This study shows that water content and substrate density affect both the storage and loss stiffnesses of articular cartilage. The results indicate that the storage stiffness, \({\mathrm{k}}^{\mathrm{^{\prime}}}\), increases with reducing tissue hydration (from 76% to 8.5% water mass fraction); but subsequently reduces as the tissue water content continues to reduce. Because, in this study, only three levels of hydration were used, we are unable to say at what level of hydration a turning point occurs. The stage of RH-1% represented a quasi-dehydrated state (approximating ≈ 0% water content). These findings suggest a non-linear relationship between \({\mathrm{k}}^{\mathrm{^{\prime}}}\) and hydration for articular cartilage. A different trend was apparent when comparing \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) and water content of cartilage; with a reduction in \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) seen for all samples when the water content was decreased. Increasing the substrate density resulted in an increase in both \({\mathrm{k}}^{\mathrm{^{\prime}}}\) and \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) of the cartilage sample. Thus, this study suggests that an increase in bone density increases osteochondral dynamic stiffness. If it is important for the osteochondral construct to maintain a constant stiffness. A response to counteract any initial increase in substrate stiffness (caused by sclerosis of bone) could be through increased swelling as this study has shown that a decrease in water content may result in an increase of \({\mathrm{k}}^{\mathrm{^{\prime}}}\).
This study has shown that there may be a non-linear relationship between hydration and \({\mathrm{k}}^{\mathrm{^{\prime}}}\). \({\mathrm{k}}^{\mathrm{^{\prime}}}\) increased as tissue water content decreased 76% to 8.5%. For both 76% and 8.5%, \({\mathrm{k}}^{\mathrm{^{\prime}}}\) was frequency-dependent. However, \({\mathrm{k}}^{\mathrm{^{\prime}}}\) subsequently decreased as hydration was further reduced to ≈ 0%. In addition, at ≈ 0% the frequency-dependency of \({\mathrm{k}}^{\mathrm{^{\prime}}}\) was less clear. This finding, therefore, implies that the ability of cartilage to store energy (available for subsequent recoil following loading), as well as the frequency-dependency of this ability to store energy for recoil, is dependent on its water content.
A reduction in \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) will result in a reduction in the energy dissipated by articular cartilage. At ≈ 0% \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) ranged from 55 N/mm to 46 N/mm, approximately a quarter of the values for \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) at 76%. If the swelling pressure of cartilage was solely responsible for the dissipation of energy, one might expect \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) to tend to zero as water content approached zero. This study has shown this is not the case, and therefore fluid interaction with collagen may be important for energy dissipation; indeed, collagen itself may have an intrinsic ability to dissipate energy. This is in agreement with Sadeghi et al. [22], who performed DMA at low frequencies (0.001 Hz) to allow time for fluid dissipative effects to occur (following time-scales of loading which mimicked those necessary to achieve peak pressure). If fluid alone was responsible for dissipation of energy, \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) would be expected to increase at low frequency; however, Sadeghi et al. demonstrated this not the case, with \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) being frequency independent at loading frequencies well below those which are relevant during normal gait.
The results reported by Pearson & Espino [20] for hyper- and hypo-hydration levels tested were likely in the range between 76% and 8.5%, given the test methods employed and the increase reported for \({\mathrm{k}}^{\mathrm{^{\prime}}}\). However, it is unlikely that their hyper-hydrated samples achieved 100% hydration or that their hypo-hydrated samples achieved the low levels used in this study at RH-30%. A comparison of the hyper-hydrated storage and loss values measured by Pearson & Espino and values at RH-100% in this study, show comparable results [20]. Pearson & Espino reported that they measured no statistically significant change in sample thickness; from their results, it is estimated that their samples underwent a 1 – 5% reduction in mass through dehydration, much lower than the 67% reduction in mass found in this study at RH-30%. This suggests that their values for hyper- and hypo-hydration would have been measured at hydration levels just above and below physiological conditions, respectively. When comparing this study to Pearson & Espino [20], it is important to note that they tested specimens on-bone, as opposed to the off-bone cartilage cores used in this study. The attachment to the underlying subchondral bone has been shown to alter \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) and its frequency-dependency [18]. Restraining cartilage may also affect its ability to dissipate energy [38].
To date, there have been few studies of the relationship between hydration and the dynamic mechanical behaviour of articular cartilage. As Pearson et al. analysed two values of hydration, it is not possible to evaluate any non-linear trends in viscoelastic properties that may be present over a wider range [20]. The effect of osmolarity on the viscoelastic properties has been studied, showing an increase in dynamic modulus as osmolarity was decreased from approximately physiological (0.2 M) to 0.0015 M [39]. Cartilage water content was not reported but it is likely that this corresponds to an approximate range of water content from physiological to hyper-hydrated. Although this is similar in range to Pearson et al., the differences in methodology limit the direct comparison of data from those studies. Certainly, the technique used in this current study enables the water content of cartilage to be directly controlled, and for its effect on the viscoelastic behaviour of cartilage to be evaluated. There remains scope to evaluate the non-linearity in viscoelastic properties in between the range of hydration parameters evaluated in our current study, and the effects of hydration on the underlying subchondral bone.
It is important to note that our current study has not directly replicated the in-vivo physiological conditions of articular cartilage. For example, this study has ignored the water exchange between living cartilage and the synovial fluid which may affect surface lubrication and, therefore, the mechanical response. Surface lubrication is tangential to this current study; with the role of surface proteins being important [40, 41] and of interest to consider for future work. However, this study has carefully controlled experimental parameters in order to reduce variability between samples tested, isolating the role of water on the viscoelastic properties of cartilage.
An increase in density of cartilage substrate has been shown to increase \({\mathrm{k}}^{\mathrm{^{\prime}}}\) and \({\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) in this study. Further, the ratio of the ability of cartilage to store/dissipate energy (\({\mathrm{k}}^{\mathrm{^{\prime}}}/{\mathrm{k}}^{\mathrm{^{\prime}}\mathrm{^{\prime}}}\) ratio) on a substrate of density 156.8 kg/m3 was 10.0 at 1 Hz and 18.2 at 88 Hz. This increased to 12.7 at 1 Hz and 19.0 at 88 Hz when the substrate density increased to 663.7 kg/m3. Therefore, the potential to store excessive energy increases with the density of underlying material. This finding is in agreement with recent studies such as that by Mahmood et al. which found an increased predisposition of cartilage to fail at frequencies above those of normal gait when combined with an increased subchondral bone density [35]. Our findings are also in broad agreement with those by Fell et al., who found a positive correlation between loss modulus of cartilage and subchondral bone density [34]. However, the findings by Fell et al. may also relate to remodelling of bone and cartilage which are not the subject of this current study.
Although the densities of substrate chosen in this study do not model the properties of healthy and osteoporotic bone, they have been chosen as they enable density to be varied in a controlled manner. It has been previously shown that a variation of subchondral bone density correlates with a variation in the mechanical properties of the corresponding cartilage [34]. This results in two dependant variables, with both the mechanical behaviour of the cartilage and the underlying subchondral bone varying between samples, making it difficult to draw conclusions about the impact of only one of these variables. Therefore, in this study, we have aimed to isolate the effect of only a change in substrate density. As the substrate used in this study is a synthetic material of a known density it has not been tested independently. Using this idealised scenario, a recent study evaluated the effect of substrate density on cartilage surface damage [35].
Both an increase in BMD (through subchondral sclerosis), and an increase in water content, are believed to occur during the early onset of OA [42]. An increase in subchondral BMD might occur in response to changes in mechanotransduction [43], however, sclerosis of subchondral cortical bone may also occur without mechanical derangement [44]. From this current study, the density of a substrate under articular cartilage clearly affects the ability to dissipate energy of the cartilage-substrate structure. In the short term, increased density of the subchondral bone aiding the dissipation of energy may be advantageous, as this may reduce damage induced in cartilage, which is less capable of repairing itself compared with bone. However, if an increase in BMD is chronic, it may in the long-term increase the predisposition to failure of cartilage. If subsequent changes in cartilage include increased water content, then cartilage might be further at risk of mechanical failure. It is important to note that stress induced damage due to repetitive, over-loading is not the only potential factor when looking at the prognosis of OA, with metabolic factors implicated in the matrix-metalloproteinase related weakening of the collagen structure [45].
This current study has shown that the water content of cartilage affects the ability of cartilage both to store and to dissipate energy. It is, therefore, likely that stress transfer between collagen and its surrounding matrix [46,47,48] (with proteoglycans attracting water), with energy stored and dissipated during this process, dictates the viscoelastic behaviour of cartilage. However, it should be noted that other factors may cause energy dissipation in the cartilage-bone construct. For example, Becher et al. have shown that the intra-articular temperature increased by 6.1 °C after 60 min of jogging [49], whilst modelling of the knee joint suggested a potential cartilage temperature increase of 1.2 °C after 10 min of loading under conditions expected whilst walking [50]. To date, cartilage temperature changes have not been measured during dynamic loading and could provide further insight in future work. Although the water content of cartilage may increase, changing the hydration of healthy cartilage is an oversimplification of the pathogenesis of OA. For example, the increased water observed during early-onset OA is due to altered synthesis of proteoglycans [51], as opposed to saturating the proteoglycans in healthy cartilage. Better understanding of the mechanisms by which water is physically held within cartilage, and their alteration during OA, may benefit further understanding of the mechanical behaviour of both healthy and OA cartilage. Although this study has not aimed to mimic the physiological conditions of cartilage, it has provided further insight into the effects of these variables on the mechanical behaviour of cartilage. The exploration and understanding of the relationships between the viscoelastic properties of cartilage, and parameters such as hydration and substrate density could aid in understanding the mechanical behaviour of osteoarthritic cartilage in a controlled manner [52] and allow a more targeted approach to cartilage repair or the design of bioinspired materials [53].
Conclusion
This study has found that a decrease in hydration will cause a decrease in the loss stiffness of articular cartilage and that a non-linear relationship may exist between hydration and the storage stiffness of cartilage. This study also found that both storage and loss stiffnesses of cartilage increase as the substrate density increases, which suggests greater likelihood of cartilage failure with increasing density of the underlying bone.