Cellular Rehab

The rehabilitation component is absolutely critical. It’s not only beneficial; it’s necessary for the therapy to work right.—Stephen Badylak,
University of Pittsburgh

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

NEW NEURONS: In the dentate gyrus of the hippocampus, mice injected with stretched muscle stem cells show an approximately threefold greater increase in immature neurons (black, left) than mice injected with untreated stem cells (right), likely as a result of growth and neurotrophic factors released into the bloodstream by the stretched cells.JENNIFER MERRITTThe 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.

I can see such a difference in the way people receive some of these ideas of regenerative rehab. It was really kind of novel as recently as 2010, whereas now it’s actu­ally part of our vernacular.—Fabrisia Ambrosio,
University of Pittsburgh

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.”

Stretch goals

CELLULAR WORKOUT: Regenerative rehabilitation promises to enhance the potential of cell- and gene-based techniques by incorporating principles of physical therapy.
See full infographic here: WEB | PDF© KIMBERLY BATTISTAMartin Childers, who is collaborating with San Francisco–based Audentes Therapeutics, says he hopes it could also improve outcomes following a gene therapy in children with a rare and fatal muscle weakness disorder called X-linked myotubular myopathy.

The disorder is caused by mutations in the MTM1 gene that encodes an enzyme needed for the development and maintenance of muscle cells. Children with the condition suffer from extreme muscle weakness, generally lacking the strength needed to move air in and out of their lungs without mechanical assistance. Audentes’s therapy will deliver a good copy of the MTM1 gene into the blood and hopefully help affected individuals respire without assistance. But after treatment, “you can’t just turn the ventilator off,” Childers says. “There’s going to have to be some rehabilitation therapies.”

Specifically, Childers plans to couple the gene therapy with breathing training. In addition to helping patients wean themselves off the ventilator, pulmonary exercise might enhance the expression of the introduced gene, he says. For now, this is only a hunch. But Audentes is preparing for the launch of a Phase 1 trial next year, and Childers is studying human tissue samples in the lab to answer this question.

Other gene therapy trials already incorporate physical therapy into their recovery protocols. At Nationwide Children’s Hospital in Columbus, Ohio, for example, Jerry Mendell and his colleagues are testing a gene-correction treatment on six- to nine-month-old babies with a severe form of spinal muscular atrophy, a genetic disease that involves the degeneration of motor neurons. The infants are born with tight joints; their legs are often fixed in a splayed, frog-like position. Mendell’s team strongly encourages parents to massage their children’s stiffened limbs on a daily basis after the gene transfer—a necessity, Mendell says, for the gene-corrected motor neurons to interface properly with the weakened muscles. Without it, “you’re not going to be able to improve function,” he says.

Mendell is also incorporating bicycle training into a small gene-therapy trial for children with Duchenne muscular dystrophy. Although mutations in the gene that codes for a muscle-associated protein called dystrophin are responsible for this disorder, Mendell is not delivering a working copy of that particular gene back into patients. Instead, he is using a gene therapy product he developed in collaboration with Cleveland-based Milo Biotechnology that includes the gene for follistatin, a protein that helps release the brakes on muscle growth and could thus prove beneficial for a variety of muscle diseases.

In an earlier trial that tested the same gene therapy on six adults with Becker muscular dystrophy, a milder condition also caused by mutations in the gene for dystrophin, Mendell noticed that the participant who had the most active lifestyle—on account of his job at a garden center—exhibited the most dramatic improvement in how far he could walk.¹² “He was one of the highest responders to the follistatin gene therapy,” Mendell says. Hoping to re-create the success, Mendell is now having the kids in his ongoing Duchenne muscular dystrophy trial ride stationary bikes for 15-minute sessions three times per week.

One umbrella

WHEELCHAIR CALISTHENICS: The University of Iowa’s Richard Shields developed a system to assess the effects of mechanical loading, vibration, and electrical stimulation on bone, muscle, nerve, and cell signaling on the lower leg. The goal is to apply optimal combinations of the three forces to enhance regenerative medicine treatments.COURTESY OF RICHARD SHIELDS“In the end,” says Boninger, regenerative rehabilitation “is about improving function in patients. So, being able to look someone in the eye and say, ‘This is why we’re doing this exercise program, and this will add to your recovery and function when you’re done’ is where I really want to get to.”

To help make that happen, several academic institutions have been shuffling departmental structures to bring stem cell scientists and rehabilitation doctors under the same administrative umbrella. These include the University of Pittsburgh and the Mayo Clinic, both leaders in the nascent hybrid discipline, though the first place to do so was Columbia University Medical Center in New York City, where the hospital renamed its rehab division the Department of Rehabilitation and Regenerative Medicine in 2010.

Five years on, however, efforts to bridge the two disciplines “remain to some degree aspirational,” admits Joel Stein, a rehabilitation specialist who chairs the Columbia department. Regenerative rehabilitation “is becoming more popular within the field as a vision for the future,” he says. “But has it translated into good, hard science that’s led to definitive new therapies? No, not yet, and it might take a while.”

“We need to be dedicating more efforts to thinking about dosing, intensity, and protocols,” Ambrosio agrees. “That means we have a lot of work ahead for us.”

In the meantime, proponents of regenerative rehabilitation continue to look to success stories like Strang’s for inspiration. At one point, Strang was unsure that he’d ever be able to walk normally again. Now a police officer at the Veterans Affairs hospital in Pittsburgh, Strang is on his feet constantly, moving about easily and pain-free. Just last month, in fact, he married his longtime girlfriend at a church outside Pittsburgh—and he walked down the aisle with no problems. 

Elie Dolgin is a news editor at STAT in Boston.

References

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2. N.E. Gentile et al., “Targeted rehabilitation after extracellular matrix scaffold transplantation for the treatment of volumetric muscle loss,” Am J Phys Med Rehabil, 93:S79-S87, 2014.
3. B.M. Sicari et al., “An acellular biologic scaffold promotes skeletal muscle formation in mice and humans with volumetric muscle loss,” Sci Transl Med, 6:234ra58, 2014.
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6. F. Ambrosio et al., “The synergistic effect of treadmill running on stem-cell transplantation to heal injured skeletal muscle,” Tissue Eng Part A, 16:839-49, 2010.
7. G. Distefano et al., “Neuromuscular electrical stimulation as a method to maximize the beneficial effects of muscle stem cells transplanted into dystrophic skeletal muscle,” PLOS ONE, 8:e54922, 2013.
8. T. Imura et al., “Interactive effects of cell therapy and rehabilitation realize the full potential of neurogenesis in brain injury model,” Neurosci Lett, 555:73-78, 2013.
9. K. Zou et al., “Mesenchymal stem cells augment the adaptive response to eccentric exercise,” Med Sci Sports Exerc, 47:315-25, 2015.
10. C.L. McHenry et al., “Potential regenerative rehabilitation technology: Implications of mechanical stimuli to tissue health,” BMC Res Notes, 7:334, 2014.
11. S. Dudley-Javoroski et al., “Bone architecture adaptations after spinal cord injury: impact of long-term vibration of a constrained lower limb,” Osteoporos Int, doi:10.1007/s00198-015-3326-4, 2015.
12. J.R. Mendell et al., “A phase 1/2a follistatin gene therapy trial for Becker muscular dystrophy,” Mol Ther, 23:192-201, 2015.