Elsevier

Journal of Biomechanics

Volume 43, Issue 7, 7 May 2010, Pages 1343-1350
Journal of Biomechanics

Electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage

https://doi.org/10.1016/j.jbiomech.2010.01.021Get rights and content

Abstract

This study presents direct experimental evidence for assessing the electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage. Immature and mature bovine cartilage samples were tested in unconfined compression and their depth-dependent equilibrium compressive modulus was determined using strain measurements with digital image correlation analysis. The electrostatic contribution was assessed by testing samples in isotonic and hypertonic saline; the combined contribution was assessed by testing untreated and proteoglycan-depleted samples.

Though it is well recognized that proteoglycans contribute significantly to the compressive stiffness of cartilage, results demonstrate that the combined electrostatic and non-electrostatic contributions may add up to more than 98% of the modulus, a magnitude not previously appreciated. Of this contribution, about two thirds arises from electrostatic effects. The compressive modulus of the proteoglycan-depleted cartilage matrix may be as low as 3 kPa, representing less than 2% of the normal tissue modulus; experimental evidence also confirms that the collagen matrix in digested cartilage may buckle under compressive strains, resulting in crimping patterns. Thus, it is reasonable to model the collagen as a fibrillar matrix that can sustain only tension. This study also demonstrates that residual stresses in cartilage do not arise exclusively from proteoglycans, since cartilage remains curled relative to its in situ geometry even after proteoglycan depletion. These increased insights on the structure–function relationships of cartilage can lead to improved constitutive models and a better understanding of the response of cartilage to physiological loading conditions.

Introduction

It is well known that proteoglycans (PGs) contribute significantly to the compressive modulus of articular cartilage (Maroudas, 1979). However, a precise quantitative assessment of this contribution is yet to be reported. Articular cartilage is mostly comprised of water (60–85% by wet weight), type II collagen (15–22% by wet weight), and PGs known as aggrecan (5–10% by wet weight; Maroudas, 1979; Mow et al., 2005). Aggrecans are macromolecules consisting of a main protein core with laterally covalently attached glycosaminoglycan (GAG) side chains, the main GAG types being chondroitin sulfate and, to a lesser extent, keratan sulfate (Muir, 1980).

Many of the studies available in the literature that have used various treatment options to digest the solid matrix have examined only partially digested or degraded tissue (Bader and Kempson, 1994; Basalo et al., 2004, 2005; Bonassar et al., 1995; Harris et al., 1972; Lotke and Granda, 1972; Lyyra et al., 1999b). While these studies are valuable to the understanding of such pathologies as osteoarthritis, where PG degradation is known to occur (Carney et al., 1984; McDevitt and Muir, 1976; Muir, 1977), from a basic physics standpoint it is also important to understand the exact contribution each constituent of the solid matrix makes to the overall properties of the tissue.

Experiments have shown that the digestion of proteoglycans with chondroitinase ABC or streptomyces hyaluronidase results in a significant reduction of the equilibrium compressive modulus of articular cartilage (Korhonen et al., 2003; Lyyra et al., 1999a; Nieminen et al., 2000; Rieppo et al., 2003; Toyras et al., 1999; Zhu et al., 1993). Unfortunately, few selective enzymatic digestion studies have quantified the changes in modulus and PG content simultaneously. Zhu et al. (1993) have observed a reduction of the compressive Young’s modulus by approximately 70% when the PG content was reduced by approximately 80%.

The fixed negative charges of PGs induce a Donnan osmotic pressurization of the tissue’s interstitial fluid (Eisenberg and Grodzinsky, 1985; Lai et al., 1991; Maroudas, 1979; Overbeek, 1956) that contributes significantly to its compressive modulus according to theory (Ateshian et al., 2004; Azeloglu et al., 2007). This electrostatic contribution has been supported by the finding that cartilage becomes softer in bathing environments of increasing ionic strengths (Chahine et al., 2004; Eisenberg and Grodzinsky, 1985; Elmore et al., 1963; Maroudas, 1975; Parsons and Black, 1979; Sokoloff, 1963). In particular, Eisenberg and Grodzinsky (1985) have shown that the equilibrium compressive modulus of articular cartilage decreases from 0.55 MPa at 0.15 M NaCl, to 0.27 MPa at 1.0 M NaCl, suggesting that the contribution from Donnan osmotic pressure under isotonic conditions represents ∼50% of the total tissue stiffness. Similarly, Chahine et al. (2004) have shown that the Donnan pressure contributes ∼60% of the total tissue stiffness, when comparing compressive equilibrium moduli at 0.15 and 2 M NaCl.

Examination of these literature results suggests that the Donnan osmotic pressure induced by the negatively charged GAGs does not account for the total contribution of PGs to the compressive modulus, since the reduction in compressive stiffness observed with partial PG digestion is greater than that achieved by neutralizing charge (and thus, Donnan) effects. Indeed, direct measurements of the osmotic pressure of chondroitin sulfate solutions in various ionic strengths of NaCl demonstrate that there exists a non-Donnan contribution to the osmotic pressure, of significant magnitude, believed to result from the configurational entropy of GAG molecules (Chahine et al., 2005; Ehrlich et al., 1998). The first aim of this study is to determine this non-Donnan contribution experimentally in situ, by comparing measurements of the equilibrium compressive modulus of articular cartilage in isotonic and hypertonic NaCl, before and after nearly complete digestion of PGs. The PG digestion protocol described by Schmidt et al. (1990) is employed, which has been shown to remove most of the initial PG content, while preserving the collagen content.

Recent advances in the constitutive modeling of articular cartilage have placed an emphasis on modeling the fibrillar nature of the collagen matrix, idealizing it as a material that can sustain only tension (Ateshian et al., 2009; Korhonen et al., 2003; Soltz and Ateshian, 2000; Soulhat et al., 1999; Wilson et al., 2007). In these fiber-reinforced models of cartilage, the PGs are generally assumed to represent the ground matrix. A corollary aim of this study is to determine whether a cartilage matrix depleted of all its PGs exhibits negligible compressive modulus compared with the normal tissue, to help ascertain the validity of the basic constitutive assumption that collagen fibers contribute only to the tensile response.

To further elucidate the potential interaction of zonal variations in PG content with treatment and bathing environment, measurements of the depth-dependent compressive properties are performed across the thickness of the articular layer. Finally, since PG content and depth-dependent properties may vary with age (Williamson et al., 2001), experiments are performed on mature and immature bovine cartilage to determine whether age-related differences may increase our insight on these phenomena.

Section snippets

Materials and methods

Details regarding materials and methods are provided in the Supplementary Data (Appendix A). Briefly, six cartilage specimens were harvested each from immature and mature bovine humeral heads for mechanical testing and biochemical analysis; additional specimens were used for histology. Cylindrical disks were sub-punched and the outer ring was used for biochemical analysis. Each disk and its outer ring was cut into half and one half was digested of its proteoglycan content using the protocol of

Results

The serial digestion dissected away 97.7% of PGs from the immature tissue and 90.4% from the mature specimens, both representing a significant depletion of PG content (Fig. 1). There was also a significant increase in water content per wet weight in the immature samples (p<0.0001), and collagen content per wet weight in the mature samples (p<0.01). Histology comparing an undigested and digested immature sample shows an even depletion of PG (Fig. 2). Digested mature samples still showed

Discussion

The primary aim of this study was to examine the electrostatic and non-electrostatic contributions of proteoglycans to the compressive modulus of articular cartilage. This investigation was motivated in part by the recognition that the osmotic pressure of chondroitin sulfate solutions in various concentrations of NaCl does not reduce to zero under hypertonic conditions (Chahine et al., 2005; Ehrlich et al., 1998), implying a significant contribution from non-electrostatic effects (Fig. 8).

Conflict of interest

None.

Acknowledgments

This study was supported with funds from the National Institute of Arthritis, Musculoskeletal and Skin Diseases of the US National Institutes of Health (AR46532).

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