Complex I Inhibition after Intra-Articular Fracture Prevents Rapid Progression of Osteoarthritis in a Porcine Model

Purpose: The rapid progression of posttraumatic osteoarthritis (PTOA) after severe injuries like intra-articular fractures (IAF) creates an urgent need for preventive PTOA therapies that can extend healthy joint lifespans. Our previous studies demonstrated protection of chondrocytes after impact using rotenone, a well characterized irreversible inhibitor of complex I of the mitochondrial electron transport chain. Amobarbital represents a more therapeutically viable inhibitor of complex I than rotenone given its reversible binding and decreased toxicity. This study tests the hypothesis that rapid development of PTOA subsequent to IAF can be prevented by inhibiting mitochondrial metabolism after injury. Methods: Our minipig model utilizes a 40 J impact to the talus to cause a distal tibial fracture without surgical disruption of the joint capsule. Fractures are repaired using human-like open reduction and internal fixation (ORIF). PTOA occurs by six months, with lesions concentrated around the central-to-anterior portion of the medial talus. Amobarbital (2.5 mM) was added to F-127/hyaluronic acid reverse thermal hydrogel. Hydrogel vehicle (n = 5) or amobarbital (n = 5) was injected intra-articularly after completion of the ORIF and again one week later. Sham animals (n = 5) receiving surgical procedures without fracture impact as well as naïve controls (n = 4) were included. After treatment, animals were put into pasture for six months then euthanized. All procedures were conducted under IACUC approved protocols. Fractured and contralateral hocks were harvested and the distal tibia and talar dome fixed in formalin, decalcified and embedded in paraffin. Sagittal sections of weight-bearing tissue 5 μm-thick were cut and stained with safranin O, fast green, and Weigert’s hematoxylin. Semi-automated Mankin scoring was done using a custom built Matlab program. Treatment effects were analyzed by one-way ANOVA. Confirmation of inhibition of complex I by amobarbital was conducted using fresh bovine chondrocytes. Excess NADH, CoQ and complex III blockade were added to lysates while oxidation of NADH was monitored spectrophotometrically at 340 nm for 3 minutes with and without rotenone. This yields a rate of rotenone-inhibitable NADH oxidation, i.e. complex I activity. Complexes II, III, and IV were assessed with similar methods but with no inhibition by amobarbital. Results: Fresh whole chondrocyte lysates demonstrated a dose responsive inhibition of complex I by amobarbital, reaching > 90% at maximal doses (Figure 1, p < 0.01 at 2.5 mM, n = 3). At 6 months, sham animals displayed no increases in talar Mankin scores (Figure 2A) while fracture (ORIF) induced a significant increase in Mankin score (Figure 2A), decreased overall cartilage thickness and safranin O staining, and caused focal areas of eburnation on the talus (Figure 2B, representative micrograph). Animals receiving amobarbital after ORIF (ORIF+Amo) showed statistically significant decreases in talar Mankin scoring (Figure 2A, p < 0.01) and thicker cartilage that stained more strongly for safranin O than fractured controls (Figure 2B, representative micrographs).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: These results support the hypothesis that a therapeutic window for blunting PTOA with amobarbital exists at the earliest stages after IAF. This approach has not yet been combined with any anti-inflammatory approaches that may augment efficacy and no dose/timing optimization studies have been done. Manipulating joint mitochondrial metabolism after traumatic injury may represent a viable pathway to preventing PTOA.