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VOLUME REGULATION BY IN SITU GROWTH PLATE CHONDROCYTES

Division of Biomedical and Clinical Laboratory Sciences, The University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD.

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.

Correspondence should be addressed to Mr Carlos Wigderowitz, Honorary Secretary BORS, University Dept of Orthopaedic & Trauma Surgery, Ninewells Hospital & Medical School, Dundee DD1 9SY.

Supported by the Arthritis Research Campaign (H0621) and the Royal College of Surgeons of Edinburgh.