Time- and depth-dependent changes to articular cartilage mitochondrial content occur during the 48 hours after injury

Abstract

Purpose: Posttraumatic osteoarthritis (PTOA) is a degenerative joint disease after injury with no viable long-term treatments beyond joint arthroplasty and analgesia. It has recently been shown that impact injury and subsequent electron transport initiate a decrease in mitochondrial content in the superficial zone of articular cartilage, suggesting mitochondria play a role early in the development of PTOA. Electron transport inhibition after trauma has shown some promise; however, in published studies this approach is only effective if applied less than 24 hours after injury. Given that dysfunctional mitochondria have been observed as late as 4 weeks after injury and it has been reported several times that end-stage OA chondrocytes have mitochondrial dysfunction, we wanted to better understand the development and extent of mitochondrial dysfunction throughout cartilage after injury. We hypothesized injured areas deeper than the superficial zone would show decreases in mitochondrial content between 24 and 48 h after injury. Methods: Healthy bovine stifle (knee) joints with no signs of osteoarthritis were obtained from a local abattoir (Bud’s Custom Meats Riverside, IA). Matched 10 mm diameter osteochondral plugs were taken from the loaded portion of the femoral condyle. Once removed, the plugs were washed twice in HBSS with antibiotics then cultured in normal media (45% DMEM, 45% F12, 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μm/mL amphotericin B) and maintained at 37°C, 5% CO2, and 5% O2. After 24 h equilibration to culture conditions, the plugs were subject to either a sham (0 J/cm2) or a 2 J/cm2 impact delivered via a custom-built drop tower utilizing a 6 mm flat, beveled, stainless steel platen. After impact, the samples were placed in fresh porcine media and returned to the incubator. Prior studies had viewed plugs from above using confocal microscopy; in this case, to view the deeper zones of cartilage plugs were bisected slightly off center of the impact into a 5.9 mm section with an Isomet 1000 (Buehler, Lake Bluff, IL), then faced by trimming 0.5 mm of the cartilage with a single straight scalpel cut. These measured cuts (the combination of the isomet blade with and the cartilage removed during facing) provided a sagittal cross-section of the plug at the center of the impact site. The plugs were washed in phenol red free DMEM/F12 media and co-stained for 30 min in the incubator with 1 mg/mL Calcein Green AM and 200 nM MitoTracker Deep Red (both Life Technologies, Waltham, MA). The samples were then washed and imaged on an Olympus FV1000 (Shinjuku, Japan) confocal microscope. Images were taken at the middle of the impact site with a 10X objective lens and images were manually segmented according to either specific zones of the cartilage (superficial, transitional, radial, or deep zone) or by 50 μm increments in depth starting from the superficial zone (Figure 1). Within each zone or depth, a custom watershed-based 3D image analysis algorithm was used to segment live cells according to Calcein Green AM. Per-cell MitoTracker staining intensity for live cells was extracted and averaged by zone depth. Intensities of cells in injured plugs were normalized to their respective sham control. Two-way ANOVA with Tukey multiple comparisons was performed to determine significance between groups. Results: Live cell mitochondrial staining intensity in the superficial and transitional zones of the cartilage significantly increased in the first 24 h after injury compared to sham (Figure 2, n = 4, p < 0.05). However, 48 h after impact, live cell mitochondrial staining decreased significantly both in the superficial zone and in the transitional zone compared to 24 h after injury (p < 0.05). Mitochondrial content at 48 h decreased below control levels in the superficial and transitional zones of three out of four specimens. Analysis by either measured depth or cartilage zone each revealed this significant change from 24 to 48 h after injury. Conclusions: We observed a significant increase in live cell mitochondrial staining 24 h after impact compared to sham; however, this time point was marked by significant inter- and intra-specimen variation as shown in the variance in Figure 2. This acute increase in staining may indicate stimulation of the chondrocytes by the mechanical injury. However, we observed a substantial and significant decrease in live chondrocyte mitochondrial staining in the superficial and transitional zones by 48 h after injury. These changes must be verified via immunohistochemistry, but when compared to prior results they suggest dynamic changes in mitochondrial dysfunction through the depth of articular cartilage after injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)