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

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 3kPa, 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.

Figure 1

Proteoglycan (PG), collagen and water as a percentage of wet weight for the four sample groups, immature untreated, immature digested, mature untreated and mature digested. (* p<0.005)

Figure 2

Safranin O staining of untreated (A) and digested (B) immature cartilage samples showing uniform depletion of PG content through the depth.

Figure 3

Curling exhibited in PG depleted mature samples. SZ = superficial zone, DZ = deep zone.

Figure 4

Sequential compression (0 - 6%) of a mature digested sample. Thin white lines indicate delineations between superficial, middle and deep zones as determined from polarized light microscopy on other, age-matched samples. Arrows indicate areas of visible crimping in the solid matrix.

Figure 5

Summary of the apparent compressive Young's modulus, evaluated from the slope of the stress versus engineering strain, for all sample groups at both 0.15M and 2M bath concentrations; *p<.0001 and **p<0.01.

Figure 6

Incremental Young's moduli for immature sample groups over the applied strain range shown for both untreated (top, n=6) and digested (bottom, n=6) tested in 0.15M (left) and 2M (right) solutions. (Note the difference in the range of the ordinate axis between untreated and digested samples.) Depth-varying moduli are locally averaged within each zone (depth): superficial, middle and deep. Significance differences in moduli within individual plots are denoted by *p < 0.05 or ** p<0.0001 for depth, and †p<0.005 and ††p<0.001 for significance in applied strain.

Figure 7

Incremental Young's moduli for mature sample groups over the applied strain range shown for both untreated (top, n=6) and digested (bottom, n=6) tested in 0.15M (left) and 2M (right) solutions. (Note the difference in the range of the ordinate axis between untreated and digested samples.) Depth-varying moduli are locally averaged within each zone (depth): superficial, middle and deep. Significance differences in moduli within individual plots are denoted by *p < 0.05 for depth, and †p<0.005 for significance in applied strain.

Figure 8

Equilibrium osmotic pressure of chondroitin sulfate solutions measured from direct membrane osmometry, as a function of fixed charge density (25°C), in various NaCl concentrations. Solid lines indicate the corresponding virial expansion polynomial fits. Reproduced from Chahine et al. (2005) with permission.