Inhibition of glycosaminoglycan incorporation influences collagen network formation during cartilage matrix production Yvonne M. Bastiaansen-Jenniskens a,b , Wendy Koevoet c , Kaspar M.B. Jansen d , Jan A.N. Verhaar a , Jeroen DeGroot b , Gerjo J.V.M. VanOsch a,c, * a Erasmus MC, University Medical Centre Rotterdam, Department of Orthopaedics, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands b TNO Quality of Life, Business Unit BioSciences, Leiden, The Netherlands c Erasmus MC, University Medical Centre Rotterdam, Department of Otorhinolaryngology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands d Delft University of Technology, Faculty 3mE, Department Precision and Microsystems Engineering, The Netherlands article info Article history: Received 26 November 2008 Available online 25 December 2008 Keywords: Glycosaminoglycan Collagen Cartilage matrix Chondrocyte abstract To understand cartilage degenerative diseases and improve repair procedures, we investigate the influ- ence of glycosaminoglycans (GAGs) on cartilage matrix biochemistry and functionality. Bovine articular chondrocytes were cultured in alginate beads with(out) para-nitrophenyl-beta-D-xyloside (PNPX) to inhi- bit GAG incorporation into newly formed proteoglycans. As expected, GAG deposition in alginate beads decreased with increasing PNPX concentration. Next to GAGs, collagen deposition and cross-linking also decreased. In the presence of PNPX, GAGs and collagen were deposited further away from the chondro- cyte than in the control and increased amounts were found in the culture medium. These changes resulted in decreased functional properties of the construct. We conclude that in our culture system, intact proteoglycans play a role in deposition of collagen and thus the formation of a functional matrix. The effect of less proteoglycans on the collagen network could explain why cartilage repair is ineffective in osteoarthritis and help us with development of new therapies. Ó 2008 Elsevier Inc. All rights reserved. Introduction The function of articular cartilage depends on structure, compo- sition and integrity of its extracellular matrix (ECM). The main ECM molecules present in cartilage are collagens and proteoglycans. Collagens are organized into a fibrous network, which defines the basic tissue architecture and provides tensile strength [1,2]. Together with type IX and XI, collagen type II forms fibrils [3,4], which are stabilized by the formation of intermolecular cross-links. A high concentration of proteoglycans (PGs) is embedded in the collagen network giving the cartilage the ability to hold water and to reverse deformation [5]. Proteoglycans consist of a protein core with carbohydrate side chains called glycosaminoglycans (GAGs). Aggrecan is the most common PG in articular cartilage with chondroitin and keratan sulfate side chains [6]. Other smaller proteoglycans and present to a lesser extent include decorin, bigly- can and perlecan [7,8]. These small proteoglycans have one or two GAG side chains such as dermatan sulfate or heparan sulfate. For all proteoglycans, these GAG side chains are covalently joined to the protein core by a glycosidic bond between xylose and the hy- droxyl group of a serine residue [9]. After xylosylation of serine, linkage region synthesis occurs by addition of two galactosyl moi- eties by galactosyltransferase I [10] and glucuronic acid by glucu- ronosyltransferase I [11]. For chondroitin sulfate, alternate transfer of N-acetylgalactosamine and glucuronic acid [12] results in elongation of side chain. With cartilage degeneration in osteoarthritis (OA), changes in structure and composition of the tissue occur. Fewer proteoglycans are present in OA cartilage [13] together with enhanced collagen degradation [14]. However, the rate of synthesis of both matrix components is increased as well [15–17]. This suggests an acti- vated repair mechanism that is however ineffective in repairing or maintaining the ECM homeostasis. In OA, the mechanical stiff- ness of cartilage decreases in relation to the extent of its degener- ation [18]. Changes in mechanical properties include a decrease of the cartilage compression stiffness and an increase in the tendency to swell compared to healthy cartilage [19,20]. Understanding matrix assembly and interaction between colla- gens and proteoglycans is necessary to understand the disease pro- cess in OA and develop solutions for the repair of articular cartilage damage. The purpose of this study was therefore to examine the effect of GAGs on matrix production and distribution and conse- quently matrix functionality, by dose-dependently inhibiting 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.12.028 * Corresponding author. Address: Department of Orthopaedics and Department of Otorhinolaryngology, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. Fax: +31 0 10 7044690. E-mail address: g.vanosch@erasmusmc.nl (G.J.V.M. VanOsch). Biochemical and Biophysical Research Communications 379 (2009) 222–226 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc