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Digital X-ray microtomography yields stunning views of limb regeneration


Employing high-tech, digital X-ray microtomography (microCT), Northwestern University scientists have discovered the way in which newts form new bone and cartilage during limb regeneration. Newts are a type of salamander, the only vertebrates capable of rebuilding lost structures such as limbs throughout their lifetimes.

Reporting in the January issue of Developmental Dynamics, Northwestern researchers Hans-Georg Simon and Stuart Stock showed that bone formation in a regenerated forelimb combines elements of embryonic development and of adult wound healing.

Results of their research, which have not been observed before in other studies, may have implications for replacement of limb parts missing from injury or birth defects, and, ultimately, for growing new tissue from parts of organs such as livers.

Simon is assistant professor of pediatrics at the Feinberg School of Medicine at Northwestern University and a developmental biologist at the Children’s Memorial Institute for Education and Research. Stock is research professor at the Feinberg School and a material scientist at the Institute for Bioengineering and Nanoscience in Advanced Medicine at Northwestern University.

MicroCT shows promise for detecting and characterizing soft tissue structures, skeletal abnormalities and tumors in live animals, Stock said. It provides high-resolution images (typically 25 micrometers or less) and rapid data acquisition (5 to 30 minutes).

"MicroCT data sets show us how mineral is distributed within bones," he said. "Mineral distribution affects the susceptibility of bone to fracture, for example, a major concern in osteoporosis."

"Because the structure of a given bone varies greatly between individuals, changes in bone are seen most clearly if the same volume of tissue is examined noninvasively at different points in time. This is what microCT is able to do," Stock said.

In the study reported in Developmental Dynamics, microCT allowed scientists to observe microscopic changes inside the regenerating forelimbs without dissecting the tissue as it is done in conventional analyses.

"Scientists previously thought that regeneration progressed in a continuous directional manner, from the amputation site to the farthest distal point," said Simon.

The Northwestern researchers found that although to the naked eye the limb appeared to regenerate from the amputation site at the upper arm to the fingertips, when they examined the mineral formation in the forming bones via microCT, they found that bone formation did not occur in the same order.

"With this microCT method, we can see things other people probably have missed in previous years," said Simon.

"Although the cartilage developed into bone in the lower limb arm from the elbow joint down to the fingers, when we zoom in on at the amputation site in the upper part of the limb arm it looks pretty similar to a normal fracture. There is a gap where bone has not yet formed, between the cut side and the new regenerated limb, which resembles normal wound healing," he said.

Using this new microCT imaging, Simon and Stock will be able to conduct further studies on regeneration. In a recent article (Developmental Biology, August 2002), Simon showed that different regulatory gene mechanisms are in force during regeneration, indicating that regeneration is not simply a reiteration of developmental gene programs.

While the same genes are employed as during embryonic development, the new studies provide additional evidence that during regeneration, several genes are regulated in a different manner.

"We are making the first baby steps to just be able to see the process," said Simon.

"Now we can watch the process of rebuilding a limb over time in one living animal, see what these genes are doing and how they instruct the growth of news structures such as cartilage and bone," he said.

Stock added, "In fact, if we do microCT at a synchrotron radiation source such as that at the Argonne National Laboratory, we have resolution 2 micrometers or less and can see details at the cellular level. The real challenge is to recognize important information contained in the Gigbytes of microCT data we produce.

"Regeneration is the most complete repair mechanism there is. If we can develop a non-invasive experimental model system using microCT, we can learn a great deal about this process, which is directly related to wound healing and repairing of broken bones in the clinic," Simon said.

"Regenerating newt cells are similar to activated stem cells. However, what makes them special is that they contain a complete blueprint of the biological structure they have to rebuild." he said.

Elizabeth Crown and Ellen Hunt | EurekAlert!
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