Contribution of Glycosaminoglycans to the Viscoelastic Tensile Behavior of Human Ligament

Introduction: Viscoelastic behavior is intrinsic to human ligament and may guard against structural failure. Glycosaminoglycans (GAGs) have been implicated as macromolecules that likely impact ligament viscoelasticity [1-2]. GAGs selfassociate [3] and may mechanically connect adjacent collagen fibrils and thus influence fibril sliding during tension. Although studies have shown GAGs to influence viscoelasticity in other connective tissues [4, 5], no study has specifically determined the effect of GAGs on ligament viscoelasticity. Therefore, the aim of this study was to determine whether GAGs influence the viscoelastic material behavior of ligament under tension. Determination of microstructural influences on tissuescale viscoelasticity can improve the innovation and evaluation of ligament treatment modalities. Materials and Methods: Eleven unpaired human MCLs were used for this experiment. Four tensile samples were extracted from each superficial MCL. Tensile loading was performed along the fiber direction in a testing chamber that maintained the buffer solution at 37°C. After tensile testing was completed, samples were removed from the material testing system and incubated for six hours in a control or chondroitinase ABC (ChABC) treatment. Control treatment was the buffer solution and ChABC treatment consisted of the buffer solution with 1 U/mL ChABC. Following incubation, mechanical testing was then repeated on each specimen using parameters identical to the pre-treatment mechanical test. Samples were immersed in the buffer solution [6] during equilibration (2 hr), preand post-treatment mechanical tests (1 hr) and incubation (6 hr). Experiments were also performed with the buffer solution plus 7.5% polyethylene glycol (PEG), which inhibits tissue swelling [7]. Independent variables included treatment, buffer solution and fiber strain level. Eight groups were tested with n=5 samples in each group, for a total of 40 samples. The testing protocol involved preconditioning, a recovery period and viscoelastic testing. A 0.1 N preload was first applied to establish a consistent reference position. To precondition the samples, the tissue was ramped at 1.0%/sec to 8% of the reference length, and held for 5 minutes. This was immediately followed by a triangular displacement profile applied to 8% of the reference length for ten cycles at a strain rate of 1%/sec. After preconditioning, the sample recovered for 10 minutes at its reference length. The specimen was then ramped at 1%/sec to a prescribed strain level (4% or 6%) and allowed to stress-relax for 10 minutes. Finally, ten cycles of sinusoidal displacement waves at 0.125% strain amplitude were applied for five frequencies (0.1, 1, 5, 10, and 15 hz). To monitor tissue hydration during testing, a strip of tissue was harvested adjacent to each tensile sample. This strip shadowed the testing protocol of the mechanically tested specimen in an unloaded state. The efficacy of ChABC treatment was determined by using dimethylmethylene blue assays [7]. Stress relaxation was calculated by normalizing stress-time results to peak stress. Phase shift and dynamic modulus were calculated by fitting the oscillation data to a four-parameter sine function in Matlab [8]. The effect of incubation was measured using paired t-tests, while the effect of independent variables was measured with ANCOVA, by controlling for pre-treatment results. Results: ChABC reduced total GAG content by 83% in the buffer solution and by 64% in the buffer+PEG solution, respectively. Ligament wet weight increased by 18 ± 2% in the buffer solution and decreased by 11 ± 2% in the buffer+PEG solution. The wet weights did not significantly change during mechanical testing.