Abstract
The meniscus is comprised largely of type I collagen, as well as fibrochondrocytes and proteoglycans. In articular cartilage and intervertebral disc, proteoglycans make a significant contribution to mechanical stiffness of the tissue via negatively charged moieties which generate Donnan osmotic pressures. To date, such a role for proteoglycans in meniscal tissue has not been established. This study aimed to investigate whether meniscal proteoglycans contribute to mechanical stiffness of the tissue via electrostatic effects.
Following local University Ethics Committee approval, discs of meniscal tissue two millimetres thick and of five millimetres diameter were obtained from 12 paired fresh frozen human menisci, from donors < 6 5 years of age, with no history of osteoarthritis or meniscal injury. Samples were taken from anterior, middle and posterior meniscal regions. Each disc was placed within a custom confined compression chamber, permeable at the top and bottom only and then bathed in one of three solutions − 0.14M PBS (mimics cellular environment), deionised water (negates effect of mobile ions) or 3M PBS (negates all ionic effects). The apparatus was mounted within a Bose Electroforce 3100 materials testing machine and a 0.3N preload was applied. The sample was allowed to reach equilibrium, before being subjected to a 10% ramp compressive strain followed by a 7200 second hold phase. Equal numbers of samples from each meniscus and meniscal region were tested in each solution. Resultant stress relaxation curves were fitted to a nonlinear poroviscoelastic model with strain dependent permeability using FEBio finite element modelling software. Goodness of fit (R2) was assessed using a coefficient of determination. All samples were assayed for proteoglycan content. Comparison of resultant mechanical parameters was undertaken using multivariate ANOVA with Bonferroni adjustment for multiple comparisons.
36 samples were tested. A significant difference (p < 0 .05) was observed in the value of the Young's modulus (E) between samples tested in deionised water compared to 0.14M/3M PBS, with the meniscus found to be stiffest in deionised water (E = 1.15 MPa) and least stiff in 3M PBS (E = 0.43 MPa), with the value of E in 0.14M PBS falling in between (0.68 MPa). No differences were observed in the zero strain permeability or the exponential strain dependent/stiffening coefficients. The viscoelastic coefficient and relaxation time values were not found to improve model fit and were thus held at zero. The mean R2 value was 0.78, indicating a good fit and did not differ significantly between solutions. Proteoglycan content was not found to differ with solution, but was found to be significantly increased in the middle region of both menisci.
Proteoglycans make a significant electrostatic contribution to mechanical stiffness of the meniscus, increasing it by 58% in the physiological condition, and are hence integral to its function. It is important to include the influence of ionic effects when modelling meniscus, particularly where fluid flow or localised strain is modelled. From a clinical perspective, it is critical that meniscal regeneration strategies such as scaffolds or allografts attempt to preserve, or compensate for, the function of proteoglycans to ensure normal meniscal function.
