The Journal of Rheumatology 2003; 30:2 326 2002-238-1 From the Orthopaedic Research Laboratory and the Division of Orthopaedic Surgery, Stanford University School of Medicine, Stanford; and the RR&D Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, California, USA. Supported by NIH Grant AR45788. M.S. Lee, MD, Assistant Professor, Chang Gung Memorial Hospital, Taiwan, ROC; M.C.D. Trindade, MS, Medical Student; T. Ikenoue, MD, Postdoctoral fellow; R.L. Smith, PhD, Professor (Research), Orthopaedic Research Laboratory; D.J. Schurman, MD, Professor; S.B. Goodman, MD, PhD, Professor, Division of Orthopaedic Surgery. Address reprint requests to Dr. R.L. Smith, Orthopaedic Research Laboratory, Stanford University School of Medicine, 300 Pasteur Drive, R144, Stanford, CA, 94305-5341, USA. E-mail: smith@rrdmail.stanford.edu Submitted March 14, 2002; revision accepted June 4, 2002. Articular chondrocytes respond to mechanical stimuli by altered matrix macromolecule expression and release of soluble mediators, including nitric oxide (NO) 1-4 . NO serves as multifunctional cellular messenger for an array of biolog- ical processes and is implicated in rheumatological diseases 5-7 . In bovine articular chondrocytes, induction of NO by shear stress modulates matrix metabolism 8 . Other types of mechanical loads may exert differential effects on articular chondrocyte metabolism through NO 9,10 . We tested effects of intermittent hydrostatic pressure (IHP) on NO release by human osteoarthritic chondrocytes induced by shear stress and matrix macromolecule gene expression following exposure to shear stress or an NO donor. MATERIALS AND METHODS Cell culture. Human osteoarthritic chondrocytes were isolated from carti- lage samples of 9 patients receiving total joint replacements as described 2 . Chondrocytes were plated at a density of 5 × 10 4 /cm 2 , maintained in DMEM/F12 supplemented with fetal bovine serum and antibiotics and placed in serum-free medium 24 h prior to loading, as described 11 . The cells were then exposed to mechanical loading in the presence of fresh serum- free medium. Each experiment was carried out in duplicate and repeated with 6 individual samples. To test the response of chondrocytes to IHP after preconditioning with an NO donor, sodium nitroprusside (SNP) (Sigma Chemical, St. Louis, MO, USA) was added to the cultures at concentrations of 20 μM or 2000 μM, prior to mechanical loading. To test the response of chondrocytes to IHP after treatment with shear stress, the cells were exposed to shear stress for 2 h (200 rpm; 1.6 Pa) followed by transfer to a pressure vessel for application of IHP. Mechanical loading. IHP (10 MPa at 1 Hz) was applied to confluent cell monolayers by placing the culture plates in heat-sealed bags filled with serum-free medium, as described 2 . The loading of IHP included test periods of 4 h per day for 1, 2 or 4 days. NO release. Nitrite, the stable end product of NO oxidation, was used as an indicator of NO synthesis. Nitrite concentration in the culture medium was measured using the Griess reaction with sodium nitrite as the standard, as described 8 . Analysis of mRNA expression. Following loading, the cells were maintained Intermittent Hydrostatic Pressure Inhibits Shear Stress- Induced Nitric Oxide Release in Human Osteoarthritic Chondrocytes in Vitro MEL S. LEE, MICHAEL C.D. TRINDADE, TAKASHI IKENOUE, DAVID J. SCHURMAN, STUART B. GOODMAN, and R. LANE SMITH ABSTRACT. Objective. To test the effects of intermittent hydrostatic pressure (IHP) on nitric oxide (NO) release induced by shear stress and matrix macromolecule gene expression in human osteoarthritic chon- drocytes in vitro. Methods. Chondrocytes isolated from cartilage samples from 9 patients with osteoarthritis were cultured and exposed to either shear stress or an NO donor. Nitrite concentration was measured using the Griess reaction. Matrix macromolecule mRNA signal levels were determined using reverse-transcriptase polymerase chain reaction and quantified by imaging analysis software. Results. Exposure to shear stress upregulated NO release in a dose and time-dependent manner. Application of IHP inhibited shear stress induced NO release but did not alter NO release from chondrocytes not exposed to shear stress. Shear stress induced NO or addition of an NO donor (sodium nitroprusside) was associated with decreased mRNA signal levels for the cartilage matrix proteins, aggrecan, and type II collagen. Intermittent hydrostatic pressure blocked the inhibitory effects of sodium nitroprusside but did not alter the inhibitory effects of shear stress on cartilage macromolecule gene expression. Conclusion. Our data show that shear stress and IHP differentially alter chondrocyte metabolism and suggest that a balance of effects between different loading forces preserve cartilage extracel- lular matrix in vivo. (J Rheumatol 2003;30:326–8) Key Indexing Terms: NITRIC OXIDE MECHANICAL LOADS OSTEOARTHRITIS CHONDROCYTES AGGRECAN COLLAGEN Personal, non-commercial use only. The Journal of Rheumatology Copyright © 2003. All rights reserved. www.jrheum.org Downloaded on October 31, 2021 from