Cartilage articulation enhances chondrocyte injury and death after impact injury

Abstract

Purpose: Post-traumatic osteoarthritis results from physical trauma to joints due to cartilage damage and inflammation. Adverse biological responses include mitochondria (MT) depolarization which occurs minutes after injury, leading to bioenergetic failure of the cell through decreased ATP production. MT depolarization also leads to caspase activation and excess reactive oxygen species production, resulting in apoptosis and cell death respectively. Previous work has shown that both impact injury and sliding cause cellular damage in healthy cartilage tissue, however it is unknown if damage would be exacerbated by impacting and then sliding injured cartilage. These combined mechanical forces would more closely simulate the mechanical environment of an injured joint that is subject to continued use after trauma. We hypothesize that sliding injured tissue leads to increased chondrocyte damage throughout the depth of the tissue. Methods: Femoral condyle cartilage of 6 neonatal bovids was collected within 24 h of sacrifice and sectioned into cylindrical plugs (6 mm diameter by 2 mm thick), using sterile practices, and incubated overnight prior to impact. Cartilage plugs were randomly assigned to one of 4 treatment groups (n ≥ 7/group): uninjured control, impact, sliding, or impact followed by sliding. A cylindrical indenter was used to impact cartilage plugs in unconfined axial compression (2.36 ± 0.31 MJ/m3). Impacts were characterized at 50 kHz using a load cell and linear variable differential transducer to measure impact force and cartilage deformation. A custom tribometer was used to slide samples at 1 mm/s for 1 h. Samples were compressed to 15% axial strain and submerged in a lubricating bath of bovine synovial fluid. Samples were axially bisected after injury, stained for either chondrocyte viability (Calcein AM and ethidium homodimer), apoptosis (CellEvent Caspase-3/7 Green), or MT polarization (MitoTracker Green and tetramethylrhodamine). The cross-section (550 μm x 725 μm) was imaged using confocal microscopy at a minimum of 3 h after injury. Bulk tissue and depth-dependent cellular response was quantified using confocal images and a custom MATLAB code. Statistical analysis was conducted using a two-way ANOVA with significance evaluated at p &lt 0.05 for both bulk tissue and depth-dependent results. Results: Bulk tissue analysis revealed that individually, impact and sliding caused increased chondrocyte death and damage for all assessments, with impact having the greater effect, however the combination further increased cell damage and death (Figure 1A-C). For cell death and apoptosis, the effects of impact and sliding were additive, while for MT depolarization the effect was more than additive (Figure 1C) (p = 0.016 for interaction via two-way ANOVA). Examining the spatial patterns of chondrocyte damage and death (Figure 2A-C) revealed that all treatments resulted in cell damage and death that was highest near the articular surface and decreased with depth from the surface. In controls cell death, apoptosis, and MT depolarization were unaffected deeper in the tissue. Sliding alone resulted in levels of cell death, apoptosis, and MT depolarization that were not different from controls deeper into the tissue (p &gt 0.05). Impact alone increased cell death, and MT depolarization in the deeper regions of the cartilage compared to controls (p &lt 0.05). The combination of impact and sliding was nominally higher than either impact or sliding alone, but the interaction was only statistically higher for MT depolarization (Figure 2C). This combination resulted in dramatically higher MT depolarization deeper in the tissue compared to the impact only group (∼60% vs ∼30%, p = 0.005). Conclusions: The goal of this study was to assess the combined effects of two types of loading experienced in joints after trauma. The data show that both impact injury and sliding affect chondrocyte damage. Damage was primarily concentrated at the articular surface, which indicates the surface acts as a protective layer to limit damage propagation deeper into the tissue. Assessments of cell death and apoptosis show additive effects of the combination treatment. However, this combination causes synergistic damage in the assessment of MT depolarization, particularly deeper in the tissue. This population of cells with MT depolarization represents a possible mechanism by which damage propagates to other areas of the tissue and joint. As such this cell population represents a potential target for therapy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)