Annals of Biomedical Engineering, Vol. 33, No. 8, August 2005 (© 2005) pp. 1071–1077 DOI: 10.1007/s10439-005-5775-y Osmolarity Regulates Gene Expression in Intervertebral Disc Cells Determined by Gene Array and Real-Time Quantitative RT-PCR LAWRENCE M. BOYD, 1 WILLIAM J. RICHARDSON, 2 JUN CHEN, 1 VIRGINIA B. KRAUS, 3 ALOK TEWARI, 1 and LORI A. SETTON 1,2 1 Department of Biomedical Engineering, Duke University, Durham, North Carolina; 2 Division of Orthopaedic Surgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina; and 3 Division of Rheumatology and Immunology, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27708 (Received 22 November 2004; accepted 16 March 2005) Abstract—Intervertebral disc (IVD) cells experience a broad range of physicochemical stimuli under physiologic conditions, including alterations in their osmotic environment. Cellular re- sponses to altered osmolarity have been documented at the tran- scriptional and post-translational level, but mainly for extracel- lular matrix proteins. In this study, the gene expression profile of human IVD cells was quantified with gene array technology following exposure to increased osmolarity in order to capture the biological responses for a broad set of targets. A total of 42 genes were identified in IVD cells as significantly changed following culture under hyper-osmotic conditions. Gene expression patterns were verified using RT-PCR. Genes identified in this study include those related to cytoskeleton remodeling and stabilization (ephrin- B2, muskelin), as well as membrane transport (ion transporter SLC21A12, osmolyte transporter SLC5A3, monocarboxylic acid SLC16A6). An unexpected finding was the differential regulation of the gene for the neurotrophin, brain-derived neurotrophic factor, by hyper-osmotic stimuli that suggests a capability of IVD cells to respond to physicochemical stimuli with factors that may regulate discogenic pain. Keywords—Osmotic pressure, Osmolarity, Intervertebral disc, Cell culture, Gene array, Microarray, Gene expression, Hyperos- molarity. INTRODUCTION The intervertebral discs contain a sparse population of cells distributed in a large volume of extracellular matrix composed primarily of water (60–99% by weight), col- lagens and negatively charged proteoglycans. 31 Loading of the intervertebral disc (IVD) under static or dynamic conditions gives rise to complex physicochemical stimuli, including interstitial hydrostatic pressures, matrix stresses and strains, as well as associated changes in tissue hydra- tion that may modify extracellular cation concentrations, Address correspondence to L.A. Setton, PhD, Department of Biomed- ical Engineering, Duke University, Room 136, Hudson Hall, Box 90281, Durham, North Carolina 27708. Electronic mail: setton@duke.edu pH, and intracellular and extracellular osmolarities. 36,37 In particular, changes in osmolarity are an important compo- nent of the physicochemical environment of the IVD as variations in disc loading lead to significant changes in disc hydration. 36 As for other cell types, isolated cells of the IVD are known to exhibit both passive and active cell volume changes following exposure to hyper- or hypo-osmotic me- dia in vitro. 21,32,33 Osmotic stimuli have also been shown to elicit calcium transients in IVD cells that are modulated, in part, by stability of the actin cytoskeleton. 32,33 These identified changes in cell volume and second messengers following altered osmotic stimuli can be expected to impact metabolic events in the cells of the intervertebral disc. Previous studies have shown that osmotic pressure is a potent regulator of biosynthesis for IVD cells in cul- ture. Fibrochondrocytes of the disc have been shown to alter post-translational biosynthesis of proteoglycans (i.e., 35 S-incorporation) following exposure to hyper- or hypo- osmotic media in both explant and isolated cell culture. 5,6,21 These studies demonstrate that maximal proteoglycan syn- thesis is achieved at osmolarities believed to represent in situ values (−430 mOsm), with decreased proteoglycan synthesis found at osmolarities above or below these values. A recent study of IVD cells in a three-dimensional alginate culture system confirmed that the biological response to al- tered osmolarity is mediated, in part, by changes at the tran- scriptional level. 10 Short periods of culture in hypo-osmotic media (4 h) resulted in increased gene expression for aggre- can and type II collagen in porcine IVD cells, whereas gene expression for the small proteoglycans, decorin and bigly- can, increased under both hypo- and hyper-osmotic condi- tions. The responses of these cells to hyper-osmotic stimuli is considered to be of particular interest, as mechanical loading of the disc in compression will generate increased proteoglycan-associated charge densities, that are associ- ated with increased osmotic pressures within the tissue. A wide range of genes may be transcriptionally ac- tivated in multiple cell types following exposure to 1071 0090-6964/05/0800-1071/1 C  2005 Biomedical Engineering Society