Rebuilding Bone

Hear the term "stem cells" and it's likely you think of human embryos and high-profile controversy. Fact is, stem-cell therapy has been quietly practiced for some 40 years.

Hematopoietic stem cells, the precursors of blood cells, were identified in bone marrow back in the 1960s. Since their discovery, bone-marrow transplants—the destruction of diseased marrow and "re-seeding" with healthy stem cells—have been used in the treatment of leukemia, lymphoma, and other blood disorders.

stem cells in a baby mouse
Christopher Niyibizi

Distribution of mesenchymal stem cells in a baby mouse 7 days after the cells were injected into the mouse's bloodstream. By tagging the cells with green fluorescent protein, Niyibizi shows they find their way into many tissues and organs.

More recently, however, researchers have found stem cells in many other types of mature tissue, including brain, blood vessel, skin, fat, muscle, and liver—and the list continues to grow. Their job, says Christopher Niyibizi, is regeneration and repair. "In the case of tissue damage or injury, they kick on and begin producing healthy new cells."

At first, these "adult" stem cells were not considered very promising for cell-based therapies. They are usually found in very small quantities, which makes them difficult to collect and purify. Unlike embryonic stem cells, they lose their potential if maintained in culture for long periods of time. Most importantly, adult stem cells are far less "plastic" than their better-known counterparts: Where embryonic stem cells have an unrestricted capacity for self-renewal and the potential to differentiate into any type of adult cell, adult stem cells are much more limited in the types of cells they can become.

On the other hand, some say, adult stem cells could bypass the immune-system challenges that are likely with transplant from donor to patient. Nor do adult cells raise the ethical concerns that arise from using human embryonic stem cells.

Spurred in part by those concerns, researchers are looking more closely at adult stem cells. Recent findings suggest that these cells may be more flexible, and therefore more useful, than previously thought.

Bone marrow extract

Christopher Niyibizi

Christopher Niyibizi

Niyibizi, an associate professor of orthopaedics and rehabilitation and biochemistry and molecular biology at the Penn State College of Medicine, is interested in their potential for regenerating bone. He works with mesenchymal stem cells taken from bone marrow and fat, which have the capacity to become bone, fat, or cartilage. "You can direct what they become, by adding specific growth factors," he explains.

A dramatic example of the short-term promise of these cells made headlines late in 2004, when German surgeons used them to repair a large hole in the skull of a 7-year-old girl who had been severely injured in an accident. Extracted from fat taken from the girl's buttocks, the cells were mixed with a small amount of powdered bone from her pelvis—"as a scaffold," Niyibizi says—and placed on the 19-square-inch area of exposed brain. Within several weeks, the German team reported, the hole was filled with new-grown bone.

It was apparently the first time stem cells had been used to grow bone in a human. But because the patient was human, it was impossible to prove that it was actually the stem cells that had done the work. To get a better view, literally, of what these cells do in the body requires recourse to animal models. Niyibizi uses one such model with mice, taking advantage of a commonly used biological marker known as green fluorescent protein.

"We infect the cells with a virus we have engineered to contain the gene that makes this protein," he explains. "Once it enters the body, the infected cell makes the protein, which causes the cell to glow green when observed under a fluorescent microscope. In this way, it can be tracked after it is injected into the body."

In a recent experiment, Niyibizi injected these tinted cells directly into the bone cavities of mice afflicted with brittle bone disease, a congenital skeletal disorder. On the desktop computer screen in his office at the Hershey Medical
Center he flashes before-and-after images. In the first, the dark outline of a mouse's femur is paper-thin, giving the knobby head of the bone a hollowed-out appearance. A second cross-section taken four weeks later shows the same cavity filled with a mix of gray and green tinted shadow. "You can see here that there was successful engraftment," Niyibizi says. "The stem cells were accepted and have started producing bone.

"We still need to do strength and structural tests to see if this bone is actually normal," he stresses. "But if it is, this could be important for treating bone loss in the elderly, especially osteoporosis."

A whole new skeleton

Mesenchymal stem cells also hold promise for repairing joint cartilage damaged by arthritis, Niyibizi says. With numerous clinical trials underway around the world, this kind of localized repair may become available as a standard
treatment option in just a few years, he says. Meanwhile he and other researchers are embarked on a more ambitious quest: to develop stem-cell therapies for the correction of genetic disorders.

Full body image of mouse’s lungs showing flourescent stem cells
Christopher Niyibizi

A composite full-body image shows the fluorescent stems cells concentrated in the mouse's lungs (top) and pelvic girdle.

His current focus is brittle bone disease. Also known as osteogenesis imperfecta, this condition results from a defect in the genes that produce type I collagen, the main component in bone. "The mineral doesn't deposit correctly and as a result the bones that are formed break very easily," Niyibizi explains. In severe cases, infants with the disease may die before or shortly after birth. Even in its milder forms, it can cause frequent fractures and stunted growth. "Surgeons try to fix these bones with metal rods, but there's really no effective treatment," he says.

A cell-therapy approach would involve transplanting mesenchymal cells from a normal-bone donor into the brittle-bone patient, with growth-factor "instructions" to regenerate the entire skeleton. Already researchers have tried this in a handful of severely afflicted children, as an adjunct to standard bone-marrow transplant, and seen significant improvement in bone growth.

Again, to better understand the cell biology, Niyibizi turned to his model, injecting stem cells tagged with green fluorescent protein directly into newborn mice with brittle bone disease. He followed the fate of these cells by imaging their locations at various points during the early weeks of development.

"The cells do go to the bones," he reports, summoning a slide that shows green-glowing shapes dimly recognizable as mammalian ribs and pelvis. "But they also go to the liver, the lung—they go all over the place." In fact, as the next slide makes clear, after 25 days most of the stem cells end up in the lungs. What this confirms, Niyibizi says, is the need to find a way to target cells specifically to bone.

One hope, he suggests, lies in searching carefully through the variety of stem cell sub-types that are present in bone marrow. "Some of these types may have a higher affinity for bone. If so, then maybe we can find out why." Already, Feng Li, a postdoctoral fellow in Niyibizi's lab, has identified a specific population of stem cells that will migrate, engraft, and synthesize normal type I collagen in the bones of brittle-bone mice. "This is quite exciting," Niyibizi says. "Now we are looking closely at the nature of this population."

Newborn mice with brittle bone disease.
Christopher Niyibizi

Newborn mice with brittle bone disease. Stem cells marked with green fluorescent protein are injected through a vein on the side of the head.

"It's also possible," he adds, "that we could actually condition stem cells to want to go to bone." In another of his mouse experiments he tried exactly that: Stem cells were injected into bone, allowed to remain for a period of two weeks—"not long enough to differentiate"—then re-drawn. This process was repeated three times before the cells were finally injected into the mouse's bloodstream. "What we found," he says, "is that these 'trained' cells will go back to where they were. Something changes. They know that bone is where they belong."

Getting past rejection

Even if stem cells can be targeted specifically, transplantation still raises the possibility of rejection. The drastic measures required to limit the body's response to foreign tissue "are in the long run not acceptable,"
Niyibizi says simply. That's why he is also exploring the use of gene therapy for skeletal regeneration.

The process involved is very similar to what he uses to get fluorescence into his test cells, Niyibizi explains. "We take the gene that the mouse is missing and we engineer it into a virus." The mouse-patient is infected, and the
virus ferries the normal gene into the abnormal cells. "Once it goes in, the infected cells start spitting out the proteins you engineered into the virus.

"We have been able to demonstrate that this is do-able," Niyibizi says. "But putting a virus into a person"—even a gutted virus, from which the disease-producing genes have been removed—"is very scary. Right now, many researchers are trying to find other vehicles. Lipids are one possibility. The problem is they don't express the gene as well."

Assuming that the right vehicle can be found, he acknowledges, there remain many other obstacles to successful gene therapy. But if he and his colleagues can one day overcome these hurdles, defective stem cells could be removed,
corrected, and returned to the same patient to produce healthy new bone.

"If it can be done, and done safely," Niyibizi says, "this would be the best approach of all."

Christopher Niyibizi, Ph.D., is associate professor of orthopaedics and rehabilitation and biochemistry and molecular biology at the Penn State College of Medicine in Hershey. He can be reached at cniyibizi@psu.edu.

Niyibizi presented a talk on the work described here at CrossOver 2005, a symposium held at University Park in October 2005 to explore the interface of life sciences and materials at Penn State. His work is supported by grants from the National Institutes of Health and the Children's Brittle Bone Foundation.

Last Updated February 26, 2007