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