52 IN VITROANDEX VIVOASSESSMENT OF GAP JUNCTION FUNCTION IN TENDON USING FRAP

Abstract

Introduction Gap junctions (GJ) are intercellular channels that are found in nearly all cell types. They play an essential role in cell-to-cell communication. By enabling the exchange of ions and small metabolites between adjacent cells, they coordinate the activities of individual cells and synchronise cellular response within a tissue. Although GJ are indispensable for many processes including impulse propagation in heart, their role and regulation in tissues like tendon has not been sufficiently studied. Tendons act as force transmitters between muscle and bone. They consist of bundles of collagen with cells (tenocytes) aligned in longitudinal rows separated by collagen fibres. Tenocytes wrap around these fibres forming an elaborate network of cell processes linked by GJ. The aim of the present study was to establish and subsequently validate a method to study gap junction-mediated intercellular communication in tendon-derived cells as well as in whole tissue samples. Fluorescence recovery after photobleaching (FRAP) is a non-invasive technique that allows quantitative assessment of GJ function in living cells. It measures the redistribution of a GJ permeable fluorescent dye following photobleaching. Methods Tenocytes were isolated by explant culture of human hamstring tendons. The cells were cultured in monolayer or 3D collagen gels, loaded with green-fluorescent dye calcein and subjected to photobleaching using Zeiss LSM710 Confocal Microscope. A time-lapse series was subsequently recorded to monitor the transfer of calcein between the cells. Mobile fraction percentage was calculated to quantify the gap junctional communication. In ex vivo experiments individual tendon fascicles were isolated from mouse tail tendons and immobilised onto an imaging dish with fibrin sealant before they were subjected to FRAP analysis. GJ were blocked by pre-treating the samples with 18β-glycyrrhetinic acid (GA) or carbenoxolone (CBX). Results Fluorescently labelled tenocytes subjected to photobleaching rapidly re-acquired the fluorescent dye from adjacent cells. The recovery however was dependent on the number of cell-to-cell connexions. In both monolayer and 3D cultures, isolated cells showed a decrease in intercellular communication when compared to cells in confluent cultures. The mobile fraction percentage for well-connected cells reached about 70%. HeLa cells, which do not communicate via gap junctions, served as a control for the experiments and accordingly remained permanently bleached. The mobile fraction values obtained for the ex vivo tissue were comparable with in vitro cultured tenocytes. Standard inhibitors of GJ activity, GA and CBX, impaired fluorescence recovery both in vitro and ex vivo . Previously, it has been described that mechanical stimulation regulates GJ function in rat tendons. In order to investigate the GJ communication in presence of mechanical loading in human, fluorescently labelled tenocytes were subjected to sheer stress using a see-saw rocker and subsequently photobleached. Fluid sheer stress reduced the florescence recovery demonstrating that mechanical loading can regulate GJ function in human tenocytes. Discussion In this study we showed that FRAP provides a tool to study the function of GJ in tendon both in vitro and ex vivo . Furthermore, our results demonstrated that GJ function in a native tendon can be quantified and experimentally manipulated in the same way as in in vitro cultured tenocytes.