Osteoporosis causes a decrease in bone mass and density. The result is bones that are porous and fragile, and therefore prone to fracture. About 25 million American men and women, mostly post-menopausal women, have osteoporosis, and they suffer about 2 million fractures a year.
Today, doctors can prescribe medications and recommend lifestyle changes to slow bone loss, but there is no treatment to add significant bone mass or strength. However, tissue engineering and other advances in medical research may offer new hope within the next decade for patients with debilitating musculoskeletal diseases such as osteoporosis and osteoarthritis, as well as traumatic injuries to bone and soft tissues in joints.
Researchers in the Georgia Tech/Emory Center for the Engineering of Living Tissues (GTEC) are seeking solutions to regenerate functional bone, cartilage and fibrocartilage.
"At GTEC, we are doing fundamental studies and developing technologies that will enable companies to develop products for clinical use," says Robert Guldberg, a GTEC research director and assistant professor in the Georgia Tech School of Mechanical Engineering. "In orthopedic tissue engineering, we hope to see these technologies lead to improved therapies that will impact patient care within five to 10 years."
One promising technology is a delivery mechanism for a localized gene therapy using LMP-1, a gene that stimulates surrounding cells to make bone. A research team led by Scott Boden, an Emory University professor of orthopedic surgery, discovered LMP-1 in 1997 and soon learned its potential. Subsequently, a Memphis, Tenn., company, Medtronic Sofamor Danek, purchased the rights from Emory to use LMP-1 to improve the success rate of spine fusion surgeries.
Boden's team is now conducting fundamental research on how LMP-1 functions, and is meanwhile collaborating with Guldberg on preliminary studies in the development of a gene therapy delivery mechanism. Boden is exploring an ex vivo approach in which the patient's own blood cells can be mixed with a virus containing the LMP-1 gene. Surgeons would either directly inject the LMP-1 cells into a skeletally deficient site or deliver the genetically engineered cells within a porous biomaterial scaffold.
"It's too early to tell what sort of increase in bone density could be expected from LMP-1 gene therapy," Boden says. "But even a 10 percent increase would be very significant clinically."
Laboratory studies are under way. The results so far indicate a moderate, but consistent increase in trabecular bone (the porous region of bone, which is affected by osteoporosis) formation in non-human limbs injected with cells genetically engineered to overexpress LMP-1, Guldberg reports.
Guldberg's part in the project is to analyze tissue-engineered bone with high-resolution micro-computed tomography (micro-CT). "We can visualize changes in the 3D microarchitecture of the bone and quantify not only the amount of new bone formation, but how it is oriented and organized," Guldberg explains.
photo by Gary Meek
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Assistant Professor Marc Levenston and graduate student Christopher Hunter have developed an in vitro model system to study integration between tissue-engineered cartilage and native tissue. Here, Hunter holds an engineered construct containing cells from cow knee cartilage. (300-dpi JPEG version - 404k)
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Preliminary studies should be completed within a year. After that, Guldberg plans to conduct mechanical tests on bone produced by the LMP-1 gene. Meanwhile, Boden is studying immune response issues stemming from the viral delivery of LMP-1. He wants to use the lowest, and therefore safest, dose of the virus that will effectively deliver the gene without triggering antibodies that will attack the virus, he explains.
Eventually, LMP-1 gene therapy could treat unhealed fractures, age-related spine degeneration, and regional or systemic osteoporosis, Boden adds.
Another gene delivery technology under development at GTEC focuses on Runx2/Cbfa1, a bone cell master gene that stimulates bone production. In the Georgia Tech School of Mechanical Engineering, Assistant Professor Andres Garcia and graduate student Benjamin Byers are engineering various types of cells -- including cells that do not typically make bone -- to express Cbfa1 and make bone in culture.
"We're focusing on stimulating cells that usually do not make bone tissue to express this master gene in order to turn on other genes responsible for bone formation and eventually enhance bone production in culture," Garcia explains.
Eventually, researchers will combine Runx2/Cbfa1-producing cells with biological and synthetic scaffolds to creates mineralized templates. Surgeons could then use these implantable constructs to treat bone defects in patients.
For now, Garcia and Byers are experimenting with delivering this gene to clinically relevant target cells in the laboratory. So far, they have found that the extent of mineralization depends on the target cell. Experiments with bone marrow and other bone-forming cells have produced excellent enhancement of bone tissue production in culture, Garcia says. The next phase of the research involves the integration of these genetically engineered cells with scaffolds of adequate biological and mechanical properties.
Optimal biological scaffolds are a key component to the success of tissue-engineered constructs, or living implants. "And a major barrier is producing scaffolds with sufficient mechanical properties to withstand in vivo forces within a patient," Guldberg explains.
photo by Gary Meek
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Graduate student Kimberly Huynh is working with Georgia Tech Assistant Professor Robert Guldberg to analyze tissue-engineered bone with high-resolution micro-computed tomography (micro-CT). Here, she holds a tray of specimen containers for the micro-CT imaging system. On the computer screen behind her is a 3D image of bone grown in an incubator in Guldberg's lab. Researchers can visualize changes in the 3D microarchitecture of the bone and quantify, not only the amount of new bone formation, but how it is oriented and organized.
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In a new manufacturing approach developed and patented by GTEC partner company BioAmide, graduate student Angela Lin is collaborating with corporate scientist Tom Barrows to coat a polymer onto small wires and then fuse them together. A pore-forming agent in the polymer is triggered when researchers submerse the polymer in hot oil. The high temperature decomposes the pore-forming agent in something like a foaming process. The result is interconnected porosity that creates a scaffold with a lot of surface area where cells can attach and grow. Researchers remove the fine stainless steel wire in the scaffold and create even larger pores that run throughout the scaffold.
"Longitudinal pores in the scaffold are the highways, and micro-pores are the parking spots for cells once they arrive," Guldberg explains. "The highways also provide nutrient pathways to cells." The intent is to provide the cells with rapid access to the scaffold interior so the cells can quickly integrate with vascular tissue.
Guldberg and Lin are quantifying the architecture of these scaffolds and comparing their mechanical properties to their porosity. "The scaffolds have stiffness and strength comparable to trabecular bone. The next step is to evaluate the function of cells seeded onto the scaffolds," Guldberg says. "We have used confocal microscopy to verify cell viability within the scaffolds at one week and have just completed a study quantifying the formation of mineralized bone within the scaffolds after four weeks. One observation we have made is that more bone forms on the outside of the scaffolds than on the inside because of nutrient diffusion limitations."
To overcome this limitation, Guldberg, graduate student Blaise Porter and post-doctoral fellow Sarah Cartmell are developing a 3D tissue culture system that pushes nutrients through scaffolds at controlled rates. They are testing the effects of perfusion rate on gene expression and viability of cells at the center of the scaffolds. Guldberg has filed a provisional patent on the system, which in addition to perfusing constructs, has the ability to simultaneously apply cyclic forces intended to simulate the mechanical environment in the patient. This system will be used to screen cell and scaffold technologies and provide a test bed for improved construct designs.
Meanwhile, Marc Levenston, an assistant professor of mechanical engineering at Georgia Tech, and graduate student Christopher Hunter are facing the challenge of integrating tissue-engineered cartilage with surrounding native tissues. Damaged cartilage does not heal well, in part, because it has no blood supply.
photo by Gary Meek
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Georgia Tech Assistant Professor Robert Guldberg, right, and graduate student Blaise Porter discuss their work on a 3D bone culture system that pushes nutrients through biological scaffolds at controlled rates. They are testing the effects of perfusion rate on gene expression and viability of cells at the center of the scaffolds. Guldberg has filed a provisional patent on the system.
(300-dpi JPEG version - 304k)
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Repaired cartilage is often mechanically inferior to normal tissue and may become painful or arthritic. Tissue-engineered cartilage, however, could provide hope for patients who have cartilage damaged because of overuse, sports injuries, rheumatoid arthritis or genetic factors.
In repairing damaged cartilage, tissue engineers face the challenge of finding ways to seamlessly integrate engineered cartilage with remaining normal tissue. Levenston and Hunter have developed an in vitro model system to study integration between tissue-engineered cartilage and native tissue. They cut a piece of cow knee cartilage into a doughnut shape. In the center, researchers place a piece of tissue-engineered cartilage created from the same animal's cells.
Together, the components create a hybrid construct that Levenston and Hunter place in an incubator and then examine for signs of tissue integration. They also apply cyclic deformation to the construct, loosely simulating the mechanical environment associated with walking.
"In a general sense, we see that living tissue affects cells in the engineered tissue regardless of mechanical loading," Levenston says. "The engineered tissue is getting signals from cells in the living tissue, and the cells are proliferating at a slower pace. Normally, cells divide a lot. So the trend we're seeing is that the cells divide slower, but produce more tissue."
The researchers are comparing different types of constructs and measuring how much force it takes to push the tissue-engineered cartilage out. "This will test the interface and tell us how much integration we are getting," Levenston explains. "Then we can screen the interfaces in advance of animal model studies." It will likely be five to 10 years before tissue-engineered cartilage therapies become widely available for human clinical use.
For more information, contact Robert Guldberg, School of Mechanical Engineering, Georgia Tech, Atlanta, GA 30332-0405. (Telephone: 404-894-6589)
(E-mail: robert.guldberg@me.gatech.edu)
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Last updated: Nov. 12, 2001
