Lack Of Specific Collagen Type Leads To Osteoarthritis

The results of their experiments with mice provide new insights that could lead to potential treatments for a disease that afflicts more than 40 million Americans, said the researchers.

The researchers found that mice lacking the gene that controls the production of type VI collagen developed osteoarthritis at a rate more than five times greater than mice with a functioning gene. Collagen is a ubiquitous protein found throughout the body in connective tissue, muscle, cartilage and bone. To date, 27 different types have been identified.

To examine structures within the cartilage of mouse joints, Leonidas Alexopoulos, Ph.D., developed a novel "micro-vacuuming" technique. With this device, Alexopoulos extracted key structures within the cartilage of mouse hip joints, which are the size of the ball in a ball-point pen, and analyzed how they responded to the stresses of everyday life.

Alexopoulos presented the results of the Duke study Feb. 20, 2005, at the 51th annual scientific meeting of the Orthopedic Research Society in Washington, D.C. Alexopoulos, now a post-doctoral fellow at the Massachusetts Institute of Technology, conducted the research in the laboratory of Farshid Guilak, Ph.D., director of orthopedic research and senior member of the Duke team. The study was funded by the National Institutes of Health.

The researchers focused their attention on the narrow region of tissue that surrounds the cartilage cells on the surface of joints and is known as the pericellular matrix (PCM). Together with cartilage cells known as chondrocytes, collagen types II, VI and IX, and other proteins, the PCM forms a structure called a chondron, which is believed to provide a "buffer" zone between the cells and the remainder of the cartilage tissue.

"The interesting thing is that type VI collagen occurs nowhere else in the cartilage but the PCM, and no one really understood why," Alexopoulos explained. "When we analyzed the PCM of mice unable to produce type VI collagen, we found that the chondrons in these mice were much softer and the joints did not respond well to mechanical pressures. The joint looked as if it osteoarthritis had developed.

"It appears now that the type VI collagen acts like a scaffold that provides structure and stiffness to the PCM," Alexopoulos continued. "With this model for osteoarthritis, we have a better understanding of how changes in the mechanical forces on the cells may lead to degeneration of the cartilage."

For their experiments, the team compared how chondrons changed over time in three different groups of mice: one group had functioning type VI collagen genes, while the two other groups were strains of "knockout" mice developed by Paolo Bonaldo, University of Padova, Italy. One group of mice had both parents with the type VI collagen gene knocked out, while the other group had only one parent without the gene. After six months, the researchers removed chondrons to determine how they responded.

"We found significant osteoarthritic and developmental differences among the three groups," Alexopoulos said. Specifically, 73 percent of the mice with two knock-out parents showed evidence of mild to severe osteoarthritis. This compared to 40 percent for mice with one knock-out parent and 13 percent for the control mice.

"These findings represent an important advance in our understanding of osteoarthritis," Guilak said. "The study provides direct evidence of the role of type VI collagen in the biomechanical properties of the PCM. While the mechanism behind the accelerated development of osteoarthritis is not yet clear, it suggests that the lack of type VI collagen negatively impacts the ability of the cartilage to respond properly to the mechanical stresses and pressures on the joint."

The experiments would not have been possible without the custom-built "microaspirator," which could extract individual, intact chondrons. Other methods of isolating chondrons, which either involve dissolving surrounding tissues with harsh enzymes or grinding the cartilage in pieces, typically yield damaged chondrons, Alexopoulos said.

"Using a tiny syringe, I was able to go across the surface of the cartilage and vacuum up the chondrons without damaging them," Alexopoulos said. "The chondrons literally popped out of the cartilage and into the syringe. From that point, it was easy to analyze their structure."

It is estimated that more than 70 percent of Americans over the age of 65 show some signs of osteoarthritis, which is characterized by the slow degeneration of the buffering layer of cartilage within joints. The other major form of arthritis, rheumatoid arthritis, occurs when the body's immune system attacks the linings of joints.

Guilak currently leads of group of clinicians and investigators from Duke and the Durham VA Medical Center who are carrying out a broad range of basic and clinical research into better understanding and treating osteoarthritis.

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The effort is funded by grants from the National Institute on Aging (NIA) and the National Institute of Arthritis and Musculoskeletal and Skin Disease (NIAMS).