Inhibition of glycosaminoglycan incorporation influences collagen network formation during cartilage matrix production

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Abstract

To understand cartilage degenerative diseases and improve repair procedures, we investigate the influence 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 inhibit 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 chondrocyte 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.

Introduction

The function of articular cartilage depends on structure, composition 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, biglycan 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 hydroxyl group of a serine residue [9]. After xylosylation of serine, linkage region synthesis occurs by addition of two galactosyl moieties by galactosyltransferase I [10] and glucuronic acid by glucuronosyltransferase 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], [16], [17]. This suggests an activated repair mechanism that is however ineffective in repairing or maintaining the ECM homeostasis. In OA, the mechanical stiffness of cartilage decreases in relation to the extent of its degeneration [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 collagens and proteoglycans is necessary to understand the disease process 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 consequently matrix functionality, by dose-dependently inhibiting GAG incorporation in the newly formed cartilage matrix using the exogenous substrate acceptor of galactosyl transferase para-nitrophenyl-beta-d-xyloside (PNPX).

Section snippets

Cell culture

Articular cartilage was harvested from the metacarpophalangeal joints of calves aged 6–12 months. Chondrocytes were isolated, suspended in 1.2% (w/v) alginate in a concentration of 4 × 106 cells per mL of alginate, and alginate beads were made as described previously [21]. Beads were cultured in 75 μl/bead DMEM/F12 with GlutaMAX supplemented with 10% fetal bovine serum (GibcoBRL), 50 μg/ml l-asorbic acid 2-phosphate (Sigma), 50 μg/ml gentamicin and 1.5 μg/ml fungizone (both GibcoBRL). Chondrocytes

Gene expression

Aggrecan core protein (AGCN) gene expression, Collagen type 2 (COL2) gene expression and SOX9 gene expression as indication for chondrocyte phenotype were not significantly changed by the presence of PNPX at day 21 of culture. Although AGCN had a trend towards upregulation with increasing PNPX concentration, it did not reach statistical significance (Fig. 1).

Matrix production and distribution

After 35 days of culture, no difference was seen in the amount of DNA between the conditions, being on average 0.23 ± 0.07 μg DNA per bead (

Discussion

Once damaged, adult articular cartilage has a poor repair capacity even though the loss of proteoglycans is considered reversible [30], [31]. Although collagen turnover is also increased in osteoarthritis, this does not lead to the formation of a functional network [17], [32]. In this study, the effect GAGs on matrix assembly and distribution and the subsequent mechanical functioning of the matrix was examined. We found that by inhibiting GAG incorporation in the newly formed cartilage matrix,

Acknowledgment

This study was supported by a grant of The Dutch Arthritis Association (Reumafonds), NR 02-2-40.

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