The purpose of the present study was to investigate the effects of mechanical strain, anisotropy, and tissue region on electrical conductivity and ion diffusivity in meniscus fibrocartilage. A one-dimensional, 4-wire conductivity experiment was employed to measure the electrical conductivity in porcine meniscus tissues from two tissue regions (horn and central), for two tissue orientations (axial and circumferential), and for three levels of compressive strain (0%, 10%, and 20%). Conductivity values were then used to estimate the relative ion diffusivity in meniscus. The water volume fraction of tissue specimens was determined using a buoyancy method. A total of 135 meniscus samples were measured; electrical conductivity values ranged from 2.47 mS/cm to 4.84 mS/cm, while relative ion diffusivity was in the range of 0.235 to 0.409. Results show that electrical conductivity and ion diffusion are significantly anisotropic (p<0.001), both being higher in the circumferential direction than in the axial direction. Additionally, the findings show that compression significantly affects the electrical conductivity with decreasing conductivity levels corresponding to increased compressive strain (p<0.001). Furthermore, there was no statistically significant effect of tissue region when comparing axial conductivity in the central and horn regions of the tissue (p=0.999). There was a positive correlation between tissue water volume fraction and both electrical conductivity and relative ion diffusivity for all groups investigated. This study provides important insight into the electromechanical and transport properties in meniscus fibrocartilage, which is essential in developing new strategies to treat and/or prevent tissue degeneration.
The fibrocartilaginous meniscus is a charged, hydrated soft tissue important for load distribution, maintaining congruency, and aiding in lubrication in the knee (Makris et al., 2011). The tissue has a composition more similar to temporomandibular joint (TMJ) cartilage than hyaline cartilage, with high water content (~70%), and the remaining comprising mostly collagen (~75% dry weight, primarily type I) with small quantities (2–3% dry weight) of proteoglycans (PGs) (Almarza and Athanasiou, 2004). PGs are large, negatively charged molecules formed by glycosaminoglycans (GAGs) linked to a core protein; the negatively charged anions attached to GAG molecules attract positive cations in the surrounding fluid thus creating the Donnan osmotic pressure. This contributes to tissue hydration and related compressive properties, as well as allowing for the mechano-electrochemical responses in the tissue (Fithian et al., 1990, Hardingham and Fosang, 1992, Mow et al., 1999 and Sweigart and Athanasiou, 2001).
Electrical conductivity is an important material property of biological tissues that depends on ion diffusivities and concentrations within the tissues, which are related to tissue composition and structure (Frank et al., 1990 and Maroudas, 1968). The electrical conductivity of several cartilaginous tissues has been investigated, see Table 1. These studies have found that conductivity is directly correlated to tissue water content (Gu and Justiz, 2002, Gu et al., 2002, Gu et al., 2004, Jackson et al., 2009, Kuo et al., 2011 and Wright et al., 2013), and is strain-dependent (Jackson et al., 2009, Kuo et al., 2011 and Wright et al., 2013). Better understanding of electromechanical properties of tissues, including conductivity and ion transport, and their relationship to tissue composition and relevant loading conditions, can provide essential information about endogenous electrical signals, which play a key role in directing resident cellular activity.
Electrical conductivity can be used to estimate the relative ion diffusivity in a tissue (Gu et al., 2004, Jackson et al., 2006, Kuo et al., 2011 and Wright et al., 2013). Elucidating transport properties in meniscus is important given that much of the adult meniscus is avascular (Makris et al., 2011). As a result, essential nutrients are supplied by vasculature in outer tissue regions and surrounding synovial fluid. Solute concentrations in the tissue are related to transport rates through the ECM (i.e., solute diffusivities). Thus, better understanding transport properties in meniscus can provide necessary information regarding the chemical environment in the tissue.
Increased knowledge of electromechanical and transport properties in meniscus is important for fully understanding structure-function relations in the tissue. Such information is valuable in developing novel strategies for meniscus repair and/or regeneration (e.g., tissue engineering or drug delivery approaches) and can be employed in theoretical modeling, used to predict the in vivo environment in the meniscus. To our knowledge, no previous study has investigated the electrical conductivity and/or ion diffusivity in meniscus fibrocartilage. We hypothesized that electrical conductivity and ion diffusivity in porcine meniscus is strain-dependent, anisotropic, and region-dependent. Therefore, our objective was to measure the electrical conductivity of porcine meniscus from two tissue regions, in two directions, and under three levels of compression. This information was then used to estimate relative ion diffusivity in the tissue.
Fifteen menisci were harvested from the knees of 9 pigs (Yorkshire, ~20–25 weeks) obtained from a local abattoir within one hour of death. Cylindrical specimens (d=5 mm, h=3.06±0.27 mm) were punched using a corneal trephine and trimmed to the desired height using a microtome with freezing stage. Specimens were harvested from either the central or horn region in either axial or circumferential orientation, see Fig. 1(a). A total of nine groups were investigated, including three orientation/regions [axial central (A–C), axial horn (A–H), circumferential central (C–C)] and three levels of compressive strain (0%, 10%, 20%). In each group, fifteen (n=15) samples were measured, for a total of 135 tissue specimens; only one conductivity measurement was taken on each sample.
The water volume fraction of undeformed specimens was measured using a buoyancy method (Gu et al., 1996). The weight of the specimen in air, Wwet, and in phosphate buffered saline (PBS), WPBS, were measured using a density determination kit of an analytical balance prior to experimentation. Following conductivity measurements, specimens were lyophilized, and the weight of the dry specimen, Wdry, was measured. The volume fraction of water, View the MathML source, of the specimens was calculated by:
where ρPBS and ρH2O are the mass densities of PBS and water, respectively. The volume fraction of water in the compressed tissue (ϕw) could be estimated based on the tissue dilatation, e (Lai et al., 1991):
For one-dimensional confined compression, the dilatation is equal to the compressive strain (e.g., for 10% compressive strain, e=−0.1).
The custom conductivity apparatus is similar to that in the literature (Gu and Justiz, 2002 and Jackson et al., 2009), see Fig. 1(c). Briefly, the apparatus consists of two stainless steel current electrodes, two Polyester-coated Ag/AgCl voltage electrodes, a metal spacer, and a non-conductive acrylic chamber. A four-wire method was applied using a sourcemeter. The resistance, Ω, across the tissue sample was measured at a low constant current of 10 µA (current density =0.051 mA/cm2). Electrical conductivity (χ) is related to resistance by:
where A is the cross sectional area and h is the thickness of the sample.
Conductivity measurements were taken at 0%, 10%, or 20% compression. Prior to measurements, tissue specimens were compressed to the desired thickness via uniaxial confined compression between two porous plates in a separate compression chamber based on initial height measurement and desired strain level, see Fig. 1(b). After compression and a brief equilibration period, the compressed sample was moved to the custom conductivity apparatus; the level of compression was maintained by controlling the distance between current electrodes using metal spacers. For each experiment, several resistance measurements were obtained at 10 min intervals to ensure the tissue had reached equilibrium in the chamber; that is, measurements were repeated until the same resistance value (within 5%) was measured for two consecutive readings, signifying equilibrium was reached (i.e., no fluid flow).
The electrical conductivity of a charged porous material is related to the diffusivity of its intrinsic cations and anions (Di, i=+,−) under zero fluid flow conditions by (Frank et al., 1990, Gu et al., 2004, Helfferich, 1962 and Maroudas, 1968):
where Fc is the Faraday constant, R is the gas constant, T is the absolute temperature, ϕw is the water volume fraction, c+ is the cation concentration, and c− is the anion concentration. Due to the low GAG content (~2–3% dry weight) in meniscus compared to other cartilaginous tissues, it was considered an uncharged tissue. The relative diffusivity (D/Do) of NaCl can be related to the conductivity measurements by (Gu et al., 2004, Kuo et al., 2011 and Wright et al., 2013):
where χo is the conductivity of the bathing solution. This value is the averaged relative diffusivity of Na+ and Cl− ions, which were assumed to carry the current as the primary ions in PBS solution (Kuo et al., 2011 and Wright et al., 2013).
A total of 9 pigs were tested (n=9) with three factors (location: A–C, A–H, and C–C) and three levels per factor (compression level: 0%, 10%, 20%). The total measurements taken were 135. Three independent variables were studied for each sample: level of compression, direction of diffusion, and regional location. Statistical significance between groups was determined by two-way repeated measures ANOVA analysis of variance tests using IBM SPSS Statistics 22 with multiple pairwise comparisons using SIDAK post-hoc methods to control for the alpha level, which was set at p<0.05; sample size was set to n=9.
One-way ANOVA was also performed to determine if water volume fraction and height measurements differed significantly between the three test groups (A–C, A–H, C–C). Regression analysis was performed to determine if relationships between electrical conductivity or ion diffusivity and water volume fraction and strain level were statistically significant.
A total of 135 specimens were measured, with an average uncompressed water volume fraction of 0.70±0.04; results for all groups are shown in Table 2. The relationship between mechanical compression and the electrical conductivity and ion diffusivity in porcine meniscus for all groups is shown in Fig. 2. Electrical conductivity and ion diffusivity significantly decreased with increasing level of compression for all three region/direction groups (p<0.001). In addition, both conductivity and relative ion diffusion in the circumferential direction were significantly greater than that in the axial direction in the central region. Two-way repeated measures ANOVA indicated that electrical conductivity and ion diffusivity were significantly affected by both compression (p<0.001) and direction of diffusion (p<0.001) when comparing A–C and C–C groups. However, two-way repeated measures ANOVA showed no significant effect of region when comparing A–C and A–H groups (p=0.999), although a significant compression effect was still seen (p<0.001). SIDAK post-hoc analysis indicated that circumferential conductivity and diffusivity was significantly (p<0.001) higher than that in the axial direction at all three levels of compression. SIDAK post-hoc analysis also showed that, for each group, when comparing both electrical conductivity and ion diffusivity at 0% and 10%, 0% and 20%, and at 10% and 20% compression, results were significantly (p<0.05) different. There was no significant difference between the height or water volume fraction of any of the groups (ANOVA, p<0.05).
The goal of this study was to determine the effect of compressive strain, anisotropy, and tissue region on the electrical conductivity and ion diffusivity in porcine meniscus. We found significant strain-dependent and anisotropic behaviors, while no significant regional variation for conductivity or ion diffusivity in meniscus tissues was seen. Overall, our values for conductivity and ion transport are comparable to those in the literature for other cartilaginous tissues, and are most similar to values for porcine TMJ (Table 1). This is likely because meniscus composition is most like that of TMJ tissue, which also has a relatively low GAG content as compared to articular cartilage or intervertebral disc (Almarza and Athanasiou, 2004).
The strain-dependent behavior of electrical conductivity and relative ion diffusivity found here is similar to results in the literature for other cartilaginous tissues (i.e., meniscus, articular cartilage, intervertebral disc, TMJ), which showed that static compression leads to reduced solute diffusivity and/or electrical conductivity (Jackson et al., 2009, Jackson et al., 2008, Kleinhans et al., 2015, Kuo et al., 2011, Quinn et al., 2000, Quinn et al., 2001, Wright et al., 2013 and Yuan et al., 2009). This change is likely due to reduced tissue water content caused by fluid exudation during compression. In fact, we found a significant positive correlation between conductivity and ion diffusivity and tissue water volume fraction, see Fig. 3; previous studies have found similar correlations (Gu and Justiz, 2002, Gu et al., 2002, Gu et al., 2004, Jackson et al., 2009, Kuo et al., 2011 and Wright et al., 2013). Overall, these findings indicate that solute transport through the tissue is hindered by mechanical loading.
Electrical conductivity and ion diffusivity in porcine meniscus was also found to be anisotropic (i.e., direction-dependent). We believe this behavior is a result of the particular organization of the tissue ECM; that is, collagen fibers in meniscus are aligned along the circumferential direction, allowing for pores that are not apparent in the axial direction. This pore structure, similar to that found for other cartilaginous tissues (ap Gwynn et al., 2002, Iatridis and ap Gwynn, 2004, Jackson et al., 2009, Kleinhans et al., 2015 and Travascio et al., 2009), may allow ions to move more freely in the circumferential direction, parallel to the collagen fiber bundles, resulting in higher conductivity and ion transport rates. Other studies have also found the diffusion coefficient to be significantly higher in the direction parallel to collagen fibers (i.e., circumferential) in other cartilaginous tissues (Leddy et al., 2006 and Stylianopoulos et al., 2010). These trends are similar to earlier findings for glucose diffusivity in porcine meniscus (Kleinhans et al., 2015).
There was no significant regional variation in electrical conductivity, or ion diffusivity, in porcine meniscus when comparing the A–C and A–H groups. Previous studies investigating mechanical properties, solute diffusivity, and fluid transport (i.e., hydraulic permeability) in meniscus have found mixed results regarding the inhomogeneity of tissue properties, see review (Makris et al., 2011). While we found no significant difference between horn and central regions, these findings may be attributed to the fact that comparisons were only made for a relatively small sample size of axially oriented tissue specimens. However, because of the anisotropic trend, conductivity and relative ion diffusivity in other directions (e.g., circumferential) may be affected by tissue region and deserves further study.
Although this study investigated porcine tissues, previous studies have shown that porcine meniscus has similar structure and composition to human meniscus (Almarza and Athanasiou, 2004, Chu et al., 2010, Joshi et al., 1995, Sweigart and Athanasiou, 2005 and Sweigart et al., 2004); thus general findings may be translated to better understanding of human tissues. More studies are needed to investigate the effects of important physiological factors, such as tissue degeneration, on these tissue properties for human meniscus. Additionally, while uniaxial confined compression does not precisely mimic the in vivo strain configurations in the meniscus, the data and trends gained here can be incorporated into computational models of the tissue to better understand in vivo conditions and predict the overall impact of mechanical loading on the tissue chemical environment. Overall, the findings of this study provide important insight and baseline quantitative information regarding the strain-dependent, anisotropic, and region-dependent behavior of electromechanical and transport properties in meniscus fibrocartilage, and establish the methods necessary to move toward full characterization of the tissue.
The authors declare no potential conflicts of interest regarding the authorship and/or publication of this research article.
This research was funded by the University of Miami College of Engineering. The authors would like to thank Dr. Soyeon Ahn for her expert advice for statistical analysis of the data. The authors would also like to thank Lukas Jaworski and Adam Bofill for assistance in apparatus development.
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