Professor Cornell University Ithaca, New York, United States
Introduction: Diseases of cartilage are the most common causes of disability in the developed world. In all its forms, the role of cartilage is entirely mechanical – supporting loads, protecting adjacent tissues, and providing a nearly frictionless surface for joint articulation. Despite having little innate capacity for self-repair, cartilage endures more than 3 million loading cycles per year at stresses of ~20 MPa with little damage or wear over decades of use. This talk will explore the molecular origins of the remarkable mechanics of cartilage using fast confocal microscopy techniques, combined with vibrational microspectroscopy to quantify structure-property relationships on the microscale.
Materials and
Methods: Articular cartilage was obtained from a variety of sources, young and adult bovine and equine knee and ankle joints, as well as healthy and osteoarthritic human knee and ankle joints. Samples of these tissues were cut into disks and mounted on a tissue deformation imaging stage mounted on a fast confocal microscope. Images of samples were obtained at various stages of deformation with simultaneous measurement of loads, which were used to calculate tissue stresses. Images were used to measure local deformations, which were used to calculate local strains. Stress and strain measurements were used to calculate shear moduli on the scale of 25 microns. Tissue samples were also imaged using FTIR microscopy and infrared spectra were used to calculate proteoglycan and collagen content on a length scale of 25 microns. Local composition and mechanics data were used to understand structure property relationships, which were compared to simulations of tissue mechanics based on rigidity percolation theory.
In parallel studies, the biological responses of tissues to mechanical loading were also observed. While tissues were mounted in custom loading stages, samples were stained with dyes to reveal cell viability, mitochondrial polarization, calcium content, and apoptosis. Local viability, mitochondrial activity, and apoptosis were compared to local strains, and machine learning algorithms were used to determine mechanobiological phenotypes.
Results, Conclusions, and Discussions: Microscale testing of articular cartilage demonstrated that tissue from all species and ages showed a similar spatial distribution of mechanics: the top 100-200 microns of the tissue were dramatically more compliant than the deeper tissue, with moduli 10-50 times lower than the deeper tissue. This region of the tissue also showed the lowest collagen and proteoglycan content, 2-4 times lower than the deeper tissue. Chondrocytes in this region of the tissue experienced higher strain under loading, which lead to increased calcium influx, mitochondrial depolarization, apoptosis, and cell death.
Spatial patterns of cartilage shear modulus were well predicted by rigidity percolation models based on local changes in composition. The 10-50 fold increase in modulus due to 2-4 fold changes in composition is consistent with the idea of a mechanical phase transition due to matrix connectivity. These data motivate a new perspective on the disease of osteoarthritis, in which cartilage tissue transitions from mechanically competent to incompetent based on changes in ECM composition related to matrix connectivity.
Applying these techniques to study single cell mechanobiology reveals the presence of normal and pathologic phenotypes in chondrocytes. These phenotypes are described by the relationships between calcium transients, which occur on the time scale of seconds, to bioenergetics changes, which occur on the time scale of hours, and cell death and apoptosis, which occur on the time scale of hours to days. Collectively, these studies give insights into the cellular and extracellular mechanisms that contribute to the development of cartilage pathology,
