High resolution imaging of the distribution and permeability of methyl viologen dication in bovine articular cartilage using scanning electrochemical microscopy

Abstract

Scanning electrochemical microscopy (SECM) has been used in the induced transfer (SECMIT) mode to image the permeability of a probe cation, methyl viologen (MV²⁺), in samples of articular cartilage. An ultramicroelectrode (UME), scanned just above the surface of a sample, is used to amperometrically detect the probe solute. The resulting depletion of MV²⁺ in solution induces the transfer of this cation from the sample into the solution for detection at the UME. The current provides quantitative information on local permeability, provided that the sample-UME distance is known. It is shown that the necessary topographical information can be obtained using the amperometric response for the oxidation of Ru(CN)⁴⁻₆, which does not permeate into the cartilage matrix. This procedure was validated by marking samples in situ, after electrochemical imaging, with subsequent examination by ex situ interferometry and optical microscopy. Wide variations in the permeability of MV²⁺ have been detected by SECMIT. These observations represent the first demonstration of the inhomogeneous permeability of a cation in cartilage on a micrometre scale. The permeability maps show similar features to the proteoglycan distribution, identified by toluidine blue staining, and it is likely that proteoglycans are the main determinant of MV²⁺ permeability in articular cartilage.

Introduction

Permeability is a key factor governing transport rates in biological membranes and tissues [1]. Articular cartilage is a specialised tissue for which permeability and fluid transport have been particularly well studied [2], [3], [4], [5], due to the widespread belief that impaired mass transport is involved in diseases such as rheumatoid arthritis and osteoarthritis [6], [7]. Cartilage comprises a porous supporting framework of collagen fibres, embedded in a gel composed of cells (chondrocytes), interstitial water and anionic proteoglycan molecules which confer a net negative fixed charge structure [8]. The exact proportion of each of the above components varies between joints, with depth within a joint and with age [9], and is altered by disease [10]. The largest variations are observed in the proportion of proteoglycans [2], [11], [12], which is of particular importance since these macromolecules influence significantly the transport of solutes and water through the cartilage matrix [13], [14], [15], [16] as well as the viscoelastic properties of the tissue [17]. Some of the transport effects observed for small ions have been explained in terms of a microscopically inhomogeneous fixed negative charge distribution [14], but the experimental techniques available at the time lacked the spatial resolution to confirm this hypothesis.

The question of heterogeneity in transport has been addressed partially by taking advantage of the variations in tissue properties which occur with depth, to compare transport through tissue slices obtained at different depths within the tissue [3], [14], [15]. However, data were still averaged across the surface of a slice, thereby neglecting any lateral inhomogeneities in transport rates.

More recently, magnetic resonance imaging (MRI) has emerged as a useful tool for the study of biological tissues such as cartilage [18]. A number of measurements of water and solute diffusion in cartilage have been reported [19], [20], [21], [22]. The diffusivities of small singly charged anions and cations were found to be lower in cartilage than in free solution [19], in agreement with the findings of Maroudas [13]. Potter et al. [22] monitored the diffusion of the divalent cation, Cu²⁺, in bovine nasal cartilage and found that the diffusion coefficient was inversely related to the fixed charge density of the tissue. The fixed charge density and proteoglycan distribution in cartilage have been mapped using MRI by investigating the electrostatic interaction of charged species, including Mn²⁺ [23], Na⁺ [24], [25] and gadolinium diethylenetriaminepentaacetic acid [26], [27], with the tissue matrix. Although MRI has the advantage of being non-invasive, with in vivo capabilities, the imaging resolution in complex tissues, such as cartilage, is limited by the low signal to noise ratio. An in plane resolution of 250–500 μm is typical [18], although higher resolutions (up to 26 μm [22]) have been reported for transport measurements.

Scanning electrochemical microscopy (SECM) is a powerful technique for examining the local transport of solutes by diffusion, convection and migration [28], [29]. In SECM, a mobile ultramicroelectrode (UME) [30], positioned close to an interface with submicron precision, can be used to probe the topography, reactivity or permeability of that interface with high spatial resolution [31], [32], [33]. SECM has been applied to the study of a number of membranes and biomaterials including skin [34], [35], [36], [37], [38], [39], dentine [40], [41], [42] and bilayer lipid membranes and cells [43], [44], [45], [46], [47]. In humid environments nanometre resolution is attainable, as evidenced by imaging studies of DNA [48]. SECM has the advantage over scanning ion conductance microscopy, which has found some application in the investigation of membrane transport [49], [50], in that it can selectively detect both neutral and charged species, rather than measure total ion currents.

We have used SECM to image osmotically driven convective transport through laryngeal cartilage [51] and were able, for the first time, to correlate local convective fluxes with sample morphology on a microscopic scale. More recently, we have mapped localised oxygen permeability in bovine articular cartilage samples [52] using an SECM induced transfer (SECMIT) approach [53]. In SECMIT, the partitioning of a target solute between a bathing solution and a tissue sample is allowed to come to equilibrium. A UME, positioned in the bathing solution close to the sample surface, is biased at a potential to deplete the local concentration of solute at the tip via diffusion-limited electrolysis [52], [53] or ion transfer [54]. This perturbs the equilibrium, driving the local transfer of solute from the tissue, thereby influencing the current flow to the UME probe. This approach allows measurements of diffusion, concentration or permeability of the solute in the tissue without the need to enter or contact the sample. This is particularly advantageous for measurements in biological tissues, where sample integrity is paramount. In our previous study, we were able to correlate spatially localised permeability with cellular topography, demonstrating that oxygen showed enhanced permeability in regions where cells were located, corresponding to areas where the distribution of collagen was most sparse.

In this paper, we report SECMIT studies aimed at quantitatively examining the permeability of a cationic species, methyl viologen (MV²⁺), in articular cartilage. This choice of solute enables us to elucidate the interaction of cations with the negatively charged cartilage matrix. The quantitative application of SECMIT requires that the topography of the cartilage sample can be measured under the same conditions as the permeability measurements. This is achieved by carrying out hindered diffusion scans [55] with a negatively charged mediator which does not permeate into the cartilage matrix [52]. To validate this method, we have used a technique to mark the area of cartilage imaged by SECM for subsequent ex situ quantitative examination of surface topography using vertical scanning interferometry.