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Injuries to the inner regions of the knee meniscus do not heal and can result in degenerative changes to the articular surface, ultimately leading to osteoarthritis. A possible stimulus to enhance meniscus healing is to use electric fields that induce galvanotaxis. In this study, a novel characterization of the effects of direct current electric fields on migration characteristics of meniscus cells was performed. Primary and passaged inner and outer meniscus cells were exposed to varying electric field strengths from 0 to 6 V/cm. Cell migration was tracked using time lapse digital photography, and cell displacement and cathodal direct velocity were quantified. Cytoskeletal staining was performed to examine actin distribution and nuclear content. Cell adhesion strength was quantified as a function of wall shear stress. Meniscus cells exhibited cathodal migration and cell elongation perpendicular to the applied electric field accompanied by actin reorganization. Outer meniscus cells migrated quicker and exhibited lower adhesion strengths when compared to inner meniscus cells. Passaged cells exhibited higher migration characteristics when compared to primary cells. Overall, this study demonstrated that electric fields can significantly enhance and direct meniscus cell migration and suggests the potential for their incorporation in strategies of meniscus repair and tissue engineering. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:103–111, 2012
The knee meniscus is a fibrocartilaginous semi-lunar shaped disc located on the tibial plateau. It serves several functions, including load transmission, shock absorption, and joint lubrication. Although several sub-types of cells are present in the meniscus,1 the tissue can be divided into an outer region containing mainly fibroblast-like cells that are elongated or spindle-shaped and an inner region containing mainly chondrocyte-like cells that are rounded or spherical. Meniscus cells are responsible for maintaining homeostasis, and their metabolic activity depends on the forces exerted on the tissue. Although the origin of the different cell types is unclear, it is thought that during embryonic development the meniscus is formed by precursor cells expressing collagen type 2a and by cells that invade the joint and are not part of the anlagen.2
About 1 million surgeries are performed each year to repair meniscal lesions and tears.3 The current gold standard is to leave small tears untreated and remove partial tears arthroscopically; however, this technique can lead to increased stresses on the articular surface. Ideally, clinical practice should aim at meniscal preservation, and repair techniques using polymer-based arrows, darts, screws, staples, and other suture devices are being investigated.4 None of these approaches, however, has achieved broad clinical acceptance as concerns of insufficient biomechanical stability and delayed or early polymer degradation are high. The potential of bioelectrical stimuli to aid in the repair process of soft tissues is largely unexplored. This is surprising since musculoskeletal tissues such as bone and muscle exhibit electrical activity during injury,5, 6 and endogenously generated electric fields are known to guide cell migration during wound healing and embryonic development.7–9 Of particular interest are direct current (DC) electric fields that induce galvanotaxis and in some instances galvanotropism. Although these effects are cell and species dependent, most cell types move either to the cathode or the anode with only a few cell types insensitive to electric fields.10
Thus, DC electric fields may induce cell migration towards the site of meniscal injury or into scaffolds for tissue engineering. In this study, we performed a novel characterization of the effects of varying DC electric fields and cell passage number on the migration characteristics of inner meniscus (IM) and outer meniscus (OM) cells in vitro. We hypothesized that meniscus cells would exhibit cathodal migration and increased cell speeds at higher electric field strengths and passage numbers. We also hypothesized that OM cells would migrate quicker than IM cells and exhibit lower adhesion strengths.
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We characterized the effects of DC electric fields on meniscus cells plated in 2D culture. Primary IM and OM cells likely contain a less homogeneous cell population than passaged cells. Thus, most experiments were performed with once passaged IM and OM cells. In response to an electric field, IM and OM cells exhibited cathodal migration and cell elongation perpendicular to the applied electric field (i.e., galvanotropism) accompanied by actin reorganization. Furthermore, higher passage cells migrated quicker than cells at lower passages. Finally, the adhesion strengths of IM cells were found to be significantly higher than that of OM cells.
Damage to the knee meniscus can lead to a poor healing response due to a limited supply of vasculature. Thus, a vicious cycle ensues in which the normal inflammatory response and clotting cascade responsible for tissue healing and repair does not occur. As such, techniques to recruit cells to the site of injury may be particularly important for these tissues, and exogenous stimuli via electric fields may provide a mode to achieve this. Meniscal allografts11 and devitalized meniscal plugs12 can be repopulated with cells when placed in vivo. External stimuli, such as radiofrequency waves, enhance and direct synovial migration to heal meniscal defects in rabbits.13 Given the correct conditions, meniscus cells close to the injury site may be able to participate in self-repair. This study examined meniscus cell migration in a 2D environment to identify appropriate exogenous stimuli necessary for enhanced and directed cell migration. Cell speed and directionality were examined at electric field intensities ≤6 V/cm as prior studies with chondrocytes showed some cell death at field strengths >6 V/cm.14 Significant increases over the controls in cell speed and directed cathodal velocity were only observed at the higher electric field strengths. Outer, fibroblast-like meniscus cells migrated more quickly than inner chondrocyte-like meniscus cells; however, distances traveled were in the range of ligament fibroblasts for the OM cells and articular chondrocytes for IM cells.14, 15 Ensuing experiments will examine meniscus cell migration in 3D within a collagen gel that would more closely mimic the native tissue.
Migration results for controls were mirrored by adhesion tests that showed IM cells exhibited higher adhesion strength than OM cells on a glass surface. This is consistent with observed cell morphology and phenotype of IM and OM cells in culture and confirms previous findings that fibroblasts migrate more quickly than chondrocytes.15 Adhesion strengths vary based on surface chemistry, plating time, and shear application time. In our experiment, the first two were kept constant (glass surface and 1 h plating), and the time of shear application was varied. Interestingly, for IM cells, no significant differences were observed among groups until shear stresses exceeded 266 dynes/cm2, suggesting that integrins responsible for cell-surface adhesions stabilize and make connections to the surface relatively quickly.16 In contrast, OM cells exposed to 10 min intervals of step increases in shear stress exhibited lower adhesion strengths when compared to respective 1 min controls at shear stresses >59 dynes/cm2. This suggests that integrins on the OM cell surface take longer to stabilize and make cell-surface connections under the influence of external fluid flow. This disparity may also be explained by intrinsic cellular differences between IM and OM cells. OM cells express MMP-2 and MMP-3 proteases that are important for matrix remodeling and may regulate integrin formation and stability.17 IM cells express α-smooth muscle actin, an important component in formation and stability of focal adhesions on the cell surface.18
Meniscus cells elongated and aligned perpendicularly to applied electric fields with the majority of cells increasing their motility towards the cathode, especially at higher electric field strengths. Due to the polar nature of water and negative surface charge associated with the chamber and cell surfaces,19, 20 in these studies, a concomitant electro-osmotic flow was directed from the anode to the cathode.20 However, the influence of these flow fields is likely minimal as cells generally align parallel to the direction of fluid flow,21 while in our experiment perpendicular alignment was observed. The mechanisms involved during this transformation include changes at both the cellular and molecular level.10 At the cellular level, cell elongation and alignment is achieved through stabilization of microtubules and reorganization of actin stress fibers.22 Actin reorganization is the major driver as its inhibition disrupts migration completely, while microtubule disruption results in reduced cell speed, but does not affect cell motility.23 At the molecular level, cytoskeletal rearrangement is likely influenced by an influx of ions such as calcium, through voltage-gated24 or stretch-activated channels.25 These ions influence actin polymerization and depolymerization dynamics and myosin contractility.10 Electric fields also redistribute and activate growth factor receptors on the cell surface.26 Cells contain charged surface molecules on their plasma membrane that may shift during electric field application, thereby altering and perhaps polarizing the plasma membrane.27 Cell stimulation may also result from upregulation of autocrine motility factor that is known to activate small Rho-like GTPases and subsequently induce actin fiber reorganization.28
The electric field experiments were performed over a 2 h period. Most studies only report cell data at the final time point; however, cell migration trends at shorter times may also be significant. We observed that OM cells traveled quickest in the last 30 min of stimulation. Acceleration in cell speed was absent in IM cells exposed to electric fields and in both IM and OM control groups where cell migration occurred at a uniform pace through the 2 h period. These findings indicate meniscus cells may require priming for longer periods to garner maximum cell displacement and directed velocity. Alternatively, other stimuli such as growth factors may be used synergistically with electric fields to enhance and direct cell migration.29
Passaged meniscus cells were used as they are common in tissue engineering studies where primary cells are scarce and must be expanded to attain sufficient cell numbers. Enhanced cell speed and directed cathodal velocity was observed with increasing passage number. This was not surprising as meniscus cells differentiate with passage towards a fibroblastic lineage,30 and fibroblasts are known to migrate quicker than chondrocytes.14, 15
Our results have direct implications for meniscus tissue engineering studies in which spatial sorting of cells within a scaffold system is important and may influence functional properties of the construct. A scaffold can be devised to include primary or passaged cells from both the IM and OM. Differences in cell migration speed between OM and IM cells may affect desired spatial cell sorting within the scaffold after electric field stimulation. This concept has been examined for fibroblast migration within a 3D-collagen gel.31 In another study by the same group comparing fibroblasts and bone marrow-derived mesenchymal stem cells, the authors showed that fibroblasts were able to reorient the collagen matrix to a greater degree than were the mesenchymal stem cells.32 This result is consistent with the higher adhesion strengths of the mesenchymal stem cells, which presumably allows them to resist reorientation in the field. Alternatively, surface properties of the scaffold may be modulated to achieve desired adhesion strengths required for optimal cell migration or reorientation. Thus, our study can be used as a platform for future meniscus tissue engineering studies utilizing primary or passaged meniscus cells for scaffold-based tissue engineering.
In conclusion, we demonstrated that applied electric fields can significantly enhance and direct IM and OM cell migration. To build on these results, future experiments should examine meniscus migration on ECM substrates and through dense matrices, as substrates can influence cell motility and galvanotactic response.15, 33 An examination of the effect of electric fields on co-cultures of meniscus cells and synoviocytes may also provide a more realistic picture, as synoviocytes aid in meniscus repair.13 In addition, in vivo techniques used in other applications34–36 to generate and quantify electric fields will need to be further developed for the meniscus repair scenario.