Enlargement of non-fibrous patches discovered in meniscus fibrocartilage may be linked to disease processes
Knee injuries are among the top five reasons people visit an orthopedic surgeon for treatments, which include 719,000 total knee replacements performed annually in the United States. Now, new research reveals underlying biomechanics that may be involved in meniscus fibrocartilage function as well as dysfunction and could guide novel treatments for some of the most debilitating and costly orthopedic problems in the U.S., including meniscus tears and age-related joint degeneration.
The knee meniscus is a piece of cartilage between the leg bones that cushions and stabilizes the joint, protecting the bones from wear and tear. In studies of samples from cows and humans, bioengineers funded by the National Institute of Biomedical Imaging and Bioengineering identified small patches of non-fibrous “islands” known as microdomains in the knee fibrocartilage that makes up the meniscus. Samples from older animals and humans consistently had larger microdomains. The discovery opens the way for a more detailed examination of fibrocartilage microstructure and how changes at the cellular and molecular levels contribute to orthopedic health and disease.
“This is impressive and important work,” says Rosemarie Hunziker, Ph.D., Director of the NIBIB Program in Tissue Engineering and Regenerative Medicine, “both in the fundamental discovery and the fact that they rapidly built a model to study it.” The discovery by researchers at the University of Delaware and the University of Pennsylvania and subsequent development of a bioengineered model of the tissue are reported in the January 4 issue of Nature Materials.1
Non-fibrous patches of microdomains grow with age and mechanical stress
The group examined cow knee tissue samples spanning from fetal development through adulthood. The microdomains, interspersed within the fibrocartilage of the meniscus, were present at the fetal stage in cow samples and grew larger in size, but not number, as the age of the animal increased. Studies of human samples revealed the same correlation between advanced age and increased size of microdomains.
In addition to the increase in microdomains with age, human donors with higher body mass index also had increases in the size of the microdomains. “Taken together these observations suggested that increased microdomain size might be an adaptive response to handle increased mechanical load accumulating over time or increased pressure on the meniscus in heavier individuals," explains Dawn Elliott, Ph.D. chair of the University of Delaware’s Department of Biomedical Engineering and co-senior author of the study. "An important question we immediately had was whether such an adaptation could be pushed to the point where it could also lead to impaired function of knee fibrocartilage.”
Investigating structure and function at the micro-level
Studies of cells in the fiber-rich regions of the tissue and in the microdomains revealed the type of information the team looks for: a critical mechano-responsive difference between the flexible fibers and the inflexible microdomains. “There was a distinct change in calcium signaling in response to stretching and increased mechanical loading in cells within the fibrous part of the tissue,” explains Robert L. Mauck, Ph.D., Department of Orthopaedic Surgery and Bioengineering at the University of Pennsylvania and co-senior author of the study, “but in cells in the non-fibrous microdomains, calcium levels remained static – these cells were not responsive to stretching or increased mechanical loading.”
Mauck explains that calcium movement in and out of the cells in response to tissue stretching is a signal for the cells to activate genes and processes that cause regeneration and remodeling of tissue. Conversely, the lack of such a response in the microdomains added evidence to the theory that microdomains could contribute to impaired function, as a knee meniscus with larger microdomains might fall short in its attempt to repair itself in response to stress.
Building tissue-engineered constructs for in-depth study and experimentation
For more detailed studies of the characteristics observed in meniscus samples, the research team engineered knee fibrocartilage in the laboratory. They called their laboratory-made knee fibrocartilage heterogeneous tissue engineered constructs or (hetTECs). “Heterogeneous” refers to the fact that the laboratory-made tissue contained the two elements observed in the native tissue: the fibrous domains interspersed with islands of the non-fibrous microdomains.
Mauck stresses how the different expertise and perspectives of the research team made constructing hetTECs possible. “Dr. Elliott’s work in tissue mechanics, essentially drilling down to reveal the micro-level structure of the tissue provides me with the details I need as a biomaterials and tissue engineering scientist to build an accurate tissue engineered model from the ground up.”
Extensive testing of the hetTECs demonstrated that they faithfully mimicked the characteristics of the native tissue, including the calcium response to stress in the fibrous areas and the lack of a response by cells in the microdomains. The team is enthusiastic about using the bioengineered hetTECs to answer questions about how the microstructural details relate to the function of knee fibrocartilage.
Mauck explains the utility of the hetTECs. “We now have an in vitro platform to study the mechanobiology of knee tissue in a controlled fashion in the lab. For example, we may find that microdomains are necessary for the proper development of fibrocartilage in the embryo but then become detrimental in aging animals. We are now performing tests that will help us unravel the mechanical details of how the structure relates to function”
But getting back to sore knees, while they are cautious about blaming microdomains for fibrocartilage degeneration without more extensive testing, the team acknowledges the powerful potential of their hetTEC constructs for identifying disease processes at the micro-level and then testing potential treatments that reverse or halt the disease process. Concludes Mauck, “ultimately, we are working to use this type of engineered model to develop new treatments for ailing knees as well as other types of connective tissues.”
The research was supported by the National Institutes of Health through grant EB02425 from the National Institute of Biomedical Imaging and Bioengineering and grant AR050950 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, which supports the Penn Center of Musculoskeletal Disorders.
1. Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage. Han WM, Heo SJ, Driscoll TP, Delucca JF, McLeod CM, Smith LJ, Duncan RL, Mauck RL, Elliott DM. Nat Mater. 2016 Jan 4