Orthopedic Regeneration With a Combination of Stem Cells, Gene Therapy, and Tissue Engineering

A Duke University research team has combined synthetic scaffolding materials with gene delivery techniques to generate replacement cartilage precisely where it’s needed in the body.

The ingenious strategy utilized by this research project circumvents the need for large quantities of growth factors, which are expensive and difficult to apply after implantation. The Duke team led by Farshid Guilak, director of orthopedic research at Duke University Medical Center, used gene therapy to make stem cells that synthesize their own growth factors.

In brief, Guilak and his collaborators used genetically engineered viruses to transfer genes to stem cells embedded in a synthetic matrix. Upon infection, the stem cells grew and differentiated as needed, but the scaffolding provided the necessary structural cues for the stem cells to move to the proper configuration and form cartilage with the proper shape and biomechanical properties.

Guilak has devoted several years to developing biodegradable synthetic scaffolds that mimic the mechanical properties of cartilage. After testing many different scaffolds, he settled on a 3D woven poly(ε-caprolactone) scaffold, which is completely biodegradable and provides an excellent structural matrix for the synthesis of cartilage.  However, an additional challenge for engineering good cartilage is to coax stem cells embed themselves in the scaffold while differentiating into cartilage-making cells, known as chondrocytes, after the scaffold has been implanted into a living organism.

One widely used strategy is to treat the stem cells with growth factors to induce chrondrocyte formation and cartilage production. Such cartilage can be implanted after it has been grown in the laboratory. However, this approach has some inherent limitations.

Guilak explained that “a major limitation in engineering tissue replacements has been the difficulty in delivering growth factors to the stem cells once they are implanted in the body.” Guilak continued: “There’s a limited amount of growth factor that you can put into the scaffolding, and once it’s released, it’s all gone. We need a method for long-term delivery of growth factors, and that’s where the gene therapy comes in.”

To tackle this perennial problem, Guilak tapped a talented colleague of his, Charles Gersbach, an assistant professor of biomedical engineering, who happens to also be a gene therapy expert.

Gersbach looked at the tissue engineering problem in an entirely new way and suggested that if the mountain will not come to Mohammed (that is to say if the growth factors cannot be given to stem cells after implantation), then Mohammed should grow his own mountain (the stem cells should be genetically engineered to make their own growth factors). Unfortunately, the conventional gene therapy methods are too complex to be commercially feasible. Typically, stem cells are collected, infected with genetically modified viruses that introduces new genes into them, grown to large numbers, and applied to synthetic cartilage scaffolds and implanted into the patient. Sounds like a headache? That’s because it is.

Fortunately, Gersbach had a slick gene therapy trick up his lab coat sleeve: “There are a few challenges with that process, one of them being that there are way too many extra steps,” said Gersbach. “So we turned to a technique I had previously developed that affixes the viruses that deliver the new genes onto a material’s surface.”

A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold.  Photo source:  http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.
A microscopic view using electron microscopy of human stem cells and viral gene carriers adhering to the fibers of a polymer scaffold. Photo source: http://www.pratt.duke.edu/news/gene-therapy-might-grow-replacement-tissue-inside-body.

This new study combines Gersbach’s gene therapy technique—dubbed biomaterial-mediated gene delivery—to induce those human mesenchymal stem cells embedded in Guilak’s synthetic cartilage scaffolding to produce growth factor proteins (in particular a molecule called transforming growth factor β3  or TGF-β3). Based on the results of their experiments, the technique works and that the resulting synthetic, composite cartilage-like material is at least as good biochemically and biomechanically as if the growth factors were introduced in the laboratory.

“We want the new cartilage to form in and around the synthetic scaffold at a rate that can match or exceed the scaffold’s degradation,” said Jonathan Brunger, a graduate student who has spent time in both Guilak’s and Gersbach’s laboratories developing and testing the new technique. “So while the stem cells are making new tissue (in the body), the scaffold can withstand the load of the joint. In the ideal case, one would eventually end up with a viable cartilage tissue substitute replacing the synthetic material.”

This particular study examines cartilage regeneration, but Guilak and Gersbach hope that their technique could be applied to the regeneration of many different kinds of tissues, especially orthopaedic tissues such as tendons, ligaments and bones. Also, because the platform comes ready to use with any stem cell, it presents an important step toward commercialization.

“One of the advantages of our method is getting rid of the growth factor delivery, which is expensive and unstable, and replacing it with scaffolding functionalized with the viral gene carrier,” said Gersbach. “The virus-laden scaffolding could be mass-produced and just sitting in a clinic ready to go. We hope this gets us one step closer to a translatable product.”

Citation: “Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.” Brunger, J.M., Huynh, N.P.T., Guenther, C.M., Perez-Pinera, P., Moutos, F.T., Sanchez-Adams, J., Gersbach C.A., and Guilak F. PNAS Plus, 2014. DOI: 10.1073/pnas.1321744111/-/DCSupplemental

Mesenchymal Stem Cell Treatment Prevent Post-Fracture Arthritis in Mice

Researchers at Duke University Medical Center have found a promising stem cell therapy that might prevent osteoarthritis after joint injury.

Joint injuries tend to raise the odds of contracting a type of arthritis known as post-traumatic arthritis (PTA). There are no available therapies that delay the onset or progression of arthritis after joint-related injuries.

However, a research team at the Duke University Health System has discovered a promising therapeutic approach for PTA that utilizes a particular type of stem cell known as a mesenchymal stem cell (MSC). MSCs have the ability to quell PTA, since these particular stem cells have the ability to suppress inflammation. Additionally, MSCs have the ability to readily differentiate into cartilage-making, bone-making, fat-making, and smooth muscle-making cells, which makes them prim candidates to regenerate damaged joints, since these stem cells have beneficial properties in other regions of the body.

This therapeutic approach was investigated in mice that had suffered the type of bone fractures (intraarticular fractures) that would usually lead to PTA. Injections of 10,000 MSCs into the joints of mice that had suffered intraarticular fractures decreased inflammation and greatly increase bone deposition during healing. Furthermore, the mice that had MSC injections into the joints did not develop PTA while those that received injections of saline into their joints largely did develop PTA. This study could potentially help produce a therapy that would be used after joint injury and before the patient suffers the initial signs of significant osteoarthritis.

Farshid Guilak, Ph.D., director of orthopedic research at Duke and senior author of the study, summarized the results of this study: “The stem cells were able to prevent post-traumatic arthritis.” The study was published on August 10 in the journal Cell Transplantation.

Another track investigated by Guilak’s lab was so-called “superhealer” strains of laboratory mice that tend to heal from bone fractures much faster than other strains. Guilak and his colleagues examined if MSCs from the MRL/MpJ (MRL) “superhealer” mouse strain would increase bone healing when compared to MSCs from C57BL/6 (B6) mice. This was to determine if the exceptional regenerative abilities of MRL mice was due to their MSCs. Unfortunately, Guilak and colleagues discovered that B6 MSCs did the job just as well, and, in fact, a little better than MSCs from MRL mice. Thus, even though they thought that superhealer mice bred for their super-healing properties would probably fare better than typical mice, in turned out that they were wrong.

“We decided to investigate two therapies for the study, said lead author Brian Diekman, Ph.D., who works as a postdoctoral research fellow in the Guilak lab. “We thought that stem cells from so-called superhealer mice would be superior at providing protection, and instead, we found that they were no better than stem cells from typical mice. We thought that maybe it would take stem cells from superhealers to gain an effect as strong as preventing arthritis after a fracture, but we were surprised – and excited – to learn that regular stem cells work just as well.”

Interestingly, certain people appear to fall into the superhealer category. They bounce back quickly and heal well naturally after a fracture, while other people eventually form cases of arthritis at the fractured joint, said Guilak, who is a professor of orthopedic surgery and biomedical engineering at Duke University.

“The ability of the superhealer mice to have superior healing after a fracture may go beyond the properties of their stem cells and be some beneficial factor, like a growth factor, that we don’t know about yet,” Guilak said.

Diekman said the team looked at markers of inflammation and saw that the stem cells affected the inflammatory environment of the joint after fracture.

“The stem cells changed the levels of certain immune factors, called cytokines, and altered the bone healing response,” said Diekman, who is also with the Duke Department of Biomedical Engineering.

According to Guilak, very few studies have purified stem cells to the degree they were purified for this study. The MSCs used in this study were from bone marrow, and they are not directly involved in the production of blood cells, even though they do play an important support role for blood cell-making stem cells in bone marrow (see C. Shi. Immunology 2012 136(2): 133-8 & also see here).

Diekman said that one of the challenges in the field is devising a precise protocol for identifying, isolating and purifying MSCs from bone marrow, since these stem cells tend to be rather rare in bone marrow.
“We found that by placing the stem cells into low-oxygen conditions, they would grow more rapidly in culture so that we could deliver enough of them to make a difference therapeutically,” Diekman said.