The 2022 Kappa Delta Anne Doner Vaughn Award was presented to Lawrence J. Bonassar, PhD, for his research on the microscale mechanics and composition of articular cartilage and their relevance to musculoskeletal disease. The Kappa Delta Awards recognize research in musculoskeletal disease or injury with great potential to advance patient care.
These discoveries by Dr. Bonassar and his colleagues will not only aid in preventing disease and identifying therapeutic windows for treatment, but also play a crucial role in determining the key components and structures in diseased tissues to be targeted for tissue preservation, repair, or regeneration.
For many types of arthritis, such as osteoarthritis (OA), damage begins at the articular cartilage, a very thin surface that covers the ends of bones. This process occurs when inflammatory mediators induce the release of enzymes, resulting in degradation of the extracellular collagen and aggrecan networks—two of the most important constituents responsible for the mechanical properties of cartilage. Aggrecan is the major proteoglycan in articular cartilage, providing the hydrated gel structure that allows cartilage to bear loads and dissipate energy.
“A striking feature of connective tissues, such as articular cartilage, is their heterogeneity of composition and structure at multiple length scales, which is a concept used in physics to define length or distance determined with the precision of one order of magnitude,” said Dr. Bonassar, the Daljit S. and Elaine Sarkaria professor at the Meinig School of Biomedical Engineering and Sibley School of Mechanical and Aerospace Engineering at Cornell University in New York.
The critical role of the articular surface
More than 15 years ago, Dr. Bonassar partnered with Itai Cohen, PhD, a professor in Cornell University’s Department of Physics who studies the behaviors of soft materials, including cartilage. Dr. Cohen had already developed microrheology techniques to examine the micromechanics of soft tissues. Microrheology is the study of mechanics (e.g., microviscosity) of complex materials at small length scales.
Drs. Bonassar and Cohen created a testing device that was small enough to fit on a microscope and could capture images at 10 to 100 milliseconds to observe how the tissue deforms on the length scale/diameter of a human hair. They discovered that the top 100 microns (a metric unit of measurement where 1 micron is equivalent to 0.001 mm) of articular cartilage has extremely different mechanical behavior than the rest of the tissue. In fact, it was 10 to 100 times more compliant (i.e., less stiff or more likely to be deformed).
“We learned that 90 percent of the energy dissipation occurred in the top 100 microns, so a very small region was doing almost all the work protecting the rest of the tissue,” said Dr. Bonassar. “Having established the important mechanical role of the most superficial region of articular cartilage, we sought to understand how the composition of the tissue (water, collagen, proteoglycan) and mechanics are connected.”
To accomplish this goal, they coupled this state-of-the-art mechanical analysis with compositional analysis using Fourier Transform Infrared (FTIR) microscopy and Raman microspectroscopy to understand how the mechanics and composition of the tissue connect. They discovered that the surface region, which acts as a shock absorber, contains a low concentration of collagen and proteoglycans. They furthered this analysis by altering the composition of the cartilage with an enzyme used to selectively remove proteoglycans from the extracellular matrix (ECM), leaving the collagen network largely intact. ECM contains structural support cells that regulate cellular growth.
“When you remove proteoglycans from the tissue, you begin a phase transition from a mechanically stiff to a mechanically floppy network,” said Dr. Bonassar. “This local subtle damage to the articular surface is what we believe represents the initial stages of damage in arthritis that starts to cascade into the progression of the disease. Essentially, when the tissue is healthy, it’s stiff and robust enough to carry load, but the top 100 microns layer of the articular cartilage is close to a tipping point. With just a little damage, it can cause a huge decrease in its ability to carry load, and once that surface damage happens and function is lost, it’s hard to get back.”
Function of cartilage implants
The next phase of research was to study implants that are used to replace cartilage utilizing the same techniques to understand how the composition impacts the mechanics of those products. Their work provided a benchmark to understand whether the function of those replacement tissues resembled the function of the native cartilage.
Cartilage implants are developed when cells are seeded on a scaffold or a sponge, allowing the cells to grow within the matrix of the scaffold. The team analyzed two cartilage replacement products using the new mechanical evaluation tool. It revealed some behavioral aspects that had never been seen before.
“One of the real challenges for companies that make these products is knowing how long to culture them and how much ECM needs to be deposited before the implants can function mechanically,” said Dr. Bonassar. “The maturation threshold is dependent on the type of scaffold and the patient’s cells. However, we were able to provide a clear roadmap for these companies to define how much matrix needs to be made by the cells. In some cases, once the scaffold is 20 percent full, it’s stiff enough to make the construct fully functional.”
Cell behavior in damaged tissue
Their research continued to focus on the biologic implications or cellular responses of tissues with mechanical injury. Drs. Bonassar and Cohen partnered with cell biologists and veterinarians, Lisa A. Fortier, DVM, PhD, James Law professor of surgery, and Michelle L. Delco, DVM, PhD, assistant research professor in the Department of Clinical Sciences, who both work at Cornell’s College of Veterinary Medicine. The partnership allowed the team to integrate a clinical perspective, as many drivers of arthritis are similar in humans and animals.
They were interested in using the tools previously developed to understand pure mechanics and determine how the mechanics drove cell behavior, as well as how the local mechanical environment instructed the chondrocytes (the cells that populate cartilage) to help or harm the tissue’s health.
Injuries, such as tears to the meniscus or anterior cruciate ligament (ACL) or a severe ankle sprain, can greatly increase a person’s chance of developing arthritis in those joints, in part due to cellular damage that occurs from the impact injury.
“We built a device that fits on a fast-imaging microscope that allowed us to deliver controlled amounts of energy to pieces of cartilage––the same impact a person might experience in an ACL or meniscus tear or a car accident,” said Dr. Bonassar. “By capturing images at milliseconds, we observed, in real time, how the tissue deforms and what happens to the cells in the regions that experienced different amounts of deformation. We discovered that the damage to the cells is directly related to how much strain the tissue experiences and is concentrated in the area of impact. For example, in a matter of minutes following an ACL tear, the chondrocytes, particularly in that top 100 microns, are damaged in a very specific way, in that their mitochondria are less efficient at doing their job.”
Drs. Fortier and Delco were interested in therapies that target mitochondria to help prevent damage. The team demonstrated that delivering a small peptide to the cartilage stabilized the mitochondria and prevented damage to the cells and tissue. The team is currently looking at these peptides as a potential therapeutic option for post-traumatic OA.
Key insights for other soft tissues
The team also applied the approaches they developed for understanding the microscale mechanics, composition, and mechanobiology of articular cartilage to answer important questions about the function of other cartilaginous and soft tissues. Key findings include:
- The mechanics of the growth plate arise from the columnar arrangements of cells. Tissue deformations are concentrated between those cellular columns, and those regions of the tissue are the most susceptible to damage.
- Articular cartilage from the temporomandibular joint is different from all other joints in the body. It contains a layer of fibrous tissue at the articular surface that is highly organized and susceptible to shear-loading.
- The attachment of the meniscus to the tibia has a complex organization designed to create a smooth profile in deformation from the stiff bone to the compliant meniscus.
Collectively, these studies have had major impacts on the cartilage and soft tissue biomechanics communities.