears to be involved in the differentiation and regulation of bone morphogenic proteins. Scientists do not fully understand how these proteins work, but somehow they control the cells that make and resorb bone. Treatment with hPTH began 56 days after the rats' ovaries were removed and continued for four weeks. We found that hPTH did increase trabecular bone volume and trabecular thickness to baseline levels or higher, although it did not re-establish the bone's original structure by recreating lost trabecular interconnections. (See images below.) This and other studies suggest that hPTH's beneficial effects on bone mass do not depend upon the presence of functioning ovaries, which is very good news for post-menopausal women. The failure of hPTH to re-establish trabecular interconnections after 50% of them had been lost may mean either that earlier intervention or prolonged treatment, or both, are required.





Other Work with the XTM
Laboratory scientists also are working with Roche Biosciences of Switzerland to study bone loss caused by continuous use of steroidal anti-inflammatories such as prednisone. Preliminary work has demonstrated that the bone loss caused by medications such as prednisone is very different from estrogen-induced bone loss. Roche has developed a new compound that they believe prevents this bone loss.
We have also used the XTM to study periodontal disease and coronary artery disease. In the future, the XTM may be used to study fracture healing, kidney stone disease, autoimmune diseases such as arthritis, or any other calcified tissues. The key to all of this work is our ability to noninvasively examine body anatomy three dimensionally. With the XTM, we can evaluate therapies and conditions that affect many common but difficult-to-solve health problems. X-ray tomographic microscopy is significantly advancing our understanding of several very important public health issues.

Key Words: computed tomography, human parathyroid hormone, osteoporosis, steroidal anti-inflammatories, x-ray tomographic microscopy (XTM).

Bibliography
Kinney, J. H., et al., "In Vivo, Three-Dimensional Microscopy of Trabecular Bone," Journal of Bone and Mineral Research 10, 2 (1995).
Lane, N. E., et al., "Intermittent Treatment with Human Parathyroid Hormone (hPTH[1-34]) Increased Trabecular Bone Volume but not Connectivity in Osteopenic Rats," Journal of Bone and Mineral Research 10, 10 (1995).
"Nondestructive Imaging with the X-Ray Tomographic Microscope," Energy & Technology Review, UCRL-52000-91-9/10, Lawrence Livermore National Laboratory (September-October 1991) pp. 31-39.

For further information contact John Kinney (510) 422-6669 (kinney3@llnl.gov).


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