ARTHRITIS & RHEUMATISM Vol. 50, No. 3, March 2004 pp 840–848 DOI 10.1002/art.20101 © 2004, American College of Rheumatology Mechanisms and Kinetics of Glycosaminoglycan Release Following In Vitro Cartilage Injury Michael A. DiMicco, 1 Parth Patwari, 2 Patrick N. Siparsky, 2 Sanjay Kumar, 3 Michael A. Pratta, 3 Michael W. Lark, 3 Young-Jo Kim, 4 and Alan J. Grodzinsky 2 Objective. Acute joint injury leads to increased risk for osteoarthritis (OA). Although the mechanisms underlying this progression are unclear, early struc- tural, metabolic, and compositional indicators of OA have been reproduced using in vitro models of cartilage injury. This study was undertaken to determine whether glycosaminoglycan (GAG) loss following in vitro carti- lage injury is mediated by cellular biosynthesis, activa- tion of enzymatic activity, or mechanical disruption of the cartilage extracellular matrix. Methods. Immature bovine cartilage was cultured for up to 10 days. After 3 days, groups of samples were subjected to injurious mechanical compression (single uniaxial unconfined compression to 50% thickness, strain rate 100% per second). GAG release to the medium was measured, and levels were compared with those in location-matched, uninjured controls. The ef- fects of medium supplementation with inhibitors of biosynthesis (cycloheximide), of matrix metalloprotein- ase (MMP) activity (CGS 27023A or GM 6001), and of aggrecanase activity (SB 703704) on GAG release after injury were assessed. Results. GAG release from injured cartilage was highest during the first 4 hours after injury, but re- mained higher than that in controls during the first 24 hours postinjury, and was not affected by inhibitors of biosynthesis or degradative enzymes. GAG release dur- ing the period 24–72 hours postinjury was similar to that in uninjured controls, but the MMP inhibitor CGS 27023A reduced cumulative GAG loss from injured samples between 1 day and 7 days postinjury. Other inhibitors of enzymatic degradation or biosynthesis had no significant effect on GAG release. Conclusion. Injurious compression of articular cartilage induces an initially high rate of GAG release from the tissue, which could not be inhibited, consistent with mechanical damage. However, the finding that MMP inhibition reduced GAG loss in the days following injury suggests a potential therapeutic intervention. Degenerative joint diseases such as osteoarthritis (OA) result in degradation of articular cartilage, char- acterized by release of macromolecular constituents from the tissue and subsequent loss of tissue integrity and mechanical properties (1). However, the precise etiology of the disease remains unknown. Consistent with observations that the in vivo mechanical environ- ment of cartilage influences its cellular biosynthesis (2,3), abnormalities in joint loading due to obesity, joint laxity (as in ligament transection [4]), or altered joint geometries (e.g., acetabular dysplasia [5]) have been recognized as risk factors for OA (6). Additionally, traumatic joint injury leads to in- creased risk of OA (6,7), though the precise mechanism by which joint injury leads to disease is poorly under- stood. In studies of synovial fluid from joints after trauma, it has been found that in the hours following injury, the level of the zymogen form of stromelysin (matrix metalloproteinase 3 [MMP-3]) in the synovial fluid was increased up to 40-fold over normal levels, and elevated levels of MMP-3 persisted for up to 20 years following injury (8–11). Further analysis of synovial fluid revealed evidence of MMP and aggrecanase activities in Supported by NIH grant AR-45579 and by GlaxoSmithKline Pharmaceuticals. 1 Michael A. DiMicco, PhD: Massachusetts Institute of Tech- nology and Children’s Hospital, Boston, Massachusetts; 2 Parth Pat- wari, MD, ScD, Patrick N. Siparsky, BS, Alan J. Grodzinsky, ScD: Massachusetts Institute of Technology, Boston; 3 Sanjay Kumar, PhD, Michael A. Pratta, MS, Michael W. Lark, PhD (current address: Centocor, Inc., Malvern, Pennsylvania): GlaxoSmithKline Pharmaceu- ticals, King of Prussia, Pennsylvania; 4 Young-Jo Kim, MD, PhD: Children’s Hospital, Boston, Massachusetts. Address correspondence and reprint requests to Michael A. DiMicco, PhD, Massachusetts Institute of Technology, Room NE47- 377, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: mdimicco@mit.edu. Submitted for publication July 27, 2003; accepted in revised form December 3, 2003. 840