Molecular Medicine Israel

Cellular Rehab

Physical therapy and exercise are critical to the success of cell therapies approaching the clinic

Ron Strang lay on his back and bent his left leg. “I could feel the difference right away,” recalls the 31-year-old ex-Marine.

The day before, Strang had undergone an experimental surgery to help repair a deep gouge in his quadriceps. He’d been injured in April 2010 while on foot patrol in Afghanistan’s Helmand Province, when a crude roadside bomb sent shrapnel tearing through his upper thigh. Ten soldiers were wounded in the blast, Strang the most grievously. A year later, even after numerous surgeries and skin grafts, he still couldn’t walk without his knee buckling. So he signed up to receive an experimental regenerative therapy.

In July 2011, Stephen Badylak, a tissue-engineering specialist at the University of Pittsburgh, transplanted a thin sheet of extracellular matrix (ECM) derived from pig bladders into Strang’s leg. The fibrous material was intended not only to provide structural support for the muscle, but also, by releasing signaling proteins, to recruit and coax stem cells in the body to differentiate into new tissue.

After such an invasive surgery, patients typically rest before starting to work the damaged limb. Three years earlier, for example, after Badylak and his colleagues had used the same kind of pig bladder–derived matrix to treat another wounded Marine, Isaias Hernandez, at the US Army Institute of Surgical Research in Houston, the patient stayed in the hospital for five days after his surgery, and it was four weeks before he started physical therapy. That operation was a success: Hernandez recovered a great deal of muscle strength, and CT scans revealed new tissue growth at the implant site.1 But Badylak saw room for improvement.

He didn’t plan to change the surgery or the pig tissue itself. Instead, he focused on the rehabilitation regimen. That’s why Strang was out of bed and down on the floor exercising just 24 hours after his surgery. Starting with his leg outstretched, the soldier slowly bent his knee to slide his heel toward his buttocks. He gnashed his teeth as he did so. But by the next physical therapy session two days later, the pain was already subsiding. And within a few days Strang climbed stairs with minimal discomfort. More than four years on, he can now jog for more than a mile.

Badylak, who last year published a case report documenting Strang’s treatment and recovery,2 credits the success of the protocol to the differentiation of a type of mesenchymal stem cell, known as a perivascular stem cell, into load-bearing muscle tissue. The pig bladder scaffold helps recruit these stem cells from blood-vessel walls to the site of injury, Badylak has shown in mice.3 And he believes it was the physical therapy that directed those cells to become muscle tissue in Strang’s thigh. “The rehabilitation component is absolutely critical,” Badylak says. “It’s not only beneficial; it’s necessary for the therapy to work right.”

Physical forces

Researchers have long recognized the influence of physical forces on molecular and cellular function. Nearly 40 years ago, Judah Folkman, a cancer biologist at Harvard Medical School, and his undergraduate assistant Anne Moscona, now an infectious-disease researcher at Weill Cornell Medicine in New York City, grew cells in petri dishes and found that as cells stretched out and flattened more and more on the plate, their rate of DNA synthesis and cell division increased.4 This revelation led to an explosion of interest in how squeezes, tugs, pushes, and pulls mold the architecture of the cell and, in turn, influence molecular processes within, such as gene expression.

For the most part, however, the field of mechanobiology has been stuck in the laboratory, with few physicians thinking about how physical stresses at the cellular level might affect clinical outcomes, and even fewer physical therapists considering the molecular milieu. As Christopher Evans, director of the Rehabilitation Medicine Research Center at the Mayo Clinic in Rochester, Minnesota, puts it: “The people doing the stem cell work have been largely ignorant of rehabilitation, and the rehabilitation medicine community hasn’t been thinking in terms of cell and molecular biology.”

With stem cell therapies and tissue engineering nearing medical prime time, that’s starting to change. A growing number of scientists, clinicians, and physical therapists are now taking an interdisciplinary approach to rehabilitation, pairing exercise with technologies that regenerate bone, muscle, cartilage, ligaments, nerves, and other tissues. They call it regenerative rehabilitation.

“This is a new future,” says Carmen Perez-Terzic, a cardiovascular disease researcher at the Mayo Clinic. “This is an area that’s going to explode in the next 5 or 10 years.”

Fusion approach

The first public call for stem cell biologists and physical therapists to integrate regenerative medicine and rehabilitation science came in a 2010 editorial by Fabrisia Ambrosio, director of the University of Pittsburgh’s Cellular Rehabilitation Laboratory, and Alan Russell, then director of Pitt’s McGowan Institute for Regenerative Medicine.5“Regenerative rehabilitation is difficult but inevitable,” Ambrosio and Russell wrote, “and now is the time to prepare specific, science-based protocols.”

Ambrosio trained as a physical therapist before earning her PhD with rehabilitation medicine specialist Michael Boninger at Pitt, where she studied how wheelchair design affects strength in people with spinal cord injuries and degenerative conditions such as multiple sclerosis. When Ambrosio started her own research group at Pitt in 2005, she began to investigate how mechanical and electrical stimulation might promote healing following stem cell transplantation.

She transplanted muscle-derived stem cells into bruised hind limbs of mice, then ran the animals on treadmills every weekday for five weeks. The active mice developed more new muscle cells than sedentary controls.6 Ambrosio’s team later demonstrated that applying low-level electrical pulses to muscles injected with stem cells improved strength and reduced fatigue in mice that experienced progressive muscle degeneration characteristic of Duchenne muscular dystrophy.7 “Using very noninvasive, clinically relevant protocols, we can actually dictate the behavior of stem cells,” she says. And that got her thinking: “All of this should lay the groundwork for how we see regenerative medicine therapies being applied in the clinic.”

Starting in 2011, Ambrosio and Boninger launched an annual Symposium on Regenerative Rehabilitation; they held the fourth conference in September at the Mayo Clinic in Minnesota. Last year, the duo also started the International Consortium for Regenerative Rehabilitation, a coalition of eight participating institutions from the U.S., Japan, and Italy that is now developing a strategic agenda for the field. And a few months ago, they secured funding to create the Alliance for Regenerative Rehabilitation Research & Training, which includes four US universities and hospitals (Pitt, Stanford University, Mayo, and the University of California, San Francisco) and will support webinars, minisabbaticals, seed grants, and more.

“This is about getting more people doing this work, understanding this work, and translating this field,” says Boninger, who is leading the alliance together with Stanford stem-cell biologist Thomas Rando. Just adding exercise to a stem cell therapy is “easy,” Boninger notes. “Doing the basic science to evaluate that is a little more challenging.”

The science may still be in its infancy, but Ambrosio says her efforts in community building are beginning to pay off. “I can see such a difference in the way people receive some of these ideas of regenerative rehab,” she says. “It was really kind of novel as recently as 2010, whereas now it’s actually part of our vernacular.”

(Re)Generating interest

Rehabilitation regimens are now being integrated into the preclinical development of regenerative treatments for heart disease, bone fractures, and even brain injuries. In Japan, for example, researchers at Hiroshima University have shown that running directs neural stem cells to properly differentiate when transplanted into mice with experimentally induced brain damage.8 “Combining cell therapy and rehabilitation is needed to correct the neural network and achieve a functional recovery,” says study author Takeshi Imura, who presented the research at Japan’s first-ever Workshop on Regenerative Rehabilitation in Kyoto last March. And earlier this year, muscle biologist Marni Boppart and her colleagues at the University of Illinois at Urbana-Champaign reported that stem cells only enhance muscle repair and growth in mice when coupled with weight-training exercise.9

In addition to exercising recipients of cell therapies, scientists are also looking to give the cells themselves a workout, by stretching stem cells in a dish ahead of transplantation. “In effect, we’re exercising the stem cells without exercising the animal,” says Boppart. In unpublished work, Boppart’s team found that old mice injected with muscle stem cells taken from young mice and stretched before injection exhibited improved blood flow, stronger muscles, and more new neurons in the brain’s hippocampus, thanks to the release of growth, neurotrophic, and immunomodulatory factors brought on by the mechanical stimulus. Stem cells not given the laboratory workout provided no such benefits.

At the Mayo Clinic, Perez-Terzic is also applying physical pressure in vitro to improve the differentiation of stem cells. Her goal is to develop new regenerative treatments for heart disease, and she is hoping to find more-efficient ways of coaxing embryonic stem cells to become heart muscle cells for transplantation. The results are preliminary, Perez-Terzic says, but so far it looks like “if you put some pressure into the system, the differentiation is much better.”

Boppart is hopeful that translating such therapies to the clinic will help patients who are unable to exercise, such as some elderly individuals or those with extreme muscle weakness. “This type of alternative stem cell therapy may provide the boost in strength necessary for someone to transition from disability to regain of function,” she says.

Richard Shields, an applied physiologist at the University of Iowa’s Carver College of Medicine, has another solution, one that doesn’t require any sort of cellular calisthenics in the laboratory. He has invented a device that can deliver different kinds of mechanical loads directly to the lower leg, even for patients confined to a wheelchair. A compression system covers the knee, while the foot rests on a vibrating platform. A doctor or physical therapist can then deliver therapeutic loads in a safe and quantifiable manner. (See illustration below.)

After testing the device on eight people with complete paralysis,10 Shields and his colleagues wondered whether delivering a controlled dose of vibration would improve bone architecture in spinal cord injury patients, many of whom eventually develop severe osteoporosis. After 12 months of regular vibration therapy, however, bone health continued to decline in all six study participants.11 “This means that people with long-term paralysis are very resistant to change [in bone density] or that the dose was not high enough,” says Shields, who is now working to refine the training regimen for better results.

Once he and his colleagues work out the kinks, Shields says he hopes that the setup will be useful to more patients than just those who are incapable of exercise. The limb-loading system offers greater control of the degree and target of stimulation than that afforded by running or weight lifting, he says—precision that could have utility for all manner of regenerative cellular treatments. “How you dose these mechanical loads is not just all or none,” he says. “The stresses have to be applied in opportune doses.”…………


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