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Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.

Original publication

DOI

10.1117/1.jbo.19.1.015001

Type

Journal article

Journal

Journal of Biomedical Optics

Publication Date

01/2014

Volume

19

Addresses

University of Oxford, Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Oxford OX3 7LD, United Kingdom.

Keywords

Tendons, Cells, Cultured, Hela Cells, Gap Junctions, Humans, Glycyrrhetinic Acid, Carbenoxolone, Collagen, Imaging, Three-Dimensional, Fluorescence Recovery After Photobleaching, Cell Culture Techniques, Immunohistochemistry, Cell Communication, Diffusion, Adult, Middle Aged, Female, Male