Measuring fixed charge density of goat articular cartilage using indentation methods and biochemical analysis

An important indicator of osteoarthritis (OA) progression is the loss of proteoglycan (PG) aggregates from the cartilage tissue. Using the indentation creep test, two analytical methods, as previously developed by Lu et al. [Lu, X. L., Miller, C., Chen, F. H., Guo, X. E., Mow, V. C., 2007. The generalized triphasic correspondence principle for simultaneous determination of the mechanical properties and proteoglycan content of articular cartilage by indentation. Journal of Biomechanics 40, 2434-2441 (EPub).], for predicting the fixed charge density (FCD) of goat knee articular cartilage in the normal (control) and degenerated states were compared: (1) a "dual-stage" method to calculate FCD from the mechanical properties of the tissue when tested in isotonic and hypertonic solutions; and (2) a "single-stage" method to predict FCD (as in (1)) assuming an intrinsic Poisson's ratio of 0.05 in the hypertonic state. A biochemical analysis using 1,9-dimethylmethylene blue (DMMB) assay was conducted to directly measure PG content, and hence FCD. The association between the FCD and the aggregate modulus of the tissue was also explored. The mean (+/-S.D.) FCD values measured using the dual-stage method were the closest (control: 0.129+/-0.039, degenerated: 0.046+/-029) to the DMMB results (control: 0.125+/-0.034, degenerated: 0.057+/-0.024) as compared to those of the single-stage method (control: 0.147+/-0.035, degenerated: 0.063+/-0.026). The single-stage method was more reliable (r(2)=0.81) when compared to the dual-stage method (r(2)=0.79). A prediction of FCD from the aggregate modulus generated the least reliable FCD prediction (r(2)=0.68). Because both the dual- and single-stage methods provided reliable FCD estimates for normal and degenerated tissue, the less time-consuming single-stage method was concluded to be the ideal technique for predicting FCD and hence PG content of the tissue.