Hemarthrosis alters mitochondrial dynamics in situ after traumatic injury

Abstract

Purpose: Hemarthrosis occurs after a variety of joint injuries and contributes to rapid progression of posttraumatic osteoarthritis (PTOA). Similarly, mitochondrial responses to traumatic injury appear to modulate rate of PTOA progression. In vitro studies have shown that chondrocyte mitochondria are impaired by a variety of signaling molecules present in whole blood and that pro-inflammatory activity is responsive to manipulations of mitochondrial function. The combination of oxidizing mitochondria and oxidizing, labile iron derived from blood already associated with trauma may cause more severe mitochondrial damage in situ than previously characterized in agarose models. This study tested whether the addition of whole blood after injury would exacerbate losses in mitochondrial content observed after impact in a chelation-inhibitable fashion. Methods: Fresh, skeletally mature, bovine stifle (knee) joints were obtained from a local abattoir (Bud’s Custom Meats, Riverside, IA) and 1 cm diameter cylindrical osteochondral explants from the loaded articular surface of the femoral condyle were harvested. Explants were equilibrated for 1 day in 45% F-12/45% Dulbecco’s modified Eagle medium/10% fetal bovine serum at 37ºC with 5% O2 and 5% CO2. Explants were impacted with a 2 J impact via drop tower to the articular surface using a flat impermeable platen (or sham impact for controls) and placed into one of three solutions: normal media, normal media + 10% whole blood from the same animal, or normal media + 10% blood + the iron chelator desferrioxamine mesylate (DFO) (100 μM, Sigma-Aldrich). Samples were returned to the incubator in these solutions and 2 h later stained in serum free media with 1 μM calcein AM, a viability dye, and 200 nM MitoTracker Deep Red, a stain for mitochondria (both ThermoFisher Scientific). Cells were visualized with an Olympus FV1000 confocal microscope with a 20X objective immersed in phenol red-free media, as well as with a 100X immersion objective placed in contact with the impacted site. Images were analyzed using a custom-written two-part analysis routine developed in Matlab. Viable cells were automatically identified from the calcein AM-stained images using intensity thresholding and area-based shape measures, and resultant cell counts were expressed as viable cell density (cells/mm2). A mask image of only viable cells was applied to the MitoTracker images to extract mitochondrial staining intensity from viable cells. Data reported are the average results from explants obtained from 4 separate animals represented in all groups. Results: Impact resulted in a ∼50% reduction in viable cell density in the zone of impact across all groups and conditions. Total viable cell mitochondrial staining decreased after impact (p < 0.05) and this was not altered with whole blood addition after impact, Figure 1. A trend towards recovery of mitochondrial content was observed with whole blood and DFO after impact; however this has not reached statistical significance at n = 4 (p = 0.2 for reversing the effect of impact). A clear difference in mitochondrial network morphology was observed comparing samples with and without whole blood, Figure 2, with samples exposed to whole blood demonstrating a much more punctate, fragmented morphology. No significant differences in cell viability were observed after including blood in our studies, suggesting that impact itself was largely determinate of cell death at sites of impact. The mitochondrial phenotype after impact appears very different in the presence of hemarthrosis, with brighter and more punctate mitochondria than impact alone. Conclusions: Iron chelation may be effective in preventing loss of overall mitochondrial intensity, i.e. mitochondrial mass, but greater numbers of replicates will be needed to reach statistical significance. While DFO provided some recovery of intensity, it did not appear to rescue the morphology changes associated with exposure to blood. This study contributes to ongoing efforts in the field to improve the physiological relevance of in vitro models of cartilage injury by demonstrating a clear effect of whole blood on intracellular responses to mechanical injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)