Mar. 9, 2005 Researchers have created a way to transform the dead bone of a transplanted skeletal graft into living tissue in an experiment involving mice. The advance, which uses gene therapy to stimulate the body into treating the foreign splint as living bone, is a promising development for the thousands of cancer and trauma patients each year who suffer with fragile and failing bone grafts. The findings were posted online Feb. 13 and will appear in the March 1 issue of Nature Medicine.
The procedure, designed by a team led by Edward M. Schwarz, Ph.D., associate professor of orthopedics and of microbiology and immunology at the University of Rochester Medical Center, is intended to eventually aid people with various cancers or injuries whose treatment involves the replacement of large sections of bone. Cancers such as osteosarcoma, one of the most common types of bone cancers, or tumors that occur adjacent to bones, often must be treated by removing the diseased section of bone and replacing it with the only alternative available – a donated section of comparable bone from a cadaver. The new splint of bone is then literally screwed into place, giving the patient most of the strength and support of the original bone. Bone, unlike any other tissue in the human body, can still perform one of its functions, structural support, even if all its cells are completely dead. A serious problem arises, however, when the bone wears over time.
"Everyday activities cause microscopic fractures in our bones," explains Schwarz. "Those fractures are normal and healthy, and our bones re-knit them constantly. But when the bone is dead, there is no healing, and those tiny fractures begin to accumulate until finally, perhaps in 10 years, the implanted section collapses, and more drastic surgery becomes necessary."
To make the transplanted bone more robust, Schwarz looked into the activity of the genes and proteins that govern its health. His team replaced sections of bones in dozens of mice, using both healthy and dead segments, then scanned the surrounding inflammatory tissue for differences in the levels of the active genes. He discovered that the genes that create two key proteins in living bone, called RANKL and VEGF, were barely expressed around the dead bone. He then modified a harmless virus to carry these genes, devised a method of freeze-drying a paste containing the virus so it could be easily handled, and painted it directly onto a bone graft during surgery.
Numerous tests in mice confirmed that the virus permeated the inflammatory tissue around the dead bone and turned on the genes. The mouse body then began to treat the implanted bone as if it were its own tissue instead of a foreign object, which would normally trigger the body to wrap the "invader" in scar tissue.
"That recognition is the key," says Schwarz. "It's at that point that the body actually begins changing the dead, foreign bone splint, into the body's own, whole, living bone."
Such a transformation can occur because mammals use their skeletons for both support and as a kind of "calcium bank." If calcium, which is necessary for such important functions as maintaining the brain and heart, runs low, cells called osteoclasts dig out the calcium from our bones. This is why doctors encourage post-menopausal women to take calcium supplements, so that the body doesn't raid their bones for the calcium it needs. The process works both ways, fortunately, as another set of cells, called osteoblasts, rebuilds the bone when the body has excess calcium. In an average year, a healthy person may remove and rebuild 10 percent of his or her bone structure.
This process of teardown and rebuilding is triggered in the dead bone when Schwarz paints it with the genetically modified virus. New blood vessels begin to grow around and into the bone splint, stripping it down in times when the body needs the calcium, and rebuilding it when calcium levels rise. The bone that is rebuilt is now fully the patient's own, as if the dead bone were a house being renovated by replacing a single brick at a time without tearing the whole structure apart.
Schwarz's studies with mice showed their dead splints were quickly converted to new, healthy bone. He projects that the bone would be completely converted in just a year, and that a human bone might be completely converted in as little as five years. The Musculoskeletal Transplant Foundation has been trying for two decades to conquer the issues complicating bone transplants, and the group has pledged to continue supporting Schwarz's research. Schwarz hopes to begin early human trials with the procedure soon.
"This technology looks like it will have a dramatic effect on success rates for cancer patients who would otherwise be facing choices as drastic as amputation," says Arthur A. Gertzman, executive vice president for research and development at the Musculoskeletal Transplant Foundation.
Other scientists have attempted to invoke a similar reaction in dead bone by infusing tissue-growing proteins directly into the bone. This has proven successful for the small bone transplants used in spinal fusion, but not for larger grafts, because the proteins' effectiveness wear off in just hours. By contrast, the gene therapy method triggers the tissue surrounding the graft to produce the proteins continuously for up to three weeks, long enough for the body to trigger the perpetual bone remodeling response. Stem cells have also seen success in this area, but Schwarz says their appeal has waned because their handling – keeping them alive and ready to use – is far harder than Schwarz's viral paste, which can be stored at room temperature and doesn't interfere with the normal grafting surgery.
"We're very excited about the prospects of this technology," says Schwarz. "Our ultimate goal is to apply this to one of the Holy Grails of orthopedics – cartilage repair. Unlike bone, damaged cartilage, even in a healthy person, can't re-grow or repair itself.
"It's a steeper challenge that would require additional technology such as light-activated gene therapy (LAGT) to site-specifically target the genes to the edge of the damaged cartilage during arthroscopic surgery, but we're looking to use the same idea of triggering the cartilage to remake itself."
Schwarz's work on bone revitalization and LAGT has just been recognized as one of the most important discoveries of the year by the Orthopedic Research Society (ORS) and the American Academy of Orthopedic Surgeons (AAOS). Next week the ORS will honor Schwarz with one of the most prestigious awards in orthopedics, the 2005 Kappa Delta Young Investigator Award. The honor carries with it a grant for $20,000 to help expand his research.
Schwarz is also founder and president of the Rochester-based biotechnology company LAGeT, a company Schwarz spun off from his university research. The firm seeks to commercialize Schwarz's research findings on light-activated gene therapy and has licensed the technology from the university.
Also taking part from the University of Rochester were Hiromu Ito, Mette Koefoed, Prarop Tiyapatanaputi, Kirill Gromov, J. Jeffrey Goater, Jonathan Carmouche, Xinping Zhang, Paul T. Rubery, Regis J. O'Keefe, and Brendan F. Boyce. Other authors include Joseph Rabinowitz and R. Jude Samulski of the University of North Carolina; Takashi Nakamura of Kyoto University in Japan; and Kjeld Soballe of University Hospital of Aarhus in Denmark. The work was funded by the National Institutes of Health, the Orthopedic Research and Education Foundation, and the Musculoskeletal Transplant Foundation.
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