A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology.

The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. To permit a detailed morphological study of hypertrophic chondrocytes using standard histological techniques, the preservation of normal chondrocyte morphology is essential. We noticed that during fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage) artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a) paraformaldehyde (PFA; 4%), (b) glutaraldehyde (GA; 2%) with PFA (2%) with ruthenium hexamine trichloride (RHT; 0.7%), (c) GA (2%) with RHT (0.7%), or (d) GA (1.3%) with RHT (0.5%) and osmolarity adjusted to a 'physiological' level of approximately 280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescently-labelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. Our data showed that the high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids ( approximately 280mOsm). These results emphasize the importance of fixative osmolarity, in order to accurately preserve the normal volume/morphology of cells within tissues.