Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC243
Enlargement of growth plate chondrocytes in agarose culture.
K. V. Sokhi1, B. Olabi2, H. R. Simpson1, A. C. Hall2, P. G. Bush3
1. Dept. Orthopaedics & Trauma, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom. 2. Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom. 3. School of Pharmacy & Biomolecular Sciences, University of Brighton, Brighton, United Kingdom.
Limb deformities cause both functional and psychological effects, and are often the result of growth plate (GP) dysfunction. Development of an artificial GP is one solution to alleviating limb growth failure. As a first step, we have attempted to culture GP chondrocytes (GPCs) in 3-Dimensional (3D) agarose gels and follow the process of chondrocyte enlargement which is fundamental to normal long bone growth (1). GPs from bovine ribs (sourced from a local abattoir) were dissected into media (α-medium with Na2 glycerol bi-phosphate (10mM), bovine serum albumin V (1mM), and L-ascorbic acid (5mg/ml)) and diced. The tissue was then incubated with collagenase (0.4% w/v; 4hrs; 37oC) to liberate chondrocytes. Isolated chondrocytes were filtered and washed by centrifugation (1000xg; 10mins), prepared at approx. 1x106/ml) and re-suspended into 4% (w/v) low melting point agarose and ‘set’ in 100µl3 disc-shaped moulds (4oC). Agarose constructs were then incubated (37oC; 5%CO2) in either standard GP media, or GP media supplemented with insulin (10µg/ml), transferrin (10µg/ml) and Na+ selenite (3x10-8M) (ITS medium) for four days. Chondrocyte volumes were quantified by imaging calcein-loaded (5µM; 30mins; 37oC) cells using 2-photon laser scanning microscopy (Zeiss Ltd., Welwyn Garden City, UK; Coherent, CA, USA) and 3D analysis software (VolocityTM, Perkin Elmer, UK)(2). The initial volume of GPCs in 3D agarose gels was approx. 1300µm3 (Fig. 1), and after 4 days of culture in standard GP medium, there was no significant change in volume (P=0.13; Student’s unpaired t-test). However, in ITS medium, there was a significant approx. 3-fold increase in volume to compared to cells at day 0 (P=0.009) and day 4 (P<0.001). Utilising this agarose gel culture method, these results show that it is possible to stimulate chondrocyte enlargement with ITS medium. The cell swelling/hypertrophy of chondrocytes was rapid and similar to that observed in vivo (3) suggesting that this methodology could be a useful first step for developing an artificial growth plate.
Where applicable, experiments conform with Society ethical requirements