Date of Degree
PhD (Doctor of Philosophy)
James A. Martin
First Committee Member
Second Committee Member
Third Committee Member
Fourth Committee Member
This dissertation project is unique in that it seeks to link two historically independent concepts: mechanical loading of cartilage (1) induces reactive oxygen species (ROS) release from specific mitochondrial complexes, and (2) results in observable metabolic alterations. It is well known that ROS are released from certain loading conditions. It has also been shown that chondrocytes respond favorably to cyclic loading at moderate stresses, as determined metabolically by proteoglycan and collagen production. However, this study aims to demonstrate that these phenomena are interdependent, and in doing so, locates both the source(s) of load-induced ROS and the resultant molecule(s) responsible for metabolic stimulation.
To further this investigation, an osteochondral explant mechanical loading platform was built that allowed the imposition of physiological stresses on cartilage explants to further characterize cartilage metabolism. National Instruments hardware and LabVIEW controls a stepper motor driven platen, which when coupled with a load cell, allows for dynamic and static compression stimulation of articular cartilage.
Firstly, static stress (0.05 – 1.0 MPa for one hour) induces ROS release, which is mitochondrial in origin, relies on an intact cytoskeletal network, and tracks linearly with bulk tissue strain (r = 0.87). Dissolution of the cytoskeleton with cytochalasin B, blocking complex I of the mitochondria with rotenone, or addition of the cell-permeable SOD mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) reduces this ROS release at 0.25 MPa.
Next, under dynamic stress (0.25 MPa/0.5 Hz for one hour), this mitochondrial ROS release was shown to be necessary for stimulating glycolytic energy production 24 hours after stress application. The ROS release from mechanical stimulation was blocked by the addition of rotenone or Mitoquinone (MitoQ10). These treatments also both blocked the increase in intracellular adenosine triphosphate (ATP) content, and therefore show that the ROS from the mitochondria are required for stimulating ATP production.
Probing the mitochondria directly with targeted inhibitors in unloaded conditions shows that forcing superoxide generation at ubiquinol: cytochrome c-oxidoreductase (complex III), and efficiently turning this superoxide into hydrogen peroxide, resulted in a dose-dependent increase in ATP content that resembles the response to loading. Here, ATP content increased with increasing doses of antimycin A, which, when accompanied with the SOD mimetic, Galera (m40401), is always higher than antimycin A alone.
Finally, if overloading proceeds for too long (three hours at 1.0 MPa or 0.25 MPa at 0.5 Hz for 7 days), ROS-related damage ensues, resulting in significantly impaired mitochondrial function and reduced intracellular ATP content. The damage and deleterious effects are negated by administration of the antioxidant, N-acetylcysteine (NAC).
Together, these results show that mechanical stimulation of cartilage produces mitochondrial ROS and resultant products, whose role in articular cartilage is complex. In short term mechanical stimulations, these ROS act to stimulate metabolism. At higher stresses, and over longer durations, ROS cause damage which results in mitochondrial dysfunction and suppressed ATP production. These findings have important implications for the progression of osteoarthritis, which has already been linked to mitochondrial dysfunction
This dissertation project seeks to explain how cartilage produces energy. Cartilage is the tissue that covers joints and allows them to move freely, and a source of energy is required for maintenance of healthy tissue. Energy production in cartilage is known to require the forces that the body imparts on it during motion, such as impacts that occur on the hip, knee, and ankle cartilage during walking. Energy production pathways are most classically characterized in oxygenated tissues, in which oxygen is used directly in a process called oxidative phosphorylation. These tissues receive oxygen from the circulating blood; however, cartilage does not have access to blood and is thus adapted to produce most of its energy through glycolysis, which is a process considered independent of oxygen consumption. Interestingly, though, oxygen is still indirectly required for glycolysis in cartilage, in that complete removal of oxygen inhibits such energy production. This dissertation demonstrates that the forces from walking cause a reaction where oxygen is converted into peroxide. This peroxide then stimulates glycolytic energy production in cartilage. Therefore, in cartilage this oxygen is not directly used to produce energy, but instead is required to produce peroxide, which ultimately acts to stimulate glycolysis to produce energy.
xiv, 108 pages
Includes bibliographical references (pages 98-108).
Copyright © 2015 Marc James Brouillette
Brouillette, Marc James. "Mechanical stimulation of cartilage induces mitochondrial reactive oxygen species production mediating metabolic responses." PhD (Doctor of Philosophy) thesis, University of Iowa, 2015.