The role of the actin cytoskeleton in chondrocyte protection following single impact

Yanitsa Nedelcheva, Stephen Getting, Mark Kerrigan

University of Westminster, London, UK

Impact trauma results in cartilage damage and a pre-disposition to osteoarthritis, which leads to joint pain, stiffness and ultimately failure. Chondrocytes, the sole resident cell-type in cartilage, are responsible for the maintenance of the extracellular matrix (ECM) in response to their physico-chemical environment. As chondrocytes do not divide, it is therefore essential to find a method to protect chondrocytes from impact and thus maintain chondrocyte viability and cartilage integrity.

Full-depth cartilage was removed from bovine metacarpal-pharengeal joints (&female;; 18-24months of age) under aseptic conditions. Explants were incubated with 5μM calcein-AM and 1μM propidium iodide for 30mins and impact experiments performed by drop-tower with a single impact of 0.07, 0.131 & 0.263J. The effect was studied by confocal laser scanning microscopy (CLSM) and quantified using an automated method with Imaris ‘Spots’. The effect of cell shrinkage and stretch-sensitive calcium channels were determined using hypertonic media (380mOsm) and 100μM GdCl3 respectively. Actin organisation was measured on Alexa-488 phalloidin labelled chondrocytes by CLSM. Cytokines Interleukin-1 Beta (IL-1β), and Monocyte Chemoattractant Protein 1 (MCP 1) levels were determined in supernatants by commercially available ELISA and nitric oxide (NO) by greiss assay. All data are expressed as Mean ± s.e.m. *p<0.05 vs. control, n = 4-6 at three determinations.

Data were analysed using Imaris 6.02 ‘Spots’ and following 20mins post impact, there was a significant decrease in cell viability (p<0.05) for both higher impact forces when compared to initial cell death and the lower impact force of 0.07J. These data compared to previously published results thus validating this technique (Bush et al., 2005). A pre-incubation in hypertonic media protected chondrocytes from cell death whereby at 20mins death was decreased from 9.22 ± 1.65% to 3.42 ± 0.60 % (p<0.01), inhibited by 100μm GdCl3 (20.45 ± 3.88% death at 20mins) thus implicating stretch-sensitive calcium channels. The polymerised actin cytoskeleton was labelled using Alexa 488-phalldoidin. Hypertonicity increased cortical actin by 29.1 ± 0.13% for all zones of cartilage (p<0.01) and was inhibited by 100μm GdCl3. Analysis of the supernatants revealed an increase in IL-1β (1.5±0.15pg/g), (MCP1 523±126 pg/g), NO (1241±173μg/ml) over the 24hour period post impact p<0.05 vs control values.

These data show a new method for the analysis of impact-mediated chondrocyte viability and that hypertonic chondro-protection is mediated by a stretch-sensitive, calcium-dependent polymerisation of the actin cytoskeleton. Cartilage impact caused the release of pro-inflammatory cytokines that will decrease cartilage viability. Therefore, therapeutic modulation of actin polymerisation will protect chondrocytes from impact damage and could offer a suitable method to decrease the pre-disposition to osteoarthritis.

Bush, P., et al., Osteoarthritis and Cartilage, 2005. 13(1): p. 54-65