Chondrocytes are responsible for the mechanical resilience of cartilage by controlling the synthesis/degradation of the extracellular matrix. In osteoarthritis (OA), increased activity of cytokines/degradative enzymes (e.g. IL-1beta, MMP-13) play a key role leading to matrix breakdown/cartilage loss. Studying early events in OA might identify targets for limiting the deleterious changes to cartilage stability. Human chondrocyte shape in situ is normally elipsoidal/spheroidal however abnormal forms within otherwise macroscopically normal cartilage are present. Changes to cell shape can alter ECM metabolism and thus these abnormal forms might be an early event in OA. We have investigated whether levels of IL-1beta and MMP-13 are altered in human chondrocytes of abnormal morphology.

Tibial plateau cartilage was obtained from patients undergoing knee arthroplasty and only areas graded 0 or 0–1 studied. The shape of fluorescently-labelled in situ chondrocytes was classified by confocal scanning laser microscopy with cartilage depth, and cells characterised as normal (no cytoplasmic processes) or abnormal (one/more cytoplasmic process). Within grade 0 cartilage about 40% of the cells demonstrated abnormal morphology with a reduced proportion in deep zones. Fluorescence immunohistochemistry of antibodies for IL-1beta or MMP-13 was studied in the same cells and quantified. There was an increase in IL-1beta fluorescence with abnormal chondrocytes within the superficial (p=0.033; 21 joints > 190 cells) and deep zones (p=0.001; 8 joints > 100 cells). There were no differences between MMP-13 labelling of normal compared to abnormal chondrocytes within either the superficial or deep zones.

Our results suggest that in relatively non-degenerate cartilage, a proportion of the chondrocyte population demonstrated abnormal morphology and that these cells have elevated levels of IL-1beta but not MMP-13. However, we do not know if chondrocyte shape alters cytokine levels, or vice versa. Additionally, the role of cartilage age is unclear, as although the cartilage samples were relatively normal they were obtained from aged individuals. Nevertheless these results show changes to chondrocyte morphology and increased levels of IL-1beta, and thus presumably matrix catabolism - in relatively normal human articular cartilage, raising the possibility that this is an early event in cartilage degeneration.

Supported by the Wellcome Trust (075753).

Introduction: In the growth plate, chondrocyte swelling (hypertrophy) is a crucial event during endochondral ossification and bone lengthening, accounting for ~80% of the increase in bone length (1,3). The swelling is dramatic (~10x) and closely regulated. Failure of chondrocyte hypertrophy may underlie the chondrodysplasias of the vertebrate skeleton (1). However, the mechanisms which control cell swelling are poorly understood although there must be a key role for chondrocyte osmolyte transporters which are sensitive to an increase in cell volume. We have used confocal scanning laser microscopy (CLSM) to study volume regulation by living in situ growth plate chondrocytes at varying degrees of hypertrophy.

Methods: Bovine growth plates were taken from the ends of young (~12d) bovine ribs. In situ growth plate chondrocytes at the proliferative through to hypertrophic stages were fluorescently-labelled (calcein-AM; 5μM), imaged (Zeiss CLSM510) and volumes determined quantitatively as described (2). An acute osmotic challenge (280-140mOsm) was delivered by perfusion to determine volume-regulatory capacity by cells in the various zones.

Results: The resting volumes of proliferative and hypertrophic cells were 550±63μm³ and 5227±1974μm³ respectively. Reducing osmolarity resulted in a rapid (within ~1min) cell swelling, proliferative and hypertrophic chondrocytes increasing in volume by 126±2% and 146±5% (n=5) respectively. Chondrocytes within the proliferative zone then recovered in volume by ~60% over the following 20mins (p=0.04), whereas no volume recovery was detected in hypertrophic cells (p=0.94).

Conclusions: For the increase in growth plate chondrocyte volume to produce hypertrophy it is essential that the membrane transporters which normally prevent cell swelling are suppressed, otherwise the increase in volume will be compromised. These results suggest that chondrocyte hypertrophy is associated with reduced activity of the swelling-stimulated osmolyte transporter whereas the pathway is active in proliferating chondrocytes. Changes in the activity of this pathway are likely to be an important component in the control of chondrocyte hypertrophy. It is clear that the contributions of other membrane transporters in mediating chondrocyte swelling must be identified in order to understand the overall hypertrophic process.