Effect of age and cytoskeletal elements on the indentation-dependent mechanical properties of chondrocytes

Abstract

Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15-2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level.

Figure 1. Experimental set up.

Image of bovine joint used for chondrocyte isolation (A). Transmission Electron Microscopy (TEM) image of the Atomic Force Microscopy (AFM) tip/bead and image of the alignment of tip/bead over a single chondrocyte (B). Schematic of measuring Force vs chondrocyte indentation using AFM (C). Schematic and representative indentation-dependent elastic modulus of an individual chondrocyte (D). Schematic of the approach used to acquire viscoelastic properties of individual chondrocytes and a representative viscoelastic force curve from an individual chondrocyte (E)

Figure 2. The effect of age on the viscoelastic properties of bovine chondrocytes.

Comparing indentation-dependent elastic modulus (A), viscosity (B), equilibrium modulus (C) and instantaneous modulus (D) of chondrocytes isolated from different aged bovine [1 d (black bars), 5 yr (red bars) and 12 yr (blue bars)]. *p<0.05 vs. 1 d (within indentation).

Figure 3. Indentation-dependent viscoelastic properties of chondrocytes are affected by cytoskeletal disrupters.

Comparing indentation dependent elastic modulus (A), viscosity (B), equilibrium modulus (C) and instantaneous modulus (D) of chondrocytes subjected to different treatment conditions [NT (control; no treatment), Acryl (acrylamide), CytoB (cytochalasin B)]. (*p<0.001 vs. control; ∼p<0.05 vs control; ∧p<0.05 vs Acryl).

Figure 4. Visualizing the effects of cytoskeletal disrupters on chondrocytes.

Confocal fluorescent images of chondrocytes treated with media (A), 5 mM acrylamide for 16 hours (B) and 5 µM cytochalasin B for 1 hour (C). Scale bar –5 µm. Analysis of actin aggregates per chondrocyte (D). More than 20 individual cells per treatment were analyzed using Image J. Asterisks indicates a significant difference (p<0.005).

Figure 5. Visualizing the effects of cytosckeletal disrupters on chondrocyte indentation.

Z- stacked confocal fluorescent images of chondrocytes compressed at a force of 0 nN and 3 nN treated with media (A), 5 mM acrylamide for 16 hours (B) and 5 µM cytochalasin B for 1 hour (C). Scale bar –5 µm.

Figure 6. Visualizing the actin cytoskeleton of compressed chondrocytes in the presence of cytosckeletal disrupters.

Cross-sectional confocal fluorescent images of chondrocytes compressed at a force of 0, 1, 2 and 3 nN treated with media (A), 5 mM acrylamide for 16 hours (B) and 5 µM cytochalasin B for 1 hour (C).